WO2017015457A1 - Ebola vaccine - Google Patents

Abstract

The disclosure relates to Ebola virus ribonucleic acid (RNA) vaccines, as well as methods of using the vaccines and compositions comprising the vaccines. The vaccines include one or more RNA polynucleotides having an open reading frame encoding Ebola virus. Methods for preparing and using such vaccines are also described.

Description

EBOLA VACCINE

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 62/195,267, filed July 21, 2015, U.S. provisional application number 62/244,773, October 22, 2015, and U.S. provisional application number 62/247,422, filed October 28, 2015, each of which is incorporated by reference herein in its entirety.

BACKGROUND

Ebola virus belongs to the Filoviridae family, similar to the Marburg virus.

Filoviruses are relatively simple viruses of 19 Kb genomes and consist of seven genes which encode nucleoprotein (NP), glycoprotein (GP), four smaller viral proteins (VP24, VP30, VP35 and VP40), and the RNA-dependent RNA polymerase (L protein) all in a single strand of negative-sensed RNA. In general, minus-strand (-) RNA viruses, such as Ebola virus, are major causes of human suffering that cause epidemics of serious human illness. In humans the diseases caused by these viruses include Ebola (Orthomyxoviridae), mumps, measles, upper and lower respiratory tract disease (Paramyxoviridae), rabies (Rhabdoviridae), hemorrhagic fever (Filoviridae, Bunyaviridae and Arenaviridae), encephalitis (Bunyaviridae) and neurological illness (Bomaviridae). Due to the severity of disease caused by filoviruses, these viruses are considered a significant world health threat. For instance they have many of the characteristics commonly associated with biological weapons since they can be grown in large quantities, can be fairly stable, are highly infectious as an aerosol, and are exceptionally deadly.

Deoxyribonucleic acid (DNA) vaccination is one technique used to stimulate humoral and cellular immune responses to foreign 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

Provided herein is a ribonucleic acid (RNA) vaccine that builds on the knowledge that RNA (e.g. , messenger RNA (mRNA)) 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 Ebola virus, comprising both cellular and humoral immunity, without risking the possibility of insertional mutagenesis, for example.

The RNA (e.g. , mRNA) 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 an Ebola virus of various genotypes, strains, and isolates. The RNA vaccines have superior properties in that they produce much larger antibody titers and produce responses earlier than commercially available anti- viral therapeutic treatments. As demonstrated in the examples, the mRNA vaccines described herein were capable of providing 100% protection against the Ebola viral infection in an animal model. 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.

Some embodiments of the present disclosure provide Ebola virus (Ebola) vaccines that include at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding at least one Ebola antigenic polypeptide or an immunogenic fragment thereof (e.g. , an immunogenic fragment capable of inducing an immune response to Ebola).

In some embodiments, the antigenic polypeptide is selected from EBOV glycoprotein (GP), surface EBOV GP, wild type EBOV pro-GP, mature EBOV GP, secreted wild type EBOV pro-GP, secreted mature EBOV GP, EBOV nucleoprotein (NP), RNA polymerase L, and EBOV matrix protein selected from VP35, VP40, VP24, and VP30, or combinations thereof.

In some embodiments, the at least one antigenic polypeptide is from Ebola virus strain subtype Zaire, strain H.sapiens-wt/GIN/2014/Kissidougou-C15; subtype Bundibugyo, strain Uganda 2007; subtype Zaire, strain Mayinga 1976; subtype Sudan, strain Gulu, or a combination thereof.

In some embodiments, the at least one RNA polynucleotide encodes an antigenic GP polypeptide having greater than 90% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and having membrane fusion activity. In some embodiments, the at least one RNA polynucleotide encodes an antigenic polypeptide having greater than 95% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and having membrane fusion activity. In some embodiments, the at least one RNA polynucleotide encodes an antigenic polypeptide having greater than 96% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and having membrane fusion activity. In some embodiments, the at least one RNA polynucleotide encodes an antigenic polypeptide having greater than 97% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and having membrane fusion activity. In some embodiments, the at least one RNA polynucleotide encodes an antigenic polypeptide having greater than 98% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and having membrane fusion activity. In some embodiments, the at least one RNA polynucleotide encodes an antigenic polypeptide having greater than 99% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and having membrane fusion activity. In some embodiments, the at least one RNA polynucleotide encodes an antigenic polypeptide having 95-99% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and having membrane fusion activity.

In some embodiments, the at least one RNA polynucleotide encodes an antigenic nucleoprotein polypeptide having greater than 90% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and mediates viral attachment. In some embodiments, the at least one RNA polynucleotide encodes an antigenic nucleoprotein polypeptide having greater than 95% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and mediates viral attachment. In some embodiments, the at least one RNA polynucleotide encodes an antigenic nucleoprotein polypeptide having greater than 96% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and mediates viral attachment. In some embodiments, the at least one RNA polynucleotide encodes an antigenic nucleoprotein polypeptide having greater than 97% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and mediates viral attachment. In some embodiments, the at least one RNA polynucleotide encodes an antigenic nucleoprotein polypeptide having greater than 98% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and mediates viral attachment. In some embodiments, the at least one RNA polynucleotide encodes an antigenic nucleoprotein polypeptide having greater than 99% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and mediates viral attachment. In some embodiments, the at least one RNA polynucleotide encodes an antigenic nucleoprotein polypeptide having 95-99% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and mediates viral attachment. In some embodiments, the at least one RNA polynucleotide encodes an antigenic matrix polypeptide having greater than 90% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and mediates viral assembly and budding. In some embodiments, the at least one RNA polynucleotide encodes an antigenic matrix polypeptide having greater than 95% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and mediates viral assembly and budding. In some embodiments, the at least one RNA polynucleotide encodes an antigenic matrix polypeptide having greater than 96% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and mediates viral assembly and budding. In some embodiments, the at least one RNA polynucleotide encodes an antigenic matrix polypeptide having greater than 97% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and mediates viral assembly and budding. In some embodiments, the at least one RNA polynucleotide encodes an antigenic matrix polypeptide having greater than 98% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and mediates viral assembly and budding. In some embodiments, the at least one RNA polynucleotide encodes an antigenic matrix polypeptide having greater than 99% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and mediates viral assembly and budding. In some embodiments, the at least one RNA polynucleotide encodes an antigenic matrix polypeptide having 95-99% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and mediates viral assembly and budding.

