US20230020362A1 - Infectious disease vaccines - Google Patents

Abstract

Aspects of the disclosure relate to nucleic acid vaccines. The vaccines include one or more RNA polynucleotides having an open reading frame encoding one or more Chikungunya antigen(s), one or more Zika virus antigens, and one or more Dengue antigens. Methods for preparing and using such vaccines are also described.

Description

RELATED APPLICATIONS
• This application is a continuation of U.S. application Ser. No. 16/009,880, filed Jun. 15, 2018, which is a continuation of U.S. application Ser. No. 15/746,286, filed Jan. 19, 2018, which is a national stage filing under 35 U.S.C. § 371 of international application number PCT/US2016/043348, filed Jul. 21, 2016, which claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application No. 62/357,806, filed Jul. 1, 2016, U.S. provisional application No. 62/351,200, filed Jun. 16, 2016, U.S. provisional application No. 62/351,244, filed Jun. 16, 2016, U.S. provisional application No. 62/351,267, filed Jun. 16, 2016, U.S. provisional application No. 62/351,148, filed Jun. 16, 2016, U.S. provisional application No. 62/351,206, filed Jun. 16, 2016, U.S. provisional application No. 62/303,666, filed Mar. 4, 2016, U.S. provisional application No. 62/303,405, filed Mar. 4, 2016, U.S. provisional application No. 62/247,551, filed Oct. 28, 2015, U.S. provisional application No. 62/247,527, filed Oct. 28, 2015, U.S. provisional application No. 62/247,660, filed Oct. 28, 2015, U.S. provisional application No. 62/247,644, filed Oct. 28, 2015, U.S. provisional application No. 62/247,581, filed Oct. 28, 2015, U.S. provisional application No. 62/245,179, filed Oct. 22, 2015, U.S. provisional application No. 62/244,995, filed Oct. 22, 2015, U.S. provisional application No. 62/244,855, filed Oct. 22, 2015, U.S. provisional application No. 62/244,859, filed Oct. 22, 2015, U.S. provisional application No. 62/245,233, filed Oct. 22, 2015, U.S. provisional application No. 62/241,699, filed Oct. 14, 2015, U.S. provisional application No. 62/199,204, filed Jul. 30, 2015, and U.S. provisional application No. 62/195,263, filed Jul. 21, 2015, each of which is incorporated by reference herein in its entirety.
BACKGROUND OF INVENTION
• Chikungunya virus (CHIKV) is a mosquito-borne virus belonging to the Alphavirus genus of the Togaviridae family that was first isolated in 1953 in Tanzania, where the virus was endemic. Outbreaks occur repeatedly in west, central, and southern Africa and have caused several human epidemics in those areas since that time. The virus is passed to humans by two species of mosquito of the genus Aedes: A. albopictus and A. aegypti. There are several Chikungunya genotypes: Indian Ocean, East/Central/South African (ECSA), Asian, West African, and Brazilian.
• Presently, CHIKV is a re-emerging human pathogen that has now established itself in Southeast Asia and has more recently spread to Europe. The Chikungunya virus (CHIKV) was introduced into Asia around 1958, and sites of endemic transmission within Southeastern Asia, including the Indian Ocean, were observed through 1996. The CHIKV epidemic moved throughout Asia, reaching Europe and Africa in the early 2000s, and was imported via travelers to North America and South America from 2005 to 2007. Sporadic outbreaks are still occurring in several countries, such as Italy, inflicting naive populations. Singapore, for instance, experienced two successive waves of Chikungunya virus outbreaks in January and August 2008. Of the two strain lineages of CHIKV, the African strain remains enzootic by cycling between mosquitoes and monkeys, but the Asian strain is transmitted directly between mosquitoes and humans. This cycle of transmission may have allowed the virus to become more pathogenic as the reservoir host was eliminated.
• In humans, CHIKV causes a debilitating disease characterized by fever, headache, nausea, vomiting, fatigue, rash, muscle pain and joint pain. Following the acute phase of the illness, patients develop severe chronic symptoms lasting from several weeks to months, including fatigue, incapacitating joint pain and polyarthritis.
• The re-emergence of CHIKV has caused millions of cases throughout countries around the Indian Ocean and in Southeast Asia. Specifically, India, Indonesia, Maldives, Myanmar and Thailand have reported over 1.9 million cases since 2005. Globally, human CHIKV epidemics from 2004-2011 have resulted in 1.4-6.5 million reported cases, including a number of deaths. Thus, CHIKV remains a public threat that constitutes a major public health problem with severe social and economic impact.
• Despite significant morbidity and some cases of mortality associated with CHIKV infection and its growing prevalence and geographic distribution, there is currently no licensed CHIKV vaccine or antiviral approved for human use. Several potential CHIKV vaccine candidates have been tested in humans and animals with varying success.
• Dengue virus (DENV) is a mosquito-borne (Aedes aegypti/Aedes albopictus) member of the family Flaviviridae (positive-sense, single-stranded RNA virus). Dengue virus is a positive-sense RNA virus of the Flavivirus genus of the Flaviviridae family, which also includes West Nile virus, Yellow Fever Virus, and Japanese Encephalitis virus. It is transmitted to humans through Stegomyia aegypti (formerly Aedes) mosquito vectors and is mainly found in the tropical and semitropical areas of the world, where it is endemic in Asia, the Pacific region, Africa, Latin America, and the Caribbean. The incidence of infections has increased 30-fold over the last 50 years (WHO, Dengue: Guidelines for diagnosis, treatment, prevention, and control (2009)) and Dengue virus is the second most common tropical infectious disease worldwide after malaria.
• There is no specific treatment for DENV infection, and control of DENV by vaccination has proved elusive, in part, because the pathogenesis of DHF/DSS is not completely understood. While infection with one serotype confers lifelong homotypic immunity, it confers only short term (approximately three to six months) cross protection against heterotypic serotypes. Also, there is evidence that prior infection with one type can produce an antibody response that can intensify, or enhance, the course of disease during a subsequent infection with a different serotype. The possibility that vaccine components could elicit enhancing antibody responses, as opposed to protective responses, has been a major concern in designing and testing vaccines to protect against dengue infections.
• In late 2015 and early 2016, the first dengue vaccine, Dengvaxia (CYD-TDV) by Sanofi Pasteur, was registered in several countries for use in individuals 9-45 years of age living in endemic areas. Issues with the vaccine include (1) weak protection against DENV1 and DENV2 (<60% efficacy); (2) relative risk of dengue hospitalization among children <9 years old (7.5× higher than placebo); (3) immunogenicity not sustained after 1-2 years (implying the need for a 4^th dose booster); and (4) lowest efficacy against DENV2, which often causes more severe conditions. This latter point is a major weakness with the Dengvaxia vaccine, signaling the need of a new, more effective vaccine effective against DENV2. Other tetravalent live-attenuated vaccines are under development in phase II and phase III clinical trials, and other vaccine candidates (based on subunit, DNA and purified inactivated virus platforms) are at earlier stages of clinical development, although the ability of these vaccine candidates to provide broad serotype protection has not been demonstrated.
• Zika virus (ZIKV) is a member of the Flaviviridae virus family and the flavivirus genus. In humans, it causes a disease known as Zika fever. It is related to dengue, yellow fever, West Nile and Japanese encephalitis, viruses that are also members of the virus family Flaviviridae. ZIKV is spread to people through mosquito bites. The most common symptoms of ZIKV disease (Zika) are fever, rash, joint pain, and red eye. The illness is usually mild with symptoms lasting from several days to a week. There is no vaccine to prevent, or medicine to treat, Zika virus.
• Deoxyribonucleic acid (DNA) vaccination is one technique used to stimulate humoral and cellular immune responses to foreign antigens, such as ZIKV antigens. The direct injection of genetically engineered DNA (e.g., naked plasmid DNA) into a living host results in a small number of its cells directly producing an antigen, resulting in a protective immunological response. With this technique, however, comes potential problems, including the possibility of insertional mutagenesis, which could lead to the activation of oncogenes or the inhibition of tumor suppressor genes.
SUMMARY OF INVENTION
• Provided herein is a ribonucleic acid (RNA) vaccine (e.g., messenger RNA (mRNA)) that can safely direct the body's cellular machinery to produce nearly any protein of interest, from native proteins to antibodies and other entirely novel protein constructs that can have therapeutic activity inside and outside of cells. The RNA vaccines of the present disclosure may be used to induce a balanced immune response against a single virus or multiple viruses, including Chikungunya virus (CHIKV), Zika Virus (ZIKV) and Dengue virus (DENV), comprising both cellular and humoral immunity, without the associated safety concerns, e.g., risking the possibility of insertional mutagenesis.
• Some embodiments of the present disclosure provide vaccines and/or combination vaccines comprising one or more RNA polynucleotides, e.g., mRNA. In some embodiments, the RNA polynucleotide(s) encode a CHIKV antigen, a ZIKV antigen, a DENV antigen, or any combination of two or three of the foregoing (e.g., CHIKV antigen/ZIKV antigen, CHIKV antigen/DENV antigen, ZIKV antigen/DENV antigen, or CHIKV/DENV/ZIKV) on either the same polynucleotide or different polynucleotides. In some embodiments, the RNA polynucleotide(s) encode a ZIKV antigen and a DENV antigen, on either the same polynucleotide or different polynucleotides.
• Thus, it should be understood the phrase “a CHIKV, DENV and/or ZIKV” is intended to encompass each individual virus in the alternative (CHIKV or DENV or ZIKV) as well as the individual combinations of CHIKV and DENV (CHIKV/DENV), CHIKV and ZIKV (CHIKV/ZIKV), ZIKV and DENV (ZIKV/DENV), and CHIKV, DENV and ZIKV (CHIKV/DENV/ZIKV).
• In some aspects, the present disclosure provides a vaccine or a combination vaccine of at least one RNA polynucleotide encoding at least one CHIKV antigenic polypeptide, at least one ZIKV antigenic polypeptide, at least one DENV antigenic polypeptide, or a combination of any two or three of the foregoing, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the RNA polynucleotides encoding the DENV antigenic polypeptide, the ZIKV antigenic polypeptide and/or the CHIKV antigenic polypeptide are mono-cistronic RNA polynucleotides. In other embodiments, the RNA polynucleotide encoding the DENV antigenic polypeptide, the ZIKV antigenic polypeptide and/or the CHIKV antigenic polypeptide is a poly-cistronic. In other embodiments, the RNA polynucleotides include combinations of mono-cistronic and poly-cistronic RNA.
• In some aspects, the present disclosure provides a vaccine or a combination vaccine of at least one RNA polynucleotide encoding at least one ZIKV antigenic polypeptide and at least one DENV antigenic polypeptide and a pharmaceutically acceptable carrier or excipient. In some embodiments, the RNA polynucleotides encoding the ZIKV antigenic polypeptide and the DENV antigenic polypeptide are mono-cistronic RNA polynucleotides. In other embodiments, the RNA polynucleotide encoding the ZIKV antigenic polypeptide and the DENV antigenic polypeptide is a poly-cistronic RNA polynucleotide. In other embodiments, the RNA polynucleotides include combinations of mono-cistronic and poly-cistronic RNA.
• In some aspects, the present disclosure provides a vaccine or a combination vaccine of at least one RNA polynucleotide encoding at least one ZIKV antigenic polypeptide and at least one CHIKV antigenic polypeptide and a pharmaceutically acceptable carrier or excipient. In some embodiments, the RNA polynucleotides encoding the ZIKV antigenic polypeptide and the CHIKV antigenic polypeptide are mono-cistronic RNA polynucleotides. In other embodiments, the RNA polynucleotide encoding the ZIKV antigenic polypeptide and the CHIKV antigenic polypeptide is a poly-cistronic RNA polynucleotide. In other embodiments, the RNA polynucleotides include combinations of mono-cistronic and poly-cistronic RNA.
• In some aspects, the present disclosure provides a vaccine or a combination vaccine of at least one RNA polynucleotide encoding at least one DENV antigenic polypeptide and at least one CHIKV antigenic polypeptide and a pharmaceutically acceptable carrier or excipient. In some embodiments, the RNA polynucleotides encoding the DENV antigenic polypeptide and the CHIKV antigenic polypeptide are mono-cistronic RNA polynucleotides.
• In other embodiments, the RNA polynucleotide encoding the DENV antigenic polypeptide and the CHIKV antigenic polypeptide is a poly-cistronic RNA polynucleotide. In other embodiments, the RNA polynucleotides include combinations of mono-cistronic and poly-cistronic RNA.
• The at least one RNA polynucleotide, e.g., mRNA, in some embodiments, encodes two or more CHIKV antigenic polypeptides, two or more ZIKV antigenic polypeptides or two or more DENV antigenic polypeptides. The at least one RNA polynucleotide, e.g., mRNA, in some embodiments, encodes two or more CHIKV antigenic polypeptides, two or more ZIKV antigenic polypeptides and two or more DENV antigenic polypeptides. In some embodiments, the at least one RNA polynucleotide, e.g., mRNA, encodes two or more ZIKV antigenic polypeptides and two or more DENV antigenic polypeptides. In some embodiments, the at least one RNA polynucleotide, e.g., mRNA, encodes two or more ZIKV antigenic polypeptides and two or more CHIKV antigenic polypeptides. In some embodiments, the at least one RNA polynucleotide, e.g., mRNA, encodes two or more CHIKV antigenic polypeptides and two or more DENV antigenic polypeptides.
• The CHIKV antigenic polypeptide may be a Chikungunya structural protein or an antigenic fragment or epitope thereof. The DENV antigenic polypeptide may be a Dengue virus (DENV) structural protein or an antigenic fragment or epitope thereof. The ZIKV antigenic polypeptide may be a Zika virus (ZIKV) structural protein (e.g., polyprotein) or an antigenic fragment or epitope thereof.
• In some embodiments, the antigenic polypeptide is a CHIKV structural protein or an antigenic fragment thereof. For example, a CHIKV structural protein may be an envelope protein (E), a 6K protein, or a capsid (C) protein. In some embodiments, the CHIKV structural protein is an envelope protein selected from E1, E2, and E3. In some embodiments, the CHIKV structural protein is E1 or E2. In some embodiments, the CHIKV structural protein is a capsid protein. In some embodiments, the antigenic polypeptide is a fragment or epitope of a CHIKV structural protein.
• In some embodiments, at least one antigenic polypeptide is a ZIKV polyprotein. In some embodiments, at least one antigenic polypeptide is a ZIKV structural polyprotein. In some embodiments, at least one antigenic polypeptide is a ZIKV nonstructural polyprotein.
• In some embodiments, at least one antigenic polypeptide is a ZIKV capsid protein, a ZIKV premembrane/membrane protein, a ZIKV envelope protein, a ZIKV non-structural protein 1, a ZIKV non-structural protein 2A, a ZIKV non-structural protein 2B, a ZIKV non-structural protein 3, a ZIKV non-structural protein 4A, a ZIKV non-structural protein 4B, or a ZIKV non-structural protein 5.
• In some embodiments, at least one antigenic polypeptide is a ZIKV capsid protein, a ZIKV premembrane/membrane protein, a ZIKV envelope protein, a ZIKV non-structural protein 1, a ZIKV non-structural protein 2A, a ZIKV non-structural protein 2B, a ZIKV non-structural protein 3, a ZIKV non-structural protein 4A, a ZIKV non-structural protein 4B, or a ZIKV non-structural protein 5.
• In some embodiments, the vaccine comprises a RNA polynucleotide having an open reading frame encoding a ZIKV capsid protein, a RNA polynucleotide having an open reading frame encoding a ZIKV premembrane/membrane protein, and a RNA polynucleotide having an open reading frame encoding a ZIKV envelope protein.
• In some embodiments, the vaccine comprises a RNA polynucleotide having an open reading frame encoding a ZIKV capsid protein and a RNA polynucleotide having an open reading frame encoding a ZIKV premembrane/membrane protein.
• In some embodiments, the vaccine comprises a RNA polynucleotide having an open reading frame encoding a ZIKV capsid protein and a RNA polynucleotide having an open reading frame encoding a ZIKV envelope protein.
• In some embodiments, the vaccine comprises a RNA polynucleotide having an open reading frame encoding a ZIKV premembrane/membrane protein and a RNA polynucleotide having an open reading frame encoding a ZIKV envelope protein.
• In some embodiments, the vaccine comprises a RNA polynucleotide having an open reading frame encoding a ZIKV capsid protein and at least one RNA polynucleotide having an open reading frame encoding any one or more of a ZIKV non-structural protein 1, 2A, 2B, 3, 4A, 4B or 5.
• In some embodiments, the vaccine comprises a RNA polynucleotide having an open reading frame encoding a ZIKV premembrane/membrane protein and at least one RNA polynucleotide having an open reading frame encoding any one or more of a ZIKV non-structural protein 1, 2A, 2B, 3, 4A, 4B or 5.
• In some embodiments, the vaccine comprises a RNA polynucleotide having an open reading frame encoding a ZIKV envelope protein and at least one RNA polynucleotide having an open reading frame encoding any one or more of a ZIKV non-structural protein 1, 2A, 2B, 3, 4A, 4B or 5.
• In some embodiments, the at least one antigenic polypeptide comprises a combination of any two or more of a ZIKV capsid protein, a ZIKV premembrane/membrane protein, a ZIKV envelope protein, a ZIKV non-structural protein 1, a ZIKV non-structural protein 2A, a ZIKV non-structural protein 2B, a ZIKV non-structural protein 3, a ZIKV non-structural protein 4A, a ZIKV non-structural protein 4B, or a ZIKV non-structural protein 5.
• In some embodiments, the at least one ZIKV antigenic polypeptide is fused to signal peptide having a sequence set forth as SEQ ID NO: 125, 126, 128 or 131. In some embodiments, the signal peptide is fused to the N-terminus of the at least one ZIKV antigenic polypeptide.
• In some embodiments, the antigenic polypeptide comprises two or more CHIKV structural proteins. In some embodiments, the two or more CHIKV structural proteins are envelope proteins. In some embodiments, the two or more CHIKV structural proteins are E1 and E2. In some embodiments, the two or more CHIKV structural proteins are E1 and E3. In some embodiments, the two or more CHIKV structural proteins are E2 and E3. In some embodiments, the two or more CHIKV structural proteins are E1, E2, and E3. In some embodiments, the two or more CHIKV structural proteins are envelope and capsid proteins. In some embodiments, the two or more CHIKV structural proteins are E1 and C. In some embodiments, the two or more CHIKV structural proteins are E2 and C. In some embodiments, the two or more CHIKV structural proteins are E3 and C. In some embodiments, the two or more CHIKV structural proteins are E1, E2, and C. In some embodiments, the two or more CHIKV structural proteins are E1, E3, and C. In some embodiments, the two or more CHIKV structural proteins are E2, E3, and C. In some embodiments, the two or more CHIKV structural proteins are E1, E2, E3, and C. In some embodiments, the two or more CHIKV structural proteins are E1, 6K, and E2. In some embodiments, the two or more CHIKV structural proteins are E2, 6K, and E3. In some embodiments, the two or more CHIKV structural proteins are E1, 6K, and E3. In some embodiments, the two or more CHIKV structural proteins are E1, E2, E3, 6K, and C. In some embodiments, the antigenic polypeptide comprises the CHIKV structural polyprotein comprising C, E3, E2, 6K, and E1. In some embodiments, the antigenic polypeptide is a fragment or epitope of two or more CHIKV structural proteins or a fragment or epitope of the polyprotein.
• In some embodiments the at least one antigenic polypeptide has greater than 90% identity to an amino acid sequence of any one of Tables 13, 15, 18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15, 17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and has membrane fusion activity. In some embodiments the at least one CHIKV antigenic polypeptide has greater than 90% identity to an amino acid sequence of any one of SEQ ID NO: 14 or 37-47 and has membrane fusion activity. In some embodiments the at least one DENV antigenic polypeptide has greater than 90% identity to an amino acid sequence of any one of SEQ ID NO: 15, 17, 19, 21, 23, 26, 29, 32, 162-298 and has membrane fusion activity. In some embodiments the at least one ZIKV antigenic polypeptide has greater than 90% identity to an amino acid sequence of any one of SEQ ID NO: 67-134 and has membrane fusion activity.
• In some embodiments the at least one antigenic polypeptide has greater than 95% identity to an amino acid sequence of any one of Tables 13, 15, 18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15, 17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and has membrane fusion activity. In some embodiments the at least one CHIKV antigenic polypeptide has greater than 95% identity to an amino acid sequence of any one of SEQ ID NO: 14 or 37-47 and has membrane fusion activity. In some embodiments the at least one DENV antigenic polypeptide has greater than 95% identity to an amino acid sequence of any one of SEQ ID NO: 15, 17, 19, 21, 23, 26, 29, 32, or 162-298 and has membrane fusion activity. In some embodiments the at least one ZIKV antigenic polypeptide has greater than 95% identity to an amino acid sequence of any one of SEQ ID NO: 67-134 and has membrane fusion activity.
• In some embodiments the at least one antigenic polypeptide has greater than 96% identity to an amino acid sequence of any one of Tables 13, 15, 18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15, 17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and has membrane fusion activity. In some embodiments the at least one CHIKV antigenic polypeptide has greater than 96% identity to an amino acid sequence of any one of SEQ ID NO: 14 or 37-47 and has membrane fusion activity. In some embodiments the at least one DENV antigenic polypeptide has greater than 96% identity to an amino acid sequence of any one of SEQ ID NO: 15, 17, 19, 21, 23, 26, 29, 32, or 162-298 and has membrane fusion activity. In some embodiments the at least one ZIKV antigenic polypeptide has greater than 96% identity to an amino acid sequence of any one of SEQ ID NO: 67-134 and has membrane fusion activity.
• In some embodiments the at least one antigenic polypeptide has greater than 97% identity to an amino acid sequence of any one of Tables 13, 15, 18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15, 17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and has membrane fusion activity. In some embodiments the at least one CHIKV antigenic polypeptide has greater than 97% identity to an amino acid sequence of any one of SEQ ID NO: 14 or 37-47 and has membrane fusion activity. In some embodiments the at least one DENV antigenic polypeptide has greater than 97% identity to an amino acid sequence of any one of SEQ ID NO: 15, 17, 19, 21, 23, 26, 29, 32, or 162-298 and has membrane fusion activity. In some embodiments the at least one ZIKV antigenic polypeptide has greater than 97% identity to an amino acid sequence of any one of SEQ ID NO: 67-134 and has membrane fusion activity.
• In some embodiments the at least one antigenic polypeptide has greater than 98% identity to an amino acid sequence of any one of Tables 13, 15, 18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15, 17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and has membrane fusion activity. In some embodiments the at least one CHIKV antigenic polypeptide has greater than 98% identity to an amino acid sequence of any one of SEQ ID NO: 14 or 37-47 and has membrane fusion activity. In some embodiments the at least one DENV antigenic polypeptide has greater than 98% identity to an amino acid sequence of any one of SEQ ID NO: 15, 17, 19, 21, 23, 26, 29, 32, or 162-298 and has membrane fusion activity. In some embodiments the at least one ZIKV antigenic polypeptide has greater than 98% identity to an amino acid sequence of any one of SEQ ID NO: 67-134 and has membrane fusion activity.
• In some embodiments the at least one antigenic polypeptide has greater than 99% identity to an amino acid sequence of any one of Tables 13, 15, 18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15, 17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and has membrane fusion activity. In some embodiments the at least one CHIKV antigenic polypeptide has greater than 99% identity to an amino acid sequence of any one of SEQ ID NO: 14 or 37-47 and has membrane fusion activity. In some embodiments the at least one DENV antigenic polypeptide has greater than 99% identity to an amino acid sequence of any one of SEQ ID NO: 15, 17, 19, 21, 23, 26, 29, 32, or 162-298 and has membrane fusion activity. In some embodiments the at least one ZIKV antigenic polypeptide has greater than 99% identity to an amino acid sequence of any one of SEQ ID NO: 67-134 and has membrane fusion activity.
• In some embodiments the at least one antigenic polypeptide has greater than 95-99% identity to an amino acid sequence of any one of Tables 13, 15, 18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15, 17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and has membrane fusion activity. In some embodiments the at least one CHIKV antigenic polypeptide has greater than 95-99% identity to an amino acid sequence of any one of SEQ ID NO: 14 or 37-47 and has membrane fusion activity. In some embodiments the at least one DENV antigenic polypeptide has greater than 95-99% identity to an amino acid sequence of any one of SEQ ID NO: 15, 17, 19, 21, 23, 26, 29, 32, or 162-298 and has membrane fusion activity. In some embodiments the at least one ZIKV antigenic polypeptide has greater than 95-99% identity to an amino acid sequence of any one of SEQ ID NO: 67-134 and has membrane fusion activity.
• In other embodiments the at least one antigenic polypeptides encode an antigenic polypeptide having an amino acid sequence of Tables 13, 15, 18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15, 17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and wherein the RNA polynucleotide is codon optimized mRNA. In yet other embodiments the at least one antigenic polypeptide has an amino acid sequence of Tables 13, 15, 18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15, 17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and wherein the RNA polynucleotide has less than 80% identity to wild-type mRNA sequence. According to some embodiments the at least one antigenic polypeptide has an amino acid sequence of Tables 13, 15, 18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15, 17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and wherein the RNA polynucleotide has greater than 80% identity to wild-type mRNA sequence, but does not include wild-type mRNA sequence.
• In some embodiments, the DENV antigen is a concatemeric DENV antigen. In some embodiments, the DENV concatemeric antigen comprises between 2-100 DENV peptide epitopes connected directly to one another or interspersed by linkers. In some embodiments, the DENV vaccine's peptide epitopes are T cell epitopes and/or B cell epitopes. In other embodiments, the DENV vaccine's peptide epitopes comprise a combination of T cell epitopes and B cell epitopes. In some embodiments, at least one of the peptide epitopes of the DENV vaccine is a T cell epitope. In some embodiments, at least one of the peptide epitopes of the DENV vaccine is a B cell epitope. In some embodiments, the T cell epitope of the DENV vaccine comprises between 8-11 amino acids. In some embodiments, the B cell epitope of the DENV vaccine comprises between 13-17 amino acids.
• In some embodiments, the RNA polynucleotide, e.g., mRNA, of a vaccine is encoded by at least one polynucleotide comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to any of the nucleotide sequences of Tables 1-4, 13, 15, 31, 34 or 38, or any one of SEQ ID NO: 1-13 (CHIKV), 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33, 34, 144-152 or 199-212 (DENV), or 48-66 (ZIKV). In some embodiments, the RNA polynucleotide, e.g., mRNA, of a vaccine is encoded by at least one polynucleotide comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to any of the CHIKV nucleotide sequences of SEQ ID NO: 1-13. In some embodiments, the RNA polynucleotide, e.g., mRNA, of a vaccine is encoded by at least one polynucleotide comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to any of the DENV nucleotide sequences of SEQ ID NO: 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33, 34, 144-152 or 199-212. In some embodiments, the RNA polynucleotide, e.g., mRNA, of a vaccine is encoded by at least one polynucleotide comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to any of the ZIKV nucleotide sequences of SEQ ID NO: 67-134.
• In other embodiments, the RNA polynucleotide comprises a polynucleotide sequence derived from an Asian strain, Brazilian strain, West African strain, ECSA strain, and Indian Ocean strain of Chikungunya.
• In some embodiments, at least one antigenic polypeptide is a ZIKV envelope protein.
• In some embodiments, at least one antigenic polypeptide is a Spondweni virus Polyprotein.
• In some embodiments, at least one antigenic polypeptide is a polyprotein obtained from ZIKV strain MR 766, ACD75819 or SPH2015.
• In some embodiments, at least one antigenic polypeptide has an amino acid sequence of any one of the sequences listed in Table 32.
• In some embodiments, at least one antigenic polypeptide has at least 95% identity to an antigenic polypeptide having an amino acid sequence of any one of the sequences listed in Table 32.
• In some embodiments, the at least one RNA polynucleotide encodes at least one antigenic polypeptide having a sequence of listed in Table 31.
• In some embodiments, the at least one RNA polynucleotide encodes at least one protein variant having at least 95% identity to an antigenic polypeptide having a sequence of listed in Table 31.
• Tables herein provide National Center for Biotechnology Information (NCBI) accession numbers of interest. It should be understood that the phrase “an amino acid sequence of Table X” (e.g., Table 33 or Table 35) refers to an amino acid sequence identified by one or more NCBI accession numbers listed in Table X. Each of the amino acid sequences, and variants having greater than 95% identity to each of the amino acid sequences encompassed by the accession numbers of Table X (e.g., Table 33 or Table 35) are included within the constructs of the present disclosure.
• In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide having at least 90% identity to an amino acid sequence of Table 32 or 33 Table 32 or 33 and having membrane fusion activity. In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide having at least 95% identity to an amino acid sequence of Table 32 or 33 and having membrane fusion activity. In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide having at least 96% identity to an amino acid sequence of Table 32 or 33 and having membrane fusion activity. In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide having at least 97% identity to an amino acid sequence of Table 32 or 33 and having membrane fusion activity. In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide having at least 98% identity to an amino acid sequence of Table 32 or 33 and having membrane fusion activity. In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide having at least 99% identity to an amino acid sequence of Table 32 or 33 and having membrane fusion activity. In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide having 95-99% identity to an amino acid sequence of Table 32 or 33 and having membrane fusion activity.
• In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide having an amino acid sequence of Table 32 or 33 and is codon optimized mRNA.
• In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide having an amino acid sequence of Table 32 or 33 and has less than 80% identity to wild-type mRNA sequence. In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide having an amino acid sequence of Table 32 or 33 and has less than 75%, 85% or 95% identity to wild-type mRNA sequence. In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide having an amino acid sequence of Table 32 or 33 and has 50-80%, 60-80%, 40-80%, 30-80%, 70-80%, 75-80% or 78-80% identity to wild-type mRNA sequence. In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide having an amino acid sequence of Table 32 or 33 and has 40-85%, 50-85%, 60-85%, 30-85%, 70-85%, 75-85%, or 80-85% identity to wild-type mRNA sequence. In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide having an amino acid sequence of Table 32 or 33 and has 40-90%, 50-90%, 60-90%, 30-90%, 70-90%, 75-90%, 80-90%, or 85-90% identity to wild-type mRNA sequence.
• In some embodiments, at least one RNA polynucleotide is encoded by a nucleic acid having at least 90% identity to a nucleic acid sequence of Table 31. In some embodiments, at least one RNA polynucleotide is encoded by a nucleic acid having at least 95% identity to a nucleic acid sequence of Table 31. In some embodiments, at least one RNA polynucleotide is encoded by a nucleic acid having at least 96% identity to a nucleic acid sequence of Table 31. In some embodiments, at least one RNA polynucleotide is encoded by a nucleic acid having at least 97% identity to a nucleic acid sequence of Table 31. In some embodiments, at least one RNA polynucleotide is encoded by a nucleic acid having at least 98% identity to a nucleic acid sequence of Table 31. In some embodiments, at least one RNA polynucleotide is encoded by a nucleic acid having at least 99% identity to a nucleic acid sequence of Table 31. In some embodiments, at least one RNA polynucleotide is encoded by a nucleic acid having 95-99% identity to a nucleic acid sequence of Table 31.
• In some embodiments, at least one mRNA polynucleotide is encoded by a nucleic acid having a sequence of Table 31 and has less than 80% identity to wild-type mRNA sequence. In some embodiments, at least one mRNA polynucleotide is encoded by a nucleic acid having a sequence of Table 31 and has less than 75%, 85% or 95% identity to a wild-type mRNA sequence. In some embodiments, at least one mRNA polynucleotide is encoded by a nucleic acid having a sequence of Table 31 and has less than 50-80%, 60-80%, 40-80%, 30-80%, 70-80%, 75-80% or 78-80% identity to wild-type mRNA sequence. In some embodiments, at least one mRNA polynucleotide is encoded by a nucleic acid having a sequence of Table 31 and has less than 40-85%, 50-85%, 60-85%, 30-85%, 70-85%, 75-85% or 80-85% identity to wild-type mRNA sequence. In some embodiments, at least one mRNA polynucleotide is encoded by a nucleic acid having a sequence of Table 31 and has less than 40-90%, 50-90%, 60-90%, 30-90%, 70-90%, 75-90%, 80-90%, or 85-90% identity to wild-type mRNA sequence.
• In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide having an amino acid sequence of Table 32 or 33 and having at least 80% identity to wild-type mRNA sequence, but does not include wild-type mRNA sequence.
• In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide that attaches to cell receptors.
• In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide that causes fusion of viral and cellular membranes.
• In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide that is responsible for binding of the ZIKV to a cell being infected.
• Some embodiments of the present disclosure provide a CHIKV vaccine that includes at least one RNA polynucleotide having an open reading frame encoding a CHIKV antigenic polypeptides, in which the RNA polynucleotide of the CHIKV vaccine includes a 5′ terminal cap. Some embodiments of the present disclosure provide a DENV vaccine that includes at least one RNA polynucleotide having an open reading frame encoding a DENV antigenic polypeptides, in which the RNA polynucleotide of the DENV vaccine includes a 5′ terminal cap. Some embodiments of the present disclosure provide a ZIKV vaccine that includes at least one RNA polynucleotide having an open reading frame encoding a ZIKV antigenic polypeptides, in which the RNA polynucleotide of the ZIKV vaccine includes a 5′ terminal cap.
• Some embodiments of the present disclosure provide a CHIKV/DENV/ZIKV combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one each of CHIKV, DENV, and ZIKV antigenic polypeptides, in which the RNA polynucleotide of the CHIKV, DENV, and ZIKV RNA vaccine includes a 5′ terminal cap. Some embodiments of the present disclosure provide a DENV/ZIKV combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one each of DENV and ZIKV antigenic polypeptides, in which the RNA polynucleotide of the DENV, and ZIKV RNA vaccine includes a 5′ terminal cap. Some embodiments of the present disclosure provide a CHIKV/ZIKV combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one each of CHIKV and ZIKV antigenic polypeptides, in which the RNA polynucleotide of the CHIKV and ZIKV RNA vaccine includes a 5′ terminal cap. Some embodiments of the present disclosure provide a CHIKV/DENV combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one each of CHIKV and DENV antigenic polypeptides, in which the RNA polynucleotide of the CHIKV and DENV RNA vaccine includes a 5′ terminal cap. In some embodiments, the 5′ terminal cap is 7mG(5′)ppp(5′)NlmpNp.
• Some embodiments of the present disclosure provide a vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV antigenic polypeptide in which the RNA polynucleotide of the CHIKV RNA vaccine includes at least one chemical modification. Some embodiments of the present disclosure provide a vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one DENV antigenic polypeptide in which the RNA polynucleotide of the DENV RNA vaccine includes at least one chemical modification. Some embodiments of the present disclosure provide a vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one ZIKV antigenic polypeptide in which the RNA polynucleotide of the ZIKV RNA vaccine includes at least one chemical modification.
• Some embodiments of the present disclosure provide a combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV antigenic polypeptide, at least one DENV antigenic polypeptide, and at least one ZIKV antigenic polypeptide in which the RNA polynucleotide of the CHIKV/DENV/ZIKV combination RNA vaccine includes at least one chemical modification. Some embodiments of the present disclosure provide a combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV antigenic polypeptide and at least one DENV antigenic polypeptide in which the RNA polynucleotide of the CHIKV/DENV combination RNA vaccine includes at least one chemical modification. Some embodiments of the present disclosure provide a combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV antigenic polypeptide and at least one ZIKV antigenic polypeptide in which the RNA polynucleotide of the CHIKV/ZIKV combination RNA vaccine includes at least one chemical modification. Some embodiments of the present disclosure provide a combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one DENV antigenic polypeptide and at least one ZIKV antigenic polypeptide in which the RNA polynucleotide of the DENV/ZIKV combination RNA vaccine includes at least one chemical modification.
• In some embodiments, the chemical modification is selected from pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, 5-methyluridine, and 2′-O-methyl uridine.
• In some embodiments, the RNA polynucleotide, e.g., mRNA including at least one chemical modification further includes a 5′ terminal cap. In some embodiments, the 5′ terminal cap is 7mG(5′)ppp(5′)NlmpNp.
• Some embodiments of the present disclosure provide a vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV antigenic polypeptide, wherein at least 80% of the uracil in the open reading frame have a chemical modification. Some embodiments of the present disclosure provide a vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one DENV antigenic polypeptide, wherein at least 80% of the uracil in the open reading frame have a chemical modification. Some embodiments of the present disclosure provide a vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one ZIKV antigenic polypeptide, wherein at least 80% of the uracil in the open reading frame have a chemical modification.
• Some embodiments of the present disclosure provide a combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV antigenic polypeptide and at least one DENV antigenic polypeptide, wherein at least 80% of the uracil in the open reading frame have a chemical modification. Some embodiments of the present disclosure provide a combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV antigenic polypeptide and at least one ZIKV antigenic polypeptide, wherein at least 80% of the uracil in the open reading frame have a chemical modification. Some embodiments of the present disclosure provide a combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one DENV antigenic polypeptide and at least one ZIKV antigenic polypeptide, wherein at least 80% of the uracil in the open reading frame have a chemical modification. Some embodiments of the present disclosure provide a combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV antigenic polypeptide, at least one DENV antigenic polypeptide, and at least one ZIKV antigenic polypeptide, wherein at least 80% of the uracil in the open reading frame have a chemical modification. In some embodiments, 100% of the uracil in the open reading frame have a chemical modification. In some embodiments, the chemical modification is in the 5-position of the uracil. In some embodiments, the chemical modification is a N1-methyl pseudouridine.
• In some embodiments of any of the combination RNA vaccines described herein, the RNA polynucleotide of the RNA vaccine is formulated in a lipid nanoparticle (LNP) carrier. In some embodiments, the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid. In some embodiments, the lipid nanoparticle carrier comprising a molar ratio of about 20-60% cationic lipid: 5-25% non-cationic lipid: 25-55% sterol; and 0.5-15% PEG-modified lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid. In some embodiments, the non-cationic lipid is a neutral lipid. In some embodiments, the sterol is a cholesterol. In some embodiments, the cationic lipid is selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In some embodiments, the lipid nanoparticle has a polydispersity value of less than 0.4. In some embodiments, the lipid nanoparticle has a net neutral charge at a neutral pH. In some embodiments, the lipid nanoparticle has a mean diameter of 50-200 nm.
• Some embodiments of the present disclosure provide a vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV antigenic polypeptide, at least one 5′ terminal cap and at least one chemical modification, formulated within a lipid nanoparticle. Some embodiments of the present disclosure provide a vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one DENV antigenic polypeptide, at least one 5′ terminal cap and at least one chemical modification, formulated within a lipid nanoparticle. Some embodiments of the present disclosure provide a vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one ZIKV antigenic polypeptide, at least one 5′ terminal cap and at least one chemical modification, formulated within a lipid nanoparticle.
• Some embodiments of the present disclosure provide a combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV antigenic polypeptide and at least one DENV antigenic polypeptide, at least one 5′ terminal cap and at least one chemical modification, formulated within a lipid nanoparticle. Some embodiments of the present disclosure provide a combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV antigenic polypeptide and at least one ZIKV antigenic polypeptide, at least one 5′ terminal cap and at least one chemical modification, formulated within a lipid nanoparticle. Some embodiments of the present disclosure provide a combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one ZIKV antigenic polypeptide and at least one DENV antigenic polypeptide, at least one 5′ terminal cap and at least one chemical modification, formulated within a lipid nanoparticle. Some embodiments of the present disclosure provide a combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV antigenic polypeptide, at least one DENV antigenic polypeptide, at least one ZIKV antigenic polypeptide, at least one 5′ terminal cap and at least one chemical modification, formulated within a lipid nanoparticle.
• Some embodiments of the present disclosure provide a vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV antigenic polypeptide, wherein the open reading frame of the RNA polynucleotide is codon-optimized. Some embodiments of the present disclosure provide a vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one DENV antigenic polypeptide, wherein the open reading frame of the RNA polynucleotide is codon-optimized. Some embodiments of the present disclosure provide a vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one ZIKV antigenic polypeptide, wherein the open reading frame of the RNA polynucleotide is codon-optimized.
• Some embodiments of the present disclosure provide a combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV antigenic polypeptide and at least one DENV antigenic polypeptide, wherein the open reading frame of the RNA polynucleotide is codon-optimized. Some embodiments of the present disclosure provide a combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV antigenic polypeptide and at least one ZIKV antigenic polypeptide, wherein the open reading frame of the RNA polynucleotide is codon-optimized. Some embodiments of the present disclosure provide a combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one ZIKV antigenic polypeptide and at least one DENV antigenic polypeptide, wherein the open reading frame of the RNA polynucleotide is codon-optimized. Some embodiments of the present disclosure provide a combination vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV antigenic polypeptide, at least one DENV antigenic polypeptide, and at least one ZIKV antigenic polypeptide, wherein the open reading frame of the RNA polynucleotide is codon-optimized.
• Some embodiments of the present disclosure provide methods of inducing an antigen specific immune response in a subject, comprising administering to the subject a combination RNA vaccine in an amount effective to produce an antigen specific immune response against CHIKV, against DENV, against ZIKV, against CHIKV and DENV, against CHIKV and ZIKV, against DENV and ZIKV, or against CHIKV, DENV and ZIKV. In some embodiments, an antigen specific immune response comprises a T cell response. In some embodiments, an antigen specific immune response comprises a B cell response. In some embodiments, an antigen specific immune response comprises both a T cell response and a B cell response. In some embodiments, a method of producing an antigen specific immune response involves a single administration of the vaccine. In other embodiments, the method further comprises administering to the subject a second dose or a booster dose of the vaccine. In other embodiments the method comprises administering more than one dose of the vaccine, for example, 2, 3, 4 or more doses of the vaccine. In some embodiments, the vaccine is administered to the subject by intradermal or intramuscular injection.
• Further provided herein are vaccines, such as any of the vaccines described herein, for use in a method of inducing an antigen specific immune response in a subject, the method comprising administering the vaccine to the subject in an effective amount to produce an antigen specific immune response.
• Further provided herein are uses of CHIKV, DENV or ZIKV RNA vaccines and CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV or CHIKV/DENV/ZIKV combination RNA vaccines in the manufacture of a medicament for use in a method of inducing an antigen specific immune response in a subject, the method comprising administering the vaccine to the subject in an amount effective to produce an antigen specific immune response.
• In other aspects of the invention is a method of preventing or treating a CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV infection comprising administering to a subject any of the vaccines described herein. In yet other aspects of the invention is a method of preventing or treating CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV
• In some embodiments, a CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine, is formulated in an effective amount to produce an antigen specific immune response in a subject.
• In some embodiments, an anti-CHIKV, an anti-DENV, an anti-ZIKV, an anti-CHIKV/anti-DENV, an anti-CHIKV/anti-ZIKV, an anti-DENV/anti-ZIKV, or an anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is increased by at least 1 log relative to a control. In some embodiments, the anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or an anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is increased by 1-3 log relative to a control. In some embodiments, the anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or an anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is increased at least 2 times relative to a control. In some embodiments, the anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or an anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is increased at least 5 times relative to a control. In some embodiments, the anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or an anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is increased at least 10 times relative to a control. In some embodiments, the anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or an anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is increased 2-10 times relative to a control.
• In some embodiments, the control is an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or an anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a subject who has not been administered a combination (or any other) vaccine. In some embodiments, the control is an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or an anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a subject who has been administered a live attenuated or inactivated CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV, vaccine. In some embodiments, the control is an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a subject who has been administered a recombinant or purified CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine.
• In some embodiments, the effective amount is a dose equivalent to an at least 2-fold reduction in the standard of care dose of a recombinant CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.
• In some embodiments, the effective amount is a dose equivalent to an at least 4-fold reduction in the standard of care dose of a recombinant CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.
• In some embodiments, the effective amount is a dose equivalent to an at least 10-fold reduction in the standard of care dose of a recombinant CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.
• In some embodiments, the effective amount is a dose equivalent to an at least 100-fold reduction in the standard of care dose of a recombinant CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.
• In some embodiments, the effective amount is a dose equivalent to an at least 1000-fold reduction in the standard of care dose of a recombinant CHIKV/DENV/ZIKV, or DENV/ZIKV, protein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.
• In some embodiments, the effective amount is a dose equivalent to a 2-1000-fold reduction in the standard of care dose of a recombinant CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.
• In some embodiments, the effective amount is a total dose of 50-1000 μg. In some embodiments, the effective amount is a total dose of 100 μg. In some embodiments, the effective amount is a dose of 25 μg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 100 μg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 400 μg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 500 μg administered to the subject a total of two times.
• Further provided herein is a method of inducing an antigen specific immune response in a subject, the method including administering to a subject the CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine in an effective amount to produce an antigen specific immune response in a subject. In some embodiments, anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV, antigenic polypeptide antibody titer produced in the subject is increased by at least 1 log relative to a control. In some embodiments, an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV, antigenic polypeptide antibody titer produced in the subject is increased by 1-3 log relative to a control. In some embodiments, the anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV, antigenic polypeptide antibody titer produced in the subject is increased at least 2 times relative to a control. In some embodiments, the anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV, antigenic polypeptide antibody titer produced in the subject is increased at least 5 times relative to a control. In some embodiments, the anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is increased at least 10 times relative to a control. In some embodiments, the anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is increased 2-10 times relative to a control.
• In some embodiments, the control is an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a subject who has not been administered CHIKV/DENV/ZIKV, or DENV/ZIKV, vaccine. In some embodiments, the control is an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a subject who has been administered a live attenuated or inactivated CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine. In some embodiments, the control is an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a subject who has been administered a recombinant or purified CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine.
• In some embodiments, the effective amount is a dose equivalent to an at least 2-fold reduction in the standard of care dose of a recombinant CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine or a live attenuated CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.
• In some embodiments, the effective amount is a dose equivalent to an at least 4-fold reduction in the standard of care dose of a recombinant CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is equivalent to anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.
• In some embodiments, the effective amount is a dose equivalent to an at least 10-fold reduction in the standard of care dose of a recombinant CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHI
• KV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.
• In some embodiments, the effective amount is a dose equivalent to an at least 100-fold reduction in the standard of care dose of a recombinant CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.
• In some embodiments, the effective amount is a dose equivalent to an at least 1000-fold reduction in the standard of care dose of a recombinant CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.
• In some embodiments, wherein the effective amount is a dose equivalent to a 2-1000-fold reduction in the standard of care dose of a recombinant CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV, protein vaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.
• In some embodiments, the effective amount is a total dose of 50-1000 μg. In some embodiments, the effective amount is a total dose of 100 μg. In some embodiments, the effective amount is a dose of 25 μg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 100 μg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 400 μg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 500 μg administered to the subject a total of two times.
• Other aspects of the present disclosure provide a CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine, which includes a signal peptide linked to a CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein. In some embodiments, the signal peptide is a IgE signal peptide. In some embodiments, the signal peptide is an IgE HC (Ig heavy chain epsilon-1) signal peptide.
• Further provided herein, is a nucleic acid encoding CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.
• Another aspect of the present disclosure provides a CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine, which includes at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a signal peptide linked to a CHIKV, DENV, and/or ZIKV antigenic peptide. In some embodiments, the CHIKV, DENV, and/or ZIKV antigenic peptide is a CHIKV, DENV, and/or ZIKV envelope protein.
• In some embodiments, the signal peptide is a IgE signal peptide. In some embodiments, the signal peptide is an IgE HC (Ig heavy chain epsilon-1) signal peptide. In some embodiments, the signal peptide has the sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 126). In some embodiments, the signal peptide is an IgGIκ signal peptide. In some embodiments, the signal peptide has the sequence METPAQLLFLLLLWLPDTTG (SEQ ID NO: 125).
• In any of the aspects and embodiments described herein the combination vaccine is a CHIKV/DENV/ZIKV, CHIKV/DENV, CHIKV/ZIKV, and/or DENV/ZIKV vaccine.
• Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways.
BRIEF DESCRIPTION OF DRAWINGS
• The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:
• FIG. 1A shows a schematic depiction of the post-translational process of CHIKV structural proteins. FIG. 1B shows a schematic depiction of the E1/E2 heterodimer that associates as a trimeric spike on the CHIKV viral surface.
• FIG. 2 shows a phylogenetic tree of chikungunya virus strains derived from complete concatenated open reading frames for the nonstructural and structural polyproteins. E1 amino acid substitutions that facilitated (Indian Ocean lineage) or prevented (Asian lineage) adaptation to Aedes albopictus are shown on the right. CAR: Central African republic; ECSA: East/Central/South Africa.
• FIG. 3 shows CHIKV envelope protein detection of lysate in HeLa cells 16 hours post-transfection.
• FIG. 4A is a graph showing the survival rates of AG129 mice vaccinated with a single 2 μg dose or two 2 μg doses of Chikungunya E1 antigen administered either intramuscularly or intradermally. FIG. 4B is a graph showing the percent weight loss of AG129 mice vaccinated with a single 2 μg dose or two 2 μg doses of Chikungunya E1 antigen administered either intramuscularly or intradermally. FIG. 4C is a graph showing the health scores of AG129 mice vaccinated with a single 2 μg dose or two 2 μg doses of Chikungunya E1 antigen administered either intramuscularly or intradermally.
• FIG. 5A is a graph showing the survival rates of AG129 mice vaccinated with a single 2 μg dose or two 2 μg doses of Chikungunya E2 antigen administered either intramuscularly or intradermally. FIG. 5B is a graph showing the percent weight loss of AG129 mice vaccinated with a single 2 μg dose or two 2 μg doses of Chikungunya E2 antigen administered either intramuscularly or intradermally. FIG. 5C is a graph showing the health scores of AG129 mice vaccinated with a single 2 μg dose or two 2 μg doses of Chikungunya E2 antigen administered either intramuscularly or intradermally.
• FIG. 6A is a graph showing the survival rates of AG129 mice vaccinated with a single 2 μg dose or two 2 μg doses of Chikungunya C-E3-E2-6K-E1 antigen administered either intramuscularly or intradermally. FIG. 6B is a graph showing the percent weight loss of AG129 mice vaccinated with a single 2 μg dose or two 2 μg doses of Chikungunya C-E3-E2-6K-E1 antigen administered either intramuscularly or intradermally. FIG. 6C is a graph showing the health scores of AG129 mice vaccinated with a single 2 μg dose or two 2 μg doses of Chikungunya C-E3-E2-6K-E1 antigen administered either intramuscularly or intradermally.
• FIG. 7 shows the study design, schedule of injection/bleeding, readout, and survival data for the 2 μg dose study of the CHIKV E1, CHIKV E2, and CHIKV E1/E2/E3/6K/C vaccines.
• FIG. 8A is a graph showing the survival rates of AG129 mice vaccinated with a single 10 μg dose or two 10 μg doses of Chikungunya E1 antigen administered either intramuscularly or intradermally. FIG. 8B is a graph showing the percent weight loss of AG129 mice vaccinated with a single 10 μg dose or two 10 μg doses of Chikungunya E1 antigen administered either intramuscularly or intradermally. FIG. 8C is a graph showing the health scores of AG129 mice vaccinated with a single 10 μg dose or two 10 μg doses of Chikungunya E1 antigen administered either intramuscularly or intradermally.
• FIG. 9A is a graph showing the survival rates of AG129 mice vaccinated with a single 10 μg dose or two 10 μg doses of Chikungunya E2 antigen administered either intramuscularly or intradermally. FIG. 9B is a graph showing the percent weight loss of AG129 mice vaccinated with a single 10 μg dose or two 10 μg doses of Chikungunya E2 antigen administered either intramuscularly or intradermally. FIG. 9C is a graph showing the health scores of AG129 mice vaccinated with a single 10 μg dose or two 10 μg doses of Chikungunya E2 antigen administered either intramuscularly or intradermally.
• FIG. 10A is a graph showing the survival rates of AG129 mice vaccinated with a single 10 μg dose or two 10 μg doses of Chikungunya C-E3-E2-6K-E1 antigen administered either intramuscularly or intradermally. FIG. 10B is a graph showing the percent weight loss of AG129 mice vaccinated with a single 10 μg dose or two 10 μg doses of Chikungunya C-E3-E2-6K-E1 antigen administered either intramuscularly or intradermally. FIG. 10C is a graph showing the health scores of AG129 mice vaccinated with a single 10 μg dose or two 10 μg doses of Chikungunya C-E3-E2-6K-E1 antigen administered either intramuscularly or intradermally.
• FIG. 11 shows the study design, schedule of injection/bleeding, readout, and survival data for the 10 μg dose study of the CHIKV E1, CHIKV E2, and CHIKV C-E3-E2-6K-E1 vaccines.
• FIG. 12 shows the results of an in vitro transfection of mRNA encoded CHIKV structural proteins. Protein detection in HeLa cell lysate 16 h post transfection is detected.
• FIGS. 13A and 13B are schematics of an exemplary DENV peptide epitope. The polypeptide of FIG. 13A includes two or more epitopes. The epitopes can be of the same sequence or different sequence and can be all T-cell epitopes, all B-cell epitopes or a combination of both. The schematic of FIG. 13B shows the peptide epitope with various end units for enhancing MHC processing of the peptides.
• FIG. 14 is a schematic of a dengue viral genome including structural and nonstructural components.
• FIG. 15 shows exemplary dengue peptide epitopes identified using a database screen.
• FIGS. 16A-16C show Dengue Virus MHC I T cell epitopes.
• FIGS. 17A-17C show Dengue Virus MHC II T cell epitopes.
• FIG. 18 is a graph depicting the results of an ELISPOT assay of dengue-specific peptides.
• FIG. 19 is a graph depicting the results of an ELISPOT assay of dengue-specific peptides.
• FIG. 20 is a schematic of a bone marrow/liver/thymus (BLT) mouse and data on human CD8 T cells stimulated with Dengue peptide epitope.
• FIG. 21 shows DENV MHC-1_V5 concatemer transfection in HeLa cells. Triple immunofluorescence using Mitotracker Red (mitochondria), anti-V5, and anti-MHC-1 antibodies plus DAPI was performed. The arrows indicate V5-MHC1 colocalization (bottom right).
• FIG. 22 shows DENV MHC-1_V5 concatemer transfection in HeLa cells. Triple immunofluorescence using Mitotracker Red (mitochondria), anti-V5, and anti-MHC-1 antibodies plus DAPI was performed. The arrows indicate regions where V5 preferentially colocalizes with MHC1 and not with Mitotracker.
• FIG. 23 shows DENV MHC-1_V5 concatemer transfection in HeLa cells. Triple immunofluorescence using Mitotracker Red (mitochondria), anti-V5, and anti-MHC-1 antibodies plus Dapi was performed. V5 has homogeneous cytoplasmic distribution preferentially colocalizes with MHC1 and not with Mitotracker.
• FIGS. 24A and 24B shows the results of an Intracellular Cytokine Staining assay performed in PBMC cells.
• FIG. 25 shows a schematic of a genomic polyprotein obtained from Zika virus, Flaviridiaie. The ZIKV genome encodes a polyprotein with three structural proteins (capsid (C), premembrane/membrane (prM), and envelope (E, a glycosylation motif previously associated with virulence)), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). The polyprotein may be cleaved by several host peptidase or proteases to generate structural or functional proteins for the virus. The respective cleavage sites of the peptidases or proteases are indicated by arrows.
• FIG. 26A shows a schematic of a ZIKV vaccine that comprises a RNA polynucleotide encoding a signal peptide fused to Zika prM protein fused to Zika E protein. FIG. 26B shows a schematic of a ZIKV vaccine that comprises a RNA polynucleotide encoding a signal peptide fused to Zika E protein. The cleavage junction is located between the signal peptide and the Zika prM protein and is conserved between Dengue and Zika.
• FIG. 27 shows a sequence alignment of currently circulating Zika Virus strains.
• FIG. 28 shows fluorescent staining of non-reduced mammalian cell lysates. Tube 1 contains lysed cell precipitate obtained from 293T cells transfected with ZIKV prME mRNA and stained with secondary antibody only (negative control). Tube 2 contains lysed cell precipitate obtained from untransfected 293T cells and stained with anti-ZIKV human serum (1:20) and goat anti-human Alexa Fluor 647 (negative control). Tube 3 contains lysed cell precipitate obtained from 293T cells transfected with ZIKV prME mRNA and stained with anti-ZIKV human serum (1:20) and goat anti-human Alexa Fluor 647. Tube 4 contains lysed cell precipitate obtained from 293T cells transfected with ZIKV prME mRNA and stained with anti-ZIKV human serum (1:200) and goat anti-human Alexa Fluor 647. The antibodies in anti-ZIKV human serum can detect non-reduced proteins expressed by prME mRNA constructs.
• FIG. 29 shows a histogram indicating intracellular detection of ZIKA prME protein using human anti-ZIKV serum.
• FIGS. 30A-30B show the results of detecting prME protein expression in mammalian cells with fluorescence-activated cell sorting (FACS) using a flow cytometer. Cells expressing prME showed higher fluorescence intensity when stained with anti-ZIKV human serum.
• FIG. 31 shows a graph of neutralizing titers from Balb/c mice immunized with ZIKV mRNA vaccine encoding prME.
• FIG. 32 shows negative stain images for Hela samples.
• FIG. 33A shows a reducing SDS-PAGE gel of Zika VLP. FIG. 33B shows a graph of neutralizing titers obtained from Balb/c mice immunized with a ZIKV mRNA vaccine.
• FIG. 34A shows FACS analyses of cells expressing DENV2 prMEs using different antibodies against Dengue envelope protein. Numbers in the upper right corner of each plot indicate mean fluorescent intensity. FIG. 34B shows a repeat of staining in triplicate and in two different cell lines (HeLa and 293T).
• FIG. 35 shows an in vitro antigen presentation assays using OVA (peptide epitope of ovalbumin) multitopes to test different DENV mRNA vaccine construct configurations.
• FIG. 36 is a graph showing the kinetics of OVA peptide presentation in Jawsii cells. All mRNAs tested are formulated in MC3 lipid nanoparticles.
• FIG. 37 is a graph showing the Mean Fluorescent Intensity (MFI) of antibody binding to DENV-1, 2, 3, and 4 prME epitopes presented on the cell surface.
• FIGS. 38A-38D are graphs showing the design and the results of a challenge study in AG129 mice. FIG. 38A shows the immunization, challenge, and serum collection schedules.
• FIG. 38B shows the survival of the AG129 mice challenged with Dengue D2Y98P virus after being immunized with the indicated DENV mRNA vaccines. All immunized mice survived 11 days post infection, while the unimmunized (control) mice died. FIGS. 38C and 38D show the weight loss of the AG129 mice post infection. Vaccine 1, 7, 8, or 9 correspond to DENV vaccine construct 22, 21, 23, or 24 of the present disclosure, respectively.
• FIG. 39 is a graph showing the results of an in vitro neutralization assay using serum from mice immunized with the DENV mRNA vaccines in FIGS. 39A-39D.
• FIGS. 40A-40I are graphs showing the results of a challenge study in AG129 mice. The challenge study design is shown in Table 40. FIGS. 40A-40F show the survival, weight loss, and heath score of the AG129 mice challenged with D2Y98P virus after being immunized with the DENV mRNA vaccine groups 1-12 in Table 40. FIGS. 40G-40I show the survival, weight loss, and heath score of the AG129 mice challenged with D2Y98P virus after being immunized with the DENV mRNA vaccine groups 13-19 in Table 40.
• FIG. 41 is a negative-stain electron microscopy image of the virus-like particles (VLPs) assembled from the antigens (prME) encoded by the DENV mRNA vaccines. DENV mRNA vaccine constructs 21-24 in Table 38 were tested. Construct 23 is the vaccine construct used by Sanofi in its DENV vaccines. Constructs 21, 22, and 24 produced more uniform VLPs, suggesting that these VLPs may be more superior in their immunogenicity than the VLPs produced from construct 23.
• FIGS. 42A-42B are graphs showing the survival curves from a CHIKV challenge study in AG129 mice immunized with CHIKV mRNA vaccines in 10 μg, 2 μg, or 0.04 μg doses. Mice were divided into 14 groups (1-4 and 7-16, n=5). FIG. 42A shows the survival curve of mice groups 1˜4 and 7-9 challenged on day 56 post immunization. FIG. 42B shows the survival curve of mice groups 10-16 challenged on day 112 post immunization. Survival curves were plotted as “percent survival” versus “days post infection.” See also Table 45 for survival percentage.
• FIGS. 43A-43B are graphs showing the weight changes post challenge in AG129 mice immunized with CHIKV mRNA vaccines. FIG. 43A shows the weight change of mice groups 1-4 and 7-9 challenged on day 56 post immunization. FIG. 43B shows the weight changes of mice groups 10-16 challenged on day 112 post immunization. Initial weights were assessed on individual mice on study Day 0 and daily thereafter. The mean percent weights for each group compared to their percent weight on Day 0 (baseline) were plotted against “days post-infection”. Error bars represent the standard deviation (SD).
• FIGS. 44A-44B are graphs showing the post challenge heath scores of AG129 mice immunized with CHIKV mRNA vaccines. FIG. 44A shows the health scores of mice groups 1-4 and 7-9 challenged on day 56 post immunization. FIG. 44B shows the health score of mice groups 10-16 challenged on day 112 post immunization. The mean health scores for each group were plotted against “days post infection” and error bars represent the SD. Mean health scores were calculated based on observations described in Table 5.
• FIGS. 45A-45C are graphs showing the antibody titers measured by ELISA assays in the serum of AG129 mice (groups 1-4 and 7-9) 28 days post immunization with CHIKV mRNA vaccines. FIG. 45A shows the serum antibody titers against CHIKV E1 protein. FIG. 45B shows the serum antibody titers against CHIKV E2 protein. FIG. 45C shows the serum antibody titers against CHIKV lysate.
• FIGS. 46A-46C are graphs showing the antibody titers measured by ELISA assays in the serum of AG129 mice (groups 10-16) 28 days post immunization with CHIKV mRNA vaccine. FIG. 45A shows the serum antibody titers against CHIKV E1 protein. FIG. 46B shows the serum antibody titers against CHIKV E2 protein. FIG. 46C shows the serum antibody titers against CHIKV lysate.
• FIGS. 47A-47C are graphs showing the antibody titers measured by ELISA assays in the serum of AG129 mice (groups 10-16) 56 days post immunization with CHIKV mRNA vaccine. FIG. 47A shows the serum antibody titers against CHIKV E1 protein. FIG. 47B shows the serum antibody titers against CHIKV E2 protein. FIG. 47C shows the serum antibody titers against CHIKV lysate.
• FIGS. 48A-48C are graphs showing the antibody titers measured by ELISA assays in the serum of AG129 mice (groups 10-16) 112 days post immunization with CHIKV mRNA vaccine. FIG. 48A shows the serum antibody titers against CHIKV E1 protein. FIG. 48B shows the serum antibody titers against CHIKV E2 protein. FIG. 48C shows the serum antibody titers against CHIKV lysate.
• FIG. 49 shows different antigens based on the Chikungunya structural protein from three different genotypes.
• FIG. 50 shows a set of graphs depicting results of an ELISA assay to identify the amount of antibodies produced in AG129 mice in response to vaccination with mRNA encoding secreted CHIKV E1 structural protein, secreted CHIKV E2 structural protein, or CHIKV full structural polyprotein C-E3-E2-6k-E1 at a dose of 10 μg or 2 μg at 28 days post immunization.
• FIG. 51 shows a set of graphs depicting results of an ELISA assay to identify the amount of antibodies produced in AG129 mice in response to vaccination with mRNA encoding secreted CHIKV E1 structural protein, secreted CHIKV E2 structural protein, or CHIKV full structural polyprotein C-E3-E2-6k-E1 at a dose of 10 μg or 2 μg at 28 days post immunization. The two panels depict different studies.
• FIG. 52 is a graph depicting comparison of ELISA titers from the data of FIG. 50 to survival in the data of FIG. 51 left panel.
• FIG. 53 shows a set of graphs depicting efficacy results in mice in response to vaccination with mRNA encoding CHIKV full structural polyprotein C-E3-E2-6k-E1 at a dose of 10 μg (left panels), 2 μg (middle panels) or 0.4 μg (right panels) at 56 days (top panels) or 112 days (bottom panels) post immunization.
• FIG. 54 shows a set of graphs depicting amount of neutralizing antibody produced in mice in response to vaccination with mRNA encoding CHIKV full structural polyprotein C-E3-E2-6k-E1 at a dose of 10 μg, 2 μg, or 0.4 μg at 56 days post immunization.
• FIG. 55 shows a set of graphs depicting binding antibody produced in mice in response to vaccination with mRNA encoding CHIKV full structural polyprotein C-E3-E2-6k-E1 at a dose of 10 μg, 2 μg, or 0.4 μg at 56 days post immunization (top panels) and the corresponding correlation between binding and neutralizing antibodies (bottom panels).
• FIG. 56 shows a set of graphs depicting amount of neutralizing antibody produced in A129 mice in response to vaccination with mRNA encoding CHIKV full structural polyprotein C-E3-E2-6k-E1 at a dose of 10 μg, 2 μg, or 0.4 μg at 56 days post immunization against three different strains of CHIKV, African-Senegal (left panel), La Reunion (middle panel) and CDC CAR (right panel).
• FIG. 57 shows a graph depicting neutralizing antibodies against CHIKV S27 strain.
• FIG. 58 is a graph depicting antibody titer against CHIKV lysate post 3rd vaccination 10 with the mRNA vaccine in Sprague Dawley rats.
• FIG. 59 shows a set of graphs depicting antibody titers following vaccination of mice with mRNA encoded CHIKV polyprotein (C-E3-E2-6K-E1).
• FIG. 60 shows a set of plots depicting cytokine secretion and T-cell activation following vaccination of mice with mRNA encoded CHIKV polyprotein (C-E3-E2-6K-E1) (SEQ ID NO: 13).
• FIGS. 61A-61B show a set of graphs depicting CD8+ T cell activation following vaccination of mice with mRNA encoded CHIKV polyprotein (C-E3-E2-6K-E1) (SEQ ID NO: 13).
DETAILED DESCRIPTION
• Embodiments of the present disclosure provide RNA (e.g., mRNA) vaccines that are useful for vaccinating against one or multiple viruses. The vaccines, including combination vaccines, of the invention encode antigens from chikungunya virus (CHIKV), Zika virus (ZIKV), Dengue virus (DENV), or any combination of two or three of the foregoing viruses. A balanced immune response, comprising both cellular and humoral immunity, can be generated against CHIKV, against DENV, against ZIKV, against CHIKV and DENV, against CHIKV and ZIKV, against DENV and ZIKV, or against CHIKV, DENV and ZIKV, using the constructs of the invention without many of the risks associated with DNA vaccines and live attenuated vaccines. The various RNA vaccines disclosed herein produced surprising efficacy in animal models of Chikungunya infection, and Dengue infection, the results of which are discussed in detail in the Examples section. Specifically, RNA polynucleotide vaccines having an open reading frame encoding for a variety of Chikungunya antigens produced significant immunity, whereas traditional Chikungunya vaccines have not (e.g. attenuated chikungunya viruses). The CHIKV RNA polynucleotide vaccines disclosed herein encoding either CHIKV-E1, CHIKV-E2 or CHIKV-C-E3-E2-6K-E1 demonstrated a survival rate of 60%-100% after two administrations. Specifically, two injections of CHIKV E1 mRNA vaccine provided nearly full protection against infection when administered intramuscularly (IM) (60% survival) or intradermally (ID) (80% survival). Two injections of CHIKV E2 mRNA vaccine or CHIKV C-E3-E2-6K-E1 vaccine provided full protection (100% survival) against infection when administered via IM or ID. Importantly, a single injection (no booster dose) of CHIKV C-E3-E2-6K-E1 vaccine provided full protection (100% survival) against infection when administered via IM or ID.
• DENV RNA vaccines and ZIKV vaccines are also disclosed herein as well as combination DENV and CHIKV, CHIKV and ZIKV, and DENV and ZIKV vaccines. The combination vaccines of CHIKV, DENV and ZIKV, DENV and ZIKV, CHIKV and ZIKV, or CHIKV and DENV can provide a means for protecting against two or more viral infections in a single vaccine.
• Chikungunya virus is a small (about 60-70 nm-diameter), spherical, enveloped, positive-strand RNA virus having a capsid with icosahedral symmetry. The virion consists of an envelope and a nucleocapsid. The virion RNA is infectious and serves as both genome and viral messenger RNA. The genome is a linear, ssRNA(+) genome of 11,805 nucleotides which encodes for two polyproteins that are processed by host and viral proteases into non-structural proteins (nsP1, nsP2, nsP3, and RdRpnsP4) necessary for RNA synthesis (replication and transcription) and structural proteins (capsid and envelope proteins C, E3, E2, 6K, and E1) which attach to host receptors and mediate endocytosis of virus into the host cell. (FIG. 1 ). The E1 and E2 glycoproteins form heterodimers that associate as 80 trimeric spikes on the viral surface covering the surface evenly. The envelope glycoproteins play a role in attachment to cells. The capsid protein possesses a protease activity that results in its self-cleavage from the nascent structural protein. Following its cleavage, the capsid protein binds to viral RNA and rapidly assembles into icosahedric core particles. The resulting nucleocapsid eventually associates with the cytoplasmic domain of E2 at the cell membrane, leading to budding and formation of mature virions.
• E2 is an envelope glycoprotein responsible for viral attachment to target host cell, by binding to the cell receptor. E2 is synthesized as a p62 precursor which is processed at the cell membrane prior to virion budding, giving rise to an E2-E1 heterodimer. The C-terminus of E2 is involved in budding by interacting with capsid proteins.
• E1 is an envelope glycoprotein with fusion activity, which fusion activity is inactive as long as E1 is bound to E2 in the mature virion. Following virus attachment to target cell and endocytosis, acidification of the endosome induces dissociation of the E1/E2 heterodimer and concomitant trimerization of the E1 subunits. The E1 trimer is fusion active and promotes the release of the viral nucleocapsid in the cytoplasm after endosome and viral membrane fusion.
• E3 is an accessory protein that functions as a membrane translocation/transport signal for E1 and E2.
• 6K is another accessory protein involved in virus glycoprotein processing, cell permeabilization, and the budding of viral particles. Like E3, it functions as a membrane transport signal for E1 and E2.
• The CHIKV structural proteins have been shown to be antigenic, which proteins, fragments, and epitopes thereof are encompassed within the invention. A phylogenetic tree of Chikungunya virus strains derived from complete concatenated open reading frames for the nonstructural and structural polyproteins shows key envelope glycoprotein E1 amino acid substitutions that facilitated (Indian Ocean lineage) or prevented (Asian lineage) adaptation to Aedes albopictus. There are membrane-bound and secreted forms of E1 and E2, as well as the full length polyprotein antigen (C-E3-E2-6K-E1), which retains the protein's native conformation. Additionally, the different Chikungunya genotypes, strains and isolates can also yield different antigens, which are functional in the constructs of the invention. For example, there are several different Chikungunya genotypes: Indian Ocean, East/Central/South African (ECSA), Asian, West African, and the Brazilian isolates (ECSA/Asian). There are three main Chikungunya genotype. These are ESCA (East-South-Central Africa); Asia; and West Africa. While sometimes names differ in publications all belong to these three geographical strains.
• Dengue virus is a mosquito-borne (Aedes aegypti/Aedes albopictus) member of the family Flaviviridae (positive-sense, single-stranded RNA virus). The dengue virus genome encodes ten genes and is translated as a single polypeptide which is cut into ten proteins: the capsid, envelope, membrane, and nonstructural proteins (NS1, NS2A, NS2B, NS3, SN4A, NS4B, and NS5 proteins). The virus' main antigen is DENe, which is a component of the viral surface and is thought to facilitate the binding of the virus to cellular receptors (Heinz et al., Virology. 1983, 126:525). There are four similar but distinct serotypes of dengue virus (DEN-1, DEN-2, DEN-3, and DEN-4), which result annually in an estimated 50-100 million cases of dengue fever and 500,000 cases of the more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (Gubler et al., Adv Virus Res. 1999, 53:35-70). The four serotypes show immunological cross-reactivity, but are distinguishable in plaque reduction neutralization tests and by their respective monoclonal antibodies. The dengue virus E protein includes a serotype-specific antigenic determinant and determinants necessary for virus neutralization (Mason et al., J Gen Virol. 1990, 71:2107-2114).
• After inoculation, the dendritic cells become infected and travel to lymph nodes. Monocytes and macrophages are also targeted shortly thereafter. Generally, the infected individual will be protected against homotypic reinfection for life; however, the individual will only be protected against other serotypes for a few weeks or months (Sabin, Am J Trop Med Hyg. 1952, 1:30-50). In fact, DHF/DSS is generally found in children and adults infected with a dengue virus serotype differing from their respective primary infection. Thus, it is necessary to develop a vaccine that provides immunity to all four serotypes.
• Along with other viruses in the Flaviviridae family, Zika virus is enveloped and icosahedral with a non-segmented, single-stranded, positive sense RNA genome. It is most closely related to the Spondweni virus and is one of the two viruses in the Spondweni virus Glade. The virus was first isolated in 1947 from a rhesus monkey in the Zika Forest of Uganda, Africa and was isolated for the first time from humans in 1968 in Nigeria. From 1951 through 1981, evidence of human infection was reported from other African countries such as Uganda, Tanzania, Egypt, Central African Republic, Sierra Leone and Gabon, as well as in parts of Asia including India, Malaysia, the Philippines, Thailand, Vietnam and Indonesia. It is transmitted by mosquitoes and has been isolated from a number of species in the genus Aedes—Aedes aegypti, Aedes africanus, Aedes apicoargenteus, Aedes furcifer, Aedes luteocephalus and Aedes vitattus. Studies show that the extrinsic incubation period in mosquitoes is about 10 days. The vertebrate hosts of the virus include monkeys and humans.
• As of early 2016, the most widespread outbreak of Zika fever, caused by the Zika virus, is ongoing primarily in the Americas. The outbreak began in April 2015 in Brazil, and subsequently spread to other countries in South America, Central America, and the Caribbean.
• The Zika virus was first linked with newborn microcephaly during the Brazil Zika virus outbreak. In 2015, there were 2,782 cases of microcephaly compared with 147 in 2014 and 167 in 2013. The Brazilian Health Ministry has reported 4783 cases of suspected microcephaly as of January 30, an increase of more than 1000 cases from a week earlier. Confirmation of many of the recent cases is pending, and it is difficult to estimate how many cases went unreported before the recent awareness of the risk of virus infections.
• What is important is not only the number of cases but also the clinical manifestation of the cases. Brazil is seeing severe cases of microcephaly, which are more likely to be paired with greater developmental delays. Most of what is being reported out of Brazil is microcephaly with other associated abnormalities. The potential consequence of this is the fact that there are likely to be subclinical cases where the neurological sequelae will only become evident as the children grow.
• Zika virus has also been associated with an increase in a rare condition known as Guillain-Barre, where the infected individual becomes essentially paralyzed. During the Zika virus outbreak in French Polynesia, 74 patients which had had Zika symptoms—out of them, 42 were diagnosed as Guillain-Barré syndrome. In Brazil, 121 cases of neurological manifestations and Guillain-Barré syndrome (GBS) were reported, all cases with a history of Zika-like symptoms.
• In some embodiments, ZIKV vaccines comprise RNA (e.g., mRNA) encoding a ZIKV antigenic polypeptide having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with ZIKV polyprotein and having ZIKV polyprotein activity, respectively. The ZIKV polyprotein is cleaved into capsid, precursor membrane, envelope, and non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5).
• A protein is considered to have ZIKV polyprotein activity if, for example, it facilitates the attachment of the viral envelope to host receptors, mediates internalization into the host cell, and aids in fusion of the virus membrane with the host's endosomal membrane.
• The RNA vaccines may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need. The RNA vaccines may be utilized to treat and/or prevent a CHIKV, DENV and/or ZIKV infection of various genotypes, strains, and isolates. The RNA vaccines have superior properties in that they produce much larger antibody titers and produce responses early than commercially available anti-viral therapeutic treatments. While not wishing to be bound by theory, it is believed that the RNA vaccines, as mRNA polynucleotides, are better designed to produce the appropriate protein conformation upon translation as the RNA vaccines co-opt natural cellular machinery. Unlike traditional vaccines which are manufactured ex vivo and may trigger unwanted cellular responses, the RNA vaccines are presented to the cellular system in a more native fashion.
• The entire contents of International Application No. PCT/US2015/02740 is incorporated herein by reference.
Nucleic Acids/Polynucleotides
• Vaccines, including combination vaccines, as provided herein, comprise at least one (one or more) ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one CHIKV antigenic polypeptide, at least one ZIKV antigenic polypeptide, at least one DENV antigenic polypeptide, at least one CHIKV antigenic polypeptide and at least one DENV antigenic polypeptide, at least one CHIKV antigenic polypeptide and at least one ZIKV antigenic polypeptide, at least one ZIKV antigenic polypeptide and at least one DENV antigenic polypeptide, or at least one CHIKV antigenic polypeptide, at least one DENV antigenic polypeptide and at least one ZIKV antigenic polypeptide. In some embodiments, the vaccine, including combination vaccines, comprise at least one RNA polynucleotide, e.g., mRNA, having an open reading frame encoding two or more different CHIKV antigenic polypeptides, ZIKV antigenic polypeptides, and/or DENV antigenic polypeptides (e.g., two, three, four, five or more different antigenic polypeptides). In some embodiments, the combination vaccine comprises at least one RNA polynucleotide having an open reading frame encoding a CHIKV antigenic polypeptide or epitope, a ZIKV antigenic polypeptide or epitope, a DENV antigenic polypeptide or epitope, or a combination of any two or three of the forgoing. The term “nucleic acid,” in its broadest sense, includes any compound and/or substance that comprises a polymer of nucleotides. These polymers are referred to as polynucleotides. As used herein the term polypeptide refers to full-length proteins, protein fragments, variants, and epitopes.
• In some embodiments, an RNA polynucleotide, e.g., mRNA, of a combination vaccine encodes at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 antigenic polypeptides. In some embodiments, an RNA polynucleotide comprises 30 to 12,000 or more nucleotides. For example, a polynucleotide may include 30 to 100, 101 to 200, 200 to 500, 200 to 1000, 200 to 1500, 200 to 2000, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, 1500 to 4000, 1500 to 5000, 2000 to 3000, 2000 to 4000, 2000 to 5000, 5000 to 7500, 7500 to 10,000, or 10,000 to 12,000 nucleotides.
• In some embodiments, the combination vaccine comprises at least one RNA polynucleotide having an open reading frame encoding a Chikungunya structural protein or an antigenic fragment or an antigenic epitope thereof. In some embodiments, the RNA polynucleotide has an open reading frame encoding a Chikungunya envelope and/or capsid antigenic polypeptide selected from a CHIKV E1, E2, E3, 6K, and capsid (C) antigenic polypeptide. In some embodiments, the RNA polynucleotide has an open reading frame encoding any combination of CHIKV E1, E2, E3, 6K, and capsid (C) antigenic polypeptides, for example, a combination selected from CHIKV E1 and E2 antigens, CHIKV E1 and E3 antigens, CHIKV E2 and E3 antigens, CHIKV E1, E2, and E3 antigens, CHIKV E1, E2, E3, and C antigens, CHIKV E1, E2, and 6K antigens, CHIKV E2, E3 and 6K antigens, CHIKV E1, E3, and 6K antigens, and CHIKV E1, E2, E3, 6K, and C antigens.
• Some embodiments of the present disclosure provide DENV vaccines that include at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one DENV antigenic polypeptide or an immunogenic fragment or epitope thereof. Some embodiments of the present disclosure provide DENV vaccines that include at least one RNA polynucleotide having an open reading frame encoding two or more DENV antigenic polypeptides or an immunogenic fragment or epitope thereof. Some embodiments of the present disclosure provide DENV vaccines that include two or more RNA polynucleotides having an open reading frame encoding two or more DENV antigenic polypeptides or immunogenic fragments or epitopes thereof. The one or more DENV antigenic polypeptides may be encoded on a single RNA polynucleotide or may be encoded individually on multiple (e.g., two or more) RNA polynucleotides.
• In some embodiments, the at least one RNA polynucleotide may encode at least one DENV antigenic polypeptide. In some instances the dengue viral antigenic polypeptide is an intact dengue virus peptide or other large antigen (i.e. greater than 25 amino acids in length). In some embodiments, the at least one RNA polynucleotide encodes a DENV capsid protein or immunogenic fragment or epitope thereof. In some embodiments, the at least one RNA polynucleotide encodes a DENV envelope protein or immunogenic fragment or epitope thereof. In some embodiments, the at least one RNA polynucleotide encodes a DENV membrane protein or immunogenic fragment or epitope thereof. In some embodiments, the at least one RNA polynucleotide encodes a DENV nonstructural protein or immunogenic fragment or epitope thereof. Large gene segments in non-structural genes, in particular may be used for antigens. In some embodiments, the DENV non-structural protein is selected from NS1, NS2A, NS2B, NS3, SN4A, NS4B, and NS5 proteins, or immunogenic fragments or epitopes thereof. In some embodiments, the DENV non-structural protein is NS3. In some embodiments, the at least one RNA polynucleotide encodes DENe, which is a component of the viral surface and is thought to facilitate the binding of the virus to cellular receptors. In any of these embodiments, the at least one RNA polynucleotide encodes a DENV polypeptide from a DENV serotype selected from DENV-1, DENV-2, DENV-3, and DENV-4. For example, the DENV polypeptide may be one or more polypeptides encoded by SEQ ID NO: 15 (DENV1), SEQ ID NO: 17 (DENV2), SEQ ID NO: 19 (DENV3), and SEQ ID NO: 21 (DENV4), In some embodiments, the DENV polypeptide is a polypeptide found in SEQ ID NO: 14 (DENV1), SEQ ID NO: 16 (DENV2), SEQ ID NO: 18 DENV3), and/or SEQ ID NO: 20 (DENV4). In some embodiments, the Dengue virus (DENV) vaccine comprises at least one (one or more) ribonucleic acid (RNA) polynucleotide having an open reading frame encoding SEQ ID NO: 23 or an immunogenic fragment or epitope thereof. In some embodiments, the Dengue virus (DENV) vaccine comprises at least one (one or more) ribonucleic acid (RNA) polynucleotide having an open reading frame encoding SEQ ID NO: 26 or an immunogenic fragment or epitope thereof. In some embodiments, the Dengue virus (DENV) vaccine comprises at least one (one or more) ribonucleic acid (RNA) polynucleotide having an open reading frame encoding SEQ ID NO: 29 or an immunogenic fragment or epitope thereof. In some embodiments, the Dengue virus (DENV) vaccine comprises at least one (one or more) ribonucleic acid (RNA) polynucleotide having an open reading frame encoding SEQ ID NO: 32 or an immunogenic fragment or epitope thereof. In some embodiments, the DENV RNA polynucleotide comprises SEQ ID NO: 25 (or is encoded by SEQ ID NO: 24) or a fragment thereof. In some embodiments, the DENV RNA polynucleotide comprises SEQ ID NO: 28 (or is encoded by SEQ ID NO: 27) or a fragment thereof. In some embodiments, the DENV RNA polynucleotide comprises SEQ ID NO: 31 (or is encoded by SEQ ID NO: 30) or a fragment thereof. In some embodiments, the DENV RNA polynucleotide comprises SEQ ID NO: 34 (or is encoded by SEQ ID NO: 33) or a fragment thereof. In some embodiments, the DENV RNA polynucleotide encodes a polypeptide comprising SEQ ID NO:23 or an immunogenic fragment or epitope thereof. In some embodiments, the DENV RNA polynucleotide encodes a polypeptide comprising SEQ ID NO: 26 or an immunogenic fragment or epitope thereof. In some embodiments, the DENV RNA polynucleotide encodes a polypeptide comprising SEQ ID NO: 29 or an immunogenic fragment or epitope thereof. In some embodiments, the DENV RNA polynucleotide encodes a polypeptide comprising SEQ ID NO: 32 or an immunogenic fragment or epitope thereof.
• Dengue virus (DENV) vaccine antigens, as provided herein, comprise at least one (one or more) ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one DENV antigenic polypeptide. In some embodiments, the DENV antigenic polypeptide is longer than 25 amino acids and shorter than 50 amino acids. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. Polypeptides may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked. Most commonly, disulfide linkages are found in multichain polypeptides. The term polypeptide may also apply to amino acid polymers in which at least one amino acid residue is an artificial chemical analogue of a corresponding naturally-occurring amino acid.
• In other embodiments the antigen is a concatemeric peptide antigen composed of multiple peptide epitopes. In some embodiments, a RNA polynucleotide of a DENV vaccine encodes 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9 or 9-10 antigenic polypeptides. In some embodiments, a RNA polynucleotide of a DENV vaccine encodes at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 antigenic polypeptides. In some embodiments, a RNA polynucleotide of a DENV vaccine encodes at least 100 or at least 200 antigenic polypeptides. In some embodiments, a RNA polynucleotide of a DENV vaccine encodes 1-10, 5-15, 10-20, 15-25, 20-30, 25-35, 30-40, 35-45, 40-50, 1-50, 1-100, 2-50 or 2-100 antigenic polypeptides.
• In order to design useful epitopes, publically available databases, such as the Immune Epitope Database (IEDB), may be used to predict immunogenic Dengue T cell epitopes showing strong homology across all 4 Dengue serotypes. For instance, the IEDB is a free database offering searching of experimental data characterizing antibody and T cell epitopes and assisting in the prediction and analysis of B cell and T cell epitopes. The Dengue peptides identified by database may be confirmed using peptides in MHC allele binding assays (such as those described in the Examples provided herein) and/or restimulation assays during the acute phase of Dengue infection (i.e. Day 7). Some examples of epitopes are shown in FIG. 15 . These epitopes may be evaluated in test mice and using an assay such as that shown in FIG. 18 .
• Some embodiments of the present disclosure provide ZIKV vaccines, including combination vaccines, that include at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one ZIKV antigenic polypeptide or an immunogenic fragment or epitope thereof. Some embodiments of the present disclosure provide ZIKV combination vaccines that include at least one RNA polynucleotide having an open reading frame encoding two or more ZIKV antigenic polypeptides or an immunogenic fragment or epitope thereof. Some embodiments of the present disclosure provide ZIKV vaccines that include two or more RNA polynucleotides having an open reading frame encoding two or more ZIKV antigenic polypeptides or immunogenic fragments or epitopes thereof. The one or more ZIKV antigenic polypeptides may be encoded on a single RNA polynucleotide or may be encoded individually on multiple (e.g., two or more) RNA polynucleotides.
• In some embodiments, the at least one RNA polynucleotide may encode at least one ZIKV antigenic polypeptide. In some instances the ZIKV antigenic polypeptide is an intact ZIKV peptide or other large antigen (i.e. greater than 25 amino acids in length). In any of these embodiments, the at least one RNA polynucleotide encodes a ZIKV polypeptide from a ZIKV serotype selected from MR 766, SPH2015 or ACD75819. For example, the ZIKV polypeptide may be one or more polypeptides encoded by SEQ ID NO: 67-134 or an immunogenic fragment or epitope thereof.
• Zika virus (ZIKV) vaccines, including combination vaccines, as provided herein, comprise at least one (one or more) ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one ZIKV antigenic polypeptide. In some embodiments, the ZIKV antigenic polypeptide is longer than 25 amino acids and shorter than 50 amino acids. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. Polypeptides may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked. Most commonly, disulfide linkages are found in multichain polypeptides. The term polypeptide may also apply to amino acid polymers in which at least one amino acid residue is an artificial chemical analogue of a corresponding naturally-occurring amino acid.
• The generation of antigens that elicit a desired immune response (e.g. B and/or T-cell responses) against targeted polypeptide sequences in vaccine development remains a challenging task. The invention involves technology that overcome hurdles associated with vaccine development. Through the use of the technology of the invention, it is possible to tailor the desired immune response by selecting appropriate T or B cell epitopes which, by virtue of the fact that they are processed intra-cellularly, are able to be presented more effectively on MHC-1 or MHC-2 molecules (depending on whether they are T or B-cell epitope, respectively). In particular, the invention involves the generation of DENV concatemers of epitopes (particularly T cell epitopes) preferably interspersed with cleavage sites by proteases that are abundant in Antigen Presenting Cells (APCs). These methods mimic antigen processing and may lead to a more effective antigen presentation than can be achieved with peptide antigens.
• The fact that the peptide epitopes of the invention are expressed from RNA as intracellular peptides provides advantages over prior art peptides that are delivered as exogenous peptides or as DNA. The RNA is delivered intra-cellularly and expresses the epitopes in proximity to the appropriate cellular machinery for processing the epitopes such that they will be recognized by the appropriate immune cells. Additionally, a targeting sequence will allow more specificity in the delivery of the peptide epitopes.
• In some embodiments the DENV mRNA vaccine of the invention is a poly-epitopic RNA. Poly-epitopes consist of strings of epitopes on the same mRNA. The RNA sequences that code for the peptide epitopes may be interspersed by sequences that code for amino acid sequences recognized by proteolytic enzymes, by other linkers or linked directly.
• A concatemeric peptide as used herein is a series of at least two peptide epitopes linked together to form the propeptide. In some embodiments a concatemeric peptide is composed of 3 or more, 4 or more, 5 or more 6 or more 7 or more, 8 or more, 9 or more peptide epitopes.
• In other embodiments the concatemeric peptide is composed of 1000 or less, 900 or less, 500 or less, 100 or less, 75 or less, 50 or less, 40 or less, 30 or less, 20 or less or 100 or less peptide epitopes. In yet other embodiments a concatemeric peptide has 3-100, 5-100, 10-100, 15-100, 20-100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100, 55-100, 60-100, 65-100, 70-100, 75-100, 80-100, 90-100, 5-50, 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 100-150, 100-200, 100-300, 100-400, 100-500, 50-500, 50-800, 50-1,000, or 100-1,000 peptide epitopes.
• An epitope, also known as an antigenic determinant, as used herein is a portion of an antigen that is recognized by the immune system in the appropriate context, specifically by antibodies, B cells, or T cells. Epitopes include B cell epitopes and T cell epitopes. B-cell epitopes are peptide sequences which are required for recognition by specific antibody producing B-cells. B cell epitopes refer to a specific region of the antigen that is recognized by an antibody. The portion of an antibody that binds to the epitope is called a paratope. An epitope may be a conformational epitope or a linear epitope, based on the structure and interaction with the paratope. A linear, or continuous, epitope is defined by the primary amino acid sequence of a particular region of a protein. The sequences that interact with the antibody are situated next to each other sequentially on the protein, and the epitope can usually be mimicked by a single peptide. Conformational epitopes are epitopes that are defined by the conformational structure of the native protein. These epitopes may be continuous or discontinuous, i.e. components of the epitope can be situated on disparate parts of the protein, which are brought close to each other in the folded native protein structure.
• T-cell epitopes are peptide sequences which, in association with proteins on APC, are required for recognition by specific T-cells. T cell epitopes are processed intracellularly and presented on the surface of APCs, where they are bound to MHC molecules including MHC class II and MHC class I.
• The present disclosure, in some aspects, relates to a process of developing T or B cell concatemeric epitopes or concatemeric epitopes composed of both B and T cell epitopes. Several tools exist for identifying various peptide epitopes. For instance, epitopes can be identified using a free or commercial database (Lonza Epibase, antitope for example). Such tools are useful for predicting the most immunogenic epitopes within a target antigen protein. The selected peptides may then be synthesized and screened in human HLA panels, and the most immunogenic sequences are used to construct the mRNA polynucleotides encoding the concatemeric antigens. One strategy for mapping epitopes of Cytotoxic T-Cells based on generating equimolar mixtures of the four C-terminal peptides for each nominal 11-mer across your an protein. This strategy would produce a library antigen containing all the possible active CTL epitopes.
• The peptide epitope may be any length that is reasonable for an epitope. In some embodiments the peptide epitope is 9-30 amino acids. In other embodiments the length is 9-22, 9-29, 9-28, 9-27, 9-26, 9-25, 9-24, 9-23, 9-21, 9-20, 9-19, 9-18, 10-22, 10-21, 10-20, 11-22, 22-21, 11-20, 12-22, 12-21, 12-20, 13-22, 13-21, 13-20, 14-19, 15-18, or 16-17 amino acids. In some embodiments, the optimal length of a peptide epitope may be obtained through the following procedure: synthesizing a V5 tag concatemer-test protease site, introducing it into DC cells (for example, using an RNA Squeeze procedure, lysing the cells, and then running an anti-V5 Western blot to assess the cleavage at protease sites.
• In some embodiments, the RNA polynucleotide of the combination vaccine is encoded by at least one nucleic acid sequence selected from SEQ ID NO: 1-13 (CHIKV), 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33, 34, 144-152 or 199-212 (DENV), and 48-66 (ZIKV). In some embodiments, the RNA polynucleotide of the combination vaccine is encoded by at least one fragment of a nucleic acid sequence selected from SEQ ID NO: 1-13 (CHIKV), 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33, 34, 144-152 or 199-212 (DENV), and 48-66 (ZIKV). In some embodiments, the RNA polynucleotide of the combination vaccine is encoded by at least one epitope sequence of a nucleic acid sequence selected from SEQ ID NO: 1-13 (CHIKV), 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33, 34, 144-152 or 199-212 (DENV), or 48-66 (ZIKV).
• In particular embodiments, the RNA polynucleotide is encoded by any of SEQ ID NO: 1, 5, 10, and 12. In particular embodiments, the RNA polynucleotide is encoded by any of SEQ ID NO: 2, 4, 6 and 11. In particular embodiments, the RNA polynucleotide is encoded by any of SEQ ID NO: 7-9. In a particular embodiment, the RNA polynucleotide is encoded by SEQ ID NO: 3. In a particular embodiment, the RNA polynucleotide is encoded by SEQ ID NO: 13.
• Nucleic acids (also referred to as polynucleotides) may be or may include, for example, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino-α-LNA having a 2′-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or chimeras or combinations thereof.
• In some embodiments, polynucleotides of the present disclosure is or functions as a messenger RNA (mRNA). As used herein the term “messenger RNA” (mRNA) refers to any polynucleotide that encodes at least one polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo.
• Traditionally, the basic components of an mRNA molecule include at least one coding region, a 5′ untranslated region (UTR), and a 3′ UTR. In some embodiments, the mRNA molecules further includes a 5′ cap. In some embodiments, the mRNA further includes a polyA tail. Polynucleotides of the present disclosure may function as mRNA but are distinguished from wild-type mRNA in their functional and/or structural design features which serve to overcome existing problems of effective polypeptide production using nucleic-acid based therapeutics. Antigenic polypeptides (antigens) of the present disclosure may be encoded by polynucleotides translated in vitro, referred to as “in vitro translated” (IVT) polynucleotides.
• The RNA polynucleotides of the present disclosure may be or comprise variant or mutant sequence. The term “polynucleotide variant” refers to a nucleotide molecule which differs in its nucleotide sequence from a native, wildtype, or reference sequence. The nucleotide sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the nucleotide sequence, as compared to the corresponding native, wildtype or reference sequence. In some embodiments, the nucleotide variants possess at least 80% identity (homology) to a native, wildtype or reference sequence, for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (homology) to a native, wildtype or reference sequence.
• In some embodiments, the RNA polynucleotide is encoded by a nucleic acid sequence having at least 80%-85% sequence identity to any of SEQ ID NO: 1-14 (CHIKV), 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33, 34, 144-152 or 199-212 (DENV), or 48-66 (ZIKV). In some embodiments, the RNA polynucleotide is encoded by a nucleic acid sequence having at least 86%-90% sequence identity to any of SEQ ID NO: 1-13 (CHIKV), 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33, 34, 144-152 or 199-212 (DENV), or 48-66 (ZIKV). In some embodiments, the RNA polynucleotide is encoded by a nucleic acid sequence having at least 91%-95% sequence identity to any of SEQ ID NO: 1-13 (CHIKV), 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33, 34, 144-152 or 199-212 (DENV), or 48-66 (ZIKV). In some embodiments, the RNA polynucleotide is encoded by a nucleic acid sequence having at least 96%-98% sequence identity to any of SEQ ID NO: 1-13 (CHIKV), 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33, 34, 144-152 or 199-212 (DENV), or 48-66 (ZIKV). In some embodiments, the RNA polynucleotide is encoded by a nucleic acid sequence having at least 99% sequence identity to any of SEQ ID NO: 1-13 (CHIKV), 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33, 34, 144-152 or 199-212 (DENV), or 48-66 (ZIKV).
• In some embodiments, a polynucleotide of the present disclosure, e.g., polynucleotide variants, have less than 80% identity (homology) to a native, wildtype or reference sequence, for example, less than 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60% or less identity (homology) to a native, wildtype or reference sequence. In some embodiments, polynucleotide of the invention, e.g., polynucleotide variants, have about 65% to about 85% identity to a native, wildtype or reference sequence, e.g., 65%-82%, 67%-81%, or 66%-80% identity to a native, wildtype or reference sequence.
• Polynucleotides of the present disclosure, in some embodiments, are codon optimized. Codon optimization methods are known in the art and may be used as provided herein. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g. glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art. Non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and/or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.
• In some embodiments, a codon optimized sequence shares less than 95% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide. In some embodiments, a codon optimized sequence shares less than 90% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide. In some embodiments, a codon optimized sequence shares less than 85% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide. In some embodiments, a codon optimized sequence shares less than 80% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide. In some embodiments, a codon optimized sequence shares less than 75% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide.
• In some embodiments, a codon optimized sequence shares between 65% and 85% (e.g., between about 67% and about 85% or between about 67% and about 80%) sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide. In some embodiments, a codon optimized sequence shares between 65% and 75 or about 80% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide.
• In some embodiments, the RNA polynucleotides of the present disclosure may further comprise sequence comprising or encoding additional sequence, for example, one or more functional domain(s), one or more further regulatory sequence(s), an engineered 5′ cap.
• Thus, in some embodiments, the RNA vaccines comprise a 5′UTR element, an optionally codon optimized open reading frame, and a 3′UTR element, a poly(A) sequence and/or a polyadenylation signal wherein the RNA is not chemically modified.
• In Vitro Transcription of RNA (e.g., mRNA)
• The combination vaccine of the present disclosure comprise at least one RNA polynucleotide, such as a mRNA (e.g., modified mRNA). mRNA, for example, is transcribed in vitro from template DNA, referred to as an “in vitro transcription template.” In some embodiments, an in vitro transcription template encodes a 5′ untranslated (UTR) region, contains an open reading frame, and encodes a 3′ UTR and a polyA tail. The particular nucleotide sequence composition and length of an in vitro transcription template will depend on the mRNA encoded by the template.
• A “5′ untranslated region” (UTR) refers to a region of an mRNA that is directly upstream (i.e., 5′) from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide.
• A “3′ untranslated region” (UTR) refers to a region of an mRNA that is directly downstream (i.e., 3′) from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide.
• An “open reading frame” is a continuous stretch of codons beginning with a start codon (e.g., methionine (ATG)), and ending with a stop codon (e.g., TAA, TAG or TGA) that encodes a polypeptide.
• A “polyA tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3′), from the 3′ UTR that contains multiple, consecutive adenosine monophosphates. A polyA tail may contain 10 to 300 adenosine monophosphates. For example, a polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a polyA tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo) the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus and translation.
• In some embodiments a codon optimized RNA may, for instance, be one in which the levels of G/C are enhanced. The G/C-content of nucleic acid molecules may influence the stability of the RNA. RNA having an increased amount of guanine (G) and/or cytosine (C) residues may be functionally more stable than nucleic acids containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. WO02/098443 discloses a pharmaceutical composition containing an mRNA stabilized by sequence modifications in the translated region. Due to the degeneracy of the genetic code, the modifications work by substituting existing codons for those that promote greater RNA stability without changing the resulting amino acid. The approach is limited to coding regions of the RNA.