In some embodiments, the at least one RNA polynucleotide encodes an antigenic polypeptide having an amino acid sequence of any one of Tables 3, 5 and 7, and wherein the RNA polynucleotide is codon optimized mRNA.

In some embodiments, the at least one RNA polynucleotide has a nucleic acid sequence encoded by a nucleic acid sequence of any one of Tables 3, 4 and 6. In other embodiments, the at least one RNA polynucleotide has a nucleic acid sequence encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the polynucleotides set forth in any one of Tables 3, 4 and 6. In other embodiments, the at least one RNA polynucleotide has a nucleic acid sequence encoded by a nucleic acid sequence having less than 80% sequence identity to the polynucleotides set forth in any one of Tables 3, 4 and 6 and encoding an Ebola immunogenic antigen.

In some embodiments, the at least one RNA polynucleotide encodes an antigenic polypeptide having an amino acid sequence of any one of Tables 3, 5 and 7, and wherein the RNA polynucleotide has less than 80% identity to wild-type mRNA sequence. In other embodiments, the at least one RNA polynucleotide encodes an antigenic polypeptide having an amino acid sequence of any one of Tables 3, 5 and 7, and wherein the RNA polynucleotide has less than 75%, 85% or 95% identity to wild-type mRNA sequence. In other embodiments, the at least one RNA polynucleotide encodes an antigenic polypeptide having an amino acid sequence of any one of Tables 3, 5 and 7, and wherein the RNA polynucleotide has 50-80%, 60- 80%, 40-80%, 30-80%, 70-80%, 75-80%, or 78-80% identity to wild-type mRNA sequence. In other embodiments, the at least one RNA polynucleotide encodes an antigenic polypeptide having an amino acid sequence of any one of Tables 3, 5 and 7, and wherein the RNA polynucleotide has 40-85%, 50- 85%, 60-85%, 30-85%, 70-85%, 75-85%, or 80-85% identity to wild-type mRNA sequence. In other embodiments, the at least one RNA polynucleotide encodes an antigenic polypeptide having an amino acid sequence of any one of Tables 3, 5 and 7, and wherein the RNA

polynucleotide 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, the at least one RNA polynucleotide encodes an antigenic polypeptide having an amino acid sequence of any one of Tables 3, 5 and 7, 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 at least one RNA polynucleotide encodes an antigenic polypeptide that attaches to cell receptors.

In some embodiments, the at least one RNA polynucleotide encodes an antigenic polypeptide that causes fusion of viral and cellular membranes.

In some embodiments, the at least one RNA polynucleotide encodes an antigenic polypeptide that mediates viral assembly and budding.

In some embodiments, the at least one RNA polynucleotide encodes an antigenic polypeptide that mediates viral attachment.

In some embodiments, the vaccines further comprise an adjuvant.

Some embodiments of the present disclosure provide an Ebola virus vaccine that includes at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding at least one Ebola antigenic polypeptide, at least one 5' terminal cap and at least one chemical modification, formulated within a lipid nanoparticle.

In some embodiments, a 5' terminal cap is 7mG(5')ppp(5')NlmpNp.

In some embodiments, at least one chemical modification is selected from

pseudouridine, Nl-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2- thio-1 -methyl- 1-deaza-pseudouridine, 2-thio-l-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-l-methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine), 5-methoxyuridine and 2'-0-methyl uridine.

In some embodiments, a lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid. In some embodiments, a cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol. In some embodiments, a cationic lipid is selected from 2,2-dilinoleyl-4-dimethylaminoethyl- [l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3- DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

Some embodiments of the present disclosure provide an Ebola virus vaccine that includes at least one RNA polynucleotide having an open reading frame encoding at least one Ebola antigenic polypeptide, wherein at least 80% of the uracil in the open reading frame have a chemical modification, optionally wherein the Ebola vaccine is formulated in a lipid nanoparticle.

In some embodiments, 100% of the uracil in the open reading frame have a chemical modification. In some embodiments, a chemical modification is in the 5-position of the uracil. In some embodiments, a chemical modification is a Nl-methyl pseudouridine.

Some embodiments of the present disclosure provide methods of inducing an antigen specific immune response in a subject, comprising administering to the subject an Ebola virus vaccine in an amount effective to produce an antigen specific immune response.

In some embodiments, an antigen specific immune response comprises a T cell response or a B cell response.

In some embodiments, a method of producing an antigen specific immune response involves a single administration of the Ebola virus vaccine. In some embodiments, a method further includes administering to the subject a booster dose of the Ebola virus vaccine.

In some embodiments, an Ebola virus vaccine is administered to the subject by intradermal or intramuscular injection.

Also provided herein are Ebola virus vaccines for use in a method of inducing an antigen specific immune response in a subject, the method comprising administering the Ebola virus vaccine to the subject in an amount effective to produce an antigen specific immune response. Further provided herein are uses of Ebola virus 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 Ebola virus vaccine to the subject in an amount effective to produce an antigen specific immune response.

A nucleic acid vaccine having one or more RNA polynucleotides having an open reading frame encoding an Ebola antigen and a pharmaceutically acceptable carrier or excipient are provided in aspects of the invention. In some embodiments the Ebola antigen is an Ebola virus (EBOV) glycoprotein (GP). In other embodiments the Ebola antigen is a surface GP. In yet other embodiments the Ebola antigen is a wild type EBOV pro-GP, a wild type EBOV pro-GP- V5, a mature EBOV GP, a mature EBOV GP-V5, a secreted wild type EBOV pro-GP, a secreted wild type EBOV pro-GP- V5, a secreted mature EBOV GP, or a secreted mature EBOV GP-V5.

In yet other embodiments the RNA polynucleotide (e.g. , mRNA) comprises a polynucleotide having the polynucleotide sequence set forth as one of SEQ ID NOs 1-8 or comprises a polynucleotide having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the polynucleotide set forth as one of SEQ ID NOs 1-8. In other embodiments the RNA polynucleotide comprises a polynucleotide having 100% sequence identity to the polynucleotide set forth as one of SEQ ID NOs 1-8. The RNA polynucleotide comprises the polynucleotide set forth as one of SEQ ID NOs 17-24 in other embodiments. In other embodiment the polynucleotide has the polynucleotide sequence set forth as one of SEQ ID NOs 1-8 or comprises a polynucleotide having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the polynucleotide set forth as one of SEQ ID NOs 17-24. In yet other embodiments the RNA polynucleotide comprises a polynucleotide encoding an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth as one of SEQ ID NOs 9- 16. The RNA polynucleotide, in some embodiments, comprises a polynucleotide sequence derived from Zaire ebolavirus.

The Ebola antigen may be a full length antigenic protein or it may be an epitope. In some embodiments, the RNA polynucleotide of the RNA vaccine includes at least one chemical modification. In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, Nl-methylpseudouridine, 2-thiouridine, 4'- thiouridine, 5-methylcytosine, 2-thio-l -methyl- 1-deaza-pseudouridine, 2-thio- l-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-l- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5- methyluridine), 5-methoxyuridine, and 2'-0-methyl uridine. In one embodiment the modifications are 1 methylpseudouridine and m5C and in another embodiment the modification is methylpseudouridine.

In some embodiments, the RNA polynucleotide of the RNA vaccine is formulated in a lipid nanoparticle (LNP) carrier. In further embodiments, the lipid nanoparticle carrier comprises 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 other embodiments, the cationic lipid is selected from the group consisting of for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3- DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In some embodiments the cationic lipid is an ionizable cationic lipid and the non- cationic lipid is a neutral lipid, and the sterol is a cholesterol.

The open reading frame of the RNA polynucleotide may be codon-optimized.

The invention in other aspects is a method of inducing an antigen specific immune response in a subject, by administering the vaccines described herein to the subject in an effective amount to produce an antigen specific immune response. In some embodiments the antigen specific immune response comprises a T cell response. In other embodiments the antigen specific immune response comprises a B cell response.

The method of producing an antigen specific immune response in some embodiments involves a single administration of the vaccine. In other embodiments the method further comprises administering a second or 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.

The vaccine in some embodiments is administered to the subject by intradermal or intramuscular injection.

In other aspects the invention is 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.

In other aspects the invention is a use of the vaccines described herein 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 effective amount to produce an antigen specific immune response. In other aspects the invention is a method of preventing or treating Ebola infection comprising administering to a subject the vaccines described herein.

Some embodiments of the present disclosure provide methods of inducing an antigen specific immune response in a subject, comprising administering to the subject an Ebola virus RNA (e.g., mRNA) vaccine in an amount effective to produce an antigen specific immune response.

In some embodiments, an antigen specific immune response comprises a T cell response or a B cell response.

In some embodiments, a method of producing an antigen specific immune response involves a single administration of a Ebola virus RNA (e.g., mRNA) vaccine. In some embodiments, a method further includes administering to the subject a booster dose of a Ebola virus RNA (e.g., mRNA) vaccine.

In some embodiments, a Ebola virus RNA vaccine is administered to the subject by intradermal or intramuscular injection.

Also provided herein are Ebola virus vaccines for use in a method of inducing an antigen specific immune response in a subject, the method comprising administering the Ebola virus vaccine to the subject in an amount effective to produce an antigen specific immune response.

Further provided herein are uses of Ebola virus 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 Ebola virus vaccine to the subject in an amount effective to produce an antigen specific immune response.

The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.

In some embodiments, the Ebola virus vaccine is formulated in an effective amount to produce an antigen specific immune response in a subject.

In some embodiments, an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is increased by at least 1 log relative to a control. In some

embodiments, the anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is increased by 1-3 log relative to a control. In some embodiments, the anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is increased at least 2 times relative to a control. In some embodiments, the anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is increased at least 5 times relative to a control. In some embodiments, the anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is increased at least 10 times relative to a control. In some embodiments, the anti- Ebola virus 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-Ebola virus antigenic polypeptide antibody titer produced in a subject who has not been administered Ebola virus vaccine. In some embodiments, the control is an anti-Ebola virus antigenic polypeptide antibody titer produced in a subject who has been administered a live attenuated or inactivated Ebola virus vaccine. In some embodiments, the control is an anti-Ebola virus antigenic polypeptide antibody titer produced in a subject who has been administered a recombinant or purified Ebola virus 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 Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus 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 Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus 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 Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus 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 Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus 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 Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus 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 Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus 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 Ebola virus vaccine in an effective amount to produce an antigen specific immune response in a subject. In some embodiments, an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is increased by at least 1 log relative to a control. In some embodiments, an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is increased by 1-3 log relative to a control. In some embodiments, the anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is increased at least 2 times relative to a control. In some

embodiments, the anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is increased at least 5 times relative to a control. In some embodiments, the anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is increased at least 10 times relative to a control. In some embodiments, the anti-Ebola virus 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-Ebola virus antigenic polypeptide antibody titer produced in a subject who has not been administered Ebola virus vaccine. In some embodiments, the control is an anti-Ebola virus antigenic polypeptide antibody titer produced in a subject who has been administered a live attenuated or inactivated Ebola virus vaccine. In some embodiments, the control is an anti-Ebola virus antigenic polypeptide antibody titer produced in a subject who has been administered a recombinant or purified Ebola virus 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 Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant Ebola virus protein vaccine or a live attenuated Ebola virus 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 Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus 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 Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus 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 Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus 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 Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus 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 Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus 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 provides an Ebola virus vaccine, which includes a signal peptide linked to an Ebola virus envelope protein. In some embodiments, the Ebola virus vaccine further comprising an Ebola virus glycoprotein.