Antigens/Antigenic Polypeptides
• In some embodiments, the Chikungunya antigenic polypeptide is a Chikungunya structural protein. The Chikungunya structural protein can be a CHIKV envelope (E) protein or a CHIKV capsid (C) protein. In some embodiments, the Chikungunya structural protein can be a CHIKV E1, E2, E3, 6K, or capsid (C) protein. In one embodiment, the Chikungunya structural protein is CHIKV E1. In another embodiment, the Chikungunya structural protein is CHIKV E2. In another embodiment, the Chikungunya structural protein is CHIKV E3. In another embodiment, the Chikungunya structural protein is CHIKV C. In another embodiment, the Chikungunya structural protein is CHIKV 6K.
• In some embodiments, the Chikungunya antigenic polypeptide comprises the sequence of two or more Chikungunya structural proteins selected from E1, E2, E3, 6K, and C. The antigenic polypeptide can comprise the sequence of any combination of CHIKV structural proteins, including, for example, CHIKV E1 and E2; CHIKV E2 and E3; CHIKV E1 and E3; CHIKV E1, E2, and E3; CHIKV E1, E2, E3, and C; CHIKV E1, E2, E3, 6K, and C; CHIKV E1, 6K, E2; CHIKV E2, 6K, E3; CHIKV E1, 6K, E3; and CHIKV E1, E2, E3, and 6K proteins. In one particular embodiment, the Chikungunya antigenic polypeptide comprises the sequence of the Chikungunya structural polyprotein: C-E3-E2-6K-E1.
• In some embodiments, the Chikungunya antigenic polypeptide is a fragment of a Chikungunya structural protein. The Chikungunya structural protein fragment can be a CHIKV envelope (E) protein fragment or a CHIKV capsid (C) protein fragment. In some embodiments, the Chikungunya structural protein fragment can be a CHIKV E1, E2, E3, 6K, or capsid (C) protein fragment. In one embodiment, the Chikungunya structural protein fragment is CHIKV E1 fragment. In another embodiment, the Chikungunya structural protein fragment is CHIKV E2 fragment. In another embodiment, the Chikungunya structural protein fragment is CHIKV E3 fragment. In another embodiment, the Chikungunya structural protein fragment is a CHIKV C fragment. In another embodiment, the Chikungunya structural protein fragment is a CHIKV 6K fragment.
• In some embodiments, the Chikungunya antigenic polypeptide comprises the sequence of two or more Chikungunya structural protein fragments selected from E1, E2, E3, 6K, and C protein fragments. The antigenic polypeptide can comprise the sequence of any combination of CHIKV structural protein fragments, including, for example, CHIKV E1 and E2 protein fragments; CHIKV E2 and E3 protein fragments; CHIKV E1 and E3 protein fragments; CHIKV E1, E2, and E3 protein fragments; CHIKV E1, E2, E3, and C protein fragments; CHIKV E1, E2, E3, 6K, and C protein fragments; CHIKV E1, 6K, and E2 protein fragments; CHIKV E2, 6K, and E3 protein fragments; CHIKV E1, 6K, and E3 protein fragments; and CHIKV E1, E2, E3, and 6K protein fragments. In one particular embodiment, the Chikungunya antigenic polypeptide comprises the sequence of a fragment of the Chikungunya structural polyprotein: C-E3-E2-6K-E1.
• In some embodiments, the Chikungunya antigenic polypeptide comprises the sequence of two or more Chikungunya structural proteins in which the proteins are a combination of full-length protein(s) and fragment(s) selected from E1, E2, E3, 6K, and C full-length protein(s) and fragment(s). The Chikungunya antigenic polypeptide may comprise the sequence of any combination of full-length protein(s) and fragment(s) including, for example, CHIKV E1 and E2 full-length protein(s) and fragment(s); CHIKV E2 and E3 full-length protein(s) and fragment(s); CHIKV E1 and E3 full-length protein(s) and fragment(s); CHIKV E1, E2, and E3 full-length protein(s) and fragment(s); CHIKV E1, E2, E3, and C full-length protein(s) and fragments; CHIKV E1, E2, E3, and 6K full-length protein(s) and fragment(s); CHIKV E1, E2, E3, 6K, and C full-length protein(s) and fragment(s); CHIKV E1, 6K, and E2 full-length protein(s) and fragment(s); CHIKV E2, 6K, and E3 full-length protein(s) and fragment(s); and CHIKV E1, 6K, and E3 full-length protein(s) and fragment(s). In one particular embodiment, the Chikungunya antigenic polypeptide comprises the sequence of the Chikungunya structural polyprotein: C-E3-E2-6K-E1 in which the proteins are a combination of full-length protein(s) and fragment(s).
• The polypeptide antigens of the present disclosure can be one or more full-length CHIKV protein antigens, one or more fragment antigens, one or more epitope antigens or any combination of sequences thereof. In some embodiments, the CHIKV antigenic polypeptide comprises 10-25 amino acids. In some embodiments, the CHIKV antigenic polypeptide comprises 26-50 amino acids. In some embodiments, the CHIKV antigenic polypeptide comprises 51-100 amino acids. In some embodiments, the CHIKV antigenic polypeptide comprises 101-200 amino acids. In some embodiments, the CHIKV antigenic polypeptide comprises 201-400 amino acids. In some embodiments, the CHIKV antigenic polypeptide comprises 401-500 amino acids. In some embodiments, the CHIKV antigenic polypeptide comprises 501-750 amino acids. In some embodiments, the CHIKV antigenic polypeptide comprises 751-1000 amino acids. In some embodiments, the CHIKV antigenic polypeptide comprises 1001-1500 amino acids. In some embodiments, the CHIKV antigenic polypeptide comprises 1501-2000 amino acids. In some embodiments, the CHIKV antigenic polypeptide comprises 2001-4000 amino acids.
• The polypeptide antigens of the present disclosure can be one or more full-length DENV protein antigens, one or more fragment antigens, one or more epitope antigens or any combination of sequences thereof. In some embodiments, the DENV antigenic polypeptide comprises 10-25 amino acids. In some embodiments, the DENV antigenic polypeptide comprises 26-50 amino acids. In some embodiments, the DENV antigenic polypeptide comprises 51-100 amino acids. In some embodiments, the DENV antigenic polypeptide comprises 101-200 amino acids. In some embodiments, the DENV antigenic polypeptide comprises 201-400 amino acids. In some embodiments, the DENV antigenic polypeptide comprises 401-500 amino acids. In some embodiments, the DENV antigenic polypeptide comprises 501-750 amino acids. In some embodiments, the DENV antigenic polypeptide comprises 751-1000 amino acids. In some embodiments, the DENV antigenic polypeptide comprises 1001-1500 amino acids. In some embodiments, the DENV antigenic polypeptide comprises 1501-2000 amino acids. In some embodiments, the DENV antigenic polypeptide comprises 2001-4000 amino acids.
• The polypeptide antigens of the present disclosure can be one or more full-length ZIKV protein antigens, one or more fragment antigens, one or more epitope antigens or any combination of sequences thereof. In some embodiments, the ZIKV antigenic polypeptide comprises 10-25 amino acids. In some embodiments, the ZIKV antigenic polypeptide comprises 26-50 amino acids. In some embodiments, the ZIKV antigenic polypeptide comprises 51-100 amino acids. In some embodiments, the ZIKV antigenic polypeptide comprises 101-200 amino acids. In some embodiments, the ZIKV antigenic polypeptide comprises 201-400 amino acids. In some embodiments, the ZIKV antigenic polypeptide comprises 401-500 amino acids. In some embodiments, the ZIKV antigenic polypeptide comprises 501-750 amino acids. In some embodiments, the ZIKV antigenic polypeptide comprises 751-1000 amino acids. In some embodiments, the ZIKV antigenic polypeptide comprises 1001-1500 amino acids. In some embodiments, the ZIKV antigenic polypeptide comprises 1501-2000 amino acids. In some embodiments, the ZIKV antigenic polypeptide comprises 2001-4000 amino acids.
• The antigenic polypeptides include gene products, naturally occurring polypeptides, synthetic or engineered polypeptides, mutant polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. Polypeptides may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked. Most commonly, disulfide linkages are found in multichain polypeptides. The term polypeptide may also apply to amino acid polymers in which at least one amino acid residue is an artificial chemical analogue of a corresponding naturally-occurring amino acid.
• The term “polypeptide variant” refers to molecules which differ in their amino acid sequence from a native, wildtype, or reference sequence. The amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native, wildtype, or reference sequence. Ordinarily, variants possess at least 50% identity (homology) to a native, wildtype, or reference sequence. In some embodiments, variants possess at least 80%, or at least 90% identical (homologous) to a native, wildtype, or reference sequence.
• Examples of natural variants that are encompassed by the present disclosure include CHIKV, DENV, and ZIKV structural polypeptides from different CHIKV genotypes, lineages, strains, and isolates. A phylogenetic tree of Chikungunya virus strains derived from complete concatenated open reading frames for the nonstructural and structural polyproteins shows key envelope glycoprotein E1 amino acid substitutions that facilitated (Indian Ocean lineage) or prevented (Asian lineage) adaptation to Aedes albopictus. There are membrane-bound and secreted forms of E1 and E2, as well as the full length polyprotein antigen, which retains the protein's native conformation. Additionally, the different Chikungunya genotypes can also yield different antigens, which are functional in the constructs of the invention. There are several Chikungunya genotypes: Indian Ocean, East/Central/South African (ECSA), Asian, West African, and the Brazilian isolates (ECSA/Asian). Thus, for example, natural variants that are encompassed by the present disclosure include, but is not limited to, CHIKV structural polypeptides from the following strains and isolates: TA53, SA76, UG82, 37997, IND-06, Ross, S27, M-713424, E1-A226V, E1-T98, IND-63-WB1, Gibbs 63-263, TH35, 1-634029, AF15561, IND-73-MHS, 653496, C0392-95, P0731460, MY0211MR/06/BP, SV0444-95, K0146-95, TSI-GSD-218-VR1, TSI-GSD-218, M127, M125, 6441-88, MY003IMR/06/BP, MY002IMR/06/BP, TR206/H804187, MY/06/37348, MY/06/37350, NC/2011-568, 1455-75, RSU1, 0706aTw, InDRE51CHIK, PR-S4, AMA2798/H804298, Hu/85/NR/001, PhH15483, 0706aTw, 0802aTw, MY019IMR/06/BP, PR-S6, PER160/H803609, 99659, JKT23574, 0811aTw, CHIK/SBY6/10, 2001908323-BDG E1, 2001907981-BDG E1, 2004904899-BDG E1, 2004904879-BDG E1, 2003902452-BDG E1, DH130003, 0804aTw, 2002918310-BDG E1, JC2012, chik-sy, 3807, 3462, Yap 13-2148, PR-S5, 0802aTw, MY0191MR/06/Bp, 0706aTw, PhH15483, Hu/85/NR/001, CHIKV-13-112A, InDRE 4CHIK, 0806aTw, 0712aTw, 3412-78, Yap 13-2039, LEIV-CHIKV/Moscow/1, DH130003, and 20039.
• In some embodiments “variant mimics” are provided. As used herein, the term “variant mimic” is one which contains at least one amino acid that would mimic an activated sequence. For example, glutamate may serve as a mimic for phosphoro-threonine and/or phosphoro-serine. Alternatively, variant mimics may result in deactivation or in an inactivated product containing the mimic, for example, phenylalanine may act as an inactivating substitution for tyrosine; or alanine may act as an inactivating substitution for serine.
• “Homology” as it applies to amino acid sequences is defined as the percentage of residues in the candidate amino acid sequence that are identical with the residues in the amino acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. It is understood that homology depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. By “homologs” as it applies to polypeptide sequences means the corresponding sequence of other species having substantial identity to a second sequence of a second species.
• “Analogs” is meant to include polypeptide variants which differ by one or more amino acid alterations, for example, substitutions, additions or deletions of amino acid residues that still maintain one or more of the properties of the parent or starting polypeptide.
• The present disclosure provides several types of compositions that are polypeptide based, including variants and derivatives. These include, for example, substitutional, insertional, deletion and covalent variants and derivatives. The term “derivative” is used synonymously with the term “variant” but generally refers to a molecule that has been modified and/or changed in any way relative to a reference molecule or starting molecule.
• As such, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and/or additions, deletions and covalent modifications with respect to reference sequences, in particular the polypeptide sequences disclosed herein, are included within the scope of this disclosure. “Substitutional variants” when referring to polypeptides are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. Substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.
• As used herein the term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.
• “Features” when referring to polypeptide or polynucleotide are defined as distinct amino acid sequence-based or nucleotide-based components of a molecule respectively. Features of the polypeptides encoded by the polynucleotides include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini or any combination thereof.
• As used herein when referring to polypeptides the term “domain” refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions).
• As used herein when referring to polypeptides the terms “site” as it pertains to amino acid based embodiments is used synonymously with “amino acid residue” and “amino acid side chain.” As used herein when referring to polynucleotides the terms “site” as it pertains to nucleotide based embodiments is used synonymously with “nucleotide.” A site represents a position within a peptide or polypeptide or polynucleotide that may be modified, manipulated, altered, derivatized or varied within the polypeptide or polynucleotide based molecules.
• As used herein the terms “termini” or “terminus” when referring to polypeptides or polynucleotides refers to an extremity of a polypeptide or polynucleotide respectively. Such extremity is not limited only to the first or final site of the polypeptide or polynucleotide but may include additional amino acids or nucleotides in the terminal regions. Polypeptide-based molecules may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These proteins have multiple N- and C-termini. Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic conjugate.
• As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of polypeptides of interest. For example, provided herein is any protein fragment (meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical) of a reference protein 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids in length. In another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 amino acids which are 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to any of the sequences described herein can be utilized in accordance with the disclosure. In some embodiments, a polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein.
• Reference molecules (polypeptides or polynucleotides) may share a certain identity with the designed molecules (polypeptides or polynucleotides). The term “identity” as known in the art, refers to a relationship between the sequences of two or more peptides, polypeptides or polynucleotides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between them as determined by the number of matches between strings of two or more amino acid residues or nucleosides. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related peptides can be readily calculated by known methods. Generally, variants of a particular polynucleotide or polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402). A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm. More recently a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm. Other tools are described herein, specifically in the definition of “identity” below.
• As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered homologous if the polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. Two protein sequences are considered homologous if the proteins are at least 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least 20 amino acids.
• The term “identity” refers to the overall relatedness between polymeric molecules, for example, between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).
• In some embodiments, the polypeptides further comprise additional sequences or functional domains. For example, the CHIKV polypeptides of the present disclosure may comprise one or more linker sequences. In some embodiments, the CHIKV of the present invention may comprise a polypeptide tag, such as an affinity tag (chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), SBP-tag, Strep-tag, AviTag, Calmodulin-tag); solubilization tag; chromatography tag (polyanionic amino acid tag, such as FLAG-tag); epitope tag (short peptide sequences that bind to high-affinity antibodies, such as V5-tag, Myc-tag, VSV-tag, Xpress tag, E-tag, S-tag, and HA-tag); fluorescence tag (e.g., GFP). In some embodiments, the CHIKV of the present invention may comprise an amino acid tag, such as one or more lysines, histidines, or glutamates, which can be added to the polypeptide sequences (e.g., at the N-terminal or C-terminal ends). Lysines can be used to increase peptide solubility or to allow for biotinylation. Protein and amino acid tags are peptide sequences genetically grafted onto a recombinant protein. Sequence tags are attached to proteins for various purposes, such as peptide purification, identification, or localization, for use in various applications including, for example, affinity purification, protein array, western blotting, immunofluorescence, and immunoprecipitation. Such tags are subsequently removable by chemical agents or by enzymatic means, such as by specific proteolysis or intein splicing.
• Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble, or linked to a solid support.
Multiprotein and Multicomponent Vaccines
• The present disclosure encompasses CHIKV vaccines, DENV vaccines, ZIKV vaccines, CHIKV/DENV vaccines, CHIKV/ZIKV vaccines, ZIKV/DENV vaccines, and CHIKV/DENV/ZIKV vaccines comprising one or multiple RNA (e.g., mRNA) polynucleotides, each encoding a single antigenic polypeptide, as well as vaccines comprising a single RNA polynucleotide encoding more than one antigenic polypeptide (e.g., as a fusion polypeptide). Thus, it should be understood that a vaccine composition comprising a RNA polynucleotide having an open reading frame encoding a first antigenic polypeptide and a RNA polynucleotide having an open reading frame encoding a second antigenic polypeptide encompasses (a) vaccines that comprise a first RNA polynucleotide encoding a first antigenic polypeptide and a second RNA polynucleotide encoding a second antigenic polypeptide, and (b) vaccines that comprise a single RNA polynucleotide encoding a first and second antigenic polypeptide (e.g., as a fusion polypeptide). RNA vaccines of the present disclosure, in some embodiments, comprise 2-10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10), or more, RNA polynucleotides having an open reading frame, each of which encodes a different antigenic polypeptide (or a single RNA polynucleotide encoding 2-10, or more, different antigenic polypeptides). In some embodiments, a RNA vaccine comprises a RNA polynucleotide having an open reading frame encoding a capsid protein, a RNA polynucleotide having an open reading frame encoding a premembrane/membrane protein, and a RNA polynucleotide having an open reading frame encoding a envelope protein. In some embodiments, a RNA vaccine comprises a RNA polynucleotide having an open reading frame encoding a capsid protein and a RNA polynucleotide having an open reading frame encoding a premembrane/membrane protein. In some embodiments, a RNA vaccine comprises a RNA polynucleotide having an open reading frame encoding a capsid protein and a RNA polynucleotide having an open reading frame encoding a envelope protein. In some embodiments, a RNA vaccine comprises a RNA polynucleotide having an open reading frame encoding a premembrane/membrane protein and a RNA polynucleotide having an open reading frame encoding a envelope protein.
Signal Peptides
• In some embodiments, a RNA polynucleotide encodes an antigenic polypeptide fused to a signal peptide (e.g., SEQ ID NO: 125, 126, 128 or 131). The signal peptide may be fused at the N-terminus or the C-terminus of the antigenic polypeptide. In some embodiments, antigenic polypeptides encoded by CHIKV, DENV and/or ZIKV nucleic acids comprise a signal peptide. Signal peptides, comprising the N-terminal 15-60 amino acids of proteins, are typically needed for the translocation across the membrane on the secretory pathway and thus universally control the entry of most proteins both in eukaryotes and prokaryotes to the secretory pathway. Signal peptides generally include of three regions: an N-terminal region of differing length, which usually comprises positively charged amino acids; a hydrophobic region; and a short carboxy-terminal peptide region. In eukaryotes, the signal peptide of a nascent precursor protein (pre-protein) directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across it. The signal peptide is not responsible for the final destination of the mature protein, however. Secretory proteins devoid of further address tags in their sequence are by default secreted to the external environment. Signal peptides are cleaved from precursor proteins by an endoplasmic reticulum (ER)-resident signal peptidase or they remain uncleaved and function as a membrane anchor. During recent years, a more advanced view of signal peptides has evolved, showing that the functions and immunodominance of certain signal peptides are much more versatile than previously anticipated.
• Proteins encoded by the ZIKV genome, e.g., the ZIKV Envelope protein, contain a signal peptide at the N-terminus to facilitate protein targeting to the ER for processing. ER processing produces a mature Envelope protein, wherein the signal peptide is cleaved, typically by a signal peptidase of the host cell. A signal peptide may also facilitate the targeting of the protein to the cell membrane.
• CHIKV vaccines, DENV vaccines, ZIKV vaccines, CHIKV/DENV vaccines, CHIKV/ZIKV vaccines, ZIKV/DENV vaccines, and CHIKV/DENV/ZIKV vaccines of the present disclosure may comprise, for example, RNA polynucleotides encoding an artificial signal peptide, wherein the signal peptide coding sequence is operably linked to and is in frame with the coding sequence of the CHIKV, DENV and/or ZIKV antigenic polypeptide. Thus, CHIKV vaccines, DENV vaccines, ZIKV vaccines, CHIKV/DENV vaccines, CHIKV/ZIKV vaccines, ZIKV/DENV vaccines, and CHIKV/DENV/ZIKV vaccines of the present disclosure, in some embodiments, produce an antigenic polypeptide comprising a CHIKV, DENV and/or ZIKV antigenic polypeptide fused to a signal peptide. In some embodiments, a signal peptide is fused to the N-terminus of the CHIKV, DENV and/or ZIKV antigenic polypeptide. In some embodiments, a signal peptide is fused to the C-terminus of the CHIKV, DENV and/or ZIKV antigenic polypeptide.
• In some embodiments, the signal peptide fused to an antigenic polypeptide is an artificial signal peptide. In some embodiments, an artificial signal peptide fused to an antigenic polypeptide encoded by a RNA vaccine is obtained from an immunoglobulin protein, e.g., an IgE signal peptide or an IgG signal peptide. In some embodiments, a signal peptide fused to an antigenic polypeptide encoded by a RNA vaccine is an Ig heavy chain epsilon-1 signal peptide (IgE HC SP) having the sequence of: MDWTWILFLVAAATRVHS (SEQ ID NO: 126). In some embodiments, a signal peptide fused to a ZIKV antigenic polypeptide encoded by the ZIKV RNA vaccine is an IgG_k chain V-III region HAH signal peptide (IgG_k SP) having the sequence of METPAQLLFLLLLWLPDTTG (SEQ ID NO: 125). In some embodiments, a signal peptide fused to an antigenic polypeptide encoded by a RNA vaccine has an amino acid sequence set forth in SEQ ID NO: 125, 126, 128 or 131. The examples disclosed herein are not meant to be limiting and any signal peptide that is known in the art to facilitate targeting of a protein to ER for processing and/or targeting of a protein to the cell membrane may be used in accordance with the present disclosure.
• A signal peptide may have a length of 15-60 amino acids. For example, a signal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. In some embodiments, a signal peptide may have a length of 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25, or 15-20 amino acids.
• Non-limiting examples of antigenic polypeptides fused to signal peptides, which are encoded by a ZIKV RNA vaccine of the present disclosure, may be found in Table 31, SEQ ID NO: 48-59.
• A signal peptide is typically cleaved from the nascent polypeptide at the cleavage junction during ER processing, as illustrated in FIG. 26 . The mature ZIKV antigenic polypeptide produce by a ZIKV RNA vaccine, for example, typically does not comprise a signal peptide.
Chemical Modifications
• In some embodiments, the RNA vaccines of the present disclosure, in some embodiments, comprise at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one CHIKV, DENV and/or ZIKV antigenic polypeptide that comprises at least one chemical modification.
• The terms “chemical modification” and “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides in at least one of their position, pattern, percent or population. Generally, these terms do not refer to the ribonucleotide modifications in naturally occurring 5′-terminal mRNA cap moieties. With respect to a polypeptide, the term “modification” refers to a modification relative to the canonical set 20 amino acids. RNA polynucleotides, as provided herein, are also considered “modified” of they contain amino acid substitutions, insertions or a combination of substitutions and insertions.
• Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), in some embodiments, comprise various (more than one) different modifications. In some embodiments, a particular region of a polynucleotide contains one, two or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide), introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified polynucleotide. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide), introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response).
• Modifications of polynucleotides include, without limitation, those described herein. Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) may comprise modifications that are naturally-occurring, non-naturally-occurring or the polynucleotide may comprise a combination of naturally-occurring and non-naturally-occurring modifications. Polynucleotides may include any useful modification, for example, of a sugar, a nucleobase, or an internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage or to the phosphodiester backbone).
• Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the polynucleotides to achieve desired functions or properties. The modifications may be present on an internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified.
• The present disclosure provides for modified nucleosides and nucleotides of a polynucleotide (e.g., RNA polynucleotides, such as mRNA polynucleotides). A “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). A nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides.
• Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base/sugar or linker may be incorporated into polynucleotides of the present disclosure.
• The skilled artisan will appreciate that, except where otherwise noted, polynucleotide sequences set forth in the instant application will recite “T”s in a representative DNA sequence but where the sequence represents RNA, the “T”s would be substituted for “U”s.
• Modifications of polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) that are useful in the vaccines of the present disclosure include, but are not limited to the following: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonyl carbamoyladenosine; N6-glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladeno sine; 1,2′-O-dimethyladenosine; 1-methyladenosine; 2′-O-methyladenosine; 2′-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine; 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine; 2′-O-methyladenosine; 2′-O-ribosyladenosine (phosphate); Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine; N6,2′-O-dimethyladenosine; N6,2′-O-dimethyladenosine; N6,N6,2′-O-trimethyladenosine; N6,N6-dimethyladenosine; N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine; N6-methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine; N1-methyl-adenosine; N6,N6 (dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; α-thio-adenosine; 2 (amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6 (isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine; 2-(propyl)adenine; 2′-Amino-2′-deoxy-ATP; 2′-Azido-2′-deoxy-ATP; 2′-Deoxy-2′-a-aminoadenosine TP; 2′-Deoxy-2′-a-azidoadenosine TP; 6 (alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8 (amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine; 8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine; 1-Deazaadenosine TP; 2′Fluoro-N6-Bz-deoxyadenosine TP; 2′-OMe-2-Amino-ATP; 2′O-methyl-N6-Bz-deoxyadenosine TP; 2′-a-Ethynyladenosine TP; 2-aminoadenine; 2-Aminoadenosine TP; 2-Amino-ATP; 2′-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP; 2′-b-Ethynyladenosine TP; 2-Bromoadenosine TP; 2′-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP; 2′-Deoxy-2′,2′-difluoroadenosine TP; 2′-Deoxy-2′-a-mercaptoadenosine TP; 2′-Deoxy-2′-a-thiomethoxyadenosine TP; 2′-Deoxy-2′-b-aminoadenosine TP; 2′-Deoxy-2′-b-azidoadenosine TP; 2′-Deoxy-2′-b-bromoadenosine TP; 2′-Deoxy-2′-b-chloroadenosine TP; 2′-Deoxy-2′-b-fluoroadenosine TP; 2′-Deoxy-2′-b-iodoadenosine TP; 2′-Deoxy-2′-b-mercaptoadenosine TP; 2′-Deoxy-2′-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP; 2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2-methoxy-adenine; 2-methylthio-adenine; 2-Trifluoromethyladenosine TP; 3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP; 3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP; 3-Deazaadenosine TP; 4′-Azidoadenosine TP; 4′-Carbocyclic adenosine TP; 4′-Ethynyladenosine TP; 5′-Homo-adenosine TP; 8-Aza-ATP; 8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP; 9-Deazaadenosine TP; 2-aminopurine; 7-deaza-2,6-diaminopurine; 7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine; 2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine; 2-thiocytidine; 3-methylcytidine; 5-formylcytidine; 5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine; 2′-O-methylcytidine; 2′-O-methylcytidine; 5,2′-O-dimethylcytidine; 5-formyl-2′-O-methylcytidine; Lysidine; N4,2′-O-dimethylcytidine; N4-acetyl-2′-O-methylcytidine; N4-methylcytidine; N4,N4-Dimethyl-2′-OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine; Pseudo-iso-cytidine; pyrrolo-cytidine; α-thio-cytidine; 2-(thio)cytosine; 2′-Amino-2′-deoxy-CTP; 2′-Azido-2′-deoxy-CTP; 2′-Deoxy-2′-a-aminocytidine TP; 2′-Deoxy-2′-a-azidocytidine TP; 3 (deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2′-O-dimethylcytidine; 5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5 (trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine; 5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine; 5-bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6-(azo)cytosine; 6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine; 1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine; 2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine; 2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine; 4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine; Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2′-anhydro-cytidine TP hydrochloride; 2′Fluor-N4-Bz-cytidine TP; 2′Fluoro-N4-Acetyl-cytidine TP; 2′-O-Methyl-N4-Acetyl-cytidine TP; 2′O-methyl-N4-Bz-cytidine TP; 2′-a-Ethynylcytidine TP; 2′-a-Trifluoromethylcytidine TP; 2′-b-Ethynylcytidine TP; 2′-b-Trifluoromethylcytidine TP; 2′-Deoxy-2′,2′-difluorocytidine TP; 2′-Deoxy-2′-a-mercaptocytidine TP; 2′-Deoxy-2′-a-thiomethoxycytidine TP; 2′-Deoxy-2′-b-aminocytidine TP; 2′-Deoxy-2′-b-azidocytidine TP; 2′-Deoxy-2′-b-bromocytidine TP; 2′-Deoxy-2′-b-chlorocytidine TP; 2′-Deoxy-2′-b-fluorocytidine TP; 2′-Deoxy-2′-b-iodocytidine TP; 2′-Deoxy-2′-b-mercaptocytidine TP; 2′-Deoxy-2′-b-thiomethoxycytidine TP; 2′-O-Methyl-5-(1-propynyl)cytidine TP; 3′-Ethynylcytidine TP; 4′-Azidocytidine TP; 4′-Carbocyclic cytidine TP; 4′-Ethynylcytidine TP; 5-(1-Propynyl)ara-cytidine TP; 5-(2-Chloro-phenyl)-2-thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidine TP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynylara-cytidine TP; 5-Ethynylcytidine TP; 5′-Homo-cytidine TP; 5-Methoxycytidine TP; 5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidine TP; Pseudoisocytidine; 7-methylguanosine; N2,2′-O-dimethylguanosine; N2-methylguanosine; Wyosine; 1,2′-O-dimethylguanosine; 1-methylguanosine; 2′-O-methylguanosine; 2′-O-ribosylguanosine (phosphate); 2′-O-methylguanosine; 2′-O-ribosylguanosine (phosphate); 7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine; Methylwyo sine; N2,7-dimethylguanosine; N2,N2,2′-O-trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine; N2,7,2′-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine; 8-oxo-guanosine; N1-methyl-guanosine; α-thio-guanosine; 2 (propyl)guanine; 2-(alkyl)guanine; 2′-Amino-2′-deoxy-GTP; 2′-Azido-2′-deoxy-GTP; 2′-Deoxy-2′-a-aminoguanosine TP; 2′-Deoxy-2′-a-azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine; 6-(methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7 (deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8 (halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; aza guanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine; 1-methyl-6-thio-guanosine; 6-methoxy-guanosine; 6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine; 6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine; 7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine; N2-methyl-6-thio-guanosine; 1-Me-GTP; 2′Fluoro-N2-isobutyl-guanosine TP; 2′O-methyl-N2-isobutyl-guanosine TP; 2′-a-Ethynylguanosine TP; 2′-a-Trifluoromethylguanosine TP; 2′-b-Ethynylguano sine TP; 2′-b-Trifluoromethylguanosine TP; 2′-Deoxy-2′,2′-difluoroguanosine TP; 2′-Deoxy-2′-a-mercaptoguanosine TP; 2′-Deoxy-2′-a-thiomethoxyguanosine TP; 2′-Deoxy-2′-b-aminoguanosine TP; 2′-Deoxy-2′-b-azidoguanosine TP; 2′-Deoxy-2′-b-bromoguanosine TP; 2′-Deoxy-2′-b-chloroguanosine TP; 2′-Deoxy-2′-b-fluoroguanosine TP; 2′-Deoxy-2′-b-iodoguanosine TP; 2′-Deoxy-2′-b-mercaptoguanosine TP; 2′-Deoxy-2′-b-thiomethoxyguanosine TP; 4′-Azidoguanosine TP; 4′-Carbocyclic guanosine TP; 4′-Ethynylguanosine TP; 5′-Homo-guanosine TP; 8-bromo-guanosine TP; 9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; Inosine; 1,2′-O-dimethylinosine; 2′-O-methylinosine; 7-methylinosine; 2′-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine; Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deaza thymidine; deoxy-thymidine; 2′-O-methyluridine; 2-thiouridine; 3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine; 5-methyluridine; 5-taurinomethyl-2-thiouridine; 5-taurinomethyluridine; Dihydrouridine; Pseudouridine; (3-(3-amino-3-carboxypropyl)uridine; 1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1-methylpseduouridine; 1-methyl-pseudouridine; 2′-O-methyluridine; 2′-O-methylpseudouridine; 2′-O-methyluridine; 2-thio-2′-O-methyluridine; 3-(3-amino-3-carboxypropyl)uridine; 3,2′-O-dimethyluridine; 3-Methyl-pseudo-Uridine TP; 4-thiouridine; 5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methyl ester; 5,2′-O-dimethyluridine; 5,6-dihydro-uridine; 5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2′-O-methyluridine; 5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine methyl ester; 5-carboxymethylaminomethyl-2′-O-methyluridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5-Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2′-O-methyluridine; 5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5-methoxyuridine; 5-methyluridine, 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-Methyldihydrouridine; 5-Oxyacetic acid-Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP; N1-methyl-pseudo-uridine; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 3-(3-Amino-3-carboxypropyl)-Uridine TP; 5-(iso-Pentenylaminomethyl)-2-thiouridine TP; 5-(iso-Pentenylaminomethyl)-2′-O-methyluridine TP; 5-(iso-Pentenylaminomethyl)uridine TP; 5-propynyl uracil; α-thio-uridine; 1 (aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil; 1 (aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1 (aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1 (aminoalkylaminocarbonylethylenyl)-pseudouracil; 1 (aminocarbonylethylenyl)-2(thio)-pseudouracil; 1 (aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1 (aminocarbonylethylenyl)-4 (thio)pseudouracil; 1 (aminocarbonylethylenyl)-pseudouracil; 1 substituted 2(thio)-pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1 substituted 4 (thio)pseudouracil; 1 substituted pseudouracil; 1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil; 1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP; 1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-Methyl-pseudo-UTP; 2 (thio)pseudouracil; 2′ deoxy uridine; 2′ fluorouridine; 2-(thio)uracil; 2,4-(dithio)psuedouracil; 2′ methyl, 2′-amino, 2′-azido, 2′-fluro-guanosine; 2′-Amino-2′-deoxy-UTP; 2′-Azido-2′-deoxy-UTP; 2′-Azido-deoxyuridine TP; 2′-O-methylpseudouridine; 2′ deoxy uridine; 2′ fluorouridine; 2′-Deoxy-2′-a-aminouridine TP; 2′-Deoxy-T-a-azidouridine TP; 2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4 (thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil; 4-thiouracil; 5 (1,3-diazole-1-alkyl)uracil; 5 (2-aminopropyl)uracil; 5 (aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5 (methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl) 2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2 (thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5 (methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5 (trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4 (dithio)pseudouracil; 5-(alkyl)-4 (thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5-(guanidiniumalkyl)uracil; 5-(halo)uracil; 5-(1,3-diazole-1-alkyl)uracil; 5-(methoxy)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl) 2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil; 5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil; 5-(methyl)-4 (thio)pseudouracil; 5-(methyl)pseudouracil; 5-(methylaminomethyl)-2 (thio)uracil; 5-(methylaminomethyl)-2,4(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine; allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil; Pseudo-UTP-1-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP; 1-carboxymethyl-pseudouridine; 1-methyl-1-deaza-pseudouridine; 1-propynyl-uridine; 1-taurinomethyl-1-methyl-uridine; 1-taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine; 2-methoxy-4-thio-pseudouridine; 2-thio-1-methyl-1-deaza-pseudouridine; 2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine; 2-thio-dihydropseudouridine; 2-thio-dihydrouridine; 2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine; 4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine; 4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine; (±)1-(2-Hydroxypropyl)pseudouridine TP; (2R)-1-(2-Hydroxypropyl)pseudouridine TP; (2S)-1-(2-Hydroxypropyl)pseudouridine TP; (E)-5-(2-Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine TP; (Z)-5-(2-Bromo-vinyl)ara-uridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP; 1-(2,2,2-Trifluoroethyl)-pseudo-UTP; 1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP; 1-(2,2-Diethoxyethyl)pseudouridine TP; 1-(2,4,6-Trimethylbenzyl)pseudouridine TP; 1-(2,4,6-Trimethyl-benzyl)pseudo-UTP; 1-(2,4,6-Trimethyl-phenyl)pseudo-UTP; 1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-UTP; 1-(2-Hydroxyethyl)pseudouridine TP; 1-(2-Methoxyethyl)pseudouridine TP; 1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP; 1-(3,4-Dimethoxybenzyl)pseudouridine TP; 1-(3-Amino-3-carboxypropyl)pseudo-UTP; 1-(3-Amino-propyl)pseudo-UTP; 1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP; 1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP; 1-(4-Amino-butyl)pseudo-UTP; 1-(4-Amino-phenyl)pseudo-UTP; 1-(4-Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzyl)pseudouridine TP; 1-(4-Chlorobenzyl)pseudouridine TP; 1-(4-Fluorobenzyl)pseudouridine TP; 1-(4-Iodobenzyl)pseudouridine TP; 1-(4-Methanesulfonylbenzyl)pseudouridine TP; 1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methoxy-benzyl)pseudo-UTP; 1-(4-Methoxy-phenyl)pseudo-UTP; 1-(4-Methylbenzyl)pseudouridine TP; 1-(4-Methyl-benzyl)pseudo-UTP; 1-(4-Nitrobenzyl)pseudouridine TP; 1-(4-Nitro-benzyl)pseudo-UTP; 1(4-Nitro-phenyl)pseudo-UTP; 1-(4-Thiomethoxybenzyl)pseudouridine TP; 1-(4-Trifluoromethoxybenzyl)pseudouridine TP; 1-(4-Trifluoromethylbenzyl)pseudouridine TP; 1-(5-Amino-pentyl)pseudo-UTP; 1-(6-Amino-hexyl)pseudo-UTP; 1,6-Dimethyl-pseudo-UTP; 1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridine TP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl} pseudouridine TP; 1-Acetylpseudouridine TP; 1-Alkyl-6-(1-propynyl)-pseudo-UTP; 1-Alkyl-6-(2-propynyl)-pseudo-UTP; 1-Alkyl-6-allyl-pseudo-UTP; 1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl-pseudo-UTP; 1-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP; 1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP; 1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP; 1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP; 1-Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP; 1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP; 1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP; 1-Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP; 1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP; 1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP; 1-Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP; 1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1-Homoallylpseudouridine TP; 1-Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP; 1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP; 1-Me-alpha-thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine TP; 1-Methoxymethylpseudouridine TP; 1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP; 1-Methyl-6-(4-morpholino)-pseudo-UTP; 1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1-Methyl-6-(substituted phenyl)pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP; 1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6-bromo-pseudo-UTP; 1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP; 1-Methyl-6-cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP; 1-Methyl-6-ethoxy-pseudo-UTP; 1-Methyl-6-ethylcarboxylate-pseudo-UTP; 1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo-UTP; 1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6-hydroxyamino-pseudo-UTP; 1-Methyl-6-hydroxy-pseudo-UTP; 1-Methyl-6-iodo-pseudo-UTP; 1-Methyl-6-iso-propyl-pseudo-UTP; 1-Methyl-6-methoxy-pseudo-UTP; 1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo-UTP; 1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP; 1-Methyl-6-trifluoromethoxy-pseudo-UTP; 1-Methyl-6-trifluoromethyl-pseudo-UTP; 1-Morpholinomethylpseudouridine TP; 1-Pentyl-pseudo-UTP; 1-Phenyl-pseudo-UTP; 1-Pivaloylpseudouridine TP; 1-Propargylpseudouridine TP; 1-Propyl-pseudo-UTP; 1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP; 1-Thiomethoxymethylpseudouridine TP; 1-Thiomorpholinomethylpseudouridine TP; 1-Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP; 1-Vinylpseudouridine TP; 2,2′-anhydro-uridine TP; 2′-bromo-deoxyuridine TP; 2′-F-5-Methyl-2′-deoxy-UTP; 2′-OMe-5-Me-UTP; 2′-OMe-pseudo-UTP; 2′-a-Ethynyluridine TP; 2′-a-Trifluoromethyluridine TP; 2′-b-Ethynyluridine TP; 2′-b-Trifluoromethyluridine TP; 2′-Deoxy-2′,2′-difluorouridine TP; 2′-Deoxy-T-a-mercaptouridine TP; 2′-Deoxy-T-a-thiomethoxyuridine TP; 2′-Deoxy-T-b-aminouridine TP; 2′-Deoxy-T-b-azidouridine TP; 2′-Deoxy-T-b-bromouridine TP; 2′-Deoxy-T-b-chlorouridine TP; 2′-Deoxy-2′-b-fluorouridine TP; 2′-Deoxy-T-b-iodouridine TP; 2′-Deoxy-T-b-mercaptouridine TP; 2′-Deoxy-T-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine; 2-methoxyuridine; 2′-O-Methyl-5-(1-propynyl)uridine TP; 3-Alkyl-pseudo-UTP; 4′-Azidouridine TP; 4′-Carbocyclic uridine TP; 4′-Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridine TP; 5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5′-Homo-uridine TP; 5-iodo-2′-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP; 5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-Uridine TP; 5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP; 6-(4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP; 6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP; 6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP; 6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP; 6-Dimethylamino-pseudo-UTP; 6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP; 6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxyamino-pseudo-UTP; 6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP; 6-Methoxy-pseudo-UTP; 6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP; 6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo-UTP; 6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine 1-(4-methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoic acid) TP; Pseudouridine TP 1-[3-(2-ethoxy)]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}] propionic acid; Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP 1-methylphosphonic acid diethyl ester; Pseudo-UTP-N1-3-propionic acid; Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-pentanoic acid; Pseudo-UTP-N1-6-hexanoic acid; Pseudo-UTP-N1-7-heptanoic acid; Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N1-p-benzoic acid; Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine; undermodified hydroxywybutosine; 4-demethylwyosine; 2,6-(diamino)purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl: 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl; 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine; 2,4,5-(trimethyl)phenyl; 2′ methyl, 2′ amino, 2′ azido, 2′-fluro-cytidine; 2′ methyl, 2′ amino, 2′ azido, 2′-fluro-adenine; 2′-methyl, 2′ amino, 2′ azido, 2′-fluro-uridine; 2′-amino-2′-deoxyribose; 2-amino-6-Chloro-purine; 2-aza-inosinyl; 2′-azido-2′-deoxyribose; 2′-fluoro-2′-deoxyribose; 2′-fluoro-modified bases; 2′-O-methyl-ribose; 2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl; 2-pyridinone; 3 nitropyrrole; 3-(methyl)-7-(propynyl)isocarbostyrilyl; 3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5 nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrilyl; 5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloro-purine; 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aza)indolyl; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl; 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl; Aminoindolyl; Anthracenyl; bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl; Isocarbostyrilyl; Isoguanisine; N2-substituted purines; N6-methyl-2-amino-purine; N6-substituted purines; N-alkylated derivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl; Nitroindazolyl; Nitropyrazolyl; Nubularine; 06-substituted purines; O-alkylated derivative; ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl; Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl; Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted 1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5′-TP; 2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine; pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP; Formycin B TP; Pyrrolosine TP; 2′-OH-ara-adenosine TP; 2′-OH-ara-cytidine TP; 2′-OH-ara-uridine TP; 2′-OH-ara-guanosine TP; 5-(2-carbomethoxyvinyl)uridine TP; and N6-(19-Amino-pentaoxanonadecyl)adenosine TP.
• In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
• In some embodiments, modified nucleobases in the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) are selected from the group consisting of pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine5-methyluridine, and 2′-O-methyl uridine. In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
• In some embodiments, modified nucleobases in the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) are selected from the group consisting of 1-methyl-pseudouridine (m1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine and α-thio-adenosine. In some embodiments, the polynucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
• In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises pseudouridine (ψ) and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 1-methyl-pseudouridine (m1ψ). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 1-methyl-pseudouridine (m1ψ) and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2-thiouridine (s2U). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises methoxy-uridine (mo5U). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2′-O-methyl uridine. In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2′-O-methyl uridine and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises N6-methyl-adenosine (m6A). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).
• In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) is uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, a polynucleotide can be uniformly modified with 5-methyl-cytidine (m5C), meaning that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m5C). Similarly, a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as any of those set forth above.
• In some embodiments, the modified nucleobase is a modified cytosine. Examples of nucleobases and nucleosides having a modified cytosine include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine.
• In some embodiments, a modified nucleobase is a modified uridine. Example nucleobases and nucleosides having a modified uridine include 5-cyano uridine or 4′-thio uridine.
• In some embodiments, a modified nucleobase is a modified adenine. Example nucleobases and nucleosides having a modified adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and 2,6-Diaminopurine.
• In some embodiments, a modified nucleobase is a modified guanine. Example nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (mil), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.
• In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U), 4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m³U), 5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine 5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U), 5-methoxycarbonylmethyl-uridine (mcm⁵U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U), 5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine (mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U), 5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U), 5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine (cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm⁵U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm⁵s²U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ), 5-methyl-2-thio-uridine (m⁵s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp³U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ), 5-(isopentenylaminomethyl)uridine (inm⁵U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um), 2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um), 5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um), 3,2′-O-dimethyl-uridine (m³Um), and 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine, deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)]uridine.
• In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m³C), N4-acetyl-cytidine (ac⁴C), 5-formylcytidine (f⁵C), N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k₂C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm), 5,2′-O-dimethylcytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm), N4,2′-O-dimethylcytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm), N4,N4,2′-O-trimethyl-cytidine (m⁴2 Cm), 1-thio-cytidine, 2′-F-ara-cytidine, 2′-F-cytidine, and 2′-0H-ara-cytidine.
• In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m¹A), 2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A), 2-methylthio-N6-methyl-adenosine (ms² m⁶A), N6-isopentenyl-adenosine (i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A), N6-(cis-hydroxyisopentenyl)adenosine (io⁶A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A), N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine (t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A), N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine (hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A), N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am), N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶2 Am), 1,2′-O-dimethyl-adenosine (m¹Am), 2′-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine, 2′-0H-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.
• In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o₂yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ₀), 7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G⁺), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m¹G), N2-methyl-guanosine (m²G), N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guanosine (m^2,7G), N2,N2,7-dimethyl-guanosine (m^2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2′-O-methyl-guanosine (Gm), N2-methyl-2′-O-methyl-guanosine (m²Gm), N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm), 1-methyl-2′-O-methyl-guanosine (m¹Gm), N2,7-dimethyl-2′-O-methyl-guanosine (m^2,7Gm), 2′-O-methyl-inosine (Im), 1,2′-O-dimethyl-inosine (m¹Im), 2′-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, 06-methyl-guanosine, 2′-F-ara-guanosine, and 2′-F-guanosine.
Methods of Treatment
• Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits and reagents for prevention and/or treatment of CHIKV, DENV, ZIKV, CHIKV/DENV (the combination of CHIKV and DENV, CHIKV/ZIKV (the combination of CHIKV and ZIKV), ZIKV and DENV (the combination of ZIKV and DENV), and CHIKV/DENV/ZIKV (the combination of CHIKV, DENV and ZIKV) in humans and other mammals. CHIKV RNA (e.g. mRNA) vaccines, DENV RNA (e.g. mRNA) vaccines, ZIKV RNA (e.g. mRNA) vaccines, CHIKV/DENV RNA (e.g. mRNA) vaccines, CHIKV/ZIKV RNA (e.g. mRNA) vaccines, ZIKV/DENV RNA (e.g. mRNA) vaccines, and CHIKV/DENV/ZIKV RNA (e.g. mRNA) vaccines can be used as therapeutic or prophylactic agents. They may be used in medicine to prevent and/or treat infectious disease. In exemplary aspects, the vaccines, of the present disclosure are used to provide prophylactic protection from CHIKV, DENV, ZIKV or any combination of two or three of the foregoing viruses. Prophylactic protection from CHIKV, DENV and/or ZIKV can be achieved following administration of a CHIKV, DENV and/or ZIKV vaccine or combination vaccine, of the present disclosure. Vaccines (including combination vaccines) can be administered once, twice, three times, four times or more but it is likely sufficient to administer the vaccine once (optionally followed by a single booster). It is possible, although less desirable, to administer the vaccine to an infected individual to achieve a therapeutic response. Dosing may need to be adjusted accordingly.
Broad Spectrum Vaccines
• It is envisioned that there may be situations where persons are at risk for infection with more than one strain of CHIKV, DENV and/or ZIKV (e.g., more than one strain of CHIKV, more than one strain of DENV, and/or more than one strain of ZIKV). RNA (e.g., mRNA) therapeutic vaccines are particularly amenable to combination vaccination approaches due to a number of factors including, but not limited to, speed of manufacture, ability to rapidly tailor vaccines to accommodate perceived geographical threat, and the like. Moreover, because the vaccines utilize the human body to produce the antigenic protein, the vaccines are amenable to the production of larger, more complex antigenic proteins, allowing for proper folding, surface expression, antigen presentation, etc. in the human subject. To protect against more than one strain of CHIKV, DENV and/or ZIKV, a vaccine (including a combination vaccine) can be administered that includes RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a first CHIKV, DENV and/or ZIKV and further includes RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a second CHIKV, DENV and/or ZIKV. RNAs (mRNAs) can be coformulated, for example, in a single lipid nanoparticle (LNP) or can be formulated in separate LNPs destined for co-administration.
• A method of eliciting an immune response in a subject against a CHIKV, DENV and/or ZIKV is provided in aspects of the invention. The method involves administering to the subject a CHIKV, DENV and/or ZIKV RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV, DENV and/or ZIKV antigenic polypeptide or an immunogenic fragment thereof, thereby inducing in the subject an immune response specific to CHIKV, DENV and/or ZIKV antigenic polypeptide or an immunogenic fragment thereof, wherein anti-antigenic polypeptide antibody titer in the subject is increased following vaccination relative to anti-antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the CHIKV, DENV and/or ZIKV. An “anti-antigenic polypeptide antibody” is a serum antibody the binds specifically to the antigenic polypeptide.
• A prophylactically effective dose is a therapeutically effective dose that prevents infection with the virus at a clinically acceptable level. In some embodiments the therapeutically effective dose is a dose listed in a package insert for the vaccine. A traditional vaccine, as used herein, refers to a vaccine other than the mRNA vaccines of the invention. For instance, a traditional vaccine includes but is not limited to live microorganism vaccines, killed microorganism vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, etc.
• In some embodiments the anti-antigenic polypeptide antibody titer in the subject is increased 1 log to 10 log following vaccination relative to anti-antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the CHIKV, DENV and/or ZIKV.
• In some embodiments the anti-antigenic polypeptide antibody titer in the subject is increased 1 log following vaccination relative to anti-antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the CHIKV, DENV and/or ZIKV.
• In some embodiments the anti-antigenic polypeptide antibody titer in the subject is increased 2 log following vaccination relative to anti-antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the CHIKV, DENV and/or ZIKV.
• In some embodiments the anti-antigenic polypeptide antibody titer in the subject is increased 3 log following vaccination relative to anti-antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the CHIKV, DENV and/or ZIKV.
• In some embodiments the anti-antigenic polypeptide antibody titer in the subject is increased 5 log following vaccination relative to anti-antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the CHIKV, DENV and/or ZIKV.
• In some embodiments the anti-antigenic polypeptide antibody titer in the subject is increased 10 log following vaccination relative to anti-antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the CHIKV, DENV and/or ZIKV.
• A method of eliciting an immune response in a subject against a CHIKV, DENV and/or ZIKV is provided in other aspects of the invention. The method involves administering to the subject a CHIKV, DENV and/or ZIKV RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV, DENV and/or ZIKV antigenic polypeptide or an immunogenic fragment thereof, thereby inducing in the subject an immune response specific to CHIKV, DENV and/or ZIKV antigenic polypeptide or an immunogenic fragment thereof, wherein the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine against the CHIKV, DENV and/or ZIKV at 2 times to 100 times the dosage level relative to the RNA vaccine.
• In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at twice the dosage level relative to the CHIKV, DENV and/or ZIKV RNA vaccine.
• In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at three times the dosage level relative to the CHIKV, DENV and/or ZIKV RNA vaccine.
• In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 4 times the dosage level relative to the CHIKV, DENV and/or ZIKV vaccine.
• In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 5 times the dosage level relative to the CHIKV, DENV and/or ZIKV RNA vaccine.
• In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 10 times the dosage level relative to the CHIKV, DENV and/or ZIKV RNA vaccine.
• In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 50 times the dosage level relative to the CHIKV, DENV and/or ZIKV RNA vaccine.
• In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 100 times the dosage level relative to the CHIKV, DENV and/or ZIKV RNA vaccine.
• In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 10 times to 1000 times the dosage level relative to the CHIKV, DENV and/or ZIKV RNA vaccine.
• In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 100 times to 1000 times the dosage level relative to the CHIKV, DENV and/or ZIKV RNA vaccine
• In other embodiments the immune response is assessed by determining [protein] antibody titer in the subject.
• In other aspects the present disclosure is a method of eliciting an immune response in a subject against a CHIKV, DENV and/or ZIKV by administering to the subject a CHIKV, DENV and/or ZIKV RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one CHIKV, DENV and/or ZIKV antigenic polypeptide or an immunogenic fragment thereof, thereby inducing in the subject an immune response specific to CHIKV, DENV and/or ZIKV antigenic polypeptide or an immunogenic fragment thereof, wherein the immune response in the subject is induced 2 days to 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the CHIKV, DENV and/or ZIKV. In some embodiments the immune response in the subject is induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine at 2 times to 100 times the dosage level relative to the RNA vaccine.
• In some embodiments the immune response in the subject is induced 2 days earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.
• In some embodiments the immune response in the subject is induced 3 days earlier relative to an immune response induced in a subject vaccinated a prophylactically effective dose of a traditional vaccine.
• In some embodiments the immune response in the subject is induced 1 week earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.
• In some embodiments the immune response in the subject is induced 2 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.
• In some embodiments the immune response in the subject is induced 3 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.
• In some embodiments the immune response in the subject is induced 5 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.
• In some embodiments the immune response in the subject is induced 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.
• Also provided herein are methods of eliciting an immune response in a subject against a CHIKV, DENV and/or ZIKV by administering to the subject a CHIKV, DENV and/or ZIKV RNA vaccine having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide does not include a stabilization element, and wherein an adjuvant is not coformulated or co-administered with the vaccine.
Therapeutic and Prophylactic Compositions
• Provided herein are compositions, methods, kits and reagents for the prevention, treatment or diagnosis of Chikungunya virus in humans and other mammals, for example. The active therapeutic agents of the present disclosure include the CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines), cells containing CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines), and antigenic polypeptides translated from the polynucleotides comprising the RNA vaccines. CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines) can be used as therapeutic or prophylactic agents. They may be used in medicine and/or for the priming of immune effector cells, for example, to activate peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (re-infused) into a subject.
• In some embodiments, a vaccines, including a combination vaccine, containing RNA polynucleotides, e.g., mRNA, as described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject), and the RNA polynucleotides are translated in vivo to produce an antigenic polypeptide.
• The CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, may be induced for translation of a polypeptide (e.g., antigen or immunogen) in a cell, tissue or organism. Such translation can be in vivo, ex vivo, in culture or in vitro. The cell, tissue or organism is contacted with an effective amount of a composition containing a CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, that contains a polynucleotide that has at least one a translatable region encoding an antigenic polypeptide.
• An “effective amount” of the CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size, and extent of modified nucleosides) and other components of the CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, and other determinants. In general, CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, provides an induced or boosted immune response as a function of antigen production in the cell, preferably more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or a peptide antigen. Increased antigen production may be demonstrated by increased cell transfection (the percentage of cells transfected with the RNA vaccine), increased protein translation from the polynucleotide, decreased nucleic acid degradation (as demonstrated, for example, by increased duration of protein translation from a modified polynucleotide), or altered antigen specific immune response of the host cell.
• In some embodiments, RNA vaccines (including polynucleotides and their encoded polypeptides) and cells comprising the RNA vaccines in accordance with the present disclosure may be used for the treatment of Chikungunya virus, Dengue virus, Zika virus, or any combination of two or three of the foregoing viruses.
• CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, may be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or early in infection during the incubation phase or during active infection after onset of symptoms. In some embodiments, the amount of RNA vaccine of the present disclosure provided to a cell, a tissue or a subject may be an amount effective for immune prophylaxis.
• CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, may be administered with other prophylactic or therapeutic compounds. As a non-limiting example, a prophylactic or therapeutic compound may be an adjuvant or a booster. As used herein, when referring to a prophylactic composition, such as a vaccine, the term “booster” refers to an extra administration of the prophylactic (vaccine) composition. A booster (or booster vaccine) may be given after an earlier administration of the prophylactic composition. The time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 15 months, 18 months, 21 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more than 99 years, and any time period in-between.
• In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, may be administered intramuscularly or intradermally, similarly to the administration of inactivated vaccines known in the art.
• The CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need. As a non-limiting example, the RNA vaccines may be utilized to treat and/or prevent infectious disease caused by Chikungunya virus. RNA vaccines have superior properties in that they produce much larger antibody titers and produce responses early than commercially available anti-virals.
• Provided herein are pharmaceutical compositions including CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines and RNA vaccine compositions and/or complexes optionally in combination with one or more pharmaceutically acceptable excipients.
• CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, may be formulated or administered alone or in conjunction with one or more other components. For instance, CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines (vaccine compositions) may comprise other components including, but not limited to, adjuvants. In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, do not include an adjuvant (they are adjuvant free).
• CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, may be formulated or administered in combination with one or more pharmaceutically-acceptable excipients. In some embodiments, vaccine compositions comprise at least one additional active substance, such as, for example, a therapeutically-active substance, a prophylactically-active substance, or a combination of both. Vaccine compositions may be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents, such as vaccine compositions, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).
• In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, are administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to the RNA vaccines or the polynucleotides contained therein, for example, RNA polynucleotides (e.g., mRNA polynucleotides) encoding CHIKV, DENV and/or ZIKV antigenic polypeptides.
• Formulations of the vaccine compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., mRNA polynucleotide) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
• Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
• CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation); (4) alter the biodistribution (e.g., target to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein (antigen) in vivo. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.
Stabilizing Elements
• Naturally-occurring eukaryotic mRNA molecules have been found to contain stabilizing elements, including, but not limited to untranslated regions (UTR) at their 5′-end (5′UTR) and/or at their 3′-end (3′UTR), in addition to other structural features, such as a 5′-cap structure or a 3′-poly(A) tail. Both the 5′UTR and the 3′UTR are typically transcribed from the genomic DNA and are elements of the premature mRNA. Characteristic structural features of mature mRNA, such as the 5′-cap and the 3′-poly(A) tail are usually added to the transcribed (premature) mRNA during mRNA processing. The 3′-poly(A) tail is typically a stretch of adenine nucleotides added to the 3′-end of the transcribed mRNA. It can comprise up to about 400 adenine nucleotides. In some embodiments the length of the 3′-poly(A) tail may be an essential element with respect to the stability of the individual mRNA.
• In some embodiments the RNA vaccine may include one or more stabilizing elements. Stabilizing elements may include for instance a histone stem-loop. A stem-loop binding protein (SLBP), a 32 kDa protein has been identified. It is associated with the histone stem-loop at the 3′-end of the histone messages in both the nucleus and the cytoplasm. Its expression level is regulated by the cell cycle; it is peaks during the S-phase, when histone mRNA levels are also elevated. The protein has been shown to be essential for efficient 3′-end processing of histone pre-mRNA by the U7 snRNP. SLBP continues to be associated with the stem-loop after processing, and then stimulates the translation of mature histone mRNAs into histone proteins in the cytoplasm. The RNA binding domain of SLBP is conserved through metazoa and protozoa; its binding to the histone stem-loop depends on the structure of the loop. The minimum binding site includes at least three nucleotides 5′ and two nucleotides 3′ relative to the stem-loop.
• In some embodiments, the RNA vaccines include a coding region, at least one histone stem-loop, and optionally, a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal generally should enhance the expression level of the encoded protein. The encoded protein, in some embodiments, is not a histone protein, a reporter protein (e.g. Luciferase, GFP, EGFP, β-Galactosidase, EGFP), or a marker or selection protein (e.g. alpha-Globin, Galactokinase and Xanthine:guanine phosphoribosyl transferase (GPT)).
• In some embodiments, the combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop, even though both represent alternative mechanisms in nature, acts synergistically to increase the protein expression beyond the level observed with either of the individual elements. It has been found that the synergistic effect of the combination of poly(A) and at least one histone stem-loop does not depend on the order of the elements or the length of the poly(A) sequence.
• In some embodiments, the RNA vaccine does not comprise a histone downstream element (HDE). “Histone downstream element” (HDE) includes a purine-rich polynucleotide stretch of approximately 15 to 20 nucleotides 3′ of naturally occurring stem-loops, representing the binding site for the U7 snRNA, which is involved in processing of histone pre-mRNA into mature histone mRNA. Ideally, the inventive nucleic acid does not include an intron.
• In some embodiments, the RNA vaccine may or may not contain a enhancer and/or promoter sequence, which may be modified or unmodified or which may be activated or inactivated. In some embodiments, the histone stem-loop is generally derived from histone genes, and includes an intramolecular base pairing of two neighbored partially or entirely reverse complementary sequences separated by a spacer, consisting of a short sequence, which forms the loop of the structure. The unpaired loop region is typically unable to base pair with either of the stem loop elements. It occurs more often in RNA, as is a key component of many RNA secondary structures, but may be present in single-stranded DNA as well. Stability of the stem-loop structure generally depends on the length, number of mismatches or bulges, and base composition of the paired region. In some embodiments, wobble base pairing (non-Watson-Crick base pairing) may result. In some embodiments, the at least one histone stem-loop sequence comprises a length of 15 to 45 nucleotides.
• In other embodiments the RNA vaccine may have one or more AU-rich sequences removed. These sequences, sometimes referred to as AURES are destabilizing sequences found in the 3′UTR. The AURES may be removed from the RNA vaccines. Alternatively the AURES may remain in the RNA vaccine.
Nanoparticle Formulations
• In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, are formulated in a nanoparticle. In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, are formulated in a lipid nanoparticle. In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, are formulated in a lipid-polycation complex, referred to as a cationic lipid nanoparticle. The formation of the lipid nanoparticle may be accomplished by methods known in the art and/or as described in U.S. Pub. No. 20120178702, herein incorporated by reference in its entirety. As a non-limiting example, the cationic lipid nanoparticle may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and/or polyarginine and the cationic peptides described in International Pub. No. WO2012013326 or US Patent Pub. No. US20130142818; each of which is herein incorporated by reference in its entirety. In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, are formulated in a lipid nanoparticle that includes a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
• A lipid nanoparticle formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, ratio of all components and biophysical parameters such as size. For example, the lipid nanoparticle formulation may be composed of 57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA. (Semple et al., Nature Biotech. 2010 28:172-176; herein incorporated by reference in its entirety). Altering the composition of the cationic lipid can more effectively deliver RNA to various antigen presenting cells (Basha et al. Mol Ther. 2011 19:2186-2200; herein incorporated by reference in its entirety).
• In some embodiments, lipid nanoparticle formulations may comprise 35 to 45% cationic lipid, 40% to 50% cationic lipid, 50% to 60% cationic lipid and/or 55% to 65% cationic lipid. In some embodiments, the ratio of lipid to RNA (e.g., mRNA) in lipid nanoparticles may be 5:1 to 20:1, 10:1 to 25:1, 15:1 to 30:1 and/or at least 30:1.
• In some embodiments, the ratio of PEG in the lipid nanoparticle formulations may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified from C14 to C18 to alter the pharmacokinetics and/or biodistribution of the lipid nanoparticle formulations. As a non-limiting example, lipid nanoparticle formulations may contain 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5% to 5.0% and/or 3.0% to 6.0% of the lipid molar ratio of PEG-c-DOMG (R-3-[(ω-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) as compared to the cationic lipid, DSPC and cholesterol. In some embodiments, the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-Dimyristoyl-sn-glycerol) and/or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationic lipid may be selected from any lipid known in the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.
• In some embodiments, the CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccine formulation is a nanoparticle that comprises at least one lipid. The lipid may be selected from, but is not limited to, DLin-DMA, Dlin-K-DMA, 98N12-5, C12-200, Dlin-MC3-DMA, Dlin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. In some embodiments, the lipid may be a cationic lipid such as, but not limited to, Dlin-DMA, Dlin-D-DMA, Dlin-MC3-DMA, Dlin-KC2-DMA, DODMA and amino alcohol lipids. The amino alcohol cationic lipid may be the lipids described in and/or made by the methods described in US Patent Publication No. US20130150625, herein incorporated by reference in its entirety. As a non-limiting example, the cationic lipid may be 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (Compound 2 in US20130150625); 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (Compound 3 in US20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (Compound 4 in US20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof.
• Lipid nanoparticle formulations typically comprise a lipid, in particular, an ionizable cationic lipid, for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Dlin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), or di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and further comprise a neutral lipid, a sterol and a molecule capable of reducing particle aggregation, for example a PEG or PEG-modified lipid.
• In some embodiments, a lipid nanoparticle formulation consists essentially of (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Dlin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG or PEG-cDMA, in a molar ratio of 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15% PEG-lipid.
• In some embodiments, a lipid nanoparticle formulation includes 25% to 75% on a molar basis of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Dlin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., 35 to 65%, 45 to 65%, 60%, 57.5%, 50% or 40% on a molar basis.
• In some embodiments, a lipid nanoparticle formulation includes 0.5% to 15% on a molar basis of the neutral lipid, e.g., 3 to 12%, 5 to 10% or 15%, 10%, or 7.5% on a molar basis. Examples of neutral lipids include, without limitation, DSPC, POPC, DPPC, DOPE and SM. In some embodiments, the formulation includes 5% to 50% on a molar basis of the sterol (e.g., 15 to 45%, 20 to 40%, 40%, 38.5%, 35%, or 31% on a molar basis. A non-limiting example of a sterol is cholesterol. In some embodiments, a lipid nanoparticle formulation includes 0.5% to 20% on a molar basis of the PEG or PEG-modified lipid (e.g., 0.5 to 10%, 0.5 to 5%, 1.5%, 0.5%, 1.5%, 3.5%, or 5% on a molar basis. In some embodiments, a PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of 2,000 Da. In some embodiments, a PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of less than 2,000, for example around 1,500 Da, around 1,000 Da, or around 500 Da. Non-limiting examples of PEG-modified lipids include PEG-distearoyl glycerol (PEG-DMG) (also referred herein as PEG-C14 or C14-PEG), PEG-cDMA (further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005) the contents of which are herein incorporated by reference in its entirety).
• In some embodiments, lipid nanoparticle formulations include 25-75% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Dlin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% of the neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG or PEG-modified lipid on a molar basis.
• In some embodiments, lipid nanoparticle formulations include 35-65% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Dlin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% of the neutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG or PEG-modified lipid on a molar basis.
• In some embodiments, lipid nanoparticle formulations include 45-65% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Dlin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% of the neutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG or PEG-modified lipid on a molar basis.
• In some embodiments, lipid nanoparticle formulations include 60% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (Dlin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.5% of the neutral lipid, 31% of the sterol, and 1.5% of the PEG or PEG-modified lipid on a molar basis.
• In some embodiments, lipid nanoparticle formulations include 50% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (Dlin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% of the neutral lipid, 38.5% of the sterol, and 1.5% of the PEG or PEG-modified lipid on a molar basis.
• In some embodiments, lipid nanoparticle formulations include 50% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (Dlin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% of the neutral lipid, 35% of the sterol, 4.5% or 5% of the PEG or PEG-modified lipid, and 0.5% of the targeting lipid on a molar basis.
• In some embodiments, lipid nanoparticle formulations include 40% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Dlin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 15% of the neutral lipid, 40% of the sterol, and 5% of the PEG or PEG-modified lipid on a molar basis.
• In some embodiments, lipid nanoparticle formulations include 57.2% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Dlin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.1% of the neutral lipid, 34.3% of the sterol, and 1.4% of the PEG or PEG-modified lipid on a molar basis.
• In some embodiments, lipid nanoparticle formulations include 57.5% of a cationic lipid selected from the PEG lipid is PEG-cDMA (PEG-cDMA is further discussed in Reyes et al. (J. Controlled Release, 107, 276-287 (2005), the contents of which are herein incorporated by reference in its entirety), 7.5% of the neutral lipid, 31.5% of the sterol, and 3.5% of the PEG or PEG-modified lipid on a molar basis.
• In some embodiments, lipid nanoparticle formulations consists essentially of a lipid mixture in molar ratios of 20-70% cationic lipid: 5-45% neutral lipid: 20-55% cholesterol: 0.5-15% PEG-modified lipid. In some embodiments, lipid nanoparticle formulations consists essentially of a lipid mixture in a molar ratio of 20-60% cationic lipid: 5-25% neutral lipid: 25-55% cholesterol: 0.5-15% PEG-modified lipid.
• In some embodiments, the molar lipid ratio is 50/10/38.5/1.5 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG, PEG-DSG or PEG-DPG), 57.2/7.1134.3/1.4 (mol % cationic lipid/neutral lipid, e.g., DPPC/Chol/PEG-modified lipid, e.g., PEG-cDMA), 40/15/40/5 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 50/10/35/4.5/0.5 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DSG), 50/10/35/5 (cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 40/10/40/10 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), 35/15/40/10 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA) or 52/13/30/5 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).
• Non-limiting examples of lipid nanoparticle compositions and methods of making them are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51: 8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (the contents of each of which are incorporated herein by reference in their entirety).
• In some embodiments, lipid nanoparticle formulations may comprise a cationic lipid, a PEG lipid and a structural lipid and optionally comprise a non-cationic lipid. As a non-limiting example, a lipid nanoparticle may comprise 40-60% of cationic lipid, 5-15% of a non-cationic lipid, 1-2% of a PEG lipid and 30-50% of a structural lipid. As another non-limiting example, the lipid nanoparticle may comprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid and 38.5% structural lipid. As yet another non-limiting example, a lipid nanoparticle may comprise 55% cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid and 32.5% structural lipid. In some embodiments, the cationic lipid may be any cationic lipid described herein such as, but not limited to, Dlin-KC2-DMA, Dlin-MC3-DMA and L319.
• In some embodiments, the lipid nanoparticle formulations described herein may be 4 component lipid nanoparticles. The lipid nanoparticle may comprise a cationic lipid, a non-cationic lipid, a PEG lipid and a structural lipid. As a non-limiting example, the lipid nanoparticle may comprise 40-60% of cationic lipid, 5-15% of a non-cationic lipid, 1-2% of a PEG lipid and 30-50% of a structural lipid. As another non-limiting example, the lipid nanoparticle may comprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid and 38.5% structural lipid. As yet another non-limiting example, the lipid nanoparticle may comprise 55% cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid and 32.5% structural lipid. In some embodiments, the cationic lipid may be any cationic lipid described herein such as, but not limited to, Dlin-KC2-DMA, Dlin-MC3-DMA and L319.
• In some embodiments, the lipid nanoparticle formulations described herein may comprise a cationic lipid, a non-cationic lipid, a PEG lipid and a structural lipid. As a non-limiting example, the lipid nanoparticle comprise 50% of the cationic lipid Dlin-KC2-DMA, 10% of the non-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% of the structural lipid cholesterol. As a non-limiting example, the lipid nanoparticle comprise 50% of the cationic lipid Dlin-MC3-DMA, 10% of the non-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% of the structural lipid cholesterol. As a non-limiting example, the lipid nanoparticle comprise 50% of the cationic lipid Dlin-MC3-DMA, 10% of the non-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DMG and 38.5% of the structural lipid cholesterol. As yet another non-limiting example, the lipid nanoparticle comprise 55% of the cationic lipid L319, 10% of the non-cationic lipid DSPC, 2.5% of the PEG lipid PEG-DMG and 32.5% of the structural lipid cholesterol.
• Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a vaccine composition may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w/w) of the active ingredient. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
• In some embodiments, the RNA vaccine composition may comprise the polynucleotide described herein, formulated in a lipid nanoparticle comprising MC3, Cholesterol, DSPC and PEG2000-DMG, the buffer trisodium citrate, sucrose and water for injection. As a non-limiting example, the composition comprises: 2.0 mg/mL of drug substance (e.g., polynucleotides encoding H10N8 influenza virus), 21.8 mg/mL of MC3, 10.1 mg/mL of cholesterol, 5.4 mg/mL of DSPC, 2.7 mg/mL of PEG2000-DMG, 5.16 mg/mL of trisodium citrate, 71 mg/mL of sucrose and 1.0 mL of water for injection.
• In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 10-500 nm, 20-400 nm, 30-300 nm, 40-200 nm. In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 50-150 nm, 50-200 nm, 80-100 nm or 80-200 nm.
• In one embodiment, the RNA vaccines of the invention may be formulated in lipid nanoparticles having a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 to about 100 nm.
• In one embodiment, the lipid nanoparticles may have a diameter from about 10 to 500 nm.
• In one embodiment, the lipid nanoparticle may have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.
Modes of Vaccine Administration
• CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, may be administered by any route which results in a therapeutically effective outcome. These include, but are not limited, to intradermal, intramuscular, and/or subcutaneous administration. The present disclosure provides methods comprising administering RNA vaccines to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, compositions are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, compositions may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
• In certain embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, may be administered at dosage levels sufficient to deliver 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005 mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kg to 0.5 mg/kg, 0.01 mg/kg to 50 mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kg to 30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kg to 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect (see e.g., the range of unit doses described in International Publication No WO2013078199, herein incorporated by reference in its entirety). The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used.
• In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, may be administered at dosage levels sufficient to deliver 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005 mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kg to 0.5 mg/kg, 0.01 mg/kg to 50 mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kg to 30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kg to 25 mg/kg, of subject body weight per day, one or more times a day, per week, per month, etc. to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect (see e.g., the range of unit doses described in International Publication No WO2013078199, herein incorporated by reference in its entirety). The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, every four weeks, every 2 months, every three months, every 6 months, etc. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used. In exemplary embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, may be administered at dosage levels sufficient to deliver 0.0005 mg/kg to 0.01 mg/kg, e.g., about 0.0005 mg/kg to about 0.0075 mg/kg, e.g., about 0.0005 mg/kg, about 0.001 mg/kg, about 0.002 mg/kg, about 0.003 mg/kg, about 0.004 mg/kg or about 0.005 mg/kg.
• In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, may be administered once or twice (or more) at dosage levels sufficient to deliver 0.025 mg/kg to 0.250 mg/kg, 0.025 mg/kg to 0.500 mg/kg, 0.025 mg/kg to 0.750 mg/kg, or 0.025 mg/kg to 1.0 mg/kg.
• In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, may be administered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later, Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0 and 10 years later) at a total dose of or at dosage levels sufficient to deliver a total dose of 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg. Higher and lower dosages and frequency of administration are encompassed by the present disclosure. For example, a CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, may be administered three or four times.
• In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, may be administered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later, Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0 and 10 years later) at a total dose of or at dosage levels sufficient to deliver a total dose of 0.010 mg, 0.025 mg, 0.100 mg or 0.400 mg.
• In some embodiments the RNA vaccine for use in a method of vaccinating a subject is administered the subject a single dosage of between 10 μg/kg and 400 μg/kg of the nucleic acid vaccine in an effective amount to vaccinate the subject. In some embodiments the RNA vaccine for use in a method of vaccinating a subject is administered the subject a single dosage of between 10 μg and 400 μg of the nucleic acid vaccine in an effective amount to vaccinate the subject.
• A RNA vaccine pharmaceutical composition described herein can be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).
• In some embodiments, a RNA (e.g., mRNA) vaccine for use in a method of vaccinating a subject is administered the subject a single dosage of 10 μg of the nucleic acid vaccine in an effective amount to vaccinate the subject. In some embodiments, a RNA vaccine for use in a method of vaccinating a subject is administered the subject a single dosage of 2 μg of the nucleic acid vaccine in an effective amount to vaccinate the subject. In some embodiments, a vaccine for use in a method of vaccinating a subject is administered the subject two dosages of 10 μg of the nucleic acid vaccine in an effective amount to vaccinate the subject. In some embodiments, a RNA vaccine for use in a method of vaccinating a subject is administered the subject two dosages of 2 μg of the nucleic acid vaccine in an effective amount to vaccinate the subject.
• A RNA (e.g. mRNA) vaccine pharmaceutical composition described herein can be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).
RNA Vaccine Formulations and Methods of Use
• Some aspects of the present disclosure provide formulations of a RNA (e.g., mRNA) vaccine, wherein the RNA vaccine is formulated in an effective amount to produce an antigen specific immune response in a subject (e.g., production of antibodies specific to a CHIKV, DENV and/or ZIKV antigenic polypeptide). “An effective amount” is a dose of an RNA (e.g., mRNA) vaccine effective to produce an antigen-specific immune response. Also provided herein are methods of inducing an antigen-specific immune response in a subject.
• In some embodiments, the antigen-specific immune response is characterized by measuring an anti-CHIKV, anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer produced in a subject administered a RNA (e.g., mRNA) vaccine as provided herein. An antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen or epitope of an antigen. Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers, for example.
• In some embodiments, an antibody titer is used to assess whether a subject has had an infection or to determine whether immunizations are required. In some embodiments, an antibody titer is used to determine the strength of an autoimmune response, to determine whether a booster immunization is needed, to determine whether a previous vaccine was effective, and to identify any recent or prior infections. In accordance with the present disclosure, an antibody titer may be used to determine the strength of an immune response induced in a subject by the RNA vaccine.
• In some embodiments, an anti-ZIKV antigenic polypeptide antibody titer produced in a subject is increased by at least 1 log relative to a control. For example, antibody titer produced in a subject may be increased by at least 1.5, at least 2, at least 2.5, or at least 3 log relative to a control. In some embodiments, the antibody titer produced in the subject is increased by 1, 1.5, 2, 2.5 or 3 log relative to a control. In some embodiments, the antibody titer produced in the subject is increased by 1-3 log relative to a control. For example, the antibody titer produced in a subject may be increased by 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5, 1.5-3, 2-2.5, 2-3, or 2.5-3 log relative to a control.
• In some embodiments, the antibody titer produced in a subject is increased at least 2 times relative to a control. For example, the antibody titer produced in a subject may be increased at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times relative to a control. In some embodiments, the antibody titer produced in the subject is increased 2, 3, 4, 5, 6, 7, 8, 9, or 10 times relative to a control. In some embodiments, the anti antibody titer produced in a subject is increased 2-10 times relative to a control. For example, the antibody titer produced in a subject may be increased 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 times relative to a control.
• A control, in some embodiments, is an anti-CHIKV, anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer produced in a subject who has not been administered a RNA (e.g., mRNA) vaccine. In some embodiments, a control is an anti-CHIKV, anti-DENV and/or anti-ZIKV antibody titer produced in a subject who has been administered a live attenuated CHIKV, DENV and/or ZIKV vaccine. An attenuated vaccine is a vaccine produced by reducing the virulence of a viable (live). An attenuated virus is altered in a manner that renders it harmless or less virulent relative to live, unmodified virus. In some embodiments, a control is an anti-CHIKV, anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer produced in a subject administered inactivated CHIKV, DENV and/or ZIKV vaccine. In some embodiments, a control is an anti-CHIKV, anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer produced in a subject administered a recombinant or purified CHIKV, DENV and/or ZIKV protein vaccine. Recombinant protein vaccines typically include protein antigens that either have been produced in a heterologous expression system (e.g., bacteria or yeast) or purified from large amounts of the pathogenic organism.
• In some embodiments, an effective amount of a RNA (e.g., mRNA) vaccine is a dose that is reduced compared to the standard of care dose of a recombinant CHIKV, DENV and/or ZIKV protein vaccine. A “standard of care,” as provided herein, refers to a medical or psychological treatment guideline and can be general or specific. “Standard of care” specifies appropriate treatment based on scientific evidence and collaboration between medical professionals involved in the treatment of a given condition. It is the diagnostic and treatment process that a physician/clinician should follow for a certain type of patient, illness or clinical circumstance. A “standard of care dose,” as provided herein, refers to the dose of a recombinant or purified CHIKV, DENV and/or ZIKV protein vaccine, or a live attenuated or inactivated CHIKV, DENV and/or ZIKV vaccine, that a physician/clinician or other medical professional would administer to a subject to treat or prevent CHIKV, DENV and/or ZIKV or a related condition, while following the standard of care guideline for treating or preventing CHIKV, DENV and/or ZIKV, or a related condition.
• In some embodiments, the anti-CHIKV, anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer produced in a subject administered an effective amount of a ZIKV RNA vaccine is equivalent to an anti-CHIKV, anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer produced in a control subject administered a standard of care dose of a recombinant or purified CHIKV, DENV and/or ZIKV protein vaccine or a live attenuated or inactivated CHIKV, DENV and/or ZIKV vaccine.
• In some embodiments, an effective amount of a RNA (e.g., mRNA) vaccine is a dose equivalent to an at least 2-fold reduction in a standard of care dose of a recombinant or purified CHIKV, DENV and/or ZIKV protein vaccine. For example, an effective amount of a CHIKV, DENV and/or ZIKV RNA vaccine may be a dose equivalent to an at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold reduction in a standard of care dose of a recombinant or purified CHIKV, DENV and/or ZIKV protein vaccine. In some embodiments, an effective amount of a CHIKV, DENV and/or ZIKV RNA vaccine is a dose equivalent to an at least at least 100-fold, at least 500-fold, or at least 1000-fold reduction in a standard of care dose of a recombinant or purified CHIKV, DENV and/or ZIKV protein vaccine. In some embodiments, an effective amount of a CHIKV, DENV and/or ZIKV RNA vaccine is a dose equivalent to a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 20-, 50-, 100-, 250-, 500-, or 1000-fold reduction in a standard of care dose of a recombinant or purified CHIKV, DENV and/or ZIKV protein vaccine. In some embodiments, the anti-CHIKV, anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer produced in a subject administered an effective amount of a CHIKV, DENV and/or ZIKV RNA vaccine is equivalent to an anti-CHIKV, anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or protein CHIKV, DENV and/or ZIKV protein vaccine or a live attenuated or inactivated CHIKV, DENV and/or ZIKV vaccine. In some embodiments, an effective amount of a RNA (e.g., mRNA) vaccine is a dose equivalent to a 2-fold to 1000-fold (e.g., 2-fold to 100-fold, 10-fold to 1000-fold) reduction in the standard of care dose of a recombinant or purified CHIKV, DENV and/or ZIKV protein vaccine, wherein the anti-CHIKV, anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-CHIKV, anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified CHIKV, DENV and/or ZIKV protein vaccine or a live attenuated or inactivated CHIKV, DENV and/or ZIKV vaccine.
• In some embodiments, the effective amount of a RNA (e.g., mRNA) vaccine is a dose equivalent to a 2 to 1000-, 2 to 900-, 2 to 800-, 2 to 700-, 2 to 600-, 2 to 500-, 2 to 400-, 2 to 300-, 2 to 200-, 2 to 100-, 2 to 90-, 2 to 80-, 2 to 70-, 2 to 60-, 2 to 50-, 2 to 40-, 2 to 30-, 2 to 20-, 2 to 10-, 2 to 9-, 2 to 8-, 2 to 7-, 2 to 6-, 2 to 5-, 2 to 4-, 2 to 3-, 3 to 1000-, 3 to 900-, 3 to 800-, 3 to 700-, 3 to 600-, 3 to 500-, 3 to 400-, 3 to 3 to 00-, 3 to 200-, 3 to 100-, 3 to 90-, 3 to 80-, 3 to 70-, 3 to 60-, 3 to 50-, 3 to 40-, 3 to 30-, 3 to 20-, 3 to 10-, 3 to 9-, 3 to 8-, 3 to 7-, 3 to 6-, 3 to 5-, 3 to 4-, 4 to 1000-, 4 to 900-, 4 to 800-, 4 to 700-, 4 to 600-, 4 to 500-, 4 to 400-, 4 to 4 to 00-, 4 to 200-, 4 to 100-, 4 to 90-, 4 to 80-, 4 to 70-, 4 to 60-, 4 to 50-, 4 to 40-, 4 to 30-, 4 to 20-, 4 to 10-, 4 to 9-, 4 to 8-, 4 to 7-, 4 to 6-, 4 to 5-, 4 to 4-, 5 to 1000-, 5 to 900-, 5 to 800-, 5 to 700-, 5 to 600-, 5 to 500-, 5 to 400-, 5 to 300-, 5 to 200-, 5 to 100-, 5 to 90-, 5 to 80-, 5 to 70-, 5 to 60-, 5 to 50-, 5 to 40-, 5 to 30-, 5 to 20-, 5 to 10-, 5 to 9-, 5 to 8-, 5 to 7-, 5 to 6-, 6 to 1000-, 6 to 900-, 6 to 800-, 6 to 700-, 6 to 600-, 6 to 500-, 6 to 400-, 6 to 300-, 6 to 200-, 6 to 100-, 6 to 90-, 6 to 80-, 6 to 70-, 6 to 60-, 6 to 50-, 6 to 40-, 6 to 30-, 6 to 20-, 6 to 10-, 6 to 9-, 6 to 8-, 6 to 7-, 7 to 1000-, 7 to 900-, 7 to 800-, 7 to 700-, 7 to 600-, 7 to 500-, 7 to 400-, 7 to 300-, 7 to 200-, 7 to 100-, 7 to 90-, 7 to 80-, 7 to 70-, 7 to 60-, 7 to 50-, 7 to 40-, 7 to 30-, 7 to 20-, 7 to 10-, 7 to 9-, 7 to 8-, 8 to 1000-, 8 to 900-, 8 to 800-, 8 to 700-, 8 to 600, 8 to 500-, 8 to 400-, 8 to 300-, 8 to 200-, 8 to 100-, 8 to 90-, 8 to 80-, 8 to 70-, 8 to 60-, 8 to 50-, 8 to 40-, 8 to 30-, 8 to 20-, 8 to 10-, 8 to 9-, 9 to 1000-, 9 to 900-, 9 to 800-, 9 to 700-, 9 to 600-, 9 to 500-, 9 to 400-, 9 to 300-, 9 to 200-, 9 to 100-, 9 to 90-, 9 to 80-, 9 to 70-, 9 to 60-, 9 to 50-, 9 to 40-, 9 to 30-, 9 to 20-, 9 to 10-, 10 to 1000-, 10 to 900-, 10 to 800-, 10 to 700-, 10 to 600-, 10 to 500-, 10 to 400-, 10 to 300-, 10 to 200-, 10 to 100-, 10 to 90-, 10 to 80-, 10 to 70-, 10 to 60-, 10 to 50-, 10 to 40-, 10 to 30-, 10 to 20-, 20 to 1000-, 20 to 900-, 20 to 800-, 20 to 700-, 20 to 600-, 20 to 500-, 20 to 400-, 20 to 300-, 20 to 200-, 20 to 100-, 20 to 90-, 20 to 80-, 20 to 70-, 20 to 60-, 20 to 50-, 20 to 40-, 20 to 30-, 30 to 1000-, 30 to 900-, 30 to 800-, 30 to 700-, 30 to 600-, 30 to 500-, 30 to 400-, 30 to 300-, 30 to 200-, 30 to 100-, 30 to 90-, 30 to 80-, 30 to 70-, 30 to 60-, 30 to 50-, 30 to 40-, 40 to 1000-, 40 to 900-, 40 to 800-, 40 to 700-, 40 to 600-, 40 to 500-, 40 to 400-, 40 to 300-, 40 to 200-, 40 to 100-, 40 to 90-, 40 to 80-, 40 to 70-, 40 to 60-, 40 to 50-, 50 to 1000-, 50 to 900-, 50 to 800-, 50 to 700-, 50 to 600-, 50 to 500-, 50 to 400-, 50 to 300-, 50 to 200-, 50 to 100-, 50 to 90-, 50 to 80-, 50 to 70-, 50 to 60-, 60 to 1000-, 60 to 900-, 60 to 800-, 60 to 700-, 60 to 600-, 60 to 500-, 60 to 400-, 60 to 300-, 60 to 200-, 60 to 100-, 60 to 90-, 60 to 80-, 60 to 70-, 70 to 1000-, 70 to 900-, 70 to 800-, 70 to 700-, 70 to 600-, 70 to 500-, 70 to 400-, 70 to 300-, 70 to 200-, 70 to 100-, 70 to 90-, 70 to 80-, 80 to 1000-, 80 to 900-, 80 to 800-, 80 to 700-, 80 to 600-, 80 to 500-, 80 to 400-, 80 to 300-, 80 to 200-, 80 to 100-, 80 to 90-, 90 to 1000-, 90 to 900-, 90 to 800-, 90 to 700-, 90 to 600-, 90 to 500-, 90 to 400-, 90 to 300-, 90 to 200-, 90 to 100-, 100 to 1000-, 100 to 900-, 100 to 800-, 100 to 700-, 100 to 600-, 100 to 500-, 100 to 400-, 100 to 300-, 100 to 200-, 200 to 1000-, 200 to 900-, 200 to 800-, 200 to 700-, 200 to 600-, 200 to 500-, 200 to 400-, 200 to 300-, 300 to 1000-, 300 to 900-, 300 to 800-, 300 to 700-, 300 to 600-, 300 to 500-, 300 to 400-, 400 to 1000-, 400 to 900-, 400 to 800-, 400 to 700-, 400 to 600-, 400 to 500-, 500 to 1000-, 500 to 900-, 500 to 800-, 500 to 700-, 500 to 600-, 600 to 1000-, 600 to 900-, 600 to 800-, 600 to 700-, 700 to 1000-, 700 to 900-, 700 to 800-, 800 to 1000-, 800 to 900-, or 900 to 1000-fold reduction in the standard of care dose of a recombinant CHIKV, DENV and/or ZIKV protein vaccine. In some embodiments, such as the foregoing, the anti-CHIKV, anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-CHIKV, anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified CHIKV, DENV and/or ZIKV protein vaccine or a live attenuated or inactivated CHIKV, DENV and/or ZIKV vaccine. In some embodiments, the effective amount is a dose equivalent to (or equivalent to an at least) 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, 110-, 120-, 130-, 140-, 150-, 160-, 170-, 1280-, 190-, 200-, 210-, 220-, 230-, 240-, 250-, 260-, 270-, 280-, 290-, 300-, 310-, 320-, 330-, 340-, 350-, 360-, 370-, 380-, 390-, 400-, 410-, 420-, 430-, 440-, 450-, 4360-, 470-, 480-, 490-, 500-, 510-, 520-, 530-, 540-, 550-, 560-, 5760-, 580-, 590-, 600-, 610-, 620-, 630-, 640-, 650-, 660-, 670-, 680-, 690-, 700-, 710-, 720-, 730-, 740-, 750-, 760-, 770-, 780-, 790-, 800-, 810-, 820-, 830-, 840-, 850-, 860-, 870-, 880-, 890-, 900-, 910-, 920-, 930-, 940-, 950-, 960-, 970-, 980-, 990-, or 1000-fold reduction in the standard of care dose of a recombinant CHIKV, DENV and/or ZIKV protein vaccine. In some embodiments, such as the foregoing, an anti-CHIKV, anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-CHIKV, anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified CHIKV, DENV and/or ZIKV protein vaccine or a live attenuated or inactivated CHIKV, DENV and/or ZIKV vaccine.
• In some embodiments, the effective amount of a RNA (e.g., mRNA) vaccine is a total dose of 50-1000 μg. In some embodiments, the effective amount of a RNA (e.g., mRNA) vaccine is a total dose of 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 50-90, 50-80, 50-70, 50-60, 60-1000, 60-900, 60-800, 60-700, 60-600, 60-500, 60-400, 60-300, 60-200, 60-100, 60-90, 60-80, 60-70, 70-1000, 70-900, 70-800, 70-700, 70-600, 70-500, 70-400, 70-300, 70-200, 70-100, 70-90, 70-80, 80-1000, 80-900, 80-800, 80-700, 80-600, 80-500, 80-400, 80-300, 80-200, 80-100, 80-90, 90-1000, 90-900, 90-800, 90-700, 90-600, 90-500, 90-400, 90-300, 90-200, 90-100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1000, 600-900, 600-900, 600-700, 700-1000, 700-900, 700-800, 800-1000, 800-900, or 900-1000 μg. In some embodiments, the effective amount of a RNA (e.g., mRNA) vaccine is a total dose of 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 μg. In some embodiments, the effective amount is a dose of 25-500 μg administered to the subject a total of two times. In some embodiments, the effective amount of a RNA (e.g., mRNA) vaccine is a dose of 25-500, 25-400, 25-300, 25-200, 25-100, 25-50, 50-500, 50-400, 50-300, 50-200, 50-100, 100-500, 100-400, 100-300, 100-200, 150-500, 150-400, 150-300, 150-200, 200-500, 200-400, 200-300, 250-500, 250-400, 250-300, 300-500, 300-400, 350-500, 350-400, 400-500 or 450-500 μg administered to the subject a total of two times. In some embodiments, the effective amount of a RNA (e.g., mRNA) vaccine is a total dose of 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg administered to the subject a total of two times.
• This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
EXAMPLES Example 1: Manufacture of Polynucleotides
• According to the present disclosure, the manufacture of polynucleotides and or parts or regions thereof may be accomplished utilizing the methods taught in International Application WO2014/152027 entitled “Manufacturing Methods for Production of RNA Transcripts”, the contents of which is incorporated herein by reference in its entirety.
• Purification methods may include those taught in International Application WO2014/152030 and WO2014/152031, each of which is incorporated herein by reference in its entirety.
• Detection and characterization methods of the polynucleotides may be performed as taught in WO2014/144039, which is incorporated herein by reference in its entirety.
• Characterization of the polynucleotides of the disclosure may be accomplished using a procedure selected from the group consisting of polynucleotide mapping, reverse transcriptase sequencing, charge distribution analysis, and detection of RNA impurities, wherein characterizing comprises determining the RNA transcript sequence, determining the purity of the RNA transcript, or determining the charge heterogeneity of the RNA transcript. Such methods are taught in, for example, WO2014/144711 and WO2014/144767, the contents of each of which is incorporated herein by reference in its entirety.
Example 2: Chimeric Polynucleotide Synthesis Introduction
• According to the present disclosure, two regions or parts of a chimeric polynucleotide may be joined or ligated using triphosphate chemistry.
• According to this method, a first region or part of 100 nucleotides or less is chemically synthesized with a 5′ monophosphate and terminal 3′-desOH or blocked OH. If the region is longer than 80 nucleotides, it may be synthesized as two strands for ligation.
• If the first region or part is synthesized as a non-positionally modified region or part using in vitro transcription (IVT), conversion the 5′-monophosphate with subsequent capping of the 3′ terminus may follow.
• Monophosphate protecting groups may be selected from any of those known in the art.
• The second region or part of the chimeric polynucleotide may be synthesized using either chemical synthesis or IVT methods. IVT methods may include an RNA polymerase that can utilize a primer with a modified cap. Alternatively, a cap of up to 130 nucleotides may be chemically synthesized and coupled to the IVT region or part.
• It is noted that for ligation methods, ligation with DNA T4 ligase, followed by treatment with DNAse should readily avoid concatenation.
• The entire chimeric polynucleotide need not be manufactured with a phosphate-sugar backbone. If one of the regions or parts encodes a polypeptide, then it is preferable that such region or part comprise a phosphate-sugar backbone.
• Ligation is then performed using any known click chemistry, orthoclick chemistry, solulink, or other bioconjugate chemistries known to those in the art.
Synthetic Route
• The chimeric polynucleotide is made using a series of starting segments. Such segments include:
• (a) Capped and protected 5′ segment comprising a normal 3′OH (SEG. 1)
• (b) 5′ triphosphate segment which may include the coding region of a polypeptide and comprising a normal 3′OH (SEG. 2)
• (c) 5′ monophosphate segment for the 3′ end of the chimeric polynucleotide (e.g., the tail) comprising cordycepin or no 3′OH (SEG. 3) After synthesis (chemical or IVT), segment 3 (SEG. 3) is treated with cordycepin and
• then with pyrophosphatase to create the 5′-monophosphate.
• Segment 2 (SEG. 2) is then ligated to SEG. 3 using RNA ligase. The ligated polynucleotide is then purified and treated with pyrophosphatase to cleave the diphosphate. The treated SEG. 2-SEG. 3 construct is then purified and SEG. 1 is ligated to the 5′ terminus. A further purification step of the chimeric polynucleotide may be performed.
• Where the chimeric polynucleotide encodes a polypeptide, the ligated or joined segments may be represented as: 5′UTR (SEG. 1), open reading frame or ORF (SEG. 2) and 3′UTR+PolyA (SEG. 3).
• The yields of each step may be as much as 90-95%.
Example 3: PCR for cDNA Production
• PCR procedures for the preparation of cDNA are performed using 2×KAPA HIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This system includes 2×KAPA ReadyMix12.5 μl; Forward Primer (10 μM) 0.75 μl; Reverse Primer (10 PM) 0.75 μl; Template cDNA —100 ng; and dH20 diluted to 25.0 μl. The reaction conditions are at 95° C. for 5 min. and 25 cycles of 98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for 45 sec, then 72° C. for 5 min. then 4° C. to termination.
• The reaction is cleaned up using Invitrogen's PURELINK™ PCR Micro Kit (Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg). Larger reactions will require a cleanup using a product with a larger capacity. Following the cleanup, the cDNA is quantified using the NANODROP™ and analyzed by agarose gel electrophoresis to confirm the cDNA is the expected size. The cDNA is then submitted for sequencing analysis before proceeding to the in vitro transcription reaction.
Example 4: In Vitro Transcription (IVT)
• The in vitro transcription reaction generates polynucleotides containing uniformly modified polynucleotides. Such uniformly modified polynucleotides may comprise a region or part of the polynucleotides of the disclosure. The input nucleotide triphosphate (NTP) mix is made in-house using natural and un-natural NTPs.
• A typical in vitro transcription reaction includes the following:
• 1 Template cDNA 1.0 μg
  2 10× transcription buffer (400 mM Tris-HCl pH 8.0, 190 mM MgCl2, 50 mM DTT, 10 mM Spermidine) 2.0 μl
  3 Custom NTPs (25 mM each) 7.2 μl
4 RNase Inhibitor 20 U
• T7 RNA polymerase 3000 U
  6 dH20 Up to 20.0 μl. and
  7 Incubation at 37° C. for 3 hr-5 hrs.
• The crude IVT mix may be stored at 4° C. overnight for cleanup the next day. 1 U of RNase-free DNase is then used to digest the original template. After 15 minutes of incubation at 37° C., the mRNA is purified using Ambion's MEGACLEAR™ Kit (Austin, Tex.) following the manufacturer's instructions. This kit can purify up to 500 ag of RNA. Following the cleanup, the RNA is quantified using the NanoDrop and analyzed by agarose gel electrophoresis to confirm the RNA is the proper size and that no degradation of the RNA has occurred.
Example 5: Exemplary Nucleic Acids Encoding CHIKV E1 RNA Polynucleotides for Use in a RNA Vaccine
• The following sequences are exemplary sequences that can be used to encode CHIKV E1 RNA polynucleotides for use in the CHIKV RNA vaccine:

• TABLE 1
  CHIKV E1 RNA polynucleotides

  		SEQ ID
  Name	Sequence	NO
  ChiK.secE1	TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA		1
  HS3UPCRfree	ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAGAC
  (CHIKV	ACCTGCACAGCTGTTGTTTCTGCTGCTGCTTTGGTTGCCCGATACCACCG
  secreted E1	GTGACTACAAAGACGACGACGATAAATACGAGCACGTGACGGTAATACCA
  antigen)	AACACTGTGGGGGTGCCATACAAGACCCTGGTAAATCGCCCAGGCTACTC
  	TCCCATGGTGCTGGAGATGGAGCTCCAGTCTGTGACCTTAGAGCCAACCC
  	TCTCACTCGACTATATCACCTGTGAATACAAAACAGTGATCCCATCCCCC
  	TACGTGAAATGTTGCGGAACTGCAGAGTGTAAGGATAAGAGTCTGCCCGA
  	TTACAGCTGCAAGGTGTTTACAGGCGTGTATCCATTTATGTGGGGAGGAG
  	CCTACTGTTTTTGCGATGCCGAAAATACTCAGCTGTCTGAAGCCCATGTG
  	GAGAAGAGTGAAAGTTGCAAGACCGAATTTGCTAGTGCCTACAGGGCACA
  	CACCGCTTCTGCCTCCGCTAAACTCCGAGTCCTTTACCAGGGCAATAATA
  	TTACGGTCGCTGCCTACGCTAACGGGGACCACGCTGTGACAGTCAAGGAC
  	GCCAAATTCGTAGTGGGCCCAATGAGCTCCGCCTGGACTCCCTTCGACAA
  	CAAAATCGTCGTGTATAAAGGCGACGTGTACAATATGGACTACCCACCCT
  	TCGGGGCTGGAAGACCGGGCCAGTTTGGAGATATCCAATCAAGGACACCC
  	GAGTCAAAGGACGTGTACGCCAATACGCAGCTGGTGCTGCAGAGACCCGC
  	CGCTGGTACCGTGCATGTGCCTTATTCCCAAGCTCCATCTGGCTTCAAGT
  	ACTGGTTGAAAGAGCGCGGTGCTTCGCTGCAGCATACAGCACCGTTCGGA
  	TGTCAGATAGCAACCAACCCTGTACGGGCTGTCAACTGTGCCGTGGGAAA
  	TATACCTATTTCCATCGACATTCCGGACGCAGCTTTCACACGTGTCGTTG
  	ATGCCCCCTCAGTGACTGACATGTCATGTGAGGTGCCTGCTTGCACCCAC
  	AGCAGCGATTTTGGCGGAGTGGCCATAATCAAGTACACCGCCTCCAAAAA
  	AGGAAAGTGTGCCGTACACTCTATGACCAACGCCGTCACAATCAGAGAAG
  	CCGACGTTGAAGTAGAGGGAAATTCACAGCTGCAAATCAGCTTCAGCACC
  	GCTCTTGCCTCTGCTGAGTTTAGGGTTCAGGTTTGCAGTACTCAGGTGCA
  	CTGTGCAGCCGCTTGCCATCCCCCCAAGGATCATATCGTGAATTATCCTG
  	CATCCCACACCACACTGGGAGTCCAGGATATCTCAACAACTGCAATGTCT
  	TGGGTGCAGAAGATCACCTGATAATAGGCTGGAGCCTCGGTGGCCATGCT
  	TCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGT
  	ACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC
  Chik-	TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA	2
  Strain37997	ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGTACGA
  -E1 (CHIKV	ACACGTAACAGTGATCCCGAACACGGTGGGAGTACCGTATAAGACTCTAG
  E1 antigen-	TCAACAGACCGGGCTACAGCCCCATGGTATTGGAGATGGAGCTTCTGTCT
  Strain	GTCACCTTGGAACCAACGCTATCGCTTGATTACATCACGTGCGAGTATAA
  37997):	AACCGTTATCCCGTCTCCGTACGTGAAATGCTGCGGTACAGCAGAGTGTA
  	AGGACAAGAGCCTACCTGATTACAGCTGTAAGGTCTTCACCGGCGTCTAC
  	CCATTCATGTGGGGCGGCGCCTACTGCTTCTGCGACACCGAAAATACGCA
  	ATTGAGCGAAGCACATGTGGAGAAGTCCGAATCATGCAAAACAGAATTTG
  	CATCAGCATACAGGGCTCATACCGCATCCGCATCAGCTAAGCTCCGCGTC
  	CTTTACCAAGGAAATAATATCACTGTGGCTGCTTATGCAAACGGCGACCA
  	TGCCGTCACAGTTAAGGACGCTAAATTCATAGTGGGGCCAATGTCTTCAG
  	CCTGGACACCTTTCGACAATAAAATCGTGGTGTACAAAGGCGACGTCTAC
  	AACATGGACTACCCGCCCTTCGGCGCAGGAAGACCAGGACAATTTGGCGA
  	CATCCAAAGTCGCACGCCTGAGAGCGAAGACGTCTATGCTAATACACAAC
  	TGGTACTGCAGAGACCGTCCGCGGGTACGGTGCACGTGCCGTACTCTCAG
  	GCACCATCTGGCTTCAAGTATTGGCTAAAAGAACGAGGGGCGTCGCTGCA
  	GCACACAGCACCATTTGGCTGTCAAATAGCAACAAACCCGGTAAGAGCGA
  	TGAACTGCGCCGTAGGGAACATGCCTATCTCCATCGACATACCGGACGCG
  	GCCTTTACCAGGGTCGTCGACGCGCCATCTTTAACGGACATGTCGTGTGA
  	GGTATCAGCCTGCACCCATTCCTCAGACTTTGGGGGCGTAGCCATCATTA
  	AATATGCAGCCAGTAAGAAAGGCAAGTGTGCAGTGCACTCGATGACTAAC
  	GCCGTCACTATTCGGGAAGCTGAAATAGAAGTAGAAGGGAACTCTCAGTT
  	GCAAATCTCTTTTTCGACGGCCCTAGCCAGCGCCGAATTTCGCGTACAAG
  	TCTGTTCTACACAAGTACACTGTGCAGCCGAGTGCCATCCACCGAAAGAC
  	CATATAGTCAATTACCCGGCGTCACACACCACCCTCGGGGTCCAAGACAT
  	TTCCGCTACGGCGATGTCATGGGTGCAGAAGATCACGGGAGGTGTGGGAC
  	TGGTTGTCGCTGTTGCAGCACTGATCCTAATCGTGGTGCTATGCGTGTCG
  	TTTAGCAGGCACTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGC
  	CCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCC
  	GTGGTCTTTGAATAAAGTCTGAGTGGGCGGC
  Chik-	TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA	3
  Strain37997	ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCTATG
  -E1 (CHIKV	GAACGAACAGCAGCCCCTGTTCTGGTTGCAGGCTCTTATCCCGCTGGCCG
  E1 antigen-	CCTTGATCGTCCTGTGCAACTGTCTGAAACTCTTGCCATGCTGCTGTAAG
  Strain	ACCCTGGCTTTTTTAGCCGTAATGAGCATCGGTGCCCACACTGTGAGCGC
  37997):	GTACGAACACGTAACAGTGATCCCGAACACGGTGGGAGTACCGTATAAGA
  	CTCTTGTCAACAGACCGGGTTACAGCCCCATGGTGTTGGAGATGGAGCTA
  	CAATCAGTCACCTTGGAACCAACACTGTCACTTGACTACATCACGTGCGA
  	GTACAAAACTGTCATCCCCTCCCCGTACGTGAAGTGCTGTGGTACAGCAG
  	AGTGCAAGGACAAGAGCCTACCAGACTACAGCTGCAAGGTCTTTACTGGA
  	GTCTACCCATTTATGTGGGGCGGCGCCTACTGCTTTTGCGACGCCGAAAA
  	TACGCAATTGAGCGAGGCACATGTAGAGAAATCTGAATCTTGCAAAACAG
  	AGTTTGCATCGGCCTACAGAGCCCACACCGCATCGGCGTCGGCGAAGCTC
  	CGCGTCCTTTACCAAGGAAACAACATTACCGTAGCTGCCTACGCTAACGG
  	TGACCATGCCGTCACAGTAAAGGACGCCAAGTTTGTCGTGGGCCCAATGT
  	CCTCCGCCTGGACACCTTTTGACAACAAAATCGTGGTGTACAAAGGCGAC
  	GTCTACAACATGGACTACCCACCTTTTGGCGCAGGAAGACCAGGACAATT
  	TGGTGACATTCAAAGTCGTACACCGGAAAGTAAAGACGTTTATGCCAACA
  	CTCAGTTGGTACTACAGAGGCCAGCAGCAGGCACGGTACATGTACCATAC
  	TCTCAGGCACCATCTGGCTTCAAGTATTGGCTGAAGGAACGAGGAGCATC
  	GCTACAGCACACGGCACCGTTCGGTTGCCAGATTGCGACAAACCCGGTAA
  	GAGCTGTAAATTGCGCTGTGGGGAACATACCAATTTCCATCGACATACCG
  	GATGCGGCCTTTACTAGGGTTGTCGATGCACCCTCTGTAACGGACATGTC
  	ATGCGAAGTACCAGCCTGCACTCACTCCTCCGACTTTGGGGGCGTCGCCA
  	TCATCAAATACACAGCTAGCAAGAAAGGTAAATGTGCAGTACATTCGATG
  	ACCAACGCCGTTACCATTCGAGAAGCCGACGTAGAAGTAGAGGGGAACTC
  	CCAGCTGCAAATATCCTTCTCAACAGCCCTGGCAAGCGCCGAGTTTCGCG
  	TGCAAGTGTGCTCCACACAAGTACACTGCGCAGCCGCATGCCACCCTCCA
  	AAGGACCACATAGTCAATTACCCAGCATCACACACCACCCTTGGGGTCCA
  	GGATATATCCACAACGGCAATGTCTTGGGTGCAGAAGATTACGGGAGGAG
  	TAGGATTAATTGTTGCTGTTGCTGCCTTAATTTTAATTGTGGTGCTATGC
  	GTGTCGTTTAGCAGGCACTAATGATAATAGGCTGGAGCCTCGGTGGCCAT
  	GCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACC
  	CGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC
  chikv-	TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA	4
  Brazillian-	ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGTACGA
  E1 (CHIKV	ACACGTAACAGTGATCCCGAACACGGTGGGAGTACCGTATAAGACTCTAG
  E1 antigen	TCAATAGACCGGGCTACAGTCCCATGGTATTGGAGATGGAACTACTGTCA
  - Brazilian	GTCACTTTGGAGCCAACGCTATCGCTTGATTACATCACGTGCGAGTACAA
  strain)	AACCGTTATCCCGTCTCCGTACGTGAAATGCTGCGGTACAGCAGAGTGCA
  	AGGACAAAAACCTACCTGACTACAGCTGTAAGGTCTTCACCGGCGTCTAC
  	CCATTTATGTGGGGCGGAGCCTACTGCTTCTGCGACGCTGAAAACACGCA
  	ATTGAGCGAAGCACACGTGGAGAAGTCCGAATCATGCAAAACAGAATTTG
  	CATCAGCATACAGGGCTCATACCGCATCCGCATCAGCTAAGCTCCGCGTC
  	CTTTACCAAGGAAATAACATCACTGTAACTGCCTATGCTAACGGCGACCA
  	TGCCGTCACAGTTAAGGACGCCAAATTCATTGTGGGGCCAATGTCTTCAG
  	CCTGGACACCTTTCGACAACAAAATTGTGGTGTACAAAGGTGACGTCTAT
  	AACATGGACTACCCGCCCTTTGGCGCAGGAAGACCAGGACAATTTGGCGA
  	TATCCAAAGTCGCACACCTGAGAGTAAAGACGTCTATGCTAATACACAAC
  	TGGTACTGCAGAGACCGGCTGCGGGTACGGTACATGTGCCATACTCTCAG
  	GCACCATCTGGCTTTAAGTATTGGCTAAAAGAACGAGGGGCGTCGCTGCA
  	GCACACAGCACCATTTGGCTGCCAAATAGCAACAAACCCGGTAAGAGCGG
  	TGAATTGCGCCGTAGGGAACATGCCCATCTCCATCGACATACCGGATGCG
  	GCCTTCATTAGGGTCGTCGACGCGCCCTCTTTAACGGACATGTCGTGCGA
  	GGTACCAGCCTGCACCCATTCCTCAGATTTCGGGGGCGTCGCCATTATTA
  	AATATGCAGCCAGCAAGAAAGGCAAGTGTGCGGTGCATTCGATGACCAAC
  	GCCGTCACAATTCGGGAAGCTGAGATAGAAGTTGAAGGGAATTCTCAGCT
  	GCAAATCTCTTTCTCGACGGCCTTGGCCAGCGCCGAATTCCGCGTACAAG
  	TCTGTTCTACACAAGTACACTGTGTAGCCGAGTGCCACCCTCCGAAGGAC
  	CACATAGTCAATTACCCGGCGTCACATACCACCCTCGGGGTCCAGGACAT
  	TTCCGCTACGGCGCTGTCATGGGTGCAGAAGATCACGGGAGGCGTGGGAC
  	TGGTTGTCGCTGTTGCAGCACTGATTCTAATCGTGGTGCTATGCGTGTCG
  	TTCAGCAGGCACTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGC
  	CCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCC
  	GTGGTCTTTGAATAAAGTCTGAGTGGGCGGC

Example 6: Exemplary Nucleic Acids Encoding CHIKV E2 RNA Polynucleotides for Use in a RNA Vaccine
• The following sequences are exemplary sequences that can be used to encode CHIKV E2 RNA polynucleotides for use in a RNA vaccine:

• TABLE 2
  CHIKV E2 RNA polynucleotides

  		SEQ ID
  Name	Sequence	NO
  ChiK.secE2	ATGGAGACCCCAGCTCAGCTTCTGTTTCTTCTCCTTCTATGGCTGCCTGA		5
  HS3UPCRfree	CACGACTGGACATCACCACCATCATCATAGTACAAAAGACAATTTCAATG
  (CHIKV	TGTACAAGGCCACCCGCCCTTATTTAGCACACTGTCCAGATTGCGGTGAG
  secreted E2	GGGCACTCCTGTCACTCTCCTATCGCCTTGGAGCGGATCCGGAATGAGGC
  antigen):	GACCGATGGAACACTGAAAATCCAGGTAAGCTTGCAGATTGGCATCAAGA
  	CTGACGATAGCCATGATTGGACCAAACTACGGTATATGGATAGCCATACA
  	CCTGCCGATGCTGAACGGGCCGGTCTGCTTGTGAGAACTAGCGCTCCATG
  	CACCATCACGGGGACAATGGGACATTTTATCCTGGCTAGATGCCCAAAGG
  	GCGAAACCCTCACCGTCGGATTCACCGACTCAAGGAAAATTTCTCACACA
  	TGTACCCATCCCTTCCACCATGAGCCACCGGTGATCGGGCGCGAACGCTT
  	CCACAGCAGGCCTCAGCATGGAAAAGAACTGCCATGCTCGACCTATGTAC
  	AGTCCACCGCCGCTACCGCCGAAGAGATCGAAGTGCATATGCCTCCCGAC
  	ACACCCGACCGAACCCTAATGACACAACAATCTGGGAATGTGAAGATTAC
  	AGTCAATGGACAGACTGTGAGGTATAAGTGTAACTGCGGTGGCTCAAATG
  	AGGGCCTCACCACAACGGATAAGGTGATCAATAACTGCAAAATTGACCAG
  	TGTCACGCGGCCGTGACCAACCATAAGAACTGGCAGTACAACTCACCCTT
  	AGTGCCTAGGAACGCTGAGCTGGGAGATCGCAAGGGGAAGATACACATTC
  	CCTTCCCGTTGGCGAATGTGACCTGCCGTGTGCCAAAAGCGAGAAATCCT
  	ACCGTAACATATGGCAAAAATCAGGTGACCATGTTGCTCTACCCGGATCA
  	CCCAACTCTGCTGAGCTATCGGAATATGGGACAAGAACCCAATTACCACG
  	AGGAATGGGTTACGCACAAGAAAGAGGTGACCCTTACAGTCCCTACTGAA
  	GGTCTGGAAGTGACCTGGGGCAATAACGAGCCTTATAAGTACTGGCCCCA
  	GATGAGTACAAACGGCACCGCCCATGGACATCCACACGAGATCATTCTGT
  	ATTACTACGAACTATATCCCACAATGACTGGCAAGCCTATACCAAACCCA
  	CTTCTCGGCCTTGATAGCACATGATAATAGGCTGGAGCCTCGGTGGCCAT
  	GCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACC
  	CGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC
  chikv-	TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA	6
  Brazillian-	ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGAGTAC
  E2 (CHIKV	CAAGGACAACTTCAATGTCTATAAAGCCACAAGACCGTACTTAGCTCACT
  E2 antigen	GTCCCGACTGTGGAGAAGGGCACTCGTGCCATAGTCCCGTAGCATTAGAA
  - Brazilian	CGCATCAGAAATGAAGCGACAGACGGGACGCTGAAAATCCAGGTCTCCTT
  strain):	GCAAATCGGAATAAAGACGGATGATAGCCACGATTGGACCAAGCTGCGTT
  	ACATGGACAACCACACGCCAGCGGACGCAGAGAGGGCGGGGCTATTTGTA
  	AGAACATCAGCACCGTGCACGATTACTGGAACAATGGGACACTTCATCCT
  	GACCCGATGTCCGAAAGGGGAAACTCTGACGGTGGGATTCACTGACAGTA
  	GGAAGATCAGTCACTCATGTACGCACCCATTTCACCACGACCCTCCTGTG
  	ATAGGCCGGGAGAAATTCCATTCCCGACCGCAGCACGGTAAAGAGCTGCC
  	TTGCAGCACGTACGTGCAGAGCACCGCCGCAACTACCGAGGAGATAGAGG
  	TACACATGCCCCCAGACACCCCTGATCGCACATTGATGTCACAACAGTCC
  	GGCAACGTAAAGATCACAGTTAATGGCCAGACGGTGCGGTACAAGTGTAA
  	TTGCGGTGGCTCAAATGAAGGACTAATAACTACAGACAAAGTGATTAATA
  	ACTGCAAAGTTGATCAATGTCATGCCGCGGTCACCAATCACAAAAAGTGG
  	CAGTACAACTCCCCTCTGGTCCCGCGTAATGCTGAACTTGGGGACCGAAA
  	AGGAAAAATCCACATCCCGTTTCCGCTGGCAAATGTAACATGCAGGGTGC
  	CTAAAGCAAGGAACCCCACCGTGACGTACGGGAAAAACCAAGTCATCATG
  	CTACTGTATCCCGACCACCCAACACTCCTGTCCTACCGGAACATGGGAGA
  	AGAACCAAACTACCAAGAAGAGTGGGTGACGCATAAGAAGGAAGTCGTGC
  	TAACCGTGCCGACTGAAGGGCTCGAGGTCACGTGGGGTAACAACGAGCCG
  	TATAAGTATTGGCCGCAGTTATCTACAAACGGTACAGCCCATGGCCACCC
  	GCATGAGATAATTCTGTATTATTATGAGCTGTACCCTACTATGACTGTAG
  	TAGTTGTGTCAGTGGCCTCGTTCGTACTCCTGTCGATGGTGGGTGTGGCA
  	GTGGGGATGTGCATGTGTGCACGACGCAGATGCATCACACCGTACGAACT
  	GACACCAGGAGCTACCGTCCCTTTCCTGCTTAGCCTAATATGCTGCATCA
  	GAACAGCTAAAGCGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTT
  	GCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCC
  	CCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC
  chikv-	TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA	7
  Brazillian-	ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGAGTAT
  E2 (CHIKV	TAAGGACCACTTCAATGTCTATAAAGCCACAAGACCGTACCTAGCTCACT
  E2 antigen	GTCCCGACTGTGGAGAAGGGCACTCGTGCCATAGTCCCGTAGCGCTAGAA
  - Brazilian	CGCATCAGAAACGAAGCGACAGACGGGACGTTGAAAATCCAGGTTTCCTT
  strain):	GCAAATCGGAATAAAGACGGATGATAGCCATGATTGGACCAAGCTGCGTT
  	ATATGGACAATCACATGCCAGCAGACGCAGAGCGGGCCGGGCTATTTGTA
  	AGAACGTCAGCACCGTGCACGATTACTGGAACAATGGGACACTTCATTCT
  	GGCCCGATGTCCGAAAGGAGAAACTCTGACGGTGGGGTTCACTGACGGTA
  	GGAAGATCAGTCACTCATGTACGCACCCATTTCACCATGACCCTCCTGTG
  	ATAGGCCGGGAAAAATTCCATTCCCGACCGCAGCACGGTAGGGAACTACC
  	TTGCAGCACGTACGCGCAGAGCACCGCTGCAACTGCCGAGGAGATAGAGG
  	TACACATGCCCCCAGACACCCCAGATCGCACATTAATGTCACAACAGTCC
  	GGCAATGTAAAGATCACAGTCAATAGTCAGACGGTGCGGTACAAGTGCAA
  	TTGTGGTGACTCAAGTGAAGGATTAACCACTACAGATAAAGTGATTAATA
  	ACTGCAAGGTCGATCAATGCCATGCCGCGGTCACCAATCACAAAAAATGG
  	CAGTATAACTCCCCTCTGGTCCCGCGTAATGCTGAATTCGGGGACCGGAA
  	AGGAAAAGTTCACATTCCATTTCCTCTGGCAAATGTGACATGCAGGGTGC
  	CTAAAGCAAGAAACCCCACCGTGACGTACGGAAAAAACCAAGTCATCATG
  	TTGCTGTATCCTGACCACCCAACGCTCCTGTCCTACAGGAATATGGGAGA
  	AGAACCAAACTATCAAGAAGAGTGGGTGACGCATAAGAAGGAGATCAGGT
  	TAACCGTGCCGACTGAGGGGCTCGAGGTCACGTGGGGTAACAATGAGCCG
  	TACAAGTATTGGCCGCAGTTATCCACAAACGGTACAGCCCACGGCCACCC
  	GCATGAGATAATTCTGTATTATTATGAGCTGTACCCAACTATGACTGCGG
  	TAGTTTTGTCAGTGGCCTCGTTCATACTCCTGTCGATGGTGGGTGTGGCA
  	GTGGGGATGTGCATGTGTGCACGACGCAGATGCATTACACCGTACGAACT
  	GACACCAGGAGCTACCGTCCCTTTCCTGCTTAGCCTAATATGCTGCATTA
  	GAACAGCTAAAGCGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTT
  	GCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCC
  	CCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC
  Chik-Strain	TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA		8
  37997-E2	ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCCATA
  (CHIKV E2	TCTAGCTCATTGTCCTGACTGCGGAGAAGGGCATTCGTGCCACAGCCCTA
  Antigen-	TCGCATTGGAGCGCATCAGAAATGAAGCAACGGACGGAACGCTGAAAATC
  Strain	CAGGTCTCTTTGCAGATCGGGATAAAGACAGATGACAGCCACGATTGGAC
  37997)	CAAGCTGCGCTATATGGATAGCCATACGCCAGCGGACGCGGAGCGAGCCG
  	GATTGCTTGTAAGGACTTCAGCACCGTGCACGATCACCGGGACCATGGGA
  	CACTTTATTCTCGCCCGATGCCCGAAAGGAGAGACGCTGACAGTGGGATT
  	TACGGACAGCAGAAAGATCAGCCACACATGCACACACCCGTTCCATCATG
  	AACCACCTGTGATAGGTAGGGAGAGGTTCCACTCTCGACCACAACATGGT
  	AAAGAGTTACCTTGCAGCACGTACGTGCAGAGCACCGCTGCCACTGCTGA
  	GGAGATAGAGGTGCATATGCCCCCAGATACTCCTGACCGCACGCTGATGA
  	CGCAGCAGTCTGGCAACGTGAAGATCACAGTTAATGGGCAGACGGTGCGG
  	TACAAGTGCAACTGCGGTGGCTCAAACGAGGGACTGACAACCACAGACAA
  	AGTGATCAATAACTGCAAAATTGATCAGTGCCATGCTGCAGTCACTAATC
  	ACAAGAATTGGCAATACAACTCCCCTTTAGTCCCGCGCAACGCTGAACTC
  	GGGGACCGTAAAGGAAAGATCCACATCCCATTCCCATTGGCAAACGTGAC
  	TTGCAGAGTGCCAAAAGCAAGAAACCCTACAGTAACTTACGGAAAAAACC
  	AAGTCACCATGCTGCTGTATCCTGACCATCCGACACTCTTGTCTTACCGT
  	AACATGGGACAGGAACCAAATTACCACGAGGAGTGGGTGACACACAAGAA
  	GGAGGTTACCTTGACCGTGCCTACTGAGGGTCTGGAGGTCACTTGGGGCA
  	ACAACGAACCATACAAGTACTGGCCGCAGATGTCTACGAACGGTACTGCT
  	CATGGTCACCCACATGAGATAATCTTGTACTATTATGAGCTGTACCCCAC
  	TATGACTGTAGTCATTGTGTCGGTGGCCTCGTTCGTGCTTCTGTCGATGG
  	TGGGCACAGCAGTGGGAATGTGTGTGTGCGCACGGCGCAGATGCATTACA
  	CCATATGAATTAACACCAGGAGCCACTGTTCCCTTCCTGCTCAGCCTGCT
  	ATGCTGCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTT
  	GGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGT
  	CTTTGAATAAAGTCTGAGTGGGCGGC

Example 7: Exemplary Nucleic Acids Encoding CHIKV E1-E2 RNA Polynucleotides for Use in a RNA Vaccine
• The following sequences are exemplary sequences that can be used to encode CHIKV E1-E2 RNA polynucleotides for use in a RNA vaccine:

• TABLE 3
  CHIKV E1-E2 RNA polynucleotides

  		SEQ ID
  Name	Sequence	NO

  chikv-	TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA	9
  Brazillian-	ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGAGTAC
  E2-E1	CAAGGACAACTTCAATGTCTATAAAGCCACAAGACCGTACTTAGCTCACT
  (CHIKV E1-	GTCCCGACTGTGGAGAAGGGCACTCGTGCCATAGTCCCGTAGCATTAGAA
  E2 Antigen-	CGCATCAGAAATGAAGCGACAGACGGGACGCTGAAAATCCAGGTCTCCTT
  Brazilian	GCAAATCGGAATAAAGACGGATGATAGCCACGATTGGACCAAGCTGCGTT
  strain) :	ACATGGACAACCACACGCCAGCGGACGCAGAGAGGGCGGGGCTATTTGTA
  	AGAACATCAGCACCGTGCACGATTACTGGAACAATGGGACACTTCATCCT
  	GACCCGATGTCCGAAAGGGGAAACTCTGACGGTGGGATTCACTGACAGTA
  	GGAAGATCAGTCACTCATGTACGCACCCATTTCACCACGACCCTCCTGTG
  	ATAGGCCGGGAGAAATTCCATTCCCGACCGCAGCACGGTAAAGAGCTGCC
  	TTGCAGCACGTACGTGCAGAGCACCGCCGCAACTACCGAGGAGATAGAGG
  	TACACATGCCCCCAGACACCCCTGATCGCACATTGATGTCACAACAGTCC
  	GGCAACGTAAAGATCACAGTTAATGGCCAGACGGTGCGGTACAAGTGTAA
  	TTGCGGTGGCTCAAATGAAGGACTAATAACTACAGACAAAGTGATTAATA
  	ACTGCAAAGTTGATCAATGTCATGCCGCGGTCACCAATCACAAAAAGTGG
  	CAGTACAACTCCCCTCTGGTCCCGCGTAATGCTGAACTTGGGGACCGAAA
  	AGGAAAAATCCACATCCCGTTTCCGCTGGCAAATGTAACATGCAGGGTGC
  	CTAAAGCAAGGAACCCCACCGTGACGTACGGGAAAAACCAAGTCATCATG
  	CTACTGTATCCCGACCACCCAACACTCCTGTCCTACCGGAACATGGGAGA
  	AGAACCAAACTACCAAGAAGAGTGGGTGACGCATAAGAAGGAAGTCGTGC
  	TAACCGTGCCGACTGAAGGGCTCGAGGTCACGTGGGGTAACAACGAGCCG
  	TATAAGTATTGGCCGCAGTTATCTACAAACGGTACAGCCCATGGCCACCC
  	GCATGAGATAATTCTGTATTATTATGAGCTGTACCCTACTATGACTGTAG
  	TAGTTGTGTCAGTGGCCTCGTTCGTACTCCTGTCGATGGTGGGTGTGGCA
  	GTGGGGATGTGCATGTGTGCACGACGCAGATGCATCACACCGTACGAACT
  	GACACCAGGAGCTACCGTCCCTTTCCTGCTTAGCCTAATATGCTGCATCA
  	GAACAGCTAAAGCGTACGAACACGTAACAGTGATCCCGAACACGGTGGGA
  	GTACCGTATAAGACTCTAGTCAATAGACCGGGCTACAGTCCCATGGTATT
  	GGAGATGGAACTACTGTCAGTCACTTTGGAGCCAACGCTATCGCTTGATT
  	ACATCACGTGCGAGTACAAAACCGTTATCCCGTCTCCGTACGTGAAATGC
  	TGCGGTACAGCAGAGTGCAAGGACAAAAACCTACCTGACTACAGCTGTAA
  	GGTCTTCACCGGCGTCTACCCATTTATGTGGGGCGGAGCCTACTGCTTCT
  	GCGACGCTGAAAACACGCAATTGAGCGAAGCACACGTGGAGAAGTCCGAA
  	TCATGCAAAACAGAATTTGCATCAGCATACAGGGCTCATACCGCATCCGC
  	ATCAGCTAAGCTCCGCGTCCTTTACCAAGGAAATAACATCACTGTAACTG
  	CCTATGCTAACGGCGACCATGCCGTCACAGTTAAGGACGCCAAATTCATT
  	GTGGGGCCAATGTCTTCAGCCTGGACACCTTTCGACAACAAAATTGTGGT
  	GTACAAAGGTGACGTCTATAACATGGACTACCCGCCCTTTGGCGCAGGAA
  	GACCAGGACAATTTGGCGATATCCAAAGTCGCACACCTGAGAGTAAAGAC
  	GTCTATGCTAATACACAACTGGTACTGCAGAGACCGGCTGCGGGTACGGT
  	ACATGTGCCATACTCTCAGGCACCATCTGGCTTTAAGTATTGGCTAAAAG
  	AACGAGGGGCGTCGCTGCAGCACACAGCACCATTTGGCTGCCAAATAGCA
  	ACAAACCCGGTAAGAGCGGTGAATTGCGCCGTAGGGAACATGCCCATCTC
  	CATCGACATACCGGATGCGGCCTTCATTAGGGTCGTCGACGCGCCCTCTT
  	TAACGGACATGTCGTGCGAGGTACCAGCCTGCACCCATTCCTCAGATTTC
  	GGGGGCGTCGCCATTATTAAATATGCAGCCAGCAAGAAAGGCAAGTGTGC
  	GGTGCATTCGATGACCAACGCCGTCACAATTCGGGAAGCTGAGATAGAAG
  	TTGAAGGGAATTCTCAGCTGCAAATCTCTTTCTCGACGGCCTTGGCCAGC
  	GCCGAATTCCGCGTACAAGTCTGTTCTACACAAGTACACTGTGTAGCCGA
  	GTGCCACCCTCCGAAGGACCACATAGTCAATTACCCGGCGTCACATACCA
  	CCCTCGGGGTCCAGGACATTTCCGCTACGGCGCTGTCATGGGTGCAGAAG
  	ATCACGGGAGGCGTGGGACTGGTTGTCGCTGTTGCAGCACTGATTCTAAT
  	CGTGGTGCTATGCGTGTCGTTCAGCAGGCACTGATAATAGGCTGGAGCCT
  	CGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCC
  	TTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC
  chikv-	TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA	10
  Brazillian-	ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGAGTAT
  E2-E1	TAAGGACCACTTCAATGTCTATAAAGCCACAAGACCGTACCTAGCTCACT
  (CHIKV E1-	GTCCCGACTGTGGAGAAGGGCACTCGTGCCATAGTCCCGTAGCGCTAGAA
  E2 Antigen-	CGCATCAGAAACGAAGCGACAGACGGGACGTTGAAAATCCAGGTTTCCTT
  Brazilian	GCAAATCGGAATAAAGACGGATGATAGCCATGATTGGACCAAGCTGCGTT
  strain):	ATATGGACAATCACATGCCAGCAGACGCAGAGCGGGCCGGGCTATTTGTA
  	AGAACGTCAGCACCGTGCACGATTACTGGAACAATGGGACACTTCATTCT
  	GGCCCGATGTCCGAAAGGAGAAACTCTGACGGTGGGGTTCACTGACGGTA
  	GGAAGATCAGTCACTCATGTACGCACCCATTTCACCATGACCCTCCTGTG
  	ATAGGCCGGGAAAAATTCCATTCCCGACCGCAGCACGGTAGGGAACTACC
  	TTGCAGCACGTACGCGCAGAGCACCGCTGCAACTGCCGAGGAGATAGAGG
  	TACACATGCCCCCAGACACCCCAGATCGCACATTAATGTCACAACAGTCC
  	GGCAATGTAAAGATCACAGTCAATAGTCAGACGGTGCGGTACAAGTGCAA
  	TTGTGGTGACTCAAGTGAAGGATTAACCACTACAGATAAAGTGATTAATA
  	ACTGCAAGGTCGATCAATGCCATGCCGCGGTCACCAATCACAAAAAATGG
  	CAGTATAACTCCCCTCTGGTCCCGCGTAATGCTGAATTCGGGGACCGGAA
  	AGGAAAAGTTCACATTCCATTTCCTCTGGCAAATGTGACATGCAGGGTGC
  	CTAAAGCAAGAAACCCCACCGTGACGTACGGAAAAAACCAAGTCATCATG
  	TTGCTGTATCCTGACCACCCAACGCTCCTGTCCTACAGGAATATGGGAGA
  	AGAACCAAACTATCAAGAAGAGTGGGTGACGCATAAGAAGGAGATCAGGT
  	TAACCGTGCCGACTGAGGGGCTCGAGGTCACGTGGGGTAACAATGAGCCG
  	TACAAGTATTGGCCGCAGTTATCCACAAACGGTACAGCCCACGGCCACCC
  	GCATGAGATAATTCTGTATTATTATGAGCTGTACCCAACTATGACTGCGG
  	TAGTTTTGTCAGTGGCCTCGTTCATACTCCTGTCGATGGTGGGTGTGGCA
  	GTGGGGATGTGCATGTGTGCACGACGCAGATGCATTACACCGTACGAACT
  	GACACCAGGAGCTACCGTCCCTTTCCTGCTTAGCCTAATATGCTGCATTA
  	GAACAGCTAAAGCGTACGAACACGTAACAGTGATCCCGAACACGGTGGGA
  	GTACCGTATAAGACTCTAGTCAACAGACCGGGCTACAGCCCCATGGTATT
  	GGAGATGGAGCTTCTGTCTGTCACCTTGGAACCAACGCTATCGCTTGATT
  	ACATCACGTGCGAGTATAAAACCGTTATCCCGTCTCCGTACGTGAAATGC
  	TGCGGTACAGCAGAGTGTAAGGACAAGAGCCTACCTGATTACAGCTGTAA
  	GGTCTTCACCGGCGTCTACCCATTCATGTGGGGCGGCGCCTACTGCTTCT
  	GCGACACCGAAAATACGCAATTGAGCGAAGCACATGTGGAGAAGTCCGAA
  	TCATGCAAAACAGAATTTGCATCAGCATACAGGGCTCATACCGCATCCGC
  	ATCAGCTAAGCTCCGCGTCCTTTACCAAGGAAATAATATCACTGTGGCTG
  	CTTATGCAAACGGCGACCATGCCGTCACAGTTAAGGACGCTAAATTCATA
  	GTGGGGCCAATGTCTTCAGCCTGGACACCTTTCGACAATAAAATCGTGGT
  	GTACAAAGGCGACGTCTACAACATGGACTACCCGCCCTTCGGCGCAGGAA
  	GACCAGGACAATTTGGCGACATCCAAAGTCGCACGCCTGAGAGCGAAGAC
  	GTCTATGCTAATACACAACTGGTACTGCAGAGACCGTCCGCGGGTACGGT
  	GCACGTGCCGTACTCTCAGGCACCATCTGGCTTCAAGTATTGGCTAAAAG
  	AACGAGGGGCGTCGCTGCAGCACACAGCACCATTTGGCTGTCAAATAGCA
  	ACAAACCCGGTAAGAGCGATGAACTGCGCCGTAGGGAACATGCCTATCTC
  	CATCGACATACCGGACGCGGCCTTTACCAGGGTCGTCGACGCGCCATCTT
  	TAACGGACATGTCGTGTGAGGTATCAGCCTGCACCCATTCCTCAGACTTT
  	GGGGGCGTAGCCATCATTAAATATGCAGCCAGTAAGAAAGGCAAGTGTGC
  	AGTGCACTCGATGACTAACGCCGTCACTATTCGGGAAGCTGAAATAGAAG
  	TAGAAGGGAACTCTCAGTTGCAAATCTCTTTTTCGACGGCCCTAGCCAGC
  	GCCGAATTTCGCGTACAAGTCTGTTCTACACAAGTACACTGTGCAGCCGA
  	GTGCCATCCACCGAAAGACCATATAGTCAATTACCCGGCGTCACACACCA
  	CCCTCGGGGTCCAAGACATTTCCGCTACGGCGATGTCATGGGTGCAGAAG
  	ATCACGGGAGGTGTGGGACTGGTTGTCGCTGTTGCAGCACTGATCCTAAT
  	CGTGGTGCTATGCGTGTCGTTTAGCAGGCACATGAGTATTAAGGACCACT
  	TCAATGTCTATAAAGCCACAAGACCGTACCTAGCTCACTGTCCCGACTGT
  	GGAGAAGGGCACTCGTGCCATAGTCCCGTAGCGCTAGAACGCATCAGAAA
  	CGAAGCGACAGACGGGACGTTGAAAATCCAGGTTTCCTTGCAAATCGGAA
  	TAAAGACGGATGATAGCCATGATTGGACCAAGCTGCGTTATATGGACAAT
  	CACATGCCAGCAGACGCAGAGCGGGCCGGGCTATTTGTAAGAACGTCAGC
  	ACCGTGCACGATTACTGGAACAATGGGACACTTCATTCTGGCCCGATGTC
  	CGAAAGGAGAAACTCTGACGGTGGGGTTCACTGACGGTAGGAAGATCAGT
  	CACTCATGTACGCACCCATTTCACCATGACCCTCCTGTGATAGGCCGGGA
  	AAAATTCCATTCCCGACCGCAGCACGGTAGGGAACTACCTTGCAGCACGT
  	ACGCGCAGAGCACCGCTGCAACTGCCGAGGAGATAGAGGTACACATGCCC
  	CCAGACACCCCAGATCGCACATTAATGTCACAACAGTCCGGCAATGTAAA
  	GATCACAGTCAATAGTCAGACGGTGCGGTACAAGTGCAATTGTGGTGACT
  	CAAGTGAAGGATTAACCACTACAGATAAAGTGATTAATAACTGCAAGGTC
  	GATCAATGCCATGCCGCGGTCACCAATCACAAAAAATGGCAGTATAACTC
  	CCCTCTGGTCCCGCGTAATGCTGAATTCGGGGACCGGAAAGGAAAAGTTC
  	ACATTCCATTTCCTCTGGCAAATGTGACATGCAGGGTGCCTAAAGCAAGA
  	AACCCCACCGTGACGTACGGAAAAAACCAAGTCATCATGTTGCTGTATCC
  	TGACCACCCAACGCTCCTGTCCTACAGGAATATGGGAGAAGAACCAAACT
  	ATCAAGAAGAGTGGGTGACGCATAAGAAGGAGATCAGGTTAACCGTGCCG
  	ACTGAGGGGCTCGAGGTCACGTGGGGTAACAATGAGCCGTACAAGTATTG
  	GCCGCAGTTATCCACAAACGGTACAGCCCACGGCCACCCGCATGAGATAA
  	TTCTGTATTATTATGAGCTGTACCCAACTATGACTGCGGTAGTTTTGTCA
  	GTGGCCTCGTTCATACTCCTGTCGATGGTGGGTGTGGCAGTGGGGATGTG
  	CATGTGTGCACGACGCAGATGCATTACACCGTACGAACTGACACCAGGAG
  	CTACCGTCCCTTTCCTGCTTAGCCTAATATGCTGCATTAGAACAGCTAAA
  	GCGTACGAACACGTAACAGTGATCCCGAACACGGTGGGAGTACCGTATAA
  	GACTCTAGTCAACAGACCGGGCTACAGCCCCATGGTATTGGAGATGGAGC
  	TTCTGTCTGTCACCTTGGAACCAACGCTATCGCTTGATTACATCACGTGC
  	GAGTATAAAACCGTTATCCCGTCTCCGTACGTGAAATGCTGCGGTACAGC
  	AGAGTGTAAGGACAAGAGCCTACCTGATTACAGCTGTAAGGTCTTCACCG
  	GCGTCTACCCATTCATGTGGGGCGGCGCCTACTGCTTCTGCGACACCGAA
  	AATACGCAATTGAGCGAAGCACATGTGGAGAAGTCCGAATCATGCAAAAC
  	AGAATTTGCATCAGCATACAGGGCTCATACCGCATCCGCATCAGCTAAGC
  	TCCGCGTCCTTTACCAAGGAAATAATATCACTGTGGCTGCTTATGCAAAC
  	GGCGACCATGCCGTCACAGTTAAGGACGCTAAATTCATAGTGGGGCCAAT
  	GTCTTCAGCCTGGACACCTTTCGACAATAAAATCGTGGTGTACAAAGGCG
  	ACGTCTACAACATGGACTACCCGCCCTTCGGCGCAGGAAGACCAGGACAA
  	TTTGGCGACATCCAAAGTCGCACGCCTGAGAGCGAAGACGTCTATGCTAA
  	TACACAACTGGTACTGCAGAGACCGTCCGCGGGTACGGTGCACGTGCCGT
  	ACTCTCAGGCACCATCTGGCTTCAAGTATTGGCTAAAAGAACGAGGGGCG
  	TCGCTGCAGCACACAGCACCATTTGGCTGTCAAATAGCAACAAACCCGGT
  	AAGAGCGATGAACTGCGCCGTAGGGAACATGCCTATCTCCATCGACATAC
  	CGGACGCGGCCTTTACCAGGGTCGTCGACGCGCCATCTTTAACGGACATG
  	TCGTGTGAGGTATCAGCCTGCACCCATTCCTCAGACTTTGGGGGCGTAGC
  	CATCATTAAATATGCAGCCAGTAAGAAAGGCAAGTGTGCAGTGCACTCGA
  	TGACTAACGCCGTCACTATTCGGGAAGCTGAAATAGAAGTAGAAGGGAAC
  	TCTCAGTTGCAAATCTCTTTTTCGACGGCCCTAGCCAGCGCCGAATTTCG
  	CGTACAAGTCTGTTCTACACAAGTACACTGTGCAGCCGAGTGCCATCCAC
  	CGAAAGACCATATAGTCAATTACCCGGCGTCACACACCACCCTCGGGGTC
  	CAAGACATTTCCGCTACGGCGATGTCATGGGTGCAGAAGATCACGGGAGG
  	TGTGGGACTGGTTGTCGCTGTTGCAGCACTGATCCTAATCGTGGTGCTAT
  	GCGTGTCGTTTAGCAGGCACTGATAATAGGCTGGAGCCTCGGTGGCCATG
  	CTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCC
  	GTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC
  Chik-Strain	TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA	11
  37997-E2-E1	ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCCATA
  (CHIKV E1-	TCTAGCTCATTGTCCTGACTGCGGAGAAGGGCATTCGTGCCACAGCCCTA
  E2 Antigen-	TCGCATTGGAGCGCATCAGAAATGAAGCAACGGACGGAACGCTGAAAATC
  Strain	CAGGTCTCTTTGCAGATCGGGATAAAGACAGATGACAGCCACGATTGGAC
  37997) :	CAAGCTGCGCTATATGGATAGCCATACGCCAGCGGACGCGGAGCGAGCCG
  	GATTGCTTGTAAGGACTTCAGCACCGTGCACGATCACCGGGACCATGGGA
  	CACTTTATTCTCGCCCGATGCCCGAAAGGAGAGACGCTGACAGTGGGATT
  	TACGGACAGCAGAAAGATCAGCCACACATGCACACACCCGTTCCATCATG
  	AACCACCTGTGATAGGTAGGGAGAGGTTCCACTCTCGACCACAACATGGT
  	AAAGAGTTACCTTGCAGCACGTACGTGCAGAGCACCGCTGCCACTGCTGA
  	GGAGATAGAGGTGCATATGCCCCCAGATACTCCTGACCGCACGCTGATGA
  	CGCAGCAGTCTGGCAACGTGAAGATCACAGTTAATGGGCAGACGGTGCGG
  	TACAAGTGCAACTGCGGTGGCTCAAACGAGGGACTGACAACCACAGACAA
  	AGTGATCAATAACTGCAAAATTGATCAGTGCCATGCTGCAGTCACTAATC
  	ACAAGAATTGGCAATACAACTCCCCTTTAGTCCCGCGCAACGCTGAACTC
  	GGGGACCGTAAAGGAAAGATCCACATCCCATTCCCATTGGCAAACGTGAC
  	TTGCAGAGTGCCAAAAGCAAGAAACCCTACAGTAACTTACGGAAAAAACC
  	AAGTCACCATGCTGCTGTATCCTGACCATCCGACACTCTTGTCTTACCGT
  	AACATGGGACAGGAACCAAATTACCACGAGGAGTGGGTGACACACAAGAA
  	GGAGGTTACCTTGACCGTGCCTACTGAGGGTCTGGAGGTCACTTGGGGCA
  	ACAACGAACCATACAAGTACTGGCCGCAGATGTCTACGAACGGTACTGCT
  	CATGGTCACCCACATGAGATAATCTTGTACTATTATGAGCTGTACCCCAC
  	TATGACTGTAGTCATTGTGTCGGTGGCCTCGTTCGTGCTTCTGTCGATGG
  	TGGGCACAGCAGTGGGAATGTGTGTGTGCGCACGGCGCAGATGCATTACA
  	CCATATGAATTAACACCAGGAGCCACTGTTCCCTTCCTGCTCAGCCTGCT
  	ATGCTGCCTATGGAACGAACAGCAGCCCCTGTTCTGGTTGCAGGCTCTTA
  	TCCCGCTGGCCGCCTTGATCGTCCTGTGCAACTGTCTGAAACTCTTGCCA
  	TGCTGCTGTAAGACCCTGGCTTTTTTAGCCGTAATGAGCATCGGTGCCCA
  	CACTGTGAGCGCGTACGAACACGTAACAGTGATCCCGAACACGGTGGGAG
  	TACCGTATAAGACTCTTGTCAACAGACCGGGTTACAGCCCCATGGTGTTG
  	GAGATGGAGCTACAATCAGTCACCTTGGAACCAACACTGTCACTTGACTA
  	CATCACGTGCGAGTACAAAACTGTCATCCCCTCCCCGTACGTGAAGTGCT
  	GTGGTACAGCAGAGTGCAAGGACAAGAGCCTACCAGACTACAGCTGCAAG
  	GTCTTTACTGGAGTCTACCCATTTATGTGGGGCGGCGCCTACTGCTTTTG
  	CGACGCCGAAAATACGCAATTGAGCGAGGCACATGTAGAGAAATCTGAAT
  	CTTGCAAAACAGAGTTTGCATCGGCCTACAGAGCCCACACCGCATCGGCG
  	TCGGCGAAGCTCCGCGTCCTTTACCAAGGAAACAACATTACCGTAGCTGC
  	CTACGCTAACGGTGACCATGCCGTCACAGTAAAGGACGCCAAGTTTGTCG
  	TGGGCCCAATGTCCTCCGCCTGGACACCTTTTGACAACAAAATCGTGGTG
  	TACAAAGGCGACGTCTACAACATGGACTACCCACCTTTTGGCGCAGGAAG
  	ACCAGGACAATTTGGTGACATTCAAAGTCGTACACCGGAAAGTAAAGACG
  	TTTATGCCAACACTCAGTTGGTACTACAGAGGCCAGCAGCAGGCACGGTA
  	CATGTACCATACTCTCAGGCACCATCTGGCTTCAAGTATTGGCTGAAGGA
  	ACGAGGAGCATCGCTACAGCACACGGCACCGTTCGGTTGCCAGATTGCGA
  	CAAACCCGGTAAGAGCTGTAAATTGCGCTGTGGGGAACATACCAATTTCC
  	ATCGACATACCGGATGCGGCCTTTACTAGGGTTGTCGATGCACCCTCTGT
  	AACGGACATGTCATGCGAAGTACCAGCCTGCACTCACTCCTCCGACTTTG
  	GGGGCGTCGCCATCATCAAATACACAGCTAGCAAGAAAGGTAAATGTGCA
  	GTACATTCGATGACCAACGCCGTTACCATTCGAGAAGCCGACGTAGAAGT
  	AGAGGGGAACTCCCAGCTGCAAATATCCTTCTCAACAGCCCTGGCAAGCG
  	CCGAGTTTCGCGTGCAAGTGTGCTCCACACAAGTACACTGCGCAGCCGCA
  	TGCCACCCTCCAAAGGACCACATAGTCAATTACCCAGCATCACACACCAC
  	CCTTGGGGTCCAGGATATATCCACAACGGCAATGTCTTGGGTGCAGAAGA
  	TTACGGGAGGAGTAGGATTAATTGTTGCTGTTGCTGCCTTAATTTTAATT
  	GTGGTGCTATGCGTGTCGTTTAGCAGGCACTAATGATAATAGGCTGGAGC
  	CTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCC
  	CCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCG
  	GC

Example 8: Exemplary Nucleic Acids Encoding CHIKV C-E3-E2-6K-E1 RNA Polynucleotides for Use in a RNA Vaccine
• The following sequence is an exemplary sequence that can be used to encode an CHIKV, DENV and/or ZIKV RNA polynucleotide C-E3-E2-6K-E1 for use in a RNA vaccine:

• TABLE 4
  CHIKV RNA polynucleotide C-E3-E2-6K-E1

  		SEQ ID
  Name	Sequence	NO
  Chik.C-E3-	TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA	12
  E2-6K-	ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAGTT
  E1_HS3UPCRf	TATCCCTACGCAGACGTTCTATAATCGGAGGTACCAGCCCAGGCCTTGGG
  ree (C-E3-	CCCCCCGCCCTACAATCCAAGTGATAAGACCACGTCCCAGGCCGCAGAGA
  E2-6K-E1	CAAGCCGGCCAATTGGCGCAACTCATCAGCGCAGTTAACAAGTTGACCAT
  Antigen)	GCGAGCGGTTCCTCAGCAGAAGCCGAGGCGGAACCGGAAGAATAAGAAAC
  	AACGCCAAAAGAAACAGGCGCCGCAGAACGACCCTAAACAGAAGAAACAA
  	CCTCCCCAGAAAAAGCCAGCTCAGAAGAAGAAGAAGCCTGGACGCCGTGA
  	AAGAATGTGCATGAAAATCGAAAATGATTGCATCTTTGAGGTGAAGCACG
  	AGGGCAAAGTGATGGGGTACGCATGCCTGGTGGGCGATAAGGTCATGAAG
  	CCAGCACATGTGAAGGGGACAATCGATAATGCTGATCTGGCCAAGCTAGC
  	TTTTAAACGTAGCTCCAAATACGATCTTGAGTGTGCCCAGATACCTGTGC
  	ACATGAAATCTGATGCAAGCAAGTTCACACACGAGAAGCCTGAGGGCTAT
  	TATAACTGGCATCATGGTGCGGTTCAGTACTCCGGCGGCCGATTTACCAT
  	TCCTACAGGGGCAGGAAAGCCGGGCGATTCGGGGAGACCCATTTTCGACA
  	ACAAAGGCCGCGTGGTAGCTATCGTGCTCGGTGGGGCTAATGAGGGTGCA
  	CGTACTGCACTTAGCGTGGTTACCTGGAATAAGGACATTGTCACAAAGAT
  	TACACCGGAGGGAGCAGAGGAATGGAGCCTGGCACTGCCCGTTCTGTGCC
  	TGCTGGCCAACACCACTTTCCCATGTAGTCAACCCCCTTGCACTCCCTGC
  	TGCTATGAGAAAGAGCCTGAGAGCACGTTACGTATGCTGGAAGATAATGT
  	CATGAGGCCCGGGTACTATCAACTGCTCAAGGCTAGTCTGACATGCTCGC
  	CCCACAGGCAGCGCAGGTCCACGAAAGATAACTTCAACGTTTACAAGGCT
  	ACTAGGCCTTATTTGGCCCACTGTCCCGATTGCGGAGAGGGACATTCTTG
  	TCATAGTCCTATTGCCTTGGAGCGAATCCGCAACGAGGCCACTGATGGAA
  	CCCTTAAGATTCAAGTATCTTTGCAGATTGGCATTAAGACAGATGATTCC
  	CATGACTGGACAAAGCTTCGGTACATGGACTCACACACGCCTGCAGATGC
  	TGAAAGGGCAGGGCTCTTGGTCAGGACCTCGGCCCCTTGTACAATTACCG
  	GGACCATGGGCCACTTCATCCTTGCACGCTGCCCTAAGGGGGAGACGCTG
  	ACGGTGGGCTTTACTGACTCGCGTAAGATCTCACACACATGTACACACCC
  	TTTCCACCACGAACCTCCAGTCATAGGGAGAGAGAGATTTCACTCTCGCC
  	CACAGCATGGCAAAGAGCTGCCATGCTCCACATATGTCCAGAGCACTGCT
  	GCTACCGCTGAAGAAATTGAGGTTCACATGCCACCCGATACACCAGACCG
  	TACTCTGATGACCCAACAGAGCGGCAACGTGAAGATTACCGTAAATGGAC
  	AGACCGTGAGATATAAGTGCAACTGTGGTGGCTCCAATGAGGGCTTAACA
  	ACAACGGATAAGGTGATTAACAATTGCAAAATAGATCAGTGCCATGCCGC
  	AGTGACCAATCACAAGAATTGGCAATACAACTCACCCCTAGTGCCGAGGA
  	ACGCAGAACTAGGCGACAGGAAAGGGAAAATCCATATACCCTTCCCCCTA
  	GCAAATGTGACCTGCCGAGTGCCCAAGGCCAGAAACCCCACGGTTACTTA
  	CGGCAAGAACCAGGTGACGATGCTTTTGTACCCAGACCATCCCACCTTGC
  	TCTCTTATAGAAACATGGGACAGGAGCCTAACTATCATGAGGAGTGGGTG
  	ACACACAAGAAAGAAGTCACCCTTACCGTGCCTACCGAAGGGCTTGAAGT
  	CACCTGGGGCAACAACGAGCCTTACAAGTATTGGCCACAGATGTCCACAA
  	ACGGAACAGCCCACGGCCACCCGCACGAGATCATACTGTATTACTATGAG
  	CTTTATCCCACAATGACTGTCGTAATTGTGAGCGTTGCCAGCTTCGTGTT
  	GCTTTCAATGGTTGGCACTGCCGTGGGGATGTGCGTGTGTGCTAGGCGCC
  	GCTGTATAACTCCTTATGAACTAACTCCAGGCGCCACCGTTCCTTTCCTG
  	CTCTCACTACTGTGTTGTGTGCGCACAACAAAGGCTGCCACCTACTACGA
  	AGCCGCCGCCTACTTATGGAATGAACAGCAGCCTCTCTTTTGGTTACAGG
  	CGCTGATTCCTCTTGCTGCCCTGATCGTGCTATGCAACTGCCTCAAGCTG
  	CTGCCCTGTTGTTGCAAGACCCTAGCTTTTCTCGCCGTGATGAGCATCGG
  	GGCACATACAGTGTCCGCCTATGAGCACGTCACCGTTATCCCGAACACCG
  	TCGGTGTGCCATATAAGACGTTAGTCAATCGACCCGGCTACTCTCCAATG
  	GTGCTGGAAATGGAACTCCAGAGTGTGACACTGGAGCCAACCTTATCCCT
  	CGATTATATTACCTGCGAATACAAGACCGTCATCCCTTCACCCTATGTCA
  	AGTGCTGTGGGACCGCTGAATGCAAAGACAAGAGCTTGCCTGATTACAGT
  	TGCAAGGTCTTCACAGGTGTGTACCCCTTCATGTGGGGGGGAGCTTATTG
  	CTTTTGTGATGCTGAGAACACCCAACTGAGCGAGGCTCACGTCGAGAAAT
  	CTGAGTCTTGCAAGACCGAGTTTGCCTCAGCTTACAGGGCCCACACGGCC
  	AGCGCATCCGCCAAATTGAGGGTACTCTACCAGGGTAATAATATCACCGT
  	TGCCGCATATGCAAACGGCGATCACGCCGTGACTGTCAAGGATGCCAAGT
  	TCGTTGTGGGCCCCATGTCTAGCGCTTGGACACCGTTCGATAATAAGATC
  	GTCGTGTACAAAGGGGACGTGTATAATATGGACTACCCACCTTTCGGGGC
  	CGGCCGACCGGGACAGTTCGGGGATATTCAGAGCCGCACACCCGAATCTA
  	AAGATGTTTACGCCAATACTCAGCTCGTCCTGCAGAGGCCCGCCGCTGGT
  	ACAGTTCACGTTCCTTACTCACAGGCACCCTCTGGGTTTAAGTATTGGCT
  	GAAAGAACGAGGTGCCAGCTTGCAGCATACAGCGCCTTTCGGATGCCAGA
  	TTGCCACTAACCCCGTACGGGCTGTCAACTGCGCGGTCGGCAATATTCCC
  	ATTAGCATTGATATCCCGGACGCAGCTTTCACCAGGGTTGTGGACGCCCC
  	GAGCGTCACCGACATGAGTTGTGAGGTGCCAGCCTGCACGCATAGCAGTG
  	ATTTCGGCGGCGTCGCCATCATTAAATATACCGCAAGCAAGAAAGGCAAG
  	TGCGCCGTCCACTCGATGACTAACGCCGTCACAATTCGGGAAGCCGATGT
  	TGAGGTCGAAGGCAACTCCCAGCTGCAGATCAGCTTCTCTACTGCTCTTG
  	CAAGCGCCGAGTTTCGAGTCCAGGTCTGCAGTACGCAGGTGCATTGTGCA
  	GCTGCCTGCCATCCACCCAAAGATCATATTGTGAATTATCCGGCGTCACA
  	TACCACACTGGGGGTCCAGGATATTAGTACAACGGCGATGTCCTGGGTGC
  	AGAAAATTACGGGAGGAGTGGGCTTAATTGTTGCCGTGGCGGCCCTGATC
  	CTGATCGTTGTGCTGTGTGTTAGCTTCTCTAGGCATGACTATAAAGATGA
  	CGATGACAAATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCC
  	CTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGT
  	GGTCTTTGAATAAAGTCTGAGTGGGCGGC
  CHIKV C-E3-	TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA	13
  E2-6K-E1	ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAGTT
  	TATCCCTACGCAGACGTTCTATAATCGGAGGTACCAGCCCAGGCCTTGGG
  	CCCCCCGCCCTACAATCCAAGTGATAAGACCACGTCCCAGGCCGCAGAGA
  	CAAGCCGGCCAATTGGCGCAACTCATCAGCGCAGTTAACAAGTTGACCAT
  	GCGAGCGGTTCCTCAGCAGAAGCCGAGGCGGAACCGGAAGAATAAGAAAC
  	AACGCCAAAAGAAACAGGCGCCGCAGAACGACCCTAAACAGAAGAAACAA
  	CCTCCCCAGAAAAAGCCAGCTCAGAAGAAGAAGAAGCCTGGACGCCGTGA
  	AAGAATGTGCATGAAAATCGAAAATGATTGCATCTTTGAGGTGAAGCACG
  	AGGGCAAAGTGATGGGGTACGCATGCCTGGTGGGCGATAAGGTCATGAAG
  	CCAGCACATGTGAAGGGGACAATCGATAATGCTGATCTGGCCAAGCTAGC
  	TTTTAAACGTAGCTCCAAATACGATCTTGAGTGTGCCCAGATACCTGTGC
  	ACATGAAATCTGATGCAAGCAAGTTCACACACGAGAAGCCTGAGGGCTAT
  	TATAACTGGCATCATGGTGCGGTTCAGTACTCCGGCGGCCGATTTACCAT
  	TCCTACAGGGGCAGGAAAGCCGGGCGATTCGGGGAGACCCATTTTCGACA
  	ACAAAGGCCGCGTGGTAGCTATCGTGCTCGGTGGGGCTAATGAGGGTGCA
  	CGTACTGCACTTAGCGTGGTTACCTGGAATAAGGACATTGTCACAAAGAT
  	TACACCGGAGGGAGCAGAGGAATGGAGCCTGGCACTGCCCGTTCTGTGCC
  	TGCTGGCCAACACCACTTTCCCATGTAGTCAACCCCCTTGCACTCCCTGC
  	TGCTATGAGAAAGAGCCTGAGAGCACGTTACGTATGCTGGAAGATAATGT
  	CATGAGGCCCGGGTACTATCAACTGCTCAAGGCTAGTCTGACATGCTCGC
  	CCCACAGGCAGCGCAGGTCCACGAAAGATAACTTCAACGTTTACAAGGCT
  	ACTAGGCCTTATTTGGCCCACTGTCCCGATTGCGGAGAGGGACATTCTTG
  	TCATAGTCCTATTGCCTTGGAGCGAATCCGCAACGAGGCCACTGATGGAA
  	CCCTTAAGATTCAAGTATCTTTGCAGATTGGCATTAAGACAGATGATTCC
  	CATGACTGGACAAAGCTTCGGTACATGGACTCACACACGCCTGCAGATGC
  	TGAAAGGGCAGGGCTCTTGGTCAGGACCTCGGCCCCTTGTACAATTACCG
  	GGACCATGGGCCACTTCATCCTTGCACGCTGCCCTAAGGGGGAGACGCTG
  	ACGGTGGGCTTTACTGACTCGCGTAAGATCTCACACACATGTACACACCC
  	TTTCCACCACGAACCTCCAGTCATAGGGAGAGAGAGATTTCACTCTCGCC
  	CACAGCATGGCAAAGAGCTGCCATGCTCCACATATGTCCAGAGCACTGCT
  	GCTACCGCTGAAGAAATTGAGGTTCACATGCCACCCGATACACCAGACCG
  	TACTCTGATGACCCAACAGAGCGGCAACGTGAAGATTACCGTAAATGGAC
  	AGACCGTGAGATATAAGTGCAACTGTGGTGGCTCCAATGAGGGCTTAACA
  	ACAACGGATAAGGTGATTAACAATTGCAAAATAGATCAGTGCCATGCCGC
  	AGTGACCAATCACAAGAATTGGCAATACAACTCACCCCTAGTGCCGAGGA
  	ACGCAGAACTAGGCGACAGGAAAGGGAAAATCCATATACCCTTCCCCCTA
  	GCAAATGTGACCTGCCGAGTGCCCAAGGCCAGAAACCCCACGGTTACTTA
  	CGGCAAGAACCAGGTGACGATGCTTTTGTACCCAGACCATCCCACCTTGC
  	TCTCTTATAGAAACATGGGACAGGAGCCTAACTATCATGAGGAGTGGGTG
  	ACACACAAGAAAGAAGTCACCCTTACCGTGCCTACCGAAGGGCTTGAAGT
  	CACCTGGGGCAACAACGAGCCTTACAAGTATTGGCCACAGATGTCCACAA
  	ACGGAACAGCCCACGGCCACCCGCACGAGATCATACTGTATTACTATGAG
  	CTTTATCCCACAATGACTGTCGTAATTGTGAGCGTTGCCAGCTTCGTGTT
  	GCTTTCAATGGTTGGCACTGCCGTGGGGATGTGCGTGTGTGCTAGGCGCC
  	GCTGTATAACTCCTTATGAACTAACTCCAGGCGCCACCGTTCCTTTCCTG
  	CTCTCACTACTGTGTTGTGTGCGCACAACAAAGGCTGCCACCTACTACGA
  	AGCCGCCGCCTACTTATGGAATGAACAGCAGCCTCTCTTTTGGTTACAGG
  	CGCTGATTCCTCTTGCTGCCCTGATCGTGCTATGCAACTGCCTCAAGCTG
  	CTGCCCTGTTGTTGCAAGACCCTAGCTTTTCTCGCCGTGATGAGCATCGG
  	GGCACATACAGTGTCCGCCTATGAGCACGTCACCGTTATCCCGAACACCG
  	TCGGTGTGCCATATAAGACGTTAGTCAATCGACCCGGCTACTCTCCAATG
  	GTGCTGGAAATGGAACTCCAGAGTGTGACACTGGAGCCAACCTTATCCCT
  	CGATTATATTACCTGCGAATACAAGACCGTCATCCCTTCACCCTATGTCA
  	AGTGCTGTGGGACCGCTGAATGCAAAGACAAGAGCTTGCCTGATTACAGT
  	TGCAAGGTCTTCACAGGTGTGTACCCCTTCATGTGGGGGGGAGCTTATTG
  	CTTTTGTGATGCTGAGAACACCCAACTGAGCGAGGCTCACGTCGAGAAAT
  	CTGAGTCTTGCAAGACCGAGTTTGCCTCAGCTTACAGGGCCCACACGGCC
  	AGCGCATCCGCCAAATTGAGGGTACTCTACCAGGGTAATAATATCACCGT
  	TGCCGCATATGCAAACGGCGATCACGCCGTGACTGTCAAGGATGCCAAGT
  	TCGTTGTGGGCCCCATGTCTAGCGCTTGGACACCGTTCGATAATAAGATC
  	GTCGTGTACAAAGGGGACGTGTATAATATGGACTACCCACCTTTCGGGGC
  	CGGCCGACCGGGACAGTTCGGGGATATTCAGAGCCGCACACCCGAATCTA
  	AAGATGTTTACGCCAATACTCAGCTCGTCCTGCAGAGGCCCGCCGCTGGT
  	ACAGTTCACGTTCCTTACTCACAGGCACCCTCTGGGTTTAAGTATTGGCT
  	GAAAGAACGAGGTGCCAGCTTGCAGCATACAGCGCCTTTCGGATGCCAGA
  	TTGCCACTAACCCCGTACGGGCTGTCAACTGCGCGGTCGGCAATATTCCC
  	ATTAGCATTGATATCCCGGACGCAGCTTTCACCAGGGTTGTGGACGCCCC
  	GAGCGTCACCGACATGAGTTGTGAGGTGCCAGCCTGCACGCATAGCAGTG
  	ATTTCGGCGGCGTCGCCATCATTAAATATACCGCAAGCAAGAAAGGCAAG
  	TGCGCCGTCCACTCGATGACTAACGCCGTCACAATTCGGGAAGCCGATGT
  	TGAGGTCGAAGGCAACTCCCAGCTGCAGATCAGCTTCTCTACTGCTCTTG
  	CAAGCGCCGAGTTTCGAGTCCAGGTCTGCAGTACGCAGGTGCATTGTGCA
  	GCTGCCTGCCATCCACCCAAAGATCATATTGTGAATTATCCGGCGTCACA
  	TACCACACTGGGGGTCCAGGATATTAGTACAACGGCGATGTCCTGGGTGC
  	AGAAAATTACGGGAGGAGTGGGCTTAATTGTTGCCGTGGCGGCCCTGATC
  	CTGATCGTTGTGCTGTGTGTTAGCTTCTCTAGGCATTGATAATAGGCTGG
  	AGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCC
  	TCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGG
  	GCGGC
  CHIKV C-E3-	SSFWTLVQKLIRLTIGKERKEEEEIEPPWSLSLRRRSIIGGTSPGLGPPA	14
  E2-6K-E1	LQSKDHVPGRRDKPANWRNSSAQLTSPCERFLSRSRGGTGRIRNNAKRNR
  	RRRTTLNRRNNLPRKSQLRRRRSLDAVKECAKSKMIASLRSTRAKWGTHA
  	WWAIRSSQHMRGQSIMLIWPSLLNVAPNTILSVPRYLCTNLMQASSHTRS
  	LRAIITGIMVRFSTPAADLPFLQGQESRAIRGDPFSTTKAAWLSCSVGLM
  	RVHVLHLAWLPGIRTLSQRLHRREQRNGAWHCPFCACWPTPLSHVVNPLA
  	LPAAMRKSLRARYVCWKIMSGPGTINCSRLVHARPTGSAGPRKITSTFTR
  	LLGLIWPTVPIAERDILVIVLLPWSESATRPLMEPLRFKYLCRLALRQMI
  	PMTGQSFGTWTHTRLQMLKGQGSWSGPRPLVQLPGPWATSSLHAALRGRR
  	RWALLTRVRSHTHVHTLSTTNLQSGERDFTLAHSMAKSCHAPHMSRALLL
  	PLKKLRFTCHPIHQTVLPNRAATRLPMDRPDISATVVAPMRAQQRIRLTI
  	AKISAMPQPITRIGNTTHPCRGTQNATGKGKSIYPSPQMPAECPRPETPR
  	LLTARTRRCFCTQTIPPCSLIETWDRSLTIMRSGHTRKKSPLPCLPKGLK
  	SPGATTSLTSIGHRCPQTEQPTATRTRSYCITMSFIPQLSLALPASCCFQ
  	WLALPWGCACVLGAAVLLMNLQAPPFLSCSHYCVVCAQQRLPPTTKPPPT
  	YGMNSSLSFGYRRFLLLPSCYATASSCCPVVARPLFSPASGHIQCPPMST
  	SPLSRTPSVCHIRRSIDPATLQWCWKWNSRVHWSQPYPSIILPANTRPSS
  	LHPMSSAVGPLNAKTRACLITVARSSQVCTPSCGGELIAFVMLRTPNARL
  	TSRNLSLARPSLPQLTGPTRPAHPPNGYSTRVIISPLPHMQTAITPLSRM
  	PSSLWAPCLALGHRSIIRSSCTKGTCIIWTTHLSGPADRDSSGIFRAAHP
  	NLKMFTPILSSSCRGPPLVQFTFLTHRHPLGLSIGKNEVPACSIQRLSDA
  	RLPLTPYGLSTARSAIFPLALISRTQLSPGLWTPRASPTVVRCQPARIAV
  	ISAASPSLNIPQARKASAPSTRLTPSQFGKPMLRSKATPSCRSASLLLLQ
  	APSFESRSAVRRCIVQLPAIHPKIILIIRRHIPHWGSRILVQRRCPGCRK
  	LREEWALLPWRPSSLCCVLASLGIDNRLEPRWPCFLPLGPPPSPSSPSCT
  	RTPVVFESLSGR

• FIG. 2 shows a phylogenetic tree of chikungunya virus strains derived from complete concatenated open reading frames for the nonstructural and structural polyproteins. E1 amino acid substitutions that facilitated (Indian Ocean lineage) or prevented (Asian lineage) adaptation to Aedes albopictus are shown on the right. CAR: Central African republic; ECSA: East/Central/South Africa
Example 9: Protocol to Determine Efficacy of mRNA-Encoded Chikungunya Antigen Candidates Against CHIKV
• Chikungunya has a polycistronic genome and different antigens, based on the Chikungunya structural protein, are possible. There are membrane-bound and secreted forms of E1 and E2, as well as the full length polyprotein antigen, which retains the protein's native conformation. Additionally, the different CHIKV genotypes can also yield different antigens.
• The efficacy of Chik candidate vaccines in AG129 mice against challenge with a lethal dose of CHIKV strain 181/25 was investigated. A129 mice, which lack IFN α/β receptor signaling, injected intradermally in the footpad with 10⁴ PFU of CHIKV 181/25 virus have a 100% survival rate post-injection. In contrast, AG129 mice, which lack IFN α/β and

  

  receptor signaling, injected intradermally in the footpad with 10⁴ PFU of CHIKV 181/25 virus do not survive past day 5 post-injection. The tested vaccines included: MC3-LNP formulated mRNA encoded CHIKV-E1, MC3-LNP formulated mRNA encoded CHIKV-E2, and MC3-LNP formulated mRNA encoded CHIKV-E1/E2/E3/C. Fifteen groups of five AG129 mice were vaccinated via intradermal (ID) or intramuscular (IM) injection with either 2 μg or 10 μg of the candidate vaccine. The vaccines were given to AG129 mice as single or two doses (second dose provided 28 days after the first dose). The positive control group was vaccinated via intranasal instillation (20 μL volume) with heat-inactivated CHIKV. Phosphate-buffered saline (PBS) was used as a negative control.
• On day 56, mice were challenged with 1×10⁴ PFU of CHIKV via ID injection in 50 μL volume and monitored for 10 days for weight loss, morbidity, and mortality. Mice that displayed severe illness, defined as >30% weight loss, a health score of 6 or above, extreme lethargy, and/or paralysis were euthanized. Notably, mice “vaccinated” with heat-inactivated CHIKV (positive control group) became morbid and were euthanized following the second dose of HI-CHIKV (they were not included in the challenge portion of the study).
• In addition, individual samples were tested for reactivity in a semi-quantitative ELISA for mouse IgG against either Chikungunya-specific E1 (groups 1-4), Chikungunya-specific E2 (groups 5-8), or Chikungunya-specific E1 and E2 proteins (groups 9-15).
• The health status is scored as indicated in the following Table 5:

• TABLE 5
  Health Status

  SCORE	INITIALS	DESCRIPTION	APPEARANCE	MOBILITY	ATTITUDE
  1	H	Healthy	Smooth Coat. Bright Eyes.	Active, Scurrying, Burrowing	Alert
  2	SR	Slightly Ruffled	Slightly Ruffled coat (usually	Active, Scurrying, Burrowing	Alert
  			only around head and neck)
  3	R	Ruffled	Ruffled Coat throughout	Active, Scurrying, Burrowing	Alert
  			body. A “wet” appearance.
  4	S	Sick	Very Ruffled coat. Slightly	Walking, but no scurrying.	Mildly
  			closed, inset eyes.		Lethargic
  5	VS	Very Sick	Very Ruffled Coat. Closed,	Slow to no movement. Will	Extremely
  		(Euthanize)	inset eyes.	return to upright position	Lethargic
  				if put on its side.
  6	E	Euthanize	Very ruffled Coat. Closed,	No movement or	Completely
  			inset eyes. Moribund	Uncontrollable, spastic	Unaware or in
  			requiring humane	movements. Will NOT return to	Noticeable
  			euthanasia.	upright position if put on its	Distress
  				side.
  7	D	Deceased	—	—	—

Example 10: Efficacy of Chikungunya E1 Antigen mRNA Vaccine Candidate
• AG129 mice (n=5 per group) were vaccinated with 2 μg or 10 μg of MC-3-LNP formulated mRNA encoding CHIKV E1. The AG129 mice were vaccinated on either Day 0 or Days 0 and 28 via IM or ID delivery. On Day 56 following final vaccination all mice were challenged with a lethal dose of CHIKV. The survival curve, percent weight loss, and health status of the mice vaccinated with 2 μg CHIKV E1 mRNA are shown in FIGS. 4A-C. The survival results are tabulated in Table 6 below. The survival curve, percent weight loss, and health status of the mice vaccinated with 10 μg CHIKV E1 mRNA are shown in FIGS. 8A-C. The survival results are tabulated in Table 7 below.

• TABLE 6
  Survival of mice vaccinated with Chikungunya
  E1 antigen mRNA - 2 μg dose

  	E1	E1	E1	E1
  days post	IM LNP	IM LNP	ID LNP	ID LNP
  infection	Day
  0	Day 0, 28	Day 0	Day 0, 28	Vehicle

  0.000	100.000	100.000	100.000	100.000	100.000
  4.000	80.000	40.000	40.000	60.000
  5.000	0.000	0.000	0.000	0.000	0.000

• TABLE 7
  Survival of mice vaccinated with Chikungunya
  E1 antigen mRNA - 10 μg dose

  	E1	E1	E1	E1
  days post	IM LNP	IM LNP	ID LNP	ID LNP
  infection	Day
  0	Day 0, 28	Day 0	Day 0, 28	Vehicle

  0.000	100.000	100.000	100.000	100.000	100.000
  4.000	60.000		80.000
  5.000	0.000	80.000	0.000		0.000
  6.000		60.000		80.000
  10.000		60.000		80.000

• As shown in Table 6, the 2 μg dose of CHIKV E1 mRNA vaccine gave no protection post-CHIKV infection challenge when administered via IM or ID with either a single dose or two doses. Likewise, the single dose of 10 μg CHIKV E1 vaccine provided little to no protection when administered via IM or ID. However, as indicated in Table 7, the 10 μg dose of CHIKV E1 mRNA vaccine provided 60% protection post-CHIKV challenge when administered via IM using two doses and provided 80% protection post-CHIKV challenge when administered via ID using two doses.
• In all experiments, the negative control mice had a ˜0% survival rate, as did the positive control mice (heat-inactivated CHIKV), which died before CHIKV challenge. Some mice died during the vaccination period.
Example 11: Efficacy of Chikungunya E2 Antigen mRNA Vaccine Candidate
• AG129 mice (n=5 per group) were vaccinated with 2 μg or 10 μg of MC-3-LNP formulated mRNA encoding CHIKV E2. The mice were vaccinated on either Day 0 or Days 0 and 28 via IM or ID delivery. On Day 56 following final vaccination all mice were challenged with a lethal dose of CHIKV. The survival curve, percent weight loss, and health status of the mice vaccinated with 2 μg CHIKV E2 mRNA are shown in FIGS. 5A-C. The survival results are tabulated in Table 8 below. The survival curve, percent weight loss, and health status of the mice vaccinated with 10 μg CHIKV E2 mRNA are shown in FIGS. 9A-C. The survival results are tabulated in Table 9 below.

• TABLE 8
  Survival of mice vaccinated with Chikungunya
  E2 antigen mRNA - 2 μg dose

  	E1	E1	E1	E1
  days post	IM LNP	IM LNP	ID LNP	ID LNP
  infection	Day
  0	Day 0, 28	Day 0	Day 0, 28	Vehicle

  0.000	100.000	100.000	100.000	100.000	100.000
  3.000	60.000
  4.000	20.000		80.000		0.000
  5.000	0.000		0.000		0.000
  6.000		80.000
  10.000		80.000		100.000

• TABLE 9
  Survival of mice vaccinated with Chikungunya
  E2 antigen mRNA - 10 μg dose

  	E2	E2	E2	E2
  days post	IM LNP	IM LNP	ID LNP	ID LNP
  infection	Day
  0	Day 0, 28	Day 0	Day 0, 28	Vehicle

  0.000	100.000	100.000	100.000	100.000	100.000
  5.000	40.000		0.000		0.000
  6.000	0.000
  10.000		100.000		100.000

• As shown in Table 8, the 2 μg dose of CHIKV E2 mRNA vaccine gave no protection post-CHIKV infection challenge when administered via IM or ID in a single dose. However, when provided in two doses, the 2 μg dose of CHIKV E2 mRNA vaccine provided 80% protection when administered via IM and 100% protection when administered via ID post-CHIKV challenge. As indicated in Table 9, the 10 μg dose of CHIKV E2 mRNA mouse provided no protection post-CHIKV challenge when administered via IM or ID in a single dose. However, administration of CHIKV E2 mRNA via IM or ID using two doses provided 100% protection post-CHIKV challenge.
• In all experiments, the negative control mice had a ˜0% survival rate, as did the positive control mice (heat-inactivated CHIKV) which died prior to CHIKV challenge. Some mice died during the vaccination period.
Example 12: Efficacy of Chikungunya C-E3-E2-6K-E1 Antigen mRNA Vaccine Candidate
• AG129 mice (n=5 per group) were vaccinated with 2 μg or 10 μg of MC-3-LNP formulated mRNA encoding CHIKV C-E3-E2-6K-E1 mRNA (SEQ ID NO:3). The AG129 mice were vaccinated on either Day 0 or Days 0 and 28 via IM or ID delivery. On Day 56 following final vaccination all mice were challenged with a lethal dose of CHIKV. The survival curve, percent weight loss, and health status of the mice vaccinated with 2 μg CHIKV C-E3-E2-6K-E1 mRNA are shown in FIGS. 6A-C. The survival results are tabulated in Table 10 below. The survival curve, percent weight loss, and health status of the mice vaccinated with 10 μg CHIKV C-E3-E2-6K-E1/E2/E3/C mRNA are shown in FIGS. 10A-C. The survival results are tabulated in Table 11 below.

• TABLE 10
  Survival of mice vaccinated with Chikungunya
  C-E3-E2-6K-E1 antigen mRNA - 2 μg

  	E1/E2/	E1/E2/	E1/E2/	E1/E2/
  	E3C	E3C	E3C	E3C
  days post	IM LNP	IM LNP	ID LNP	ID LNP
  infection	Day
  0	Day 0, 28	Day 0	Day 0, 28	Vehicle

  0.000	100.000	100.000	100.000	100.000	100.000
  5.000			80.000		0.000
  10.000	100.000	100.000	80.000	100.000

• TABLE 11
  Survival of mice vaccinated with Chikungunya
  C-E3-E2-6K-E1 antigen mRNA - 10 μg

  	E1/E2/	E1/E2/	E1/E2/	E1/E2/
  	E3C	E3C	E3C	E3C
  days post	IM LNP	IM LNP	ID LNP	ID LNP
  infection	Day
  0	Day 0, 28	Day 0	Day 0, 28	Vehicle

  0.000	100.000	100.000	100.000	100.000	100.000
  5.000					0.000
  10.000	100.000	100.000	100.000	100.000

• As shown in Table 10, the 2 μg dose of C-E3-E2-6K-E1 mRNA vaccine provided 100% protection post-CHIKV challenge when administered via IM in a single dose and provided 80% protection post-CHIKV challenge when administered via ID in a single dose. The 2 μg dose of C-E3-E2-6K-E1 mRNA vaccine provided 100% protection post-CHIKV challenge when administered via IM or ID in two doses. As shown in Table 11, the 10 μg dose of C-E3-E2-6K-E1 mRNA vaccine provided 100% protection post-CHIKV infection challenge when administered via IM or ID in either a single dose or in two doses.
• In all experiments, the negative control mice had a ˜0% survival rate, as did the positive control mice (heat-inactivated CHIKV) which died prior to CHIKV challenge. Some mice died during the vaccination period.
Example 13: Summary of Survival Data Using Chikungunya an n mRNA Vaccine Candidates CHIKV E1, CHIKV E2, and CHIKV C-E3-E2-6K-E1
• Table 12 shows the survival data of the mice vaccinated with the CHIKV mRNA antigens used in the studies reported in Examples 10-12.

• TABLE 12
  Summary of Day 6 post-injection survival data

  Dose

  10	Dose 2
  		ug/mouse	ug/mouse
  G#	Antigen/route/regime	(survival %)	(survival %)

  1	Chik-E1-IM- single dose	0	0
  2	Chik-E1-IM- two doses	60	0
  3	Chik-E1-ID- single dose	0	0
  4	Chik-E1-ID- two doses	80	0
  5	Chik-E2-IM- single dose	0	0
  6	Chik-E2-IM- two doses	100	80
  7	Chik-E2-ID- single dose	0	0
  8	Chik-E2-ID- two doses	100	100
  9	Chik-E1-E2-E3-C-6KIM- single dose	100	100
  10	Chik-E1-E2-E3-C-6KIM- two doses	100	100
  11	Chik-E1-E2-E3-C-6KID- single dose	100	80
  12	Chik-E1-E2-E3-C-6KID- two doses	100	100
  13	HI CHIKV (+)	0	0
  14	HI CHIKV (+)	0	0
  15	Control (−)	0	0

Example 14: In Vitro Transfection of mRNA-Encoded Chikungunya Virus Envelope Protein
• The in vitro transfection of mRNA encoding Notch and a PBS control were performed in 150k HeLa cells/well transfected with 1 μg mRNA+2 μL LF2000/well in a 24 well plate. Lysate containing proteins expressed from the CHIKV envelope mRNAs transfected in HeLa cells were collected 16 hours post-transfection and then detected by Western blotting with a V5 tag-HRP antibody. The successful detection of a CHIKV envelope protein is shown in FIG. 3 .
Example 15: Detection of Immunity (Mouse IgG) Against Either Chikungunya-Specific E1, Chikungunya-Specific E2, or Chikungunya-Specific E1 and E2 Proteins
• Serum samples from mice vaccinated with the CHIKV E1, E2, or E1-E2-E3-C vaccine described in Examples 11-13 were tested using a semi-quantitative ELISA for the detection of mouse IgG against either Chikungunya-specific E1, Chikungunya-specific E2, or Chikungunya-specific E1 and E2 proteins.
• Fifteen groups of five mice were vaccinated via intradermal (ID) or intramuscular (IM) injection with either 2 μg or 10 μg of the candidate vaccine. The vaccines were given to AG129 mice as single or two doses (second dose provided 28 days after the first dose). On day 56, mice were challenged with 1×104 PFU of CHIKV via ID injection in 50 μL volume and monitored for 10 days for weight loss, morbidity, and mortality. Mice were bled on day 7 and day 28 post-vaccination via the peri-orbital sinus (retro-orbital bleed). In addition, mice surviving the CHIKV challenge were bled 10 days post-challenge.
• The individual samples were tested for reactivity in a semi-quantitative ELISA for mouse IgG against either Chikungunya-specific E1, Chikungunya-specific E2, or Chikungunya-specific E1 and E2 proteins. The results are shown in FIGS. 50-52 .
• The data depicting the results of the ELISA assay to identify the amount of antibodies produced in AG129 mice in response to vaccination with mRNA encoding secreted CHIKV E1 structural protein, secreted CHIKV E2 structural protein, or CHIKV full structural polyprotein C-E3-E2-6k-E1 at a dose of 10 μg or 2 μg at 28 days post immunization is shown in FIGS. 50-51 . The 10 μg of mRNA encoding CHIKV polyprotein produced significant levels of antibody in both studies. The data depicting a comparison of ELISA titers from the data of FIG. 50 to survival in the data of FIG. 51 left panel is shown in FIG. 52 . As shown in the survival results, the animals vaccinated with either dose (single or double administration) of mRNA encoding CHIKV polyprotein had 100% survival rates.
Example 16: Efficacy of Chikungunya Polyprotein (C-E3-E2-6K-E1) mRNA Vaccine Candidate
• AG129 mice (n=5 per group) were vaccinated with either 10 μg, 2 μg or 0.4 μg of MC-3-LNP formulated mRNA encoded CHIKV polyprotein (C-E3-E2-6K-E1) (SEQ ID NO: 13). The mice were vaccinated on either Day 0 or Days 0 and 28 via IM delivery. In one study, all mice were challenged on day 56 with a lethal dose of CHIKV following final vaccination. In another study, all mice were challenged on day 84 with a lethal dose of CHIKV following final vaccination. The survival curve, percent weight loss, and health status of the mice vaccinated with 10 μg, 2 μg or 0.4 μg mRNA were determined as described previously in Examples 10-12. The survival rates, neutralizing antibodies and binding antibodies were assessed. Neutralizing antibodies were also identified against three different strains of CHIKV.
• The survival rates of the mice vaccinated with mRNA encoding CHIKV C-E3-E2-6k-E1 is shown in FIG. 53 . The data depicts vaccination at a dose of 10 μg (left panels), 2 μg (middle panels) or 0.4 μg (right panels) at 56 days (top panels) or 112 days (bottom panels) post immunization. These data demonstrate that a single 2 μg dose of the mRNA vaccine afforded 100% protection for at least 112 days (16 weeks.) Following 5 the study out further, the data demonstrated that a single 2 μg dose of the mRNA vaccine afforded 100% protection for at least 140 days (20 weeks.)
• The neutralizing antibody and binding antibody produced in treated mice is shown in FIGS. 54 and 55 respectively. As can be seen in FIGS. 54 and 55 , the levels of neutralizing Ab were dependent or dose and regimen with the highest titers evident with 10 μg dosed twice (days 0 and 28). Plaque reduction neutralization tests (PRNT50 and PRNT80) were used to quantify the titer of neutralizing antibody for the virus. Antigen binding Ab was determined by ELISA. The corresponding correlation between binding Ab and neutralizing antibodies is shown in the bottom panels of FIG. 55 . Following the study out to 16 weeks showed that the highest E1 titers were achieved when 10 μg mRNA vaccine was dosed twice.
• The data depicting neutralizing antibodies against three different strains of CHIKV is shown in FIG. 56 . The neutralizing antibodies were tested against three different strains of CHIKV, African-Senegal (left panel), La Reunion (middle panel) and CDC CAR (right panel). FIG. 56 shows that the polyprotein-encoding mRNA vaccine elicited broadly neutralizing antibodies against the three strains tested. Sera were further tested against Chik S27 strain (Chikungunya virus (strain S27-African prototype). The data depicting neutralizing antibodies against CHIKV S27 strain is shown in FIG. 57 . These data collectively show that the polyprotein encoding mRNA vaccine elicited broadly neutralizing antibodies against all four strains tested. The vaccine induced neutralizing antibodies against multiple strains of Chikungunya. The prime and boost with the 10 μg dose produced the most robust neutralizing antibody response followed by the single dose with 10 μg.
Example 17: Transfection of mRNA Encoded CHIKV Structural Proteins
• In vitro transfection of mRNA encoding CHIKV structural proteins and PBS control were performed in 400 k HeLa cells transfected with 1.25 μg mRNA lipoplexed with 5ul LF2000/well in 6 well plate. Protein detection in HeLa cell lysate 16 h post transfection was measured. Lysates which contain proteins expressed from the CHIKV mRNAs transfected in HeLa were collected 16 h post transfection. Proteins were detected by WB with anti Flag or and V5 antibody.
• FIG. 12 show the results of the assay. mRNA encoded CHIKV structural proteins. Protein production in the HeLa cell lysate 16 h post transfection was detected.
Example 18: Exemplary Dengue Sequences
• The following are nucleic acid (SEQ ID NO: 16, 18, 20, and 22) and amino acid (SEQ ID NO: 15, 17, 19, and 21) sequences for each of DEN-1, DEN-2, DEN-3, and DEN-4.