Further provided herein, is a nucleic acid encoding the Ebola virus vaccine.

Another aspcet of the present disclosure provides an Ebola virus vaccine, which includes at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a signal peptide linked to an Ebola virus antigenic peptide. In some embodiments, the Ebola virus antigenic peptide is an Ebola virus envelope protein.

In some embodiments, the efficacy of the vaccine against Ebola virus is greater than

60%. Vaccine efficacy refers to the percent reduction of disease incidence in a vaccinated group compared to an unvaccinated group under optimal conditions. In some embodiments, the efficacy of the vaccine against Ebola virus is greater than 65%. In some embodiments, the efficacy of the vaccine against Ebola virus is greater than 71%. In some embodiments, the efficacy of the vaccine against Ebola virus is greater than 75%. In some embodiments, the efficacy of the vaccine against Ebola virus is greater than 80%. In some embodiments, the efficacy of the vaccine against Ebola virus is greater than 85%. In some embodiments, the the efficacy of the vaccine against Ebola virus is greater than 90%. In some embodiments, the vaccine immunizes a subject against Ebola virus for more than 2 years. In some embodiments, the vaccine immunizes a subject against Ebola virus for more than 3 years. In some embodiments, the vaccine immunizes a subject against Ebola virus for more than 4 years. In some embodiments, the vaccine immunizes a subject against Ebola virus for more than 5 years.

In some embodiments, the subject sustains immunity against Ebola virus for more than 2 years. A subject may be assessed for immunity to Ebola virus using standard test, such as a blood test for antibodies (e.g., neturalizing antibodies) to Ebola virus antigens. In some embodiments, the subject sustains immunity against Ebola virus for more than 3 years. In some embodiments, the subject sustains immunity against Ebola virus for more than 4 years. In some embodiments, the subject sustains immunity against Ebola virus for more than 5 years.

In some embodiments, the subject is older than 45 years. In some embodiments, the subject is older than 60 years. In some embodiments, the subject is younger than 9 years. In some embodiments, the subject is younger than 5 years. In some embodiments, the subject is younger than 1 year.

In some embodiments, the subject is immunosuppressed.

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 THE DRAWINGS

The foregoing and other objects, features and advantages will be apparent from the following description of particular embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the invention.

FIG. 1 shows a schematic depiction of the structure of Ebola glycoprotein (GP) and antigen constructs.

FIG. 2 shows the study design for the immunogenicity evaluation. FIG. 3 is a graph depicting the initial anti-Ebola GP response at Day 10 after a single primary challenge. The positive control indicates the OD of the standard curve from 10 U/ml to 1 U/ml of mouse anti-Ebola GP mAb.

FIG. 4 shows the anti-Ebola GP antibody titer of selected antigens on Day 21 and Day 23 (n=3 animals per group).

FIG. 5 shows the anti-Ebola GP response at Day 21 post-vaccination.

FIG. 6 shows the in vitro neutralization activity of serum samples in the Delta Vp30 Ebola virus system.

FIG. 7 shows a schematic of an Ebola vaccine study in a Guinea Pig model.

FIG. 8 is a set of graphs depicting data in terms of survival, weight, temperature and score from the vaccination study shown in FIG. 7. Vaccination conferred 100% protection against 10E3 PFUs of gp-adapted Ebola (Zaire species, Mayinga strain).

DETAILED DESCRIPTION

Embodiments of the present disclosure provide RNA (e.g., mRNA) vaccines that include polynucleotide encoding an Ebola virus antigen. Ebola virus RNA vaccines, as provided herein may be used to induce a balanced immune response, comprising both cellular and humoral immunity, without many of the risks associated with DNA vaccination.

It was discovered quite surprisingly, according to the invention that vaccination with the mRNA vaccine of the invention conferred 100% protection against 10E3 PFUs of gp- adapted Ebola (Zaire species, Mayinga strain) in a guinea pig model of Ebola virus infection. In contrast to the vaccines of the invention, untreated animals succumbed to the infection completely by day 10 post infection.

In some embodiments, the amino acid sequence of the Ebola antigen or fragment thereof comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identify with any of the amino acid sequences provided in Tables 3, 5 and 7. In other embodiments, the nucleic acid sequence of the mRNA encoding the Ebola antigen or fragment thereof comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identify with any of the nucleic acid sequences provided in Tables 3, 4 and 6. In other embodiments, the nucleic acid sequence of the mRNA encoding the Ebola antigen or fragment thereof is encoded by a nucleic acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identify with any of the nucleic acid sequences provided in Tables 3, 4 and 6. Thus, the RNA (e.g. , mRNA) vaccines of the present invention comprise one or more polynucleotides, e.g., polynucleotide constructs, which encode one or more wild type or engineered antigens. Exemplary polynucleotides, e.g., polynucleotide constructs, include antigen-encoding mRNA polynucleotides. In an exemplary aspect, polynucleotides of the invention, e.g., antigen-encoding RNA polynucleotides, may include at least one chemical modification. In exemplary aspects, polynucleotides of the invention, e.g., antigen-encoding RNA polynucleotides, may be fully modified (e.g., chemically modified) with respect to one or more nucleobases.

In some embodiments, the RNA vaccine of the invention is a polynucleotide encoding an Ebola virus antigen. There are five Ebola viruses within the genus Ebolavirus. Four of the five known ebolaviruses cause a severe and often fatal hemorrhagic fever in humans and other mammals, known as Ebola virus disease (EVD). The Ebola glycoprotein (GP) is the only virally expressed protein on the virion surface, where it is essential for the attachment to host cells and catalyzes membrane fusion. As a result, the Ebola GP is a critical component of vaccines, as well as a target of neutralizing antibodies and inhibitors of attachment and fusion. Pre-GP is cleaved by furin at a multi-basic motif into two subunits, GP1 and GP2, which remain associated through a disulfide linkage between Cys53 of GP1 and Cys609 of GP2. The heterodimer (GP1 and GP2) then assembles into a 450-kDa trimer (3 GP1 and 3 GP2) at the surface of nascent virions, where it exerts its functions.

In some embodiments the Ebola antigen in the nucleic acid vaccine is an EBOV glycoprotein (GP). In other embodiments the Ebola antigen is a surface GP. Exemplary Ebola GP and antigen constructs tested herein are shown in FIG. 1 and Table 1.

The Ebola antigen may be a wild type EBOV pro-GP or a mature EBOV GP.

"Mature" EBOV GP has been engineered to include a human signal peptide. Alternatively the Ebola antigen may be a secreted wild type EBOV pro-GP or mature EBOV GP.

"Secreted" EBOV GP has been engineered to remove the transmembrane domain, i.e., residues 651-676. The constructs may also include V5.

The following are nucleic acid sequences (SEQ ID NOs 1-8) for the open reading frames of the RNA polynucleotides or (SEQ ID Nos 17-24) for the RNA polynucleotides and amino acid (SEQ ID NOs 9- 16) sequences for each of the exemplary constructs. Table 3

SEQ Nucleic Acid Sequence

ID NO.