• TABLE 13
  DENV polynucleotide sequences and amino acid sequences

  		SEQ ID
  Name	Sequence	NO
  DEN-1	MNNQRKKTGRPSFNMLKRARNRVSTVSQLAKRFSKGLLSGQGPMKLVMAF	15
  (NC_001477.	IAFLRFLAIPPTAGILARWGSFKKNGAIKVLRGFKKEISNMLNIMNRRKR
  1)	SVTMLLMLLPTALAFHLTTRGGEPHMIVSKQERGKSLLFKTSAGVNMCTL
  	IAMDLGELCEDTMTYKCPRITETEPDDVDCWCNATETWVTYGTCSQTGEH
  	RRDKRSVALAPHVGLGLE
  	TRTETWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQKGII
  	FILLMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKDK
  	PTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQGEATLVEEQDT
  	NFVCRRTFVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYS
  	VIVTVHTGDQHQVGNETTEHGTTATITPQAPTSEIQLTDYGALTLDCSPR
  	TGLDFNEMVLLTMKKKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLV
  	TFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLK
  	MDKLILKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFS
  	SQDEKGVTQNGRLITANPIVTDKEKPVNIEAEPPFGESYIVVGAGEKALK
  	LSWFKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSVGKLIHQ
  	IFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMVTLY
  	LGVMVQADSGCVINWKGRELKCGSGIFVTNEVHTWTEQYKFQADSPKRLS
  	AAIGKAWEEGVCGIRSATRLENIMWKQISNELNHILLENDMKFTVVVGDV
  	SGILAQGKKMIRPQPMEHKYSWKSWGKAKIIGADVQNTTFIIDGPNTPEC
  	PDNQRAWNIWEVEDYGFGIFTTNIWLKLRDSYTQVCDHRLMSAAIKDSKA
  	VHADMGYWIESEKNETWKLARASFIEVKTCIWPKSHTLWSNGVLESEMII
  	PKIYGGPISQHNYRPGYFTQTAGPWHLGKLELDFDLCEGTTVVVDEHCGN
  	RGPSLRTTTVTGKTIHEWCCRSCTLPPLRFKGEDGCWYGMEIRPVKEKEE
  	NLVKSMVSAGSGEVDSFSLGLLCISIMIEEVMRSRWSRKMLMTGTLAVFL
  	LLTMGQLTWNDLIRLCIMVGANASDKMGMGTTYLALMATFRMRPMFAVGL
  	LFRRLTSREVLLLTVGLSLVASVELPNSLEELGDGLAMGIMMLKLLTDFQ
  	SHQLWATLLSLTFVKTTFSLHYAWKTMAMILSIVSLFPLCLSTTSQKTTW
  	LPVLLGSLGCKPLTMFLITENKIWGRKSWPLNEGIMAVGIVSILLSSLLK
  	NDVPLAGPLIAGGMLIACYVISGSSADLSLEKAAEVSWEEEAEHSGASHN
  	ILVEVQDDGTMKIKDEERDDTLTILLKATLLAISGVYPMSIPATLFVWYF
  	WQKKKQRSGVLWDTPSPPEVERAVLDDGIYRILQRGLLGRSQVGVGVFQE
  	GVFHTMWHVTRGAVLMYQGKRLEPSWASVKKDLISYGGGWRFQGSWNAGE
  	EVQVIAVEPGKNPKNVQTAPGTFKTPEGEVGAIALDFKPGTSGSPIVNRE
  	GKIVGLYGNGVVTTSGTYVSAIAQAKASQEGPLPEIEDEVFRKRNLTIMD
  	LHPGSGKTRRYLPAIVREAIKRKLRTLVLAPTRVVASEMAEALKGMPIRY
  	QTTAVKSEHTGKEIVDLMCHATFTMRLLSPVRVPNYNMIIMDEAHFTDPA
  	SIAARGYISTRVGMGEAAAIFMTATPPGSVEAFPQSIQDEERDIPERSWN
  	SGYDWITDFPGKTVWFVPSIKSGNDIANCLRKNGKRVVQLSRKTFDTEYQ
  	KTKNNDWDYVVTTDISEMGANFRADRVIDPRRCLKPVILKDGPERVILAG
  	PMPVTVASAAQRRGRIGRNQNKEGDQYIYMGQPLNNDEDHAHWTEAKMLL
  	DNINTPEGIIPALFEPEREKSAAIDGEYRLRGEARKTFVELMRRGDLPVW
  	LSYKVASEGFQYSDRRWCFDGERNNQVLEENMDVEIWTKEGERKKLRPRW
  	LDARTYSDPLALREFKEFAAGRRSVSGDLILEIGKLPQHLTQRAQNALDN
  	LVMLHNSEQGGKAYRHAMEELPDTIETLMLLALIAVLTGGVTLFFLSGRG
  	LGKTSIGLLCVIASSALLWMASVEPHWIAASIILEFFLMVLLIPEPDRQR
  	TPQDNQLAYVVIGLLFMILTVAANEMGLLETTKKDLGIGHAAAENHHHAA
  	MLDVDLHPASAWTLYAVATTIITPMMRHTIENTTANISLTAIANQAAILM
  	GLDKGWPISKMDIGVPLLALGCYSQVNPLTLTAAVLMLVAHYAIIGPGLQ
  	AKATREAQKRTAAGIMKNPTVDGIVAIDLDPVVYDAKFEKQLGQIMLLIL
  	CTSQILLMRTTWALCESITLATGPLTTLWEGSPGKFWNTTIAVSMANIFR
  	GSYLAGAGLAFSLMKSLGGGRRGTGAQGETLGEKWKRQLNQLSKSEFNTY
  	KRSGIIEVDRSEAKEGLKRGETTKHAVSRGTAKLRWFVERNLVKPEGKVI
  	DLGCGRGGWSYYCAGLKKVTEVKGYTKGGPGHEEPIPMATYGWNLVKLYS
  	GKDVFFTPPEKCDTLLCDIGESSPNPTIEEGRTLRVLKMVEPWLRGNQFC
  	IKILNPYMPSVVETLEQMQRKHGGMLVRNPLSRNSTHEMYWVSCGTGNIV
  	SAVNMTSRMLLNRFTMAHRKPTYERDVDLGAGTRHVAVEPEVANLDIIGQ
  	RIENIKNEHKSTWHYDEDNPYKTWAYHGSYEVKPSGSASSMVNGVVRLLT
  	KPWDVIPMVTQIAMTDTTPFGQQRVFKEKVDTRTPKAKRGTAQIMEVTAR
  	WLWGFLSRNKKPRICTREEFTRKVRSNAAIGAVFVDENQWNSAKEAVEDE
  	RFWDLVHRERELHKQGKCATCVYNMMGKREKKLGEFGKAKGSRAIWYMWL
  	GARFLEFEALGFMNEDHWFSRENSLSGVEGEGLHKLGYILRDISKIPGGN
  	MYADDTAGWDTRITEDDLQNEAKITDIMEPEHALLATSIFKLTYQNKVVR
  	VQRPAKNGTVMDVISRRDQRGSGQVGTYGLNTFTNMEAQLIRQMESEGIF
  	SPSELETPNLAERVLDWLKKHGTERLKRMAISGDDCVVKPIDDRFATALT
  	ALNDMGKVRKDIPQWEPSKGWNDWQQVPFCSHHFHQLIMKDGREIVVPCR
  	NQDELVGRARVSQGAGWSLRETACLGKSYAQMWQLMYFHRRDLRLAANAI
  	CSAVPVDWVPTSRTTWSIHAH
  	HQWMTTEDMLSVWNRVWIEENPWMEDKTHVSSWEDVPYLGKREDQWCGSL
  	IGLTARATWATNIQVAINQVRRLIGNENYLDFMTSMKRFKNESDPEGALW
  DEN-1	agttgttagtctacgtggaccgacaagaacagtttcgaatcggaagcttg	16
  (NC_001477.	cttaacgtagttctaacagttttttattagagagcagatctctgatgaac
  1)	aaccaacggaaaaagacgggtcgaccgtctttcaatatgctgaaacgcgc
  	gagaaaccgcgtgtcaactgtttcacagttggcgaagagattctcaaaag
  	gattgctttcaggccaaggacccatgaaattggtgatggcttttatagca
  	ttcctaagatttctagccatacctccaacagcaggaattttggctagatg
  	gggctcattcaagaagaatggagcgatcaaagtgttacggggtttcaaga
  	aagaaatctcaaacatgttgaacataatgaacaggaggaaaagatctgtg
  	accatgctcctcatgctgctgcccacagccctggcgttccatctgaccac
  	ccgagggggagagccgcacatgatagttagcaagcaggaaagaggaaaat
  	cacttttgtttaagacctctgcaggtgtcaacatgtgcacccttattgca
  	atggatttgggagagttatgtgaggacacaatgacctacaaatgcccccg
  	gatcactgagacggaaccagatgacgttgactgttggtgcaatgccacgg
  	agacatgggtgacctatggaacatgttctcaaactggtgaacaccgacga
  	gacaaacgttccgtcgcactggcaccacacgtagggcttggtctagaaac
  	aagaaccgaaacgtggatgtcctctgaaggcgcttggaaacaaatacaaa
  	aagtggagacctgggctctgagacacccaggattcacggtgatagccctt
  	tttctagcacatgccataggaacatccatcacccagaaagggatcatttt
  	tattttgctgatgctggtaactccatccatggccatgcggtgcgtgggaa
  	taggcaacagagacttcgtggaaggactgtcaggagctacgtgggtggat
  	gtggtactggagcatggaagttgcgtcactaccatggcaaaagacaaacc
  	aacactggacattgaactcttgaagacggaggtcacaaaccctgccgtcc
  	tgcgcaaactgtgcattgaagctaaaatatcaaacaccaccaccgattcg
  	agatgtccaacacaaggagaagccacgctggtggaagaacaggacacgaa
  	ctttgtgtgtcgacgaacgttcgtggacagaggctggggcaatggttgtg
  	ggctattcggaaaaggtagcttaataacgtgtgctaagtttaagtgtgtg
  	acaaaactggaaggaaagatagtccaatatgaaaacttaaaatattcagt
  	gatagtcaccgtacacactggagaccagcaccaagttggaaatgagacca
  	cagaacatggaacaactgcaaccataacacctcaagctcccacgtcggaa
  	atacagctgacagactacggagctctaacattggattgttcacctagaac
  	agggctagactttaatgagatggtgttgttgacaatgaaaaaaaaatcat
  	ggctcgtccacaaacaatggtttctagacttaccactgccttggacctcg
  	ggggcttcaacatcccaagagacttggaatagacaagacttgctggtcac
  	atttaagacagctcatgcaaaaaagcaggaagtagtcgtactaggatcac
  	aagaaggagcaatgcacactgcgttgactggagcgacagaaatccaaacg
  	tctggaacgacaacaatttttgcaggacacctgaaatgcagattaaaaat
  	ggataaactgattttaaaagggatgtcatatgtaatgtgcacagggtcat
  	tcaagttagagaaggaagtggctgagacccagcatggaactgttctagtg
  	caggttaaatacgaaggaacagatgcaccatgcaagatccccttctcgtc
  	ccaagatgagaagggagtaacccagaatgggagattgataacagccaacc
  	ccatagtcactgacaaagaaaaaccagtcaacattgaagcggagccacct
  	tttggtgagagctacattgtggtaggagcaggtgaaaaagctttgaaact
  	aagctggttcaagaagggaagcagtatagggaaaatgtttgaagcaactg
  	cccgtggagcacgaaggatggccatcctgggagacactgcatgggacttc
  	ggttctataggaggggtgttcacgtctgtgggaaaactgatacaccagat
  	ttttgggactgcgtatggagttttgttcagcggtgtttcttggaccatga
  	agataggaatagggattctgctgacatggctaggattaaactcaaggagc
  	acgtccctttcaatgacgtgtatcgcagttggcatggtcacactgtacct
  	aggagtcatggttcaggcggactcgggatgtgtaatcaactggaaaggca
  	gagaactcaaatgtggaagcggcatttttgtcaccaatgaagtccacacc
  	tggacagagcaatataaattccaggccgactcccctaagagactatcagc
  	ggccattgggaaggcatgggaggagggtgtgtgtggaattcgatcagcca
  	ctcgtctcgagaacatcatgtggaagcaaatatcaaatgaattaaaccac
  	atcttacttgaaaatgacatgaaatttacagtggtcgtaggagacgttag
  	tggaatcttggcccaaggaaagaaaatgattaggccacaacccatggaac
  	acaaatactcgtggaaaagctggggaaaagccaaaatcataggagcagat
  	gtacagaataccaccttcatcatcgacggcccaaacaccccagaatgccc
  	tgataaccaaagagcatggaacatttgggaagttgaagactatggatttg
  	gaattttcacgacaaacatatggttgaaattgcgtgactcctacactcaa
  	gtgtgtgaccaccggctaatgtcagctgccatcaaggatagcaaagcagt
  	ccatgctgacatggggtactggatagaaagtgaaaagaacgagacttgga
  	agttggcaagagcctccttcatagaagttaagacatgcatctggccaaaa
  	tcccacactctatggagcaatggagtcctggaaagtgagatgataatccc
  	aaagatatatggaggaccaatatctcagcacaactacagaccaggatatt
  	tcacacaaacagcagggccgtggcacttgggcaagttagaactagatttt
  	gatttatgtgaaggtaccactgttgttgtggatgaacattgtggaaatcg
  	aggaccatctcttagaaccacaacagtcacaggaaagacaatccatgaat
  	ggtgctgtagatcttgcacgttaccccccctacgtttcaaaggagaagac
  	gggtgctggtacggcatggaaatcagaccagtcaaggagaaggaagagaa
  	cctagttaagtcaatggtctctgcagggtcaggagaagtggacagttttt
  	cactaggactgctatgcatatcaataatgatcgaagaggtaatgagatcc
  	agatggagcagaaaaatgctgatgactggaacattggctgtgttcctcct
  	tctcacaatgggacaattgacatggaatgatctgatcaggctatgtatca
  	tggttggagccaacgcttcagacaagatggggatgggaacaacgtaccta
  	gctttgatggccactttcagaatgagaccaatgttcgcagtcgggctact
  	gtttcgcagattaacatctagagaagttcttcttcttacagttggattga
  	gtctggtggcatctgtagaactaccaaattccttagaggagctaggggat
  	ggacttgcaatgggcatcatgatgttgaaattactgactgattttcagtc
  	acatcagctatgggctaccttgctgtctttaacatttgtcaaaacaactt
  	tttcattgcactatgcatggaagacaatggctatgatactgtcaattgta
  	tctctcttccctttatgcctgtccacgacttctcaaaaaacaacatggct
  	tccggtgttgctgggatctcttggatgcaaaccactaaccatgtttctta
  	taacagaaaacaaaatctggggaaggaaaagctggcctctcaatgaagga
  	attatggctgttggaatagttagcattcttctaagttcacttctcaagaa
  	tgatgtgccactagctggcccactaatagctggaggcatgctaatagcat
  	gttatgtcatatctggaagctcggccgatttatcactggagaaagcggct
  	gaggtctcctgggaagaagaagcagaacactctggtgcctcacacaacat
  	actagtggaggtccaagatgatggaaccatgaagataaaggatgaagaga
  	gagatgacacactcaccattctcctcaaagcaactctgctagcaatctca
  	ggggtatacccaatgtcaataccggcgaccctctttgtgtggtatttttg
  	gcagaaaaagaaacagagatcaggagtgctatgggacacacccagccctc
  	cagaagtggaaagagcagtccttgatgatggcatttatagaattctccaa
  	agaggattgttgggcaggtctcaagtaggagtaggagtttttcaagaagg
  	cgtgttccacacaatgtggcacgtcaccaggggagctgtcctcatgtacc
  	aagggaagagactggaaccaagttgggccagtgtcaaaaaagacttgatc
  	tcatatggaggaggttggaggtttcaaggatcctggaacgcgggagaaga
  	agtgcaggtgattgctgttgaaccggggaagaaccccaaaaatgtacaga
  	cagcgccgggtaccttcaagacccctgaaggcgaagttggagccatagct
  	ctagactttaaacccggcacatctggatctcctatcgtgaacagagaggg
  	aaaaatagtaggtctttatggaaatggagtggtgacaacaagtggtacct
  	acgtcagtgccatagctcaagctaaagcatcacaagaagggcctctacca
  	gagattgaggacgaggtgtttaggaaaagaaacttaacaataatggacct
  	acatccaggatcgggaaaaacaagaagataccttccagccatagtccgtg
  	aggccataaaaagaaagctgcgcacgctagtcttagctcccacaagagtt
  	gtcgcttctgaaatggcagaggcgctcaagggaatgccaataaggtatca
  	gacaacagcagtgaagagtgaacacacgggaaaggagatagttgacctta
  	tgtgtcacgccactttcactatgcgtctcctgtctcctgtgagagttccc
  	aattataatatgattatcatggatgaagcacattttaccgatccagccag
  	catagcagccagagggtatatctcaacccgagtgggtatgggtgaagcag
  	ctgcgattttcatgacagccactccccccggatcggtggaggcctttcca
  	cagagcaatgcagttatccaagatgaggaaagagacattcctgaaagatc
  	atggaactcaggctatgactggatcactgatttcccaggtaaaacagtct
  	ggtttgttccaagcatcaaatcaggaaatgacattgccaactgtttaaga
  	aagaatgggaaacgggtggtccaattgagcagaaaaacttttgacactga
  	gtaccagaaaacaaaaaataacgactgggactatgttgtcacaacagaca
  	tatccgaaatgggagcaaacttccgagccgacagggtaatagacccgagg
  	cggtgcctgaaaccggtaatactaaaagatggcccagagcgtgtcattct
  	agccggaccgatgccagtgactgtggctagcgccgcccagaggagaggaa
  	gaattggaaggaaccaaaataaggaaggcgatcagtatatttacatggga
  	cagcctctaaacaatgatgaggaccacgcccattggacagaagcaaaaat
  	gctccttgacaacataaacacaccagaagggattatcccagccctctttg
  	agccggagagagaaaagagtgcagcaatagacggggaatacagactacgg
  	ggtgaagcgaggaaaacgttcgtggagctcatgagaagaggagatctacc
  	tgtctggctatcctacaaagttgcctcagaaggcttccagtactccgaca
  	gaaggtggtgctttgatggggaaaggaacaaccaggtgttggaggagaac
  	atggacgtggagatctggacaaaagaaggagaaagaaagaaactacgacc
  	ccgctggctggatgccagaacatactctgacccactggctctgcgcgaat
  	tcaaagagttcgcagcaggaagaagaagcgtctcaggtgacctaatatta
  	gaaatagggaaacttccacaacatttaacgcaaagggcccagaacgcctt
  	ggacaatctggttatgttgcacaactctgaacaaggaggaaaagcctata
  	gacacgccatggaagaactaccagacaccatagaaacgttaatgctccta
  	gctttgatagctgtgctgactggtggagtgacgttgttcttcctatcagg
  	aaggggtctaggaaaaacatccattggcctactctgcgtgattgcctcaa
  	gtgcactgttatggatggccagtgtggaaccccattggatagcggcctct
  	atcatactggagttctttctgatggtgttgcttattccagagccggacag
  	acagcgcactccacaagacaaccagctagcatacgtggtgataggtctgt
  	tattcatgatattgacagtggcagccaatgagatgggattactggaaacc
  	acaaagaaggacctggggattggtcatgcagctgctgaaaaccaccatca
  	tgctgcaatgctggacgtagacctacatccagcttcagcctggactctct
  	atgcagtggccacaacaattatcactcccatgatgagacacacaattgaa
  	aacacaacggcaaatatttccctgacagctattgcaaaccaggcagctat
  	attgatgggacttgacaagggatggccaatatcaaagatggacataggag
  	ttccacttctcgccttggggtgctattctcaggtgaacccgctgacgctg
  	acagcggcggtattgatgctagtggctcattatgccataattggacccgg
  	actgcaagcaaaagctactagagaagctcaaaaaaggacagcagccggaa
  	taatgaaaaacccaactgtcgacgggatcgttgcaatagatttggaccct
  	gtggtttacgatgcaaaatttgaaaaacagctaggccaaataatgttgtt
  	gatactttgcacatcacagatcctcctgatgcggaccacatgggccttgt
  	gtgaatccatcacactagccactggacctctgactacgctttgggaggga
  	tctccaggaaaattctggaacaccacgatagcggtgtccatggcaaacat
  	ttttaggggaagttatctagcaggagcaggtctggccttttcattaatga
  	aatctctaggaggaggtaggagaggcacgggagcccaaggggaaacactg
  	ggagaaaaatggaaaagacagctaaaccaattgagcaagtcagaattcaa
  	cacttacaaaaggagtgggattatagaggtggatagatctgaagccaaag
  	aggggttaaaaagaggagaaacgactaaacacgcagtgtcgagaggaacg
  	gccaaactgaggtggtttgtggagaggaaccttgtgaaaccagaagggaa
  	agtcatagacctcggttgtggaagaggtggctggtcatattattgcgctg
  	ggctgaagaaagtcacagaagtgaaaggatacacgaaaggaggacctgga
  	catgaggaaccaatcccaatggcaacctatggatggaacctagtaaagct
  	atactccgggaaagatgtattctttacaccacctgagaaatgtgacaccc
  	tcttgtgtgatattggtgagtcctctccgaacccaactatagaagaagga
  	agaacgttacgtgttctaaagatggtggaaccatggctcagaggaaacca
  	attttgcataaaaattctaaatccctatatgccgagtgtggtagaaactt
  	tggagcaaatgcaaagaaaacatggaggaatgctagtgcgaaatccactc
  	tcaagaaactccactcatgaaatgtactgggtttcatgtggaacaggaaa
  	cattgtgtcagcagtaaacatgacatctagaatgctgctaaatcgattca
  	caatggctcacaggaagccaacatatgaaagagacgtggacttaggcgct
  	ggaacaagacatgtggcagtagaaccagaggtggccaacctagatatcat
  	tggccagaggatagagaatataaaaaatgaacacaaatcaacatggcatt
  	atgatgaggacaatccatacaaaacatgggcctatcatggatcatatgag
  	gtcaagccatcaggatcagcctcatccatggtcaatggtgtggtgagact
  	gctaaccaaaccatgggatgtcattcccatggtcacacaaatagccatga
  	ctgacaccacaccctttggacaacagagggtgtttaaagagaaagttgac
  	acgcgtacaccaaaagcgaaacgaggcacagcacaaattatggaggtgac
  	agccaggtggttatggggttttctctctagaaacaaaaaacccagaatct
  	gcacaagagaggagttcacaagaaaagtcaggtcaaacgcagctattgga
  	gcagtgttcgttgatgaaaatcaatggaactcagcaaaagaggcagtgga
  	agatgaacggttctgggaccttgtgcacagagagagggagcttcataaac
  	aaggaaaatgtgccacgtgtgtctacaacatgatgggaaagagagagaaa
  	aaattaggagagttcggaaaggcaaaaggaagtcgcgcaatatggtacat
  	gtggttgggagcgcgctttttagagtttgaagcccttggtttcatgaatg
  	aagatcactggttcagcagagagaattcactcagtggagtggaaggagaa
  	ggactccacaaacttggatacatactcagagacatatcaaagattccagg
  	gggaaatatgtatgcagatgacacagccggatgggacacaagaataacag
  	aggatgatcttcagaatgaggccaaaatcactgacatcatggaacctgaa
  	catgccctattggccacgtcaatctttaagctaacctaccaaaacaaggt
  	agtaagggtgcagagaccagcgaaaaatggaaccgtgatggatgtcatat
  	ccagacgtgaccagagaggaagtggacaggttggaacctatggcttaaac
  	accttcaccaacatggaggcccaactaataagacaaatggagtctgaggg
  	aatcttttcacccagcgaattggaaaccccaaatctagccgaaagagtcc
  	tcgactggttgaaaaaacatggcaccgagaggctgaaaagaatggcaatc
  	agtggagatgactgtgtggtgaaaccaatcgatgacagatttgcaacagc
  	cttaacagctttgaatgacatgggaaaggtaagaaaagacataccgcaat
  	gggaaccttcaaaaggatggaatgattggcaacaagtgcctttctgttca
  	caccatttccaccagctgattatgaaggatgggagggagatagtggtgcc
  	atgccgcaaccaagatgaacttgtaggtagggccagagtatcacaaggcg
  	ccggatggagcttgagagaaactgcatgcctaggcaagtcatatgcacaa
  	atgtggcagctgatgtacttccacaggagagacttgagattagcggctaa
  	tgctatctgttcagccgttccagttgattgggtcccaaccagccgcacca
  	cctggtcgatccatgcccaccatcaatggatgacaacagaagacatgttg
  	tcagtgtggaatagggtttggatagaggaaaacccatggatggaggacaa
  	gactcatgtgtccagttgggaagacgttccatacctaggaaaaagggaag
  	atcaatggtgtggttccctaataggcttaacagcacgagccacctgggcc
  	accaacatacaagtggccataaaccaagtgagaaggctcattgggaatga
  	gaattatctagacttcatgacatcaatgaagagattcaaaaacgagagtg
  	atcccgaaggggcactctggtaagccaactcattcacaaaataaaggaaa
  	ataaaaaatcaaacaaggcaagaagtcaggccggattaagccatagcacg
  	gtaagagctatgctgcctgtgagccccgtccaaggacgtaaaatgaagtc
  	aggccgaaagccacggttcgagcaagccgtgctgcctgtagctccatcgt
  	ggggatgtaaaaacccgggaggctgcaaaccatggaagctgtacgcatgg
  	ggtagcagactagtggttagaggagacccctcccaagacacaacgcagca
  	gcggggcccaacaccaggggaagctgtaccctggtggtaaggactagagg
  	ttagaggagaccccccgcacaacaacaaacagcatattgacgctgggaga
  	gaccagagatcctgctgtctctacagcatcattccaggcacagaacgcca
  	aaaaatggaatggtgctgttgaatcaacaggttct
  DEN-2	MNNQRKKAKNTPFNMLKRERNRVSTVQQLTKRFSLGMLQGRGPLKLFMAL	17
  (NC_001474.	VAFLRFLTIPPTAGILKRWGTIKKSKAINVLRGFRKEIGRMLNILNRRRR
  2)	SAGMIIMLIPTVMAFHLTTRNGEPHMIVSRQEKGKSLLFKTEDGVNMCTL
  	MAMDLGELCEDTITYKCPLLRQNEPEDIDCWCNSTSTWVTYGTCTTMGEH
  	RREKRSVALVPHVGMGLETRTETWMSSEGAWKHVQRIETWILRHPGFTMM
  	AAILAYTIGTTHFQRALIFILLTAVTPSMTMRCIGMSNRDFVEGVSGGSW
  	VDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTT
  	ESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFR
  	CKKNMEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSI
  	TEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPW
  	LPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEI
  	QMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTI
  	VIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAE
  	PPFGDSYIIIGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAW
  	DFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGMNS
  	RSTSLSVTLVLVGIVTLYLGVMVQADSGCVVSWKNKELKCGSGIFITDNV
  	HTWTEQYKFQPESPSKLASAIQKAHEEGICGIRSVTRLENLMWKQITPEL
  	NHILSENEVKLTIMTGDIKGIMQAGKRSLRPQPTELKYSWKTWGKAKMLS
  	TESHNQTFLIDGPETAECPNTNRAWNSLEVEDYGFGVFTTNIWLKLKEKQ
  	DVFCDSKLMSAAIKDNRAVHADMGYWIESALNDTWKIEKASFIEVKNCHW
  	PKSHTLWSNGVLESEMIIPKNLAGPVSQHNYRPGYHTQITGPWHLGKLEM
  	DFDFCDGTTVVVTEDCGNRGPSLRTTTASGKLITEWCCRSCTLPPLRYRG
  	EDGCWYGMEIRPLKEKEENLVNSLVTAGHGQVDNFSLGVLGMALFLEEML
  	RTRVGTKHAILLVAVSFVTLITGNMSFRDLGRVMVMVGATMTDDIGMGVT
  	YLALLAAFKVRPTFAAGLLLRKLTSKELMMTTIGIVLLSQSTIPETILEL
  	TDALALGMMVLKMVRNMEKYQLAVTIMAILCVPILQNAWKVSCTILAVVS
  	VSPLLLTSSQQKTDWIPLALTIKGLNPTAIFLTTLSRTSKKRSWPLNEAI
  	MAVGMVSILASSLLKNDIPMTGPLVAGGLLTVCYVLTGRSADLELERAAD
  	VKWEDQAEISGSSPILSITISEDGSMSIKNEEEEQTLTILIRTGLLVISG
  	LFPVSIPITAAAWYLWEVKKQRAGVLWDVPSPPPMGKAELEDGAYRIKQK
  	GILGYSQIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKKDLIS
  	YGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGLFKTNAGTIGAVSL
  	DFSPGTSGSPIIDKKGKVVGLYGNGVVTRSGAYVSAIAQTEKSIEDNPEI
  	EDDIFRKRRLTIMDLHPGAGKTKRYLPAIVREAIKRGLRTLILAPTRVVA
  	AEMEEALRGLPIRYQTPAIRAEHTGREIVDLMCHATFTMRLLSPVRVPNY
  	NLIIMDEAHFTDPASIAARGYISTRVEMGEAAGIFMTATPPGSRDPFPQS
  	NAPIIDEEREIPERSWNSGHEWVTDFKGKTVWFVPSIKAGNDIAACLRKN
  	GKKVIQLSRKTFDSEYVKTRTNDWDFVVTTDISEMGANFKAERVIDPRRC
  	MKPVILTDGEERVILAGPMPVTHSSAAQRRGRIGRNPKNENDQYIYMGEP
  	LENDEDCAHWKEAKMLLDNINTPEGIIPSMFEPEREKVDAIDGEYRLRGE
  	ARKTFVDLMRRGDLPVWLAYRVAAEGINYADRRWCFDGVKNNQILEENVE
  	VEIWTKEGERKKLKPRWLDARIYSDPLALKEFKEFAAGRKSLTLNLITEM
  	GRLPTFMTQKARDALDNLAVLHTAEAGGRAYNHALSELPETLETLLLLTL
  	LATVTGGIFLFLMSGRGIGKMTLGMCCIITASILLWYAQIQPHWIAASII
  	LEFFLIVLLIPEPEKQRTPQDNQLTYVVIAILTVVAATMANEMGFLEKTK
  	KDLGLGSIATQQPESNILDIDLRPASAWTLYAVATTFVTPMLRHSIENSS
  	VNVSLTAIANQATVLMGLGKGWPLSKMDIGVPLLAIGCYSQVNPITLTAA
  	LFLLVAHYAIIGPGLQAKATREAQKRAAAGIMKNPTVDGITVIDLDPIPY
  	DPKFEKQLGQVMLLVLCVTQVLMMRTTWALCEALTLATGPISTLWEGNPG
  	RFWNTTIAVSMANIFRGSYLAGAGLLFSIMKNTTNTRRGTGNIGETLGEK
  	WKSRLNALGKSEFQIYKKSGIQEVDRTLAKEGIKR
  	GETDHHAVSRGSAKLRWFVERNMVTPEGKVVDLGCGRGGWSYYCGGLKNV
  	REVKGLTKGGPGHEEPIPMSTYGWNLVRLQSGVDVFFIPPEKCDTLLCDI
  	GESSPNPTVEAGRTLRVLNLVENWLNNNTQFCIKVLNPYMPSVIEKMEAL
  	QRKYGGALVRNPLSRNSTHEMYWVSNASGNIVSSVNMISRMLINRFTMRY
  	KKATYEPDVDLGSGTRNIGIESEIPNLDIIGKRIEKIKQEHETSWHYDQD
  	HPYKTWAYHGSYETKQTGSASSMVNGVVRLLTKPWDVVPMVTQMAMTDTT
  	PFGQQRVFKEKVDTRTQEPKEGTKKLMKITAEWLWKELGKKKTPRMCTRE
  	EFTRKVRSNAALGAIFTDENKWKSAREAVEDSRFWELVDKERNLHLEGKC
  	ETCVYNMMGKREKKLGEFGKAKGSRAIWYMWLGARFLEFEALGFLNEDHW
  	FSRENSLSGVEGEGLHKLGYILRDVSKKEGGAMYADDTAGWDTRITLEDL
  	KNEEMVTNHMEGEHKKLAEAIFKLTYQNKVVRVQRPTPRGTVMDIISRRD
  	QRGSGQVGTYGLNTFTNMEAQLIRQMEGEGVFKSIQHLTITEEIAVQNWL
  	ARVGRERLSRMAISGDDCVVKPLDDRFASALTALNDMGKIRKDIQQWEPS
  	RGWNDWTQVPFCSHHFHELIMKDGRVLVVPCRNQDELIGRARISQGAGWS
  	LRETACLGKSYAQMWSLMYFHRRDLRLAANAICSAVPSHWVPTSRTTWSI
  	HAKHEWMTTEDMLTVWNRVWIQENPWMEDKTPVESWEEIPYLGKREDQWC
  	GSLIGLTSRATWAKNIQAAINQVRSLIGNEEYTDYMPSMKRFRREEEEAG
  	VLW
  DEN-2	agttgttagtctacgtggaccgacaaagacagattctttgagggagctaa	18
  (NC_001474.	gctcaacgtagttctaacagttttttaattagagagcagatctctgatga
  2)	ataaccaacggaaaaaggcgaaaaacacgcctttcaatatgctgaaacgc
  	gagagaaaccgcgtgtcgactgtgcaacagctgacaaagagattctcact
  	tggaatgctgcagggacgaggaccattaaaactgttcatggccctggtgg
  	cgttccttcgtttcctaacaatcccaccaacagcagggatattgaagaga
  	tggggaacaattaaaaaatcaaaagctattaatgttttgagagggttcag
  	gaaagagattggaaggatgctgaacatcttgaataggagacgcagatctg
  	caggcatgatcattatgctgattccaacagtgatggcgttccatttaacc
  	acacgtaacggagaaccacacatgatcgtcagcagacaagagaaagggaa
  	aagtcttctgtttaaaacagaggatggcgtgaacatgtgtaccctcatgg
  	ccatggaccttggtgaattgtgtgaagacacaatcacgtacaagtgtccc
  	cttctcaggcagaatgagccagaagacatagactgttggtgcaactctac
  	gtccacgtgggtaacttatgggacgtgtaccaccatgggagaacatagaa
  	gagaaaaaagatcagtggcactcgttccacatgtgggaatgggactggag
  	acacgaactgaaacatggatgtcatcagaaggggcctggaaacatgtcca
  	gagaattgaaacttggatcttgagacatccaggcttcaccatgatggcag
  	caatcctggcatacaccataggaacgacacatttccaaagagccctgatt
  	ttcatcttactgacagctgtcactccttcaatgacaatgcgttgcatagg
  	aatgtcaaatagagactttgtggaaggggtttcaggaggaagctgggttg
  	acatagtcttagaacatggaagctgtgtgacgacgatggcaaaaaacaaa
  	ccaacattggattttgaactgataaaaacagaagccaaacagcctgccac
  	cctaaggaagtactgtatagaggcaaagctaaccaacacaacaacagaat
  	ctcgctgcccaacacaaggggaacccagcctaaatgaagagcaggacaaa
  	aggttcgtctgcaaacactccatggtagacagaggatggggaaatggatg
  	tggactatttggaaagggaggcattgtgacctgtgctatgttcagatgca
  	aaaagaacatggaaggaaaagttgtgcaaccagaaaacttggaatacacc
  	attgtgataacacctcactcaggggaagagcatgcagtcggaaatgacac
  	aggaaaacatggcaaggaaatcaaaataacaccacagagttccatcacag
  	aagcagaattgacaggttatggcactgtcacaatggagtgctctccaaga
  	acgggcctcgacttcaatgagatggtgttgctgcagatggaaaataaagc
  	ttggctggtgcacaggcaatggttcctagacctgccgttaccatggttgc
  	ccggagcggacacacaagggtcaaattggatacagaaagagacattggtc
  	actttcaaaaatccccatgcgaagaaacaggatgttgttgttttaggatc
  	ccaagaaggggccatgcacacagcacttacaggggccacagaaatccaaa
  	tgtcatcaggaaacttactcttcacaggacatctcaagtgcaggctgaga
  	atggacaagctacagctcaaaggaatgtcatactctatgtgcacaggaaa
  	gtttaaagttgtgaaggaaatagcagaaacacaacatggaacaatagtta
  	tcagagtgcaatatgaaggggacggctctccatgcaagatcccttttgag
  	ataatggatttggaaaaaagacatgtcttaggtcgcctgattacagtcaa
  	cccaattgtgacagaaaaagatagcccagtcaacatagaagcagaacctc
  	cattcggagacagctacatcatcataggagtagagccgggacaactgaag
  	ctcaactggtttaagaaaggaagttctatcggccaaatgtttgagacaac
  	aatgaggggggcgaagagaatggccattttaggtgacacagcctgggatt
  	ttggatccttgggaggagtgtttacatctataggaaaggctctccaccaa
  	gtctttggagcaatctatggagctgccttcagtggggtttcatggactat
  	gaaaatcctcataggagtcattatcacatggataggaatgaattcacgca
  	gcacctcactgtctgtgacactagtattggtgggaattgtgacactgtat
  	ttgggagtcatggtgcaggccgatagtggttgcgttgtgagctggaaaaa
  	caaagaactgaaatgtggcagtgggattttcatcacagacaacgtgcaca
  	catggacagaacaatacaagttccaaccagaatccccttcaaaactagct
  	tcagctatccagaaagcccatgaagagggcatttgtggaatccgctcagt
  	aacaagactggagaatctgatgtggaaacaaataacaccagaattgaatc
  	acattctatcagaaaatgaggtgaagttaactattatgacaggagacatc
  	aaaggaatcatgcaggcaggaaaacgatctctgcggcctcagcccactga
  	gctgaagtattcatggaaaacatggggcaaagcaaaaatgctctctacag
  	agtctcataaccagacctttctcattgatggccccgaaacagcagaatgc
  	cccaacacaaatagagcttggaattcgttggaagttgaagactatggctt
  	tggagtattcaccaccaatatatggctaaaattgaaagaaaaacaggatg
  	tattctgcgactcaaaactcatgtcagcggccataaaagacaacagagcc
  	gtccatgccgatatgggttattggatagaaagtgcactcaatgacacatg
  	gaagatagagaaagcctctttcattgaagttaaaaactgccactggccaa
  	aatcacacaccctctggagcaatggagtgctagaaagtgagatgataatt
  	ccaaagaatctcgctggaccagtgtctcaacacaactatagaccaggcta
  	ccatacacaaataacaggaccatggcatctaggtaagcttgagatggact
  	ttgatttctgtgatggaacaacagtggtagtgactgaggactgcggaaat
  	agaggaccctctttgagaacaaccactgcctctggaaaactcataacaga
  	atggtgctgccgatcttgcacattaccaccgctaagatacagaggtgagg
  	atgggtgctggtacgggatggaaatcagaccattgaaggagaaagaagag
  	aatttggtcaactccttggtcacagctggacatgggcaggtcgacaactt
  	ttcactaggagtcttgggaatggcattgttcctggaggaaatgcttagga
  	cccgagtaggaacgaaacatgcaatactactagttgcagtttcttttgtg
  	acattgatcacagggaacatgtcctttagagacctgggaagagtgatggt
  	tatggtaggcgccactatgacggatgacataggtatgggcgtgacttatc
  	ttgccctactagcagccttcaaagtcagaccaacttttgcagctggacta
  	ctcttgagaaagctgacctccaaggaattgatgatgactactataggaat
  	tgtactcctctcccagagcaccataccagagaccattcttgagttgactg
  	atgcgttagccttaggcatgatggtcctcaaaatggtgagaaatatggaa
  	aagtatcaattggcagtgactatcatggctatcttgtgcgtcccaaacgc
  	agtgatattacaaaacgcatggaaagtgagttgcacaatattggcagtgg
  	tgtccgtttccccactgctcttaacatcctcacagcaaaaaacagattgg
  	ataccattagcattgacgatcaaaggtctcaatccaacagctatttttct
  	aacaaccctctcaagaaccagcaagaaaaggagctggccattaaatgagg
  	ctatcatggcagtcgggatggtgagcattttagccagttctctcctaaaa
  	aatgatattcccatgacaggaccattagtggctggagggctcctcactgt
  	gtgctacgtgctcactggacgatcggccgatttggaactggagagagcag
  	ccgatgtcaaatgggaagaccaggcagagatatcaggaagcagtccaatc
  	ctgtcaataacaatatcagaagatggtagcatgtcgataaaaaatgaaga
  	ggaagaacaaacactgaccatactcattagaacaggattgctggtgatct
  	caggactttttcctgtatcaataccaatcacggcagcagcatggtacctg
  	tgggaagtgaagaaacaacgggccggagtattgtgggatgttccttcacc
  	cccacccatgggaaaggctgaactggaagatggagcctatagaattaagc
  	aaaaagggattcttggatattcccagatcggagccggagtttacaaagaa
  	ggaacattccatacaatgtggcatgtcacacgtggcgctgttctaatgca
  	taaaggaaagaggattgaaccatcatgggcggacgtcaagaaagacctaa
  	tatcatatggaggaggctggaagttagaaggagaatggaaggaaggagaa
  	gaagtccaggtattggcactggagcctggaaaaaatccaagagccgtcca
  	aacgaaacctggtcttttcaaaaccaacgccggaacaataggtgctgtat
  	ctctggacttttctcctggaacgtcaggatctccaattatcgacaaaaaa
  	ggaaaagttgtgggtctttatggtaatggtgttgttacaaggagtggagc
  	atatgtgagtgctatagcccagactgaaaaaagcattgaagacaacccag
  	agatcgaagatgacattttccgaaagagaagactgaccatcatggacctc
  	cacccaggagcgggaaagacgaagagataccttccggccatagtcagaga
  	agctataaaacggggtttgagaacattaatcttggcccccactagagttg
  	tggcagctgaaatggaggaagcccttagaggacttccaataagataccag
  	accccagccatcagagctgagcacaccgggcgggagattgtggacctaat
  	gtgtcatgccacatttaccatgaggctgctatcaccagttagagtgccaa
  	actacaacctgattatcatggacgaagcccatttcacagacccagcaagt
  	atagcagctagaggatacatctcaactcgagtggagatgggtgaggcagc
  	tgggatttttatgacagccactcccccgggaagcagagacccatttcctc
  	agagcaatgcaccaatcatagatgaagaaagagaaatccctgaacgttcg
  	tggaattccggacatgaatgggtcacggattttaaagggaagactgtttg
  	gttcgttccaagtataaaagcaggaaatgatatagcagcttgcctgagga
  	aaaatggaaagaaagtgatacaactcagtaggaagacctttgattctgag
  	tatgtcaagactagaaccaatgattgggacttcgtggttacaactgacat
  	ttcagaaatgggtgccaatttcaaggctgagagggttatagaccccagac
  	gctgcatgaaaccagtcatactaacagatggtgaagagcgggtgattctg
  	gcaggacctatgccagtgacccactctagtgcagcacaaagaagagggag
  	aataggaagaaatccaaaaaatgagaatgaccagtacatatacatggggg
  	aacctctggaaaatgatgaagactgtgcacactggaaagaagctaaaatg
  	ctcctagataacatcaacacgccagaaggaatcattcctagcatgttcga
  	accagagcgtgaaaaggtggatgccattgatggcgaataccgcttgagag
  	gagaagcaaggaaaacctttgtagacttaatgagaagaggagacctacca
  	gtctggttggcctacagagtggcagctgaaggcatcaactacgcagacag
  	aaggtggtgttttgatggagtcaagaacaaccaaatcctagaagaaaacg
  	tggaagttgaaatctggacaaaagaaggggaaaggaagaaattgaaaccc
  	agatggttggatgctaggatctattctgacccactggcgctaaaagaatt
  	taaggaatttgcagccggaagaaagtctctgaccctgaacctaatcacag
  	aaatgggtaggctcccaaccttcatgactcagaaggcaagagacgcactg
  	gacaacttagcagtgctgcacacggctgaggcaggtggaagggcgtacaa
  	ccatgctctcagtgaactgccggagaccctggagacattgcttttactga
  	cacttctggctacagtcacgggagggatctttttattcttgatgagcgga
  	aggggcatagggaagatgaccctgggaatgtgctgcataatcacggctag
  	catcctcctatggtacgcacaaatacagccacactggatagcagcttcaa
  	taatactggagttttttctcatagttttgcttattccagaacctgaaaaa
  	cagagaacaccccaagacaaccaactgacctacgttgtcatagccatcct
  	cacagtggtggccgcaaccatggcaaacgagatgggtttcctagaaaaaa
  	cgaagaaagatctcggattgggaagcattgcaacccagcaacccgagagc
  	aacatcctggacatagatctacgtcctgcatcagcatggacgctgtatgc
  	cgtggccacaacatttgttacaccaatgttgagacatagcattgaaaatt
  	cctcagtgaatgtgtccctaacagctatagccaaccaagccacagtgtta
  	atgggtctcgggaaaggatggccattgtcaaagatggacatcggagttcc
  	ccttctcgccattggatgctactcacaagtcaaccccataactctcacag
  	cagctcttttcttattggtagcacattatgccatcatagggccaggactc
  	caagcaaaagcaaccagagaagctcagaaaagagcagcggcgggcatcat
  	gaaaaacccaactgtcgatggaataacagtgattgacctagatccaatac
  	cttatgatccaaagtttgaaaagcagttgggacaagtaatgctcctagtc
  	ctctgcgtgactcaagtattgatgatgaggactacatgggctctgtgtga
  	ggctttaaccttagctaccgggcccatctccacattgtgggaaggaaatc
  	cagggaggttttggaacactaccattgcggtgtcaatggctaacattttt
  	agagggagttacttggccggagctggacttctcttttctattatgaagaa
  	cacaaccaacacaagaaggggaactggcaacataggagagacgcttggag
  	agaaatggaaaagccgattgaacgcattgggaaaaagtgaattccagatc
  	tacaagaaaagtggaatccaggaagtggatagaaccttagcaaaagaagg
  	cattaaaagaggagaaacggaccatcacgctgtgtcgcgaggctcagcaa
  	aactgagatggttcgttgagagaaacatggtcacaccagaagggaaagta
  	gtggacctcggttgtggcagaggaggctggtcatactattgtggaggact
  	aaagaatgtaagagaagtcaaaggcctaacaaaaggaggaccaggacacg
  	aagaacccatccccatgtcaacatatgggtggaatctagtgcgtcttcaa
  	agtggagttgacgttttcttcatcccgccagaaaagtgtgacacattatt
  	gtgtgacataggggagtcatcaccaaatcccacagtggaagcaggacgaa
  	cactcagagtccttaacttagtagaaaattggttgaacaacaacactcaa
  	ttttgcataaaggttctcaacccatatatgccctcagtcatagaaaaaat
  	ggaagcactacaaaggaaatatggaggagccttagtgaggaatccactct
  	cacgaaactccacacatgagatgtactgggtatccaatgcttccgggaac
  	atagtgtcatcagtgaacatgatttcaaggatgttgatcaacagatttac
  	aatgagatacaagaaagccacttacgagccggatgttgacctcggaagcg
  	gaacccgtaacatcgggattgaaagtgagataccaaacctagatataatt
  	gggaaaagaatagaaaaaataaagcaagagcatgaaacatcatggcacta
  	tgaccaagaccacccatacaaaacgtgggcataccatggtagctatgaaa
  	caaaacagactggatcagcatcatccatggtcaacggagtggtcaggctg
  	ctgacaaaaccttgggacgtcgtccccatggtgacacagatggcaatgac
  	agacacgactccatttggacaacagcgcgtttttaaagagaaagtggaca
  	cgagaacccaagaaccgaaagaaggcacgaagaaactaatgaaaataaca
  	gcagagtggctttggaaagaattagggaagaaaaagacacccaggatgtg
  	caccagagaagaattcacaagaaaggtgagaagcaatgcagccttggggg
  	ccatattcactgatgagaacaagtggaagtcggcacgtgaggctgttgaa
  	gatagtaggttttgggagctggttgacaaggaaaggaatctccatcttga
  	aggaaagtgtgaaacatgtgtgtacaacatgatgggaaaaagagagaaga
  	agctaggggaattcggcaaggcaaaaggcagcagagccatatggtacatg
  	tggcttggagcacgcttcttagagtttgaagccctaggattcttaaatga
  	agatcactggttctccagagagaactccctgagtggagtggaaggagaag
  	ggctgcacaagctaggttacattctaagagacgtgagcaagaaagaggga
  	ggagcaatgtatgccgatgacaccgcaggatgggatacaagaatcacact
  	agaagacctaaaaaatgaagaaatggtaacaaaccacatggaaggagaac
  	acaagaaactagccgaggccattttcaaactaacgtaccaaaacaaggtg
  	gtgcgtgtgcaaagaccaacaccaagaggcacagtaatggacatcatatc
  	gagaagagaccaaagaggtagtggacaagttggcacctatggactcaata
  	ctttcaccaatatggaagcccaactaatcagacagatggagggagaagga
  	gtctttaaaagcattcagcacctaacaatcacagaagaaatcgctgtgca
  	aaactggttagcaagagtggggcgcgaaaggttatcaagaatggccatca
  	gtggagatgattgtgttgtgaaacctttagatgacaggttcgcaagcgct
  	ttaacagctctaaatgacatgggaaagattaggaaagacatacaacaatg
  	ggaaccttcaagaggatggaatgattggacacaagtgcccttctgttcac
  	accatttccatgagttaatcatgaaagacggtcgcgtactcgttgttcca
  	tgtagaaaccaagatgaactgattggcagagcccgaatctcccaaggagc
  	agggtggtctttgcgggagacggcctgtttggggaagtcttacgcccaaa
  	tgtggagcttgatgtacttccacagacgcgacctcaggctggcggcaaat
  	gctatttgctcggcagtaccatcacattgggttccaacaagtcgaacaac
  	ctggtccatacatgctaaacatgaatggatgacaacggaagacatgctga
  	cagtctggaacagggtgtggattcaagaaaacccatggatggaagacaaa
  	actccagtggaatcatgggaggaaatcccatacttggggaaaagagaaga
  	ccaatggtgcggctcattgattgggttaacaagcagggccacctgggcaa
  	agaacatccaagcagcaataaatcaagttagatcccttataggcaatgaa
  	gaatacacagattacatgccatccatgaaaagattcagaagagaagagga
  	agaagcaggagttctgtggtagaaagcaaaactaacatgaaacaaggcta
  	gaagtcaggtcggattaagccatagtacggaaaaaactatgctacctgtg
  	agccccgtccaaggacgttaaaagaagtcaggccatcataaatgccatag
  	cttgagtaaactatgcagcctgtagctccacctgagaaggtgtaaaaaat
  	ccgggaggccacaaaccatggaagctgtacgcatggcgtagtggactagc
  	ggttagaggagacccctcccttacaaatcgcagcaacaatgggggcccaa
  	ggcgagatgaagctgtagtctcgctggaaggactagaggttagaggagac
  	ccccccgaaacaaaaaacagcatattgacgctgggaaagaccagagatcc
  	tgctgtctcctcagcatcattccaggcacagaacgccagaaaatggaatg
  	gtgctgttgaatcaacaggttct
  DEN-3	MNNQRKKTGKPSINMLKRVRNRVSTGSQLAKRFSKGLLNGQGPMKLVMAF	19
  (NC_001475.	IAFLRFLAIPPTAGVLARWGTFKKSGAIKVLKGFKKEISNMLSIINQRKK
  2)	TSLCLMMILPAALAFHLTSRDGEPRMIVGKNERGKSLLFKTASGINMCTL
  	IAMDLGEMCDDTVTYKCPHITEVEPEDIDCWCNLTSTWVTYGTCNQAGEH
  	RRDKRSVALAPHVGMGLDTRTQTWMSAEGAWRQVEKVETWALRHPGFTIL
  	ALFLAHYIGTSLTQKVVIFILLMLVTPSMTMRCVGVGNRDFVEGLSGATW
  	VDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEGKITNITT
  	DSRCPTQGEAVLPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQ
  	CLEPIEGKVVQYENLKYTVIITVHTGDQHQVGNETQGVTAEITPQASTTE
  	AILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWAS
  	GATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQN
  	SGGTSIFAGHLKCRLKMDKLELKGMSYAMCTNTFVLKKEVSETQHGTILI
  	KVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPP
  	FGESNIVIGIGDNALKINWYKKGSSIGKMFEATERGARRMAILGDTAWDF
  	GSVGGVLNSLGKMVHQIFGSAYTALFSGVSWVMKIGIGVLLTWIGLNSKN
  	TSMSFSCIAIGIITLYLGAVVQADMGCVINWKGKELKCGSGIFVTNEVHT
  	WTEQYKFQADSPKRLATAIAGAWENGVCGIRSTTRMENLLWKQIANELNY
  	ILWENNIKLTVVVGDTLGVLEQGKRTLTPQPMELKYSWKTWGKAKIVTAE
  	TQNSSFIIDGPNTPECPSASRAWNVWEVEDYGFGVFTTNIWLKLREVYTQ
  	LCDHRLMSAAVKDERAVHADMGYWIESQKNGSWKLEKASLIEVKTCTWPK
  	SHTLWTNGVLESDMIIPKSLAGPISQHNYRPGYHTQTAGPWHLGKLELDF
  	NYCEGTTVVITESCGTRGPSLRTTTVSGKLIHEWCCRSCTLPPLRYMGED
  	GCWYGMEIRPISEKEENMVKSLVSAGSGKVDNFTMGVLCLAILFEEVLRG
  	KFGKKHMIAGVFFTFVLLLSGQITWRDMAHTLIMIGSNASDRMGMGVTYL
  	ALIATFKIQPFLALGFFLRKLTSRENLLLGVGLAMATTLQLPEDIEQMAN
  	GVALGLMALKLITQFETYQLWTALVSLTCSNTIFTLTVAWRTATLILAGV
  	SLLPVCQSSSMRKTDWLPMTVAAMGVPPLPLFIFSLKDTLKRRSWPLNEG
  	VMAVGLVSILASSLLRNDVPMAGPLVAGGLLIACYVITGTSADLTVEKAP
  	DVTWEEEAEQTGVSHNLMITVDDDGTMRIKDDETENILTVLLKTALLIVS
  	GIFPYSIPATLLVWHTWQKQTQRSGVLWDVPSPPETQKAELEEGVYRIKQ
  	QGIFGKTQVGVGVQKEGVFHTMWHVTRGAVLTHNGKRLEPNWASVKKDLI
  	SYGGGWRLSAQWQKGEEVQVIAVEPGKNPKNFQTTPGTFQTTTGEIGAIA
  	LDFKPGTSGSPIINREGKVVGLYGNGVVTKNGGYVSGIAQTNAEPDGPTP
  	ELEEEMFKKRNLTIMDLHPGSGKTRKYLPAIVREAIKRRLRTLILAPTRV
  	VAAEMEEALKGLPIRYQTTATKSEHTGREIVDLMCHATFTMRLLSPVRVP
  	NYNLIIMDEAHFTDPASIAARGYISTRVGMGEAAAIFMTATPPGTADAFP
  	QSNAPIQDEERDIPERSWNSGNEWITDFAGKTVWFVPSIKAGNDIANCLR
  	KNGKKVIQLSRKTFDTEYQKTKLNDWDFVVTTDISEMGANFKADRVIDPR
  	RCLKPVILTDGPERVILAGPMPVTAASAAQRRGRVGRNPQKENDQYIFTG
  	QPLNNDEDHAHWTEAKMLLDNINTPEGIIPALFEPEREKSAAIDGEYRLK
  	GESRKTFVELMRRGDLPVWLAHKVASEGIKYTDRKWCFDGQRNNQILEEN
  	MDVEIWTKEGEKKKLRPRWLDARTYSDPLALKEFKDFAAGRKSIALDLVT
  	EIGRVPSHLAHRTRNALDNLVMLHTSEDGGRAYRHAVEELPETMETLLLL
  	GLMILLTGGAMLFLISGKGIGKTSIGLICVIASSGMLWMAEVPLQWIASA
  	IVLEFFMMVLLIPEPEKQRTPQDNQLAYVVIGILTLAATIAANEMGLLET
  	TKRDLGMSKEPGVVSPTSYLDVDLHPASAWTLYAVATTVITPMLRHTIEN
  	STANVSLAAIANQAVVLMGLDKGWPISKMDLGVPLLALGCYSQVNPLTLT
  	AAVLLLITHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGIMTIDLDSV
  	IFDSKFEKQLGQVMLLVLCAVQLLLMRTSWALCEALTLATGPITTLWEGS
  	PGKFWNTTIAVSMANIFRGSYLAGAGLAFSIMKSVGTGKRGTGSQGETLG
  	EKWKKKLNQLSRKEFDLYKKSGITEVDRTEAKEGLKRGETTHHAVSRGSA
  	KLQWFVERNMVVPEGRVIDLGCGRGGWSYYCAGLKKVTEVRGYTKGGPGH
  	EEPVPMSTYGWNIVKLMSGKDVFYLPPEKCDTLLCDIGESSPSPTVEESR
  	TIRVLKMVEPWLKNNQFCIKVLNPYMPTVIEHLERLQRKHGGMLVRNPLS
  	RNSTHEMYWISNGTGNIVSSVNMVSRLLLNRFTMTHRRPTIEKDVDLGAG
  	TRHVNAEPETPNMDVIGERIKRIKEEHNSTWHYDDENPYKTWAYHGSYEV
  	KATGSASSMINGVVKLLTKPWDVVPMVTQMAMTDTTPFGQQRVFKEKVDT
  	RTPRPMPGTRKAMEITAEWLWRTLGRNKRPRLCTREEFTKKVRTNAAMGA
  	VFTEENQWDSAKAAVEDEEFWKLVDRERELHKLGKCGSCVYNMMGKREKK
  	LGEFGKAKGSRAIWYMWLGARYLEFEALGFLNEDHWFSRENSYSGVEGEG
  	LH
  	KLGYILRDISKIPGGAMYADDTAGWDTRITEDDLHNEEKIIQQMDPEHRQ
  	LANAIFKLTYQNKVVKVQRPTPTGTVMDIISRKDQRGSGQLGTYGLNTFT
  	NMEAQLVRQMEGEGVLTKADLENPHLLEKKITQWLETKGVERLKRMAISG
  	DDCVVKPIDDRFANALLALNDMGKVRKDIPQWQPSKGWHDWQQVPFCSHH
  	FHELIMKDGRKLVVPCRPQDELIGRARISQGAGWSLRETACLGKAYAQMW
  	SLMYFHRRDLRLASNAICSAVPVHWVPTSRTTWSIHAHHQWMTTEDMLTV
  	WNRVWIEENPWMEDKTPVTTWENVPYLGKREDQWCGSLIGLTSRATWAQN
  	IPTAIQQVRSLIGNEEFLDYMPSMKRFRKEEESEGAIW
  DEN-3	agttgttagtctacgtggaccgacaagaacagtttcgactcggaagcttg	20
  (NC_001475.	cttaacgtagtgctgacagttttttattagagagcagatctctgatgaac
  2)	aaccaacggaagaagacgggaaaaccgtctatcaatatgctgaaacgcgt
  	gagaaaccgtgtgtcaactggatcacagttggcgaagagattctcaaaag
  	gactgctgaacggccagggaccaatgaaattggttatggcgttcatagct
  	ttcctcagatttctagccattccaccaacagcaggagtcttggctagatg
  	gggaaccttcaagaagtcgggggccattaaggtcctgaaaggcttcaaga
  	aggagatctcaaacatgctgagcataatcaaccaacggaaaaagacatcg
  	ctctgtctcatgatgatattgccagcagcacttgctttccacttgacttc
  	acgagatggagagccgcgcatgattgtggggaagaatgaaagaggtaaat
  	ccctactttttaagacagcctctggaatcaacatgtgcacactcatagcc
  	atggatttgggagagatgtgtgatgacacggtcacttacaaatgccccca
  	cattaccgaagtggaacctgaagacattgactgctggtgcaaccttacat
  	caacatgggtgacttatggaacgtgcaatcaagctggagagcatagacgc
  	gacaagagatcagtggcgttagctccccatgtcggcatgggactggacac
  	acgcacccaaacctggatgtcggctgaaggagcttggagacaagtcgaga
  	aggtagagacatgggcccttaggcacccagggttcaccatactagcccta
  	tttctcgcccattacataggcacttccctgacccagaaggtggttatttt
  	catattattaatgctggtcaccccatccatgacaatgagatgtgtgggag
  	taggaaacagagattttgtggaagggctatcaggagctacgtgggttgac
  	gtggtgctcgagcacggggggtgtgtgactaccatggctaagaacaagcc
  	cacgctggatatagagcttcagaagaccgaggccacccaactggcgaccc
  	taaggaagctatgcattgaggggaaaattaccaacataacaactgactca
  	agatgtcctacccaaggggaagcggttttgcctgaggagcaggaccagaa
  	ctacgtgtgtaagcatacatacgtagacagaggttgggggaacggttgtg
  	gtttgtttggcaaaggaagcttggtaacatgtgcgaaatttcaatgcctg
  	gaaccaatagagggaaaagtggtgcaatatgagaacctcaaatacaccgt
  	catcattacagtgcacacaggagaccaacaccaggtgggaaatgaaacgc
  	aaggagtcacggctgagataacacctcaggcatcaaccactgaagccatc
  	ttgcctgaatatggaacccttgggctagaatgctcaccacggacaggttt
  	ggatttcaatgaaatgatcttactaacaatgaagaacaaagcatggatgg
  	tacatagacaatggttctttgacctacctctaccatgggcatcaggagct
  	acaacagaaacaccaacctggaacaggaaggagcttcttgtgacattcaa
  	aaacgcacatgcgaaaaaacaagaagtagttgtccttggatcgcaagagg
  	gagcaatgcataccgcactgacaggagctacagaaatccaaaactcagga
  	ggcacaagcattttcgcggggcacttaaaatgtagacttaagatggacaa
  	attggaactcaaggggatgagctatgcaatgtgcacgaatacctttgtgt
  	tgaagaaagaagtctcagaaacgcagcacgggacaatactcattaaggtt
  	gagtacaaaggggaagatgcaccttgcaagattcccttttccacagagga
  	tggacaagggaaagctcataatggcagactgatcacagccaaccctgtgg
  	tgactaagaaggaggagcctgtcaatattgaggctgaacctccttttggg
  	gaaagcaatatagtaattggaattggagacaacgccttgaaaatcaactg
  	gtacaagaaggggagctcgattgggaagatgttcgaggccactgaaaggg
  	gtgcaaggcgcatggccatcttgggagacacagcttgggactttggatca
  	gtgggtggtgttctgaactcattaggcaaaatggtgcaccaaatatttgg
  	aagtgcttatacagccctgttcagtggagtctcttgggtgatgaaaattg
  	gaataggtgtcctcttgacttggatagggttgaattcaaaaaacacatcc
  	atgtcattttcatgcattgcgataggaatcattacactctatctgggagc
  	tgtggtacaagctgacatggggtgtgtcataaactggaagggcaaagaac
  	tcaaatgtggaagcggaattttcgtcaccaatgaggtccatacctggaca
  	gagcaatacaaattccaagcagactccccaaaaagattggcaacagccat
  	tgcaggcgcctgggagaatggagtgtgtggaattaggtcaacaaccagaa
  	tggagaatctcttgtggaagcaaatagccaatgaactgaactacatatta
  	tgggaaaacaatatcaaattaacggtagttgtgggcgatacacttggggt
  	cttagagcaagggaaaagaacactaacaccacaacccatggagctaaaat
  	actcatggaaaacgtggggaaaggcaaaaatagtgacagctgaaacacaa
  	aattcctctttcataatagacgggccaaacacaccggagtgtccaagtgc
  	ctcaagagcatggaatgtgtgggaggtggaagattacgggttcggagtct
  	tcacaaccaacatatggctgaaactccgagaggtctacacccaactatgt
  	gaccataggctaatgtcggcagctgtcaaggatgagagggccgtgcatgc
  	cgacatgggctactggatagaaagccaaaagaatggaagttggaagctag
  	aaaaagcatccctcatagaggtaaaaacctgcacatggccaaaatcacac
  	actctctggactaatggtgtgctagagagtgacatgatcatcccaaagag
  	tctagctggtcctatctcacaacacaactacaggcccgggtaccacaccc
  	aaacggcaggaccctggcacttaggaaaattggagctggacttcaactac
  	tgtgaaggaacaacagttgtcatcacagaaagctgtgggacaagaggccc
  	atcattgagaacaacaacagtgtcagggaagttgatacacgaatggtgtt
  	gccgctcgtgcacacttccccccctgcgatacatgggagaagacggctgc
  	tggtatggcatggaaatcagacccatcagtgagaaagaagagaacatggt
  	aaagtctttagtctcagcgggaagtggaaaggtggacaacttcacaatgg
  	gtgtcttgtgtttggcaatcctctttgaagaggtgttgagaggaaaattt
  	gggaagaaacacatgattgcaggggttttctttacgtttgtgctccttct
  	ctcagggcaaataacatggagagacatggcgcacacactaataatgatcg
  	ggtccaacgcctctgacaggatgggaatgggcgtcacctacctagctcta
  	attgcaacatttaaaatccagccattcttggctttgggatttttcctaag
  	aaagctgacatctagagaaaatttattgttaggagttgggttggccatgg
  	caacaacgttacaactgccagaggacattgaacaaatggcaaatggagtc
  	gctctggggctcatggctcttaaactgataacacaatttgaaacatacca
  	attgtggacggcattagtctccttaacgtgttcaaacacaatttttacgt
  	tgactgttgcctggagaacagccactctgattttggccggagtttcgctt
  	ttaccagtgtgccagtcttcaagcatgaggaaaacagattggctcccaat
  	gacagtggcagctatgggagttccaccccttccactttttatttttagct
  	tgaaagacacactcaaaaggagaagctggccactgaatgaaggggtgatg
  	gctgttgggcttgtgagcattctggccagttctctccttagaaatgatgt
  	gcccatggctggaccattagtggccgggggcttgctgatagcgtgctacg
  	tcataactggcacgtcagcggacctcactgtagaaaaagccccagatgta
  	acatgggaggaagaggctgagcagacaggagtgtcccacaacttaatgat
  	cacagttgatgatgatggaacaatgagaataaaagatgatgagactgaga
  	acatcctaacagtgcttttaaaaacagcattactaatagtatcaggcatt
  	tttccatactccatacccgcaacattgttggtctggcacacttggcaaaa
  	acaaacccaaagatccggcgttttatgggacgtacccagccccccagaga
  	cacagaaagcagaactggaagaaggggtttataggatcaaacagcaagga
  	atttttgggaaaacccaagtaggggttggagtacagaaagaaggagtctt
  	ccacaccatgtggcacgtcacaagaggggcagtgttgacacataatggga
  	aaagactggaaccaaactgggctagtgtgaaaaaagatctgatttcatat
  	ggaggaggatggagactgagcgcacaatggcaaaagggggaggaggtgca
  	ggttattgccgtagagccagggaagaacccaaagaactttcaaaccacgc
  	caggcactttccagactactacaggggaaataggagcaattgcactggat
  	ttcaagcctggaacttcaggatctcctatcataaatagagagggaaaggt
  	agtgggactgtatggcaatggagtggttacaaagaatggtggctatgtca
  	gcggaatagcgcaaacaaatgcagaaccagatggaccgacaccagagttg
  	gaagaagagatgttcaaaaagcgaaacctgaccataatggatcttcatcc
  	tgggtcaggaaagacacggaaataccttccagctattgtcagagaggcaa
  	tcaagagacgtttaagaaccttaattttggcaccgacaagggtggttgca
  	gctgagatggaagaagcattgaaagggctcccaataaggtaccaaacaac
  	agcaacaaaatctgaacacacaggaagagagattgttgatctaatgtgcc
  	acgcaacgttcacaatgcgtttgctgtcaccagttagggttccaaattac
  	aacttgataataatggatgaggcccatttcacagacccagccagtatagc
  	ggctagagggtacatatcaactcgtgttggaatgggagaggcagccgcaa
  	tcttcatgacagcaacaccccctggaacagctgatgcctttcctcagagc
  	aacgctccaattcaagatgaagaaagggacataccagaacgctcatggaa
  	ttcaggcaatgaatggattaccgacttcgctgggaaaacggtgtggtttg
  	tccctagcattaaagccggaaatgacatagcaaactgcttgcgaaaaaac
  	gggaaaaaagtcattcaacttagtaggaagacttttgacacagaatatca
  	gaagactaaactgaatgattgggactttgtggtgacaactgacatttcag
  	aaatgggggccaatttcaaagcagatagagtgatcgacccaagaagatgt
  	ctcaaaccagtgatcttgacagatggaccagagcgggtgatcctggccgg
  	accaatgccagtcaccgcggcgagtgctgcgcaaaggagagggagagttg
  	gcaggaacccacaaaaagagaatgaccagtacatattcacgggccagcct
  	ctcaacaatgatgaagaccatgctcactggacagaagcaaaaatgctgct
  	ggacaacatcaacacaccagaagggattataccagctctctttgaaccag
  	aaagggagaagtcagccgccatagacggtgagtatcgcctgaagggtgag
  	tccaggaagactttcgtggaactcatgaggaggggtgaccttccagtttg
  	gttagcccataaagtagcatcagaaggaatcaaatacacagatagaaaat
  	ggtgctttgatgggcaacgcaataatcaaattttagaggagaacatggat
  	gtggaaatttggacaaaggaaggagaaaagaaaaaattgagacctaggtg
  	gcttgatgcccgcacttattcagatccattggcactcaaggaattcaagg
  	actttgcggctggcagaaagtcaatcgcccttgatcttgtgacagaaata
  	ggaagagtgccttcacatctagcccacagaacaagaaacgctctggacaa
  	tctggtgatgctgcatacgtcagaagatggcggtagggcttacaggcatg
  	cggtggaggaactaccagaaacaatggaaacactcctactcttgggacta
  	atgatcttgttgacaggtggagcaatgcttttcttgatatcaggtaaagg
  	gattggaaagacttcaataggactcatttgtgtaatcgcttccagcggca
  	tgttgtggatggccgaagttccactccaatggatcgcgtcggctatagtc
  	ctggagttttttatgatggtgttgctcataccagaaccagaaaagcagag
  	aaccccccaagacaaccaactcgcatatgtcgtgataggcatacttacat
  	tggctgcaacaatagcagccaatgaaatgggactgctggaaaccacaaag
  	agagacttaggaatgtctaaggagccaggtgttgtttctccaaccagcta
  	tttggatgtggacttgcacccagcatcagcctggacattgtacgccgtgg
  	ccactacagtaataacaccaatgttaagacataccatagagaattctaca
  	gcaaatgtgtccctggcagctatagccaaccaggcagtggtcctgatggg
  	tttggacaaaggatggccaatatcaaaaatggacttaggcgtgccactac
  	tggcactgggttgctattcacaagtgaacccactgactctaactgcggca
  	gtacttttgctaatcacacattatgctatcataggtccaggattgcaagc
  	aaaagccacccgtgaagctcagaaaaggacagctgctggaataatgaaga
  	atccaacagtggatgggataatgacaatagacctagattctgtaatattt
  	gattcaaaatttgaaaaacaactgggacaggttatgctcctggttttgtg
  	cgcagtccaactcttgctaatgagaacatcatgggccttgtgtgaagctt
  	taactctagctacaggaccaataacaacactctgggaaggatcacctggt
  	aagttctggaacaccacgatagctgtttccatggcgaacatttttagagg
  	gagctatttagcaggagctgggcttgctttttctattatgaaatcagttg
  	gaacaggaaaaagaggaacaggctcacaaggtgaaactttaggagaaaaa
  	tggaaaaagaaattaaatcaattatcccggaaagagtttgacctttacaa
  	gaaatctggaatcactgaagtggatagaacagaagccaaagaagggttga
  	aaagaggagagacaacacatcatgccgtgtcccgaggtagcgcaaaactt
  	caatggtttgtggaaagaaacatggtcgttcccgaaggaagagtcataga
  	cttgggctgtggaagaggaggctggtcatattactgtgcaggactgaaaa
  	aagtcacagaagtgcgaggatacacaaaaggcggtccaggacacgaagaa
  	ccagtacctatgtctacatatggatggaacatagttaagttaatgagcgg
  	aaaggatgtgttctatctcccacctgaaaagtgtgataccctgttgtgtg
  	acattggagaatcttcaccaagcccaacagtggaagagagcagaactata
  	agagttttgaagatggttgaaccatggctaaaaaacaaccagttttgcat
  	taaagttttgaacccttacatgccaactgtgattgagcacctagaaagac
  	tacaaaggaaacatggaggaatgcttgtgagaaatccactttcacgaaac
  	tccacgcacgaaatgtactggatatctaatggcacaggtaacattgtctc
  	ttcagtcaacatggtgtctagattgctactgaacaggttcacgatgacac
  	acaggagacccaccatagagaaagatgtggatttaggagcaggaactcga
  	catgttaatgcggaaccagaaacacccaacatggatgtcattggggaaag
  	aataaaaaggatcaaggaggagcataattcaacatggcactatgatgacg
  	aaaacccctacaaaacgtgggcttaccatggatcctatgaagtcaaagcc
  	acaggctcagcctcctccatgataaatggagtcgtgaaactcctcaccaa
  	accatgggatgtggtgcccatggtgacacagatggcaatgacagacacaa
  	ctccatttggccagcagagagtctttaaagagaaagtggacaccaggacg
  	cccaggcccatgccagggacaagaaaggctatggagatcacagcggagtg
  	gctctggagaaccctgggaaggaacaaaagacccagattatgcacaaggg
  	aagagtttacaaaaaaggtcagaactaacgcagccatgggcgccgttttc
  	acagaggagaaccaatgggacagtgcgaaagctgctgttgaggatgaaga
  	attttggaaacttgtggacagagaacgtgaactccacaaattgggcaaat
  	gtggaagctgcgtttataacatgatgggcaagagagagaaaaaacttgga
  	gagtttggcaaagcaaaaggcagtagagctatatggtacatgtggttggg
  	agccaggtaccttgagttcgaagcccttggattcttaaatgaagaccact
  	ggttctcgcgtgaaaactcttacagtggagtagaaggagaaggactgcac
  	aagctaggctacatattaagggacatttccaagatacccggaggagccat
  	gtatgctgatgacacagctggttgggacacaagaataacagaagatgacc
  	tgcacaatgaggaaaagatcatacagcaaatggaccctgaacacaggcag
  	ttagcgaacgctatattcaagctcacataccaaaacaaagtggtcaaagt
  	tcaacgaccgactccaacgggcacggtaatggatattatatctaggaaag
  	accaaaggggcagtggacaactgggaacttatggcctgaatacattcacc
  	aacatggaagcccagttagtcagacaaatggaaggagaaggtgtgctgac
  	aaaggcagacctcgagaaccctcatctgctagagaagaaaatcacacaat
  	ggttggaaaccaaaggagtggagaggttaaaaagaatggccattagcggg
  	gatgattgcgtggtgaaaccaatcgatgacaggttcgctaatgccctgct
  	tgctttgaacgatatgggaaaggttcggaaagacatacctcaatggcagc
  	catcaaagggatggcatgattggcaacaggttcctttctgctcccaccac
  	tttcatgaattgatcatgaaagatggaagaaagttggtggttccctgcag
  	accccaggacgaactaataggaagagcaagaatctctcaaggagcgggat
  	ggagccttagagaaactgcatgtctggggaaagcctacgcccaaatgtgg
  	agtctcatgtattttcacagaagagatctcagattagcatccaacgccat
  	atgttcagcagtaccagtccactgggttcccacaagtagaacgacatggt
  	ctattcatgctcaccatcagtggatgactacagaagacatgcttactgtt
  	tggaacagggtgtggatagaggaaaatccatggatggaagacaaaactcc
  	agttacaacttgggaaaatgttccatatctaggaaagagagaagaccaat
  	ggtgtggatcacttattggtctcacttccagagcaacctgggcccagaac
  	atacccacagcaattcaacaggtgagaagccttataggcaatgaagagtt
  	cctggactacatgccttcaatgaagagattcaggaaggaagaggagtcgg
  	agggagccatttggtaaacgtaggaagtggaaaagaggctaactgtcagg
  	ccaccttaagccacagtacggaagaagctgtgctgcctgtgagccccgtc
  	caaggacgttaaaagaagaagtcaggccccaaagccacggtttgagcaaa
  	ccgtgctgcctgtagctccgtcgtggggacgtaaaacctgggaggctgca
  	aactgtggaagctgtacgcacggtgtagcagactagcggttagaggagac
  	ccctcccatgacacaacgcagcagcggggcccgagcactgagggaagctg
  	tacctccttgcaaaggactagaggttagaggagaccccccgcaaataaaa
  	acagcatattgacgctgggagagaccagagatcctgctgtctcctcagca
  	tcattccaggcacagaacgccagaaaatggaatggtgctgttgaatcaac
  	aggttct
  DEN-4	MNQRKKVVRPPFNMLKRERNRVSTPQGLVKRFSTGLFSGKGPLRMVLAFI	21
  (NC_002640.	TFLRVLSIPPTAGILKRWGQLKKNKAIKILIGFRKEIGRMLNILNGRKRS
  1)	TITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLI
  	AMDLGEMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGERR
  	REKRSVALTPHSGMGLETRAETWMSSEGAWKHAQRVESWILRNPGFALLA
  	GFMAYMIGQTGIQRTVFFVLMMLVAPSYGMRCVGVGNRDFVEGVSGGAWV
  	DLVLEHGGCVTTMAQGKPTLDFELTKTTAKEVALLRTYCIEASISNITTA
  	TRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAKFSC
  	SGKITGNLVQIENLEYTVVVTVHNGDTHAVGNDTSNHGVTAMITPRSPSV
  	EVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWT
  	AGADTSEVHWNYKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVD
  	SGDGNHMFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTV
  	VKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEP
  	PFGDSYIVIGVGNSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAWD
  	FGSVGGLFTSLGKAVHQVFGSVYTTMFGGVSWMIRILIGFLVLWIGTNSR
  	NTSMAMTCIAVGGITLFLGFTVQADMGCVASWSGKELKCGSGIFVVDNVH
  	TWTEQYKFQPESPARLASAILNAHKDGVCGIRSTTRLENVMWKQITNELN
  	YVLWEGGHDLTVVAGDVKGVLTKGKRALTPPVSDLKYSWKTWGKAKIFTP
  	EARNSTFLIDGPDTSECPNERRAWNSLEVEDYGFGMFTTNIWMKFREGSS
  	EVCDHRLMSAAIKDQKAVHADMGYWIESSKNQTWQIEKASLIEVKTCLWP
  	KTHTLWSNGVLESQMLIPKSYAGPFSQHNYRQGYATQTVGPWHLGKLEID
  	FGECPGTTVTIQEDCDHRGPSLRTTTASGKLVTQWCCRSCTMPPLRFLGE
  	DGCWYGMEIRPLSEKEENMVKSQVTAGQGTSETFSMGLLCLTLFVEECLR
  	RRVTRKHMILVVVITLCAIILGGLTWMDLLRALIMLGDTMSGRIGGQIHL
  	AIMAVFKMSPGYVLGVFLRKLTSRETALMVIGMAMTTVLSIPHDLMELID
  	GISLGLILLKIVTQFDNTQVGTLALSLTFIRSTMPLVMAWRTIMAVLFVV
  	TLIPLCRTSCLQKQSHWVEITALILGAQALPVYLMTLMKGASRRSWPLNE
  	GIMAVGLVSLLGSALLKNDVPLAGPMVAGGLLLAAYVMSGSSADLSLEKA
  	ANVQWDEMADITGSSPIVEVKQDEDGSFSIRDVEETNMITLLVKLALITV
  	SGLYPLAIPVTMTLWYMWQVKTQRSGALWDVPSPAATKKAALSEGVYRIM
  	QRGLFGKTQVGVGIHMEGVFHTMWHVTRGSVICHETGRLEPSWADVRNDM
  	ISYGGGWRLGDKWDKEEDVQVLAIEPGKNPKHVQTKPGLFKTLTGEIGAV
  	TLDFKPGTSGSPIINRKGKVIGLYGNGVVTKSGDYVSAITQAERIGEPDY
  	EVDEDIFRKKRLTIMDLHPGAGKTKRILPSIVREALKRRLRTL1LAPTRV
  	VAAEMEEALRGLPIRYQTPAVKSEHTGREIVDLMCHATFTTRLLSSTRVP
  	NYNLIVMDEAHFTDPSSVAARGYISTRVEMGEAAAIFMTATPPGATDPFP
  	QSNSPIEDIEREIPERSWNTGFDWITDYQGKTVWFVPSIKAGNDIANCLR
  	KSGKKVIQLSRKTFDTEYPKTKLTDWDFVVTTDISEMGANFRAGRVIDPR
  	RCLKPVILPDGPERVILAGPIPVTPASAAQRRGRIGRNPAQEDDQYVFSG
  	DPLKNDEDHAHWTEAKMLLDNIYTPEGIIPTLFGPEREKTQAIDGEFRLR
  	GEQRKTFVELMRRGDLPVWLSYKVASAGISYEDREWCFTGERNNQILEEN
  	MEVEIWTREGEKKKLRPRWLDARVYADPMALKDFKEFASGRKSITLDILT
  	EIASLPTYLSSRAKLALDNIVMLHTTERGGRAYQHALNELPESLETLMLV
  	ALLGAMTAGIFLFFMQGKGIGKLSMGLITIAVASGLLWVAEIQPQWIAAS
  	IILEFFLMVLLIPEPEKQRTPQDNQLIYVILTILTIIGLIAANEMGLIEK
  	TKTDFGFYQVKTETTILDVDLRPASAWTLYAVATTILTPMLRHTIENTSA
  	NLSLAAIANQAAVLMGLGKGWPLHRMDLGVPLLAMGCYSQVNPTTLTASL
  	VMLLVHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGITVIDLEPISYD
  	PKFEKQLGQVMLLVLCAGQLLLMRTTWAFCEVLTLATGPILTLWEGNPGR
  	FWNTTIAVSTANIFRGSYLAGAGLAFSLIKNAQTPRRGTGTTGETLGEKW
  	KRQLNSLDRKEFEEYKRSGILEVDRTEAKSALKDGSKIKHAVSRGSSKIR
  	WIVERGMVKPKGKVVDLGCGRGGWSYYMATLKNVTEVKGYTKGGPGHEEP
  	IPMATYGWNLVKLHSGVDVFYKPTEQVDTLLCDIGESSSNPTIEEGRTLR
  	VLKMVEPWLSSKPEFCIKVLNPYMPTVIEELEKLQRKHGGNLVRCPLSRN
  	STHEMYWVSGASGNIVSSVNTTSKMLLNRFTTRHRKPTYEKDVDLGAGTR
  	SVSTETEKPDMTIIGRRLQRLQEEHKETWHYDQENPYRTWAYHGSYEAPS
  	TGSASSMVNGVVKLLTKPWDVIPMVTQLAMTDTTPFGQQRVFKEKVDTRT
  	PQPKPGTRMVMTTTANWLWALLGKKKNPRLCTREEFISKVRSNAAIGAVF
  	QEEQGWTSASEAVNDSRFWELVDKERALHQEGKCESCVYNMMGKREKKLG
  	EFGRAKGSRAIWYMWLGARFLEFEALGFLNEDHWFGRENSWSGVEGEGLH
  	RLGYILEEIDKKDGDLMYADDTAGWDTRITEDDLQNEELITEQMAPHHKI
  	LAKAIFKLTYQNKVVKVLRPTPRGAVMDIISRKDQRGSGQVGTYGLNTFT
  	NMEVQLIRQMEAEGVITQDDMQNPKGLKERVEKWLKECGVDRLKRMAISG
  	DDCVVKPLDERFGTSLLFLNDMGKVRKDIPQWEPSKGWKNWQEVPFCSHH
  	FHKIFMKDGRSLVVPCRNQDELIGRARISQGAGW
  	SLRETACLGKAYAQMWSLMYFHRRDLRLASMAICSAVPTEWFPTSRTTWS
  	IHAHHQWMTTEDMLKVWNRVWIEDNPNMTDKTPVHSWEDIPYLGKREDLW
  	CGSLIGLSSRATWAKNIHTAITQVRNLIGKEEYVDYMPVMKRYSAPSESE
  	GVL
  DEN-4	agttgttagtctgtgtggaccgacaaggacagttccaaatcggaagcttg	22
  (NC_002640.	cttaacacagttctaacagtttgtttgaatagagagcagatctctggaaa
  1)	aatgaaccaacgaaaaaaggtggttagaccacctttcaatatgctgaaac
  	gcgagagaaaccgcgtatcaacccctcaagggttggtgaagagattctca
  	accggacttttttctgggaaaggacccttacggatggtgctagcattcat
  	cacgtttttgcgagtcctttccatcccaccaacagcagggattctgaaga
  	gatggggacagttgaagaaaaataaggccatcaagatactgattggattc
  	aggaaggagataggccgcatgctgaacatcttgaacgggagaaaaaggtc
  	aacgataacattgctgtgcttgattcccaccgtaatggcgttttccctca
  	gcacaagagatggcgaacccctcatgatagtggcaaaacatgaaaggggg
  	agacctctcttgtttaagacaacagaggggatcaacaaatgcactctcat
  	tgccatggacttgggtgaaatgtgtgaggacactgtcacgtataaatgcc
  	ccctactggtcaataccgaacctgaagacattgattgctggtgcaacctc
  	acgtctacctgggtcatgtatgggacatgcacccagagcggagaacggag
  	acgagagaagcgctcagtagctttaacaccacattcaggaatgggattgg
  	aaacaagagctgagacatggatgtcatcggaaggggcttggaagcatgct
  	cagagagtagagagctggatactcagaaacccaggattcgcgctcttggc
  	aggatttatggcttatatgattgggcaaacaggaatccagcgaactgtct
  	tctttgtcctaatgatgctggtcgccccatcctacggaatgcgatgcgta
  	ggagtaggaaacagagactttgtggaaggagtctcaggtggagcatgggt
  	cgacctggtgctagaacatggaggatgcgtcacaaccatggcccagggaa
  	aaccaaccttggattttgaactgactaagacaacagccaaggaagtggct
  	ctgttaagaacctattgcattgaagcctcaatatcaaacataactacggc
  	aacaagatgtccaacgcaaggagagccttatctgaaagaggaacaggacc
  	aacagtacatttgccggagagatgtggtagacagagggtggggcaatggc
  	tgtggcttgtttggaaaaggaggagttgtgacatgtgcgaagttttcatg
  	ttcggggaagataacaggcaatttggtccaaattgagaaccttgaataca
  	cagtggttgtaacagtccacaatggagacacccatgcagtaggaaatgac
  	acatccaatcatggagttacagccatgataactcccaggtcaccatcggt
  	ggaagtcaaattgccggactatggagaactaacactcgattgtgaaccca
  	ggtctggaattgactttaatgagatgattctgatgaaaatgaaaaagaaa
  	acatggctcgtgcataagcaatggtttttggatctgcctcttccatggac
  	agcaggagcagacacatcagaggttcactggaattacaaagagagaatgg
  	tgacatttaaggttcctcatgccaagagacaggatgtgacagtgctggga
  	tctcaggaaggagccatgcattctgccctcgctggagccacagaagtgga
  	ctccggtgatggaaatcacatgtttgcaggacatcttaagtgcaaagtcc
  	gtatggagaaattgagaatcaagggaatgtcatacacgatgtgttcagga
  	aagttttcaattgacaaagagatggcagaaacacagcatgggacaacagt
  	ggtgaaagtcaagtatgaaggtgctggagctccgtgtaaagtccccatag
  	agataagagatgtaaacaaggaaaaagtggttgggcgtatcatctcatcc
  	acccctttggctgagaataccaacagtgtaaccaacatagaattagaacc
  	cccctttggggacagctacatagtgataggtgttggaaacagcgcattaa
  	cactccattggttcaggaaagggagttccattggcaagatgtttgagtcc
  	acatacagaggtgcaaaacgaatggccattctaggtgaaacagcttggga
  	ttttggttccgttggtggactgttcacatcattgggaaaggctgtgcacc
  	aggtttttggaagtgtgtatacaaccatgtttggaggagtctcatggatg
  	attagaatcctaattgggttcttagtgttgtggattggcacgaactcgag
  	gaacacttcaatggctatgacgtgcatagctgttggaggaatcactctgt
  	ttctgggcttcacagttcaagcagacatgggttgtgtggcgtcatggagt
  	gggaaagaattgaagtgtggaagcggaatttttgtggttgacaacgtgca
  	cacttggacagaacagtacaaatttcaaccagagtccccagcgagactag
  	cgtctgcaatattaaatgcccacaaagatggggtctgtggaattagatca
  	accacgaggctggaaaatgtcatgtggaagcaaataaccaacgagctaaa
  	ctatgttctctgggaaggaggacatgacctcactgtagtggctggggatg
  	tgaagggggtgttgaccaaaggcaagagagcactcacacccccagtgagt
  	gatctgaaatattcatggaagacatggggaaaagcaaaaatcttcacccc
  	agaagcaagaaatagcacatttttaatagacggaccagacacctctgaat
  	gccccaatgaacgaagagcatggaactctcttgaggtggaagactatgga
  	tttggcatgttcacgaccaacatatggatgaaattccgagaaggaagttc
  	agaagtgtgtgaccacaggttaatgtcagctgcaattaaagatcagaaag
  	ctgtgcatgctgacatgggttattggatagagagctcaaaaaaccagacc
  	tggcagatagagaaagcatctcttattgaagtgaaaacatgtctgtggcc
  	caagacccacacactgtggagcaatggagtgctggaaagccagatgctca
  	ttccaaaatcatatgcgggccctttttcacagcacaattaccgccagggc
  	tatgccacgcaaaccgtgggcccatggcacttaggcaaattagagataga
  	ctttggagaatgccccggaacaacagtcacaattcaggaggattgtgacc
  	atagaggcccatctttgaggaccaccactgcatctggaaaactagtcacg
  	caatggtgctgccgctcctgcacgatgcctcccttaaggttcttgggaga
  	agatgggtgctggtatgggatggagattaggcccttgagtgaaaaagaag
  	agaacatggtcaaatcacaggtgacggccggacagggcacatcagaaact
  	ttttctatgggtctgttgtgcctgaccttgtttgtggaagaatgcttgag
  	gagaagagtcactaggaaacacatgatattagttgtggtgatcactcttt
  	gtgctatcatcctgggaggcctcacatggatggacttactacgagccctc
  	atcatgttgggggacactatgtctggtagaataggaggacagatccacct
  	agccatcatggcagtgttcaagatgtcaccaggatacgtgctgggtgtgt
  	ttttaaggaaactcacttcaagagagacagcactaatggtaataggaatg
  	gccatgacaacggtgctttcaattccacatgaccttatggaactcattga
  	tggaatatcactgggactaattttgctaaaaatagtaacacagtttgaca
  	acacccaagtgggaaccttagctctttccttgactttcataagatcaaca
  	atgccattggtcatggcttggaggaccattatggctgtgttgtttgtggt
  	cacactcattcctttgtgcaggacaagctgtcttcaaaaacagtctcatt
  	gggtagaaataacagcactcatcctaggagcccaagctctgccagtgtac
  	ctaatgactcttatgaaaggagcctcaagaagatcttggcctcttaacga
  	gggcataatggctgtgggtttggttagtctcttaggaagcgctcttttaa
  	agaatgatgtccctttagctggcccaatggtggcaggaggcttacttctg
  	gcggcttacgtgatgagtggtagctcagcagatctgtcactagagaaggc
  	cgccaacgtgcagtgggatgaaatggcagacataacaggctcaagcccaa
  	tcgtagaagtgaagcaggatgaagatggctctttctccatacgggacgtc
  	gaggaaaccaatatgataacccttttggtgaaactggcactgataacagt
  	gtcaggtctctaccccttggcaattccagtcacaatgaccttatggtaca
  	tgtggcaagtgaaaacacaaagatcaggagccctgtgggacgtcccctca
  	cccgctgccactaaaaaagccgcactgtctgaaggagtgtacaggatcat
  	gcaaagagggttattcgggaaaactcaggttggagtagggatacacatgg
  	aaggtgtatttcacacaatgtggcatgtaacaagaggatcagtgatctgc
  	cacgagactgggagattggagccatcttgggctgacgtcaggaatgacat
  	gatatcatacggtgggggatggaggcttggagacaaatgggacaaagaag
  	aagacgttcaggtcctcgccatagaaccaggaaaaaatcctaaacatgtc
  	caaacgaaacctggccttttcaagaccctaactggagaaattggagcagt
  	aacattagatttcaaacccggaacgtctggttctcccatcatcaacagga
  	aaggaaaagtcatcggactctatggaaatggagtagttaccaaatcaggt
  	gattacgtcagtgccataacgcaagccgaaagaattggagagccagatta
  	tgaagtggatgaggacatttttcgaaagaaaagattaactataatggact
  	tacaccccggagctggaaagacaaaaagaattcttccatcaatagtgaga
  	gaagccttaaaaaggaggctacgaactttgattttagctcccacgagagt
  	ggtggcggccgagatggaagaggccctacgtggactgccaatccgttatc
  	agaccccagctgtgaaatcagaacacacaggaagagagattgtagacctc
  	atgtgtcatgcaaccttcacaacaagacttttgtcatcaaccagggttcc
  	aaattacaaccttatagtgatggatgaagcacatttcaccgatccttcta
  	gtgtcgcggctagaggatacatctcgaccagggtggaaatgggagaggca
  	gcagccatcttcatgaccgcaacccctcccggagcgacagatccctttcc
  	ccagagcaacagcccaatagaagacatcgagagggaaattccggaaaggt
  	catggaacacagggttcgactggataacagactaccaagggaaaactgtg
  	tggtttgttcccagcataaaagctggaaatgacattgcaaattgtttgag
  	aaagtcgggaaagaaagttatccagttgagtaggaaaacctttgatacag
  	agtatccaaaaacgaaactcacggactgggactttgtggtcactacagac
  	atatctgaaatgggggccaattttagagccgggagagtgatagaccctag
  	aagatgcctcaagccagttatcctaccagatgggccagagagagtcattt
  	tagcaggtcctattccagtgactccagcaagcgctgctcagagaagaggg
  	cgaataggaaggaacccagcacaagaagacgaccaatacgttttctccgg
  	agacccactaaaaaatgatgaagatcatgcccactggacagaagcaaaga
  	tgctgcttgacaatatctacaccccagaagggatcattccaacattgttt
  	ggtccggaaagggaaaaaacccaagccattgatggagagtttcgcctcag
  	aggggaacaaaggaagacttttgtggaattaatgaggagaggagaccttc
  	cggtgtggctgagctataaggtagcttctgctggcatttcttacgaagat
  	cgggaatggtgcttcacaggggaaagaaataaccaaattttagaagaaaa
  	catggaggttgaaatttggactagagagggagaaaagaaaaagctaaggc
  	caagatggttagatgcacgtgtatacgctgaccccatggctttgaaggat
  	ttcaaggagtttgccagtggaaggaagagtataactctcgacatcctaac
  	agagattgccagtttgccaacttacctttcctctagggccaagctcgccc
  	ttgataacatagtcatgctccacacaacagaaagaggagggagggcctat
  	caacacgccctgaacgaacttccggagtcactggaaacactcatgcttgt
  	agctttactaggtgctatgacagcaggcatcttcctgtttttcatgcaag
  	ggaaaggaatagggaaattgtcaatgggtttgataaccattgcggtggct
  	agtggcttgctctgggtagcagaaattcaaccccagtggatagcggcctc
  	aatcatactagagttttttctcatggtactgttgataccggaaccagaaa
  	aacaaaggaccccacaagacaatcaattgatctacgtcatattgaccatt
  	ctcaccatcattggtctaatagcagccaacgagatggggctgattgaaaa
  	aacaaaaacggattttgggttttaccaggtaaaaacagaaaccaccatcc
  	tcgatgtggacttgagaccagcttcagcatggacgctctatgcagtagcc
  	accacaattctgactcccatgctgagacacaccatagaaaacacgtcggc
  	caacctatctctagcagccattgccaaccaggcagccgtcctaatggggc
  	ttggaaaaggatggccgctccacagaatggacctcggtgtgccgctgtta
  	gcaatgggatgctattctcaagtgaacccaacaaccttgacagcatcctt
  	agtcatgcttttagtccattatgcaataataggcccaggattgcaggcaa
  	aagccacaagagaggcccagaaaaggacagctgctgggatcatgaaaaat
  	cccacagtggacgggataacagtaatagatctagaaccaatatcctatga
  	cccaaaatttgaaaagcaattagggcaggtcatgctactagtcttgtgtg
  	ctggacaactactcttgatgagaacaacatgggctttctgtgaagtcttg
  	actttggccacaggaccaatcttgaccttgtgggagggcaacccgggaag
  	gttttggaacacgaccatagccgtatccaccgccaacattttcaggggaa
  	gttacttggcgggagctggactggctttttcactcataaagaatgcacaa
  	acccctaggaggggaactgggaccacaggagagacactgggagagaagtg
  	gaagagacagctaaactcattagacagaaaagagtttgaagagtataaaa
  	gaagtggaatactagaagtggacaggactgaagccaagtctgccctgaaa
  	gatgggtctaaaatcaagcatgcagtatcaagagggtccagtaagatcag
  	atggattgttgagagagggatggtaaagccaaaagggaaagttgtagatc
  	ttggctgtgggagaggaggatggtcttattacatggcgacactcaagaac
  	gtgactgaagtgaaagggtatacaaaaggaggtccaggacatgaagaacc
  	gattcccatggctacttatggttggaatttggtcaaactccattcagggg
  	ttgacgtgttctacaaacccacagagcaagtggacaccctgctctgtgat
  	attggggagtcatcttctaatccaacaatagaggaaggaagaacattaag
  	agttttgaagatggtggagccatggctctcttcaaaacctgaattctgca
  	tcaaagtccttaacccctacatgccaacagtcatagaagagctggagaaa
  	ctgcagagaaaacatggtgggaaccttgtcagatgcccgctgtccaggaa
  	ctccacccatgagatgtattgggtgtcaggagcgtcgggaaacattgtga
  	gctctgtgaacacaacatcaaagatgttgttgaacaggttcacaacaagg
  	cataggaaacccacttatgagaaggacgtagatcttggggcaggaacgag
  	aagtgtctccactgaaacagaaaaaccagacatgacaatcattgggagaa
  	ggcttcagcgattgcaagaagagcacaaagaaacctggcattatgatcag
  	gaaaacccatacagaacctgggcgtatcatggaagctatgaagctccttc
  	gacaggctctgcatcctccatggtgaacggggtggtaaaactgctaacaa
  	aaccctgggatgtgattccaatggtgactcagttagccatgacagataca
  	accccttttgggcaacaaagagtgttcaaagagaaggtggataccagaac
  	accacaaccaaaacccggtacacgaatggttatgaccacgacagccaatt
  	ggctgtgggccctccttggaaagaagaaaaatcccagactgtgcacaagg
  	gaagagttcatctcaaaagttagatcaaacgcagccataggcgcagtctt
  	tcaggaagaacagggatggacatcagccagtgaagctgtgaatgacagcc
  	ggttttgggaactggttgacaaagaaagggccctacaccaggaagggaaa
  	tgtgaatcgtgtgtctataacatgatgggaaaacgtgagaaaaagttagg
  	agagtttggcagagccaagggaagccgagcaatctggtacatgtggctgg
  	gagcgcggtttctggaatttgaagccctgggttttttgaatgaagatcac
  	tggtttggcagagaaaattcatggagtggagtggaaggggaaggtctgca
  	cagattgggatatatcctggaggagatagacaagaaggatggagacctaa
  	tgtatgctgatgacacagcaggctgggacacaagaatcactgaggatgac
  	cttcaaaatgaggaactgatcacggaacagatggctccccaccacaagat
  	cctagccaaagccattttcaaactaacctatcaaaacaaagtggtgaaag
  	tcctcagacccacaccgcggggagcggtgatggatatcatatccaggaaa
  	gaccaaagaggtagtggacaagttggaacatatggtttgaacacattcac
  	caacatggaagttcaactcatccgccaaatggaagctgaaggagtcatca
  	cacaagatgacatgcagaacccaaaagggttgaaagaaagagttgagaaa
  	tggctgaaagagtgtggtgtcgacaggttaaagaggatggcaatcagtgg
  	agacgattgcgtggtgaagcccctagatgagaggtttggcacttccctcc
  	tcttcttgaacgacatgggaaaggtgaggaaagacattccgcagtgggaa
  	ccatctaagggatggaaaaactggcaagaggttcctttttgctcccacca
  	ctttcacaagatctttatgaaggatggccgctcactagttgttccatgta
  	gaaaccaggatgaactgatagggagagccagaatctcgcagggagctgga
  	tggagcttaagagaaacagcctgcctgggcaaagcttacgcccagatgtg
  	gtcgcttatgtacttccacagaagggatctgcgtttagcctccatggcca
  	tatgctcagcagttccaacggaatggtttccaacaagcagaacaacatgg
  	tcaatccacgctcatcaccagtggatgaccactgaagatatgctcaaagt
  	gtggaacagagtgtggatagaagacaaccctaatatgactgacaagactc
  	cagtccattcgtgggaagatataccttacctagggaaaagagaggatttg
  	tggtgtggatccctgattggactttcttccagagccacctgggcgaagaa
  	cattcatacggccataacccaggtcaggaacctgatcggaaaagaggaat
  	acgtggattacatgccagtaatgaaaagatacagtgctccttcagagagt
  	gaaggagttctgtaattaccaacaacaaacaccaaaggctattgaagtca
  	ggccacttgtgccacggtttgagcaaaccgtgctgcctgtagctccgcca
  	ataatgggaggcgtaataatccccagggaggccatgcgccacggaagctg
  	tacgcgtggcatattggactagcggttagaggagacccctcccatcactg
  	ataaaacgcagcaaaagggggcccgaagccaggaggaagctgtactcctg
  	gtggaaggactagaggttagaggagacccccccaacacaaaaacagcata
  	ttgacgctgggaaagaccagagatcctgctgtctctgcaacatcaatcca
  	ggcacagagcgccgcaagatggattggtgttgttgatccaacaggttct

Example 19: Dengue Virus RNA Vaccine Immunogenicity in Mice
• This study provides a preliminary analysis of the immunogenicity of a nucleic acid mRNA vaccine using a dengue virus (DENV) serotype 2 antigen in BALB/c mice. The study utilizes 44 groups of 10 BALB/c female (5) and male (5) mice (440 total, 6-8 weeks of age at study initiation, see Table 10 for design summary). In this study, construct numbers used are referenced and found in Table 14.