ATGGAGACTCCAGCCCAGCTGCTGTTCTTACTCTTACTCTGGCTGCCTGATACCACGGGAATACCGTTAG

GCGTTATTCATAATTCGACACTCCAGGTGTCAGACGTGGATAAGCTGGTCTGCCGCGATAAGCTGAGTA

GTACGAACCAGCTGAGATCCGTCGGTCTAAACCTTGAGGGAAATGGTGTGGCCACTGACGTCCCCAGC

GCGACCAAGCGGTGGGGGTTCAGGTCTGGTGTCCCACCTAAAGTAGTGAACTACGAAGCAGGTGAGT

GGGCAGAAAACTGCTACAACTTAGAAATCAAAAAGCCAGATGGATCTGAATGCCTGCCCGCCGCGCCA

GATGGGATCCGCGGCTTCCCAAGGTGCCGCTACGTACACAAGGTGAGCGGAACCGGGCCTTGTGCCGG

GGATTTCGCTTTTCATAAAGAGGGGGCGTTCTTTTTGTATGATAGATTGGCGAGCACCGTTATCTACCGC

GGTACCACCTTCGCCGAAGGCGTGGTGGCCTTTCTTATCCTGCCTCAGGCCAAAAAGGACTTCTTCTCCA

GTCACCCGTTGCGCGAACCCGTCAATGCTACCGAGGACCCATCTTCAGGATACTATTCCACCACTATACG

CTATCAGGCCACAGGCTTCGGGACCAATGAAACTGAGTACCTCTTTGAGGTCGACAACCTTACATATGT

CCAACTCGAATCACGCTTCACTCCTCAATTTCTGCTTCAGTTGAACGAGACAATATATACATCAGGCAAG

CGATCTAATACTACCGGCAAGCTGATATGGAAGGTGAATCCAGAGATCGATACCACAATCGGCGAGTG

GGCCTTTTGGGAGACTAAGAAAAACTTGACTCGCAAAATCCGGAGTGAGGAGCTGAGTTTCACTGTGG

TGTCTAATGGTGCGAAGAATATAAGCGGCCAATCCCCTGCTAGGACCTCTTCGGATCCCGGTACGAACA

CAACAACTGAGGACCACAAGATTATGGCCAGCGAAAATTCTAGCGCAATGGTACAGGTGCATTCACAG

GGCCGGGAGGCGGCAGTCTCTCACCTAACGACACTGGCGACCATATCGACCTCTCCACAGTCACTGACA

ACTAAGCCTGGACCCGATAATTCCACACACAACACCCCCGTCTACAAGTTAGATATATCTGAGGCAACTC

AAGTAGAACAACATCACCGGCGCACAGACAATGACTCCACCGCCTCTGACACCCCTAGCGCCACTACGG

CAGCAGGGCCACCTAAGGCAGAGAACACCAACACATCGAAATCAACCGACTTTCTGGACCCGGCAACT

ACCACATCTCCCCAGAATCATTCAGAAACAGCCGGAAACAATAATACACATCACCAAGATACAGGCGAG

GAGAGTGCCAGCTCCGGAAAGTTGGGTCTCATAACCAACACAATTGCTGGCGTTGCCGGCCTTATCACA

GGTGGCAGACGGACAAGGAGAGAAGCTATTGTGAACGCCCAGCCTAAGTGCAACCCAAATCTCCACTA

CTGGACGACACAGGATGAAGGGGCAGCGATCGGGCTGGCCTGGATACCTTACTTTGGACCCGCAGCAG

AGGGCATCTATATCGAGGGCTTGATGCACAACCAAGACGGGCTGATCTGCGGCCTGCGCCAGCTGGCC

AACGAAACTACACAGGCCCTGCAGCTGTTTCTGCGGGCTACAACTGAGCTCCGGACTTTTAGTATTCTG

AATCGAAAGGCCATAGATTTCCTTCTGCAGAGATGGGGTGGCACATGTCACATCCTTGGCCCCGATTGC

TGCATCGAACCCCACGATTGGACAAAAAACATTACCGATAAGATAGACCAGATCATACACGATTTCGTT

GACAAGACATTGCCCGACCAAGGAGATAATGACAATTGGTGGACAGGATGGAGGCAGTGGATACCAG

CTGGCATAGGCGTGACCGGGGTCATCATCGCAGTGATTGCCTTGTTTTGCATTTGCAAGTTCGTGTTC

(SEQ I D NO. 1) ATGGAGACCCCCGCTCAGCTCCTGTTTCTGCTCCTGCTATGGTTACCAGATACAACTGGTATTCCGCTCG

GTGTTATACACAATTCCACACTCCAGGTCTCAGATGTAGATAAATTAGTTTGTCGGGATAAACTGTCTAG

TACCAATCAGCTTAGGTCTGTCGGTCTTAATCTCGAAGGAAACGGCGTTGCCACCGATGTGCCTAGCGC

TACGAAGAGATGGGGCTTTCGAAGCGGAGTGCCCCCAAAGGTGGTCAACTATGAAGCTGGAGAATGG

GCCGAGAACTGCTATAACCTCGAAATAAAAAAGCCTGACGGCTCTGAGTGTCTGCCAGCCGCCCCAGAT

GGAATTAGAGGGTTTCCACGATGCAGGTACGTACATAAAGTCAGCGGCACCGGTCCATGCGCGGGCGA

CTTTGCATTTCACAAAGAGGGAGCCTTCTTCCTATACGACAGGTTAGCCAGCACAGTGATATACAGGGG

GACTACGTTCGCTGAGGGCGTCGTTGCA 1 1 1 1 I GA I 1 1 ACCACAAGCAAAAAAAGAC 1 1 1 1 1 CAGTTCT

CATCCTCTGCGAGAACCCGTAAATGCTACTGAGGACCCCTCCTCAGGCTACTATTCTACCACAATTCGTT

ATC AG G CG AC AG G CTTTG GTACT AATG A A ACCG AGT ATC 1 1 1 1 CGAAGTAGACAATCTGACGTACGTGC

AGCTCGAATCACGTTTCACCCCACAATTCCTCCTGCAACTCAACGAGACAATCTATACATCCGGTAAACG

CAGTAACACTACTGGGAAACTCATCTGGAAGGTGAATCCTGAGATAGACACTACTATCGGCGAGTGGG

CA I 1 1 1 GGGAAACCAAAAAAAATCTGACTCGTAAGATTAGATCAGAAGAGTTATCCTTTACTGTCGTCA

GTAATGGAGCCAAAAACATCAGTGGGCAGTCCCCGGCTCGGACGAGCTCCGACCCCGGCACCAATACA

ACTACTGAAGATCATAAGATCATGGCATCCGAAAATAGTTCCGCCATGGTACAGGTGCACAGCCAGGG

CAGAGAAGCCGCTGTCTCACACCTTACTACGCTGGCTACTATAAGCACCTCGCCTCAATCCCTGACCACA

AAGCCTGGTCCCGACAACTCCACCCATAACACCCCTGTTTACAAGCTGGACATTAGCGAAGCAACTCAG

GTCGAACAGCATCACAGGCGTACAGATAACGATAGCACCGCAAGTGATACCCCATCTGCCACGACCGC

CGCCGGACCCCCTAAGGCAGAGAACACCAACACATCGAAGTCAACAGA I 1 1 1 CTTGACCCAGCCACTAC

CACATCACCCCAGAATCACTCTGAAACTGCTGGCAACAATAATACTCATCACCAGGATACTGGCGAAGA

GAGCGCCAGCTCTGGCAAACTGGGATTGATTACAAACACGATTGCAGGAGTTGCCGGTCTCATCACCG

GAGGTAGAAGAACCCGCCGGGAGGCTATTGTGAACGCCCAGCCAAAGTGTAACCCTAACCTTCATTACT

GGACCACTCAGGATGAAGGAGCTGCAATTGGCCTCGCCTGGATTCCCTACTTCGGACCAGCCGCCGAG

GGTATTTACATCGAGGGCCTCATGCATAACCAGGATGGCCTGATCTGCGGCCTAAGGCAGTTGGCCAA

CGAAACGACGCAAGCGCTCCAGTTATTTCTTCGAGCAACTACAGAACTACGCAC I 1 1 1 I CAATCCTGAAC

CGAAAAGCCATCGACTTTCTCCTGCAGAGATGGGGCGGCACTTGTCACATTCTGGGGCCTGATTGTTGT

ATCGAGCCACATGACTGGACCAAAAATATCACGGACAAGATCGATCAGATCATTCATGACTTTGTGGAT

AAAACACTGCCAGACCAAGGGGATAACGATAACTGGTGGACTGGCTGGCGGCAGTGGATCCCCGCAG

GTATTG G G GTC ACTG G AGTA ATTATCG CCGTG ATTG CTCTG 1 1 1 I GCA I 1 I GCAAG I I CG I 1 1 I CGGTAA

ACCCATTCCAAATCCCCTACTAGGCCTGGACAGCACA(SEQ I D NO. 2)