• TABLE 14
  Dengue Antigen polynucleotides

  			ORF	mRNA	Protein
  Construct			SEQ	SEQ	SEQ
  Number	Gene ID	Description	ID NO	ID NO	ID NO	Construct
  1	131502	Dengue 2,	24	25	23	DEN2_D2Y98P_PrME_Hs3
  		D2Y98P strain,
  		PrME
  		transmembrane
  		antigen
  2	131503	Dengue 2,	27	28	26	DEN2_D2Y98P_PrME80_Hs3
  		D2Y98P strain,
  		PrME secreted
  		antigen
  3	131507	Dengue 2,	30	31	29	DEN2_D2Y98P_PrME80_ScFv.aDEC205.FLAG_Hs3
  		D2Y98P strain,
  		PrME secreted
  		antigen with
  		dendritic
  		targeting ScFv
  		against mouse
  		DEC205
  4	120554	Dengue strain 2	33	34	32	DEN2_DIII_Ferritin_Hs3
  		domain
  3
  		ferritin

  
  The sequences are shown below:

• TABLE 15
  		SEQ ID
  Name	Sequence	NO
  	MDAMKRGLCCVLLLCGAVFVSPFHLTTRNGEPHMIVSRQEKGKSLLFKTE	23
  	NGVNMCTLMAMDLGELCEDTITYNCPLLRQNEPEDIDCWCNSTSTWVTYG
  	TCTATGEHRREKRSVALVPHVGMGLETRTETWMSSEGAWKHAQRIETWVL
  	RHPGFTIMAAILAYTIGTTYFQRVLIFILLTAVAPSMTMRCIGISNRDFV
  	EGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKHPATLRKYCIE
  	AKLTNTTTASRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGG
  	IVTCAMFTCKKNMEGKIVQPENLEYTIVITPHSGEEGNDTGKHGKEIKVT
  	PQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLD
  	LPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALT
  	GATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAET
  	QHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPV
  	NIEAEPPFGDSYIIIGVEPGQLKLSWFKKGSSIGQMFETTMRGAKRMAIL
  	GDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVVITW
  	IGMNSRSTSLSVSLVLVGVVTLYLGVMVQA
  	ATGGATGCTATGAAAAGAGGCCTGTGTTGTGTGTTGCTGTTGTGCGGAGC	24
  	TGTGTTTGTGTCACCTTTCCACCTGACTACCCGCAATGGTGAGCCCCATA
  	TGATTGTGTCGCGCCAGGAGAAGGGGAAGTCCCTCCTGTTCAAAACTGAA
  	AACGGCGTGAACATGTGTACCCTGATGGCCATGGACCTTGGAGAACTGTG
  	CGAGGACACCATCACCTACAATTGTCCGCTCCTGCGCCAAAACGAACCAG
  	AAGATATCGACTGCTGGTGCAATTCCACTTCAACCTGGGTTACCTACGGA
  	ACTTGCACCGCCACGGGAGAACACAGAAGAGAAAAGCGCTCGGTGGCGCT
  	GGTGCCTCATGTCGGAATGGGACTGGAGACTCGGACGGAGACTTGGATGT
  	CCTCGGAGGGAGCATGGAAACATGCCCAACGGATCGAAACTTGGGTGCTG
  	AGGCACCCTGGATTCACCATCATGGCAGCGATCCTCGCCTACACTATAGG
  	TACTACCTACTTTCAAAGGGTGCTGATCTTCATTCTCCTCACCGCAGTGG
  	CCCCTTCAATGACCATGAGGTGCATTGGGATCTCGAACCGGGACTTCGTC
  	GAAGGAGTGTCCGGAGGTAGCTGGGTCGACATCGTCCTGGAACACGGAAG
  	CTGCGTGACTACTATGGCGAAGAACAAGCCAACCTTGGACTTCGAGCTTA
  	TCAAGACCGAGGCGAAGCACCCGGCCACTCTGAGAAAGTACTGCATCGAG
  	GCTAAGCTCACCAACACGACCACTGCCTCGCGATGCCCAACTCAGGGAGA
  	ACCGTCACTGAACGAAGAACAGGATAAACGCTTTGTGTGCAAGCATAGCA
  	TGGTGGATAGAGGCTGGGGAAACGGCTGTGGACTCTTCGGAAAGGGTGGA
  	ATTGTGACGTGCGCAATGTTCACTTGCAAGAAGAATATGGAAGGGAAGAT
  	CGTCCAGCCGGAGAACCTGGAATACACTATCGTGATCACCCCGCACTCAG
  	GCGAGGAGAACGCAGTGGGCAACGATACCGGGAAGCACGGGAAGGAAATC
  	AAGGTGACCCCGCAGTCGTCCATTACCGAGGCCGAACTCACCGGATACGG
  	CACTGTGACTATGGAATGCTCGCCACGGACCGGGCTGGATTTCAATGAGA
  	TGGTGCTCTTGCAAATGGAGAACAAAGCCTGGCTGGTCCACCGCCAGTGG
  	TTCCTCGACCTCCCCCTTCCGTGGCTGCCGGGAGCTGACACCCAAGGATC
  	CAACTGGATCCAAAAAGAAACCCTTGTCACGTTTAAGAATCCACATGCCA
  	AAAAGCAGGACGTGGTCGTGCTCGGAAGCCAGGAAGGAGCCATGCACACT
  	GCGCTGACTGGAGCAACCGAAATTCAAATGTCGAGCGGCAACCTCCTCTT
  	CACTGGACATCTGAAGTGCCGGCTGCGCATGGACAAACTGCAACTTAAGG
  	GCATGTCATACTCGATGTGTACCGGCAAATTCAAGGTGGTGAAGGAGATC
  	GCGGAGACTCAGCACGGGACCATCGTCATCCGGGTCCAGTATGAGGGTGA
  	TGGTTCCCCCTGCAAGATCCCTTTCGAAATCATGGATCTGGAGAAACGTC
  	ACGTGCTGGGCCGGCTGATCACTGTGAATCCGATCGTTACGGAGAAAGAC
  	AGCCCGGTGAACATCGAAGCTGAACCGCCGTTTGGGGATAGCTACATTAT
  	CATCGGCGTGGAACCAGGCCAGCTCAAGTTGTCGTGGTTCAAGAAAGGAT
  	CCAGCATCGGACAGATGTTCGAAACCACTATGCGCGGAGCCAAACGCATG
  	GCTATCCTGGGGGACACGGCCTGGGACTTCGGGTCGCTGGGTGGTGTGTT
  	CACCTCCATTGGAAAGGCGCTCCATCAGGTGTTTGGTGCGATCTACGGCG
  	CCGCATTCTCCGGAGTGTCATGGACCATGAAGATCCTCATCGGAGTCGTC
  	ATCACCTGGATCGGCATGAATTCTCGGTCCACTTCCTTGAGCGTCAGCCT
  	GGTGCTGGTCGGAGTTGTGACTCTGTACCTTGGAGTGATGGTCCAGGCC
  	GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUG	25
  	GAUGCUAUGAAAAGAGGCCUGUGUUGUGUGUUGCUGUUGUGCGGAGCUGU
  	GUUUGUGUCACCUUUCCACCUGACUACCCGCAAUGGUGAGCCCCAUAUGA
  	UUGUGUCGCGCCAGGAGAAGGGGAAGUCCCUCCUGUUCAAAACUGAAAAC
  	GGCGUGAACAUGUGUACCCUGAUGGCCAUGGACCUUGGAGAACUGUGCGA
  	GGACACCAUCACCUACAAUUGUCCGCUCCUGCGCCAAAACGAACCAGAAG
  	AUAUCGACUGCUGGUGCAAUUCCACUUCAACCUGGGUUACCUACGGAACU
  	UGCACCGCCACGGGAGAACACAGAAGAGAAAAGCGCUCGGUGGCGCUGGU
  	GCCUCAUGUCGGAAUGGGACUGGAGACUCGGACGGAGACUUGGAUGUCCU
  	CGGAGGGAGCAUGGAAACAUGCCCAACGGAUCGAAACUUGGGUGCUGAGG
  	CACCCUGGAUUCACCAUCAUGGCAGCGAUCCUCGCCUACACUAUAGGUAC
  	UACCUACUUUCAAAGGGUGCUGAUCUUCAUUCUCCUCACCGCAGUGGCCC
  	CUUCAAUGACCAUGAGGUGCAUUGGGAUCUCGAACCGGGACUUCGUCGAA
  	GGAGUGUCCGGAGGUAGCUGGGUCGACAUCGUCCUGGAACACGGAAGCUG
  	CGUGACUACUAUGGCGAAGAACAAGCCAACCUUGGACUUCGAGCUUAUCA
  	AGACCGAGGCGAAGCACCCGGCCACUCUGAGAAAGUACUGCAUCGAGGCU
  	AAGCUCACCAACACGACCACUGCCUCGCGAUGCCCAACUCAGGGAGAACC
  	GUCACUGAACGAAGAACAGGAUAAACGCUUUGUGUGCAAGCAUAGCAUGG
  	UGGAUAGAGGCUGGGGAAACGGCUGUGGACUCUUCGGAAAGGGUGGAAUU
  	GUGACGUGCGCAAUGUUCACUUGCAAGAAGAAUAUGGAAGGGAAGAUCGU
  	CCAGCCGGAGAACCUGGAAUACACUAUCGUGAUCACCCCGCACUCAGGCG
  	AGGAGAACGCAGUGGGCAACGAUACCGGGAAGCACGGGAAGGAAAUCAAG
  	GUGACCCCGCAGUCGUCCAUUACCGAGGCCGAACUCACCGGAUACGGCAC
  	UGUGACUAUGGAAUGCUCGCCACGGACCGGGCUGGAUUUCAAUGAGAUGG
  	UGCUCUUGCAAAUGGAGAACAAAGCCUGGCUGGUCCACCGCCAGUGGUUC
  	CUCGACCUCCCCCUUCCGUGGCUGCCGGGAGCUGACACCCAAGGAUCCAA
  	CUGGAUCCAAAAAGAAACCCUUGUCACGUUUAAGAAUCCACAUGCCAAAA
  	AGCAGGACGUGGUCGUGCUCGGAAGCCAGGAAGGAGCCAUGCACACUGCG
  	CUGACUGGAGCAACCGAAAUUCAAAUGUCGAGCGGCAACCUCCUCUUCAC
  	UGGACAUCUGAAGUGCCGGCUGCGCAUGGACAAACUGCAACUUAAGGGCA
  	UGUCAUACUCGAUGUGUACCGGCAAAUUCAAGGUGGUGAAGGAGAUCGCG
  	GAGACUCAGCACGGGACCAUCGUCAUCCGGGUCCAGUAUGAGGGUGAUGG
  	UUCCCCCUGCAAGAUCCCUUUCGAAAUCAUGGAUCUGGAGAAACGUCACG
  	UGCUGGGCCGGCUGAUCACUGUGAAUCCGAUCGUUACGGAGAAAGACAGC
  	CCGGUGAACAUCGAAGCUGAACCGCCGUUUGGGGAUAGCUACAUUAUCAU
  	CGGCGUGGAACCAGGCCAGCUCAAGUUGUCGUGGUUCAAGAAAGGAUCCA
  	GCAUCGGACAGAUGUUCGAAACCACUAUGCGCGGAGCCAAACGCAUGGCU
  	AUCCUGGGGGACACGGCCUGGGACUUCGGGUCGCUGGGUGGUGUGUUCAC
  	CUCCAUUGGAAAGGCGCUCCAUCAGGUGUUUGGUGCGAUCUACGGCGCCG
  	CAUUCUCCGGAGUGUCAUGGACCAUGAAGAUCCUCAUCGGAGUCGUCAUC
  	ACCUGGAUCGGCAUGAAUUCUCGGUCCACUUCCUUGAGCGUCAGCCUGGU
  	GCUGGUCGGAGUUGUGACUCUGUACCUUGGAGUGAUGGUCCAGGCCUGAU
  	AAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCC
  	CAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAA
  	GUCUGAGUGGGCGGC
  	MDAMKRGLCCVLLLCGAVFVSPFHLTTRNGEPHMIVSRQEKGKSLLFKTE	26
  	NGVNMCTLMAMDLGELCEDTITYNCPLLRQNEPEDIDCWCNSTSTWVTYG
  	TCTATGEHRREKRSVALVPHVGMGLETRTETWMSSEGAWKHAQRIETWVL
  	RHPGFTIMAAILAYTIGTTYFQRVLIFILLTAVAPSMTMRCIGISNRDFV
  	EGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKHPATLRKYCIE
  	AKLTNTTTASRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGG
  	IVTCAMFTCKKNMEGKIVQPENLEYTIVITPHSGEEGNDTGKHGKEIKVT
  	PQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLD
  	LPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALT
  	GATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAET
  	QHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPV
  	NIEAEPPFGDSYIIIGVEPGQLKLSWFKKG
  	ATGGATGCTATGAAAAGAGGCCTGTGTTGTGTGTTGCTGTTGTGCGGAGC	27
  	TGTGTTTGTGTCACCTTTCCACCTGACTACCCGCAATGGTGAGCCCCATA
  	TGATTGTGTCGCGCCAGGAGAAGGGGAAGTCCCTCCTGTTCAAAACTGAA
  	AACGGCGTGAACATGTGTACCCTGATGGCCATGGACCTTGGAGAACTGTG
  	CGAGGACACCATCACCTACAATTGTCCGCTCCTGCGCCAAAACGAACCAG
  	AAGATATCGACTGCTGGTGCAATTCCACTTCAACCTGGGTTACCTACGGA
  	ACTTGCACCGCCACGGGAGAACACAGAAGAGAAAAGCGCTCGGTGGCGCT
  	GGTGCCTCATGTCGGAATGGGACTGGAGACTCGGACGGAGACTTGGATGT
  	CCTCGGAGGGAGCATGGAAACATGCCCAACGGATCGAAACTTGGGTGCTG
  	AGGCACCCTGGATTCACCATCATGGCAGCGATCCTCGCCTACACTATAGG
  	TACTACCTACTTTCAAAGGGTGCTGATCTTCATTCTCCTCACCGCAGTGG
  	CCCCTTCAATGACCATGAGGTGCATTGGGATCTCGAACCGGGACTTCGTC
  	GAAGGAGTGTCCGGAGGTAGCTGGGTCGACATCGTCCTGGAACACGGAAG
  	CTGCGTGACTACTATGGCGAAGAACAAGCCAACCTTGGACTTCGAGCTTA
  	TCAAGACCGAGGCGAAGCACCCGGCCACTCTGAGAAAGTACTGCATCGAG
  	GCTAAGCTCACCAACACGACCACTGCCTCGCGATGCCCAACTCAGGGAGA
  	ACCGTCACTGAACGAAGAACAGGATAAACGCTTTGTGTGCAAGCATAGCA
  	TGGTGGATAGAGGCTGGGGAAACGGCTGTGGACTCTTCGGAAAGGGTGGA
  	ATTGTGACGTGCGCAATGTTCACTTGCAAGAAGAATATGGAAGGGAAGAT
  	CGTCCAGCCGGAGAACCTGGAATACACTATCGTGATCACCCCGCACTCAG
  	GCGAGGAGAACGCAGTGGGCAACGATACCGGGAAGCACGGGAAGGAAATC
  	AAGGTGACCCCGCAGTCGTCCATTACCGAGGCCGAACTCACCGGATACGG
  	CACTGTGACTATGGAATGCTCGCCACGGACCGGGCTGGATTTCAATGAGA
  	TGGTGCTCTTGCAAATGGAGAACAAAGCCTGGCTGGTCCACCGCCAGTGG
  	TTCCTCGACCTCCCCCTTCCGTGGCTGCCGGGAGCTGACACCCAAGGATC
  	CAACTGGATCCAAAAAGAAACCCTTGTCACGTTTAAGAATCCACATGCCA
  	AAAAGCAGGACGTGGTCGTGCTCGGAAGCCAGGAAGGAGCCATGCACACT
  	GCGCTGACTGGAGCAACCGAAATTCAAATGTCGAGCGGCAACCTCCTCTT
  	CACTGGACATCTGAAGTGCCGGCTGCGCATGGACAAACTGCAACTTAAGG
  	GCATGTCATACTCGATGTGTACCGGCAAATTCAAGGTGGTGAAGGAGATC
  	GCGGAGACTCAGCACGGGACCATCGTCATCCGGGTCCAGTATGAGGGTGA
  	TGGTTCCCCCTGCAAGATCCCTTTCGAAATCATGGATCTGGAGAAACGTC
  	ACGTGCTGGGCCGGCTGATCACTGTGAATCCGATCGTTACGGAGAAAGAC
  	AGCCCGGTGAACATCGAAGCTGAACCGCCGTTTGGGGATAGCTACATTAT
  	CATCGGCGTGGAACCAGGCCAGCTCAAGTTGTCGTGGTTCAAGAAAGGA
  	GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUG	28
  	GAUGCUAUGAAAAGAGGCCUGUGUUGUGUGUUGCUGUUGUGCGGAGCUGU
  	GUUUGUGUCACCUUUCCACCUGACUACCCGCAAUGGUGAGCCCCAUAUGA
  	UUGUGUCGCGCCAGGAGAAGGGGAAGUCCCUCCUGUUCAAAACUGAAAAC
  	GGCGUGAACAUGUGUACCCUGAUGGCCAUGGACCUUGGAGAACUGUGCGA
  	GGACACCAUCACCUACAAUUGUCCGCUCCUGCGCCAAAACGAACCAGAAG
  	AUAUCGACUGCUGGUGCAAUUCCACUUCAACCUGGGUUACCUACGGAACU
  	UGCACCGCCACGGGAGAACACAGAAGAGAAAAGCGCUCGGUGGCGCUGGU
  	GCCUCAUGUCGGAAUGGGACUGGAGACUCGGACGGAGACUUGGAUGUCCU
  	CGGAGGGAGCAUGGAAACAUGCCCAACGGAUCGAAACUUGGGUGCUGAGG
  	CACCCUGGAUUCACCAUCAUGGCAGCGAUCCUCGCCUACACUAUAGGUAC
  	UACCUACUUUCAAAGGGUGCUGAUCUUCAUUCUCCUCACCGCAGUGGCCC
  	CUUCAAUGACCAUGAGGUGCAUUGGGAUCUCGAACCGGGACUUCGUCGAA
  	GGAGUGUCCGGAGGUAGCUGGGUCGACAUCGUCCUGGAACACGGAAGCUG
  	CGUGACUACUAUGGCGAAGAACAAGCCAACCUUGGACUUCGAGCUUAUCA
  	AGACCGAGGCGAAGCACCCGGCCACUCUGAGAAAGUACUGCAUCGAGGCU
  	AAGCUCACCAACACGACCACUGCCUCGCGAUGCCCAACUCAGGGAGAACC
  	GUCACUGAACGAAGAACAGGAUAAACGCUUUGUGUGCAAGCAUAGCAUGG
  	UGGAUAGAGGCUGGGGAAACGGCUGUGGACUCUUCGGAAAGGGUGGAAUU
  	GUGACGUGCGCAAUGUUCACUUGCAAGAAGAAUAUGGAAGGGAAGAUCGU
  	CCAGCCGGAGAACCUGGAAUACACUAUCGUGAUCACCCCGCACUCAGGCG
  	AGGAGAACGCAGUGGGCAACGAUACCGGGAAGCACGGGAAGGAAAUCAAG
  	GUGACCCCGCAGUCGUCCAUUACCGAGGCCGAACUCACCGGAUACGGCAC
  	UGUGACUAUGGAAUGCUCGCCACGGACCGGGCUGGAUUUCAAUGAGAUGG
  	UGCUCUUGCAAAUGGAGAACAAAGCCUGGCUGGUCCACCGCCAGUGGUUC
  	CUCGACCUCCCCCUUCCGUGGCUGCCGGGAGCUGACACCCAAGGAUCCAA
  	CUGGAUCCAAAAAGAAACCCUUGUCACGUUUAAGAAUCCACAUGCCAAAA
  	AGCAGGACGUGGUCGUGCUCGGAAGCCAGGAAGGAGCCAUGCACACUGCG
  	CUGACUGGAGCAACCGAAAUUCAAAUGUCGAGCGGCAACCUCCUCUUCAC
  	UGGACAUCUGAAGUGCCGGCUGCGCAUGGACAAACUGCAACUUAAGGGCA
  	UGUCAUACUCGAUGUGUACCGGCAAAUUCAAGGUGGUGAAGGAGAUCGCG
  	GAGACUCAGCACGGGACCAUCGUCAUCCGGGUCCAGUAUGAGGGUGAUGG
  	UUCCCCCUGCAAGAUCCCUUUCGAAAUCAUGGAUCUGGAGAAACGUCACG
  	UGCUGGGCCGGCUGAUCACUGUGAAUCCGAUCGUUACGGAGAAAGACAGC
  	CCGGUGAACAUCGAAGCUGAACCGCCGUUUGGGGAUAGCUACAUUAUCAU
  	CGGCGUGGAACCAGGCCAGCUCAAGUUGUCGUGGUUCAAGAAAGGAUGAU
  	AAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCC
  	CAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAA
  	GUCUGAGUGGGCGGC
  	MDAMKRGLCCVLLLCGAVFVSPFHLTTRNGEPHMIVSRQEKGKSLLFKTE	29
  	NGVNMCTLMAMDLGELCEDTITYNCPLLRQNEPEDIDCWCNSTSTWVTYG
  	TCTATGEHRREKRSVALVPHVGMGLETRTETWMSSEGAWKHAQRIETWVL
  	RHPGFTIMAAILAYTIGTTYFQRVLIFILLTAVAPSMTMRCIGISNRDFV
  	EGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKHPATLRKYCIE
  	AKLTNTTTASRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGG
  	IVTCAMFTCKKNMEGKIVQPENLEYTIVITPHSGEEGNDTGKHGKEIKVT
  	PQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLD
  	LPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALT
  	GATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAET
  	QHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPV
  	NIEAEPPFGDSYIIIGVEPGQLKLSWFKKGGGGGSGGGGSGGGGSEVKLQ
  	QSGTEVVKPGASVKLSCKASGYIFTSYDIDWVRQTPEQGLEWIGWIFPGE
  	GSTEYNEKFKGRATLSVDKSSSTAYMELTRLTSEDSAVYFCARGDYYRRY
  	FDLWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSFLSTSLGNSITI
  	TCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDY
  	IFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLEIKRDYKDDDDK
  	ATGGATGCTATGAAAAGAGGCCTGTGTTGTGTGTTGCTGTTGTGCGGAGC	30
  	TGTGTTTGTGTCACCTTTCCACCTGACTACCCGCAATGGTGAGCCCCATA
  	TGATTGTGTCGCGCCAGGAGAAGGGGAAGTCCCTCCTGTTCAAAACTGAA
  	AACGGCGTGAACATGTGTACCCTGATGGCCATGGACCTTGGAGAACTGTG
  	CGAGGACACCATCACCTACAATTGTCCGCTCCTGCGCCAAAACGAACCAG
  	AAGATATCGACTGCTGGTGCAATTCCACTTCAACCTGGGTTACCTACGGA
  	ACTTGCACCGCCACGGGAGAACACAGAAGAGAAAAGCGCTCGGTGGCGCT
  	GGTGCCTCATGTCGGAATGGGACTGGAGACTCGGACGGAGACTTGGATGT
  	CCTCGGAGGGAGCATGGAAACATGCCCAACGGATCGAAACTTGGGTGCTG
  	AGGCACCCTGGATTCACCATCATGGCAGCGATCCTCGCCTACACTATAGG
  	TACTACCTACTTTCAAAGGGTGCTGATCTTCATTCTCCTCACCGCAGTGG
  	CCCCTTCAATGACCATGAGGTGCATTGGGATCTCGAACCGGGACTTCGTC
  	GAAGGAGTGTCCGGAGGTAGCTGGGTCGACATCGTCCTGGAACACGGAAG
  	CTGCGTGACTACTATGGCGAAGAACAAGCCAACCTTGGACTTCGAGCTTA
  	TCAAGACCGAGGCGAAGCACCCGGCCACTCTGAGAAAGTACTGCATCGAG
  	GCTAAGCTCACCAACACGACCACTGCCTCGCGATGCCCAACTCAGGGAGA
  	ACCGTCACTGAACGAAGAACAGGATAAACGCTTTGTGTGCAAGCATAGCA
  	TGGTGGATAGAGGCTGGGGAAACGGCTGTGGACTCTTCGGAAAGGGTGGA
  	ATTGTGACGTGCGCAATGTTCACTTGCAAGAAGAATATGGAAGGGAAGAT
  	CGTCCAGCCGGAGAACCTGGAATACACTATCGTGATCACCCCGCACTCAG
  	GCGAGGAGAACGCAGTGGGCAACGATACCGGGAAGCACGGGAAGGAAATC
  	AAGGTGACCCCGCAGTCGTCCATTACCGAGGCCGAACTCACCGGATACGG
  	CACTGTGACTATGGAATGCTCGCCACGGACCGGGCTGGATTTCAATGAGA
  	TGGTGCTCTTGCAAATGGAGAACAAAGCCTGGCTGGTCCACCGCCAGTGG
  	TTCCTCGACCTCCCCCTTCCGTGGCTGCCGGGAGCTGACACCCAAGGATC
  	CAACTGGATCCAAAAAGAAACCCTTGTCACGTTTAAGAATCCACATGCCA
  	AAAAGCAGGACGTGGTCGTGCTCGGAAGCCAGGAAGGAGCCATGCACACT
  	GCGCTGACTGGAGCAACCGAAATTCAAATGTCGAGCGGCAACCTCCTCTT
  	CACTGGACATCTGAAGTGCCGGCTGCGCATGGACAAACTGCAACTTAAGG
  	GCATGTCATACTCGATGTGTACCGGCAAATTCAAGGTGGTGAAGGAGATC
  	GCGGAGACTCAGCACGGGACCATCGTCATCCGGGTCCAGTATGAGGGTGA
  	TGGTTCCCCCTGCAAGATCCCTTTCGAAATCATGGATCTGGAGAAACGTC
  	ACGTGCTGGGCCGGCTGATCACTGTGAATCCGATCGTTACGGAGAAAGAC
  	AGCCCGGTGAACATCGAAGCTGAACCGCCGTTTGGGGATAGCTACATTAT
  	CATCGGCGTGGAACCAGGCCAGCTCAAGTTGTCGTGGTTCAAGAAAGGAG
  	GAGGTGGAGGATCCGGAGGCGGAGGGTCGGGCGGTGGTGGATCGGAGGTC
  	AAACTGCAGCAATCAGGGACCGAAGTCGTGAAGCCGGGGGCTTCAGTCAA
  	GCTGTCCTGCAAGGCCAGCGGCTATATCTTCACTAGCTACGACATCGATT
  	GGGTGCGGCAGACTCCGGAGCAAGGACTCGAGTGGATTGGGTGGATCTTT
  	CCGGGCGAGGGATCAACCGAGTACAACGAAAAATTTAAGGGACGGGCAAC
  	GCTGTCCGTGGACAAGAGCTCATCTACGGCGTACATGGAGCTGACGCGGC
  	TCACGTCAGAGGATTCCGCCGTCTACTTCTGTGCCAGGGGCGACTACTAC
  	CGGCGCTACTTTGATCTGTGGGGACAAGGAACGACCGTGACTGTCTCATC
  	AGGCGGCGGCGGATCGGGAGGAGGCGGATCGGGTGGCGGTGGTTCGGACA
  	TTCAGATGACTCAATCGCCCAGCTTCCTGTCGACCTCACTGGGGAATTCT
  	ATTACGATCACTTGTCACGCTTCGCAGAACATCAAGGGTTGGCTGGCTTG
  	GTACCAGCAGAAAAGCGGTAACGCCCCGCAACTGCTCATCTACAAGGCAT
  	CGTCCCTGCAATCGGGAGTGCCGTCACGCTTTTCAGGATCGGGCTCCGGA
  	ACCGATTACATCTTTACCATCAGCAACCTGCAGCCGGAAGACATCGCCAC
  	TTACTACTGTCAACACTATCAGAGCTTTCCGTGGACCTTTGGAGGGGGGA
  	CCAAATTGGAGATCAAGCGCGACTACAAGGATGACGATGACAAA
  	GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUG	31
  	GAUGCUAUGAAAAGAGGCCUGUGUUGUGUGUUGCUGUUGUGCGGAGCUGU
  	GUUUGUGUCACCUUUCCACCUGACUACCCGCAAUGGUGAGCCCCAUAUGA
  	UUGUGUCGCGCCAGGAGAAGGGGAAGUCCCUCCUGUUCAAAACUGAAAAC
  	GGCGUGAACAUGUGUACCCUGAUGGCCAUGGACCUUGGAGAACUGUGCGA
  	GGACACCAUCACCUACAAUUGUCCGCUCCUGCGCCAAAACGAACCAGAAG
  	AUAUCGACUGCUGGUGCAAUUCCACUUCAACCUGGGUUACCUACGGAACU
  	UGCACCGCCACGGGAGAACACAGAAGAGAAAAGCGCUCGGUGGCGCUGGU
  	GCCUCAUGUCGGAAUGGGACUGGAGACUCGGACGGAGACUUGGAUGUCCU
  	CGGAGGGAGCAUGGAAACAUGCCCAACGGAUCGAAACUUGGGUGCUGAGG
  	CACCCUGGAUUCACCAUCAUGGCAGCGAUCCUCGCCUACACUAUAGGUAC
  	UACCUACUUUCAAAGGGUGCUGAUCUUCAUUCUCCUCACCGCAGUGGCCC
  	CUUCAAUGACCAUGAGGUGCAUUGGGAUCUCGAACCGGGACUUCGUCGAA
  	GGAGUGUCCGGAGGUAGCUGGGUCGACAUCGUCCUGGAACACGGAAGCUG
  	CGUGACUACUAUGGCGAAGAACAAGCCAACCUUGGACUUCGAGCUUAUCA
  	AGACCGAGGCGAAGCACCCGGCCACUCUGAGAAAGUACUGCAUCGAGGCU
  	AAGCUCACCAACACGACCACUGCCUCGCGAUGCCCAACUCAGGGAGAACC
  	GUCACUGAACGAAGAACAGGAUAAACGCUUUGUGUGCAAGCAUAGCAUGG
  	UGGAUAGAGGCUGGGGAAACGGCUGUGGACUCUUCGGAAAGGGUGGAAUU
  	GUGACGUGCGCAAUGUUCACUUGCAAGAAGAAUAUGGAAGGGAAGAUCGU
  	CCAGCCGGAGAACCUGGAAUACACUAUCGUGAUCACCCCGCACUCAGGCG
  	AGGAGAACGCAGUGGGCAACGAUACCGGGAAGCACGGGAAGGAAAUCAAG
  	GUGACCCCGCAGUCGUCCAUUACCGAGGCCGAACUCACCGGAUACGGCAC
  	UGUGACUAUGGAAUGCUCGCCACGGACCGGGCUGGAUUUCAAUGAGAUGG
  	UGCUCUUGCAAAUGGAGAACAAAGCCUGGCUGGUCCACCGCCAGUGGUUC
  	CUCGACCUCCCCCUUCCGUGGCUGCCGGGAGCUGACACCCAAGGAUCCAA
  	CUGGAUCCAAAAAGAAACCCUUGUCACGUUUAAGAAUCCACAUGCCAAAA
  	AGCAGGACGUGGUCGUGCUCGGAAGCCAGGAAGGAGCCAUGCACACUGCG
  	CUGACUGGAGCAACCGAAAUUCAAAUGUCGAGCGGCAACCUCCUCUUCAC
  	UGGACAUCUGAAGUGCCGGCUGCGCAUGGACAAACUGCAACUUAAGGGCA
  	UGUCAUACUCGAUGUGUACCGGCAAAUUCAAGGUGGUGAAGGAGAUCGCG
  	GAGACUCAGCACGGGACCAUCGUCAUCCGGGUCCAGUAUGAGGGUGAUGG
  	UUCCCCCUGCAAGAUCCCUUUCGAAAUCAUGGAUCUGGAGAAACGUCACG
  	UGCUGGGCCGGCUGAUCACUGUGAAUCCGAUCGUUACGGAGAAAGACAGC
  	CCGGUGAACAUCGAAGCUGAACCGCCGUUUGGGGAUAGCUACAUUAUCAU
  	CGGCGUGGAACCAGGCCAGCUCAAGUUGUCGUGGUUCAAGAAAGGAGGAG
  	GUGGAGGAUCCGGAGGCGGAGGGUCGGGCGGUGGUGGAUCGGAGGUCAAA
  	CUGCAGCAAUCAGGGACCGAAGUCGUGAAGCCGGGGGCUUCAGUCAAGCU
  	GUCCUGCAAGGCCAGCGGCUAUAUCUUCACUAGCUACGACAUCGAUUGGG
  	UGCGGCAGACUCCGGAGCAAGGACUCGAGUGGAUUGGGUGGAUCUUUCCG
  	GGCGAGGGAUCAACCGAGUACAACGAAAAAUUUAAGGGACGGGCAACGCU
  	GUCCGUGGACAAGAGCUCAUCUACGGCGUACAUGGAGCUGACGCGGCUCA
  	CGUCAGAGGAUUCCGCCGUCUACUUCUGUGCCAGGGGCGACUACUACCGG
  	CGCUACUUUGAUCUGUGGGGACAAGGAACGACCGUGACUGUCUCAUCAGG
  	CGGCGGCGGAUCGGGAGGAGGCGGAUCGGGUGGCGGUGGUUCGGACAUUC
  	AGAUGACUCAAUCGCCCAGCUUCCUGUCGACCUCACUGGGGAAUUCUAUU
  	ACGAUCACUUGUCACGCUUCGCAGAACAUCAAGGGUUGGCUGGCUUGGUA
  	CCAGCAGAAAAGCGGUAACGCCCCGCAACUGCUCAUCUACAAGGCAUCGU
  	CCCUGCAAUCGGGAGUGCCGUCACGCUUUUCAGGAUCGGGCUCCGGAACC
  	GAUUACAUCUUUACCAUCAGCAACCUGCAGCCGGAAGACAUCGCCACUUA
  	CUACUGUCAACACUAUCAGAGCUUUCCGUGGACCUUUGGAGGGGGGACCA
  	AAUUGGAGAUCAAGCGCGACUACAAGGAUGACGAUGACAAAUGAUAAUAG
  	GCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCC
  	CCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUG
  	AGUGGGCGGC
  	MDWTWILFLVAAATRVHSKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQT	32
  	EGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDS
  	YIIIGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILSGGDIIKLLNE
  	QVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLN
  	ENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSK
  	DHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIA
  	KSRKS
  	ATGGATTGGACCTGGATCTTGTTTCTCGTCGCCGCAGCCACTCGCGTTCA	33
  	TAGCAAAGGAATGTCATACTCCATGTGCACGGGAAAATTCAAGGTGGTCA
  	AAGAGATCGCGGAGACTCAGCACGGCACCATCGTCATTCGCGTGCAAACT
  	GAAGGAGATGGATCTCCCTGCAAGATCCCGTTCGAGATCATGGACCTGGA
  	AAAGAGACACGTCCTCGGTAGACTGATCACCGTGAACCCGATCGTGACGG
  	AGAAGGATTCCCCGGTGAATATTGAAGCAGAGCCTCCATTTGGGGACTCA
  	TACATTATCATTGGGGTCGAGCCGGGCCAGCTGAAGCTGAATTGGTTTAA
  	GAAGGGCTCGTCAATCGGACAGATGTTCGAAACTACTATGAGGGGTGCAA
  	AGCGGATGGCGATCCTCTCGGGCGGAGATATCATCAAACTCCTTAACGAA
  	CAGGTGAACAAGGAGATGCAGTCCTCAAACCTTTACATGAGCATGTCGTC
  	CTGGTGTTACACCCATAGCCTGGACGGCGCTGGATTGTTCCTGTTTGACC
  	ATGCAGCGGAGGAATACGAACACGCCAAGAAGCTCATCATCTTCCTGAAC
  	GAGAATAACGTGCCAGTGCAACTGACCTCCATCTCGGCTCCTGAGCACAA
  	GTTCGAAGGACTCACCCAGATCTTCCAAAAGGCCTACGAACACGAACAGC
  	ACATCAGCGAATCCATCAACAATATCGTGGACCATGCTATCAAAAGCAAA
  	GACCATGCGACCTTCAACTTCCTGCAATGGTATGTCGCCGAACAGCACGA
  	AGAGGAGGTGCTGTTCAAGGACATTCTCGACAAAATCGAATTGATAGGGA
  	ACGAAAATCACGGTCTGTACCTGGCCGATCAATACGTGAAGGGAATTGCC
  	AAGTCGCGGAAGTCGT
  	GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUG	34
  	GAUUGGACCUGGAUCUUGUUUCUCGUCGCCGCAGCCACUCGCGUUCAUAG
  	CAAAGGAAUGUCAUACUCCAUGUGCACGGGAAAAUUCAAGGUGGUCAAAG
  	AGAUCGCGGAGACUCAGCACGGCACCAUCGUCAUUCGCGUGCAAACUGAA
  	GGAGAUGGAUCUCCCUGCAAGAUCCCGUUCGAGAUCAUGGACCUGGAAAA
  	GAGACACGUCCUCGGUAGACUGAUCACCGUGAACCCGAUCGUGACGGAGA
  	AGGAUUCCCCGGUGAAUAUUGAAGCAGAGCCUCCAUUUGGGGACUCAUAC
  	AUUAUCAUUGGGGUCGAGCCGGGCCAGCUGAAGCUGAAUUGGUUUAAGAA
  	GGGCUCGUCAAUCGGACAGAUGUUCGAAACUACUAUGAGGGGUGCAAAGC
  	GGAUGGCGAUCCUCUCGGGCGGAGAUAUCAUCAAACUCCUUAACGAACAG
  	GUGAACAAGGAGAUGCAGUCCUCAAACCUUUACAUGAGCAUGUCGUCCUG
  	GUGUUACACCCAUAGCCUGGACGGCGCUGGAUUGUUCCUGUUUGACCAUG
  	CAGCGGAGGAAUACGAACACGCCAAGAAGCUCAUCAUCUUCCUGAACGAG
  	AAUAACGUGCCAGUGCAACUGACCUCCAUCUCGGCUCCUGAGCACAAGUU
  	CGAAGGACUCACCCAGAUCUUCCAAAAGGCCUACGAACACGAACAGCACA
  	UCAGCGAAUCCAUCAACAAUAUCGUGGACCAUGCUAUCAAAAGCAAAGAC
  	CAUGCGACCUUCAACUUCCUGCAAUGGUAUGUCGCCGAACAGCACGAAGA
  	GGAGGUGCUGUUCAAGGACAUUCUCGACAAAAUCGAAUUGAUAGGGAACG
  	AAAAUCACGGUCUGUACCUGGCCGAUCAAUACGUGAAGGGAAUUGCCAAG
  	UCGCGGAAGUCGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGC
  	CCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC
  	GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCU

• Mice were vaccinated on weeks 0 and 3 via intramuscular (IM) or intradermal (ID) routes. One group remained unvaccinated and one was administered 10⁵ plaque-forming units (PFU) live DENV2, D2Y98P isolate via intravenous (IV) injection as a positive control. Serum was collected from each mouse on weeks 1, 3, and 5; bleeds on weeks 1 and 3 were in-life samples (tail vein or submandibular bleeds) and week 5 will be a terminal (intracardiac) bleed. Individual serum samples were stored at −80° C. until analysis by neutralization or microneutralization assay. Pooled samples from each group at the week 5 time points were tested by Western blot for reactivity with viral lysate.

• TABLE 16
  Detailed experimental design (treatment, readouts)

  		Vaccine (n =
  		10, female)
  		mice/group)
  	Mouse	Delivered		Formulation/
  Group	Strain	week		0 and 3	Chemistry	Route	Dose	Readouts

  1	Female	N/A		N/A	N/A	Serum
  2	BALB/c	DEN2Y98-PrME	N1-methyl-	ID	0.4	samples
  3	6-8	(construct 1	pseudouridine/	IM	mg/kg	collected
  	weeks	from Table 14)	5-methyl-		in LNP	on weeks
  4	of age		cytosine	ID	0.08	1, 3, and 5.
  5				IM	mg/kg	Serum
  					in LNP	analyzed
  6				ID	0.016	via
  7				IM	mg/kg	Western
  					in LNP	blot
  8			N1-methyl-	ID	0.4
  9			pseudouridine	IM	mg/kg
  					in LNP
  10				ID	0.08
  11				IM	mg/kg
  					in LNP
  11				ID	0.016
  12				IM	mg/kg
  					in LNP
  13		DEN2Y98-PrME80	N1-methyl-	ID	0.4
  14		(construct 2	pseudouridine/	IM	mg/kg
  		from Table 14)	5-methyl-		in LNP
  15			cytosine	ID	0.08
  16				IM	mg/kg
  					in LNP
  17				ID	0.016
  18				IM	mg/kg
  					in LNP
  19			N1-methyl-	ID	0.4
  20			pseudouridine	IM	mg/kg
  					in LNP
  21				ID	0.08
  22				IM	mg/kg
  					in LNP
  23				ID	0.016
  24				IM	mg/kg
  					in LNP
  25		DEN2Y98-	N1-methyl-	ID	0.4
  26		PrME80-DC	pseudouridine/	IM	mg/kg
  		(construct 3	5-methyl-		in LNP
  27		from Table 14)	cytosine	ID	0.08
  28				IM	mg/kg
  					in LNP
  29				ID	0.016
  30				IM	mg/kg
  					in LNP
  31			N1-methyl-	ID	0.4
  32			pseudouridine	IM	mg/kg
  					in LNP
  33				ID	0.08
  34				IM	mg/kg
  					in LNP
  35				ID	0.016
  36				IM	mg/kg
  					in LNP
  37		DEN2-DIII-	N1-methyl-	ID	0.4
  38		Ferritin	pseudouridine	IM	mg/kg
  		(construct 4			in LNP
  39		from Table 14)		ID	0.08
  40				IM	mg/kg
  					in LNP
  41				ID	0.016
  42				IM	mg/kg
  					in LNP
  43		Control,	—	IV	105 PFU
  		D2Y98P
  		live virus

  
  Signal was detected in groups 5, 15, 39, and 44 (live virus control) by a band that appeared between 50 and 60 kDa in the Western blot data. The data suggests that a mRNA vaccine to a single dengue viral antigen can produce antibody in preliminary studies.
• In order to provide a Dengue vaccine having enhanced immunogenicity, RNA vaccines for concatemeric antigens were designed and tested according to the invention. These vaccines, which have significantly enhanced activity, in comparison to the single protein antigens described herein, are described below.
Example 20: In Silico Prediction of T Cell Epitopes for RNA Vaccine Design
• Several peptide epitopes from Dengue virus were generated and tested for antigenic activity. The peptide epitopes are designed to maximize MHC presentation. In general the process of MHC class I presentation is quite inefficient, with only 1 peptide of 10,000 degraded molecules actually being presented. Additionally the priming of CD8 T cell with APCs having insufficient densities of surface peptide/MHC class I complexes results in weak responders exhibiting impaired cytokine secretion and a decrease memory pool. Thus, the process of designing highly effective peptide epitopes is important to the immunogenicity of the ultimate vaccine.
• In silico prediction of desirable peptide epitopes was performed using Immune Epitope Database. Using this database several immunogenic Dengue T cell epitopes showing strong homology across all 4 Dengue serotypes were predicted. Examples of these epitopes are shown in FIGS. 16A-16C and 17A-17C.
Example 21: Prediction of DENV T Cell Epitopes for RNA Vaccine Design
• The design of optimized vaccination systems to prevent or treat conditions that have failed to respond to more traditional treatments or early vaccination strategies relies on the identification of the antigens or epitopes that play a role in these conditions and which the immune system can effectively target. T cell epitopes (e.g., MHC peptide binding) for the various alleles shown in Table 17 were determined using Rapid Epitope Discovery System (ProImmune REVEAL & ProVE®). This system is used to identify those candidate epitopes that actually cause relevant immune responses from the numerous other potential candidates identified using algorithms to predict MHC-peptide binding. The REVEAL binding assay determines the ability of each candidate peptide to bind to one or more MHC I class alleles and stabilize the MHC-peptide complex. The assay identifies the most likely immunogenic peptides in a protein sequence by comparing the binding to that of a high affinity T cell epitope and detecting the presence or absence of the native conformation of the MHC-peptide complex. The epitope peptides are further tested using the assays described herein to confirm their immunogenic activity.

• TABLE 17
  Alleles Tested
  Allele

  	A*01:01
  	A*02:01
  	A*03:01
  	A*11:01
  	A*24:02
  	B*07:02
  	B*27:05
  	H-2Kb

• TABLE 18
  ProImmune REVEAL® binding assay data for A*01: 01

  Peptide I.D.	SEQ ID NO	REVEAL® score at 0 h
  TTDISEMGA	217	68.4

• TABLE 19
  ProImmune REVEAL® binding assay data for A*02: 01

  Peptide I.D.	SEQ ID NO	REVEAL® score at 0 h

  TMWHVTRGA	218	112.0
  MWHVTRGAV	219	62.7
  GLYGNGVVT	220	87.7
  TLILAPTRV	221	104.2
  LILAPTRVV	222	106.4
  ILAPTRVVA	223	95.7
  VVAAEMEEA	224	92.2
  IVDLMCHAT	225	62.7
  LMCHATFTM	226	72.9
  MGEAAAIFM	227	50.6
  GEAAAIFMT	228	74.3
  KTVWFVPSI	229	115.9
  LMRRGDLPV	230	82.3
  TLLCDIGES	231	63.9
  LLCDIGESS	232	93.9
  AMTDTTPFG	233	91.9
  GQQRVFKEK	234	47.1
  KLTYQNKVV	235	92.3
  AISGDDCVV	236	91.1
  LMYFHRRDL	237	97.8

• TABLE 20
  ProImmune REVEAL® binding assay data for A*03: 01

  Peptide I.D.	SEQ ID NO	REVEAL® score at 0 h
  RTLILAPTR	238	91.4
  TLILAPTRV	239	55.2
  MCHATFTMR	240	86.8
  TVWFVPSIK	241	53.6
  GQQRVFKEK	242	59.6
  CVYNMMGKR	243	81.6

• TABLE 21
  ProImmune REVEAL® binding assay data for A*11: 01

  Peptide I.D.	SEQ ID NO	REVEAL® score at 0 h

  HTMWHVTRG	244	56.3
  RTLILAPTR	245	89.9
  TLILAPTRV	246	59.0
  MCHATFTMR	247	91.0
  ATFTMRLLS	248	58.5
  GEAAAIFMT	249	50.3
  KTVWFVPSI	250	50.8
  TVWFVPSIK	251	92.2
  GQQRVFKEK	252	85.5
  CVYNMMGKR	253	113.2
  VYNMMGKRE	254	62.5
  YNMMGKREK	255	80.9
  NMMGKREKK	256	77.9
  GTYGLNTFT	257	63.6
  ISGDDCVVK	258	88.7

• TABLE 22
  ProImmune REVEAL® binding assay data for A*24: 02

  Peptide I.D.	SEQ ID NO	REVEAL® score at 0 h

  LMCHATFTM	259	99.5
  CHATFTMRL	260	75.9
  GEAAAIFMT	261	58.9
  KTVWFVPSI	262	89.1
  HWTEAKMLL	263	103.2
  WTEAKMLLD	264	94.7
  LGCGRGGWS	265	74.8
  MAMTDTTPF	266	51.3
  MYADDTAGW	267	76.8
  VGTYGLNTF	268	96.0
  YFHRRDLRL	269	87.5

• TABLE 23
  ProImmune REVEAL® binding assay data for B*07: 02

  Peptide I.D.	SEQ ID NO	REVEAL® score at 0 h

  FKPGTSGSP	270	50.4
  KPGTSGSPI	271	112.1
  IPERSWNSG	272	45.2
  PERVILAGP	273	56.1
  LMRRGDLPV	274	178.9
  PLSRNSTHE	275	65.0
  LSRNSTHEM	276	124.5
  SRNSTHEMY	277	52.0
  MAMTDTTPF	278	117.4
  TPFGQQRVF	279	112.7
  LMYFHRRDL	280	119.6

• TABLE 24
  ProImmune REVEAL® binding assay data for B*27: 05

  Peptide I.D.	SEQ ID NO	REVEAL® score at 0 h

  LRTLILAPT	281	58.7
  LMCHATFTM	282	98.2
  ARGYISTRV	283	125.3
  RRGDLPVWL	284	144.8
  GQQRVFKEK	285	95.4
  SRAIWYMWL	286	53.9
  FKLTYQNKV	287	53.7

• TABLE 25
  ProImmune REVEAL® binding assay data for H-2 Kb

  Peptide I.D.	SEQ ID NO	REVEAL® score at 0 h

  FKPGTSGSP	288	45.7
  LAPTRVVAA	289	102.5
  LMCHATFTM	290	59.0
  CHATFTMRL	291	60.3
  HATFTMRLL	292	69.5
  ATFTMRLLS	293	55.6
  KTVWFVPSI	294	54.4
  LSRNSTHEM	295	51.1
  QQRVFKEKV	296	63.4
  YGLNTFTNM	297	75.4
  LMYFHRRDL	298	54.9

Example 22: Activity Testing for Predicted Peptide Epitopes
• Exemplary peptide epitopes selected using the methods described above were further characterized. These peptide epitopes were confirmed to have activity using in vitro HLA binding assays (human lymphocyte binding assays). Peptides (9 aa peptides from the dengue antigen) were screened for their ability to bind to HLA. The analysis of the homology, affinity, frequency and design of these peptides is shown in FIGS. 16A-16C and 17A-17C.
Example 23: In Vivo Analysis of Mimectopes of Predicted Human Epitopes RNA Vaccines Methods
• IFNγ ELISpot. Mouse IFNγ ELISpot assays were performed using IFNγ coated Millipore IP Opaque plates according to the manufacturer's mouse IFNγ ELISPOT guidelines. Briefly, the plates were blocked using complete RPMI (R10) and incubated for 30 minutes prior to plating cells. Peptides (284-292, 408-419 or 540-548) were diluted to 5 different concentrations for stimulation at 5, −6, −7, −8, or −9 from an original stock concentration of 10 mM (−2). Mouse splenocytes (200,000-250,000 cells) were plated in appropriate wells with peptide, PMA+Ionomycin or R10 media alone. Cells were stimulated in a total volume of 125 μL per well. Plates were then incubated at 37° C., 5% CO₂ for 18-24 hrs. Plates were developed following the manufacturer's instructions. Plates were counted and quality controlled using the automated ELISPOT reader CTL ImmunoSpot/FluoroSpot.
• Intracellular Cytokine Staining (ICS). Intracellular Cytokine Staining (ICS). For intracellular cytokine staining, individual splenocytes, were resuspended at a concentration of 1.5×106 cells per mL. Peptides (284-292, 408-419 or 540-548) were made into 5 dilutions from a stock concentration of 10 mM⁽⁻²⁾. The final concentrations of each peptide were −5, −6, −7, −8, or −9 in their respective wells. Cells were stimulated in a final volume of 200 uL within a 96 well culture plate. After the addition of Golgi plug (0.2 uL per well), cells were incubated at 37° C., 5% CO₂ for 5 hours. Following stimulation, cells were surface stained, fixed, washed and put at 4° C. overnight. Intracellular staining was performed the following day, resulting in full panel of Live/Dead (Invitrogen), αCD3, αCD4, αCD8, αCD45, αCCR7, αCD44, αCD25, αIL-2, αIFNγ, and αTNFα (BD Biosciences). Cells were acquired in a 96-U bottom plate using BD LSR Fortessa HTS (BD Biosciences).
Results
• The exemplary peptide epitopes selected using the methods described herein were used to produce tests mouse mimectopes of the predicted human epitopes. These mimectopes were analyzed for in vivo activity using restimulation assays during the acute phase of Dengue infection (Day 7). The methods were performed on dengue-infected IFNαβ/γ-receptor-deficient mice (AG129). Seven days post infection splenocytes were harvested and subjected to an ELISPOT assay to quantify secretion of cytokines by T cells (CD8) as described above. Briefly, the isolated splenocytes were stimulated with the test peptides and tested for T cell activation. If the peptide is an appropriate antigen, some cells would be present antigen during infection and would be capable of stimulating T cells. The methods for analyzing the T cell activation were performed as follows:
• T cells (at a known concentration) were incubated with a specific antigen in a cell culture well the activated T cells were transferred to ELISPOT plates (precoated with anti-cytokine antibody)
  the cells were incubated such that cytokines could be secreted
  the cells were washed off the plate and enzyme coupled secondary Ig was added
  the plates were washed and substrate was added
  positive spots were scored under microscope.
• The data is shown in FIGS. 18-19 . FIGS. 18 and 19 are graphs depicting the results of an ELISPOT assay of dengue-specific peptides measuring IFN-γ (spots per million splenocytes).
• A schematic of an assay on a BLT Mouse Model (Bone Marrow/Liver/Thymus) is shown in FIG. 20 . The results of a histogram analysis of human CD8 T cells stimulated with peptide epitope is also shown in FIG. 20 .
• The following two sequences were used as controls:

• (SEQ ID NO: 35)

  (V5)8-cathb: Kozak Start GKPIPNPLLGLDST-GFLG-

  GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-

  GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-

  GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-

  GKPIPNPLLGLDST Stop

  (SEQ ID NO: 36)

  (v5)8-cathb + MHCi: Kozak Start GKPIPNPLLGLDST-

  GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-

  GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-

  GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-

  GKPIPNPLLGLDST Stop

  
  Some results are shown in Table 26:

• TABLE 26
  Results
  A*02:01

  Peptide ID	REVEAL® Score

  5.	KQWFLDLPL (SEQ ID NO: 213)	86.0
  6.	RQWFLDLPL (SEQ ID NO: 214)	77.7
  7.	RQWFFDLPL (SEQ ID NO: 215)	80.5
  8.	TALTGATEI (SEQ ID NO: 216)	0.9

  Positive Control	100.	+/-

Example 24: AG129 Mouse Challenge of Mimectopes of Predicted Human Epitopes from DENV2
• A study is performed on AG129 mouse using a cocktail of 2 peptide epitopes. The immunogenicity of the peptide epitopes is determined in AG129 mice against challenge with a lethal dose of mouse-adapted DENV 2 strain D2Y98P. AG129 mice, which lack IFN α/β and

  

  receptor signaling, injected intradermally in the footpad with 10⁴ PFU of DENV do not survive past day 5 post-injection. AG129 mice are vaccinated via intramuscular (IM) injection with either 2 μg or 10 μg of a cocktail of 2 peptide epitopes. The vaccines are given to AG129 mice with a prime and a boost (day 0 and day 28). The positive control group is vaccinated with heat-inactivated DENV 2. Phosphate-buffered saline (PBS) is used as a negative control. On day 56, mice are challenged with mouse-adapted DENV 2 and monitored for 10 days for weight loss, morbidity, and mortality. Mice that display severe illness, defined as >30% weight loss, a health score of 6 or above, extreme lethargy, and/or paralysis are euthanized.
Example 25: “Humanized” DENV Peptides Mouse Immunogenicity Study
• A study analyzing immunogenicity of the peptide epitopes on humanized mice is performed. A single-dose cocktail (30 μg) containing 3 different peptide epitopes are delivered by IM route of immunization with prime and boost (day 0, day 28). A T cell (ELISPOT and ICS) characterization may be performed on Day 7, Day 28, and Day 56.
Example 26: Testing of Non-Human Primate (NHP) Mimectopes of Predicted DENV Human Epitopes
• Non-human primate (NHP) mimectopes to the human epitopes may also be developed and tested for activity in NHP assays. The NHP mimectopes are designed based on the human antigen sequence. These mimectopes may be analyzed for in vivo activity in an NHP model using, for instance, restimulation assays. Once the NHPs have been infected, immune cells may be isolated and tested for sensitivity of activation by the particular mimectopes.
Example 27: Targeting of DENV Concatemeric Constructs Using Cytoplasmic Domain of MHC I
• MHC-1_V5 concatemer constructs were developed and transfected in HeLa cells. Triple immunofluorescence using Mitotracker Red (mitochondria), anti-V5, and anti-MHC-1 antibodies plus Dapi was performed. The data is shown in FIGS. 21-23 . FIG. 21 shows MHC-1_V5 concatemer transfection in HeLa cells. The arrows indicate V5-MHC1 colocalization (bottom right). FIG. 22 shows MHC-1_V5 concatemer transfection. The arrows indicate regions where V5 preferentially colocalizes with MHC1 and not with Mitotracker. FIG. 23 shows V5 concatemer transfection in HeLa cells. V5 has homogeneous cytoplasmic distribution preferentially colocalizes with MHC1 and not with Mitotracker. These data demonstrate that the V5 concatemer with the cytoplasmic domain from MHC class I colocalizes with MHC class I expression (FIG. 21 ), while the V5 concatemer without this sequence is only found in the cytoplasm (FIG. 23 ) following transfection in HeLa cells.
Example 28: In Vivo Analysis of DENV Concatemeric mRNA Epitope Construct
• • The Dengue concatemers used in this study consist of 8 repeats of the peptide TALGATET (SEQ ID NO: 299), a mouse CD8 T cell epitope found in the DENV2 envelope. The peptide repeats were linked via cathepsin B cleavage sites and modified with the various sequences as follows:
    (1) TALGATEI (SEQ ID NO: 299) peptide concatemer with no modification
    (2) TALGATEI (SEQ ID NO: 299) peptide concatemer with IgKappa signal peptide
    (3) TALGATEI (SEQ ID NO: 299) peptide concatemer with PEST sequence
    (4) TALGATEI (SEQ ID NO: 299) peptide concatemer with IgKappa signal peptide and PEST sequence
    (5) TALGATEI (SEQ ID NO: 299) peptide concatemer with MHC class I cytoplasmic domain
    (6) TALGATEI (SEQ ID NO: 299) peptide concatemer with IgKappa signal peptide and MHC class I cytoplasmic domain
(7) Heat-inactivated DENV2 (D2Y98P)
• (8) No immunization
• The immunogenicity of the peptide concatemeric candidate vaccines were determined in AG129 mice against challenge with a lethal dose of DENV strain D2Y98P. AG129 mice, which lack IFN α/β and

  

  receptor signaling, injected intradermally in the footpad with 10⁴ PFU of DENV do not survive past day 5 post-injection. (In this study, the mice died due to a problem with the heat-attenuation). The tested vaccines included constructs (1)-(8) disclosed above. AG129 mice were vaccinated via intramuscular (IM) injection with either 2 μg or 10 μg of the candidate vaccine. The vaccines were given to AG129 mice as a prime and a boost (second dose provided 28 days after the first dose). The positive control group was vaccinated with heat-inactivated DENV 2. Phosphate-buffered saline (PBS) was used as a negative control.
• On day 56, mice were challenged with mouse-adapted DENV 2 and monitored for 10 days for weight loss, morbidity, and mortality. Mice that displayed severe illness, defined as >30% weight loss, a health score of 6 or above, extreme lethargy, and/or paralysis were euthanized. Notably, mice “vaccinated” with heat-inactivated DENV (positive control group) became morbid and died (they were not included in the challenge portion of the study).
• In addition, individual serum samples were collected prior to challenge on day 54 and PBMCs were isolated and frozen for subsequent testing.
• The AG129 mice PBMCs were thawed and stimulated with TALGATEI (SEQ ID NO: 299) peptide for 5 hours in a standard intracellular cytokine assay. For intracellular cytokine staining, PBMCs were thawed and suspended in media. The TALGATEI (SEQ ID NO: 299) peptide was administered to stimulate the cells. After the addition of Golgi plug, cells were incubated at 37° C., 5% CO2 for 5 hours. Following stimulation, cells were surface stained, fixed, washed and put at 4° C. overnight. Intracellular staining was performed the following day and assayed via ELISPOT assay to quantify secretion of cytokines by T cells (CD8) as described above to determine T cell activation. If the peptide is an appropriate antigen, some cells would be present antigen during infection and would be capable of stimulating T cells. The results are shown in FIGS. 24A and 24B, which demonstrate that each of the peptides (1)-(6) stimulate T cell activation.
Example 29: Exemplary CHIKV Polypeptides
• The amino acids presented in the Table 27 are exemplary CHIKV antigenic polypeptides. To the extent that any exemplary antigenic peptide described herein includes a flag tag or V5, or a polynucleotide encodes a flag tag or V5, the skilled artisan understands that such flag tag or V5 is excluded from the antigenic polynucleotide in a vaccine formulation. Thus, any of the polynucleotides encoding proteins described herein are encompassed within the compositions of the invention without the flag tag or V5 sequence.

• TABLE 27
  Antigen
  identifier	Amino acid sequence
  SE_chikv-	MYEHVTVIPNTVGVPYKTLVNRPGYSPMVLEMELLSVTLEPTLSLDYITCEYKTVIPSPYVK
  Brazillian-	CCGTAECKDKSLPDYSCKVFTGVYPFMWGGAYCFCDTENTQLSEAHVEKSESCKTEFASAYR
  E1_KP164567-	AHTASASAKLRVLYQGNNITVAAYANGDHAVTVKDAKFIVGPMSSAWTPFDNKIVVYKGDVY
  71_72	NMDYPPFGAGRPGQFGDIQSRTPESEDVYANTQLVLQRPSAGTVHVPYSQAPSGFKYWLKER
  	GASLQHTAPFGCQIATNPVRAMNCAVGNMPISIDIPDAAFTRVVDAPSLTDMSCEVSACTHS
  	SDFGGVAIIKYAASKKGKCAVHSMTNAVTIREAEIEVEGNSQLQISFSTALASAEFRVQVCS
  	TQVHCAAECHPPKDHIVNYPASHTTLGVQDISATAMSWVQKITGGVGLVVAVAALILIVVLC
  	VSFSRH (SEQ ID NO. 37)
  SE_chikv-	MYEHVTVIPNTVGVPYKTLVNRPGYSPMVLEMELLSVTLEPTLSLDYITCEYKTVIPSPYVK
  Brazillian-	CCGTAECKDKNLPDYSCKVFTGVYPFMWGGAYCFCDAENTQLSEAHVEKSESCKTEFASAYR
  E1_KP164568-	AHTASASAKLRVLYQGNNITVTAYANGDHAVTVKDAKFIVGPMSSAWTPFDNKIVVYKGDVY
  69_70	NMDYPPFGAGRPGQFGDIQSRTPESKDVYANTQLVLQRPAAGTVHVPYSQAPSGFKYWLKER
  	GASLQHTAPFGCQIATNPVRAVNCAVGNMPISIDIPDAAFIRVVDAPSLTDMSCEVPACTHS
  	SDFGGVAIIKYAASKKGKCAVHSMTNAVTIREAEIEVEGNSQLQISFSTALASAEFRVQVCS
  	TQVHCVAECHPPKDHIVNYPASHTTLGVQDISATALSWVQKITGGVGLVVAVAALILIVVLC
  	VSFSRH (SEQ ID NO. 38)
  SE_chikv-	MSIKDHFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDS
  Brazillian-	HDWTKLRYMDNHMPADAERAGLFVRTSAPCTITGTMGHFILARCPKGETLTVGFTDGRKISH
  E2-	SCTHPFHHDPPVIGREKFHSRPQHGRELPCSTYAQSTAATAEEIEVHMPPDTPDRTLMSQQS
  E1_KP164567-	GNVKITVNSQTVRYKCNCGDSSEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAE
  71_72	FGDRKGKVHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEE
  	WVTHKKEIRLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTAVVL
  	SVASFILLSMVGVAVGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKAYEHVTVIPNT
  	VGVPYKTLVNRPGYSPMVLEMELLSVTLEPTLSLDYITCEYKTVIPSPYVKCCGTAECKDKS
  	LPDYSCKVFTGVYPFMWGGAYCFCDTENTQLSEAHVEKSESCKTEFASAYRAHTASASAKLR
  	VLYQGNNITVAAYANGDHAVTVKDAKFIVGPMSSAWTPFDNKIVVYKGDVYNMDYPPFGAGR
  	PGQFGDIQSRTPESEDVYANTQLVLQRPSAGTVHVPYSQAPSGFKYWLKERGASLQHTAPFG
  	CQIATNPVRAMNCAVGNMPISIDIPDAAFTRVVDAPSLTDMSCEVSACTHSSDFGGVAIIKY
  	AASKKGKCAVHSMTNAVTIREAEIEVEGNSQLQISFSTALASAEFRVQVCSTQVHCAAECHP
  	PKDHIVNYPASHTTLGVQDISATAMSWVQKITGGVGLVVAVAALILIVVLCVSFSRHMSIKD
  	HFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTK
  	LRYMDNHMPADAERAGLFVRTSAPCTITGTMGHFILARCPKGETLTVGFTDGRKISHSCTHP
  	FHHDPPVIGREKFHSRPQHGRELPCSTYAQSTAATAEEIEVHMPPDTPDRTLMSQQSGNVKI
  	TVNSQTVRYKCNCGDSSEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAEFGDRK
  	GKVHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVTHK
  	KEIRLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTAVVLSVASF
  	ILLSMVGVAVGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKAYEHVTVIPNTVGVPY
  	KTLVNRPGYSPMVLEMELLSVTLEPTLSLDYITCEYKTVIPSPYVKCCGTAECKDKSLPDYS
  	CKVFTGVYPFMWGGAYCFCDTENTQLSEAHVEKSESCKTEFASAYRAHTASASAKLRVLYQG
  	NNITVAAYANGDHAVTVKDAKFIVGPMSSAWTPFDNKIVVYKGDVYNMDYPPFGAGRPGQFG
  	DIQSRTPESEDVYANTQLVLQRPSAGTVHVPYSQAPSGFKYWLKERGASLQHTAPFGCQIAT
  	NPVRAMNCAVGNMPISIDIPDAAFTRVVDAPSLTDMSCEVSACTHSSDFGGVAIIKYAASKK
  	GKCAVHSMTNAVTIREAEIEVEGNSQLQISFSTALASAEFRVQVCSTQVHCAAECHPPKDHI
  	VNYPASHTTLGVQDISATAMSWVQKITGGVGLVVAVAALILIVVLCVSFSRH (SEQ ID
  	NO. 39)
  SQ-031495	MSTKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDS
  SE_chikv-	HDWTKLRYMDNHTPADAERAGLFVRTSAPCTITGTMGHFILTRCPKGETLTVGFTDSRKISH
  Brazillian-	SCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDRTLMSQQS
  E2-	GNVKITVNGQTVRYKCNCGGSNEGLITTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAE
  E1_KP164568-	LGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEE
  69_70	WVTHKKEVVLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVVV
  	SVASFVLLSMVGVAVGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKAYEHVTVIPNT
  	VGVPYKTLVNRPGYSPMVLEMELLSVTLEPTLSLDYITCEYKTVIPSPYVKCCGTAECKDKN
  	LPDYSCKVFTGVYPFMWGGAYCFCDAENTQLSEAHVEKSESCKTEFASAYRAHTASASAKLR
  	VLYQGNNITVTAYANGDHAVTVKDAKFIVGPMSSAWTPFDNKIVVYKGDVYNMDYPPFGAGR
  	PGQFGDIQSRTPESKDVYANTQLVLQRPAAGTVHVPYSQAPSGFKYWLKERGASLQHTAPFG
  	CQIATNPVRAVNCAVGNMPISIDIPDAAFIRVVDAPSLTDMSCEVPACTHSSDFGGVAIIKY
  	AASKKGKCAVHSMTNAVTIREAEIEVEGNSQLQISFSTALASAEFRVQVCSTQVHCVAECHP
  	PKDHIVNYPASHTTLGVQDISATALSWVQKITGGVGLVVAVAALILIVVLCVSFSRH (SEQ
  	ID NO. 40)
  SE_chikv-	MSIKDHFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDS
  Brazillian-	HDWTKLRYMDNHMPADAERAGLFVRTSAPCTITGTMGHFILARCPKGETLTVGFTDGRKISH
  E2_KP164567-	SCTHPFHHDPPVIGREKFHSRPQHGRELPCSTYAQSTAATAEEIEVHMPPDTPDRTLMSQQS
  71_72	GNVKITVNSQTVRYKCNCGDSSEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAE
  	FGDRKGKVHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEE
  	WVTHKKEIRLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTAVVL
  	SVASFILLSMVGVAVGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKA (SEQ ID
  	NO. 41)
  SE_chikv-	MSTKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDS
  Brazillian-	HDWTKLRYMDNHTPADAERAGLFVRTSAPCTITGTMGHFILTRCPKGETLTVGFTDSRKISH
  E2_KP164568-	SCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDRTLMSQQS
  69_70	GNVKITVNGQTVRYKCNCGGSNEGLITTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAE
  	LGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEE
  	WVTHKKEVVLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVVV
  	SVASFVLLSMVGVAVGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKA (SEQ ID
  	NO. 42)
  SE_CHIKV_C_E3	MEFIPTQTFYNRRYQPRPWAPRPTIQVIRPRPRPQRQAGQLAQLISAVNKLTMRAVPQQKPR
  _E2_6K_E1_no	RNRKNKKQRQKKQAPQNDPKQKKQPPQKKPAQKKKKPGRRERMCMKIENDCIFEVKHEGKVM
  Flag or V5 or	GYACLVGDKVMKPAHVKGTIDNADLAKLAFKRSSKYDLECAQIPVHMKSDASKFTHEKPEGY
  HA (Strain	YNWHHGAVQYSGGRFTIPTGAGKPGDSGRPIFDNKGRVVAIVLGGANEGARTALSVVTWNKD
  37997	IVTKITPEGAEEWSLALPVLCLLANTTFPCSQPPCTPCCYEKEPESTLRMLEDNVMRPGYYQ
  Senegal)	LLKASLTCSPHRQRRSTKDNFNVYKATRPYLAHCPDCGEGHSCHSPIALERIRNEATDGTLK
  	IQVSLQIGIKTDDSHDWTKLRYMDSHTPADAERAGLLVRTSAPCTITGTMGHFILARCPKGE
  	TLTVGFTDSRKISHTCTHPFHHEPPVIGRERFHSRPQHGKELPCSTYVQSTAATAEEIEVHM
  	PPDTPDRTLMTQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKIDQCHAAVTNHK
  	NWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVTMLLYPDHPTLL
  	SYRNMGQEPNYHEEWVTHKKEVTLTVPTEGLEVTWGNNEPYKYWPQMSTNGTAHGHPHEIIL
  	YYYELYPTMTVVIVSVASFVLLSMVGTAVGMCVCARRRCITPYELTPGATVPFLLSLLCCVR
  	TTKAATYYEAAAYLWNEQQPLFWLQALIPLAALIVLCNCLKLLPCCCKTLAFLAVMSIGAHT
  	VSAYEHVTVIPNTVGVPYKTLVNRPGYSPMVLEMELQSVTLEPTLSLDYITCEYKTVIPSPY
  	VKCCGTAECKDKSLPDYSCKVFTGVYPFMWGGAYCFCDAENTQLSEAHVEKSESCKTEFASA
  	YRAHTASASAKLRVLYQGNNITVAAYANGDHAVTVKDAKFVVGPMSSAWTPFDNKIVVYKGD
  	VYNMDYPPFGAGRPGQFGDIQSRTPESKDVYANTQLVLQRPAAGTVHVPYSQAPSGFKYWLK
  	ERGASLQHTAPFGCQIATNPVRAVNCAVGNIPISIDIPDAAFTRVVDAPSVTDMSCEVPACT
  	HSSDFGGVAIIKYTASKKGKCAVHSMTNAVTIREADVEVEGNSQLQISFSTALASAEFRVQV
  	CSTQVHCAAACHPPKDHIVNYPASHTTLGVQDISTTAMSWVQKITGGVGLIVAVAALILIVV
  	LCVSFSRH (SEQ ID NO. 43)
  SE_CHIKV_C_E3	MEFIPTQTFYNRRYQPRPWAPRPTIQVIRPRPRPQRQAGQLAQLISAVNKLTMRAVPQQKPR
  _E2_6K_E1-no	RNRKNKKQRQKKQAPQNDPKQKKQPPQKKPAQKKKKPGRRERMCMKIENDCIFEVKHEGKVM
  Flag or V5 or	GYACLVGDKVMKPAHVKGTIDNADLAKLAFKRSSKYDLECAQIPVHMKSDASKFTHEKPEGY
  HA_DX	YNWHHGAVQYSGGRFTIPTGAGKPGDSGRPIFDNKGRVVAIVLGGANEGARTALSVVTWNKD
  	IVTKITPEGAEEWSLALPVLCLLANTTFPCSQPPCTPCCYEKEPESTLRMLEDNVMRPGYYQ
  	LLKASLTCSPHRQRRSTKDNFNVYKATRPYLAHCPDCGEGHSCHSPIALERIRNEATDGTLK
  	IQVSLQIGIKTDDSHDWTKLRYMDSHTPADAERAGLLVRTSAPCTITGTMGHFILARCPKGE
  	TLTVGFTDSRKISHTCTHPFHHEPPVIGRERFHSRPQHGKELPCSTYVQSTAATAEEIEVHM
  	PPDTPDRTLMTQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKIDQCHAAVTNHK
  	NWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVTMLLYPDHPTLL
  	SYRNMGQEPNYHEEWVTHKKEVTLTVPTEGLEVTWGNNEPYKYWPQMSTNGTAHGHPHEIIL
  	YYYELYPTMTVVIVSVASFVLLSMVGTAVGMCVCARRRCITPYELTPGATVPFLLSLLCCVR
  	TTKAATYYEAAAYLWNEQQPLFWLQALIPLAALIVLCNCLKLLPCCCKTLAFLAVMSIGAHT
  	VSAYEHVTVIPNTVGVPYKTLVNRPGYSPMVLEMELQSVTLEPTLSLDYITCEYKTVIPSPY
  	VKCCGTAECKDKSLPDYSCKVFTGVYPFMWGGAYCFCDAENTQLSEAHVEKSESCKTEFASA
  	YRAHTASASAKLRVLYQGNNITVAAYANGDHAVTVKDAKFVVGPMSSAWTPFDNKIVVYKGD
  	VYNMDYPPFGAGRPGQFGDIQSRTPESKDVYANTQLVLQRPAAGTVHVPYSQAPSGFKYWLK
  	ERGASLQHTAPFGCQIATNPVRAVNCAVGNIPISIDIPDAAFTRVVDAPSVTDMSCEVPACT
  	HSSDFGGVAIIKYTASKKGKCAVHSMTNAVTIREADVEVEGNSQLQISFSTALASAEFRVQV
  	CSTQVHCAAACHPPKDHIVNYPASHTTLGVQDISTTAMSWVQKITGGVGLIVAVAALILIVV
  	LCVSFSRH (SEQ ID NO. 44)
  SE_CHIKV_E1_	MYEHVTVIPNTVGVPYKTLVNRPGYSPMVLEMELQSVTLEPTLSLDYITCEYKTVIPSPYVK
  no Flag or V5	CCGTAECKDKSLPDYSCKVFTGVYPFMWGGAYCFCDAENTQLSEAHVEKSESCKTEFASAYR
  	AHTASASAKLRVLYQGNNITVAAYANGDHAVTVKDAKFVVGPMSSAWTPFDNKIVVYKGDVY
  	NMDYPPFGAGRPGQFGDIQSRTPESKDVYANTQLVLQRPAAGTVHVPYSQAPSGFKYWLKER
  	GASLQHTAPFGCQIATNPVRAVNCAVGNIPISIDIPDAAFTRVVDAPSVTDMSCEVPACTHS
  	SDFGGVAIIKYTASKKGKCAVHSMTNAVTIREADVEVEGNSQLQISFSTALASAEFRVQVCS
  	TQVHCAAACHPPKDHIVNYPASHTTLGVQDISTTAMSWVQKITGGVGLIVAVAALILIVVLC
  	VSFSRH (SEQ ID NO. 45)
  CHIKV_E2_6K_	MSTKDNFNVYKATRPYLAHCPDCGEGHSCHSPIALERIRNEATDGTLKIQVSLQIGIKTDDS
  E1_no Flag or	HDWTKLRYMDSHTPADAERAGLLVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISH
  V5	TCTHPFHHEPPVIGRERFHSRPQHGKELPCSTYVQSTAATAEEIEVHMPPDTPDRTLMTQQS
  	GNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKIDQCHAAVTNHKNWQYNSPLVPRNAE
  	LGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVTMLLYPDHPTLLSYRNMGQEPNYHEE
  	WVTHKKEVTLTVPTEGLEVTWGNNEPYKYWPQMSTNGTAHGHPHEIILYYYELYPTMTVVIV
  	SVASFVLLSMVGTAVGMCVCARRRCITPYELTPGATVPFLLSLLCCVRTTKAATYYEAAAYL
  	WNEQQPLFWLQALIPLAALIVLCNCLKLLPCCCKTLAFLAVMSIGAHTVSAYEHVTVIPNTV
  	GVPYKTLVNRPGYSPMVLEMELQSVTLEPTLSLDYITCEYKTVIPSPYVKCCGTAECKDKSL
  	PDYSCKVFTGVYPFMWGGAYCFCDAENTQLSEAHVEKSESCKTEFASAYRAHTASASAKLRV
  	LYQGNNITVAAYANGDHAVTVKDAKFVVGPMSSAWTPFDNKIVVYKGDVYNMDYPPFGAGRP
  	GQFGDIQSRTPESKDVYANTQLVLQRPAAGTVHVPYSQAPSGFKYWLKERGASLQHTAPFGC
  	QIATNPVRAVNCAVGNIPiSIDIPDAAFTRVVDAPSVTDMSCEVPACTHSSDFGGVAIIKYT
  	ASKKGKCAVHSMTNAVTIREADVEVEGNSQLQISFSTALASAEFRVQVCSTQVHCAAACHPP
  	KDHIVNYPASHTTLGVQDISTTAMSWVQKITGGVGLIVAVAALILIVVLCVSFSRH (SEQ
  	ID NO. 46)
  SE_CHIKV_E2_	MSTKDNFNVYKATRPYLAHCPDCGEGHSCHSPIALERIRNEATDGTLKIQVSLQIGIKTDDS
  no Flag or	HDWTKLRYMDSHTPADAERAGLLVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISH
  V5	TCTHPFHHEPPVIGRERFHSRPQHGKELPCSTYVQSTAATAEEIEVHMPPDTPDRTLMTQQS
  	GNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKIDQCHAAVTNHKNWQYNSPLVPRNAE
  	LGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVTMLLYPDHPTLLSYRNMGQEPNYHEE
  	WVTHKKEVTLTVPTEGLEVTWGNNEPYKYWPQMSTNGTAHGHPHEIILYYYELYPTMTVVIV
  	SVASFVLLSMVGTAVGMCVCARRRCITPYELTPGATVPFLLSLLCCVRTTKA (SEQ ID
  	NO. 47)

Example 30. ZIKV Vaccines
• The design of preferred Zika vaccine mRNA constructs of the invention encode prME proteins from the Zika virus intended to produce significant immunogenicity. The open reading frame comprises a signal peptide (to optimize expression into the endoplasmic reticulum) followed by the Zika prME polyprotein sequence. The particular prME sequence used is from a Micronesian strain (2007) that most closely represents a consensus of contemporary strain prMEs. This construct has 99% prME sequence identity to the current Brazilian isolates.
• Within the Zika family, there is a high level of homology within the prME sequence (>90%) across all strains so far isolated (See Table 28 below). The high degree of homology is also preserved when comparing the original isolates from 1947 to the more contemporary strains circulating in Brazil in 2015, suggesting that there is “drift” occurring from the original isolates. Furthermore, attenuated virus preparations have provided cross-immunization to all other strains tested, including Latin American/Asian, and African.
• Overall, this data suggests that cross-protection of all Zika strains is possible with a vaccine based on prME.

• TABLE 28
  Zika virus prME homology

  Zika virus

  Pairwise AA % identity

  Country of

  Year of

  to Brazilian isolates

  Strain	isolation	isolation	prME	Genome

  South

  Suriname

  	2015	100.0%	99.0%
  American
  Asian	Cambodia
  	2010	99.4%	99.1%
  	French Polynesia	2013	99.7%	99.4%
  	Micronesia
  	2007	98.8%	97.1%
  African	Senegal	2002	92.5%	89.9%
  	Ugnada	1947	91.0%	87.3%

• In fact, the prM/M and E proteins of ZIKV have a very high level (99%) of sequence conservation between the currently circulating Asiatic and Brazilian viral strains. The sequence alignment of the prM/M and E proteins is shown in FIG. 27 .
• The M and E proteins are on the surface of the viral particle. Neutralizing antibodies predominantly bind to the E protein, the preM/M protein functions as a chaperone for proper folding of E protein and prevent premature fusion of E protein within acidic compartments along the cellular secretory pathway.
• Described herein are examples of ZIKV vaccine designs comprising mRNA encoding the both prM/M and E proteins or E protein alone (FIGS. 26A and 26B). FIG. 26A depicts mRNA encoding an artificial signal peptide fused to prM protein fused to E protein. FIG. 2B depicts mRNA encoding an artificial signal peptide fused to E protein.
• ZIKV vaccine constructs can encode the prME or E proteins from different strains, for example, Brazil_isolate_ZikaSPH2015 or ACD75819_Micronesia, having a signal peptide fused to the N-termini of the antigenic protein(s). In this example, ZIKV vaccines comprise mRNAs encoding antigenic polypeptides having amino acid sequences of SEQ ID NO: 50-59. The examples are not meant to be limiting.
Example 31. Expression of ZIKV prME Protein in Mammalian Cells Using ZIKV mRNA Vaccine Construct
• The ZIKV prME mRNA vaccine construct were tested in mammalian cells (239T cells) for the expression of ZIKV prME protein. 293T cells were plated in 24-well plates and were transfected with 2 μg of ZIKV prME mRNA using a Lipofectamine transfection reagent. The cells were incubated for the expression of the ZIKV prME proteins before they were lysed in an immunoprecipitation buffer containing protease inhibitor cocktails. Reducing agent was not added to the lysis buffer to ensure that the cellular proteins were in a non-reduced state. Cell lysates were centrifuged at 8,000×g for 20 mins to collect lysed cell precipitate. The cell precipitates were then stained with anti ZIKV human serum and goat anti-human Alexa Fluor 647. Fluorescence was detected as an indication of prME expression (FIG. 28 ).
• The expression of ZIKV prME protein was also detected by fluorescence-activated cell sorting (FACS) using a flow cytometer. 293F cells (2×10⁶ cells/ml, 30 ml) were transfected with 120 μg PEI, 1 ml of 150 mM NaCl, and 60 μg prME mRNA. Transfected cells were incubated for 48 hours at 37° C. in a shaker at 130 rpm and under 5% CO₂. The cells were then washed with PBS buffer containing 2% FBS and fixed in a fixation buffer (PBS buffer containing formalin) for 20 minutes at room temperature. The fixed cells were permeabilized in a permeabilization buffer (PBS+1% Triton X100+1 μl of Golgi plug/ml of cells). The permeabilized cells were then stained with anti-ZIKV human serum (1:20 dilution) and goat anti-human Alexa Fluor 647 secondary antibody, before they were sorted on a flow cytometer. As shown in FIG. 29 , FIG. 30A and FIG. 30B, cells transfected with prME mRNA and stained with the anti-ZIKA human serum shifted to higher fluorescent intensity, indicating that prME expressed from the ZIKV mRNA vaccine constructs in the transfected cells.
Example 32. Expression, Purification and Characterization of Zika VLPs
• VLPs were made in HeLa cells and in HEK293t cells and purified via PEG precipitation or ultracentrifugation, respectively. Cells were cultured in culture media. Prior to transfection, cells were passaged twice in virus growth media+10% FBS to media adaptation.
• Cells were seeded the day before transfection into T-175 flask. 100 μg of prME-encoding mRNA was transfected using 100 μg pf lipofectamine as per manufacturer's protocol. 6 hours post transfection, monolayers were washed twice with 1×PBS and 20 mL of virus growth media was added. Supernatant was collected 24-48 hours post transfection by centrifugation at 2000×g for 10 mins and 0.22 μm filtration.
• For VLP purification via PEG precipitation, VLP's were concentrated using Biovision PEG precipitation kit as per manufacturer's protocol. In brief, supernatant with VLP's was mixed with PEG8000 and incubated at 4° C. for 16 hours. After incubation, mixture was centrifuged at 3000×g for 30 mins. Pellet containing concentrated VLP's was collected and suspended into PBS. VLP's were further buffer exchanged into PBS (1:500) using amicon ultra 100MWCO filter. Purified samples were negative stained (FIG. 32 ).
• Expression of prME from the vaccine mRNA constructs on the invention was demonstrated to result in the production of virus like particles (VLPs) that are expected to present to the immune system as identical to Zika virus particles. FIG. 32 shows negative stain electron micrographs of supernatants from HeLa cells transfected with mRNA encoding Zika prME. The virus-like particles (VLPs), purified by PEG precipitation, have highly uniform size (˜35-40 nm) and morphology. The bumpy appearance of the VLP surface appears to reflect mostly immature morphology due to expression from HeLa cells, which have very low expression of furin, a host protease that is required for maturation the viral envelope. Upon maturation, these VLPs will have an exterior structure essentially identical to wild type viral particles, thus eliciting a broad immune response to future Zika virus exposure.
• For VLP purification via ultracentrifugation, 293T cells were transfected with Zika prME mRNA as described herein. Supernatant was collected 24 hours after changing the media as described herein. (30 hours post transfection) VLP's were concentrated using Biovision PEG virus precipitation kit into 500 μL volume. VLP were further purified using a 10-50% sucrose gradient. Sample layer was seen between 20-30% sucrose layers and collected. VLP's were buffered exchanged into PBS by 1:1000 dilution using a 100MWCO amicon ultra filter. VLP's concentrated after PEG precipitation and ultracentrifuge purified VLP were analyzed on a reducing SDS-PAGE gel for purity (FIG. 33 ).
Example 33: Immunogenicity Studies Study A
• The instant study was designed to test the immunogenicity in Balb/c mice of candidate ZIKV vaccines comprising a mRNA polynucleotide encoding ZIKV prME. Four groups of Balb/c mice (n=5) were immunized intramuscularly (IM) with 10 μg (n=2) or 2 μg (n=2) of the candidate vaccine. One group of mice was administered PBS intramuscularly as a control. All mice were administered an initial dose of vaccine (Groups 1-4) or PBS (Group 5) on Day 0, and then the mice in Groups 1 and 3 were administered a boost dose on Day 21, while the mice in Group 5 were administered PBS on Day 21. All mice were bled on Day 41. See Table 29. Anti-Zika neutralization IgG titer was determined on Day −1, Day 28 and Day 41 (FIG. 33B).