ATGGAAACACCAGCCCAGCTGCTGTTTCTCCTGCTGTTGTGGTTGCCAGACACTACAGGAATCCCCCTTG

GTGTGATTCATAACTCCACGCTTCAAGTGTCGGATGTAGACAAACTTGTGTGTCGGGATAAGCTTTCGT

CTACAAACCAATTAAGATCGGTGGGACTGAACCTGGAAGGAAATGGAGTGGCCACTGACGTCCCCTCC

GCGACCAAACGGTGGGGATTTCGGTCAGGCGTGCCCCCCAAAGTCGTCAATTACGAGGCCGGAGAGTG

GGCAGAAAACTGTTATAACCTCGAAATCAAGAAGCCCGATGGATCGGAATGCTTGCCGGCAGCCCCTG

ACGGGATCCGCGGGTTCCCGAGGTGCAGATACGTGCACAAAGTCTCCGGCACCGGACCTTGCGCCGGC

GACTTCGCGTTTCACAAAGAAGGCGCA 1 1 1 1 1 1 1 GTACGATCGACTTGCAAGCACTGTTATATATAGAG

GGACGACATTCGCTGAAGGCGTTGTGGCATTTCTGATACTGCCCCAAGCTAAAAAGGA I 1 1 1 1 1 I AGCT

CACATCCACTACGGGAACCGGTCAATGCTACTGAAGATCCATCAAGCGGCTATTATTCAACCACCATAA

G GTATC AG G CG AC AG G 1 1 1 1 GGCACTAATGAAACCGAGTACCTCTTTGAAGTAGACAATCTCACTTACG

TGCAACTTGAGTCCCGATTTACCCCTCAGTTTCTGCTGCAATTAAACGAGACTATTTACACTTCCGGCAA

AAGGAGTAACACGACAGGCAAACTGATCTGGAAGGTCAACCCTGAGATCGATACCACGATTGGCGAGT

GGGCATTCTGGGAGACTAAAAAAAACCTGACCAGGAAGATACGGTCCGAAGAGCTGTCTTTCACAGTC

GTTAGCAACGGTGCCAAGAACATTTCCGGCCAGTCCCCTGCCCGGACCTCGAGTGATCCCGGAACCAAT

ACGACAACCGAGGATCATAAGATCATGGCCAGCGAAAATAGCAGCGCAATGGTACAAGTTCATTCCCA

AGGGCGAGAAGCCGCGGTGTCCCACCTGACTACTCTGGCCACTATTTCCACAAGCCCTCAGTCGTTAAC

AACC AA ACCTG G G CCTG AC A ATTC A ACCC ATAAC AC ACCTGT AT AT AAG CTG G ATATTTC AG AG G C A AC

CCAAGTGGAACAGCACCATAGGAGAACGGACAATGACTCTACAGCCTCCGACACCCCCTCTGCAACGA

CCGCTGCCGGACCACCCAAGGCTGAAAACACCAACACATCCAAGAGTACCGA I 1 1 1 1 1 GGACCCGGCGA

CCACTACGAGTCCTCAGAATCACAGTGAAACGGCAGGGAATAACAACACCCACCATCAGGATACGGGC

GAAGAGTCTGCCAGTTCTGGCAAGTTGGGGCTCATCACGAACACCATTGCCGGGGTGGCTGGTCTTATC ACTGGAGGTCGCAGGACGAGACGTGAGGCTATAGTGAACGCACAGCCAAAGTGTAATCCCAACCTTCA TTACTGGACCACACAGGACGAGGGAGCTGCAATTGGGTTGGCCTGGATCCCTTACTTTGGACCCGCAG CCGAAGGCATCTACATCGAAGGCCTAATGCACAATCAGGACGGCCTTATCTGCGGATTGCGACAGCTTG CCAATGAAACAACCCAGGCTCTTCAACTGTTTCTAAGAGCCACAACTGAGTTGAGGAC I 1 1 1 AG CATCH GAATAGGAAAGCCATAGACTTCTTGTTACAGAGGTGGGGGGGCACATGTCACATCCTCGGACCCGACT GTTGTATAGAGCCTCATGACTGGACCAAGAACATCACAGATAAGATTGACCAGATTATCCATGA I I M G TCGATAAAACACTTCCCGACCAAGGGGATAATGATAACTGGTGGACCGGCTGGCGCCAG(SEQ ID NO. 3)