• TABLE 29
  ZIKV mRNA Vaccine Immunogenicity Study

  	Study
  	design
  	BALB/C		Immunization

  Group	Vaccine	N	Dose	Route	Prime	Boost	Endpoint
  1	Zika	5	10 ug	IM	Day	Day	Terminal
  	prME
  				0	21	bleeds
  	vaccine						on Day 41.
  2	Zika	5	10 ug	IM	Day	NA	Anti Zika
  	prME
  				0		neutralizing
  	vaccine						IgG titer.
  3	Zika	5	2 ug	IM	Day	Day
  	prME
  				0	21
  	vaccine
  4	Zika	5	2 ug	IM	Day	NA
  	prME
  				0
  	vaccine
  5	PBS	5	NA	IM	Day	Day
  					0	21

• Day 42 neutralizing titers reached EC50s of 427 for 2 μg and 690 for 10 μg. The control serum in this experiment was from naturally infected immunocompromised mice (Ifnar1−/−, derived from B/6 lineage) in which high viral loads would be achieved.
Study B
• The instant study is designed to test the immunogenicity in mice of candidate ZIKV vaccines comprising a mRNA polynucleotide encoding ZIKV polyprotein. Mice are immunized intravenously (IV), intramuscularly (IM), or intradermally (ID) with candidate vaccines. Up to three immunizations are given at 3-week intervals (i.e., at weeks 0, 3, 6, and 9), and sera are collected after each immunization until weeks 33-51. Serum antibody titers against ZIKV polyprotein are determined by ELISA.
Example 34: ZIKV Rodent Challenge Study A
• The instant study was designed to test the efficacy in AG129 mice of candidate ZIKV vaccines against a lethal challenge using a ZIKV vaccine comprising mRNA encoding ZIKV prME. Four groups of AG129 mice (n=8) were immunized intramuscularly (IM) with 10 μg (n=2) or 2 μg (n=2) of the candidate vaccine. One group of mice was administered PBS intramuscularly as a control. All mice were administered an initial dose of vaccine (Groups 1-4) or PBS (Group 5) on Day 0, and then the mice in Groups 1 and 3 were administered a boost dose on Day 21, while the mice in Group 5 were administered PBS on Day 21. All mice were challenged with a lethal dose of ZIKV in Day 42. All mice were then monitored for survival and weight loss. Anti-Zika neutralization IgG titer was determined on Day −1, Day 28 and Day 41, and viral load was determined 5 days post challenge.

• TABLE 30
  ZIKV In vivo challenge

  	Study
  	design
  	AG129		Immunization	Chal-	End-

  Group	Vaccine	n	Dose	Route	Prime	Boost	lenge	point
  1	Zika	8	10 ug	IM	Day	Day	Day	Monitor
  	prME
  				0	21	42	for
  	vaccine							survival
  2	Zika	8	10 ug	IM	Day	NA		and
  	prME				0			weight
  	vaccine							loss.
  3	Zika	8	2 ug	IM	Day	Day		Viral
  	prME
  				0	21		load at
  	vaccine							Day	5
  4	Zika	8	2 ug	IM	Day	NA
  	prME
  				0
  	vaccine
  5	PBS	8	NA	IM	Day	Day
  					0	21

Study B
• The instant study is designed to test the efficacy in AG129 mice of candidate ZIKV vaccines against a lethal challenge using a ZIKV vaccine comprising mRNA encoding ZIKV polyprotein. Animals are challenged with a lethal dose of the ZIKV. Animals are immunized intravenously (IV), intramuscularly (IM), or intradermally (ID) at week 0 and week 3 with candidate ZIKV vaccines with and without adjuvant. The animals are then challenged with a lethal dose of ZIKV on week 7 via IV, IM or ID. Endpoint is day 13 post infection, death or euthanasia. Animals displaying severe illness as determined by >30% weight loss, extreme lethargy or paralysis are euthanized. Body temperature and weight are assessed and recorded daily.
• In experiments where a lipid nanoparticle (LNP) formulation is used, the formulation may include a cationic lipid, non-cationic lipid, PEG lipid and structural lipid in the ratios 50:10:1.5:38.5. The cationic lipid is DLin-KC2-DMA or DLin-MC3-DMA (50 mol %), the non-cationic lipid is DSPC (10 mol %), the PEG lipid is PEG-DOMG or PEG-DMG (1.5 mol %) and the structural lipid is cholesterol (38.5 mol %), for example.

• TABLE 31
  ZIKV Nucleic Acid Sequences

  		SEQ
  		ID
  Description	Sequence	NO:
  Zika virus	ATGAAAAACCCAAAGAAGAAATCCGGAGGATTCCGGATTGTCAATATGCTAAAAC	48
  strain MR 766	GCGGAGTAGCCCGTGTAAACCCCTTGGGAGGTTTGAAGAGGCTGCCAGCCGGACT
  polyprotein	TCTGCTGGGTCATGGACCCATCAGAATGGTTTTGGCGATATTAGCCTTTTTGAGA
  gene,	TTCACAGCAATCAAGCCATCACTGGGCCTCATCAACAGATGGGGTACCGTGGGGA
  complete cds	AAAAAGAGGCTATGGAAATAATAAAAAAATTTAAGAAAGATCTTGCTGCCATGTT
  GenBank	GAGAATAATCAATGCTAGGAAGGAGAGGAAGAGACGTGGCGCAGACACCAGCATC
  Accession:	GGAATCGTTGGCCTCCTGTTGACTACAGCCATGGCAGCAGAGATCACTAGACGTG
  DQ859059	GGAGTGCATACTACATGTACTTGGATAGGAGCGATGCAGGGAAGGCCATTTCTTT
  	CGCTACCACATTGGGGGTGAACAAATGCCATGTGCAGATCATGGACCTCGGGCAC
  	ATGTGTGACGCCACCATGAGCTATGAATGCCCTATGCTGGACGAGGGGGTGGAAC
  	CAGATGACGTCGATTGCTGGTGCAACACGACATCAACTTGGGTTGTGTACGGAAC
  	CTGTCATCATAAAAAAGGTGAAGCACGGCGATCTAGAAGAGCCGTCACGCTCCCA
  	TCTCACTCCACAAGGAAATTGCAAACGCGGTCGCAGACTTGGCTAGAATCAAGAG
  	AATACACAAAGCACCTGATCAAGGTTGAAAATTGGATATTCAGGAACCCTGGTTT
  	TACGCTAGTGGCTGTCGCCATCGCCTGGCTTTTGGGAAGCTCGACGAGCCAAAAA
  	GTCATATACTTGGTCATGATACTGCTGATTGCCCCGGCATACAGTATCAGGTGCA
  	TAGGAGTCAGCAATAGAGACTTCGTGGAGGGCATGTCAGGTGGGACCTGGGTTGA
  	CGTTGTCCTGGAACATGGAGGCTGCGTCACCGTGATGGCACAGGACAAGCCAACA
  	GTTGACATAGAGCTGGTCACAACAACGGTTAGTAACATGGCCGAGGTGAGATCCT
  	ATTGTTACGAGGCATCAATATCGGACATGGCTTCGGACAGTCGCTGCCCAACACA
  	AGGTGAAGCCTACCTTGACAAGCAATCAGACACTCAATATGTTTGCAAAAGAACA
  	TTGGTGGACAGAGGTTGGGGAAATGGGTGTGGACTCTTTGGCAAAGGGAGTTTGG
  	TGACATGTGCTAAGTTCACGTGCTCCAAGAAGATGACTGGGAAGAGCATTCAGCC
  	GGAGAACCTGGAGTATCGGATAATGCTATCAGTGCATGGCTCCCAGCACAGTGGG
  	ATGATTGTTAATGATGAAAACAGAGCGAAGGTCGAGGTTACGCCCAATTCACCAA
  	GAGCAGAAGCAACCCTGGGAGGCTTTGGAAGCTTAGGACTTGATTGTGAACCAAG
  	GACAGGCCTTGACTTTTCAGATCTGTATTACCTAACCATGAATAACAAGCATTGG
  	TTGGTGCACAAAGAGTGGTTTCATGACATCCCATTGCCCTGGCATGCTGGGGCAG
  	ACACTGGAACTCCACATTGGAACAACAAGGAGGCATTAGTGGAATTCAAGGACGC
  	CCACGCCAAGAGGCAAACCGTCGTGGTTTTGGGGAGCCAGGAAGGAGCCGTCCAC
  	ACGGCTCTTGCTGGAGCTCTAGAGGCTGAGATGGATGGTGCAAAGGGAAGGCTAT
  	TCTCTGGCCACTTGAAATGTCGCTTAAAAATGGACAAGCTTAGATTGAAGGGCGT
  	GTCATATTCCTTGTGCACCGCGGCATTCACATTCACCAAGGTCCCGGCTGAAACA
  	CTACATGGAACAGTCACAGTGGAGGTGCAGTATGCAGGGACAGATGGACCCTGCA
  	AGGTCCCAGCCCAGATGGCGGTGGACATGCAGACCTTGACCCCAGTCGGAAGGCT
  	GATAACCGCCAACCCCGTGATTACTGAAAGCACTGAGAATTCAAAGATGATGTTG
  	GAGCTCGACCCACCATTTGGGGATTCTTACATTGTCATAGGAGTTGGGGATAAGA
  	AAATCACCCATCACTGGCATAGGAGTGGCAGCACCATTGGAAAAGCATTTGAAGC
  	CACTGTGAGAGGCGCTAAGAGAATGGCAGTCCTGGGGGACACAGCTTGGGACTTT
  	GGATCAGTCGGAGGTGTGTTTAACTCATTGGGCAAGGGCATTCATCAGATTTTTG
  	GAGCAGCTTTCAAATCACTGTTTGGAGGAATGTCCTGGTTCTCACAGATCCTCAT
  	AGGCACTCTGCTGGTGTGGTTAGGTCTGAACACAAAGAATGGGTCTATCTCCCTC
  	ACATGCTTAGCCCTGGGGGGAGTGATGATCTTCCTCTCCACGGCTGTTTCTGCTG
  	ACGTGGGGTGCTCGGTGGACTTCTCAAAAAAAGAAACGAGATGTGGCACGGGGGT
  	GTTCGTCTACAATGACGTTGAAGCCTGGAGGGACCGGTACAAGTACCATCCTGAC
  	TCCCCTCGTAGACTGGCAGCAGCCGTTAAGCAAGCTTGGGAAGAGGGGATTTGTG
  	GGATCTCCTCTGTTTCTAGAATGGAAAACATAATGTGGAAATCAGTGGAAGGAGA
  	GCTCAATGCAATCCTAGAGGAGAATGGAGTCCAACTGACAGTTGTTGTGGGATCT
  	GTAAAAAACCCCATGTGGAGAGGCCCACAAAGATTGCCAGTGCCTGTGAATGAGC
  	TGCCCCATGGCTGGAAAGCCTGGGGGAAATCGTACTTTGTTAGGGCGGCAAAGAC
  	CAACAACAGTTTTGTTGTCGACGGTGACACATTGAAGGAATGTCCGCTCAAGCAC
  	AGAGCATGGAACAGCTTCCTCGTGGAGGATCACGGGTTTGGGGTCTTCCACACCA
  	GTGTTTGGCTTAAGGTTAGAGAAGATTACTCACTGGAGTGTGACCCAGCCGTCAT
  	AGGAACAGCTGTTAAGGGAAAGGAGGCCGCGCACAGTGATCTAGGCTATTGGATT
  	GAAAGTGAAAAGAATGACACATGGAGGCTGAAGAGGGCTCATTTGATTGAGATGA
  	AAACATGTGAGTGGCCAAAGTCTCACACACTGTGGACAGATGGAGTGGAAGAAAG
  	TGATCTGATCATACCCAAGTCTTTAGCTGGTCCACTCAGCCACCACAACACCAGA
  	GAGGGTTACAGAACTCAAGTGAAAGGGCCATGGCATAGTGAGGAGCTTGAAATCC
  	GATTTGAGGAATGTCCAGGTACCAAGGTTCATGTGGAGGAGACATGCGGAACGAG
  	AGGACCATCTCTGAGATCAACCACTGCAAGCGGAAGGGTCATTGAGGAATGGTGC
  	TGTAGGGAATGCACAATGCCCCCACTATCGTTCCGAGCAAAAGATGGCTGCTGGT
  	ATGGAATGGAGATAAGGCCTAGGAAAGAACCAGAGAGCAACTTAGTGAGGTCAAT
  	GGTGACAGCGGGATCAACCGATCATATGGATCATTTTTCTCTTGGAGTGCTTGTG
  	ATTCTACTCATGGTGCAGGAAGGGTTGAAGAAGAGAATGACCACAAAGATCATCA
  	TGAGCACATCAATGGCAGTGCTGGTGGCCATGATCTTGGGAGGATTCTCAATGAG
  	TGACCTGGCTAAGCTTGTGATCCTGATGGGGGCCACTTTCGCAGAAATGAACACT
  	GGAGGAGACGTAGCTCACTTGGCATTAGTAGCGGCATTTAAAGTCAGACCAGCCT
  	TGCTGGTCTCATTTATCTTCAGAGCCAACTGGACACCTCGTGAGAGCATGCTGCT
  	AGCCTTGGCTTCGTGTCTTCTGCAAACTGCGATCTCCGCTCTTGAAGGCGACTTG
  	ATGGTCCTCGTTAATGGATTTGCTTTGGCCTGGTTGGCAATACGTGCAATGGCCG
  	TGCCACGCACTGACAACATCGCTCTAGCAATTCTGGCTGCTCTAACACCACTAGC
  	CCGAGGCACACTGCTCGTGGCATGGAGAGCGGGCCTCGCCACTTGTGGAGGGTTC
  	ATGCTCCTCTCCCTGAAAGGGAAAGGTAGTGTGAAGAAGAACCTGCCATTCGTCG
  	CGGCCTTGGGATTGACCGCTGTGAGAATAGTGGACCCCATTAATGTGGTGGGACT
  	ACTGTTACTCACAAGGAGTGGGAAGCGGAGCTGGCCCCCTAGTGAAGTGCTCACT
  	GCTGTCGGCCTGATATGTGCATTGGCCGGAGGGTTTGCCAAGGCAGACATAGAGA
  	TGGCTGGGCCCATGGCGGCAGTGGGCCTGCTAATTGTCAGTTATGTGGTCTCGGG
  	AAAGAGTGTAGATATGTACATTGAAAGAGCAGGTGACATCACATGGGAGAAAGAC
  	GCGGAAGTCACTGGAAACAGTCCTCGGCTTGACGTGGCACTAGATGAGAGTGGTG
  	ATTTCTCTCTGGTGGAGGAAGATGGTCCACCCATGAGAGAGATCATACTTAAGGT
  	GGTCTTGATGGCCATCTGTGGCATGAACCCAATAGCCATACCTTTTGCTGCAGGA
  	GCGTGGTATGTGTATGTGAAGACTGGGAAAAGGAGTGGTGCCCTCTGGGACGTGC
  	CTGCTCCGAAAGAAGTGAAAAAAGGAGAGACCACAGATGGAGTGTACAGAGTGAT
  	GACTCGCAGACTGCTGGGTTCAACACAAGTTGGAGTGGGAGTCATGCAGGAGGGA
  	GTCTTCCACACCATGTGGCACGTCACAAAAGGGGCCGCATTGAGGAGCGGTGAAG
  	GGAGACTTGATCCATACTGGGGGGATGTCAAGCAGGACTTGGTGTCATATTGTGG
  	GCCTTGGAAGCTGGACGCAGCTTGGGACGGAGTTAGTGAGGTGCAGCTTCTGGCC
  	GTACCCCCTGGAGAGAGAGCCAGAAACATTCAGACTCTGCCTGGAATATTTAAGA
  	CAAAGGATGGGGACATCGGAGCAGTTGCTTTGGACTATCCTGCAGGAACCTCAGG
  	ATCTCCGATCCTAGACAAATGCGGGAGAGTGATAGGACTCTATGGCAATGGGGTT
  	GTGATCAAGAACGGAAGCTATGTTAGTGCTATAACCCAGGGAAAGAGGGAGGAGG
  	AGACTCCGGTTGAGTGTTTTGAACCCTCGATGCTGAAGAAGAAGCAGCTAACTGT
  	CCTGGACCTGCATCCAGGGGCTGGGAAAACCAGGAGAGTTCTTCCTGAAATAGTC
  	CGTGAAGCTATAAAGAAGAGACTCCGCACGGTGATCTTGGCACCAACCAGGGTCG
  	TCGCTGCTGAGATGGAGGAAGCCCTGAGAGGACTTCCGGTGCGTTACATGACAAC
  	AGCAGTCAAGGTCACCCATTCTGGGACAGAAATCGTTGATTTGATGTGCCATGCC
  	ACCTTCACTTCACGCCTACTACAACCCATTAGAGTCCCTAATTACAACCTCTACA
  	TCATGGATGAAGCCCATTTCACAGACCCCTCAAGCATAGCTGCAAGAGGATATAT
  	ATCAACAAGGGTTGAGATGGGCGAGGCAGCAGCCATCTTTATGACTGCCACACCA
  	CCAGGAACCCGCGATGCGTTTCCAGATTCCAACTCACCAATCATGGACACAGAAG
  	TGGAAGTCCCAGAGAGAGCCTGGAGCTCAGGCTTTGATTGGGTGACGGACCATTC
  	TGGGAAAACAGTTTGGTTCGTTCCAAGCGTGAGGAATGGAAATGAAATCGCAGCC
  	TGTCTGACAAAGGCTGGAAAGCGGGTTATACAGCTTAGTAGGAAAACTTTTGAGA
  	CAGAGTTTCAGAAAACAAAAAATCAAGAGTGGGACTTTGTCATAACAACTGACAT
  	CTCAGAGATGGGTGCCAACTTCAAGGCTGACCGGGTTATAGATTCCAGGAGATGC
  	CTAAAGCCAGTTATACTTGATGGTGAGAGAGTCATCTTGGCTGGGCCCATGCCTG
  	TCACGCATGCTAGCGCTGCTCAGAGGAGAGGACGTATAGGCAGGAACCCCAACAA
  	GCCTGGAGATGAGTACATGTATGGAGGTGGGTGTGCGGAGACTGATGAAGACCAT
  	GCACATTGGCTTGAAGCAAGAATGCTTCTTGACAACATTTACCTCCAGGATGGCC
  	TCATAGCCTCGCTCTATCGACCTGAGGCCGACAAGGTAGCCGCCATTGAGGGAGA
  	GTTTAAGCTGAGGACAGAGCAAAGGAAGACCTTTGTGGAACTCATGAAGAGAGGA
  	GATCTTCCCGTTTGGTTGGCCTACCAGGTTGCATCTGCCGGAATAACTTATACAG
  	ACAGAAGATGGTGTTTTGATGGCACAACCAACAACACCATAATGGAAGACAGTGT
  	ACCAGCAGAGGTGTGGACCAAGTATGGAGAGAAGAGAGTGCTCAAACCAAGATGG
  	ATGGACGCCAGGGTCTGCTCAGATCATGCGGCCCTGAAGTCGTTCAAAGAATTCG
  	CCGCTGGGAAAAGAGGAGCGGCTTTGGGAGTAATGGAGGCCCTGGGAACATTACC
  	AGGACACATGACAGAGAGGTTTCAGGAAGCCATTGATAACCTCGCTGTGCTCATG
  	CGAGCAGAGACTGGAAGCAGGCCCTACAAGGCAGCGGCAGCCCAATTGCCGGAGA
  	CCCTAGAGACCATCATGCTTTTAGGCCTGCTGGGAACAGTATCGCTGGGGATCTT
  	TTTTGTCTTGATGAGGAACAAGGGCATCGGGAAGATGGGCTTTGAAATGGTAACC
  	CTTGGGGCCAGCGCATGGCTCATGTGGCTCTCAGAAATCGAACCAGCCAGAATTG
  	CATGTGTCCTTATTGTTGTGTTTTTATTACTGGTGGTGCTAATACCAGAGCCAGA
  	GAAGCAAAGATCCCCCCAGGACAATCAGATGGCAATCATTATTATGGTGGCAGTG
  	GGCCTTTTGGGGTTGATAACTGCAAATGAACTTGGATGGCTGGAGAGAACAAAAA
  	ATGACATAGCTCATCTGATGGGAAAGAGAGAAGAGGGAACAACCGTGGGATTCTC
  	AATGGACATCGATCTGCGACCAGCCTCCGCATGGGCTATTTATGCCGCATTGACA
  	ACCCTCATCACCCCAGCCGTCCAGCACGCGGTAACTACCTCGTACAACAACTACT
  	CCTTAATGGCGATGGCCACACAAGCTGGAGTGCTGTTTGGCATGGGCAAAGGGAT
  	GCCATTTTATGCATGGGACTTAGGAGTCCCGTTGCTAATGATGGGCTGCTACTCA
  	CAACTAACACCCCTGACCCTGATAGTAGCCATCATTTTGCTTGTGGCACATTACA
  	TGTACTTGATCCCAGGCCTACAGGCAGCAGCAGCACGCGCTGCCCAGAAGAGAAC
  	AGCAGCCGGCATCATGAAGAATCCCGTTGTGGATGGAATAGTGGTAACTGACATT
  	GACACAATGACAATTGACCCCCAAGTGGAGAAGAAGATGGGACAAGTGCTACTTA
  	TAGCAGTGGCTGTCTCCAGTGCTGTGTTGCTGCGGACCGCTTGGGGATGGGGGGA
  	GGCTGGAGCTTTGATCACAGCAGCAACTTCCACCCTGTGGGAAGGCTCCCCAAAC
  	AAATACTGGAACTCCTCCACAGCCACCTCACTGTGCAACATCTTCAGAGGAAGTT
  	ACTTGGCAGGAGCTTCCCTTATTTACACAGTGACAAGAAATGCCGGCCTGGTTAA
  	GAGACGTGGAGGTGGAACGGGAGAAACTCTGGGAGAGAAGTGGAAAGCCCGCCTG
  	AATCAGATGTCGGCCTTGGAGTTCTACTCTTACAAAAAGTCAGGCATCACTGAAG
  	TATGTAGAGAGGAGGCTCGCCGCGCCCTCAAGGATGGAGTGGCCACAGGAGGACA
  	TGCTGTATCCCGAGGAAGCGCAAAACTCAGATGGTTGGTGGAGAGAGGATATCTG
  	CAGCCCTATGGAAAGGTTGTTGATCTCGGATGCGGCAGAGGGGGCTGGAGTTATT
  	ATGCCGCCACCATCCGCAAAGTGCAGGAGGTGAGAGGATACACAAAGGGAGGTCC
  	CGGTCATGAAGAGCCCATGCTGGTGCAAAGCTATGGGTGGAACATAATTCGTCTC
  	AAGAGTGGAGTGGACGTCTTCCACATGGCGGCTGAGTCGTGTGACACTTTGCTGT
  	GTGACATAGGTGAGTCATCATCCAGTCCTGAAGTGGAGGAGACGCGAACACTCAG
  	AGTGCTCTCCATGGTGGGGGACTGGCTTGAGAAGAGACCAGGGGCCTTCTGCATA
  	AAGGTGTTATGCCCATACACCAGCACCATGATGGAGACCATGGAGCGACTGCAAC
  	GTAGGTATGGGGGAGGACTAGTCAGAGTGCCACTGTCCCGCAATTCTACACATGA
  	GATGTATTGGGTCTCTGGAGCAAAAAGTAACATCATAAAAAGTGTGTCCACCACA
  	AGTCAGCTCCTCCTGGGACGCATGGATGGGCCCAGGAGGCCAGTGAAGTATGAGG
  	AGGATGTGAACCTCGGCTCAGGCACACGAGCTGTGGCAAGCTGTGCTGAGGCTCC
  	CAACATGAAGGTCATTGGTAGGCGCATTGAGAGAATCCGTAGTGAACATGCAGAA
  	ACATGGTTCTTTGATGAAAACCATCCATACAGGACATGGGCCTACCACGGGAGCT
  	ACGAAGCCCCCACGCAAGGGTCAGCATCTTCCCTCGTGAATGGGGTTGTTAGACT
  	CCTGTCAAAGCCCTGGGATGTGGTGACTGGAGTTACAGGAATAGCTATGACTGAC
  	ACCACACCGTACGGCCAACAAAGAGTCTTCAAAGAAAAAGTGGACACCAGGGTGC
  	CAGACCCTCAAGAAGGTACTCGCCAGGTAATGAACATGGTCGCTTCCTGGCTGTG
  	GAAGGAGCTGGGAAAACGTAAGCGGCCACGTGTCTGCACCAAAGAAGAGTTCATC
  	AACAAGGTGCGCAGCAATGCAGCACTGGGAGCAATATTTGAAGAGGAAAAAGAAT
  	GGAAGACGGCTGTGGAAGCTGTGAATGATCCAAGGTTTTGGGCCCTAGTGGATAA
  	GGAAAGAGAACACCACCTGAGAGGAGAGTGCCATAGTTGTGTGTACAACATGATG
  	GGAAAAAGAGAAAAGAAGCAAGGGGAATTCGGGAAAGCAAAAGGCAGTCGCGCCA
  	TCTGGTACATGTGGTTGGGAGCCAGATTCTTGGAGTTTGAAGCCCTTGGATTCTT
  	GAACGAGGACCATTGGATGGGAAGAGAAAACTCAGGAGGTGGTGTCGAAGGGTTG
  	GGACTGCAAAGACTTGGATACGTTCTAGAAGAAATGAGCCGGGCACCAGGAGGAA
  	AGATGTATGCAGATGACACCGCTGGCTGGGACACCCGCATTAGCAAGTTTGATTT
  	GGAGAATGAAGCCTTGATTACTAACCAAATGGATGAAGGGCACAGAACTCTGGCG
  	TTGGCCGTGATTAAGTACACATACCAAAACAAAGTGGTGAAGGTCCTCAGACCAG
  	CTGAAGGAGGAAAAACAGTCATGGACATCATTTCAAGACAAGACCAGAGGGGGAG
  	CGGACAAGTTGTCACTTATGCTCTCAACACATTTACCAACTTGGTGGTGCAGCTC
  	ATCCGGAACATGGAGGCTGAGGAAGTGTTAGAGATGCAAGACTTATGGCTGTTGA
  	GGAAGCCAGAGAAAGTAACCAGATGGCTGCAGAGTAGCGGATGGGACAGACTCAA
  	ACGAATGGCAGTCAGTGGTGATGACTGTGTTGTAAAGCCAATTGATGACAGGTTT
  	GCACACGCCCTCAGGTTCTTGAATGATATGGGGAAAGTTAGGAAAGACACACAGG
  	AATGGAAACCCTCAACTGGATGGAGCAACTGGGAAGAAGTCCCGTTCTGCTCCCA
  	CCACTTTAACAAGCTGCACCTCAAAGACGGGAGATCCATTGTGGTCCCTTGCCGC
  	CACCAAGATGAACTGATTGGCCGGGCTCGCGTTTCGCCGGGGGCAGGATGGAGCA
  	TCCGGGAGACTGCCTGTCTTGCAAAATCATATGCACAGATGTGGCAGCTTCTTTA
  	TTTCCACAGAAGAGACCTCCGACTGATGGCCAATGCCATTTGCTCGGCCGTGCCA
  	GTTGACTGGGTCCCAACTGGGAGAACTACCTGGTCAATCCATGGAAAGGGAGAAT
  	GGATGACTACTGAGGACATGCTCATGGTGTGGAATAGAGTGTGGATTGAGGAGAA
  	TGATCACATGGAGGACAAGACCCCTGTAACAAAATGGACAGACATTCCCTATTTG
  	GGAAAAAGGGAGGACTTATGGTGTGGATCCCTTATAGGACACAGACCTCGCACCA
  	CTTGGGCTGAGAACATCAAAGACACAGTCAGCATGGTGCGCAGAATCATAGGTGA
  	TGAAGAAAAGTACATGGACTACCTATCCACTCAAGTTCGCTACTTGGGTGAGGAA
  	GGGTCTACACCTGGAGTGCTGTAA
  IgE HC signal	TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAG	49
  peptide_prM-E	AGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGACTGGACCTGGATC
  #1	CTGTTCCTGGTGGCCGCTGCCACAAGAGTGCACAGCGTGGAAGTGACCAGACGGG
  (Brazil_isolate_	GCAGCGCCTACTACATGTACCTGGACAGAAGCGACGCCGGCGAGGCCATCAGCTT
  ZikaSPH2015,	TCCAACCACCCTGGGCATGAACAAGTGCTACATCCAGATCATGGACCTGGGCCAC
  Sequence,	ATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGGACGAGGGCGTGGAAC
  NT (5′ UTR,	CCGACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGTGTACGGCAC
  ORF, 3′ UTR)	CTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCT
  	AGCCACAGCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAG
  	AGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTT
  	TGCCCTGGCTGCCGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCCCAGAAA
  	GTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGTA
  	TCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGA
  	CGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCC
  	GTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCT
  	ACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACA
  	GGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACC
  	CTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCG
  	TGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCC
  	CGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCTCCCAGCACAGCGGC
  	ATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAA
  	TCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGG
  	CCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACC
  	ATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGC
  	CCTGGCATGCTGGCGCTGATACAGGCACCCCCCACTGGAACAACAAAGAGGCTCT
  	GGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCT
  	CAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATG
  	GCGCCAAAGGCAGACTGTCCTCCGGCCACCTGAAGTGCCGGCTGAAGATGGACAA
  	GCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACC
  	AAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCG
  	GCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCT
  	GACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAG
  	AACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGA
  	TCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGATCCGGCAGCACCAT
  	CGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGC
  	GATACCGCCTGGGATTTTGGCTCTGTGGGCGGAGCCCTGAACAGCCTGGGAAAGG
  	GCATCCACCAGATCTTCGGCGCTGCCTTCAAGAGCCTGTTCGGCGGCATGAGCTG
  	GTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAG
  	AACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGCGGCGTGCTGATCTTTCTGA
  	GCACAGCCGTGTCCGCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGC
  	CCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGT
  	CTTTGAATAAAGTCTGAGTGGGCGGC
  IgE HC signal	ATGGACTGGACCTGGATCCTGTTCCTGGTGGCCGCTGCCACAAGAGTGCACAGCG	50
  peptide_prM-E	TGGAAGTGACCAGACGGGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGC
  #1	CGGCGAGGCCATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAG
  (Brazil_isolate_	ATCATGGACCTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGC
  ZikaSPH2015),	TGGACGAGGGCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCAC
  ORF	CTGGGTGGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGA
  Sequence, NT	CGGGCCGTGACACTGCCTAGCCACAGCACCAGAAAGCTGCAGACCCGGTCCCAGA
  	CCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGAT
  	CTTCCGGAACCCCGGCTTTGCCCTGGCTGCCGCTGCTATTGCTTGGCTGCTGGGC
  	AGCAGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTG
  	CCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAG
  	CGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATG
  	GCCCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATA
  	TGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGA
  	CAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAG
  	TACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGT
  	TTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGAC
  	CGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCAC
  	GGCTCCCAGCACAGCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGA
  	ACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGG
  	CGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGC
  	GACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGT
  	TCCACGACATCCCCCTGCCCTGGCATGCTGGCGCTGATACAGGCACCCCCCACTG
  	GAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACC
  	GTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCC
  	TGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCTCCGGCCACCTGAAGTG
  	CCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACC
  	GCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTG
  	TGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGC
  	CGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTG
  	ATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCG
  	GCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCA
  	CAGATCCGGCAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAG
  	AGAATGGCCGTGCTGGGCGATACCGCCTGGGATTTTGGCTCTGTGGGCGGAGCCC
  	TGAACAGCCTGGGAAAGGGCATCCACCAGATCTTCGGCGCTGCCTTCAAGAGCCT
  	GTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGG
  	CTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGCG
  	GCGTGCTGATCTTTCTGAGCACAGCCGTGTCCGCC
  IgE HC signal	G*GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAC	51
  peptide_prM-E	TGGACCTGGATCCTGTTCCTGGTGGCCGCTGCCACAAGAGTGCACAGCGTGGAAG
  #1	TGACCAGACGGGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGCCGGCGA
  (Brazil_isolate_	GGCCATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAGATCATG
  ZikaSPH2015),	GACCTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGGACG
  mRNA	AGGGCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGT
  Sequence	GGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCC
  (T100 tail)	GTGACACTGCCTAGCCACAGCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGC
  	TGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCG
  	GAACCCCGGCTTTGCCCTGGCTGCCGCTGCTATTGCTTGGCTGCTGGGCAGCAGC
  	ACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACA
  	GCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGG
  	CACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAG
  	GATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCG
  	AAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAG
  	ATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTG
  	TGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCA
  	AGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAA
  	GAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCTCC
  	CAGCACAGCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGG
  	CCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTT
  	TGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTG
  	TACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACG
  	ACATCCCCCTGCCCTGGCATGCTGGCGCTGATACAGGCACCCCCCACTGGAACAA
  	CAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTG
  	GTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAG
  	CCGAAATGGATGGCGCCAAAGGCAGACTGTCCTCCGGCCACCTGAAGTGCCGGCT
  	GAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCC
  	TTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAG
  	TGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGA
  	TATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACC
  	GAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACT
  	CCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGATC
  	CGGCAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATG
  	GCCGTGCTGGGCGATACCGCCTGGGATTTTGGCTCTGTGGGCGGAGCCCTGAACA
  	GCCTGGGAAAGGGCATCCACCAGATCTTCGGCGCTGCCTTCAAGAGCCTGTTCGG
  	CGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGC
  	CTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGCGGCGTGC
  	TGATCTTTCTGAGCACAGCCGTGTCCGCCTGATAATAGGCTGGAGCCTCGGTGGC
  	CATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCG
  	TACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAA
  	AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
  	AAAAAAAAAAAAAAAAAAAAAAAAAAAATCTAG
  IgE HC signal	TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAG	52
  peptide_prM-E	AGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGACTGGACCTGGATC
  #2	CTGTTCCTGGTGGCCGCTGCCACAAGAGTGCACAGCACCAGAAGAGGCAGCGCCT
  (Brazil_isolate_	ACTACATGTACCTGGACAGAAGCGACGCCGGCGAGGCCATCAGCTTTCCAACCAC
  ZikaSPH2015),	CCTGGGCATGAACAAGTGCTACATCCAGATCATGGACCTGGGCCACATGTGCGAC
  Sequence,	GCCACCATGAGCTACGAGTGCCCCATGCTGGACGAGGGCGTGGAACCCGACGATG
  NT (5′ UTR,	TGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGTGTACGGCACCTGTCACCA
  ORF, 3′ UTR)	CAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGCCACTCC
  	ACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGTACACCA
  	AGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGC
  	TGCCGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCCCAGAAAGTGATCTAC
  	CTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGT
  	CCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCT
  	GGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCCGTGGACATC
  	GAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACG
  	AGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGC
  	CTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGAT
  	AGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCG
  	CCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCT
  	GGAATACCGGATCATGCTGAGCGTGCACGGCTCCCAGCACAGCGGCATGATCGTG
  	AACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCA
  	ACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTG
  	CGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAAC
  	AAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATG
  	CTGGCGCTGATACAGGCACCCCCCACTGGAACAACAAAGAGGCTCTGGTGGAATT
  	CAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGC
  	GCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAG
  	GCAGACTGTCCTCCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCT
  	GAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCC
  	GCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACG
  	GCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGT
  	GGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAG
  	ATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGG
  	GAGAGAAGAAGATCACCCACCACTGGCACAGATCCGGCAGCACCATCGGCAAGGC
  	CTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGCGATACCGCC
  	TGGGATTTTGGCTCTGTGGGCGGAGCCCTGAACAGCCTGGGAAAGGGCATCCACC
  	AGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCA
  	GATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAACGGCAGC
  	ATCTCCCTGACCTGCCTGGCTCTGGGCGGCGTGCTGATCTTTCTGAGCACAGCCG
  	TGTCCGCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGC
  	CTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATA
  	AAGTCTGAGTGGGCGGC
  IgE HC signal	ATGGACTGGACCTGGATCCTGTTCCTGGTGGCCGCTGCCACAAGAGTGCACAGCA	53
  peptide_prM-E	CCAGAAGAGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGCCGGCGAGGC
  #2	CATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAGATCATGGAC
  (Brazil_Isolate_	CTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGGACGAGG
  ZikaSPH2015),	GCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGT
  ORF	GTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTG
  Sequence, NT	ACACTGCCTAGCCACTCCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGG
  	AAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAA
  	CCCCGGCTTTGCCCTGGCTGCCGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACC
  	TCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCA
  	TCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCAC
  	ATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGAT
  	AAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAG
  	TGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATG
  	CCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGC
  	AAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGG
  	GCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAG
  	CATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCTCCCAG
  	CACAGCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCA
  	AGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGG
  	ATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTAC
  	TACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACA
  	TCCCCCTGCCCTGGCATGCTGGCGCTGATACAGGCACCCCCCACTGGAACAACAA
  	AGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTG
  	CTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCG
  	AAATGGATGGCGCCAAAGGCAGACTGTCCTCCGGCCACCTGAAGTGCCGGCTGAA
  	GATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTC
  	ACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGC
  	AGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATAT
  	GCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAG
  	AGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCT
  	ACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGATCCGG
  	CAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCC
  	GTGCTGGGCGATACCGCCTGGGATTTTGGCTCTGTGGGCGGAGCCCTGAACAGCC
  	TGGGAAAGGGCATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGG
  	CATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTG
  	AACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGCGGCGTGCTGA
  	TCTTTCTGAGCACAGCCGTGTCCGCC
  IgE HC signal	G*GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAC	54
  peptide_prM-E	TGGACCTGGATCCTGTTCCTGGTGGCCGCTGCCACAAGAGTGCACAGCACCAGAA
  #2	GAGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGCCGGCGAGGCCATCAG
  (Brazil_isolate_	CTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAGATCATGGACCTGGGC
  ZikaSPH2015),	CACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGGACGAGGGCGTGG
  mRNA	AACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGTGTACGG
  Sequence	CACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTG
  (T100 tail)	CCTAGCCACTCCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCA
  	GAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGG
  	CTTTGCCCTGGCTGCCGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCCCAG
  	AAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGT
  	GTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGT
  	GGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCC
  	GCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGA
  	GCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTAC
  	ACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGG
  	ACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCC
  	TCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCA
  	GCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCTCCCAGCACAGC
  	GGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGG
  	AAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCT
  	GGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTG
  	ACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCC
  	TGCCCTGGCATGCTGGCGCTGATACAGGCACCCCCCACTGGAACAACAAAGAGGC
  	TCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGA
  	TCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGG
  	ATGGCGCCAAAGGCAGACTGTCCTCCGGCCACCTGAAGTGCCGGCTGAAGATGGA
  	CAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTC
  	ACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACG
  	CCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGAC
  	CCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACC
  	GAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCG
  	TGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGATCCGGCAGCAC
  	CATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTG
  	GGCGATACCGCCTGGGATTTTGGCTCTGTGGGCGGAGCCCTGAACAGCCTGGGAA
  	AGGGCATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGGCATGAG
  	CTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACC
  	AAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGCGGCGTGCTGATCTTTC
  	TGAGCACAGCCGTGTCCGCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCT
  	TGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGT
  	GGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAA
  	AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
  	AAAAAAAAAAAAAAAAAAATCTAG
  HuIgGk signal	TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAG	55
  peptide_prME	AGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAAACCCCTGCCCAG
  #1	CTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGATACCACCGGCGTGGAAGTGACCA
  (Brazil_isolate_	GAAGAGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGCCGGCGAGGCCAT
  ZikaSPH2015),	CAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAGATCATGGACCTG
  Sequence,	GGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGGACGAGGGCG
  NT (5′ UTR,	TGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGTGTA
  ORF, 3′ UTR)	CGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACA
  	CTGCCTAGCCACTCCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAA
  	GCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCC
  	CGGCTTTGCCCTGGCCGCTGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCC
  	CAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCC
  	GGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATG
  	GGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAG
  	CCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGC
  	GGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCC
  	TACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAG
  	CGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCA
  	GCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCAT
  	CCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAGCAC
  	TCCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGG
  	TGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATC
  	TCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTAC
  	CTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCC
  	CCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGAACAACAAAGA
  	GGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTG
  	GGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAA
  	TGGATGGCGCCAAAGGCAGACTGTCCAGCGGCCACCTGAAGTGCCGGCTGAAGAT
  	GGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACC
  	TTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGT
  	ACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCA
  	GACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGC
  	ACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACA
  	TCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACCGCAGCGGCAG
  	CACAATCGGCAAGGCCTTTGAAGCCACAGTGCGGGGAGCCAAGAGAATGGCCGTG
  	CTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACTCTCTGG
  	GCAAGGGAATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGGCAT
  	GAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAAC
  	ACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGAGGCGTGCTGATCT
  	TTCTGAGCACCGCCGTGTCTGCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCT
  	TCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCC
  	CGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC
  HuIgGk signal	ATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGATACCA	56
  peptide_prME	CCGGCGTGGAAGTGACCAGAAGAGGCAGCGCCTACTACATGTACCTGGACAGAAG
  #1	CGACGCCGGCGAGGCCATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTAC
  (Brazil_isolate_	ATCCAGATCATGGACCTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCC
  ZikaSPH2015),	CCATGCTGGACGAGGGCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCAC
  ORF	CAGCACCTGGGTGGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGG
  Sequence, NT	TCCAGACGGGCCGTGACACTGCCTAGCCACTCCACCAGAAAGCTGCAGACCCGGT
  	CCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAA
  	CTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCCGCTGCTGCTATTGCTTGGCTG
  	CTGGGCAGCAGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCG
  	CCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGG
  	CATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACA
  	GTGATGGCCCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGT
  	CCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGC
  	CAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGAC
  	ACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCG
  	GCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAA
  	GATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGC
  	GTGCACGGCAGCCAGCACTCCGGCATGATCGTGAACGACACCGGCCACGAGACAG
  	ACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCAC
  	ACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGAT
  	TTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAG
  	AGTGGTTCCACGACATCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACC
  	CCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGG
  	CAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTG
  	GCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCAGCGGCCACCT
  	GAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTG
  	TGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCG
  	TGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCA
  	GATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAAC
  	CCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCC
  	CCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCA
  	CTGGCACCGCAGCGGCAGCACAATCGGCAAGGCCTTTGAAGCCACAGTGCGGGGA
  	GCCAAGAGAATGGCCGTGCTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCG
  	GAGCCCTGAACTCTCTGGGCAAGGGAATCCACCAGATCTTCGGAGCCGCCTTTAA
  	GAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTC
  	GTGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTC
  	TGGGAGGCGTGCTGATCTTTCTGAGCACCGCCGTGTCTGCC
  HuIgGk signal	G*GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAA	57
  peptide prME	ACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGATACCACCGGCG
  #1	TGGAAGTGACCAGAAGAGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGC
  (Brazil_isolate_	CGGCGAGGCCATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAG
  ZikaSPH2015),	ATCATGGACCTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGC
  mRNA	TGGACGAGGGCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCAC
  Sequence	CTGGGTGGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGA
  (T100 tail)	CGGGCCGTGACACTGCCTAGCCACTCCACCAGAAAGCTGCAGACCCGGTCCCAGA
  	CCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGAT
  	CTTCCGGAACCCCGGCTTTGCCCTGGCCGCTGCTGCTATTGCTTGGCTGCTGGGC
  	AGCAGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTG
  	CCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAG
  	CGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATG
  	GCCCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATA
  	TGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGA
  	CAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAG
  	TACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGT
  	TTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGAC
  	CGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCAC
  	GGCAGCCAGCACTCCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGA
  	ACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGG
  	CGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGC
  	GACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGT
  	TCCACGACATCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTG
  	GAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACC
  	GTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCC
  	TGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCAGCGGCCACCTGAAGTG
  	CCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACC
  	GCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTG
  	TGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGC
  	CGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTG
  	ATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCG
  	GCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCA
  	CCGCAGCGGCAGCACAATCGGCAAGGCCTTTGAAGCCACAGTGCGGGGAGCCAAG
  	AGAATGGCCGTGCTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCC
  	TGAACTCTCTGGGCAAGGGAATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCT
  	GTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGG
  	CTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGAG
  	GCGTGCTGATCTTTCTGAGCACCGCCGTGTCTGCCTGATAATAGGCTGGAGCCTC
  	GGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTG
  	CACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAA
  	AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
  	AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATCTAG
  HuIgGk signal	TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAG	58
  peptide_prME	AGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAAACCCCTGCCCAG
  #
  2	CTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGATACCACCGGCACCAGAAGAGGCA
  (Brazil_isolate_	GCGCCTACTACATGTACCTGGACAGAAGCGACGCCGGCGAGGCCATCAGCTTTCC
  ZikaSPH2015),	AACCACCCTGGGCATGAACAAGTGCTACATCCAGATCATGGACCTGGGCCACATG
  Sequence,	TGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGGACGAGGGCGTGGAACCCG
  NT (5′ UTR,	ACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGTGTACGGCACCTG
  ORF, 3′ UTR)	TCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGC
  	CACTCCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGT
  	ACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGC
  	CCTGGCCGCTGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCCCAGAAAGTG
  	ATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCG
  	GCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGT
  	GGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCCGTG
  	GACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACT
  	GCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGG
  	CGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTG
  	GTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGA
  	CCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGA
  	GAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAGCACTCCGGCATG
  	ATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCA
  	CCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCT
  	GGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATG
  	AACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCT
  	GGCATGCCGGCGCTGATACAGGCACACCCCACTGGAACAACAAAGAGGCTCTGGT
  	GGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAG
  	GAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCG
  	CCAAAGGCAGACTGTCCAGCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCT
  	GCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAG
  	ATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCA
  	CCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGAC
  	CCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAAC
  	AGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCG
  	GCGTGGGAGAGAAGAAGATCACCCACCACTGGCACCGCAGCGGCAGCACAATCGG
  	CAAGGCCTTTGAAGCCACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGAGAT
  	ACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACTCTCTGGGCAAGGGAA
  	TCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGGCATGAGCTGGTT
  	CAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAAC
  	GGCAGCATCTCCCTGACCTGCCTGGCTCTGGGAGGCGTGCTGATCTTTCTGAGCA
  	CCGCCGTGTCTGCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCC
  	TTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTT
  	TGAATAAAGTCTGAGTGGGCGGC
  HuIgGk signal	ATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGATACCA	59
  peptide_prME	CCGGCACCAGAAGAGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGCCGG
  #
  2	CGAGGCCATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAGATC
  (Brazil_isolate_	ATGGACCTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGG
  ZikaSPH2015),	ACGAGGGCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCACCTG
  ORF	GGTGGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGG
  Sequence, NT	GCCGTGACACTGCCTAGCCACTCCACCAGAAAGCTGCAGACCCGGTCCCAGACCT
  	GGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTT
  	CCGGAACCCCGGCTTTGCCCTGGCCGCTGCTGCTATTGCTTGGCTGCTGGGCAGC
  	AGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCT
  	ACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGG
  	CGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCC
  	CAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGG
  	CCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAG
  	CAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTAC
  	GTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTG
  	GCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGG
  	CAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGC
  	AGCCAGCACTCCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACC
  	GGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGG
  	CTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGAC
  	CTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCC
  	ACGACATCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGAA
  	CAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTG
  	GTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGG
  	AAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCAGCGGCCACCTGAAGTGCCG
  	GCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCC
  	GCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGG
  	AAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGT
  	GGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATC
  	ACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCG
  	ACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACCG
  	CAGCGGCAGCACAATCGGCAAGGCCTTTGAAGCCACAGTGCGGGGAGCCAAGAGA
  	ATGGCCGTGCTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGA
  	ACTCTCTGGGCAAGGGAATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTT
  	CGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTG
  	GGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGAGGCG
  	TGCTGATCTTTCTGAGCACCGCCGTGTCTGCC
  HuIgGk signal	G*GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAA	60
  peptide_prME	ACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGATACCACCGGCA
  #2	CCAGAAGAGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGCCGGCGAGGC
  (Brazil_isolate_	CATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAGATCATGGAC
  ZikaSPH2015),	CTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGGACGAGG
  mRNA	GCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGT
  Sequence	GTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTG
  (T100 tall)	ACACTGCCTAGCCACTCCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGG
  	AAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAA
  	CCCCGGCTTTGCCCTGGCCGCTGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACC
  	TCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCA
  	TCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCAC
  	ATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGAT
  	AAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAG
  	TGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATG
  	CCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGC
  	AAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGG
  	GCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAG
  	CATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAG
  	CACTCCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCA
  	AGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGG
  	ATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTAC
  	TACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACA
  	TCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGAACAACAA
  	AGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTG
  	CTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCG
  	AAATGGATGGCGCCAAAGGCAGACTGTCCAGCGGCCACCTGAAGTGCCGGCTGAA
  	GATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTC
  	ACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGC
  	AGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATAT
  	GCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAG
  	AGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCT
  	ACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACCGCAGCGG
  	CAGCACAATCGGCAAGGCCTTTGAAGCCACAGTGCGGGGAGCCAAGAGAATGGCC
  	GTGCTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACTCTC
  	TGGGCAAGGGAATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGG
  	CATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTG
  	AACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGAGGCGTGCTGA
  	TCTTTCTGAGCACCGCCGTGTCTGCCTGATAATAGGCTGGAGCCTCGGTGGCCAT
  	GCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTAC
  	CCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAA
  	AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
  	AAAAAAAAAAAAAAAAAAAAAAAAATCTAG
  HuIgGk signal	TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAG	61
  peptide_E	AGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAAACCCCTGCCCAG
  (Brazil_isolate_	CTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACCGGCATCAGATGCATCG
  ZikaSPH2015),	GCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGT
  Sequence,	GGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCCGTG
  NT (5′ UTR,	GACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACT
  ORF, 3′ UTR)	GCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGG
  	CGAGGCCTACCTGGACAAGCAGAGCGACACCCAGTACGTGTGCAAGCGGACCCTG
  	GTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGA
  	CCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGA
  	GAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAGCACTCCGGCATG
  	ATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCA
  	CCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCT
  	GGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATG
  	AACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCT
  	GGCATGCCGGCGCTGATACAGGCACACCCCACTGGAACAACAAAGAGGCTCTGGT
  	GGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAG
  	GAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCG
  	CCAAAGGCAGACTGAGCAGCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCT
  	GCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAG
  	ATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCA
  	CCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGAC
  	CCCCGTGGGCAGGCTGATCACAGCCAACCCTGTGATCACCGAGAGCACCGAGAAC
  	AGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCG
  	GCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGAAGCGGCAGCACCATCGG
  	CAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGAGAT
  	ACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACTCTCTGGGCAAGGGAA
  	TCCACCAGATCTTCGGCGCTGCCTTCAAGAGCCTGTTCGGCGGCATGAGCTGGTT
  	CAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAAC
  	GGCAGCATCTCCCTGACCTGCCTGGCTCTGGGAGGCGTGCTGATCTTTCTGAGCA
  	CCGCCGTGTCTGCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCC
  	TTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTT
  	TGAATAAAGTCTGAGTGGGCGGC
  HuIgGk signal	ATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCA	62
  peptide_E	CCGGCATCAGATGCATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGG
  (Brazil_isolate_	CGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCC
  ZikaSPH2015),	CAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGG
  ORF	CCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAG
  Sequence, NT	CAGATGCCCTACACAGGGCGAGGCCTACCTGGACAAGCAGAGCGACACCCAGTAC
  	GTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTG
  	GCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGG
  	CAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGC
  	AGCCAGCACTCCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACC
  	GGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGG
  	CTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGAC
  	CTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCC
  	ACGACATCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGAA
  	CAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTG
  	GTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGG
  	AAGCCGAAATGGATGGCGCCAAAGGCAGACTGAGCAGCGGCCACCTGAAGTGCCG
  	GCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCC
  	GCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGG
  	AAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGT
  	GGATATGCAGACCCTGACCCCCGTGGGCAGGCTGATCACAGCCAACCCTGTGATC
  	ACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCG
  	ACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAG
  	AAGCGGCAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGA
  	ATGGCCGTGCTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGA
  	ACTCTCTGGGCAAGGGAATCCACCAGATCTTCGGCGCTGCCTTCAAGAGCCTGTT
  	CGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTG
  	GGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGAGGCG
  	TGCTGATCTTTCTGAGCACCGCCGTGTCTGCC
  HuIgGk signal	G*GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAA	63
  peptide_E	ACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACCGGCA
  (Brazil_isolate_	TCAGATGCATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCAC
  ZikaSPH2015),	ATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGAT
  mRNA	AAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAG
  Sequence	TGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATG
  (T100 tall)	CCCTACACAGGGCGAGGCCTACCTGGACAAGCAGAGCGACACCCAGTACGTGTGC
  	AAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGG
  	GCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAG
  	CATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAG
  	CACTCCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCA
  	AGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGG
  	ATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTAC
  	TACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACA
  	TCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGAACAACAA
  	AGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTG
  	CTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCG
  	AAATGGATGGCGCCAAAGGCAGACTGAGCAGCGGCCACCTGAAGTGCCGGCTGAA
  	GATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTC
  	ACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGC
  	AGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATAT
  	GCAGACCCTGACCCCCGTGGGCAGGCTGATCACAGCCAACCCTGTGATCACCGAG
  	AGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCT
  	ACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGAAGCGG
  	CAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCC
  	GTGCTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACTCTC
  	TGGGCAAGGGAATCCACCAGATCTTCGGCGCTGCCTTCAAGAGCCTGTTCGGCGG
  	CATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTG
  	AACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGAGGCGTGCTGA
  	TCTTTCTGAGCACCGCCGTGTCTGCCTGATAATAGGCTGGAGCCTCGGTGGCCAT
  	GCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTAC
  	CCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAA
  	AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
  	AAAAAAAAAAAAAAAAAAAAAAAAATCTAG
  Zika_RIO-	ATGCTGGGCAGCAACAGCGGCCAGAGAGTGGTGTTCACCATCCTGCTGCTGCTGG	64
  U1_JEVsp	TGGCCCCTGCCTACAGCGCCGAAGTGACAAGAAGAGGCAGCGCCTACTACATGTA
  Zika PRME	CCTGGACCGGAACGATGCCGGCGAGGCCATCAGCTTTCCAACCACCCTGGGCATG
  Strain	AACAAGTGCTACATCCAGATCATGGACCTGGGCCACATGTGCGACGCCACCATGA
  ascension id:	GCTACGAGTGCCCCATGCTGGACGAGGGCGTGGAACCCGACGATGTGGACTGCTG
  ANG09399 with	GTGCAATACCACCAGCACCTGGGTGGTGTACGGCACCTGTCACCACAAGAAGGGC
  JEV PRM	GAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGCCACAGCACCAGAAAGC
  signal	TGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGAT
  sequence	CCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCTGCCGCTGCT
  (optimized)	ATTGCTTGGCTGCTGGGCTCTAGCACCAGCCAGAAAGTGATCTACCTCGTGATGA
  	TCCTGCTGATCGCCCCAGCCTACTCCATCCGGTGTATCGGCGTGTCCAACCGGGA
  	CTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGC
  	GGCTGCGTGACAGTGATGGCCCAGGACAAGCCCACCGTGGACATCGAGCTCGTGA
  	CCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCAT
  	CAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGAC
  	AAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGACAGGGGCTGGG
  	GCAATGGCTGTGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGC
  	CTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGG
  	ATCATGCTGAGCGTGCACGGCTCCCAGCACAGCGGCATGATCGTGAACGACACCG
  	GCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAG
  	AGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGA
  	ACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAACACTGGC
  	TGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCTGGCGCTGA
  	TACAGGCACCCCCCACTGGAACAACAAAGAGGCCCTGGTGGAATTCAAGGACGCC
  	CACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATA
  	CAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGAG
  	CAGCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTG
  	TCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACAC
  	TGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAA
  	AGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGGCTG
  	ATCACAGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGG
  	AACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAA
  	GATCACCCACCACTGGCACAGAAGCGGCAGCACCATCGGCAAGGCCTTTGAGGCT
  	ACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGCGATACCGCCTGGGATTTTG
  	GCTCTGTGGGCGGAGCCCTGAACAGCCTGGGAAAGGGCATCCACCAGATCTTCGG
  	AGCCGCCTTTAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATC
  	GGCACCCTGCTGATGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGA
  	TGTGCCTGGCTCTGGGCGGCGTGCTGATCTTTCTGAGCACAGCCGTGTCCGCC
  Zika_ RIO-	ATGAAGTGCCTGCTGTACCTGGCCTTCCTGTTCATCGGCGTGAACTGCGCCGAAG	65
  U1-_VSVgSp	TGACCAGAAGAGGCAGCGCCTACTACATGTACCTGGACCGGAACGATGCCGGCGA
  Zika PRME	GGCCATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAGATCATG
  Strain	GACCTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGGACG
  ascension id:	AGGGCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGT
  ANG09399 with	GGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCC
  VSV g protein	GTGACACTGCCTAGCCACAGCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGC
  signal	TGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCG
  sequence	GAACCCCGGCTTTGCCCTGGCCGCTGCTGCTATTGCTTGGCTGCTGGGCAGCAGC
  (optimized)	ACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACA
  	GCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGG
  	CACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAG
  	GACAAGCCCACCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCG
  	AAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAG
  	ATGCCCTACACAGGGCGAGGCCTACCTGGACAAGCAGTCCGACACCCAGTACGTG
  	TGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCA
  	AGGGCAGCCTCGTGACCTGTGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAA
  	GAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGC
  	CAGCACTCCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGG
  	CCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTT
  	TGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTG
  	TACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACG
  	ACATCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGAACAA
  	CAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTG
  	GTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAG
  	CCGAAATGGATGGCGCCAAAGGCAGACTGTCCAGCGGCCACCTGAAGTGCAGACT
  	GAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCC
  	TTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAG
  	TGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCAGCTCAGATGGCCGTGGA
  	TATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACC
  	GAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACT
  	CCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGAAG
  	CGGCAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATG
  	GCCGTGCTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACT
  	CTCTGGGCAAGGGAATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGG
  	CGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTGATGTGGCTGGGC
  	CTGAACACCAAGAACGGCAGCATCTCCCTGATGTGCCTGGCTCTGGGAGGCGTGC
  	TGATCTTCCTGAGCACAGCCGTGTCTGCC
  ZIKA_PRME_DSP	ATGGACTGGACCTGGATCCTGTTCCTGGTGGCCGCTGCCACAAGAGTGCACAGCG	66
  _N154A	TGGAAGTGACCAGACGGGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGC
  Zika PRME	CGGCGAGGCCATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAG
  Strain	ATCATGGACCTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGC
  ascension id:	TGGACGAGGGCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCAC
  ACD75819 with	CTGGGTGGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGA
  IgE signal	CGGGCCGTGACACTGCCTAGCCACAGCACCAGAAAGCTGCAGACCCGGTCCCAGA
  peptide	CCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGAT
  (optimized)	CTTCCGGAACCCCGGCTTTGCCCTGGCTGCCGCTGCTATTGCTTGGCTGCTGGGC
  	AGCAGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTG
  	CCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAG
  	CGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATG
  	GCCCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATA
  	TGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGA
  	CAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAG
  	TACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGT
  	TTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGAC
  	CGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCAC
  	GGCTCCCAGCACAGCGGCATGATCGTGGCCGACACCGGCCACGAGACAGACGAGA
  	ACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGG
  	CGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGC
  	GACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGT
  	TCCACGACATCCCCCTGCCCTGGCATGCTGGCGCTGATACAGGCACCCCCCACTG
  	GAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACC
  	GTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCC
  	TGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCTCCGGCCACCTGAAGTG
  	CCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACC
  	GCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTG
  	TGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGC
  	CGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTG
  	ATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCG
  	GCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCA
  	CAGATCCGGCAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAG
  	AGAATGGCCGTGCTGGGCGATACCGCCTGGGATTTTGGCTCTGTGGGCGGAGCCC
  	TGAACAGCCTGGGAAAGGGCATCCACCAGATCTTCGGCGCTGCCTTCAAGAGCCT
  	GTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGG
  	CTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGCG
  	GCGTGCTGATCTTTCTGAGCACAGCCGTGTCCGCC

• TABLE 32
  ZIKV Amino Acid Sequences

  		SEQ
  		ID
  Description	Sequence	NO:

  FSM|ACD75819	MKNPKEEIRRIRIVNMLKRGVARVSPFGGLKRLPAGLLLGHGPIRMVLAI	67
  polyprotein	LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKE
  	KKRRGTDTSVGIVGLLLTTAMAVEVTRRGSAYYMYLDRSDAGEAISFPTT
  	LGMNKCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVY
  	GTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENW
  	IFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRD
  	FVEGMSGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYC
  	YEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGK
  	GSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHET
  	DENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKH
  	WLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGS
  	QEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTA
  	AFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLIT
  	ANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKA
  	FEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGM
  	SWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA
  MR_766|ABI54475	MKNPKKKSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI	68
  	LAFLRFTAIKPSLGLINRWGTVGKKEAMEIIKKFKKDLAAMLRIINARKE
  	RKRRGADTSIGIVGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATT
  	LGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVY
  	GTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENW
  	IFRNPGFTLVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRD
  	FVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYC
  	YEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGK
  	GSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDENRAK
  	VEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKE
  	WFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVH
  	TALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTK
  	VPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVIT
  	ESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVR
  	GAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQI
  	LIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA
  SM_6_V_1|ABI54480	MKNPKRAGSSRLVNMLRRGAARVIPPGGGLKRLPVGLLLGRGPIKMILAI	69
  	LAFLRFTAIKPSTGLINRWGKVGKKEAIKILTKFKADVGTMLRIINNRKT
  	KKRGVETGIVFLALLVSIVAVEVTKKGDTYYMFADKKDAGKVVTFETESG
  	PNRCSIQAMDIGHMCPATMSYECPVLEPQYEPEDVDCWCNSTAAWIVYGT
  	CTHKTTGETRRSRRSITLPSHASQKLETRSSTWLESREYSKYLIKVENWI
  	LRNPGYALVAAVIGWTLGSSRSQKIIFVTLLMLVAPAYSIRCIGIGNRDF
  	IEGMSGGTWVDIVLEHGGCVTVMSNDKPTLDFELVTTTASNMAEVRSYCY
  	EANISEMASDSRCPTQGEAYLDKMADSQFVCKRGYVDRGWGNGCGLFGKG
  	SIVTCAKFTCVKKLTGKSIQPENLEYRVLVSVHASQHGGMINNDTNHQHD
  	KENRARIDITASAPRVEVELGSFGSFSMECEPRSGLNFGDLYYLTMNNKH
  	WLVNRDWFHDLSLPWHTGATSNNHHWNNKEALVEFREAHAKKQTAVVLGS
  	QEGAVHAALAGALEAESDGHKATIYSGHLKCRLKLDKLRLKGMSYALCTG
  	AFTFARTPSETIHGTATVELQYAGEDGPCKVPIVITSDTNSMASTGRLIT
  	ANPVVTESGANSKMMVEIDPPFGDSYIIVGTGTTKITHHWHRAGSSIGRA
  	FEATMRGAKRMAVLGDTAWDFGSVGGMFNSVGKFVHQVFGSAFKALFGGM
  	SWFTQLLIGFLLIWMGLNARGGTVAMSFMGIGAMLIFLATSVSG
  MR_766|AAV34151	MKNPKEEIRRIRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI	70
  	LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKE
  	RKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATT
  	LGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVY
  	GTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENW
  	IFRNPGFALVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRD
  	FVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYC
  	YEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGK
  	GSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIGYETDEDR
  	AKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVH
  	KEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGA
  	VHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTF
  	TKVPAETLHGTVTVEVQYAGTDGPCKIPVQMAVDMQTLTPVGRLITANPV
  	ITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEAT
  	VRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFS
  	QILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA
  MR_766|YP_002790881	MKNPKEEIRRIRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI	71
  	LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKE
  	RKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATT
  	LGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVY
  	GTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENW
  	IFRNPGFALVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRD
  	FVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYC
  	YEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGK
  	GSLVTCAKFTCKKMTGKSIQPENLEYRIMLSVHGSQHSGMIGYETDEDRA
  	KVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHK
  	EWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAV
  	HTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFT
  	KVPAETLHGTVTVEVQYAGTDGPCKIPVQMAVDMQTLTPVGRLITANPVI
  	TESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATV
  	RGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQ
  	ILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA
  ARB7701|AHF49785	MKNPKKKSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI	72
  	LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKE
  	RKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATN
  	LGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVY
  	GTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENW
  	IFRNPGFALAAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRD
  	FVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYC
  	YEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGK
  	GSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDIGHET
  	DENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKH
  	WLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGS
  	QEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTA
  	AFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLIT
  	ANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKA
  	FEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGVHQIFGAAFKSLFGGM
  	SWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA
  ARB15076|AHF49784	MKNPKKKSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI	73
  	LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKE
  	RKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATN
  	LGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVY
  	GTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENW
  	IFRNPGFALAAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRD
  	FVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYC
  	YEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGK
  	GSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDENRAK
  	VEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKE
  	WFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVH
  	TALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTK
  	VPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVIT
  	ESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVR
  	GAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQI
  	LIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA
  ARB13565|AHF49783	MKNPKKKSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI	74
  	LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKE
  	RKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATN
  	LGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVY
  	GTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENW
  	IFRNPGFALAAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRD
  	FVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYC
  	YEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGK
  	GSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDIGHET
  	DENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKH
  	WLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGS
  	QEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTA
  	AFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLIT
  	ANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKA
  	FEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGVHQIFGAAFKSLFGGM
  	SWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA
  ArB1362|AHL43500	MKNPKKKSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI	75
  	LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKE
  	RKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATT
  	LGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVY
  	GTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENW
  	IFRNPGFALAAVAIAWLLGSSTSQKVIYLIMILLIAPAYSIRCIGVSNRD
  	FVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYC
  	YEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGK
  	GSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDXXXXX
  	XXNRAEVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKH
  	WLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGS
  	QEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTA
  	AFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLIT
  	ANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKA
  	FEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGM
  	SWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA
  ArD7117|AHL43501	MKNPKKRSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI	76
  	LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKE
  	RKRRGADTSIGIVGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATT
  	LGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVY
  	GTCQHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENW
  	IFRNPGFALVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRD
  	FVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYC
  	YEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGK
  	GSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDIGHET
  	DENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKH
  	WLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGS
  	QEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTA
  	VCTAAKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLIT
  	ANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKA
  	FEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGM
  	SWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA
  ArD157995|AHL43503	MKNPKKKSGRFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI	77
  	LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKE
  	RKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATT
  	LGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVY
  	GTCHHKKGETRRSRRSVSLRYHYTRKLQTRSQTWLESREYKKHLIMVENW
  	IFRNPGFAIVSVAITWLMGSLTSQKVIYLVMIVLIVPAYSISCIGVSNRD
  	LVEGMSGGTWVDVVLEHGGCVTEMAQDKPTVDIELVTMTVSNMAEVRSYC
  	YEASLSDMASASRCPTQGEPSLDKQSDTQSVCKRTLGDRGWGNGCGIFGK
  	GSLVTCSKFTCCKKMPGKSIQPENLEYRIMLPVHGSQHSGMIVNDIGHET
  	DENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKH
  	WLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGS
  	QEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTA
  	AFTFTKVPAETLHGTVTVEVQSAGTDGPCKVPAQMAVDMQTLTPVGRLIT
  	ANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKA
  	FEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGM
  	SWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA
  ArD128000|AHL43502	MKNPKRKSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI	78
  	LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKE
  	RKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATT
  	LGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVY
  	GTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENW
  	IFRNPGFALAAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRD
  	FVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYC
  	YEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGK
  	GSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMXXXXXGHET
  	DENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKH
  	RLVRKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGS
  	QEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTA
  	AFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLIT
  	ANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWLKKGSSIGKA
  	FEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGVHQIFGAAFKSLFGGM
  	SWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA
  ArD158084|AHL43504	MKNPKKKSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI	79
  	LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKE
  	RKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATT
  	LGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVY
  	GTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENW
  	IFRNPGFALVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRD
  	FVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYC
  	YEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGK
  	GSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDIGHET
  	DENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKH
  	WLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGS
  	QEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTA
  	AFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLIT
  	ANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKA
  	FEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGM
  	SWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA
  H/PF/2013|AHZ13508	MKNPKKKSGGFRIVNMLKRGVARVSPFGGLKRLPAGLLLGHGPIRMVLAI	80
  	LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKE
  	KKRRGADTSVGIVGLLLTTAMAAEVTRRGSAYYMYLDRNDAGEAISFPTT
  	LGMNKCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVY
  	GTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENW
  	IFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRD
  	FVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYC
  	YEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGK
  	GSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHET
  	DENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKH
  	WLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGS
  	QEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTA
  	AFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLIT
  	ANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKA
  	FEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGM
  	SWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA
  MR766_NIID|BAP47441	MKNPKKKSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI	81
  	LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKE
  	RKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATT
  	LGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVY
  	GTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENW
  	IFRNPGFALVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRD
  	FVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYC
  	YEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGK
  	GSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMTVNDIGYET
  	DENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKH
  	WLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGS
  	QEGAVHTALAGALEAEMDGAKGKLFSGHLKCRLKMDKLRLKGVSYSLCTA
  	AFTFTKVPAETLHGTVTVEVQYAGTDGPCKIPVQMAVDMQTLTPVGRLIT
  	ANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKA
  	FEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGM
  	SWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA
  prME	VEVTKKGDTYYMFADKKDAGKVVTFETESGPNRCSIQAMDIGHMCPATMS	82
  ABI54480_SouthAfrica	YECPVLEPQYEPEDVDCWCNSTAAWIVYGTCTHKTTGETRRSRRSITLPS
  	HASQKLETRSSTWLESREYSKYLIKVENWILRNPGYALVAAVIGWTLGSS
  	RSQKIIFVTLLMLVAPAYSIRCIGIGNRDFIEGMSGGTWVDIVLEHGGCV
  	TVMSNDKPTLDFELVTTTASNMAEVRSYCYEANISEMASDSRCPTQGEAY
  	LDKMADSQFVCKRGYVDRGWGNGCGLFGKGSIVTCAKFTCVKKLTGKSIQ
  	PENLEYRVLVSVHASQHGGMINNDTNHQHDKENRARIDITASAPRVEVEL
  	GSFGSFSMECEPRSGLNFGDLYYLTMNNKHWLVNRDWFHDLSLPWHTGAT
  	SNNHHWNNKEALVEFREAHAKKQTAVVLGSQEGAVHAALAGALEAESDGH
  	KATIYSGHLKCRLKLDKLRLKGMSYALCTGAFTFARTPSETIHGTATVEL
  	QYAGEDGPCKVPIVITSDTNSMASTGRLITANPVVTESGANSKMMVEIDP
  	PFGDSYIIVGTGTTKITHHWHRAGSSIGRAFEATMRGAKRMAVLGDTAWD
  	FGSVGGMFNSVGKFVHQVFGSAFKALFGGMSWFTQLLIGFLLIWMGLNAR
  	GGTVAMSFMGIGAMLIFLATSVSG
  prME	AEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMS	83
  AAV34151_Uganda_NHP	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  	STRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALVAVAIAWLLGSST
  	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIGYETDEDRAKVEVTPNSPRAEATLGGFGSL
  	GLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHW
  	NNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFS
  	GHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTD
  	GPCKIPVQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSY
  	IVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGG
  	VFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISL
  	TCLALGGVMIFLSTAVSA
  prME	AEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS	84
  AHZ13508_FrenchPoly_	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  2013	STRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSST
  	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGG
  	FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
  	TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
  	RLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQY
  	AGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
  	GDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
  	SVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNG
  	SISLMCLALGGVLIFLSTAVSA
  prME	AEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMS	85
  gAHL43504	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  	STRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALVAVAIAWLLGSST
  	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIVNDIGHETDENRAKVEVTPNSPRAEATLGG
  	FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
  	TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
  	RLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQY
  	AGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
  	GDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
  	SVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNG
  	SISLTCLALGGVMIFLSTAVSA
  prME	AEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMS	86
  AHL43503	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGETRRSRRSVSLRYH
  	YTRKLQTRSQTWLESREYKKHLIMVENWIFRNPGFAIVSVAITWLMGSLT
  	SQKVIYLVMIVLIVPAYSISCIGVSNRDLVEGMSGGTWVDVVLEHGGCVT
  	EMAQDKPTVDIELVTMTVSNMAEVRSYCYEASLSDMASASRCPTQGEPSL
  	DKQSDTQSVCKRTLGDRGWGNGCGIFGKGSLVTCSKFTCCKKMPGKSIQP
  	ENLEYRIMLPVHGSQHSGMIVNDIGHETDENRAKVEVTPNSPRAEATLGG
  	FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
  	TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
  	RLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQS
  	AGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
  	GDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
  	SVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNG
  	SISLTCLALGGVMIFLSTAVSA
  prME	AAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATM	87
  AHL43502	SYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPS
  	HSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALAAVAIAWLLGSS
  	TSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCV
  	TVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAY
  	LDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQ
  	PENLEYRIMLSVHGSQHSGMXXXXXGHETDENRAKVEVTPNSPRAEATLG
  	GFGSLGLDCEPRTGLDFSDLYYLTMNNKHRLVRKEWFHDIPLPWHAGADT
  	GTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAK
  	GRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQ
  	YAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPP
  	FGDSYIVIGVGDKKITHHWLKKGSSIGKAFEATVRGAKRMAVLGDTAWDF
  	GSVGGVFNSLGKGVHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKN
  	GSISLTCLALGGVMIFLSTAVSA
  prME	AEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMS	88
  AHL43501	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCQHKKGEARRSRRAVTLPSH
  	STRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALVAVAIAWLLGSST
  	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIVNDIGHETDENRAKVEVTPNSPRAEATLGG
  	FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
  	TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
  	RLFSGHLKCRLKMDKLRLKGVSYSLCTAVCTAAKVPAETLHGTVTVEVQY
  	AGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
  	GDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
  	SVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNG
  	SISLTCLALGGVMIFLSTAVSA
  prME	AEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMS
  	89
  AHL43500	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  	STRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALAAVAIAWLLGSST
  	SQKVIYLIMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIVNDXXXXXXXNRAEVEVTPNSPRAEATLGG
  	FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
  	TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
  	RLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQY
  	AGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
  	GDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
  	SVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNG
  	SISLTCLALGGVMIFLSTAVSA
  prME	AEITRRGSAYYMYLDRSDAGKAISFATNLGVNKCHVQIMDLGHMCDATMS	90
  AHF49785	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  	STRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALAAVAIAWLLGSST
  	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIVNDIGHETDENRAKVEVTPNSPRAEATLGG
  	FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
  	TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
  	RLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQY
  	AGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
  	GDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
  	SVGGVFNSLGKGVHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNG
  	SISLTCLALGGVMIFLSTAVSA
  prME	AEITRRGSAYYMYLDRSDAGKAISFATNLGVNKCHVQIMDLGHMCDATMS	91
  AHF49784_1976	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  	STRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALAAVAIAWLLGSST
  	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIVNDENRAKVEVTPNSPRAEATLGGFGSLGL
  	DCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNN
  	KEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGH
  	LKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGP
  	CKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIV
  	IGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVF
  	NSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTC
  	LALGGVMIFLSTAVSA
  prME	AEITRRGSAYYMYLDRSDAGKAISFATNLGVNKCHVQIMDLGHMCDATMS	92
  AHF49783	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  	STRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALAAVAIAWLLGSST
  	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIVNDIGHETDENRAKVEVTPNSPRAEATLGG
  	FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
  	TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
  	RLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQY
  	AGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
  	GDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
  	SVGGVFNSLGKGVHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNG
  	SISLTCLALGGVMIFLSTAVSA
  prME	VEVTRRGSAYYMYLDRSDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS	93
  ACD75819_Micronesia	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  	STRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSST
  	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGG
  	FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
  	TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
  	RLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQY
  	AGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
  	GDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
  	SVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNG
  	SISLTCLALGGVLIFLSTAVSA
  prME	AEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMS	94
  ABI54475	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  	STRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFTLVAVAIAWLLGSST
  	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIVNDENRAKVEVTPNSPRAEATLGGFGSLGL
  	DCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNN
  	KEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGH
  	LKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGP
  	CKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIV
  	IGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVF
  	NSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTC
  	LALGGVMIFLSTAVSA
  prME	AEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMS	95
  YP_002790881	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  	STRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALVAVAIAWLLGSST
  	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIGYETDEDRAKVEVTPNSPRAEATLGGFGSL
  	GLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHW
  	NNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFS
  	GHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTD
  	GPCKIPVQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSY
  	IVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGG
  	VFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISL
  	TCLALGGVMIFLSTAVSA
  prME	AEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMS	96
  BAP4744	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  	STRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALVAVAIAWLLGSST
  	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMTVNDIGYETDENRAKVEVTPNSPRAEATLGG
  	FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
  	TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
  	KLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQY
  	AGTDGPCKIPVQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
  	GDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
  	SVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNG
  	SISLTCLALGGVMIFLSTAVSA
  prME	AEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS	97
  KU365780_2015_Brazil_	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  isolate_BeH815744	STRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSST
  	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGG
  	FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
  	TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
  	RLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQY
  	AGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
  	GDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
  	SVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNG
  	SISLMCLALGGVLIFLSTAVSA
  prME	AEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS	98
  KU365779_2015_	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  Brazil_isolate_	STRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSST
  BeH819966	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGG
  	FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
  	TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
  	RLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQY
  	AGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
  	GDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
  	SVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNG
  	SISLMCLALGGVLIFLSTAVSA
  prME	AEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS	99
  KU365778_2015_	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  Brazil_isolate_	STRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSST
  BeH819015	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGG
  	FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
  	TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
  	RLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQY
  	AGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
  	GDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
  	SVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNG
  	SISLMCLALGGVLIFLSTAVSA
  prME	AEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS	100
  KU365777_2015_	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  Brazil_isolate_	STRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSST
  BeH818995	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGG
  	FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
  	TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
  	RLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQY
  	AGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
  	GDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
  	SVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNG
  	SISLMCLALGGVLIFLSTAVSA
  prME	AEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS	101
  KU321639_2015_	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  Brazil_isolate_	STRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSST
  ZikaSPH2015	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDIVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGG
  	FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
  	TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
  	RLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQY
  	AGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
  	GDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
  	SVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNG
  	SISLMCLALGGVLIFLSTAVSA
  prME	AEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHTCDATMS	102
  KU312312_2015_	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  Suriname_isolate_	STRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSST
  Z1106033	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGG
  	FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
  	TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
  	RLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQY
  	AGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
  	GDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
  	SVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNAKNG
  	SISLMCLALGGVLIFLSTAVSA
  Premembrane/membrane	AEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS	103
  protein	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  KU321639_2015_	STRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSST
  Brazil_isolate_	SQKVIYLVMILLIAPAYS
  ZikaSPH2015
  Envelop protein	IRCIGVSNRDFVEGMSGGTWVDIVLEHGGCVTVMAQDKPTVDIELVTTTV	104
  KU321639_2015_	SNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRG
  Brazil_isolate_	WGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSG
  ZikaSPH2015	MIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSD
  	LYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHA
  	KRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRL
  	KGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQ
  	TLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHW
  	HRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFG
  	AAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLST
  	AVSA
  Capsid protein	MKNPKKKSGGFRIVNMLKRGVARVSPFGGLKRLPAGLLLGHGPIRMVLAI	105
  KU321639_2015_	LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKE
  Brazil_isolate_	KKRRGADTSVGIVGLLLTTAMAAEV
  ZikaSPH2015
  Non-structural	VGCSVDFSKKETRCGTGVFVYNDVEAWRDRYKYHPDSPRRLAAAVKQAWE	106
  protein 1	DGICGISSVSRMENIMWRSVEGELNAILEENGVQLTVVVGSVKNPMWRGP
  KU321639_2015_	QRLPVPVNELPHGWKAWGKSHFVRAAKTNNSFVVDGDTLKECPLKHRAWN
  Brazil_isolate_	SFLVEDHGFGVFHTSVWLKVREDYSLECDPAVIGTAVKGKEAVHSDLGYW
  ZikaSPH2015	IESEKNDTWRLKRAHLIEMKTCEWPKSHTLWTDGIEESDLIIPKSLAGPL
  	SHHNTREGYRTQMKGPWHSEELEIRFEECPGTKVHVEETCGTRGPSLRST
  	TASGRVIEEWCCRECTMPPLSFRAKDGCWYGMEIRPRKEPESNLVRSMVT
  	AGSTDHMDHFSL
  Non-structural	GVLVILLMVQEGLKKRMTTKIIISTSMAVLVAMILGGFSMSDLAKLAILM	107
  protein 2A	GATFAEMNTGGDVAHLALIAAFKVRPALLVSFIFRANWTPRESMLLALAS
  KU321639_2015_	CLLQTAISALEGDLMVLINGFALAWLAIRAMVVPRTDNITLAILAALTPL
  Brazil_isolate_	ARGTLLVAWRAGLATCGGFMLLSLKGKGSVKKNLPFVMALGLTAVRLVDP
  ZikaSPH2015	INVVGLLLLTRSGKRSWP
  Non-structural	PSEVLTAVGLICALAGGFAKADIEMAGPMAAVGLLIVSYVVSGKSVDMYI	108
  protein 2B	ERAGDITWEKDAEVTGNSPRLDVALDESGDFSLVEDDGPPMREIILKVVL
  KU321639_2015_	MTICGMNPIAIPFAAGAWYVYVKTGKRSGALWDVPAPKEVKKGE
  Brazil_isolate_
  ZikaSPH2015

• 		SEQ
  		ID
  Description	Sequence	NO:
  Non-structural	TTDGVYRVMTRRLLGSTQVGVGVMQEGVFHTMWHVTKGSALRSGEGRLDP	109
  protein 3	YWGDVKQDLVSYCGPWKLDAAWDGHSEVQLLAVPPGERARNIQTLPGIFK
  KU321639_2015_	TKDGDIGAVALDYPAGTSGSPILDKCGRVIGLYGNGVVIKNGSYVSAITQ
  Brazil_isolate_	GRREEETPVECFEPSMLKKKQLTVLDLHPGAGKTRRVLPEIVREAIKTRL
  ZikaSPH2015	RTVILAPTRVVAAEMEEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFT
  	SRLLQPIRVPNYNLYIMDEAHFTDPSSIAARGYISTRVEMGEAAAIFMTA
  	TPPGTRDAFPDSNSPIMDTEVEVPERAWSSGFDWVTDYSGKTVWFVPSVR
  	NGNEIAACLTKAGKRVIQLSRKTFETEFQKTKHQEWDFVVTTDISEMGAN
  	FKADRVIDSRRCLKPVILDGERVILAGPMPVTHASAAQRRGRIGRNPNKP
  	GDEYLYGGGCAETDEDHAHWLEARMLLDNIYLQDGLIASLYRPEADKVAA
  	IEGEFKLRTEQRKTFVELMKRGDLPVWLAYQVASAGITYTDRRWCFDGTT
  	NNTIMEDSVPAEVWTRHGEKRVLKPRWMDARVCSDHAALKSFKEFAAGKR
  	GAA
  Non-structural	FGVMEALGTLPGHMTERFQEAIDNLAVLMRAETGSRPYKAAAAQLPETLE	110
  protein 4A	TIMLLGLLGTVSLGIFFVLMRNKGIGKMGFGMVTLGASAWLMWLSEIEPA
  KU321639_2015_	RIACVLIVVFLLLVVLIPEPEKQRSPQDNQMAIIIMVAVGLLGLITA
  Brazil_isolate_
  ZikaSPH2015
  Non-structural	NELGWLERTKSDLSHLMGRREEGATMGFSMDIDLRPASAWAIYAALTTFI	111
  protein 4B	TPAVQHAVTTSYNNYSLMAMATQAGVLFGMGKGMPFYAWDFGVPLLMIGC
  KU321639_2015_	YSQLTPLTLIVAIILLVAHYMYLIPGLQAAAARAAQKRTAAGIMKNPVVD
  Brazil_isolate_	GIVVTDIDTMTIDPQVEKKMGQVLLMAVAVSSAILSRTAWGWGEAGALIT
  ZikaSPH2015	AATSTLWEGSPNKYWNSSTATSLCNIFRGSYLAGASLIYTVTRNAGLVKR
  	RGGGTGETLGEKWKARLNQMSALEFYSYKKSGITEVCREEARRALKDGVA
  	TGGHAVSRGSAKLRWLVERGYLQPYGKVIDLGCGRGGWSYYAATIRKVQE
  	VKGYTKGGPGHEEPVLVQSYGWNIVRLKSGVDVFHMAAEPCDTLLCDIGE
  	SSSSPEVEEARTLRVLSMVGDWLEKRPGAFCIKVLCPYTSTMMETLERLQ
  	RRYGGGLVRVPLSRNSTHEMYWVSGAKSNTIKSVSTTSQLLLGRMDGPRR
  	PV
  Non-structural	KYEEDVNLGSGTRAVVSCAEAPNMKIIGNRIERIRSEHAETWFFDENHPY	112
  protein 5	RTWAYHGSYEAPTQGSASSLINGVVRLLSKPWDVVTGVTGIAMTDTTPYG
  KU321639_2015_	QQRVFKEKVDTRVPDPQEGTRQVMSMVSSWLWKELGKHKRPRVCTKEEFI
  Brazil_isolate_	NKVRSNAALGAIFEEEKEWKTAVEAVNDPRFWALVDKEREHHLRGECQSC
  ZikaSPH2015	VYNMMGKREKKQGEFGKAKGSRAIWYMWLGARFLEFEALGFLNEDHWMGR
  	ENSGGGVEGLGLQRLGYVLEEMSRIPGGRMYADDTAGWDTRISRFDLENE
  	ALITNQMEKGHRALALAIIKYTYQNKVVKVLRPAEKGKTVMDIISRQDQR
  	GSGQVVTYALNTFTNLVVQLIRNMEAEEVLEMQDLWLLRRSEKVTNWLQS
  	NGWDRLKRMAVSGDDCVVKPIDDRFAHALRFLNDMGKVRKDTQEWKPSTG
  	WDNWEEVPFCSHHFNKLHLKDGRSIVVPCRHQDELIGRARVSPGAGWSIR
  	ETACLAKSYAQMWQLLYFHRRDLRLMANAICSSVPVDWVPTGRTTWSIHG
  	KGEWMTTEDMLVVWNRVWIEENDHMEDKTPVTKWTDIPYLGKREDLWCGS
  	LIGHRPRTTWAENIKNTVNMVRRIIGDEEKYMDYLSTQVRYLGEEGSTPG
  	VL
  Signal	METPAQLLFLLLLWLPDTTGAEVTRRGSAYYMYLDRNDAGEAISFPTTLG	113
  peptide_prM-E	MNKCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGT
  	CHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIF
  	RNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFV
  	EGMSGGTWVDIVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYE
  	ASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGS
  	LVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDE
  	NRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWL
  	VHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQE
  	GAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAF
  	TFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITAN
  	PVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFE
  	ATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSW
  	FSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA
  Signal peptide_E	METPAQLLFLLLLWLPDTTGIRCIGVSNRDFVEGMSGGTWVDIVLEHGGC	114
  	VTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEA
  	YLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKS1
  	QPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATL
  	GGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGAD
  	TGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGA
  	KGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEV
  	QYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDP
  	PFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWD
  	FGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTK
  	NGSISLMCLALGGVLIFLSTAVSA
  IgE HC signal	MDWTWILFLVAAATRVHSVEVTRRGSAYYMYLDRSDAGEAISFPTTLGMN	115
  peptide_prM-E #1	KCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCH
  (Brazil_isolate_	HKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRN
  ZikaSPH2015)	PGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEG
  	MSGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEAS
  	ISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLV
  	TCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENR
  	AKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVH
  	KEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGA
  	VHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTF
  	TKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPV
  	ITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEAT
  	VRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFS
  	QILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA
  IgE HC signal	MDWTWILFLVAAATRVHSVEVTRRGSAYYMYLDRSDAGEAISFPTTLGMN	116
  peptide_prM-E #1	KCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCH
  (ACD75819_Micronesia)	HKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRN
  	PGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEG
  	MSGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEAS
  	ISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLV
  	TCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENR
  	AKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVH
  	KEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGA
  	VHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTF
  	TKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPV
  	ITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEAT
  	VRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFS
  	QILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA
  IgE HC signal	MDWTWILFLVAAATRVHSTRRGSAYYMYLDRSDAGEAISFPTTLGMNKCY	117
  peptide_prM-E #2	IQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKK
  (Brazil_isolate_	GEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGF
  ZikaSPH2015)	ALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSG
  	GTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISD
  	MASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCA
  	KFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKV
  	EITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEW
  	FHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHT
  	ALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKI
  	PAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITE
  	STENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRG
  	AKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQIL
  	IGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA
  HuIgGk signal	METPAQLLFLLLLWLPDTTGVEVTRRGSAYYMYLDRSDAGEAISFPTTLG	118
  peptide_prME #1	MNKCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGT
  (Brazil_isolate_	CHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIF
  ZikaSPH2015)	RNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFV
  	EGMSGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYE
  	ASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGS
  	LVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDE
  	NRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWL
  	VHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQE
  	GAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAF
  	TFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITAN
  	PVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFE
  	ATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSW
  	FSQILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA
  HuIgGk signal	METPAQLLFLLLLWLPDTTGTRRGSAYYMYLDRSDAGEAISFPTTLGMNK	119
  peptide_prME #2	CYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHH
  (Brazil_isolate_	KKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNP
  ZikaSPH2015)	GFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGM
  	SGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASI
  	SDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVT
  	CAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRA
  	KVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHK
  	EWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAV
  	HTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFT
  	KIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVI
  	TESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATV
  	RGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQ
  	ILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA
  HuIgGk signal	METPAQLLFLLLLWLPDTTGIRCIGVSNRDFVEGMSGGTWVDVVLEHGGC	120
  peptide_E	VTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEA
  (Brazil_isolate_	YLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKS1
  ZikaSPH2015)	QPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATL
  	GGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGAD
  	TGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGA
  	KGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEV
  	QYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDP
  	PFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWD
  	FGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTK
  	NGSISLTCLALGGVLIFLSTAVSA
  IgE HC signal	MDWTWILFLVAAATRVHSTRRGSAYYMYLDRSDAGEAISFPTTLGMNKCY	121
  peptide_prM-E #2	IQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKK
  (ACD75819_Micronesia)	GEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGF
  	ALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSG
  	GTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISD
  	MASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCA
  	KFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKV
  	EITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEW
  	FHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHT
  	ALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKI
  	PAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITE
  	STENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRG
  	AKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQIL
  	IGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA
  HuIgGk signal	METPAQLLFLLLLWLPDTTGVEVTRRGSAYYMYLDRSDAGEAISFPTTLG	122
  peptide_prME #1,	MNKCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGT
  (ACD75819_Micronesia)	CHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIF
  	RNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFV
  	EGMSGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYE
  	ASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGS
  	LVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDE
  	NRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWL
  	VHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQE
  	GAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAF
  	TFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITAN
  	PVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFE
  	ATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSW
  	FSQILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA
  HuIgGk signal	METPAQLLFLLLLWLPDTTGTRRGSAYYMYLDRSDAGEAISFPTTLGMNK	123
  peptide_prME #2,	CYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHH
  (ACD75819_Micronesia)	KKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNP
  	GFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGM
  	SGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASI
  	SDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVT
  	CAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRA
  	KVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHK
  	EWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAV
  	HTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFT
  	KIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVI
  	TESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATV
  	RGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQ
  	ILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA
  HuIgGk signal	METPAQLLFLLLLWLPDTTGIRCIGVSNRDFVEGMSGGTWVDVVLEHGGC	124
  peptide_E,	VTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEA
  (ACD75819_Micronesia)	YLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKS1
  	QPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATL
  	GGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGAD
  	TGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGA
  	KGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEV
  	QYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDP
  	PFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWD
  	FGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTK
  	NGSISLTCLALGGVLIFLSTAVSA
  HuIgGk signal	METPAQLLFLLLLWLPDTTG	125
  peptide
  IgE heavy chain	MDWTWILFLVAAATRVHS	126
  epsilon -1 signal
  peptide
  Zika_RIO-U1_JEVsp	AEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS	127
  Zika PRME Strain	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  ascension id:	STRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSST
  ANG09399	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  	VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGG
  	FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
  	TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
  	RLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQY
  	AGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
  	GDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
  	SVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNG
  	SISLMCLALGGVLIFLSTAVSA
  Japanese	MLGSNSGQRVVFTILLLLVAPAYS	128
  encephalitis PRM
  signal sequence
  Zika_RIO-U1_JEVsp	MLGSNSGQRVVFTILLLLVAPAYSAEVTRRGSAYYMYLDRNDAGEAISFP	129
  Zika PRME Strain	TTLGMNKCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWV
  ascension id:	VYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVE
  ANG09399 with JEV	NWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSN
  PRM signal	RDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRS
  sequence	YCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLF
  	GKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGH
  	ETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNN
  	KHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVL
  	GSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLC
  	TAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRL
  	ITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIG
  	KAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFG
  	GMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA
  Zika_RIO-	EVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMSY	130
  U1¬_VSVgSp	ECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHS
  Zika PRME Strain	TRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTS
  ascension id:	QKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTV
  ANG09399	MAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLD
  	KQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPE
  	NLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGF
  	GSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGT
  	PHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGR
  	LSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYA
  	GTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFG
  	DSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGS
  	VGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGS
  	ISLMCLALGGVLIFLSTAVSA
  VSV g protein	MKCLLYLAFLFIGVNCA	131
  signal sequence
  Zika_ RIO-	MKCLLYLAFLFigVNCAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKC	132
  U1¬_VSVgSp	YIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHK
  Zika PRME Strain	KGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPG
  ascension id:	FALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMS
  ANG09399 with VSV	GGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASIS
  g protein signal	DMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTC
  sequence	AKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAK
  	VEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKE
  	WFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVH
  	TALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTK
  	IPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVIT
  	ESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVR
  	GAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQI
  	LIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA
  ZIKA_PRME_DSP_N154A	VEVTRRGSAYYMYLDRSDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS	133
  (glycosylation	YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
  mutant)	STRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSST
  Zika PRME Strain	SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
  ascension id:	VMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL
  ACD75819	DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQP
  	ENLEYRIMLSVHGSQHSGMIVADTGHETDENRAKVEITPNSPRAEATLGG
  	FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
  	TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
  	RLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQY
  	AGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
  	GDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
  	SVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNG
  	SISLTCLALGGVLIFLSTAVSA
  ZIKA_PRME_DSP_N154A	MDWTWILFLVAAATRVHSVEVTRRGSAYYMYLDRSDAGEAISFPTTLGMN	134
  (glycosylation	KCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCH
  mutant with	HKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRN
  signal peptide)	PGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEG
  Zika PRME Strain	MSGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEAS
  ascension id:	ISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLV
  ACD75819 with IgE	TCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVADTGHETDENR
  signal peptide	AKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVH
  	KEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGA
  	VHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTF
  	TKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPV
  	ITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEAT
  	VRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFS
  	QILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA

• TABLE 33
  ZIKV NCBI Accession Numbers (Amino Acid Sequences)

  	Name	GenBank Accession
  	polyprotein [Zika virus]	YP_002790881.1
  	polyprotein [Zika virus]	BAP47441.1
  	polyprotein [Zika virus]	AEN75263.1
  	polyprotein [Zika virus]	AHL43504.1
  	polyprotein [Zika virus]	AEN75266.1
  	polyprotein [Zika virus]	AHF49784.1
  	polyprotein [Zika virus]	AHF49783.1
  	polyprotein [Zika virus]	AHF49785.1
  	polyprotein [Zika virus]	ABI54475.1
  	polyprotein [Zika virus]	AHL43501.1
  	polyprotein [Zika virus]	AHL43500.1
  	polyprotein [Zika virus]	AHL43502.1
  	polyprotein [Zika virus]	AEN75265.1
  	polyprotein [Zika virus]	AHL43503.1
  	polyprotein [Zika virus]	AEN75264.1
  	polyprotein [Zika virus]	AHZ13508.1
  	polyprotein [Zika virus]	ACD75819.1
  	polyprotein [Zika virus]	AFD30972.1
  	polyprotein [Zika virus]	AAK91609.1
  	envelope protein [Zika virus]	AHL43462.1
  	envelope protein [Zika virus]	AHL43464.1
  	envelope protein [Zika virus]	AHL43461.1
  	envelope protein [Zika virus]	AHL43460.1
  	envelope protein [Zika virus]	AHL43463.1
  	envelope protein [Zika virus]	AHL43444.1
  	envelope protein [Zika virus]	AHL43451.1
  	envelope protein [Zika virus]	AHL43437.1
  	envelope protein [Zika virus]	AHL43455.1
  	envelope protein [Zika virus]	AHL43448.1
  	envelope protein [Zika virus]	AHL43439.1
  	envelope protein [Zika virus]	AHL43468.1
  	E protein [Zika virus]	AIC06934.1
  	envelope protein [Zika virus]	AHL43450.1
  	envelope protein [Zika virus]	AHL43442.1
  	envelope protein [Zika virus]	AHL43458.1
  	envelope glycoprotein [Zika virus]	AHL16749.1
  	envelope protein [Zika virus]	AHL43453.1
  	envelope protein [Zika virus]	AHL43443.1
  	envelope protein [Zika virus]	AHL43438.1
  	envelope protein [Zika virus]	AHL43441.1
  	envelope protein [Zika virus]	AHL43457.1
  	envelope protein [Zika virus]	AAK91609.1
  	polyprotein [Zika virus]	AHL43505.1

Example 35: Surface Expressed DENV2 prME Antigens
• The DENV2 prME polypeptide antigen sequences provided in Table 34 were tested to confirm that the DENV prME protein antigen is translated, properly folded and expressed on the surface of cells. For the polypeptide sequences, the bolded sequence is Dengue signal sequence, the underlined sequence is DENV2 precursor membrane sequence, and the unmarked sequence is DENV2 envelope sequence. The sequences encoding the polypeptides are codon-optimized. HeLa cells were transfected with DNA encoding the prMEs from nine different Dengue 2 isolates. After 24 hours, surface expression of the prME was detected using three different antibodies followed by goat-anti-human AF700 secondary antibody and subjecting the cells to FACS analyses. Each of the three antibodies are broadly neutralizing DENV2 prME antibodies that have in vivo efficacy against Dengue virus. D88 binds to DIII of Envelope protein for all 4 Dengue serotypes (US20150225474). 2D22 binds to DIII of Envelope protein for Dengue 2 serotype. 5J7 binds to 3 domains of Envelope protein for Dengue 3 serotype. FIG. 34B shows that two of the DENV2 prME antigens are recognized by the D88 and 2D22 antibodies. These results show that the two DENV2 prME antigens identified as Thailand/01 68/1979 and Peru/IQT29 13/1996 are expressed at the cell surface in a conformationally correct form and are excellent vaccine candidates (FIG. 34A). FIG. 34B shows a repeat of staining in triplicate and in two different cell lines (HeLa and 293T). These results confirm proper conformation of expressed DENV2 prME antigens (in particular, the prME antigens from Thailand/01 68/1979 and Peru/IQT29 13/1996) and also evidence at least non-inferior and even superior DENV2 antigenicity as compared to Dengvaxia (CYD-TDV), a live attenuated tetravalent chimeric vaccine. Antigen expressed from the mRNA encoding Dengue 2 prME from Peru/IQT2913/1996 shows the best binding to 2 different DENV2 antibodies in 293T cells and in HeLa cells (D88—binds all 4 serotypes 2D22—binds Dengue 2). This construct has a single amino acid difference from the Dengue 2 Envelope III Domain immunodeterminant region (see bold, underline in SEQ ID NO: 168, Table 34).

• TABLE 34
  Example DENV2 PrME Polypeptide

  Sequence
  Name

  	5′ UTR	ORF	3′ UTR	Polypeptide
  Dengue 2	TCAAGCTT	ATGCTGAATATTCTGAACCGCCG	TGATAATA	MLNILNRRRRTA
  prME	TTGGACCC	CCGCCGGACTGCCGGGATTATAA	GGCTGGAG	GIIIMMIPTVMA
  (Thailand/	TCGTACAG	TTATGATGATTCCCACCGTGATG	CCTCGGTG	FHLTTRNGEPHM
  0168/1979)	AAGCTAAT	GCCTTCCACCTGACCACCCGGAA	GCCATGCT	IVSRQEKGKSLL
  	ACGACTCA	CGGGGAACCACATATGATCGTGT	TCTTGCCC	FKTEDGVNMCTL
  	CTATAGGG	CCAGACAGGAGAAGGGAAAGTCC	CTTGGGCC	MAMDLGELCEDT
  	AAATAAGA	CTGCTGTTCAAGACCGAGGACGG	TCCCCCCA	ITYKCPLLRQNE
  	GAGAAAAG	CGTGAACATGTGCACCCTCATGG	GCCCCTCC	PEDIDCWCNSTS
  	AAGAGTAA	CTATGGACCTGGGCGAACTCTGC	TCCCCTTC	TWVTYGTCTTTG
  	GAAGAAAT	GAGGACACCATCACCTACAAGTG	CTGCACCC	EHRREKRSVALV
  	ATAAGAGC	CCCCCTGTTGAGGCAGAACGAGC	GTACCCCC	PHVGMGLETRTE
  	CACC	CGGAGGATATTGACTGCTGGTGC	GTGGTCTT	TWMSSEGAWKHA
  	(SEQ ID	AATTCGACCAGCACCTGGGTCAC	TGAATAAA	QRIETWILRHPG
  	NO: 135)	CTACGGGACTTGCACCACAACCG	GTCTGAGT	FTIMAAILAYTI
  		GAGAACATCGGCGCGAAAAGCGC	GGGCGGC	GTTHFQRALIFI
  		AGCGTGGCTTTGGTGCCTCACGT	(SEQ ID	LLTAVAPSMTMR
  		CGGAATGGGGCTGGAGACTAGAA	NO: 153)	CIGISNRDFVEG
  		CCGAGACTTGGATGTCGTCGGAA		VSGGSWVDIVLE
  		GGGGCCTGGAAACACGCACAGCG		HGSCVTTMAKNK
  		CATCGAAACTTGGATACTCAGGC		PTLDFELIKTEA
  		ATCCCGGCTTCACCATTATGGCC		KQPATLRKYCIE
  		GCGATCCTGGCATACACCATCGG		AKLTNTTTESRC
  		TACTACCCACTTCCAACGGGCCC		PTQGEPSLNEEQ
  		TGATCTTTATCCTCCTGACCGCT		DKRFVCKHSMVD
  		GTCGCACCATCCATGACCATGCG		RGWGNGCGLFGK
  		GTGTATCGGTATCAGCAACAGGG		GGIVTCAMFTCK
  		ACTTCGTGGAGGGAGTGTCGGGA		KNMEGKIVQPEN
  		GGATCCTGGGTGGATATTGTGCT		LEYTIVVTPHSG
  		GGAACACGGTTCCTGCGTCACTA		EEHAVGNDTGKH
  		CCATGGCGAAGAACAAGCCTACC		GKEIKVTPQSSI
  		CTGGACTTTGAGCTGATCAAAAC		TEAELTGYGTVT
  		TGAGGCCAAGCAGCCGGCCACCC		MECSPRTGLDFN
  		TGCGCAAGTACTGCATCGAAGCC		EMVLLQMENKAW
  		AAGCTGACCAATACCACTACCGA		LVHRQWFLDLPL
  		ATCCCGCTGTCCGACCCAAGGGG		PWLPGADTQGSN
  		AGCCCTCCCTGAATGAGGAGCAG		WIQKETLVTFKN
  		GACAAGCGCTTCGTCTGCAAGCA		PHAKKQDVVVLG
  		TTCAATGGTCGACCGCGGCTGGG		SQEGAMHTALTG
  		GAAACGGCTGGGGACTGTTCGGA		ATEIQMSSGNLL
  		AAGGGCGGCATTGTGACCTGTGC		FTGHLKCRLRMD
  		CATGTTCACTTGCAAGAAGAACA		KLQLKGMSYSMC
  		TGGAAGGAAAGATCGTGCAGCCC		TGKFKVVKEIAE
  		GAAAACCTGGAGTATACCATCGT		TQHGTIVIRVQY
  		CGTGACCCCGCACTCCGGGGAAG		EGDGSPCKIPFE
  		AACACGCTGTGGGAAACGACACC		IMDLEKRHVLGR
  		GGAAAGCACGGAAAGGAGATCAA		LITVNPIVTEKD
  		AGTGACCCCACAGTCGAGCATTA		SPVNIEAEPPFG
  		CCGAGGCCGAACTTACTGGTTAC		DSYIIIGVEPGQ
  		GGCACTGTGACGATGGAATGTTC		LKLNWFKKGSSI
  		ACCGAGAACTGGACTGGATTTCA		GQMFETTMRGAK
  		ACGAAATGGTGCTGCTCCAAATG		RMAILGDTAWDF
  		GAAAACAAGGCCTGGCTGGTGCA		GSLGGVFTSIGK
  		CCGCCAGTGGTTTCTTGACCTCC		ALHQVFGAIYGA
  		CTCTCCCTTGGCTGCCTGGAGCA		AFSGVSWTMKIL
  		GACACTCAGGGTTCCAACTGGAT		IGVIITWIGMNS
  		TCAGAAGGAAACACTCGTGACCT		RSTSLSVSLVLV
  		TCAAGAACCCTCACGCGAAGAAG		GIVTLYLGVMVQ
  		CAGGATGTGGTCGTGCTGGGAAG		A (SEQ ID
  		CCAGGAGGGAGCGATGCATACCG		NO: 162)
  		CCCTCACCGGCGCGACGGAGATT
  		CAGATGTCCAGCGGAAACCTTCT
  		GTTCACCGGACACCTGAAGTGCA
  		GACTGAGGATGGACAAGCTGCAG
  		CTCAAGGGAATGTCCTACTCCAT
  		GTGCACTGGAAAGTTCAAGGTCG
  		TGAAGGAGATTGCCGAAACTCAG
  		CATGGTACCATCGTGATCCGGGT
  		GCAATATGAAGGGGACGGATCCC
  		CGTGCAAGATCCCTTTCGAAATC
  		ATGGACTTGGAGAAGCGACACGT
  		GCTGGGCAGACTGATCACAGTCA
  		ACCCCATCGTGACTGAGAAGGAT
  		TCACCCGTGAACATTGAAGCCGA
  		GCCGCCTTTCGGCGATAGCTACA
  		TCATCATTGGCGTGGAACCGGGA
  		CAGCTTAAGCTCAACTGGTTCAA
  		GAAGGGTTCCTCGATCGGTCAAA
  		TGTTTGAAACCACGATGCGGGGT
  		GCCAAACGGATGGCCATTCTGGG
  		AGACACCGCCTGGGATTTCGGCT
  		CCTTGGGCGGAGTGTTCACTTCT
  		ATCGGAAAGGCGCTGCACCAAGT
  		GTTCGGAGCCATCTACGGCGCCG
  		CGTTCTCGGGCGTCAGCTGGACC
  		ATGAAGATTCTGATCGGGGTCAT
  		CATCACTTGGATTGGGATGAACT
  		CACGGTCCACCTCCCTGAGCGTG
  		TCCCTTGTCCTGGTCGGCATCGT
  		GACCCTGTACCTCGGAGTGATGG
  		TGCAGGCTTAG (SEQ ID NO:
  		144)
  Dengue 2	TCAAGCTT	ATGCTTAACATTCTCAACCGCCG	TGATAATA	MLNILNRRRRTA
  prME	TTGGACCC	CCGGAGAACTGCTGGTATTATCA	GGCTGGAG	GIIIMMIPTVMA
  (Thailand/	TCGTACAG	TTATGATGATTCCCACTGTGATG	CCTCGGTG	FHLTTRNGEPHM
  16681/1984)	AAGCTAAT	GCCTTCCACCTGACCACGCGGAA	GCCATGCT	IVGRQEKGKSLL
  	ACGACTCA	CGGCGAACCCCATATGATTGTCG	TCTTGCCC	FKTEDGVNMCTL
  	CTATAGGG	GTCGGCAGGAAAAGGGGAAGTCC	CTTGGGCC	MAIDLGELCEDT
  	AAATAAGA	CTGCTGTTCAAAACTGAGGACGG	TCCCCCCA	ITYKCPLLRQNE
  	GAGAAAAG	AGTGAACATGTGCACCCTCATGG	GCCCCTCC	PEDIDCWCNSTS
  	AAGAGTAA	CTATTGACCTGGGAGAGCTGTGC	TCCCCTTC	TWVTYGTCATTG
  	GAAGAAAT	GAAGATACTATCAGGTACAAGTG	CTGCACCC	EHRREKRSVALV
  	ATAAGAGC	CCCCCTGCTGCGCCAGAACGAGC	GTACCCCC	PHVGMGLETRTE
  	CACC	CTGAGGACATTGACTGCTGGTGC	GTGGTCTT	TWMSSEGAWKHV
  	(SEQ ID	AACTCCACGTCAACCTGGGTCAC	TGAATAAA	QRIETWILRHPG
  	NO: 136)	CTACGGAACTTGCGCGACTACCG	GTCTGAGT	FTIMAAILAYTI
  		GCGAACATCGCAGAGAAAAGAGA	GGGCGGC	GTTHFQRALIFI
  		AGCGTGGCCCTCGTGCCGCACGT	(SEQ ID	LLTAVAPSMTMR
  		CGGGATGGGGCTGGAAACCCGGA	NO: 154)	CIGMSNRDFVEG
  		CCGAAACCTGGATGTCCTCGGAA		VSGGSWVDIVLE
  		GGCGCCTGGAAGCACGTGCAGAG		HGSCVTTMAKNK
  		GATCGAAACTTGGATCCTCCGGC		PTLDFELIKTEA
  		ACCCGGGATTCACCATCATGGCC		KQPATLRKYCIE
  		GCCATCCTCGCTTACACAATCGG		AKLTNTTTESRC
  		AACCACTCACTTTCAACGCGCCC		PTQGEPSLNEEQ
  		TGATCTTCATCCTGCTTACCGCC		DKRFVCKHSMVD
  		GTGGCCCCGTCCATGACCATGCG		RGWGNGCGLFGK
  		CTGCATTGGAATGTCAAACCGGG		GGIVTCAMFRCK
  		ACTTCGTCGAGGGAGTCTCCGGA		KNMEGKVVQPEN
  		GGAAGCTGGGTGGACATCGTGCT		LEYTIVITPHSG
  		GGAGCACGGCAGCTGTGTGACCA		EEHAVGNDTGKH
  		CCATGGCCAAGAACAAGCCAACT		GKEIKITPQSST
  		CTTGATTTCGAACTGATCAAGAC		TEAELTGYGTVT
  		CGAGGCCAAGCAGCCTGCCACTC		MECSPRTGLDFN
  		TGAGGAAGTACTGTATCGAAGCG		EMVLLQMENKAW
  		AAGCTGACCAACACCACTACCGA		LVHRQWFLDLPL
  		ATCCCGCTGCCCGACCCAGGGCG		PWLPGADTQGSN
  		AACCTTCCTTGAACGAAGAACAG		WIQKETLVTFKN
  		GACAAGAGATTCGTGTGCAAGCA		PHAKKQDVVVLG
  		TAGCATGGTCGACAGGGGATGGG		SQEGAMHTALTG
  		GGAACGGATGTGGACTCTTTGGG		ATEIQMSSGNLL
  		AAGGGCGGAATCGTCACCTGTGC		FTGHLKCRLRMD
  		GATGTTCCGGTGCAAGAAGAACA		KLQLKGMSYSMC
  		TGGAGGGGAAGGTCGTGCAGCCC		TGKFKVVKEIAE
  		GAAAATCTCGAGTACACTATCGT		TQHGTIVIRVQY
  		GATCACCCCGCATTCCGGAGAGG		EGDGSPCKIPFE
  		AGCACGCCGTGGGCAACGACACC		IMDLEKRHVLGR
  		GGGAAGCACGGAAAGGAGATCAA		LITVNPIVTEKD
  		AATTACCCCTCAATCCTCCACCA		SPVNIEAEPPFG
  		CCGAAGCCGAATTGACTGGTTAC		DSYIIIGVEPGQ
  		GGTACCGTGACTATGGAGTGCTC		LKLNWFKKGSSI
  		GCCGCGGACTGGCTTGGACTTCA		GQMFETTMRGAK
  		ACGAGATGGTGCTGCTGCAAATG		RMAILGDTAWDF
  		GAGAACAAGGCCTGGCTGGTGCA		GSLGGVFTSIGK
  		CCGGCAGTGGTTTCTTGATCTGC		ALHQVFGAIYGA
  		CTCTGCCTTGGCTGCCCGGAGCC		AFSGVSWTMKIL
  		GACACCCAGGGTAGCAATTGGAT		IGVIITWIGMNS
  		CCAGAAAGAGACACTCGTGACCT		RSTSLSVTLVLV
  		TTAAGAACCCGCACGCAAAGAAG		GIVTLYLGVMVQ
  		CAGGATGTCGTGGTCCTGGGAAG		A (SEQ ID
  		CCAAGAAGGGGCAATGCATACCG		NO: 163)
  		CACTCACTGGAGCCACTGAAATC
  		CAGATGTCCTCCGGCAATCTGCT
  		GTTCACCGGCCATCTGAAGTGCC
  		GACTGCGCATGGACAAGCTCCAG
  		CTTAAGGGAATGTCCTACTCCAT
  		GTGTACTGGAAAGTTCAAAGTCG
  		TGAAGGAAATTGCCGAAACCCAG
  		CACGGCACCATAGTGATCCGGGT
  		GCAGTACGAGGGCGACGGCTCAC
  		CCTGCAAAATCCCGTTCGAGATT
  		ATGGATCTCGAAAAGCGCCACGT
  		GCTGGGCAGACTGATTACCGTGA
  		ACCCTATCGTGACCGAGAAGGAT
  		TCCCCAGTGAACATCGAGGCCGA
  		ACCGCCCTTCGGAGACTCGTATA
  		TCATCATCGGCGTGGAGCCCGGC
  		CAGCTGAAGCTGAACTGGTTCAA
  		GAAGGGGTCGAGCATCGGCCAGA
  		TGTTCGAGACTACCATGCGCGGC
  		GCGAAGAGGATGGCGATCCTGGG
  		GGATACCGCTTGGGACTTCGGTT
  		CCCTCGGCGGGGTGTTCACCTCG
  		ATTGGGAAGGCCCTCCACCAAGT
  		GTTCGGTGCAATCTACGGAGCGG
  		CGTTCAGCGGAGTGTCGTGGACC
  		ATGAAGATTCTGATCGGCGTGAT
  		CATCACCTGGATTGGCATGAACT
  		CCCGGTCTACTAGCCTGTCGGTG
  		ACCCTGGTGCTGGTCGGAATCGT
  		GACCTTGTACCTGGGAGTGATGG
  		TGCAAGCTTAG (SEQ ID NO:
  		145)
  Dengue 2	TCAAGCTT	ATGCTGAACATCCTGAACCGCAG	TGATAATA	MLNILNRRRRTA
  prME	TTGGACCC	AAGGAGAACCGCCGGTATTATTA	GGCTGGAG	GIIIMMIPTVMA
  (Jamaica/	TCGTACAG	TTATGATGATCCCCACCGTGATG	CCTCGGTG	FHLTTRNGEPHM
  1409/1983)	AAGCTAAT	GCATTCCACCTGACTACCCGCAA	GCCATGCT	IVGRQEKGKSLL
  	ACGACTCA	CGGAGAGCCGCATATGATCGTGG	TCTTGCCC	FKTEDGVNMCTL
  	CTATAGGG	GCCGCCAGGAAAAGGGAAAGTCC	CTTGGGCC	MAIDLGELCEDT
  	AAATAAGA	CTGCTGTTCAAGACTGAGGACGG	TCCCCCCA	ITYKCPLLRQNE
  	GAGAAAAG	CGTGAACATGTGCACTCTCATGG	GCCCCTCC	PEDIDCWCNSTS
  	AAGAGTAA	CCATCGACCTCGGCGAACTGTGC	TCCCCTTC	TWVTYGTCATTG
  	GAAGAAAT	GAGGACACCATTACTTACAAGTG	CTGCACCC	EHRREKRSVALV
  	ATAAGAGC	CCCGCTGCTGAGACAGAACGAGC	GTACCCCC	PHVGMGLETRTE
  	CACC	CTGAGGACATCGACTGTTGGTGT	GTGGTCTT	TWMSSEGAWKHV
  	(SEQ ID	AACTCGACCTCCACCTGGGTCAC	TGAATAAA	QRIETWILRHPG
  	NO: 137)	CTACGGAACGTGCGCCACAACCG	GTCTGAGT	FTIMAAILAYTI
  		GAGAACACCGCCGGGAAAAGCGG	GGGCGGC	GTTHFQRALIFI
  		AGCGTGGCTCTGGTGCCGCACGT	(SEQ ID	LLTAVAPSMTMR
  		CGGAATGGGTCTGGAGACTAGAA	NO: 155)	CIGISNRDFVEG
  		CCGAAACCTGGATGTCATCCGAG		VSGGSWVDIVLE
  		GGGGCATGGAAACATGTGCAGCG		HGSCVTTMAKNK
  		AATCGAGACTTGGATCCTGAGAC		PTLDFELIKTEA
  		ACCCGGGCTTCACTATCATGGCG		KQPATLRKYCIE
  		GCCATCCTTGCCTACACCATTGG		AKLTNTTTESRC
  		CACTACTCACTTCCAACGGGCGC		PTQGEPSLNEEQ
  		TGATCTTCATACTGCTCACCGCG		DKRFLCKHSMVD
  		GTGGCCCCCTCCATGACGATGCG		RGWGNGCGLFGK
  		CTGCATCGGAATCTCCAACCGGG		GGIVTCAMFTCK
  		ACTTCGTGGAGGGCGTCAGCGGA		KNMEGKVVLPEN
  		GGCAGCTGGGTGGACATCGTGTT		LEYTIVITPHSG
  		GGAGCACGGAAGCTGCGTGACCA		EEHAVGNDTGKH
  		CCATGGCCAAGAACAAGCCCACT		GKEIKITPQSSI
  		CTTGATTTTGAGCTGATCAAGAC		TEAELTGYGTVT
  		GGAAGCAAAGCAGCCGGCCACTC		MECSPRTGLDFN
  		TGAGGAAGTACTGCATCGAGGCC		EMVLLQMEDKAW
  		AAGCTCACCAACACAACCACCGA		LVHRQWFLDLPL
  		ATCTCGGTGCCCGACCCAAGGAG		PWLPGADTQGSN
  		AGCCATCACTGAACGAGGAACAG		WIQKETLVTFKN
  		GACAAGAGATTCCTGTGCAAACA		PHAKKQDVVVLG
  		TTCGATGGTGGACAGGGGATGGG		SQEGAMHTALTG
  		GAAATGGTTGCGGCCTGTTCGGC		ATEIQMSSGNLL
  		AAAGGAGGCATTGTGACCTGTGC		FTGHLKCRLRMD
  		GATGTTCACTTGCAAGAAAAACA		KLQLKGMSYSMC
  		TGGAGGGGAAGGTCGTGTTGCCG		TGKFKIVKEIAE
  		GAGAACCTGGAGTACACTATCGT		TQHGTIVIRVQY
  		GATTACCCCGCACTCCGGGGAGG		EGDGSPCKIPFE
  		AACATGCCGTGGGAAATGACACC		IMDLEKRHVLGR
  		GGAAAGCACGGGAAGGAAATCAA		LITVNPIVTEKD
  		AATCACGCCTCAGTCCTCAATCA		SPVNIEAEPPFG
  		CCGAAGCCGAGCTTACCGGCTAC		DSYIIIGVEPGQ
  		GGTACCGTGACCATGGAGTGCAG		LKLNWFKKGSSI
  		CCCTCGGACTGGACTGGACTTCA		GQMFETTMRGAK
  		ACGAGATGGTGCTGCTGCAAATG		RMAILGDTAWDF
  		GAAGATAAGGCCTGGCTGGTGCA		GSLGGVFTSIGK
  		CCGGCAGTGGTTCTTGGATTTGC		ALHQVFGAIYGA
  		CACTGCCTTGGCTGCCCGGCGCG		AFSGVSWTMKIL
  		GATACCCAGGGTTCCAACTGGAT		IGVIITWIGMNS
  		TCAGAAGGAAACCCTCGTGACCT		RSTSLSVSLVLV
  		TCAAGAATCCTCACGCCAAGAAG		GVVTLYLGAMVQ
  		CAGGACGTGGTGGTGCTGGGTTC		A (SEQ ID
  		CCAAGAAGGGGCCATGCATACTG		NO: 164)
  		CCCTCACTGGAGCGACCGAAATC
  		CAGATGTCGTCCGGCAACCTCCT
  		GTTCACCGGCCACCTGAAGTGCC
  		GCCTGCGGATGGACAAGTTGCAG
  		CTGAAGGGAATGAGCTACTCGAT
  		GTGTACCGGAAAGTTCAAGATCG
  		TGAAGGAAATCGCCGAAACCCAG
  		CACGGAACCATCGTCATTAGAGT
  		GCAGTACGAAGGGGACGGCAGCC
  		CGTGCAAGATCCCCTTCGAAATT
  		ATGGACCTGGAGAAGCGCCACGT
  		GCTCGGAAGGCTCATCACTGTCA
  		ACCCAATCGTCACCGAAAAGGAC
  		TCCCCTGTGAACATCGAAGCAGA
  		GCCCCCTTTCGGGGACTCCTACA
  		TTATTATCGGCGTGGAGCCCGGC
  		CAGCTGAAGCTGAACTGGTTCAA
  		GAAGGGATCCTCGATCGGACAGA
  		TGTTCGAAACCACCATGCGGGGA
  		GCCAAGCGGATGGCTATTCTGGG
  		AGATACCGCTTGGGATTTCGGCT
  		CCCTCGGCGGCGTCTTTACTTCC
  		ATCGGGAAAGCGCTCCACCAAGT
  		GTTTGGAGCCATCTACGGTGCCG
  		CTTTTTCCGGGGTGTCATGGACC
  		ATGAAGATTCTTATCGGGGTCAT
  		TATTACTTGGATCGGCATGAACT
  		CCCGGAGCACCTCGCTGTCCGTG
  		AGCCTCGTGCTCGTGGGGGTGGT
  		CACTCTGTATCTTGGTGCCATGG
  		TGCAGGCCTAG (SEQ ID NO:
  		146)
  Dengue 2	TCAAGCTT	ATGCTTAACATCCTGAATAGAAG	TGATAATA	MLNILNRRRRTA
  prME	TTGGACCC	AAGAAGAACCGCCGGCATTATCA	GGCTGGAG	GIIIMMIPTVMA
  (Thailand/	TCGTACAG	TTATGATGATACCCACCGTGATG	CCTCGGTG	FHLTTRNGEPHM
  NGS-	AAGCTAAT	GCCTTCCACCTGACTACTCGCAA	GCCATGCT	IVSRQEKGKSLL
  C/1944)	ACGACTCA	CGGAGAGCCTCATATGATCGTGT	TCTTGCCC	FKTEDGVNMCTL
  	CTATAGGG	CGCGGCAGGAGAAGGGAAAGTCC	CTTGGGCC	MAMDLGELCEDT
  	AAATAAGA	CTGCTGTTTAAGACGGAGGACGG	TCCCCCCA	ITYKCPFLKQNE
  	GAGAAAAG	CGTGAACATGTGCACTCTTATGG	GCCCCTCC	PEDIDCWCNSTS
  	AAGAGTAA	CAATGGACCTTGGAGAGCTGTGC	TCCCCTTC	TWVTYGTCTTTG
  	GAAGAAAT	GAGGATACCATCACCTACAAGTG	CTGCACCC	EHRREKRSVALV
  	ATAAGAGC	TCCGTTCCTGAAGCAAAACGAGC	GTACCCCC	PHVGMGLETRTE
  	CACC	CTGAGGATATTGACTGCTGGTGC	GTGGTCTT	TWMSSEGAWKHA
  	(SEQ ID	AACTCCACCTCAACCTGGGTCAC	TGAATAAA	QRIETWILRHPG
  	NO: 138)	ATATGGGACCTGTACCACTACTG	GTCTGAGT	FTIMAAILAYTI
  		GCGAACACCGCCGCGAGAAAAGA	GGGCGGC	GTTHFQRALIFI
  		AGCGTGGCGTTGGTGCCTCACGT	(SEQ ID	LLTAVAPSMTMR
  		CGGCATGGGTCTGGAAACTCGGA	NO: 156)	CIGISNRDFVEG
  		CCGAAACTTGGATGAGCTCAGAG		VSGGSWVDIVLE
  		GGGGCATGGAAGCACGCCCAGAG		HGSCVTTMAKNK
  		GATTGAAACCTGGATTCTGCGCC		PTLDFELIETEA
  		ACCCTGGATTCACCATCATGGCG		KQPATLRKYCIE
  		GCTATTCTGGCGTACACTATTGG		AKLTNTTTDSRC
  		AACCACCCACTTTCAGCGGGCCC		PTQGEPSLNEEQ
  		TTATCTTCATCCTCCTCACTGCC		DKRFVCKHSMVD
  		GTGGCGCCCTCCATGACTATGCG		RGWGNGCGLFGK
  		GTGTATCGGAATTTCCAACCGCG		GGIVTCAMFTCK
  		ACTTCGTGGAAGGAGTGTCCGGA		KNMKGKVVQPEN
  		GGCTCCTGGGTCGACATTGTGCT		LEYTIVITPHSG
  		GGAACATGGTTCATGCGTGACCA		EEHAVGNDTGKH
  		CGATGGCCAAGAACAAGCCCACC		GKEIKITPQSSI
  		CTCGACTTCGAGCTGATCGAGAC		TEAELTGYGTVT
  		TGAAGCCAAGCAGCCGGCCACTC		MECSPRTGLDFN
  		TGCGGAAGTACTGTATCGAGGCC		EMVLLQMENKAW
  		AAGCTCACCAACACCACCACCGA		LVHRQWFLDLPL
  		TTCCCGCTGCCCGACCCAAGGAG		PWLPGADTQGSN
  		AACCTTCGCTCAACGAGGAGCAG		WIQKETLVTFKN
  		GACAAGCGGTTCGTGTGCAAGCA		PHAKKQDVVVLG
  		CAGCATGGTCGACAGGGGATGGG		SQEGAMHTALTG
  		GGAATGGATGCGGTCTGTTCGGA		ATEIQMSSGNLL
  		AAGGGAGGCATTGTGACTTGTGC		FTGHLKCRLRMD
  		AATGTTCACTTGCAAGAAGAACA		KLQLKGMSYSMC
  		TGAAGGGGAAGGTCGTGCAGCCG		TGKFKVVKEIAE
  		GAAAACCTGGAGTACACCATCGT		TQHGTIVIRVQY
  		GATCACCCCTCATTCGGGCGAAG		EGDGSPCKIPFE
  		AACACGCTGTGGGGAATGATACC		IMDLEKRHVLGR
  		GGAAAGCACGGAAAGGAAATTAA		LITVNPIVTEKD
  		GATCACACCCCAATCCAGCATCA		SPVNIEAEPPFG
  		CTGAGGCAGAACTGACCGGCTAC		DSYIIIGVEPGQ
  		GGCACTGTGACCATGGAGTGCTC		LKLNWFKKGSSI
  		GCCTCGGACTGGCCTGGACTTCA		GQMIETTMRGAK
  		ACGAGATGGTGCTGCTCCAAATG		RMAILGDTAWDF
  		GAAAACAAGGCCTGGCTGGTGCA		GSLGGVFTSIGK
  		CAGACAGTGGTTCCTCGATTTGC		ALHQVFGAIYGA
  		CCTTGCCGTGGCTCCCTGGCGCC		AFSGVSWIMKIL
  		GACACCCAGGGATCTAACTGGAT		IGVIITWIGMNS
  		CCAGAAGGAAACCCTTGTGACCT		RSTSLSVSLVLV
  		TCAAGAACCCGCACGCTAAGAAA		GVVTLYLGVMVQ
  		CAGGATGTGGTGGTGCTGGGAAG		A (SEQ ID
  		CCAGGAAGGAGCAATGCATACCG		NO: 165)
  		CGCTCACGGGTGCCACCGAGATC
  		CAGATGAGCTCCGGGAACCTCCT
  		GTTCACCGGTCACCTGAAGTGCC
  		GACTCCGCATGGACAAACTGCAG
  		CTCAAGGGGATGTCCTACTCCAT
  		GTGCACCGGGAAATTCAAGGTCG
  		TGAAGGAGATCGCTGAGACTCAG
  		CACGGTACTATCGTGATCCGGGT
  		GCAGTATGAGGGAGATGGGAGCC
  		CGTGCAAAATCCCATTTGAGATC
  		ATGGACTTGGAAAAGCGCCATGT
  		GCTGGGTCGGCTGATTACCGTGA
  		ACCCAATCGTCACCGAAAAGGAC
  		AGCCCCGTCAACATTGAAGCCGA
  		ACCACCCTTCGGAGACTCGTACA
  		TCATCATTGGCGTGGAACCGGGC
  		CAGCTGAAGCTGAACTGGTTCAA
  		AAAGGGGTCCTCTATCGGCCAAA
  		TGATCGAAACCACCATGCGGGGA
  		GCTAAGCGGATGGCGATTTTGGG
  		AGACACTGCGTGGGACTTTGGCT
  		CACTGGGGGGAGTGTTCACCAGC
  		ATCGGCAAAGCCCTGCACCAAGT
  		GTTCGGTGCCATCTACGGAGCCG
  		CCTTCAGCGGAGTGTCCTGGATC
  		ATGAAGATCCTGATCGGCGTGAT
  		CATTACCTGGATCGGCATGAACT
  		CCAGGTCCACCTCGCTCTCCGTG
  		TCGCTGGTGCTGGTCGGGGTCGT
  		GACCCTGTACCTGGGAGTGATGG
  		TCCAGGCCTGA (SEQ ID NO:
  		147)
  Dengue 2	TCAAGCTT	ATGTTGAATATCCTGAACCGCCG	TGATAATA	MLNILNRRRRTA
  prME	TTGGACCC	CCGGAGAACTGCCGGAATTATCA	GGCTGGAG	GIIIMMIPTVMA
  (PuertoRico/	TCGTACAG	TTATGATGATCCCTACCGTGATG	CCTCGGTG	FHLTTRNGEPHM
  PR159-	AAGCTAAT	GCGTTCCACCTTACTACCCGGAA	GCCATGCT	IVSRQEKGKSLL
  S1/1969)	ACGACTCA	CGGGGAGCCTCACATGATCGTGT	TCTTGCCC	FKTKDGTNMCTL
  	CTATAGGG	CACGCCAGGAGAAGGGGAAATCC	CTTGGGCC	MAMDLGELCEDT
  	AAATAAGA	CTGCTGTTCAAGACCAAGGACGG	TCCCCCCA	ITYKCPFLKQNE
  	GAGAAAAG	TACCAACATGTGTACCCTGATGG	GCCCCTCC	PEDIDCWCNSTS
  	AAGAGTAA	CGATGGACCTCGGAGAGCTGTGC	TCCCCTTC	TWVTYGTCTTTG
  	GAAGAAAT	GAGGACACCATCACCTACAAATG	CTGCACCC	EHRREKRSVALV
  	ATAAGAGC	CCCGTTCCTGAAGCAGAACGAGC	GTACCCCC	PHVGMGLETRTE
  	CACC	CGGAAGATATTGACTGTTGGTGC	GTGGTCTT	TWMSSEGAWKHA
  	(SEQ ID	AACTCCACCTCCACTTGGGTCAC	TGAATAAA	QRIETWILRHPG
  	NO: 139)	CTACGGAACTTGCACCACTACTG	GTCTGAGT	FTIMAAILAYTI
  		GGGAGCATAGACGGGAGAAGCGC	GGGCGGC	GTTHFQRVLIFI
  		TCCGTGGCCCTGGTGCCGCACGT	(SEQ ID	LLTAIAPSMTMR
  		CGGCATGGGACTGGAAACCAGAA	NO: 157)	CIGISNRDFVEG
  		CCGAGACTTGGATGTCCAGCGAA		VSGGSWVDIVLE
  		GGCGCCTGGAAGCACGCCCAGCG		HGSCVTTMAKNK
  		GATTGAAACTTGGATCCTGAGGC		PTLDFELIKTEA
  		ACCCGGGTTTTACCATTATGGCC		KQPATLRKYCIE
  		GCTATCTTGGCATACACCATCGG		AKLTNTTTDSRC
  		CACCACCCACTTCCAACGCGTCC		PTQGEPTLNEEQ
  		TGATCTTCATCCTGCTGACCGCC		DKRFVCKHSMVD
  		ATTGCGCCCTCCATGACCATGCG		RGWGNGCGLFGK
  		GTGCATCGGAATCAGCAACCGCG		GGIVTCAMFTCK
  		ACTTCGTGGAAGGCGTCAGCGGC		KNMEGKIVQPEN
  		GGTTCTTGGGTGGACATCGTGTT		LEYTVVITPHSG
  		GGAGCACGGATCGTGCGTGACCA		EEHAVGNDTGKH
  		CCATGGCCAAGAACAAGCCGACC		GKEVKITPQSSI
  		CTCGATTTCGAGCTGATCAAGAC		TEAELTGYGTVT
  		TGAAGCCAAGCAGCCAGCTACCC		MECSPRTGLDFN
  		TGCGGAAGTATTGCATCGAAGCC		EMVLLQMKDKAW
  		AAGCTCACTAATACTACGACCGA		LVHRQWFLDLPL
  		CAGCCGGTGTCCGACCCAAGGAG		PWLPGADTQGSN
  		AGCCCACCCTGAATGAGGAACAA		WIQKETLVTFKN
  		GACAAGCGCTTCGTGTGCAAGCA		PHAKKQDVVVLG
  		TTCCATGGTGGACCGGGGCTGGG		SQEGAMHTALTG
  		GAAACGGCTGCGGACTGTTCGGG		ATEIQMSSGNLL
  		AAAGGAGGAATTGTGACTTGCGC		FTGHLKCRLRMD
  		CATGTTCACTTGCAAGAAGAACA		KLQLKGMSYSMC
  		TGGAGGGGAAGATCGTCCAGCCT		TGKFKVVKEIAE
  		GAGAACCTCGAGTACACGGTCGT		TQHGTIVIRVQY
  		GATTACTCCGCACTCGGGAGAAG		EGDGSPCKTPFE
  		AACACGCCGTGGGCAACGACACC		IMDLEKRHVLGR
  		GGAAAGCATGGGAAGGAAGTGAA		LTTVNPIVTEKD
  		AATCACGCCCCAATCGTCGATTA		SPVNIEAEPPFG
  		CCGAGGCTGAGCTGACCGGCTAC		DSYIIIGVEPGQ
  		GGCACCGTGACCATGGAGTGCTC		LKLDWFKKGSSI
  		CCCGAGGACCGGACTGGACTTCA		GQMFETTMRGAK
  		ACGAAATGGTGCTGCTGCAGATG		RMAILGDTAWDF
  		AAGGACAAGGCCTGGCTGGTGCA		GSLGGVFTSIGK
  		CCGCCAGTGGTTCCTCGACCTCC		ALHQVFGAIYGA
  		CACTCCCCTGGCTGCCCGGAGCG		AFSGVSWTMKIL
  		GATACGCAGGGATCCAACTGGAT		IGVIITWIGMNS
  		CCAGAAGGAAACTCTTGTGACCT		RSTSLSVSLVLV
  		TCAAGAACCCTCATGCCAAGAAG		GIVTLYLGVMVQ
  		CAGGACGTGGTGGTCCTGGGATC		A (SEQ ID
  		CCAAGAGGGCGCGATGCACACCG		NO: 166)
  		CACTGACCGGCGCCACCGAAATT
  		CAGATGTCCTCCGGAAACCTCCT
  		GTTCACTGGCCACCTGAAGTGCA
  		GACTCCGCATGGACAAGCTGCAG
  		CTCAAGGGGATGAGCTACTCCAT
  		GTGTACCGGAAAATTCAAGGTCG
  		TGAAGGAAATTGCAGAAACACAG
  		CATGGGACAATTGTCATTCGGGT
  		CCAGTACGAGGGCGATGGTTCAC
  		CGTGCAAGACTCCATTCGAGATC
  		ATGGATCTGGAGAAAAGACACGT
  		GCTGGGTCGGCTGACTACCGTGA
  		ACCCAATCGTGACTGAGAAGGAC
  		TCCCCCGTGAACATCGAAGCCGA
  		GCCTCCTTTTGGCGATTCCTACA
  		TCATCATTGGAGTGGAACCCGGA
  		CAGCTTAAGTTGGATTGGTTCAA
  		GAAGGGCTCCTCGATCGGACAGA
  		TGTTCGAAACCACCATGCGCGGT
  		GCCAAGCGAATGGCCATCCTGGG
  		GGACACCGCCTGGGACTTCGGTA
  		GCCTGGGCGGAGTGTTTACCTCA
  		ATTGGAAAGGCTCTGCACCAAGT
  		GTTTGGGGCGATCTACGGAGCGG
  		CCTTCAGCGGTGTCTCCTGGACT
  		ATGAAGATTCTCATCGGAGTGAT
  		AATCACCTGGATCGGCATGAACA
  		GCCGGTCAACCAGCCTGTCCGTG
  		TCCCTGGTGCTGGTCGGCATCGT
  		GACTCTCTACCTCGGAGTGATGG
  		TGCAGGCCTAG (SEQ ID NO:
  		148)
  Dengue 2	TCAAGCTT	ATGCTCAACATACTGAACAGACG	TGATAATA	MLNILNRRRRTA
  prME	TTGGACCC	GAGAAGGACCGCCGGTATTATTA	GGCTGGAG	GIIIMMIPTVMA
  (16681-	TCGTACAG	TCATGATGATCCCTACTGTGATG	CCTCGGTG	FHLTTRNGEPHM
  PDK53)	AAGCTAAT	GCATTCCACCTGACAACCCGCAA	GCCATGCT	IVSRQEKGKSLL
  	ACGACTCA	CGGAGAGCCCCACATGATCGTGT	TCTTGCCC	FKTEVGVNMCTL
  	CTATAGGG	CACGCCAGGAGAAAGGGAAGTCA	CTTGGGCC	MAMDLGELCEDT
  	AAATAAGA	CTGCTGTTCAAGACCGAAGTCGG	TCCCCCCA	ITYKCPLLRQNE
  	GAGAAAAG	CGTGAACATGTGTACCCTGATGG	GCCCCTCC	PEDIDCWCNSTS
  	AAGAGTAA	CGATGGATCTTGGCGAACTGTGC	TCCCCTTC	TWVTYGTCTTMG
  	GAAGAAAT	GAGGACACCATCAGGTACAAGTG	CTGCACCC	EHRREKRSVALV
  	ATAAGAGC	CCCCCTGTTGCGGCAAAACGAAC	GTACCCCC	PHVGMGLETRTE
  	CACC	CAGAGGACATCGACTGCTGGTGT	GTGGTCTT	TWMSSEGAWKHV
  	(SEQ ID	AACTCCACCTCGACCTGGGTCAC	TGAATAAA	QRIETWILRHPG
  	NO: 140)	CTACGGAACCTGTACCACTATGG	GTCTGAGT	FTMMAAILAYTI
  		GGGAACACCGGCGGGAGAAGCGC	GGGCGGC	GTTHFQRALILI
  		TCCGTGGCGCTCGTGCCTCATGT	(SEQ ID	LLTAVTPSMTMR
  		CGGCATGGGACTGGAGACTCGGA	NO: 158)	CIGMSNRDFVEG
  		CTGAAACCTGGATGTCGTCGGAG		VSGGSWVDIVLE
  		GGGGCCTGGAAGCACGTCCAGCG		HGSCVTTMAKNK
  		GATCGAGACTTGGATCCTTCGCC		PTLDFELIKTEA
  		ATCCGGGCTTCACCATGATGGCC		KQPATLRKYCIE
  		GCCATCCTGGCCTACACCATCGG		AKLTNTTTESRC
  		AACCACCCATTTCCAACGGGCCC		PTQGEPSLNEEQ
  		TGATCCTGATCCTGTTGACTGCC		DKRFVCKHSMVD
  		GTGACCCCCTCCATGACTATGCG		RGWGNGCGLFGK
  		GTGCATTGGGATGTCGAACAGGG		GGIVTCAMFRCK
  		ATTTCGTGGAGGGAGTCAGCGGT		KNMEGKVVQPEN
  		GGCAGCTGGGTGGACATCGTGCT		LEYTIVITPHSG
  		GGAACATGGATCCTGCGTGACTA		EEHAVGNDTGKH
  		CCATGGCAAAGAACAAGCCAACC		GKEIKITPQSSI
  		CTCGATTTCGAACTGATCAAGAC		TEAELTGYGTIT
  		CGAGGCGAAACAGCCGGCGACCC		MECSPRTGLDFN
  		TGAGGAAGTACTGCATCGAGGCC		EIVLLQMENKAW
  		AAGCTCACCAACACCACTACCGA		LVHRQWFLDLPL
  		GAGCAGATGCCCTACCCAAGGGG		PWLPGADTQGSN
  		AACCTTCCCTGAACGAGGAGCAG		WIQKETLVTFKN
  		GACAAGAGATTCGTCTGCAAGCA		PHAKKQDVVVLG
  		CTCCATGGTGGACCGCGGCTGGG		SQEGAMHTALTG
  		GAAACGGATGCGGACTCTTCGGA		ATEIQMSSGNLL
  		AAGGGCGGTATTGTGACCTGTGC		FTGHLKCRLRMD
  		CATGTTCCGCTGCAAGAAAAACA		KLQLKGMSYSMC
  		TGGAAGGGAAAGTGGTGCAGCCC		TGKFKVVKEIAE
  		GAGAACCTCGAGTACACTATCGT		TQHGTIVIRVQY
  		GATCACACCGCACAGCGGAGAAG		EGDGSPCKIPFE
  		AACACGCCGTGGGCAACGACACT		IMDLEKRHVLGR
  		GGAAAGCACGGGAAGGAAATCAA		LITVNPIVTEKD
  		GATCACCCCGCAATCCTCAATCA		SPVNIEAEPPFG
  		CTGAGGCTGAGTTGACCGGCTAC		DSYIIIGVEPGQ
  		GGGACTATTACCATGGAATGCTC		LKLNWFKKGSSI
  		CCCACGAACGGGACTGGACTTCA		GQMFETTMRGAK
  		ACGAAATTGTGTTGCTCCAAATG		RMAILGDTAWDF
  		GAAAACAAGGCCTGGCTCGTGCA		GSLGGVFTSIGK
  		CCGGCAGTGGTTCCTGGATCTGC		ALHQVFGAIYGA
  		CCCTGCCGTGGCTGCCGGGTGCC		AFSGVSWTMKIL
  		GACACTCAGGGGAGCAACTGGAT		IGVIITWIGMNS
  		TCAGAAGGAAACCCTTGTGACCT		RSTSLSVTLVLV
  		TCAAGAACCCCCACGCAAAGAAG		GIVTLYLGVMVQ
  		CAGGACGTGGTGGTGCTGGGTAG		A (SEQ ID
  		CCAAGAAGGCGCCATGCACACGG		NO: 167)
  		CCCTGACCGGAGCGACCGAGATC
  		CAGATGTCCAGCGGAAATCTGCT
  		CTTTACTGGTCATCTGAAGTGCA
  		GACTTCGGATGGACAAGCTGCAA
  		CTGAAGGGAATGTCCTACTCAAT
  		GTGCACTGGAAAGTTCAAGGTCG
  		TGAAGGAGATCGCCGAAACCCAG
  		CACGGGACTATCGTCATCCGCGT
  		GCAGTACGAAGGAGATGGCTCCC
  		CGTGCAAGATCCCTTTCGAAATC
  		ATGGACCTGGAGAAGCGCCACGT
  		GTTGGGGCGCCTTATTACTGTGA
  		ACCCCATCGTGACCGAGAAGGAC
  		TCCCCTGTCAACATCGAGGCTGA
  		ACCGCCATTCGGAGATTCCTATA
  		TCATTATCGGAGTGGAACCGGGC
  		CAGCTCAAGCTGAATTGGTTCAA
  		GAAGGGATCCTCGATTGGCCAGA
  		TGTTCGAAACGACTATGCGGGGC
  		GCTAAGCGCATGGCCATCCTGGG
  		CGATACTGCCTGGGATTTTGGTT
  		CTCTGGGCGGAGTGTTCACCTCC
  		ATTGGAAAGGCCCTGCACCAAGT
  		GTTCGGCGCCATCTACGGTGCCG
  		CGTTTAGCGGTGTCTCATGGACC
  		ATGAAAATCCTCATTGGCGTGAT
  		CATTACCTGGATTGGCATGAACT
  		CCAGAAGCACTTCCCTGTCCGTG
  		ACCCTGGTGCTCGTCGGAATTGT
  		GACACTCTACCTCGGAGTGATGG
  		TGCAGGCTTGA (SEQ ID NO:
  		149)
  	TCAAGCTT	ATGCTGAACATTTTGAACAGACG	TCAAGCTT	MLNILNRRRRTA
  Dengue 2	TTGGACCC	CCGAAGGACCGCAGGCATTATCA	TTGGACCC	GIIIMMIPTVMA
  prME	TCGTACAG	TTATGATGATCCCTACCGTGATG	TCGTACAG	FHLTTRNGEPHM
  (Peru/IQT2913/	AAGCTAAT	GCCTTCCATCTGACTACTAGGAA	AAGCTAAT	IVSRQEKGKSLL
  1996)	ACGACTCA	CGGAGAGCCACATATGATCGTGT	ACGACTCA	FKTKDGTNMCTL
  	CTATAGGG	CGCGCCAGGAAAAGGGAAAGAGC	CTATAGGG	MAMDLGELCEDT
  	AAATAAGA	CTGCTTTTTAAAACCAAGGACGG	AAATAAGA	ITYKCPFLKQNE
  	GAGAAAAG	CACGAACATGTGCACCCTTATGG	GAGAAAAG	PEDIDCWCNSTS
  	AAGAGTAA	CCATGGACCTGGGGGAGTTGTGC	AAGAGTAA	TWVTYGTCTTTG
  	GAAGAAAT	GAGGACACCATCACCTACAAGTG	GAAGAAAT	EHRREKRSVALV
  	ATAAGAGC	CCCGTTCCTGAAGCAAAACGAGC	ATAAGAGC	PHVGMGLETRTE
  	CACC	CCGAAGATATTGACTGCTGGTGC	CACC	TWMSSEGAWKHA
  	(SEQ ID	AACTCCACCTCCACCTGGGTCAC	(SEQ ID	QRIETWILRHPG
  	NO: 141)	TTATGGGACTTGCACCACCACCG	NO: 159)	FTIMAAILAYTI
  		GCGAACATCGCAGAGAAAAGAGA		GTTHFQRVLIFI
  		AGCGTGGCCCTGGTCCCCCACGT		LLTAIAPSMTMR
  		CGGGATGGGCCTCGAGACTCGGA		CIGISNRDFVEG
  		CCGAAACTTGGATGTCATCAGAG		VSGGSWVDIVLE
  		GGCGCATGGAAGCATGCTCAGCG		HGSCVTTMAKNK
  		GATCGAAACCTGGATCCTGAGAC		PTLDFELIKTEA
  		ACCCTGGTTTCACAATTATGGCC		KQPATLRKYCIE
  		GCCATTCTTGCGTACACGATCGG		AKLTNTTTDSRC
  		AACGACTCATTTCCAACGCGTGC		PTQGEPTLNEEQ
  		TGATCTTCATTCTCCTGACCGCT		DKRFVCKHSMVD
  		ATTGCGCCGTCCATGACTATGCG		RGWGNGCGLFGK
  		GTGCATCGGAATCTCAAACCGGG		GGIVTCAMFTCK
  		ACTTCGTGGAAGGAGTGTCGGGA		KNMEGKIVQPEN
  		GGATCCTGGGTGGACATTGTGCT		LEYTVVITPHSG
  		GGAGCACGGTTCCTGCGTCACCA		EEHAVGNDTGKH
  		CCATGGCCAAAAACAAGCCTACC		GKEVKITPQSSI
  		CTGGACTTCGAGCTGATCAAGAC		TEAELTGYGTVT
  		TGAGGCCAAGCAGCCCGCGACCC		MECSPRTGLDFN
  		TCCGGAAGTACTGCATCGAGGCC		EMVLLQMEDKAW
  		AAGTTGACCAACACTACTACCGA		LVHRQWFLDLPL
  		TTCCCGGTGCCCGACCCAAGGAG		PWLPGADTQGSN
  		AACCAACTCTGAACGAAGAACAG		WIQKETLVTFKN
  		GATAAGCGGTTTGTGTGCAAGCA		PHAKKQDVVVLG
  		CTCAATGGTGGACAGGGGATGGG		SQEGAMHTALTG
  		GCAACGGCTGTGGACTGTTCGGA		ATEIQMSSGNLL
  		AAGGGTGGTATTGTGACCTGTGC		FTGHLKCRLRMD
  		AATGTTTACCTGTAAAAAGAATA		KLQLKGMSYSMC
  		TGGAGGGGAAGATCGTGCAGCCT		TGKFKIVKEIAE
  		GAAAATCTCGAGTACACTGTCGT		TQHGTIVIRVQY
  		CATCACCCCGCACTCGGGAGAGG		EGDGSPCKIPFE
  		AGCACGCTGTGGGCAACGACACC		IMDLEKRHVLGR
  		GGAAAGCACGGAAAGGAGGTCAA		LITVNPIVTEKD
  		GATAACCCCGCAATCCTCCATTA		SPVNIEAEPPFG
  		CGGAAGCCGAACTGACTGGTTAC		DSYIIIG A EPGQ
  		GGCACCGTGACTATGGAGTGCTC		LKLDWFKKGSSI
  		CCCTCGGACCGGCCTGGACTTCA		GQMFETTMRGAK
  		ACGAAATGGTGCTGCTCCAAATG		RMAILGDTAWDF
  		GAAGATAAGGCCTGGCTGGTGCA		GSLGGVFTSIGK
  		CAGGCAGTGGTTCCTGGATCTCC		ALHQVFGAIYGA
  		CGCTGCCGTGGCTGCCTGGCGCT		AFSGVSWTMKIL
  		GACACTCAGGGAAGCAACTGGAT		IGVIITWIGMNS
  		CCAGAAGGAAACCCTCGTGACCT		RSTSLSVSLVLV
  		TTAAGAACCCCCACGCCAAGAAG		GIVTLYLGVMVQ
  		CAGGATGTGGTGGTGTTGGGAAG		A (SEQ ID
  		CCAGGAGGGGGCCATGCATACTG		NO: 168)
  		CCCTCACCGGCGCGACCGAAATC
  		CAGATGTCGTCCGGCAATCTGCT
  		GTTCACCGGACACCTCAAGTGTC
  		GCCTTCGGATGGACAAGCTGCAG
  		CTGAAGGGAATGAGCTACAGCAT
  		GTGCACCGGGAAGTTCAAGATCG
  		TGAAGGAAATCGCCGAAACCCAG
  		CACGGAACCATCGTGATCCGGGT
  		GCAGTACGAGGGCGACGGTTCTC
  		CCTGCAAAATCCCCTTCGAAATC
  		ATGGATCTGGAGAAGAGACACGT
  		CCTGGGTCGCCTGATCACCGTGA
  		ACCCCATTGTGACTGAGAAGGAC
  		TCCCCAGTGAACATCGAAGCGGA
  		GCCCCCATTCGGAGACAGCTACA
  		TTATCATTGGTGCCGAACCGGGG
  		CAGCTGAAACTGGACTGGTTCAA
  		GAAGGGCAGCTCGATTGGCCAAA
  		TGTTCGAAACGACAATGCGGGGC
  		GCAAAGCGCATGGCCATCCTGGG
  		AGACACTGCCTGGGACTTCGGGT
  		CCCTTGGGGGGGTGTTCACCTCG
  		ATCGGAAAAGCCTTGCACCAAGT
  		GTTCGGCGCAATCTACGGCGCCG
  		CGTTCTCGGGAGTCTCCTGGACT
  		ATGAAGATCCTGATCGGTGTCAT
  		CATCACCTGGATCGGGATGAACT
  		CCCGGTCCACTTCCCTCTCGGTG
  		TCACTCGTGCTTGTGGGAATTGT
  		CACCCTGTACCTCGGAGTGATGG
  		TGCAGGCCTGA (SEQ ID NO:
  		150)
  Dengue 2	TCAAGCTT	ATGCTGAATATTCTGAACCGACG	TGATAATA	MLNILNRRRRTA
  prME	TTGGACCC	CCGCCGCACTGCCGGAATCATTA	GGCTGGAG	GIIIMMIPTVMA
  (Thailand/	TCGTACAG	TCATGATGATCCCTACCGTGATG	CCTCGGTG	FHLTTRNGEPHM
  PUO-	AAGCTAAT	GCGTTCCATCTCACCACTCGGAA	GCCATGCT	IVSRQEKGKSLL
  218/1980)	ACGACTCA	TGGCGAACCCCATATGATCGTGT	TCTTGCCC	FKTEDGVNMCTL
  	CTATAGGG	CGAGACAGGAAAAGGGAAAGAGC	CTTGGGCC	MAMDLGELCEDT
  	AAATAAGA	CTTTTGTTCAAAACTGAAGATGG	TCCCCCCA	ITYKCPLLRQNE
  	GAGAAAAG	AGTGAACATGTGCACTCTCATGG	GCCCCTCC	PEDIDCWCNSTS
  	AAGAGTAA	CAATGGATCTGGGCGAACTGTGC	TCCCCTTC	TWVTYGTCTTTG
  	GAAGAAAT	GAAGATACCATCACTTACAAGTG	CTGCACCC	EHRREKRSVALV
  	ATAAGAGC	TCCGCTGTTGAGACAGAACGAGC	GTACCCCC	PHVGMGLETRTE
  	CACC	CTGAGGACATCGACTGCTGGTGT	GTGGTCTT	TWMSSEGAWKHA
  	(SEQ ID	AACAGCACTTCCACCTGGGTCAC	TGAATAAA	QRIEIWILRHPG
  	NO: 142)	CTACGGCACTTGCACTACCACCG	GTCTGAGT	FTIMAAILAYTI
  		GAGAACACCGGCGCGAGAAGAGG	GGGCGGC	GTTHFQRALIFI
  		AGCGTGGCTCTTGTGCCGCACGT	(SEQ ID	LLTAVAPSMTMR
  		CGGCATGGGACTCGAGACTCGGA	NO: 160)	CIGISNRDFVEG
  		CCGAAACCTGGATGTCATCCGAA		VSGGSWVDIVLE
  		GGAGCCTGGAAACACGCCCAACG		HGSCVTTMAKNK
  		GATCGAAATTTGGATCCTGAGAC		PTLDFELIKTEA
  		ACCCCGGTTTCACTATCATGGCC		KQPATLRKYCIE
  		GCAATCCTGGCGTACACTATTGG		AKLTNTTTESRC
  		CACCACGCACTTCCAGAGGGCCC		PTQGEPSLNEEQ
  		TCATTTTCATCCTCCTGACTGCC		DKRFVCKHSMVD
  		GTGGCGCCATCCATGACCATGAG		RGWGNGCGLFGK
  		ATGTATTGGCATTTCCAACCGCG		GGIVTCAMFTCK
  		ATTTCGTGGAGGGAGTGTCCGGA		KNMEGKVVQPEN
  		GGATCCTGGGTCGACATCGTGCT		LEYTIVVTPHSG
  		GGAACACGGATCTTGCGTCACCA		EEHAVGNDTGKH
  		CCATGGCTAAGAACAAGCCCACC		GKEIKVTPQSSI
  		CTCGACTTCGAGCTGATCAAGAC		TEAELTGYGTVT
  		AGAAGCCAAGCAGCCGGCCACCC		MECSPRTGLDFN
  		TCCGCAAGTATTGCATTGAAGCC		EMVLLQMENKAW
  		AAGCTTACCAACACCACCACCGA		LVHRQWFLDLPL
  		GTCGCGGTGCCCAACCCAAGGAG		PWLPGADTQGSN
  		AGCCGAGCCTCAATGAGGAACAG		WIQKETLVTFKN
  		GACAAGCGCTTCGTGTGCAAACA		PHAKKQDVVVLG
  		CAGCATGGTCGACCGGGGTTGGG		SQEGAMHTALTG
  		GCAACGGATGTGGCCTGTTCGGG		ATEIQMSSGNLL
  		AAGGGTGGCATTGTGACTTGCGC		FTGHLKCRLRMD
  		AATGTTCACTTGCAAGAAGAACA		KLQLKGMSYSMC
  		TGGAGGGGAAAGTGGTGCAACCC		TGKFKVVKEIAE
  		GAGAACCTGGAGTACACCATCGT		TQHGTIVIRVQY
  		CGTGACCCCACACTCCGGAGAGG		EGDGSPCKIPFE
  		AGCACGCCGTGGGAAACGACACG		IMDLEKRHVLGR
  		GGGAAGCATGGAAAGGAGATCAA		LITVNPIVTEKD
  		GGTCACACCCCAATCATCTATTA		SPVNIEAEPPFG
  		CCGAGGCCGAACTGACCGGATAC		DSYIIIGVEPGQ
  		GGTACTGTGACGATGGAGTGCAG		LKLNWFKKGSSI
  		CCCGAGGACTGGACTGGACTTCA		GQMFETTMRGAK
  		ACGAAATGGTGCTGCTGCAAATG		RMAILGDTAWDF
  		GAGAACAAGGCCTGGCTCGTGCA		GSLGGVFTSIGK
  		CCGGCAGTGGTTTCTGGATCTCC		ALHQVFGAIYGA
  		CACTGCCGTGGTTGCCGGGAGCC		AFSGVSWTMKIL
  		GACACCCAGGGGTCGAACTGGAT		IGVIITWIGMNS
  		CCAGAAGGAAACTCTTGTGACGT		RSTSLSVSLVLV
  		TTAAGAATCCTCACGCGAAGAAG		GIVTLYLGVMVQ
  		CAGGACGTGGTGGTCCTGGGATC		A (SEQ ID
  		GCAGGAAGGAGCTATGCACACCG		NO: 169)
  		CTCTGACCGGCGCCACTGAGATC
  		CAGATGTCCTCGGGCAACCTCCT
  		GTTCACCGGTCATCTGAAGTGCC
  		GGCTGCGGATGGACAAATTGCAG
  		CTGAAGGGGATGTCCTACTCCAT
  		GTGCACCGGGAAGTTCAAGGTCG
  		TGAAGGAGATCGCGGAAACTCAG
  		CACGGCACCATTGTCATTAGAGT
  		GCAGTACGAGGGAGATGGTTCAC
  		CGTGCAAGATACCGTTCGAAATC
  		ATGGACCTGGAAAAGAGACATGT
  		CTTGGGACGCCTGATCACTGTGA
  		ACCCTATCGTGACCGAAAAGGAC
  		TCCCCTGTGAACATCGAGGCGGA
  		GCCGCCTTTCGGCGACTCCTACA
  		TCATTATCGGAGTGGAGCCCGGG
  		CAGCTGAAGCTCAACTGGTTTAA
  		GAAGGGGTCCAGCATCGGCCAGA
  		TGTTCGAAACCACCATGCGGGGG
  		GCGAAGAGGATGGCGATCCTGGG
  		AGACACCGCCTGGGATTTCGGTT
  		CACTGGGCGGAGTGTTCACCTCC
  		ATCGGAAAGGCCCTGCACCAAGT
  		GTTCGGCGCAATCTACGGTGCTG
  		CCTTCTCGGGAGTCTCCTGGACC
  		ATGAAGATCCTGATCGGCGTGAT
  		TATCACATGGATCGGCATGAACA
  		GCCGGTCAACCTCCCTTTCCGTG
  		TCCCTGGTGCTGGTCGGCATCGT
  		GACTCTGTACCTGGGCGTGATGG
  		TGGAGGCCTGA (SEQ ID NO:
  		151)
  Dengue 2	TCAAGCTT	ATGCTGAACATTCTGAACCGGAG	TGATAATA	MLNILNRRRRTA
  prME	TTGGACCC	AAGAAGAACCGCCGGCATTATTA	GGCTGGAG	GIIIMMIPTVMA
  (D2Y98P)	TCGTACAG	TCATGATGATTCCCACTGTGATG	CCTCGGTG	FHLTTRNGEPHM
  with	AAGCTAAT	GCATTTCACCTGACCACCCGGAA	GCCATGCT	IVSRQEKGKSLL
  native	ACGACTCA	CGGAGAACCTCATATGATCGTGT	TCTTGCCC	FKTENGVNMCTL
  leader	CTATAGGG	CGAGACAGGAGAAGGGAAAGTCC	CTTGGGCC	MAMDLGELCEDT
  	AAATAAGA	CTGCTGTTCAAGACAGAAAACGG	TCCCCCCA	ITYNCPLLRQNE
  	GAGAAAAG	AGTGAACATGTGCACCCTGATGG	GCCCCTCC	PEDIDCWCNSTS
  	AAGAGTAA	CCATGGATCTCGGCGAACTGTGC	TCCCCTTC	TWVTYGTCTATG
  	GAAGAAAT	GAGGATACTATCACCTACAACTG	CTGCACCC	EHRREKRSVALV
  	ATAAGAGC	TCCGTTGCTGCGCCAAAACGAGC	GTACCCCC	PHVGMGLETRTE
  	CACC	CGGAGGACATCGACTGCTGGTGT	GTGGTCTT	TWMSSEGAWKHA
  	(SEQ ID	AACTCCACGTCGACCTGGGTCAC	TGAATAAA	QRIETWVLRHPG
  	NO: 143)	CTACGGCACTTGCACCGCGACCG	GTCTGAGT	FTIMAAILAYTI
  		GCGAACACAGAAGAGAGAAACGC	GGGCGGC	GTTYFQRVLIFI
  		TCCGTCGCTCTGGTGCCGCACGT	(SEQ ID	LLTAVAPSMTMR
  		CGGGATGGGGCTTGAAACCCGGA	NO: 161)	CIGISNRDFVEG
  		CTGAAACCTGGATGAGCTCGGAG		VSGGSWVDIVLE
  		GGCGCTTGGAAGCATGCCCAGCG		HGSCVTTMAKNK
  		CATCGAAACTTGGGTGCTGAGGC		PTLDFELIKTEA
  		ATCCAGGCTTCACAATCATGGCC		KHPATLRKYCIE
  		GCCATCCTCGCGTACACCATCGG		AKLTNTTTASRC
  		TACTACGTACTTCCAGCGGGTGT		PTQGEPSLNEEQ
  		TGATCTTCATTCTGCTGACCGCC		DKRFVCKHSMVD
  		GTGGCCCCTAGCATGACCATGCG		RGWGNGCGLFGK
  		GTGCATCGGGATCTCCAACCGCG		GGIVTCAMFTCK
  		ATTTCGTGGAGGGGGTGTCCGGT		KNMEGKIVQPEN
  		GGAAGCTGGGTGGACATTGTGCT		LEYTIVITPHSG
  		GGAGCACGGCTCGTGCGTGACCA		EENAVGNDTGKH
  		CCATGGCCAAGAACAAGCCCACC		GKEIKVTPQSSI
  		CTTGATTTTGAGCTGATCAAGAC		TEAELTGYGTVT
  		CGAAGCGAAACACCCCGCGACCC		MECSPRTGLDFN
  		TCCGGAAGTACTGCATTGAAGCC		EMVLLQMENKAW
  		AAGCTCACCAACACTACCACGGC		LVHRQWFLDLPL
  		CTCCCGGTGCCCTACCCAAGGAG		PWLPGADTQGSN
  		AACCTTCCTTGAACGAAGAACAG		WIQKETLVTFKN
  		GACAAGCGCTTCGTGTGCAAGCA		PHAKKQDVVVLG
  		TTCAATGGTGGACCGGGGCTGGG		SQEGAMHTALTG
  		GAAATGGCTGTGGCCTCTTCGGA		ATEIQMSSGNLL
  		AAAGGCGGAATTGTGACTTGCGC		FTGHLKCRLRMD
  		AATGTTCACTTGCAAGAAGAACA		KLQLKGMSYSMC
  		TGGAGGGAAAGATTGTGCAGCCC		TGKFKVVKEIAE
  		GAGAACCTCGAGTACACTATTGT		TQHGTIVIRVQY
  		CATCACTCCCCACTCCGGCGAAG		EGDGSPCKIPFE
  		AAAACGCTGTCGGCAACGACACC		IMDLEKRHVLGR
  		GGAAAGCATGGAAAGGAGATCAA		LITVNPIVTEKD
  		GGTCACCCCGCAATCCTCAATTA		SPVNIEAEPPFG
  		CTGAGGCAGAACTGACCGGTTAC		DSYIIIGVEPGQ
  		GGAACTGTGACTATGGAGTGTTC		LKLSWFKKGSSI
  		CCCTCGCACCGGCCTCGATTTCA		GQMFETTMRGAK
  		ACGAGATGGTGCTGCTGCAAATG		RMAILGDTAWDF
  		GAGAACAAGGCCTGGCTGGTGCA		GSLGGVFTSIGK
  		CCGGCAGTGGTTCCTCGATTTGC		ALHQVFGAIYGA
  		CCCTGCCGTGGCTGCCGGGAGCC		AFSGVSWTMKIL
  		GACACTCAGGGATCCAACTGGAT		IGVVITWIGMNS
  		CCAGAAAGAAACCCTCGTGACCT		RSTSLSVSLVLV
  		TCAAAAACCCCGAGGCGAAGAAG		GVVTLYLGVMVQ
  		CAGGACGTGGTGGTGCTGGGTTC		A (SEQ ID
  		CCAAGAAGGGGCGATGCATACCG		NO: 170)
  		CCCTGACTGGTGCTACCGAAATC
  		CAGATGTCAAGCGGAAATCTCCT
  		GTTTACCGGTCACCTGAAGTGCA
  		GGCTCCGGATGGACAAGTTGCAG
  		CTGAAGGGGATGTCGTACAGCAT
  		GTGTACTGGGAAGTTCAAGGTCG
  		TGAAGGAGATTGCCGAAACCCAG
  		CACGGAACCATAGTCATCAGGGT
  		CCAGTACGAGGGCGACGGCAGCC
  		CTTGCAAGATCCCGTTCGAGATC
  		ATGGATCTGGAGAAGCGACACGT
  		GCTGGGCCGGCTTATCACTGTGA
  		ATCCAATCGTGACCGAGAAAGAC
  		TCGCCCGTGAACATCGAAGCCGA
  		GCCGCCGTTCGGCGACTCATACA
  		TCATCATCGGCGTGGAACCAGGA
  		CAGCTGAAGCTGTCATGGTTCAA
  		GAAGGGTTCCAGCATTGGTCAGA
  		TGTTCGAAACAACGATGCGCGGA
  		GCCAAGCGCATGGCTATCCTTGG
  		GGACACCGCCTGGGACTTCGGGT
  		CGCTGGGAGGAGTGTTTACCAGC
  		ATCGGAAAGGCCCTGCACCAAGT
  		GTTCGGTGCCATCTACGGAGCCG
  		CATTTTCCGGAGTGTCGTGGACT
  		ATGAAGATTCTGATCGGCGTCGT
  		GATTACCTGGATCGGGATGAACT
  		CCAGGTCTACTTCCCTCTCCGTG
  		AGCCTGGTGCTGGTCGGCGTGGT
  		CACCCTGTATCTGGGCGTGATGG
  		TCCAGGCTTAG (SEQ ID NO:
  		152)

• TABLE 35
  Full-length Dengue Amino Acid Sequences (Homo sapiens strains; Brazil, Cuba and U.S.)

  GenBank					Collection
  Accession	Length	Type	Country	Genome Region	Date	Virus Name
  AGN94866	3392	1	Brazil	UTR5CMENS1NS	2010	Dengue virus 1
  				2ANS2BNS3NS4		isolate 12898/BR-
  				A2KNS4BNS5UT		PE/10, complete
  				R3		genome
  AGN94867	3392	1	Brazil	UTR5CMENS1NS	2010	Dengue virus 1
  				2ANS2BNS3NS4		isolate 13501/BR-
  				A2KNS4BNS5UT		PE/10, complete
  				R3		genome
  AGN94868	3392	1	Brazil	UTR5CMENS1NS	2010	Dengue virus 1
  				2ANS2BNS3NS4		isolate 13671/BR-
  				A2KNS4BNS5UT		PE/10, complete
  				R3		genome
  AGN94869	3392	1	Brazil	UTR5CMENS1NS	2010	Dengue virus 1
  				2ANS2BNS3NS4		isolate 13861/BR-
  				A2KNS4BNS5UT		PE/10, complete
  				R3		genome
  AGN94870	3392	1	Brazil	UTR5CMENS1NS	2010	Dengue virus 1
  				2ANS2BNS3NS4		isolate 14985/BR-
  				A2KNS4BNS5UT		PE/10, complete
  				R3		genome
  AGN94871	3392	1	Brazil	UTR5CMENS1NS	1996	Dengue virus 1
  				2ANS2BNS3NS4		isolate 21814/BR-
  				A2KNS4BNS5UT		PE/96, complete
  				R3		genome
  AGN94872	3392	1	Brazil	UTR5CMENS1NS	1997	Dengue virus 1
  				2ANS2BNS3NS4		isolate 40604/BR-
  				A2KNS4BNS5UT		PE/97, complete
  				R3		genome
  AGN94873	3392	1	Brazil	UTR5CMENS1NS	1997	Dengue virus 1
  				2ANS2BNS3NS4		isolate 41111/BR-
  				A2KNS4BNS5UT		PE/97, complete
  				R3		genome
  AGN94874	3392	1	Brazil	UTR5CMENS1NS	1998	Dengue virus 1
  				2ANS2BNS3NS4		isolate 52082/BR-
  				A2KNS4BNS5UT		PE/98, complete
  				R3		genome
  AGN94875	3392	1	Brazil	UTR5CMENS1NS	1999	Dengue virus 1
  				2ANS2BNS3NS4		isolate 59049/BR-
  				A2KNS4BNS5UT		PE/99, complete
  				R3		genome
  AGN94876	3392	1	Brazil	UTR5CMENS1NS	2000	Dengue virus 1
  				2ANS2BNS3NS4		isolate 70523/BR-
  				A2KNS4BNS5UT		PE/00, complete
  				R3		genome
  AGN94877	3392	1	Brazil	UTR5CMENS1NS	2001	Dengue virus 1
  				2ANS2BNS3NS4		isolate 74488/BR-
  				A2KNS4BNS5UT		PE/01, complete
  				R3		genome
  AGN94878	3392	1	Brazil	UTR5CMENS1NS	2001	Dengue virus 1
  				2ANS2BNS3NS4		isolate 75861/BR-
  				A2KNS4BNS5UT		PE/01, complete
  				R3		genome
  AGN94879	3392	1	Brazil	UTR5CMENS1NS	2002	Dengue virus 1
  				2ANS2BNS3NS4		isolate 88463/BR-
  				A2KNS4BNS5UT		PE/02, complete
  				R3		genome
  AGN94865	3392	1	Brazil	UTR5CMENS1NS	2010	Dengue virus 1
  				2ANS2BNS3NS4		isolate 9808/BR-
  				A2KNS4BNS5UT		PE/10, complete
  				R3		genome
  ACO06150	3392	1	Brazil	UTR5CMENS1NS	2000	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/BR/BID-
  				R3		V2374/2000,
  						complete genome
  ACO06151	3392	1	Brazil	UTR5CMENS1NS	2000	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/BR/BID-
  				R3		V2375/2000,
  						complete genome
  ACO06153	3392	1	Brazil	UTR5CMENS1NS	2001	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/BR/BID-
  				R3		V2378/2001,
  						complete genome
  ACO06155	3392	1	Brazil	UTR5CMENS1NS	2002	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/BR/BID-
  				R3		V2381/2002,
  						complete genome
  ACO06157	3392	1	Brazil	UTR5CMENS1NS	2003	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/BR/BID-
  				R3		V2384/2003,
  						complete genome
  ACO06161	3392	1	Brazil	UTR5CMENS1NS	2004	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/BR/BID-
  				R3		V2389/2004,
  						complete genome
  ACO06164	3392	1	Brazil	UTR5CMENS1NS	2005	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/BR/BID-
  				R3		V2392/2005,
  						complete genome
  ACO06167	3392	1	Brazil	UTR5CMENS1NS	2006	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/BR/BID-
  				R3		V2395/2006,
  						complete genome
  ACO06170	3392	1	Brazil	UTR5CMENS1NS	2007	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/BR/BID-
  				R3		V2398/2007,
  						complete genome
  ACO06173	3392	1	Brazil	UTR5CMENS1NS	2008	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/BR/BID-
  				R3		V2401/2008,
  						complete genome
  ACY70762	3392	1	Brazil	UTR5CMENS1NS	2008	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		1/BR/BID-
  						V3490/2008,
  						complete genome
  ACJ12617	3392	1	Brazil	UTR5CMENS1NS		Dengue virus 1
  				2ANS2BNS3NS4		isolate DF01-
  				A2KNS4BNS5UT		HUB01021093,
  				R3		complete genome
  AHC08447	3392	1	Brazil	CMENS1NS2ANS	2011	Dengue virus 1
  				2BNS3NS4A2KN		strain
  				S4BNS5		1266/2011/BR/RJ/
  						2011 polyprotein
  						gene, partial cds
  AHC08446	3392	1	Brazil	CMENS1NS2ANS	2010	Dengue virus 1
  				2BNS3NS4A2KN		strain
  				S4BNS5		242/2010/BR/RJ/
  						2010 polyprotein
  						gene, partial cds
  AHC08448	3392	1	Brazil	CMENS1NS2ANS	1988	Dengue virus 1
  				2BNS3NS4A2KN		strain
  				S4BNS5		36034/BR/RJ/
  						1988 polyprotein
  						gene, partial cds
  AHC08449	3392	1	Brazil	CMENS1NS2ANS	1989	Dengue virus 1
  				2BNS3NS4A2KN		strain
  				S4BNS5		38159/BR/RJ/
  						1989 polyprotein
  						gene, partial cds
  AHC08450	3392	1	Brazil	CMENS1NS2ANS	2000	Dengue virus 1
  				2BNS3NS4A2KN		strain
  				S4BNS5		66694/BR/ES/
  						2000 polyprotein
  						gene, partial cds
  AHC08451	3392	1	Brazil	CMENS1NS2ANS	2001	Dengue virus 1
  				2BNS3NS4A2KN		strain
  				S4BNS5		68826/BR/RJ/
  						2001 polyprotein
  						gene, partial cds
  AGN94880	3391	2	Brazil	UTR5CMENS1NS	2010	Dengue virus 2
  				2ANS2BNS3NS4		isolate 13858/BR-
  				A2KNS4BNS5UT		PE/10, complete
  				R3		genome
  AGN94881	3391	2	Brazil	UTR5CMENS1NS	2010	Dengue virus 2
  				2ANS2BNS3NS4		isolate 14905/BR-
  				A2KNS4BNS5UT		PE/10, complete
  				R3		genome
  AGN94882	3391	2	Brazil	UTR5CMENS1NS	2010	Dengue virus 2
  				2ANS2BNS3NS4		isolate 19190/BR-
  				A2KNS4BNS5UT		PE/10, complete
  				R3		genome
  AGN94884	3391	2	Brazil	UTR5CMENS1NS	1995	Dengue virus 2
  				2ANS2BNS3NS4		isolate 3275/BR-
  				A2KNS4BNS5UT		PE/95, complete
  				R3		genome
  AGN94885	3391	2	Brazil	UTR5CMENS1NS	1995	Dengue virus 2
  				2ANS2BNS3NS4		isolate 3311/BR-
  				A2KNS4BNS5UT		PE/95, complete
  				R3		genome
  AGN94886	3391	2	Brazil	UTR5CMENS1NS	1995	Dengue virus 2
  				2ANS2BNS3NS4		isolate 3337/BR-
  				A2KNS4BNS5UT		PE/95, complete
  				R3		genome
  AGN94887	3391	2	Brazil	UTR5CMENS1NS	1997	Dengue virus 2
  				2ANS2BNS3NS4		isolate 37473/BR-
  				A2KNS4BNS5UT		PE/97, complete
  				R3		genome
  AGN94888	3391	2	Brazil	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate 47913/BR-
  				A2KNS4BNS5UT		PE/98, complete
  				R3		genome
  AGN94889	3391	2	Brazil	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate 51347/BR-
  				A2KNS4BNS5UT		PE/98, complete
  				R3		genome
  AGN94890	3391	2	Brazil	UTR5CMENS1NS	1999	Dengue virus 2
  				2ANS2BNS3NS4		isolate 57135/BR-
  				A2KNS4BNS5UT		PE/99, complete
  				R3		genome
  AGN94891	3391	2	Brazil	UTR5CMENS1NS	2000	Dengue virus 2
  				2ANS2BNS3NS4		isolate 72144/BR-
  				A2KNS4BNS5UT		PE/00, complete
  				R3		genome
  AGN94892	3391	2	Brazil	UTR5CMENS1NS	2002	Dengue virus 2
  				2ANS2BNS3NS4		isolate 87086/BR-
  				A2KNS4BNS5UT		PE/02, complete
  				R3		genome
  AGN94883	3391	2	Brazil	UTR5CMENS1NS	2010	Dengue virus 2
  				2ANS2BNS3NS4		isolate 9479/BR-
  				A2KNS4BNS5UT		PE/10, complete
  				R3		genome
  AGK36299	3391	2	Brazil	CMENS1NS2ANS	Mar. 30, 2010	Dengue virus 2
  				2BNS3NS4A2KN		isolate ACS380,
  				S4BNS5UTR3		complete genome
  AGK36289	3391	2	Brazil	UTR5CMENS1NS	Mar. 1, 2010	Dengue virus 2
  				2ANS2BNS3NS4		isolate ACS46,
  				A2KNS4BNS5UT		complete genome
  				R3
  AGK36290	3391	2	Brazil	UTR5CMENS1NS	Mar. 1, 2010	Dengue virus 2
  				2ANS2BNS3NS4		isolate ACS46_II,
  				A2KNS4BNS5UT		complete genome
  				R3
  AGK36291	3391	2	Brazil	UTR5CMENS1NS	Apr. 12, 2010	Dengue virus 2
  				2ANS2BNS3NS4		isolate ACS538,
  				A2KNS4BNS5UT		complete genome
  				R3
  AGK36292	3391	2	Brazil	UTR5CMENS1NS	May 4, 2010	Dengue virus 2
  				2ANS2BNS3NS4		isolate ACS542,
  				A2KNS4BNS5UT		complete genome
  				R3
  AGK36294	3391	2	Brazil	UTR5CMENS1NS	May 4, 2010	Dengue virus 2
  				2ANS2BNS3NS4		isolate ACS721,
  				A2KNS4BNS5UT		complete genome
  				R3
  ACO06152	3391	2	Brazil	UTR5CMENS1NS	2000	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V2376/2000,
  						complete genome
  AET43250	3391	2	Brazil	CMENS1NS2ANS	2000	Dengue virus 2
  				2BNS3NS4A2KN		isolate DENV-
  				S4BNS5UTR3		2/BR/BID-
  						V2377/2000,
  						complete genome
  ACO06154	3391	2	Brazil	UTR5CMENS1NS	2001	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V2379/2001,
  						complete genome
  ACO06156	3391	2	Brazil	UTR5CMENS1NS	2002	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V2382/2002,
  						complete genome
  ACW82928	3391	2	Brazil	UTR5CMENS1NS	2003	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V2385/2003,
  						complete genome
  ACO06158	3391	2	Brazil	UTR5CMENS1NS	2003	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V2386/2003,
  						complete genome
  ACO06162	3391	2	Brazil	UTR5CMENS1NS	2004	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V2390/2004,
  						complete genome
  ACO06165	3391	2	Brazil	UTR5CMENS1NS	2005	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V2393/2005,
  						complete genome
  ACO06168	3391	2	Brazil	UTR5CMENS1NS	2006	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V2396/2006,
  						complete genome
  ACO06171	3391	2	Brazil	UTR5CMENS1NS	2007	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V2399/2007,
  						complete genome
  ACS32031	3391	2	Brazil	UTR5CMENS1NS	2008	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V2402/2008,
  						complete genome
  ACW82873	3391	2	Brazil	UTR5CMENS1NS	2008	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V3481/2008,
  						complete genome
  ACW82874	3391	2	Brazil	UTR5CMENS1NS	2008	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V3483/2008,
  						complete genome
  ACW82875	3391	2	Brazil	UTR5CMENS1NS	2008	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V3486/2008,
  						complete genome
  ACY70763	3391	2	Brazil	UTR5CMENS1NS	2008	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		2/BR/BID-
  						V3495/2008,
  						complete genome
  ADI80655	3391	2	Brazil	UTR5CMENS1NS	2008	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		2/BR/BID-
  						V3637/2008,
  						complete genome
  ACY70778	3391	2	Brazil	UTR5CMENS1NS	2008	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V3638/2008,
  						complete genome
  ACY70779	3391	2	Brazil	UTR5CMENS1NS	2008	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		2/BR/BID-
  						V3640/2008,
  						complete genome
  ACY70780	3391	2	Brazil	UTR5CMENS1NS	2008	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		2/BR/BID-
  						V3644/2008,
  						complete genome
  ACY70781	3391	2	Brazil	UTR5CMENS1NS	2008	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V3645/2008,
  						complete genome
  ACY70782	3391	2	Brazil	UTR5CMENS1NS	2008	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V3648/2008,
  						complete genome
  ACY70783	3391	2	Brazil	UTR5CMENS1NS	2008	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V3650/2008,
  						complete genome
  ACY70784	3391	2	Brazil	UTR5CMENS1NS	2008	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/BR/BID-
  				R3		V3653/2008,
  						complete genome
  AGK36297	3391	2	Brazil	UTR5CMENS1NS	Apr. 15, 2010	Dengue virus 2
  				2ANS2BNS3NS4		isolate DGV106,
  				A2KNS4BNS5UT		complete genome
  				R3
  AGK36295	3391	2	Brazil	UTR5CMENS1NS	Feb. 24, 2010	Dengue virus 2
  				2ANS2BNS3NS4		isolate DGV34,
  				A2KNS4BNS5UT		complete genome
  				R3
  AGK36293	3391	2	Brazil	UTR5CMENS1NS	Feb. 24, 2010	Dengue virus 2
  				2ANS2BNS3NS4		isolate DGV37,
  				A2KNS4BNS5UT		complete genome
  				R3
  AGK36298	3391	2	Brazil	UTR5CMENS1NS	Mar. 9, 2010	Dengue virus 2
  				2ANS2BNS3NS4		isolate DGV69,
  				A2KNS4BNS5UT		complete genome
  				R3
  AGK36296	3391	2	Brazil	UTR5CMENS1NS	Mar. 24, 2010	Dengue virus 2
  				2ANS2BNS3NS4		isolate DGV91,
  				A2KNS4BNS5UT		complete genome
  				R3
  AFV95788	3391	2	Brazil	CMENS1NS2ANS	2008	Dengue virus 2
  				2BNS3NS4A2KN		strain
  				S4BNS5		BR0337/2008/RJ/
  						2008 polyprotein
  						gene, partial cds
  AFV95787	3391	2	Brazil	CMENS1NS2ANS	2008	Dengue virus 2
  				2BNS3NS4A2KN		strain
  				S4BNS5		BR0450/2008/RJ/
  						2008 polyprotein
  						gene, partial cds
  ADV39968	3391	2	Brazil	CMENS1NS2ANS	2008	Dengue virus 2
  				2BNS3NS4A2KN		strain
  				S4BNS5		BR0690/RJ/2008
  						polyprotein gene,
  						complete cds
  ADV71220	3391	2	Brazil	CMENS1NS2ANS	1990	Dengue virus 2
  				2BNS3NS4A2KN		strain
  				S4BNS5		BR39145/RJ/90
  						polyprotein gene,
  						partial cds
  ADV71215	3391	2	Brazil	CMENS1NS2ANS	1990	Dengue virus 2
  				2BNS3NS4A2KN		strain
  				S4BNS5		BR41768/RJ/90
  						polyprotein gene,
  						partial cds
  ADV71216	3391	2	Brazil	CMENS1NS2ANS	1991	Dengue virus 2
  				2BNS3NS4A2KN		strain
  				S4BNS5		BR42727/RJ/91
  						polyprotein gene,
  						partial cds
  ADV71217	3391	2	Brazil	CMENS1NS2ANS	1994	Dengue virus 2
  				2BNS3NS4A2KN		strain
  				S4BNS5		BR48622/CE/94
  						polyprotein gene,
  						partial cds
  ADV71218	3391	2	Brazil	CMENS1NS2ANS	1998	Dengue virus 2
  				2BNS3NS4A2KN		strain
  				S4BNS5		BR61310/RJ/98
  						polyprotein gene,
  						partial cds
  ADV71219	3391	2	Brazil	CMENS1NS2ANS	1999	Dengue virus 2
  				2BNS3NS4A2KN		strain
  				S4BNS5		BR64905/RJ/99
  						polyprotein gene,
  						partial cds
  AFH53774	3390	2	Brazil	UTR5CMENS1NS		Dengue virus 2
  				2ANS2BNS3NS4		strain JHA1,
  				A2KNS4BNS5		partial genome
  AGN94893	3390	3	Brazil	UTR5CMENS1NS	2003	Dengue virus 3
  				2ANS2BNS3NS4		isolate
  				A2KNS4BNS5UT		101905/BR-
  				R3		PE/03, complete
  						genome
  AGN94902	3390	3	Brazil	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate 129/BR-
  				A2KNS4BNS5UT		PE/04, complete
  				R3		genome
  AGN94899	3390	3	Brazil	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate 145/BR-
  				A2KNS4BNS5UT		PE/04, complete
  				R3		genome
  AGN94903	3390	3	Brazil	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate 161/BR-
  				A2KNS4BNS5UT		PE/04, complete
  				R3		genome
  AGN94896	3390	3	Brazil	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate 206/BR-
  				A2KNS4BNS5UT		PE/05, complete
  				R3		genome
  AGN94904	3390	3	Brazil	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate 249/BR-
  				A2KNS4BNS5UT		PE/05, complete
  				R3		genome
  AGN94901	3390	3	Brazil	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate 255/BR-
  				A2KNS4BNS5UT		PE/05, complete
  				R3		genome
  AGN94905	3390	3	Brazil	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate 263/BR-
  				A2KNS4BNS5UT		PE/05, complete
  				R3		genome
  AGN94898	3390	3	Brazil	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate 277/BR-
  				A2KNS4BNS5UT		PE/05, complete
  				R3		genome
  AGN94906	3390	3	Brazil	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate 283/BR-
  				A2KNS4BNS5UT		PE/05, complete
  				R3		genome
  AGN94907	3390	3	Brazil	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate 314/BR-
  				A2KNS4BNS5UT		PE/06, complete
  				R3		genome
  AGN94897	3390	3	Brazil	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate 339/BR-
  				A2KNS4BNS5UT		PE/05, complete
  				R3		genome
  AGN94908	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate 411/BR-
  				A2KNS4BNS5UT		PE/06, complete
  				R3		genome
  AGN94909	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate 418/BR-
  				A2KNS4BNS5UT		PE/06, complete
  				R3		genome
  AGN94910	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate 420/BR-
  				A2KNS4BNS5UT		PE/06, complete
  				R3		genome
  AGN94911	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate 423/BR-
  				A2KNS4BNS5UT		PE/06, complete
  				R3		genome
  AGN94912	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate 424/BR-
  				A2KNS4BNS5UT		PE/06, complete
  				R3		genome
  AGN94900	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate 603/BR-
  				A2KNS4BNS5UT		PE/06, complete
  				R3		genome
  AGN94895	3390	3	Brazil	UTR5CMENS1NS	2002	Dengue virus 3
  				2ANS2BNS3NS4		isolate 81257/BR-
  				A2KNS4BNS5UT		PE/02, complete
  				R3		genome
  AGN94894	3390	3	Brazil	UTR5CMENS1NS	2002	Dengue virus 3
  				2ANS2BNS3NS4		isolate 85469/BR-
  				A2KNS4BNS5UT		PE/02, complete
  				R3		genome
  AFK83756	3390	3	Brazil	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate
  				A2KNS4BNS5UT		D3BR/ACN/2007,
  				R3		complete genome
  AFK83755	3390	3	Brazil	UTR5CMENS1NS	2009	Dengue virus 3
  				2ANS2BNS3NS4		isolate
  				A2KNS4BNS5UT		D3BR/AL95/2009,
  				R3		complete genome
  AFK83754	3390	3	Brazil	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate
  				A2KNS4BNS5UT		D3BR/BR8/04,
  				R3		complete genome
  AFK83753	3390	3	Brazil	UTR5CMENS1NS	2002	Dengue virus 3
  				2ANS2BNS3NS4		isolate
  				A2KNS4BNS5UT		D3BR/BV4/02,
  				R3		complete genome
  AFK83762	3390	3	Brazil	UTR5CMENS1NS	2002	Dengue virus 3
  				2ANS2BNS3NS4		isolate
  				A2KNS4BNS5UT		D3BR/CU6/02,
  				R3		complete genome
  AFK83759	3390	3	Brazil	UTR5CMENS1NS	2003	Dengue virus 3
  				2ANS2BNS3NS4		isolate
  				A2KNS4BNS5UT		D3BR/MR9/03,
  				R3		complete genome
  AFK83761	3390	3	Brazil	UTR5CMENS1NS	2003	Dengue virus 3
  				2ANS2BNS3NS4		isolate
  				A2KNS4BNS5UT		D3BR/PV1/03,
  				R3		complete genome
  AFK83760	3390	3	Brazil	UTR5CMENS1NS	2002	Dengue virus 3
  				2ANS2BNS3NS4		isolate
  				A2KNS4BNS5UT		D3BR/SL3/02,
  				R3		complete genome
  AHG23238	3390	3	Brazil	UTR5CMENS1NS	2002	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V2383/2002,
  						complete genome
  ACO06159	3390	3	Brazil	UTR5CMENS1NS	2003	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V2387/2003,
  						complete genome
  ACO06160	3390	3	Brazil	UTR5CMENS1NS	2003	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V2388/2003,
  						complete genome
  ACO06163	3390	3	Brazil	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V2391/2004,
  						complete genome
  ACO06166	3390	3	Brazil	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V2394/2005,
  						complete genome
  ACO06169	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V2397/2006,
  						complete genome
  ACO06172	3390	3	Brazil	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V2400/2007,
  						complete genome
  ACO06174	3390	3	Brazil	UTR5CMENS1NS	2008	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V2403/2008,
  						complete genome
  ACQ44485	3390	3	Brazil	UTR5CMENS1NS	2001	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V2977/2001,
  						complete genome
  ACQ44486	3390	3	Brazil	UTR5CMENS1NS	2003	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V2983/2003,
  						complete genome
  ACY70743	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V3417/2006,
  						complete genome
  ACY70744	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3423/2006,
  						complete genome
  ACY70745	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V3424/2006,
  						complete genome
  ACY70746	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3427/2006,
  						complete genome
  ACY70747	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3429/2006,
  						complete genome
  ACY70748	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3430/2006,
  						complete genome
  ACW82870	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V3431/2006,
  						complete genome
  ACY70749	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3434/2006,
  						complete genome
  ACY70750	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3435/2006,
  						complete genome
  ACY70751	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3441/2006,
  						complete genome
  ACY70752	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3442/2006,
  						complete genome
  ACW82871	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V3444/2006,
  						complete genome
  ACY70753	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3446/2006,
  						complete genome
  ACY70754	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V3451/2006,
  						complete genome
  ACY70755	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V3456/2006,
  						complete genome
  ACY70756	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V3457/2006,
  						complete genome
  ACY70757	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3460/2006,
  						complete genome
  ACW82872	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V3463/2006,
  						complete genome
  ACY70758	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3464/2006,
  						complete genome
  ACY70759	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3465/2006,
  						complete genome
  ACY70760	3390	3	Brazil	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3469/2007,
  						complete genome
  ACY70761	3390	3	Brazil	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3470/2007,
  						complete genome
  ACY70764	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3584/2006,
  						complete genome
  ACY70765	3390	3	Brazil	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3585/2007,
  						complete genome
  ACY70766	3390	3	Brazil	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3588/2007,
  						complete genome
  ACY70767	3390	3	Brazil	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3589/2007,
  						complete genome
  ACY70768	3390	3	Brazil	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3590/2007,
  						complete genome
  ACY70769	3390	3	Brazil	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3591/2007,
  						complete genome
  ACY70770	3390	3	Brazil	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3593/2007,
  						complete genome
  ACY70771	3390	3	Brazil	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V3597/2007,
  						complete genome
  ACY70772	3390	3	Brazil	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3598/2007,
  						complete genome
  ACY70773	3390	3	Brazil	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3601/2007,
  						complete genome
  ACY70774	3390	3	Brazil	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3605/2007,
  						complete genome
  ACY70775	3390	3	Brazil	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		3/BR/BID-
  						V3606/2007,
  						complete genome
  ACY70776	3390	3	Brazil	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V3609/2007,
  						complete genome
  ACY70777	3390	3	Brazil	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/BR/BID-
  				R3		V3615/2007,
  						complete genome
  AEV42062	3390	3	Brazil	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate
  				A2KNS4BNS5UT		DENV3/BR/
  				R3		D3LIMHO/2006,
  						complete genome
  AGH08164	3390	3	Brazil	UTR5CMENS1NS	2002	Dengue virus 3
  				2ANS2BNS3NS4		strain 95016/BR-
  				A2KNS4BNS5UT		PE/02, complete
  				R3		genome
  AEX91754	3387	4	Brazil	UTR5CMENS1NS	Sep. 8, 2010	Dengue virus 4
  				2ANS2BNS3NS4		isolate
  				A2KNS4BNS5UT		Br246RR/10,
  				R3		complete genome
  AIQ84223	3387	4	Brazil	UTR5CMENS1NS	Mar. 28, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR12_TVP17898/
  				R3		2012
  						isolate serum_12,
  						complete genome
  AIQ84224	3387	4	Brazil	UTR5CMENS1NS	Mar. 30, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR20_TVP17906/
  				R3		2012
  						isolate serum_20,
  						complete genome
  AIQ84225	3387	4	Brazil	UTR5CMENS1NS	Mar. 30, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR23_TVP17909/
  				R3		2012
  						isolate serum_23,
  						complete genome
  AIQ84226	3387	4	Brazil	UTR5CMENS1NS	Apr. 19, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR24_TVP17910/
  				R3		2012
  						isolate serum_24,
  						complete genome
  AIQ84227	3387	4	Brazil	UTR5CMENS1NS	Apr. 12, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR27_TVP17913/
  				R3		2012
  						isolate serum_27,
  						complete genome
  AIQ84228	3387	4	Brazil	UTR5CMENS1NS	Apr. 19, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR28_TVP17914/
  				R3		2012
  						isolate serum_28,
  						complete genome
  AIQ84220	3387	4	Brazil	UTR5CMENS1NS	Apr. 23, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR2_TVP17888/
  				R3		2012
  						isolate serum_2,
  						complete genome
  AIQ84245	3387	4	Brazil	UTR5CMENS1NS	Apr. 20, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR33_TVP17919/
  				R3		2012
  						isolate serum_33,
  						complete genome
  AIQ84244	3387	4	Brazil	UTR5CMENS1NS	Mar. 30, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR35_TVP17921/
  				R3		2012
  						isolate serum_35,
  						complete genome
  AIQ84243	3387	4	Brazil	UTR5CMENS1NS	Apr. 3, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR40_TVP17926/
  				R3		2012
  						isolate serum_40,
  						complete genome
  AIQ84242	3387	4	Brazil	UTR5CMENS1NS	Apr. 5, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR44_TVP17930/
  				R3		2012
  						isolate serum_44,
  						complete genome
  AIQ84241	3387	4	Brazil	UTR5CMENS1NS	Mar. 23, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR47_TVP17933/
  				R3		2012
  						isolate serum_47,
  						complete genome
  AIQ84240	3387	4	Brazil	UTR5CMENS1NS	Mar. 21, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR48_TVP17934/
  				R3		2012
  						isolate serum_48,
  						complete genome
  AIQ84239	3387	4	Brazil	UTR5CMENS1NS	Mar. 12, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR50_TVP18148/
  				R3		2012
  						isolate serum_50,
  						complete genome
  AIQ84238	3387	4	Brazil	UTR5CMENS1NS	Mar. 20, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR52_TVP17938/
  				R3		2012
  						isolate serum_52,
  						complete genome
  AIQ84237	3387	4	Brazil	UTR5CMENS1NS	Mar. 14, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR53_TVP17939/
  				R3		2012
  						isolate serum_53,
  						complete genome
  AIQ84236	3387	4	Brazil	UTR5CMENS1NS	Mar. 14, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR55_TVP17941/
  				R3		2012
  						isolate serum_55,
  						complete genome
  AIQ84235	3387	4	Brazil	UTR5CMENS1NS	Mar. 14, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR60_TVP17946/
  				R3		2012
  						isolate serum_60,
  						complete genome
  AIQ84234	3387	4	Brazil	UTR5CMENS1NS	Apr. 19, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR73_TVP17951/
  				R3		2012
  						isolate serum_73,
  						complete genome
  AIQ84233	3387	4	Brazil	UTR5CMENS1NS	Apr. 19, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR76_TVP17953/
  				R3		2012
  						isolate serum_76,
  						complete genome
  AIQ84232	3387	4	Brazil	UTR5CMENS1NS	Feb. 3, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR84_TVP17961/
  				R3		2012
  						isolate serum_84,
  						complete genome
  AIQ84221	3387	4	Brazil	UTR5CMENS1NS	Apr. 23, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR8_TVP17894/
  				R3		2012
  						isolate serum_8,
  						complete genome
  AIQ84231	3387	4	Brazil	UTR5CMENS1NS	Feb. 3, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR91_TVP17968/
  				R3		2012
  						isolate serum_91,
  						complete genome
  AIQ84230	3387	4	Brazil	UTR5CMENS1NS	Feb. 29, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR92_TVP17969/
  				R3		2012
  						isolate serum_92,
  						complete genome
  AIQ84229	3387	4	Brazil	UTR5CMENS1NS	Feb. 16, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR94_TVP17971/
  				R3		2012
  						isolate serum_94,
  						complete genome
  AIQ84222	3387	4	Brazil	UTR5CMENS1NS	Apr. 18, 2012	Dengue virus 4
  				2ANS2BNS3NS4		strain DENV-4/MT/
  				A2KNS4BNS5UT		BR9_TVP17895/
  				R3		2012
  						isolate serum_9,
  						complete genome
  AEW50182	3387	4	Brazil	UTR5CMENS1NS	Mar. 26, 1982	Dengue virus 4
  				2ANS2BNS3NS4		strain H402276,
  				A2KNS4BNS5		complete genome
  AFX65866	3387	4	Brazil	UTR5CMENS1NS	Jul. 17, 2010	Dengue virus 4
  				2ANS2BNS3NS4		strain H772846,
  				A2KNS4BNS5UT		complete genome
  				R3
  AFX65867	3387	4	Brazil	UTR5CMENS1NS	Jul. 18, 2010	Dengue virus 4
  				2ANS2BNS3NS4		strain H772852,
  				A2KNS4BNS5UT		complete genome
  				R3
  AEW50183	3387	4	Brazil	UTR5CMENS1NS	Jul. 21, 2010	Dengue virus 4
  				2ANS2BNS3NS4		strain H772854,
  				A2KNS4BNS5		complete genome
  AFX65868	3387	4	Brazil	UTR5CMENS1NS	Aug. 20, 2010	Dengue virus 4
  				2ANS2BNS3NS4		strain H773583,
  				A2KNS4BNS5UT		complete genome
  				R3
  AFX65869	3387	4	Brazil	UTR5CMENS1NS	Aug. 24, 2010	Dengue virus 4
  				2ANS2BNS3NS4		strain H774846,
  				A2KNS4BNS5UT		complete genome
  				R3
  AFX65870	3387	4	Brazil	UTR5CMENS1NS	Nov. 10, 2010	Dengue virus 4
  				2ANS2BNS3NS4		strain H775222,
  				A2KNS4BNS5UT		complete genome
  				R3
  AFX65871	3387	4	Brazil	UTR5CMENS1NS	Jan. 12, 2011	Dengue virus 4
  				2ANS2BNS3NS4		strain H778494,
  				A2KNS4BNS5UT		complete genome
  				R3
  AFX65872	3387	4	Brazil	UTR5CMENS1NS	Jan. 11, 2011	Dengue virus 4
  				2ANS2BNS3NS4		strain H778504,
  				A2KNS4BNS5UT		complete genome
  				R3
  AFX65873	3387	4	Brazil	UTR5CMENS1NS	Jan. 20, 2011	Dengue virus 4
  				2ANS2BNS3NS4		strain H778887,
  				A2KNS4BNS5UT		complete genome
  				R3
  AFX65874	3387	4	Brazil	UTR5CMENS1NS	Jan. 14, 2011	Dengue virus 4
  				2ANS2BNS3NS4		strain H779228,
  				A2KNS4BNS5UT		complete genome
  				R3
  AFX65875	3387	4	Brazil	UTR5CMENS1NS	Jan. 24, 2011	Dengue virus 4
  				2ANS2BNS3NS4		strain H779652,
  				A2KNS4BNS5UT		complete genome
  				R3
  AFX65876	3387	4	Brazil	UTR5CMENS1NS	Nov. 29, 2010	Dengue virus 4
  				2ANS2BNS3NS4		strain H780090,
  				A2KNS4BNS5UT		complete genome
  				R3
  AFX65877	3387	4	Brazil	UTR5CMENS1NS	Nov. 21, 2010	Dengue virus 4
  				2ANS2BNS3NS4		strain H780120,
  				A2KNS4BNS5UT		complete genome
  				R3
  AFX65878	3387	4	Brazil	UTR5CMENS1NS	Jan. 29, 2011	Dengue virus 4
  				2ANS2BNS3NS4		strain H780556,
  				A2KNS4BNS5UT		complete genome
  				R3
  AFX65879	3387	4	Brazil	UTR5CMENS1NS	Jan. 29, 2011	Dengue virus 4
  				2ANS2BNS3NS4		strain H780563,
  				A2KNS4BNS5UT		complete genome
  				R3
  AFX65880	3387	4	Brazil	UTR5CMENS1NS	Jan. 13, 2011	Dengue virus 4
  				2ANS2BNS3NS4		strain H780571,
  				A2KNS4BNS5UT		complete genome
  				R3
  AFX65881	3387	4	Brazil	UTR5CMENS1NS	Mar. 18, 2011	Dengue virus 4
  				2ANS2BNS3NS4		strain H781363,
  				A2KNS4BNS5UT		complete genome
  				R3
  AIK23224	3391	2	Cuba	CMENS1NS2ANS	1981	Dengue virus 2
  				2BNS3NS4A2KN		isolate
  				S4BNS5		Cuba_A115_1981
  						polyprotein gene,
  						complete cds
  AIK23223	3391	2	Cuba	CMENS1NS2ANS	1981	Dengue virus 2
  				2BNS3NS4A2KN		isolate
  				S4BNS5		Cuba_A132_1981
  						polyprotein gene,
  						complete cds
  AIK23222	3391	2	Cuba	CMENS1NS2ANS	1981	Dengue virus 2
  				2BNS3NS4A2KN		isolate
  				S4BNS5		Cuba_A15_1981
  						polyprotein gene,
  						complete cds
  AIK23225	3391	2	Cuba	CMENS1NS2ANS	1981	Dengue virus 2
  				2BNS3NS4A2KN		isolate
  				S4BNS5		Cuba_A169_1981
  						polyprotein gene,
  						complete cds
  AIK23226	3391	2	Cuba	CMENS1NS2ANS	1981	Dengue virus 2
  				2BNS3NS4A2KN		isolate
  				S4BNS5		Cuba_A35_1981
  						polyprotein gene,
  						complete cds
  AAW31409	3391	2	Cuba	UTR5CMENS1NS		Dengue virus type
  				2ANS2BNS3NS4		2 strain
  				A2KNS4BNS5UT		Cuba115/97,
  				R3		complete genome
  AAW31407	3391	2	Cuba	UTR5CMENS1NS		Dengue virus type
  				2ANS2BNS3NS4		2 strain
  				A2KNS4BNS5UT		Cuba13/97,
  				R3		complete genome
  AAW31411	3391	2	Cuba	UTR5CMENS1NS		Dengue virus type
  				2ANS2BNS3NS4		2 strain
  				A2KNS4BNS5UT		Cuba165/97,
  				R3		complete genome
  AAW31412	3391	2	Cuba	UTR5CMENS1NS	1997	Dengue virus type
  				2ANS2BNS3NS4		2 strain
  				A2KNS4BNS5UT		Cuba205/97,
  				R3		complete genome
  AAW31408	3391	2	Cuba	UTR5CMENS1NS		Dengue virus type
  				2ANS2BNS3NS4		2 strain
  				A2KNS4BNS5UT		Cuba58/97,
  				R3		complete genome
  AAW31410	3391	2	Cuba	UTR5CMENS1NS	1997	Dengue virus type
  				2ANS2BNS3NS4		2 strain
  				A2KNS4BNS5UT		Cuba89/97,
  				R3		complete genome
  AFJ91714	3392	1	USA	UTR5CMENS1NS	2010, October	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/BOL-KW010,
  				R3		complete genome
  ACA48834	3392	1	USA	UTR5CMENS1NS	1998	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V1162/1998,
  						complete genome
  ACJ04186	3392	1	USA	UTR5CMENS1NS	1995	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V1734/1995,
  						complete genome
  ACJ04190	3392	1	USA	UTR5CMENS1NS	1998	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V1738/1998,
  						complete genome
  ACH99678	3392	1	USA	UTR5CMENS1NS	1998	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V1739/1998,
  						complete genome
  ACH99679	3392	1	USA	UTR5CMENS1NS	1998	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V1740/1998,
  						complete genome
  ACJ04191	3392	1	USA	UTR5CMENS1NS	1998	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V1741/1998,
  						complete genome
  ACJ04192	3392	1	USA	UTR5CMENS1NS	1998	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V1742/1998,
  						complete genome
  ACH99680	3392	1	USA	UTR5CMENS1NS	1995	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V1743/1995,
  						complete genome
  ACH99681	3392	1	USA	UTR5CMENS1NS	1995	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V1744/1995,
  						complete genome
  ACJ04215	3392	1	USA	UTR5CMENS1NS	1998	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2093/1998,
  						complete genome
  ACJ04216	3392	1	USA	UTR5CMENS1NS	1995	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2094/1995,
  						complete genome
  ACJ04217	3392	1	USA	UTR5CMENS1NS	1994	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2095/1994,
  						complete genome
  ACL99012	3392	1	USA	UTR5CMENS1NS	1993	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2096/1993,
  						complete genome
  ACL99013	3392	1	USA	UTR5CMENS1NS	1986	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2097/1986,
  						complete genome
  ACJ04221	3392	1	USA	UTR5CMENS1NS	1994	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2127/1994,
  						complete genome
  ACJ04222	3392	1	USA	UTR5CMENS1NS	1995	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2128/1995,
  						complete genome
  ACJ04223	3392	1	USA	UTR5CMENS1NS	1995	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2129/1995,
  						complete genome
  ACL99002	3392	1	USA	UTR5CMENS1NS	1995	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2130/1995,
  						complete genome
  ACJ04224	3392	1	USA	UTR5CMENS1NS	1996	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2131/1996,
  						complete genome
  ACJ04225	3392	1	USA	UTR5CMENS1NS	1993	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2132/1993,
  						complete genome
  ACJ04226	3392	1	USA	UTR5CMENS1NS	1993	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2133/1993,
  						complete genome
  ACJ04227	3392	1	USA	UTR5CMENS1NS	1993	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2134/1993,
  						complete genome
  ACL99003	3392	1	USA	UTR5CMENS1NS	1992	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2135/1992,
  						complete genome
  ACJ04228	3392	1	USA	UTR5CMENS1NS	1992	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2136/1992,
  						complete genome
  ACJ04229	3392	1	USA	UTR5CMENS1NS	1992	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2137/1992,
  						complete genome
  ACK28188	3392	1	USA	UTR5CMENS1NS	1996	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2138/1996,
  						complete genome
  ACJ04230	3392	1	USA	UTR5CMENS1NS	1996	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2139/1996,
  						complete genome
  ACJ04231	3392	1	USA	UTR5CMENS1NS	1996	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2140/1996,
  						complete genome
  ACK28189	3392	1	USA	UTR5CMENS1NS	1987	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2142/1987,
  						complete genome
  ACJ04232	3392	1	USA	UTR5CMENS1NS	1987	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V2143/1987,
  						complete genome
  ACA48858	3392	1	USA	UTR5CMENS1NS	2006	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V852/2006,
  						complete genome
  ACA48859	3392	1	USA	UTR5CMENS1NS	1998	Dengue virus 1
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		1/US/BID-
  				R3		V853/1998,
  						complete genome
  ACF49259	3392	1	USA	UTR5CMENS1NS	1944	Dengue virus 1
  				2ANS2BNS3NS4		isolate
  				A2KNS4BNS5UT		US/Hawaii/1944,
  				R3		complete genome
  AIU47321	3392	1	USA	UTR5CMENS1NS	1944	Dengue virus 1
  				2ANS2BNS3NS4		strain Hawaii,
  				A2KNS4BNS5UT		complete genome
  				R3
  ACA48811	3391	2	USA	UTR5CMENS1NS	2006	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1031/2006,
  						complete genome
  ACA48812	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1032/1998,
  						complete genome
  ACA48813	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1033/1998,
  						complete genome
  ACA48814	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1034/1998,
  						complete genome
  ACA48815	3391	2	USA	UTR5CMENS1NS	2006	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1035/2006,
  						complete genome
  ACA48816	3391	2	USA	UTR5CMENS1NS	2006	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1036/2006,
  						complete genome
  ACA48817	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1038/1998,
  						complete genome
  ACA48818	3391	2	USA	UTR5CMENS1NS	2006	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1039/2006,
  						complete genome
  ACA48819	3391	2	USA	UTR5CMENS1NS	2006	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1040/2006,
  						complete genome
  ACA48820	3391	2	USA	UTR5CMENS1NS	2006	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1041/2006,
  						complete genome
  ACA48821	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1042/1998,
  						complete genome
  ACA48823	3391	2	USA	UTR5CMENS1NS	2005	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1045/2005,
  						complete genome
  ACA58330	3391	2	USA	UTR5CMENS1NS	2004	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1046/2004,
  						complete genome
  ACA48824	3391	2	USA	UTR5CMENS1NS	1999	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1048/1999,
  						complete genome
  ACA48827	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1052/1998,
  						complete genome
  ACA48828	3391	2	USA	UTR5CMENS1NS	1996	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1054/1996,
  						complete genome
  ACB29511	3391	2	USA	UTR5CMENS1NS	1996	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1055/1996,
  						complete genome
  ACA58331	3391	2	USA	UTR5CMENS1NS	1994	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1057/1994,
  						complete genome
  ACA58332	3391	2	USA	UTR5CMENS1NS	1994	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1058/1994,
  						complete genome
  ACA48829	3391	2	USA	UTR5CMENS1NS	1989	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1060/1989,
  						complete genome
  ACD13309	3391	2	USA	UTR5CMENS1NS	1989	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1061/1989,
  						complete genome
  ACA48832	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1084/1998,
  						complete genome
  ACA58337	3391	2	USA	UTR5CMENS1NS	1994	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1085/1994,
  						complete genome
  ACA58338	3391	2	USA	UTR5CMENS1NS	1991	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1087/1991,
  						complete genome
  ACB29512	3391	2	USA	UTR5CMENS1NS	1986	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1163/1986,
  						complete genome
  ACA48835	3391	2	USA	UTR5CMENS1NS	1986	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1164/1986,
  						complete genome
  ACA48836	3391	2	USA	UTR5CMENS1NS	1987	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1165/1987,
  						complete genome
  ACA48837	3391	2	USA	UTR5CMENS1NS	1987	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1166/1987,
  						complete genome
  ACA48838	3391	2	USA	UTR5CMENS1NS	1987	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1167/1987,
  						complete genome
  ACA48839	3391	2	USA	UTR5CMENS1NS	1987	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1168/1987,
  						complete genome
  ACA48840	3391	2	USA	UTR5CMENS1NS	1987	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1169/1987,
  						complete genome
  ACA48841	3391	2	USA	UTR5CMENS1NS	1987	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1170/1987,
  						complete genome
  ACA48842	3391	2	USA	UTR5CMENS1NS	1987	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1171/1987,
  						complete genome
  ACA48843	3391	2	USA	UTR5CMENS1NS	1987	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1172/1987,
  						complete genome
  ACA48844	3391	2	USA	UTR5CMENS1NS	1987	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1174/1987,
  						complete genome
  ACA48845	3391	2	USA	UTR5CMENS1NS	1988	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1175/1988,
  						complete genome
  ACA48846	3391	2	USA	UTR5CMENS1NS	1988	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1176/1988,
  						complete genome
  ACA48847	3391	2	USA	UTR5CMENS1NS	1989	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1177/1989,
  						complete genome
  ACA48848	3391	2	USA	UTR5CMENS1NS	1989	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1178/1989,
  						complete genome
  ACA48849	3391	2	USA	UTR5CMENS1NS	1989	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1179/1989,
  						complete genome
  ACA48850	3391	2	USA	UTR5CMENS1NS	1989	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1180/1989,
  						complete genome
  ACA48851	3391	2	USA	UTR5CMENS1NS	1989	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1181/1989,
  						complete genome
  ACA48852	3391	2	USA	UTR5CMENS1NS	1989	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1182/1989,
  						complete genome
  ACA48853	3391	2	USA	UTR5CMENS1NS	1990	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1183/1990,
  						complete genome
  ACA48854	3391	2	USA	UTR5CMENS1NS	1990	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1188/1990,
  						complete genome
  ACA48855	3391	2	USA	UTR5CMENS1NS	1990	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1189/1990,
  						complete genome
  ACB29513	3391	2	USA	UTR5CMENS1NS	1993	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1356/1993,
  						complete genome
  ACA48856	3391	2	USA	UTR5CMENS1NS	1993	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1360/1993,
  						complete genome
  ACB29514	3391	2	USA	UTR5CMENS1NS	1995	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1367/1995,
  						complete genome
  ACB29515	3391	2	USA	UTR5CMENS1NS	1995	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1368/1995,
  						complete genome
  ACD13310	3391	2	USA	UTR5CMENS1NS	1995	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1372/1995,
  						complete genome
  ACB29516	3391	2	USA	UTR5CMENS1NS	1995	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1373/1995,
  						complete genome
  ACB29517	3391	2	USA	UTR5CMENS1NS	1996	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1376/1996,
  						complete genome
  ACB87126	3391	2	USA	UTR5CMENS1NS	1996	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1378/1996,
  						complete genome
  ACB29518	3391	2	USA	UTR5CMENS1NS	1996	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1383/1996,
  						complete genome
  ACB87127	3391	2	USA	UTR5CMENS1NS	1996	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1385/1996,
  						complete genome
  ACD13396	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1387/1998,
  						complete genome
  ACD13311	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1388/1998,
  						complete genome
  ACB29519	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1392/1998,
  						complete genome
  ACB29520	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1393/1998,
  						complete genome
  ACB87128	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1394/1998,
  						complete genome
  ACB29521	3391	2	USA	UTR5CMENS1NS	1997	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1395/1997,
  						complete genome
  ACB29522	3391	2	USA	UTR5CMENS1NS	1997	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1396/1997,
  						complete genome
  ACB29523	3391	2	USA	UTR5CMENS1NS	1997	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1397/1997,
  						complete genome
  ACB29524	3391	2	USA	UTR5CMENS1NS	1997	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1398/1997,
  						complete genome
  ACB29525	3391	2	USA	UTR5CMENS1NS	1997	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1399/1997,
  						complete genome
  ACB29526	3391	2	USA	UTR5CMENS1NS	1997	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1401/1997,
  						complete genome
  ACB29527	3391	2	USA	UTR5CMENS1NS	1997	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1404/1997,
  						complete genome
  ACB29528	3391	2	USA	UTR5CMENS1NS	1997	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1409/1997,
  						complete genome
  ACB87129	3391	2	USA	UTR5CMENS1NS	2007	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1410/2007,
  						complete genome
  ACB87130	3391	2	USA	UTR5CMENS1NS	2007	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1411/2007,
  						complete genome
  ACB87131	3391	2	USA	UTR5CMENS1NS	2007	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1412/2007,
  						complete genome
  ACB87132	3391	2	USA	UTR5CMENS1NS	2007	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1413/2007,
  						complete genome
  ACD13348	3391	2	USA	UTR5CMENS1NS	1996	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1424/1996,
  						complete genome
  ACD13349	3391	2	USA	UTR5CMENS1NS	1999	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1425/1999,
  						complete genome
  ACD13350	3391	2	USA	UTR5CMENS1NS	1999	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1426/1999,
  						complete genome
  ACD13351	3391	2	USA	UTR5CMENS1NS	1999	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1427/1999,
  						complete genome
  ACD13352	3391	2	USA	UTR5CMENS1NS	1999	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1428/1999,
  						complete genome
  ACD13353	3391	2	USA	UTR5CMENS1NS	2004	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/B ID-
  				R3		V1431/2004,
  						complete genome
  ACD13354	3391	2	USA	UTR5CMENS1NS	2004	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1432/2004,
  						complete genome
  ACD13397	3391	2	USA	UTR5CMENS1NS	2004	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1434/2004,
  						complete genome
  ACD13398	3391	2	USA	UTR5CMENS1NS	2004	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1435/2004,
  						complete genome
  ACD13399	3391	2	USA	UTR5CMENS1NS	2004	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1436/2004,
  						complete genome
  ACD13400	3391	2	USA	UTR5CMENS1NS	2005	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1439/2005,
  						complete genome
  ACE63530	3391	2	USA	UTR5CMENS1NS	2005	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1440/2005,
  						complete genome
  ACD13401	3391	2	USA	UTR5CMENS1NS	2005	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1441/2005,
  						complete genome
  ACE63543	3391	2	USA	UTR5CMENS1NS	2005	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1442/2005,
  						complete genome
  ACD13406	3391	2	USA	UTR5CMENS1NS	2000	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1461/2000,
  						complete genome
  ACD13407	3391	2	USA	UTR5CMENS1NS	2000	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1462/2000,
  						complete genome
  ACD13408	3391	2	USA	UTR5CMENS1NS	2000	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1463/2000,
  						complete genome
  ACD13409	3391	2	USA	UTR5CMENS1NS	2000	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1464/2000,
  						complete genome
  ACD13411	3391	2	USA	UTR5CMENS1NS	2001	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1467/2001,
  						complete genome
  ACD13412	3391	2	USA	UTR5CMENS1NS	2001	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1468/2001,
  						complete genome
  ACD13413	3391	2	USA	UTR5CMENS1NS	2001	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1469/2001,
  						complete genome
  ACD13414	3391	2	USA	UTR5CMENS1NS	2001	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1470/2001,
  						complete genome
  ACD13415	3391	2	USA	UTR5CMENS1NS	2001	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1471/2001,
  						complete genome
  ACD13416	3391	2	USA	UTR5CMENS1NS	2001	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1472/2001,
  						complete genome
  ACD13395	3391	2	USA	UTR5CMENS1NS	2003	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1482/2003,
  						complete genome
  ACD13419	3391	2	USA	UTR5CMENS1NS	2003	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1483/2003,
  						complete genome
  ACD13420	3391	2	USA	UTR5CMENS1NS	2003	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1484/2003,
  						complete genome
  ACD13421	3391	2	USA	UTR5CMENS1NS	2003	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1486/2003,
  						complete genome
  ACD13422	3391	2	USA	UTR5CMENS1NS	2003	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1487/2003,
  						complete genome
  ACD13424	3391	2	USA	UTR5CMENS1NS	2003	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1492/2003,
  						complete genome
  ACD13425	3391	2	USA	UTR5CMENS1NS	2003	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1493/2003,
  						complete genome
  ACD13426	3391	2	USA	UTR5CMENS1NS	2004	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1494/2004,
  						complete genome
  ACD13427	3391	2	USA	UTR5CMENS1NS	2004	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1495/2004,
  						complete genome
  ACD13428	3391	2	USA	UTR5CMENS1NS	2004	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1496/2004,
  						complete genome
  ACD13429	3391	2	USA	UTR5CMENS1NS	2005	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V1497/2005,
  						complete genome
  AEH59341	3390	2	USA	CMENS1NS2ANS	2009	Dengue virus 2
  				2BNS3NS4A2KN		isolate DENV-
  				S4BNS5		2/US/BID-
  						V4824/2009,
  						complete genome
  AEH59346	3391	2	USA	UTR5CMENS1NS	2006	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		2/US/BID-
  						V5411/2006,
  						complete genome
  AEH59345	3391	2	USA	UTR5CMENS1NS	2007	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5		2/US/BID-
  						V5412/2007,
  						complete genome
  ACA58343	3391	2	USA	UTR5CMENS1NS	2006	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V585/2006,
  						complete genome
  ACA48986	3391	2	USA	UTR5CMENS1NS	2006	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V587/2006,
  						complete genome
  ACA48987	3391	2	USA	UTR5CMENS1NS	2006	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V588/2006,
  						complete genome
  ACA48988	3391	2	USA	UTR5CMENS1NS	2006	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V589/2006,
  						complete genome
  ACA48989	3391	2	USA	UTR5CMENS1NS	2002	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V591/2002,
  						complete genome
  ACA48990	3391	2	USA	UTR5CMENS1NS	2002	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V592/2002,
  						complete genome
  ACA48991	3391	2	USA	UTR5CMENS1NS	2005	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V593/2005,
  						complete genome
  ACA48992	3391	2	USA	UTR5CMENS1NS	2006	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V594/2006,
  						complete genome
  ACA48993	3391	2	USA	UTR5CMENS1NS	2006	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V595/2006,
  						complete genome
  ACA48994	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V596/1998,
  						complete genome
  ACA48995	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V597/1998,
  						complete genome
  ACA48996	3391	2	USA	UTR5CMENS1NS	1999	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V598/1999,
  						complete genome
  ACA48997	3391	2	USA	UTR5CMENS1NS	1999	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V599/1999,
  						complete genome
  ACA48998	3391	2	USA	UTR5CMENS1NS	2005	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V600/2005,
  						complete genome
  ACA48999	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V675/1998,
  						complete genome
  ACA49000	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V676/1998,
  						complete genome
  ACA49001	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V677/1998,
  						complete genome
  ACA49002	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V678/1998,
  						complete genome
  ACA49003	3391	2	USA	UTR5CMENS1NS	1994	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V679/1994,
  						complete genome
  ACA49004	3391	2	USA	UTR5CMENS1NS	1994	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V680/1994,
  						complete genome
  ACA49005	3391	2	USA	UTR5CMENS1NS	1998	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V681/1998,
  						complete genome
  ACA49006	3391	2	USA	UTR5CMENS1NS	1994	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V682/1994,
  						complete genome
  ACA49007	3391	2	USA	UTR5CMENS1NS	1994	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V683/1994,
  						complete genome
  ACA49008	3391	2	USA	UTR5CMENS1NS	1994	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V684/1994,
  						complete genome
  ACA49009	3391	2	USA	UTR5CMENS1NS	1988	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V685/1988,
  						complete genome
  ACA49010	3391	2	USA	UTR5CMENS1NS	1989	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V686/1989,
  						complete genome
  ACA49011	3391	2	USA	UTR5CMENS1NS	1989	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V687/1989,
  						complete genome
  ACA49012	3391	2	USA	UTR5CMENS1NS	1989	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V688/1989,
  						complete genome
  ACA49013	3391	2	USA	UTR5CMENS1NS	1989	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V689/1989,
  						complete genome
  ACA49014	3391	2	USA	UTR5CMENS1NS	1988	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V690/1988,
  						complete genome
  ACA48857	3391	2	USA	UTR5CMENS1NS	1990	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V851/1990,
  						complete genome
  ACA48860	3391	2	USA	UTR5CMENS1NS	2001	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V854/2001,
  						complete genome
  ACA48861	3391	2	USA	UTR5CMENS1NS	1992	Dengue virus 2
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		2/US/BID-
  				R3		V855/1992,
  						complete genome
  ACA48822	3390	3	USA	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1043/2006,
  						complete genome
  ACA58329	3390	3	USA	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1044/2006,
  						complete genome
  ACA48825	3390	3	USA	UTR5CMENS1NS	1998	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1049/1998,
  						complete genome
  ACA48826	3390	3	USA	UTR5CMENS1NS	1998	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1050/1998,
  						complete genome
  ACA48830	3390	3	USA	UTR5CMENS1NS	1998	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1075/1998,
  						complete genome
  ACA58333	3390	3	USA	UTR5CMENS1NS	1999	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1076/1999,
  						complete genome
  ACA58334	3390	3	USA	UTR5CMENS1NS	2000	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1077/2000,
  						complete genome
  ACA48831	3390	3	USA	UTR5CMENS1NS	2003	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1078/2003,
  						complete genome
  ACA58335	3390	3	USA	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1079/2006,
  						complete genome
  ACA58336	3390	3	USA	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1080/2006,
  						complete genome
  ACA48833	3390	3	USA	UTR5CMENS1NS	1998	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1088/1998,
  						complete genome
  ACA58339	3390	3	USA	UTR5CMENS1NS	2003	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1089/2003,
  						complete genome
  ACA58340	3390	3	USA	UTR5CMENS1NS	1998	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1090/1998,
  						complete genome
  ACA58341	3390	3	USA	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1091/2004,
  						complete genome
  ACA58342	3390	3	USA	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1092/2004,
  						complete genome
  ACB87133	3390	3	USA	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1415/2007,
  						complete genome
  ACB87134	3390	3	USA	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1416/2007,
  						complete genome
  ACB87135	3390	3	USA	UTR5CMENS1NS	2007	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1417/2007,
  						complete genome
  ACD13402	3390	3	USA	UTR5CMENS1NS	1998	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1447/1998,
  						complete genome
  ACE63531	3390	3	USA	UTR5CMENS1NS	1998	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1448/1998,
  						complete genome
  ACE63532	3390	3	USA	UTR5CMENS1NS	1998	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1449/1998,
  						complete genome
  ACH99660	3390	3	USA	UTR5CMENS1NS	1998	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1450/1998,
  						complete genome
  ACE63544	3390	3	USA	UTR5CMENS1NS	1999	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1451/1999,
  						complete genome
  ACE63545	3390	3	USA	UTR5CMENS1NS	1999	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1452/1999,
  						complete genome
  ACE63533	3390	3	USA	UTR5CMENS1NS	1999	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1453/1999,
  						complete genome
  ACE63534	3390	3	USA	UTR5CMENS1NS	1999	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1454/1999,
  						complete genome
  ACD13403	3390	3	USA	UTR5CMENS1NS	1999	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1455/1999,
  						complete genome
  ACD13405	3390	3	USA	UTR5CMENS1NS	2000	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1460/2000,
  						complete genome
  ACE63528	3390	3	USA	UTR5CMENS1NS	2000	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1465/2000,
  						complete genome
  ACD13410	3390	3	USA	UTR5CMENS1NS	1999	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1466/1999,
  						complete genome
  ACD13417	3391	3	USA	UTR5CMENS1NS	2002	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1473/2002,
  						complete genome
  ACD13418	3390	3	USA	UTR5CMENS1NS	2002	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1475/2002,
  						complete genome
  ACD13392	3390	3	USA	UTR5CMENS1NS	2002	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1476/2002,
  						complete genome
  ACH61690	3390	3	USA	UTR5CMENS1NS	2002	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1477/2002,
  						complete genome
  ACJ04182	3390	3	USA	UTR5CMENS1NS	2002	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1478/2002,
  						complete genome
  ACD13393	3390	3	USA	UTR5CMENS1NS	2003	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1480/2003,
  						complete genome
  ACD13394	3390	3	USA	UTR5CMENS1NS	2003	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1481/2003,
  						complete genome
  ACE63529	3390	3	USA	UTR5CMENS1NS	2003	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1490/2003,
  						complete genome
  ACD13423	3390	3	USA	UTR5CMENS1NS	2003	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1491/2003,
  						complete genome
  ACH99651	3390	3	USA	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1604/2004,
  						complete genome
  ACO06143	3390	3	USA	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1605/2004,
  						complete genome
  ACH61715	3390	3	USA	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1606/2004,
  						complete genome
  ACH61716	3390	3	USA	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1607/2004,
  						complete genome
  ACH61717	3390	3	USA	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1608/2004,
  						complete genome
  ACH61718	3390	3	USA	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1609/2004,
  						complete genome
  ACH61719	3390	3	USA	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1610/2004,
  						complete genome
  ACO06144	3390	3	USA	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1611/2004,
  						complete genome
  ACH61720	3390	3	USA	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1612/2004,
  						complete genome
  ACH61721	3390	3	USA	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1613/2004,
  						complete genome
  ACH99652	3390	3	USA	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1614/2004,
  						complete genome
  ACJ04178	3390	3	USA	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1615/2004,
  						complete genome
  ACH99653	3390	3	USA	UTR5CMENS1NS	2004	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1616/2004,
  						complete genome
  ACH99654	3390	3	USA	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1617/2005,
  						complete genome
  ACH99655	3390	3	USA	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1618/2005,
  						complete genome
  ACH99656	3390	3	USA	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1619/2005,
  						complete genome
  ACH99657	3390	3	USA	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1620/2005,
  						complete genome
  ACH99658	3390	3	USA	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1621/2005,
  						complete genome
  ACH99665	3390	3	USA	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1622/2005,
  						complete genome
  ACH99666	3390	3	USA	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1623/2005,
  						complete genome
  ACH99667	3390	3	USA	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1624/2005,
  						complete genome
  ACH99668	3390	3	USA	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1625/2005,
  						complete genome
  ACH99669	3390	3	USA	UTR5CMENS1NS	2005	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1626/2005,
  						complete genome
  ACJ04183	3390	3	USA	UTR5CMENS1NS	2003	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1729/2003,
  						complete genome
  ACJ04184	3390	3	USA	UTR5CMENS1NS	2003	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1730/2003,
  						complete genome
  ACH99676	3390	3	USA	UTR5CMENS1NS	2003	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1731/2003,
  						complete genome
  ACJ04185	3390	3	USA	UTR5CMENS1NS	2002	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1732/2002,
  						complete genome
  ACH99677	3390	3	USA	UTR5CMENS1NS	1999	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1733/1999,
  						complete genome
  ACJ04187	3390	3	USA	UTR5CMENS1NS	1999	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1735/1999,
  						complete genome
  ACJ04188	3390	3	USA	UTR5CMENS1NS	1999	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1736/1999,
  						complete genome
  ACJ04189	3390	3	USA	UTR5CMENS1NS	1999	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V1737/1999,
  						complete genome
  ACL98985	3390	3	USA	UTR5CMENS1NS	1999	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2098/1999,
  						complete genome
  ACL98986	3390	3	USA	UTR5CMENS1NS	1998	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2099/1998,
  						complete genome
  ACL99014	3390	3	USA	UTR5CMENS1NS	2000	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2100/2000,
  						complete genome
  ACL98987	3390	3	USA	UTR5CMENS1NS	2000	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2103/2000,
  						complete genome
  ACJ04218	3390	3	USA	UTR5CMENS1NS	2000	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2104/2000,
  						complete genome
  ACJ04219	3390	3	USA	UTR5CMENS1NS	2000	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2105/2000,
  						complete genome
  ACL98988	3390	3	USA	UTR5CMENS1NS	2000	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2106/2000,
  						complete genome
  ACL98989	3390	3	USA	UTR5CMENS1NS	2000	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2107/2000,
  						complete genome
  ACL98990	3390	3	USA	UTR5CMENS1NS	2000	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2108/2000,
  						complete genome
  ACL98991	3390	3	USA	UTR5CMENS1NS	2000	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2110/2000,
  						complete genome
  ACL98992	3390	3	USA	UTR5CMENS1NS	2000	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2111/2000,
  						complete genome
  ACL98993	3390	3	USA	UTR5CMENS1NS	2000	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2112/2000,
  						complete genome
  ACL98994	3390	3	USA	UTR5CMENS1NS	2000	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2113/2000,
  						complete genome
  ACL98995	3390	3	USA	UTR5CMENS1NS	2001	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2114/2001,
  						complete genome
  ACL98996	3390	3	USA	UTR5CMENS1NS	2001	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2115/2001,
  						complete genome
  ACL98997	3390	3	USA	UTR5CMENS1NS	2001	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2117/2001,
  						complete genome
  ACL98998	3390	3	USA	UTR5CMENS1NS	2001	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2118/2001,
  						complete genome
  ACL98999	3390	3	USA	UTR5CMENS1NS	2002	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2119/2002,
  						complete genome
  ACJ04220	3390	3	USA	UTR5CMENS1NS	2002	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2120/2002,
  						complete genome
  ACL99000	3390	3	USA	UTR5CMENS1NS	2002	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2122/2002,
  						complete genome
  ACK28187	3390	3	USA	UTR5CMENS1NS	2002	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2123/2002,
  						complete genome
  ACL99001	3390	3	USA	UTR5CMENS1NS	2006	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V2126/2006,
  						complete genome
  ACA48862	3390	3	USA	UTR5CMENS1NS	2003	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V858/2003,
  						complete genome
  ACA48863	3390	3	USA	UTR5CMENS1NS	1998	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/US/BID-
  				R3		V859/1998,
  						complete genome
  AFZ40124	3390	3	USA	UTR5CMENS1NS	1963	Dengue virus 3
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		3/USA/633798/
  				R3		1963,
  						complete genome
  ACH61714	3387	4	USA	UTR5CMENS1NS	1998	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V1082/1998,
  						complete genome
  ACH61687	3387	4	USA	UTR5CMENS1NS	1986	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V1083/1986,
  						complete genome
  ACH61688	3387	4	USA	UTR5CMENS1NS	1998	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V1093/1998,
  						complete genome
  ACH61689	3387	4	USA	UTR5CMENS1NS	1998	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V1094/1998,
  						complete genome
  ACS32012	3387	4	USA	UTR5CMENS1NS	1994	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2429/1994,
  						complete genome
  ACS32013	3387	4	USA	UTR5CMENS1NS	1994	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2430/1994,
  						complete genome
  ACS32014	3387	4	USA	UTR5CMENS1NS	1995	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2431/1995,
  						complete genome
  ACS32037	3387	4	USA	UTR5CMENS1NS	1995	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2432/1995,
  						complete genome
  ACO06140	3387	4	USA	UTR5CMENS1NS	1995	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2433/1995,
  						complete genome
  ACO06145	3387	4	USA	UTR5CMENS1NS	1995	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2434/1995,
  						complete genome
  ACS32015	3387	4	USA	UTR5CMENS1NS	1996	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2435/1996,
  						complete genome
  ACS32016	3387	4	USA	UTR5CMENS1NS	1996	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2436/1996,
  						complete genome
  ACS32017	3387	4	USA	UTR5CMENS1NS	1996	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2437/1996,
  						complete genome
  ACS32018	3387	4	USA	UTR5CMENS1NS	1996	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2438/1996,
  						complete genome
  ACS32019	3387	4	USA	UTR5CMENS1NS	1996	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2439/1996,
  						complete genome
  ACO06146	3387	4	USA	UTR5CMENS1NS	1996	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2440/1996,
  						complete genome
  ACQ44402	3387	4	USA	UTR5CMENS1NS	1998	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2441/1998,
  						complete genome
  ACQ44403	3387	4	USA	UTR5CMENS1NS	1998	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2442/1998,
  						complete genome
  ACO06147	3387	4	USA	UTR5CMENS1NS	1998	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2443/1998,
  						complete genome
  ACQ44404	3387	4	USA	UTR5CMENS1NS	1998	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2444/1998,
  						complete genome
  ACQ44405	3387	4	USA	UTR5CMENS1NS	1998	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2445/1998,
  						complete genome
  ACQ44406	3387	4	USA	UTR5CMENS1NS	1999	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2446/1999,
  						complete genome
  ACQ44407	3387	4	USA	UTR5CMENS1NS	1999	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2447/1999,
  						complete genome
  ACQ44408	3387	4	USA	UTR5CMENS1NS	1999	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V2448/1999,
  						complete genome
  ACJ04171	3387	4	USA	UTR5CMENS1NS	1994	Dengue virus 4
  				2ANS2BNS3NS4		isolate DENV-
  				A2KNS4BNS5UT		4/US/BID-
  				R3		V860/1994,
  						complete genome

• TABLE 36
  DENY POLYPEPTIDE SEQUENCES

  SEQ ID
  NO:	Accession No.	Sequence
  171	gi|158348409|ref|	MNNQRKKTGRPSFNMLKRARNRVSTVSQLAKRFSKGLL
  	NP_722466.2|	SGQGPMKLVMAFIAFLRFLAIPPTAGILARWGSFKKNGAI
  	capsid protein	KVLRGFKKEISNMLNIMNRRKR
  	[Dengue virus 1]
  172	gi|164654862|ref|	MNNQRKKTGKPSINMLKRVRNRVSTGSQLAKRFSKGLL
  	YP_001531164.2|	NGQGPMKLVMAFIAFLRFLAIPPTAGVLARWGTFKKSGA
  	Capsid protein	IKVLKGFKKEISNMLSIINQRKK
  	[Dengue virus 3]
  173	gi|159024809|ref|	MNNQRKKAKNTPFNMLKRERNRVSTVQQLTKRFSLGM
  	NP_739591.2|	LQGRGPLKLFMALVAFLRFLTIPPTAGILKRWGTIKKSKA
  	Capsid protein	INVLRGFRKEIGRMLNILNRRRR
  	[Dengue virus 2]
  174	gi|158348408|ref|	MNNQRKKTGRPSFNMLKRARNRVSTVSQLAKRFSKGLL
  	NP_722457.2|	SGQGPMKLVMAFIAFLRFLAIPPTAGILARWGSFKKNGAI
  	anchored capsid	KVLRGFKKEISNMLNIMNRRKRSVTMLLMLLPTALA
  	protein [Dengue
  	virus 1]
  175	gi|164654854|ref|	MNNQRKKTGKPSINMLKRVRNRVSTGSQLAKRFSKGLL
  	YP_001531165.2|	NGQGPMKLVMAFIAFLRFLAIPPTAGVLARWGTFKKSGA
  	Anchored capsid	IKVLKGFKKEISNMLSIINQRKKTSLCLMMILPAALA
  	protein [Dengue
  	virus 3]
  176	gi|159024808|ref|	MNNQRKKAKNTPFNMLKRERNRVSTVQQLTKRFSLGM
  	NP_739581.2|	LQGRGPLKLFMALVAFLRFLTIPPTAGILKRWGTIKKSKA
  	Anchored capsid	INVLRGFRKEIGRMLNILNRRRRSAGMIIMLIPTVMA
  	protein [Dengue
  	virus 2]
  177	gi|73671168|ref|	MNQRKKVVRPPFNMLKRERNRVSTPQGLVKRFSTGLFS
  	NP_740314.1|	GKGPLRMVLAFITFLRVLSIPPTAGILKRWGQLKKNKAIK
  	anchored capsid	ILIGFRKEIGRMLNILNGRKRSTITLLCLIPTVMA
  	(anchC) protein
  	[Dengue virus 4]
  178	gi|73671167|ref|	MNQRKKVVRPPFNMLKRERNRVSTPQGLVKRFSTGLFS
  	NP_740313.1|	GKGPLRMVLAFITFLRVLSIPPTAGILKRWGQLKKNKAIK
  	virion capsid	ILIGFRKEIGRMLNILNGRKR
  	(virC) protein
  	[Dengue virus 4]
  Envelope	gi|164654853|ref|	MRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKN
  Protein	YP_001531168.2|	KPTLDIELQKTEATQLATLRKLCIEGKITNITTDSRCPTQG
  179	Envelope	EAVLPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVT
  	protein [Dengue	CAKFQCLEPIEGKVVQYENLKYTVIITVHTGDQHQVGNE
  	virus 3]	TQGVTAEITPQASTTEAILPEYGTLGLECSPRTGLDFNEMI
  		LLTMKNKAWMVHRQWFFDLPLPWASGATTETPTWNRK
  		ELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQN
  		SGGTSIFAGHLKCRLKMDKLELKGMSYAMCTNTFVLKK
  		EVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGR
  		LITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDNALKINW
  		YKKGSSIGKMFEATERGARRMAILGDTAWDFGSVGGVL
  		NSLGKMVHQIFGSAYTALFSGVSWVMKIGIGVLLTWIGL
  		NSKNTSMSFSCIAIGIITLYLGAVVQA
  180	gi|158828123|ref|	MRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKD
  	NP_722460.2|	KPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQG
  	envelope protein	EATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSLITCA
  	[Dengue virus 1]	KFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETT
  		EHGTTATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNEM
  		VLLTMKKKSWLVHKQWFLDLPLPWTSGASTSQETWNR
  		QDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQ
  		TSGTTTIFAGHLKCRLKMDKLILKGMSYVMCTGSFKLEK
  		EVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNG
  		RLITANPIVTDKEKPVNIEAEPPFGESYIVVGAGEKALKLS
  		WFKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGV
  		FTSVGKLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGL
  		NSRSTSLSMTCIAVGMVTLYLGVMVQA
  181	gi|159024812|ref|	MRCIGMSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNK
  	NP_739583.2|	PTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRCPTQGE
  	Envelope protein	PSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCA
  	[Dengue virus 2]	MFRCKKNMEGKVVQPENLEYTIVITPHSGEEHAVGNDTG
  		KHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEM
  		VLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQ
  		KETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQ
  		MSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKV
  		VKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVL
  		GRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLN
  		WFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGV
  		FTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGM
  		NSRSTSLSVTLVLVGIVTLYLGVMVQA
  182	tr|Q9IZI6|Q9IZI6_	MRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQ
  	9FLAV	GKPTLDFELTKTTAKEVALLRTYCIEASISNITTATRCPTQ
  	Envelope protein	GEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVV
  	(Fragment)	TCAKFSCSGKITGNLVRIENLEYTVVVTVHNGDTHAVGN
  	OS = Dengue virus	DTSNHGVTAMITPRSPSVEVKLPDYGELTLDCEPRSGIDF
  	4 GN = E PE = 4	NEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEVH
  	SV = 1	WNYKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGA
  		TEVDSGDGNHMFAGHLKCEVRMEKLRIKGMSYTMCSG
  		KFSIDKEMAETQHGTTVVKVKYEGAGAPCKVPIEIRDVN
  		KEKVVGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGNS
  		ALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGS
  		VGGLFTSLGKAVHQVFGSVYTTMFGGVSWMIRILIGFLV
  		LWIGTNSRNTSMAMTCIAVGGITLFLGF
  183	gi|73671170|ref|	SVALTPHSGMGLETRAETWMSSEGAWKHAQRVESWILR
  	NP_740316.1|	NPGFALLAGFMAYMIGQTGIQRTVFFVLMMLVAPSYG
  	membrane (M)
  	protein [Dengue
  	virus 4]
  184	gi|158828127|ref|	SVALAPHVGMGLDTRTQTWMSAEGAWRQVEKVETWA
  	YP_001531167.1|	LRHPGFTILALFLAHYIGTSLTQKVVIFILLMLVTPSMT
  	Membrane
  	glycoprotein
  	[Dengue virus 3]
  185	gi|158828122|ref|	SVALAPHVGLGLETRTETWMSSEGAWKQIQKVETWALR
  	NP_722459.2|	HPGFTVIALFLAHAIGTSITQKGIIFILLMLVTPSMA
  	membrane
  	glycoprotein
  	[Dengue virus 1]
  186	gi|159024811|ref|	SVALVPHVGMGLETRTETWMSSEGAWKHVQRIETWILR
  	NP_739592.2|	HPGFTMMAAILAYTIGTTHFQRALIFILLTAVTPSMT
  	Membrane
  	glycoprotein
  	[Dengue virus 2]

Example 36. OVA Multitope in vitro Screening Assay Kinetic Analysis
• As depicted in FIG. 35 , antigen surface presentation is an inefficient process in the antigen presenting cells (APC). Peptides generated from proteasome degradation of the antigens are presented with low efficiency (only 1 peptide of 10000 degraded molecules is actually presented). Thus, priming of CD8 T cells with APCs provides insufficient densities of surface peptide/MHC I complexes, resulting in weak responders exhibiting impaired cytokine secretion and decreased memory pool. To improve DENV mRNA vaccines encoding concatemeric DENV antigens, an in vitro assay was designed to test the linkers used to connect peptide repeats, the number of peptide repeats, and sequences known to enhance antigen presentation.
• mRNA constructs encoding one or more OVA epitopes were configured with different linker sequences, protease cleavage sites, and antigen presentation enhancer sequences. Their respective sequences were as shown in Table 37. To perform the assay, 200 ng of each MC3-formulated mRNA construct was transfected into JAWSII cells in a 24-well plate. Cells were isolated at 6, 24, and 48 hours post transfection and stained with fluorescently-labeled Anti-Mouse OVA257-264 (SIINFEKL) peptide bound to H-2Kb. Staining was analyzed on a LSRFortessa flow cytometer. Samples were run in triplicate. The Mean Fluorescent Intensity (MFI) for each mRNA construct was measured and shown in FIG. 36 . Constructs 2, 3, 7, 9, and 10 showed enhanced surface presentation of the OVA epitope, indicating that the configurations of these constructs may be used for DENV mRNA vaccine. Construct 5 comprises a single OVA peptide and a KDEL sequence that is known to prevent the secretion of a protein. Construct 5 showed little surface antigen presentation because the secretion of the peptide was inhibited.
Example 37. Antibody Binding to DENV-1, 2, 3, and 4 prME Epitopes
• DENV mRNA vaccines encoding concatemeric antigen epitopes were tested for binding to antibodies known to recognize one or more DENV serotypes. To test antibody binding to the epitopes, 200 ng of DENV mRNA vaccines encoding different Dengue prME epitopes were transfected into HeLa cells in 24-well plates using the TransitIT-mRNA Transfection Kit (Mirus Bio). The DENV mRNA vaccine constructs are shown in Table 34. Transfections were done in triplicate. After 24 hours, surface expression was detected using four different antibodies (10 μg/mL) followed by either goat-anti-human or anti-mouse AF700 secondary antibody (1/500). Signal generated from antibody binding are shown as Mean Fluorescent Intensity (MFI) (FIG. 37 ). Antibody D88 is known to recognize all 4 serotypes and bound to all antigen epitopes encoded by the DENV mRNA vaccine constructs tested. Antibody 2D22 is known to recognize only DENV 2 and preferentially bound to construct 21, which encodes DENV 2 antigen epitopes. Antibody 2D22 also showed weak binding to epitopes of other DENV serotypes. Antibody 5J7 is known to recognize only DENV 3 and only bound to antigen epitopes encoded by constructs 13, 19, and 20, which encode DENV 3 antigen epitopes. Antibody 1-11 is known to bind strongly to DENV 1 and 2, to bind weakly to DENV 3 and to bind little DENV 4. Antibody 1-11 bound to DENV 1, 2, and 3, and binding to DENV 3 antigen epitopes was stronger than binding to DENV 1 or 2 (FIG. 37 ).

• TABLE 37
  mRNA constructs that encode one or more OVA epitopes

  	# of
  	Peptides/		Antigen Presentation		SEQ ID
  Construct	Repeats	Linker	Enhancer Sequence	Amino acid Sequence	NO:

  1	8 OVA	G/S	—	MLESIINFEKLTEGGGGS	187
  	(8 mer)			GGGGSLESIINFEKLTEG
  	Repeats			GGGSGGGGSLESIINFEK
  	(Flanking			LTEGGGGSGGGGSLESII
  	AA)			NFEKLTEGGGGSGGGGSL
  				ESIINFEKLTEGGGGSGG
  				GGSLESIINFEKLTEGGG
  				GSGGGGSLESIINFEKLT
  				EGGGGSGGGGSLESIINF
  				EKLTE
  2	8 OVA	Cathepsin	—	MLESIINFEKLTEGFLGL	188
  	(8 mer)	B		ESIINFEKLTEGFLGLES
  	Repeats	Cleavage		IINFEKLTEGFLGLESII
  	(Flanking	Site		NFEKLTEGFLGLESIINF
  	AA)	(GFLG)		EKLTEGFLGLESIINFEK
  				LTEGFLGLESIINFEKLT
  				EGFLGLESIINFEKLTE
  3	8 OVA	—	Human MHCI	MRVTAPRTVLLLLSAALA	189
  	(8 mer)		Secretion	LTETWALESIINFEKLTE
  	Repeats		Peptide/Cytoplasmic	LESIINFEKLTELESIIN
  	(Flanking		Domain	FEKLTELESIINFEKLTE
  	AA)			LESIINFEKLTELESIIN
  				FEKLTELESIINFEKLTE
  				LESIINFEKLTEGSIVGI
  				VAGLAVLAVVVIGAVVAT
  				VMCRRKSSGGKGGSYSQA
  				ASSDSAQGSDVSLTA
  4	8 OVA	Cathepsin	Human MHCI	MRVTAPRTVLLLLSAALA	190
  	(8 mer)	B	Secretion	LTETWALESIINFEKLTE
  	Repeats	Cleavage	Peptide/Cytoplasmic	GFLGLESIINFEKLTEGF
  	(Flanking	Site	Domain	LGLESIINFEKLTEGFLG
  	AA)	(GFLG)		LESIINFEKLTEGFLGLE
  				SIINFEKLTEGFLGLESI
  				INFEKLTEGFLGLESIIN
  				FEKLTEGFLGLESIINFE
  				KLTEGSIVGIVAGLAVLA
  				VVVIGAVVATVMCRRKSS
  				GGKGGSYSQAASSDSAQG
  				SDVSLTA
  5	Single OVA	—	KDEL	MSIINFEKLKDEL	191
  6	Single OVA	—	Human MHCI	MRVTAPRTVLLLLSAALA	192
  	(Flanking		Secretion	LTETWALESIINFEKLTE
  	AA)		Peptide/Cytoplasmic	GSIVGIVAGLAVLAVVVI
  			Domain	GAVVATVMCRRKSSGGKG
  				GSYSQAASSDSAQGSDVS
  				LTA
  7	8 OVA	Cathepsin	Murine Ig Kappa	METDTLLLWVLLLWVPGS	193
  	(8 mer)	B	Signal Peptide(Igκ)	TGDSIINFEKLGFLGSII
  	Repeats	Cleavage		NFEKLGFLGSIINFEKLG
  		Site		FLGSIINFEKLGFLGSII
  		(GFLG)		NFEKLGFLGSIINFEKLG
  				FLGSIINFEKLGFLGSII
  				NFEKL
  8	8 OVA	G/S	Human MHCI	MRVTAPRTVLLLLSAALA	194
  	(8 mer)		Secretion	LTETWALESIINFEKLTE
  	Repeats		Peptide/Cytoplasmic	GGGGSGGGGSLESIINFE
  	(Flanking		Domain	KLTEGGGGSGGGGSLESI
  	AA)			INFEKLTEGGGGSGGGGS
  				LESIINFEKLTEGGGGSG
  				GGGSLESIINFEKLTEGG
  				GGSGGGGSLESIINFEKL
  				TEGGGGSGGGGSLESIIN
  				FEKLTEGGGGSGGGGSLE
  				SIINFEKLTEGSIVGIVA
  				GLAVLAVVVIGAVVATVM
  				CRRKSSGGKGGSYSQAAS
  				SDSAQGSDVSLTA
  9	8 OVA	—	—	MLESIINFEKLTELESII	195
  	(8 mer)			NFEKLTELESIINFEKLT
  	Repeats			ELESIINFEKLTELESII
  	(Flanking			NFEKLTELESIINFEKLT
  	AA)			ELESIINFEKLTELESII
  				NFEKLTE
  10	Single OVA	—	—	MSIINFEKL	196
  11	8 OVA	Cathepsin	Murine Ig Kappa	METDTLLLWVLLLWVPGS	197
  	(8 mer)	B	Signal Peptide(Igκ)	TGDHPFTEDDAVDPNDSD
  	Repeats	Cleavage	and PEST	IDPESRSIINFEKLGFLG
  		Site		SIINFEKLGFLGSIINFE
  		(GFLG)		KLGFLGSIINFEKLGFLG
  				SIINFEKLGFLGSIINFE
  				KLGFLGSIINFEKLGFLG
  				SIINFEKL
  12	8 OVA	Cathepsin	Murine MHC Class I	MSIINFEKLGFLGSIINF	198
  	(8 mer)	B	Cytoplasmic Domain	EKLGFLGSIINFEKLGFL
  	Repeats	Cleavage	(MITD)	GSIINFEKLGFLGSIINF
  		Site		EKLGFLGSIINFEKLGFL
  		(GFLG)		GSIINFEKLGFLGSIINF
  				EKLPPPSTVSNMIIIEVL
  				IVLGAVINIGAMVAFVLK
  				SKRKIGGKGGVYALAGGS
  				NSIHGSALFLEAFKA

• TABLE 38
  DENV mRNA vaccine constructs tested for
  antibody binding or in challenge studies

  Con-		SEQ
  struct	mRNA Name	ID NO

  13	DEN3_prME_PaH881/88_AF349753.1	199
  14	DEN1_prME_West_Pac_AY145121.1	200
  15	DEN1_prME_PUO-359_AAN32784.1	201
  16	DEN4_prME_DHF_Patient_JN638571.1	202
  17	DEN4_prME_DENV4/CN/GZ29/2010_KP723482.1	203
  18	DEN4_prME_rDEN4_AF326825.1	204
  19	DEN3_prME_L11439.1	205
  20	DEN3_prME_D3/Hu/TL129NIID/2005_AB214882	206
  21	DENV2_prME_Peru_IQT2913_1996	207
  22	DENV2_prME_Thailand-168_1979	208
  23	DENV2_prME_Thailand_PUO-218_1980	209
  	(Sanofi strain)
  24	DEN2_D2Y98P_PRME80_Hs3_LSP	210
  25	Non-H2Kb multitope	211
  26	H2Kb multitope	212

Example 38. DENV prME Challenge Study in Cynomolgus (Cyno) Monkey Model
• Shown in Table 39 is the design of DENV prME challenge study in cynomolgus (cyno) money. Indicated DENV mRNA vaccine encoding prME antigen epitopes, or vaccines thereof, are used to immunize cyno. The vaccines are formulated in lipid nanoparticles (e.g., MC3 formulation) and administered to the cyno monkeys intramuscularly on day 0, 21, and 42. Dosages of the vaccines are 250 μg or 5 μg per immunization. In experiments where a combination of different DENV mRNA vaccines are used, 250 μg or 5 μg of each mRNA vaccine is used. FLAG-tagged H10N8 flu vaccine is used as control at a dosage of 250 μg per immunization. Naïve cyno monkeys without immunization are also used as control. Cyno monkey sera are collected on days 20, 41, 62, and 92 post initial immunization and used for serotype-specific neutralization assays.
• Immunized cyno monkeys are challenged on day 63 post initial immunization with indicated DENV viruses. Cyno monkey sera are collected on days 62 (pre-challenge), 63-66, 68, 70, 72, 76, and 92 (end of life) to determine serum viral load.

• TABLE 39
  DENV prME Challenge Study Design in Cynomolgus (cyno) Monkey

  Group		Vaccine
  n = 3	Vaccine	Schedule	Dosage/Route	Challenge

  1	Dengue 1	Day 0, 21, 42	IM, LNP	Challenge with
  	prME		250 μg	Dengue	1/03135 s.c
  2	(Construct		IM, LNP	(5log PFU)
  	15)		5 μg
  3	Dengue 2	Day 0, 21, 42	IM, LNP	Challenge with
  	prME		250 μg	Dengue	2/99345 s.c
  4	(Construct		IM, LNP	(5log PFU)
  	21)		5 μg
  5	Dengue 3	Day 0, 21, 42	IM, LNP	Challenge with
  	prME		250 μg	Dengue	3/16562 s.c
  6	(Construct		IM, LNP	(5log PFU)
  	19)		5 μg
  7	Dengue 4	Day 0, 21, 42	IM, LNP	Challenge with
  	prME		250 μg	Dengue	4/1036 s.c
  8	(Construct		IM, LNP	(5log PFU)
  	17)		5 μg
  9	prME Combo	Day			0, 21, 42	IM, LNP	Challenge with
  	(Post-		1000 μg Total	Dengue	1/03135 s.c
  	Formulation		(250 μg of each)	(5log PFU)
  10	Mix)		IM, LNP
  	(Constructs		20 μg Total
  	15, 17, 19,		(5 μg of each)
  	and 21)
  11	prME Combo	Day			0, 21, 42	IM, LNP	Challenge with
  	(Post-		1000 μg Total	Dengue	2/99345 s.c
  	Formulation		(250 μg of each)	(5log PFU)
  12	Mix)		IM, LNP
  	(Constructs		20 μg Total
  	15, 17, 19,		(5 μg of each)
  	and 21)
  13	prME Combo	Day			0, 21, 42	IM, LNP	Challenge with
  	(Post-		1000 μg Total	Dengue	3/16562 s.c
  	Formulation		(250 μg of each)	(5log PFU)
  14	Mix)		IM, LNP
  	(Constructs		20 μg Total
  	15, 17, 19,		(5 μg of each)
  	and 21)
  15	prME Combo	Day			0, 21, 42	IM, LNP	Challenge with
  	(Post-		1000 μg Total	Dengue	4/1036 s.c
  	Formulation		(250 μg of each)	(5log PFU)
  16	Mix)		IM, LNP
  	(Constructs		20 μg Total
  	15, 17, 19,		(5 μg of each)
  	and 21)
  17	prME Combo	Day			0, 21, 42	IM, LNP	Challenge with
  	(Post-		1000 μg Total	Dengue	2/99345 s.c
  	Formulation		(250 μg of each)	(5log PFU)
  	Mix)
  	(Constructs
  	15, 17, 19,
  	and 22)
  18	H10N8- FLAG	Day			0, 21, 42	IM, LNP	Challenge with
  			250 μg	Dengue	2/99345 s.c
  				(5log PFU)
  19	Naive	—	—	Challenge with
  				Dengue 1/03135 s.c
  				(5log PFU)
  20	Naive	—	—	Challenge with
  				Dengue 2/99345 s.c
  				(5log PFU)
  21	Naive	—	—	Challenge with
  				Dengue 3/16562 s.c
  				(5log PFU)
  22	Naive	—	—	Challenge with
  				Dengue 4/1036 s.c
  				(5log PFU)
  Collect serum on day 20, 41, 62, and 92 for serotype-specific neutralization assay
  Collect serum on day 62 (per-challenge), 63-66, 68, 70, 72, 76, and 92 (end of In-life) to determine serum viral load

Example 39: Dengue 2 prME Challenge Study in AG129 Mice
• The instant study was designed to evaluate the efficacy of four DENV mRNA vaccine constructs (constructs 21-24 in Table 38) in AG129 mice challenge assays. The schedule of the challenge study was shown in FIG. 38A. The DENV mRNA vaccines were formulated in lipid nanoparticles (e.g., MC3 formulation) and administered to the AG129 mice intramuscularly on days 0 and 21. Dosage of the vaccines were 2 ag or 10 ag per immunization. Heat inactivated D2Y98P strain was used as a negative control to vaccinate the mice. Naïve AG129 mice without immunization were also used as control.
• Immunized AG129 mice were challenged on day 42 post initial immunization with Dengue D2Y98P virus (s.c., 1e5 PFU per mouse). AG129 mice sera were collected on days 20 and 41 post initial immunization and used for serotype-specific neutralization assays. Mice immunized with any of the four DENV mRNA vaccine constructs survived, while the control mice died. These data demonstrate that, after lethal challenge, there was 100% protection provided by each mRNA vaccine construct, regardless of dose. The weights and health of the mice were monitored and the results were plotted in FIGS. 38C-38D.
• Mice sera collected from mice immunized with 2 μg of the DENV mRNA vaccines were able to neutralize several DENV 2 strains and variations in the neutralization ability between the tested mRNA vaccines and between different DENV 2 strains were observed (FIG. 39 ).
Example 40: DENV prME Challenge Study in AG129 Mice Model
• Shown in Table 40 is the design of a DENV prME challenge study in AG129 mice, including the mRNA constructs tested, the vaccination schedule, the dosage, the challenge strains, and the serum collection schedule.
• Indicated DENV mRNA vaccine encoding prME antigen epitopes, or vaccines thereof, were used to immunize AG129 mice. The vaccines were formulated in lipid nanoparticles (e.g., MC3 formulation) and administered to the mice intramuscularly on days 0 and 21. Dosages of the vaccines were 2 μg or 10 μg per immunization. In experiments where a combination of different DENV mRNA vaccines were used, 2 μg of each mRNA vaccine was used. Naïve AG129 mice without immunization were used as control. AG129 mice sera were collected on days 20 and 41 post initial immunization and used for serotype-specific neutralization assays.
• Immunized AG129 mice were challenged on day 42 post initial immunization with Dengue D2Y98P virus (s.c., 1e5 PFU per mouse). The weights and health of the mice were monitored for 14 days post infection and the results were plotted in FIGS. 40A-40I.

• TABLE 40
  DENV prME Challenge Study Design in AG129 Mice

  Group		Vaccine
  n = 5	Vaccine	Schedule	Dosage/Route	Serum/PBMCs	Challenge	Readout

  1	Dengue 1	Day 0, 21	IM, LNP,	Collect serum on	Challenge with	Monitor
  	prME		10 μg	day 20 and 41 for	1e5 PFU per	weights
  	(Construct 15)			serotype-specific	mouse of	and health
  2		Day 0, 21	IM, LNP,	neutralization	D2Y98P SC	for 14
  			2 μg	assay	injection	days p.i.
  					(Day 42)
  3	Dengue 2	Day 0, 21	IM, LNP,
  	prME		10 μg
  4	(Construct 21)	Day 0, 21	IM, LNP,
  			2 μg
  5	Dengue 3	Day 0, 21	IM, LNP,
  	prME		10 μg
  6	(Construct 19)	Day 0, 21	IM, LNP,
  			2 μg
  7	Dengue 4	Day 0, 21	IM, LNP,
  	prME		10 μg
  8	(Construct 17)	Day 0, 21	IM, LNP,
  			2 μg
  9	H2Kb	Day 0, 21	IM, LNP,	Collect and
  	Multitope		10 μg	cryopreserve
  10	(Construct 25)	Day 0, 21	IM, LNP,	PBMCs on day 20
  			2 μg	and 41; Ship to
  11	Non-H2Kb	Day 0, 21	IM, LNP,	Valera
  	Multitope		10 μg
  12	(Construct 26)	Day 0, 21	IM, LNP,
  			2 μg
  13	prME Combo +	Day 0, 21	IM, LNP,	Collect serum on
  	H2Kb		10 μg Total	day 20 and 41 for
  	Multitope		(2 μg of each)	serotype-specific
  	(Constructs 15,			neutralization
  	17, 19, and 21)			assay
  	(Post7)
  14	prME Combo +	Day 0, 21	IM, LNP,
  	non-H2Kb		10 μg Total
  	Multitope		(2 μg of each)
  	(Constructs 15,
  	17, 19, 21, and
  	26) (Post7)
  15	prME Combo	Day 0, 21	IM, LNP,
  	(Constructs 15,		8 μg Total
  	17, 19, and 21)		(2 μg of each)
  	(Post7)
  16	prME Combo +	Day 0, 21	IM, LNP,
  	H2Kb		10 μg Total
  	Multitope		(2 μg of each)
  	(Constructs 15,
  	17, 19, 21 and
  	25) (Post1)
  17	prME Combo +	Day 0, 21	IM, LNP,
  	non-H2Kb		10 μg Total
  	Multitope		(2 μg of each)
  	(Constructs 15,
  	17, 19, 21, and
  	26) (Post1)
  18	prME Combo	Day 0, 21	IM, LNP,
  	(Constructs 15,		8 μg Total
  	17, 19, and 21)		(2 μg of each)
  	(Post1)
  19	Dengue 2	Day 0, 21	IM, LNP,	Collect serum on
  	prME		2 μg	day 20 and 41 for
  	(Construct 22)			Dengue 2-specific
  20	Naive	Day 0, 21	Tris/Sucrose	neutralization
  				assay

Example 41: Virus-Like Particles
• The antigens produced from the DENV prME mRNA vaccines of the present disclosure, when expressed, are able to assemble into virus-like particles (VLPs). The instant study was designed to evaluate the immunogenicity of the VLPs by negative stain electron microscope imaging. As shown in FIG. 41 , DENV mRNA vaccine constructs 21-24 were expressed and VLPs were assembled an isolated. The VLPs were visualized under negative stain electron microscopy. Construct 23 is the vaccine construct used by Sanofi in its DENV vaccines. Constructs 21, 22, and 24 produced more uniform VLPs, suggesting that these VLPs may be more superior in their immunogenicity than the VLPs produced from construct 23.
Example 42: Efficacy of CHIKV mRNA Vaccine X Against CHIKV in AG129 Mice Study Design
• Chikungunya virus (CHIKV) 181/25 strain is an attenuated vaccine strain that was developed by the US Army via multiple plaque-to-plaque passages of the 15561 Southeast Asian human isolate (Levitt et al.). It is well tolerated in humans and is highly immunogenic. It produces small plaques and has decreased virulence in infant mice and nonhuman primates. When the attenuated virus is administered to immunodeficient AG129 mice (lacking the IFN-α/β and γ receptors) the mice succumb to a lethal disease within 3-4 days with ruffled fur and weight loss (Partidos, et al. 2011 Vaccine).
• This instant study was designed to evaluate the efficacy of CHIKV candidate vaccines as described herein in AG129 mice (Table 41). The study consisted of 14 groups of female 6-8 week old AG129 mice (Table 41). Groups 1-4, 7-8, and 10-15 were vaccinated with CHIKV vaccine X via the intramuscular (IM; 0.05 mL) route on Day 0 and select groups received an additional boost on Day 28. Control Groups 9 and 16 received vehicle (PBS) only on Days 0 and 28 via IM route (0.05 mL). Regardless of vaccination schedule, Groups 1-4 and 7-9 were challenged on Day 56 while Groups 10-16 were challenged on Day 112 using the CHIKV 181/25 strain (stock titer 3.97×10⁷ PFU/mL, challenge dose 1×10⁴ PFU/mouse). For virus challenge, all mice received a lethal dose (1×10⁴ PFU) of Chikungunya (CHIK) strain 181/25 via intradermal (ID) route (0.050 mL via footpad). All mice were monitored for 10 days post infection for weight loss, morbidity, and mortality. Each mice was assigned a heath score based on Table 5. Mice displaying severe illness as determined by >30% weight loss, a health score of higher than 5, extreme lethargy, and/or paralysis were euthanized with a study endpoint of day 10 post virus challenge. Test bleeds via retro-orbital (RO) collection were performed on mice from all groups on Days −3, 28, and 56. Mice from Groups 10-16 were also bled on Days 84 & 112. Mice that survived challenge were also terminally bled on Day 10 post challenge. Serum samples from mice (Days −3, 28, 56, 84, 112 and surviving mice) were kept frozen (−80° C.) and stored until they were tested for reactivity in a semi quantitative ELISA for mouse IgG against either E1, E2 or CHIKV lysate.
Experimental Procedure Intramuscular (IM) Injection of Mice
• 1. Restrain the animal either manually, chemically, or with a restraint device.
• 2. Insert the needle into the muscle. Pull back slightly on the plunger of the syringe to check proper needle placement. If blood is aspirated, redirect the needle and recheck placement again.
• 3. Inject appropriate dose and withdraw needle. Do not exceed maximum volume. If the required volume exceeds the maximum volume allowed, multiple sites may be used with each receiving no more than the maximum volume.
• 4. The injection site may be massaged gently to disperse the injected material.
Intradermal (ID) Injections of Mice
• 1. Restrain the animal either manually, chemically, or with a restraint device.
• 2. Carefully clip the hair from the intended injection site. This procedure can be done upon animals arriving or the day before any procedures or treatments are required.
• 3. Lumbar area is the most common site for ID injections in all species, but other areas can be used as well.
• 4. Pinch or stretch the skin between your fingers (or tweezers) to isolate the injection site.
• 5. With the beveled edge facing up, insert the needle just under the surface between the layers of skin. Inject the appropriate dose and withdraw needle. A small bleb will form when an ID injection is given properly.
• 6. If the required volume exceeds the maximum volume allowed, multiple sites may be used with each receiving no more than the maximum volume.
Retro-Orbital Bleeding in Mice
• 1. Place the mice in the anesthesia chamber and open oxygen line and set to 2.5% purge. Start flow of anesthesia at 5% isoflurane.
• 2. Once the animal becomes sedate, turn anesthesia to 2.5%-3% isoflurane and continue to expose the animal to the anesthesia. Monitor the animal to avoid breathing becoming slow.
• 3. Remove the small rodent from anesthesia chamber and place on its back while restraining with left hand and scruff the back of the animal's neck, so it is easy to restrain and manipulate while performing the procedure with the right hand.
• 4. With a small motion movement, place the capillary tube in the corner of the animal's eye close to the nostril, and rotate or spin the Hematocrit glass pipette until blood start flowing out. Collect the appropriate amount of blood needed into the appropriate labeled vial.
• 5. Monitor the animal after retro-orbital bleeding is done for at least 10-15 seconds to ensure hemostasis.
• 6. Place the animal back to its original cage and monitor for any other problems or issues caused while manipulating animal due to the procedure.
Observation of Mice
• 1. Mice were observed through 10 days post infection (11 days total, 0-10 days post infection).
• 2. Mice were weighed daily on an Ohause scale and the weights are recorded.
• 3. Survival and health of each mouse were evaluated once time a day using a scoring system of 1-7 described in Table 5.
Infection
• On either Day 56 (Groups 1-4, 7-9) or Day 112 (Groups 10-16) groups of 5 female 6-8 week old AG129 mice were infected via intradermal injection with 1×10⁴ PFU/mouse of the 181/25 strain of Chikungunya diluted in PBS. The total inoculation volume was 0.05 mL administered in the rear footpad of each animal. Mice were anesthetized lightly using 2-5% v/v of isoflurane at ˜2.5 L/min of 02 (VetEquip IMPAC6) immediately prior to infection.
Dose Administration
• In this study mice were administered 0.04 μg, 2 μg, or 10 μg of various formulations of the CHIKV vaccine X or vehicle alone (PBS) on either Day 0 or on Days 0 and 28 via the intramuscular route (0.05 mL). The material was pre-formulated by the Client and diluted in PBS by IBT prior to dosing as per instructions provided by the Client.
Results
• Mice were immunized once (Day 0) or twice (Days 0 & 28) with either 0.04 μg, 2 μg, or 10 μg of Chikungunya vaccine X and were challenged with CHIKV strain 181/25 on either Day 56 (Groups 1-4, 7-9) or on Day 112 (Groups 10-16). Mice were monitored for a total of 10 days post infection for health and weight changes. Mice that received either 2 μg or 10 μg of the CHIKV vaccine X either once (Day 0) or twice (Days 0 and 28) were fully protected (100%) regardless of whether the mice were challenged 56 days or 112 days after the initial vaccination (FIGS. 42A-42B, Table 44). Mice receiving 0.04 μg of the CHIKV vaccine were not protected at all from lethal CHIKV infection. This efficacy data is supported by the health scores observed in the vaccinated mice in that the protected mice displayed little to no adverse health effects of a CHIKV infection (FIGS. 44A-44B). Weight loss is not a strong indicator of disease progression in the CHIKV AG129 mouse model (FIGS. 43A-43B).
• Mice immunized with the CHIKV vaccine X showed increased antibody titers against CHIKV E1, E2 and CHIKV lysate as compared to the vehicle only (PBS) treated groups. Serum binding against the virus lysate yielded the highest antibody titers for all vaccinated groups (FIGS. 45A-45C, 46A-46C, 47A-47C, 48A-48C). Overall, the antibody titers were dose dependent with the highest titers observed in serum from mice vaccinated with 10 μg of CHIKV vaccine X while the lowest titers were observed in serum from mice vaccinated with 0.04 μg of the CHIKV vaccine X. Similarly, higher titers were observed in serum from mice vaccinated twice (Days 0 and 28) as compared to serum from mice vaccinated only once (Day 0). Serum obtained on Day 112 post initial vaccination still yielded increased antibody titers in mice that received either 10 ag or 2 ag of CHIKV vaccine X (FIGS. 47A-47C).
• Serum from mice groups 10-16, 112 days post immunization were also tested in a Plaque Reduction Neutralization Test (PRNT). Serum from each mice was diluted from 1/20 to 1/40960 and assessed for its ability to reduce CHIKV plaque formation. The results were shown in Table 46.

• TABLE 41
  CHIKV Challenge Study Design in AG129 mice

  Group*			Dose
  (n = 5)	Vaccine	Schedule	(IM route)	Challenge	Bleeds

  1

  VAL-

  Day 0

  10

  μg

  Challenge with

  Pre-bleed for

  2

  181388

  Day 0 & 28

  1 × 104 PFU per

  serum via RO

  3

  Day 0

  2

  μg

  mouse of CHIK

  route on days −3,

  4		Day 0 & 28		181/25 via ID	28, 56, (all
  				injection	groups) & 84, 112
  				on day 56.	(groups 10-16

  7

  Day 0

  0-4

  μg

  Weights and

  only).

  8		Day 0 & 28		health for 10	Terminal bleed
  9	PBS	Day	0 & 28	—	days following	surviving mice on
  				infection.	day 10 post

  10

  VAL-

  Day 0

  10

  μg

  Challenge with

  challenge.

  11

  181388

  Day 0 & 28

  1 × 104 PFU per

  Serum stored

  12

  Day 0

  2

  μg

  mouse of CHIK

  at −80° C.

  13

  Day 0 & 28

  181/25 via ID

  14

  Day 0

  0-4

  μg

  injection on day

  15		Day 0 & 28		112. Weights and
  16	PBS	Day	0 & 28	—	health for 10
  				days following
  				infection.
  *No group 5 or 6 in this study

• TABLE 42
  Equipment and Software

  Item	Vendor	Cat#/Model
  Syringes	BD	Various
  Animal Housing	InnoVive	Various
  Scale	Ohause	AV2101
  Prism software	GraphPad	N/A
  Microplate Washer	BioTek	ELx405
  Plate reader with SoftMax Pro	Molecular Devices	VersaMax
  version 5.4.5

• TABLE 43
  ELISA Reagents

  		Storage
  Name	Supplier cat#	Temperature	Notes
  DPBS
  1X, sterile	Corning 21-031-	Ambient	For dilution of coating antigen
  	CM or equivalent
  StartingBlock T20	Thermo Scientific	2-8° C.	For blocking non-specific
  (PBS) Blocking	37539		binding and use as diluent of
  Buffer			Standards, unknown test sera
  			and detection antibody
  SureBlue Reserve	KPL 53-00-02 or	2-8° C.	N/A
  TMB Microwell	equivalent
  Peroxidase
  Substrate (1-
  Component)
  DPBS powder, non-	Corning 55-031-PB	2-8° C.	Use deionized water to
  sterile	or equivalent		dissolved DPBS powder from
  			one bottle to a final volume of
  			10 liters of 1X DPBS
  TWEEN-20	Sigma-Aldrich	Ambient	Add	5 mL TWEEN-20 to 10
  	P1379-500ML or		liters of 1X DPBS and mix
  	equivalent		well to prepare DPBS +
  			0.05% TWEEN-20 Wash
  			Buffer for automatic plate
  			washer

• TABLE 44
  ELISA Reagents

  Critical Reagent Please note: Coating antigens and standards	Supplier cat#	Storage
  are stored as single-use aliquots.	and/or lot#	Temperature	Assay Parameter

  Coating antigens	CHIKV recombinant E1 glycoprotein,	IBT Bioservices,	−70° C. or below	400 ng/well
  	expressed in 293 mammalian cells IBT's	lot 08.11.2015
  	BCA = 0.351 mg/mL
  	CHIKV recombinant E2 glycoprotein,	ImmunoDx, cat#	−70° C. or below	400 ng/well
  	expressed in E. coli IBT's BCA = 1.291	80002, lot
  	mg/mL	10MY4
  	CHIKV 181/25 lysate from sucrose-	IBT Bioservices,	−70° C. or below	300 ng/well
  	purified viruses, lysed by sonication	lot 11.23.2015
  	IBT's BCA = 1.316 mg/mL
  Standards	Anti-E1 positive control Pooled mouse	IBT Bioservices	−70° C. or below	Assigned, 30,812
  	serum from survivors of BS-1842 group			Antibody Units/mL
  	4 (vaccinated with E1 mRNA 10 μg, ID,			against E1 protein
  	LNP on study days 0 and 28) day 66
  	terminal bleeds (10 days after CHIKV
  	infection)
  	Anti-E2 positive control, Pooled mouse	IBT Bioservices	−70° C. or below	Assigned, 16912
  	serum from survivors of BS-1842 group			Antibody Units/mL
  	8 (vaccinated with E2 mRNA 10 μg, ID,			against E2 protein
  	LNP on study days 0 and 28) day 66			Assigned 14,200
  	terminal bleeds (10 days after CHIKV			Antibody Units/mL
  	infection)
  Detection antibody	Anti-mouse IgG (H + L)-HRP	KPL, cat# 474-	2-8° C.	1:6000 dilution
  		1806, lot 140081

• TABLE 45
  Survival Percentage

  		10 μg		2 μg		0.4 μg
  Days
  	10 μg	Day	0	2 μg	Day	0	0.4 μg	Day	0
  p.i.	Day	0	& 28	Day 0	& 28	Day 0	& 28	PBS

  a. Groups 1-4 and 7-9, Day 56 Challenge

  0	100	100	100	100	100	100	100
  3						80
  4					0	40	80
  5						0	0
  10	100	100	100	100

  b. Groups 10-16, Day 112 Challenge

  0	100	100	100	100	100	100	100
  3					80	80
  4					20	20	50
  5					0	0	0
  10	100	100	100	100

• TABLE 46
  CHIKV Plaque Reduction Neutralization Test (PRNT)
  Serum dilutions from 1/20 to 1/40960

  		Expt info
  	Vaccination	CHIKV strain	sample	PRNT80	PRNT50
  GP#	regimen	37997	ID	titer	titer

  10	Day 0,	CHIKV	1	1/160	1/640
  	IM/10 μg	37997	2	1/320	1/320
  		working stock	3	1/160	1/640
  		titer =	4	1/160	1/1280
  		780 PFU/ml	5	1/320	1/1280
  11	Day 0/Day 28,		1	1/640	1/2560
  	IM/10 μg		2	1/1280	1/1280
  			3	1/320	1/2560
  			4	1/640	1/5120
  			5	1/1280	1/5120
  12	Day 0,		1	1/20	1/80
  	IM/2 μg		2	1/40	1/320
  			3	<1/20	1/160
  		PRNT80	4	<1/20	1/160
  		cutoff	5	<1/20	1/20
  13	Day 0,	8 PFU	1	1/80	1/320
  	Day 28,		2	1/80	1/640
  	IM/2 μg		3	1/20	1/320
  			4	1/20	1/320
  			5	1/320	1/640
  14	Day 0,		1	<1/20	80
  	IM/0.4 μg		2	<1/20	<1/20
  			3	<1/20	<1/20
  			4	<1/20	<1/20
  			5	<1/20	<1/20
  15	Day 0,	PRNT50	1	<1/20	<1/20
  	Day 28,	cutoff	2	<1/20	80
  	IM/0.4 μg	20 PFU	3	<1/20	<1/20
  			4	<1/20	<1/20
  			5	<1/20	<1/20
  16	Vehicle		1	<1/20	<1/20
  	Day 0/Day 28		2	<1/20	<1/20
  			3	<1/20	<1/20
  			4	<1/20	<1/20
  			5	<1/20	<1/20

Example 43: Immunogenicity of Chikungunya Polyprotein (C-E3-E2-6K-E1) mRNA Vaccine Candidate in Rats
• Sprague Dawley rats (n=5) were vaccinated with 20 g of MC-3-LNP formulated mRNA 30 encoded CHIKV polyprotein (C-E3-E2-6K-E1) (SEQ ID NO: 13). The rats were vaccinated on either Day 0 or Days 0 and 14 or Days 0, 14 and 28 via IM delivery. Sera was collected on days −3, 14, 28 and 42 for ELISA testing. FIG. 58 demonstrated that there was at least a two log increase in antibody titer against CHIKV lysate post 3rd vaccination with the mRNA vaccine in normal rats.
Example 44: Evaluation of T Cell Activation of Chikungunya P 5 Polyprotein (C-E3-E2-6K-E1) mRNA Vaccine Candidate
• C57BL/6 mice (n=6 experimental group; n=3 control group) were vaccinated with 10 g of MC-3-LNP formulated mRNA encoded CHIKV polyprotein (C-E3-E2-6K-E1) (SEQ ID NO: 13). The mice were vaccinated on either Day 0 or Days 0 and 28 (boost) via IM delivery. Sera was collected on days 3, 28 and 42 for ELISA testing. Animals were sacrificed on day 42 and spleens were harvested for immunological evaluation of T cells. Splenic cells were isolated and analyzed by FACS. Briefly, spleens were removed, cells isolated, and stimulated in vitro with immunogenic peptides found within either C, E1, or E2 region of CHIKV that are known to be CD8 epitopes in B6 mice. The readout for this assay was cytokine secretion (IFN-gamma and TNF-alpha), which reveals whether the vaccine induced antigen-specific T cell responses. No CD8 T cell responses were detected using the E2 or C peptide (baseline levels of IFN-gamma and TNF-alpha), whereas there was a response to the E1-corresponding peptide (average of about 0.4% IFN-gamma and 0.1% TNF). The peptides were used to stimulate T cells used in the study were E1=HSMTNAVTI (SEQ ID NO: 300), E2=IILYYYELY (SEQ ID NO: 301), and C=ACLVGDKVM (SEQ ID NO: 302).
• FIG. 59 shows that the polyprotein-encoding CHIKV polyprotein vaccine elicited high antibody titers against the CHIKV glycoproteins. FIGS. 60 and 61A-61B show T cell activation by E1 peptide.
EQUIVALENTS
• All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
• The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
• In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims (23)

1.-111. (canceled)

112. A Dengue virus (DENV) messenger ribonucleic acid (mRNA) vaccine, comprising:

an mRNA polynucleotide comprising an open reading frame encoding a DENV polypeptide; and

a lipid nanoparticle comprising 20-60 mol % cationic lipid, 5-25 mol % neutral lipid, 25-55 mol % sterol, and 0.5-15 mol % polyethylene glycol (PEG)-modified lipid.

113. The DENV mRNA vaccine of claim 112, wherein the DENV polypeptide is selected from a DENV envelope (E) protein, a DENV membrane (M) protein, a DENV precursor membrane (prM) protein, a DENV capsid (C) protein, and a DENV prME protein.

114.-165. (canceled)

166. The DENV mRNA vaccine of claim 112, wherein the mRNA polynucleotide comprises a chemical modification.

167. The DENV mRNA vaccine of claim 166, wherein the chemical modification is selected from pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2′-O-methyl uridine.

168. (canceled)

169. The DENV mRNA vaccine of claim 167, wherein the chemical modification is a N1-methylpseudouridine.

170. (canceled)

171. The DENV mRNA vaccine of claim 169, wherein 100% of the uracil in the open reading frame has a chemical modification.

172.-182. (canceled)

183. The DENV mRNA vaccine of claim 112, wherein the cationic lipid is an ionizable cationic lipid and the sterol is cholesterol.

184.-190. (canceled)

191. A method of inducing an immune response in a subject, the method comprising administering to the subject the DENV mRNA vaccine of claim 112 in an amount effective to produce an antigen-specific immune response in the subject.

192.-477. (canceled)

478. A Dengue virus (DENV) messenger ribonucleic acid (mRNA) vaccine, comprising:

an mRNA polynucleotide comprising an open reading frame encoding a DENV polypeptide; and

a lipid nanoparticle comprising 20-60 mol % ionizable cationic lipid, 5-25 mol % neutral lipid, 25-55 mol % cholesterol, and 0.5-15 mol % polyethylene glycol (PEG)-modified lipid,

wherein 100% of the uracil in the open reading frame has a chemical modification.

479. The DENV mRNA vaccine of claim 478, wherein the chemical modification is a N1-methylpseudouridine.

480. The DENV mRNA vaccine of claim 478, wherein the DENV polypeptide comprises a DENV envelope (E) protein.

481. The DENV mRNA vaccine of claim 478, wherein the DENV polypeptide comprises a DENV membrane (M) protein.

482. The DENV mRNA vaccine of claim 478, wherein the DENV polypeptide comprises a DENV precursor membrane (prM) protein.

483. The DENV mRNA vaccine of claim 478, wherein the DENV polypeptide comprises a DENV prME protein.

484. The DENV mRNA vaccine of claim 478, wherein the lipid nanoparticle comprises 40-50 mol % of the ionizable cationic lipid, 5-10 mol % of the neutral lipid, and 1-3 mol % of the polyethylene glycol (PEG)-modified lipid.

485. A method of inducing an immune response in a subject, the method comprising administering to the subject the DENV mRNA vaccine of claim 478 in an amount effective to produce an antigen-specific immune response in the subject.

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