A 1 GGAAACGCCAGCACAA 1 1 AC I 1 1 1 I C I GC I 1 1 I GC I G I GGC 1 CCC 1 GACACAAC 1 GGACA 1 CACCACC

ATCATCATGGCAAACCTATACCTAACCCTCTGCTGGGTCTGGACTCTACCGGTGATTGGAAATGGGATG

GAGGCCTGGTCCCTCGGGGCTCTGATGAGATGTTGCGAGAGCTGCAGGAGACTAACGCAGCACTCCAG

GACGTCCGCGAACTTCTCCGACAGCAGGTTAGACAGATCACCTTCCTTAGATGTTTACTTATGGGGGGG

AGACTGCTCTGCAGATTGGAAGAGCTGGAGAGGCGTCTGGAAGAGCTGGAGCGGCGCCTTGAGGAAC

TGGAACGGGCCATTAATACAGTTGATGAGTTGGCCGCCTTGAGGAGACGTCTGGAGGAACTAGCACGC

GGCTCTATACCTCTAGGGGTTATCCATAATTCCACGCTCCAGGTTAGTGACGTTGACAAGCTGGTGTGC

CGCGATAAATTATCTTCCACGAACCAGTTGAGATCCGTTGGACTAAATCTTGAGGGAAACGGCGTTGCC

ACAGACGTGCCATCTGCCACCAAAAGGTGGGGTTTCCGCTCCGGTGTGCCCCCCAAAGTGGTGAATTAC

GAGGCAGGGGAATGGGCGGAAAACTGTTACAATCTGGAAATTAAGAAGCCTGACGGCAGTGAGTGTC

TGCCGGCCGCTCCTGACGGAATAAGAGGGTTCCCAAGGTGTAGATACGTGCACAAGGTCTCTGGAACC

GGACCTTGCGCCGGGGACTTCGCCTTCCACAAAGAAGGTGCA I 1 1 1 1 CCTATATGACCGCTTAGCCTCG

ACAGTGATTTACAGGGGCACCACTTTCGCAGAGGGGGTAGTCGCTTTCTTAA I 1 1 1 GCCTCAGGCGAAA

AAGGAC I 1 1 1 1 1 1 C 1 AGTCACCCCCTCAGGGAGCCTGTAAACGCAACCGAGGACCCAAGTAGCGGTTAC

TACTCCACAACGATCAGGTACCAGGCCACCGGATTTGGCACAAACGAAACGGAATACCTATTTGAAGTC

GACAACCTGACCTACGTCCAGCTGGAGTCCCGATTCACGCCCCAGTTCCTACTGCAGCTGAATGAGACA

ATTTACACTTCTGGTAAAAGAAGTAACACCACTGGAAAACTCATCTGGAAGGTCAATCCAGAAATTGAT

ACAACAATCGGTGAGTGGGC 1 1 1 1 1 GGGAGACGAAAAAAAACTTGACCAGAAAAATCAGGTCCGAGGA

GCTGAGTTTCACGGTAGTTAGCAACGGAGCGAAAAATATCTCAGGACAGAGTCCAGCCCGAACCTCTTC

GGATCCAGGCACTAATACGACAACAGAAGACCATAAGATTATGGCTAGTGAGAACTCATCTGCTATGGT

CCAGGTGCACTCGCAAGGACGCGAAGCAGCTGTGTCACATCTGACCACCCTTGCAACTATCAGCACAAG

TCCTCAGAGCTTAACCACCAAGCCTGGTCCCGACAATTCTACACACAATACTCCTGTTTACAAATTAGAC

ATCAGTGAAGCCACCCAGGTTGAACAACACCACCGGCGGACGGACAATGACTCAACGGCATCTGACAC

CCCATCCGCCACAACGGCTGCCGGGCCACCAAAAGCTGAGAATACCAACACATCAAAGTCAACCGACTT

CTTAGACCCCGCTACAACCACCTCCCCCCAAAATCATTCCGAAACTGCCGGTAACAACAACACCCACCAC

CAGGATACCGGGGAGGAAAGCGCCAGCAGCGGCAAGCTAGGCCTGATCACAAACACAATCGCCGGGG

TCGCAGGCCTAATCACCGGAGGCAGACGTACACGTCGGGAGGCTATTGTTAATGCCCAGCCTAAATGC

AATCCGAATCTTCATTATTGGACCACCCAGGATGAAGGAGCTGCCATTGGACTGGCTTGGATCCCTTAC

TTTGGCCCCGCCGCTGAGGGAATTTATATAGAGGGGCTGATGCACAACCAAGATGGACTAATTTGTGG

ACTCCGCCAGCTGGCCAATGAAACCACCCAAGCTTTACAACTCTTTCTGAGAGCAACAACAGAACTTCG

GACA I 1 1 1 C 1 ATATTGAATAGAAAGGCTATTGATTTCCTTCTCCAGAGATGGGGTGGTACCTGCCACATA

CTCGGTCCAGATTGTTGCATAGAACCTCATGACTGGACCAAAAATATTACTGACAAAATCGATCAGATC

ATTCACGATTTCGTGGATAAGACATTACCCGACCAAGGCGACAACGACAACTGGTGGACTGGATGGAG

GCAG (SEQ I D NO. 4)

A 1 GGGAG 1 CACCGGCA M C I GCAGC 1 CCCCAGGGA 1 CG 1 1 1 CAAAAGAAC 1 1 CC 1 1 1 1 1 1 C 1 1 G 1 AA TCATCCTGTTCCAAAGGACG 1 1 1 1 CCATCCCCCTGGGAGTTATACACAACAGTACATTGCAGGTCTCTGA CGTAG AC A AACTCGTCTG CCG G G AC AA ACTTTCT AG C ACTA ATC AG CTG CG GTCG GT AG GTCTG A ACTT GGAAGGTAATGGTGTCGCCACAGATGTGCCATCCGCCACCAAACGCTGGGGGTTCCGGTCAGGCGTGC CCCCAAAAGTCGTCAACTATGAAGCAGGCGAATGGGCGGAAAACTGTTATAATCTCGAGATCAAAAAA CCTGACGGGTCCGAGTGTTTGCCCGCCGCTCCTGACGGCATCAGAGGCTTCCCTAGGTGCAGATATGTC CACAAGGTATCAGGTACCGGCCCATGTGCTGGGGACTTCGCTTTCCACAAGGAAGGAGCTTTCTTTCTC TACGACAGGCTGGCCTCTACCGTCATATATAGGGGAACTACCTTCGCCGAAGGCGTTGTTGCC 1 1 1 1 1 G ATCCTGCCTCAGGCCAAAAAAGACTTC 1 1 1 I C I 1 CCCATCCCCTGCGGGAGCCAGTTAACGCTACCGAGG ACCCGAGCTCTGGCTATTACAGTACCACCATCCGGTATCAAGCTACCGGATTCGGCACGAACGAAACCG

AGTACCTATTCGAGGTGGACAACCTGACATATGTTCAACTGGAGTCCCGGTTCACCCCCCAGTTTCTGCT

GCAGTTGAACGAGACCATCTACACAAGCGGTAAAAGGTCTAATACCACTGGAAAACTGATCTGGAAAG

TAAACCCGGAGATAGATACCACCATTGGAGAATGGGCATTCTGGGAGACCAAGAAAAATTTGACCAGG

AAAATACGCTCTGAAGAACTGTCCTTTACGGTGGTGTCTAATGGGGCGAAGAACATCTCCGGACAGAG

CCCTGCGCGCACATCCTCAGATCCAGGCACAAATACCACAACAGAGGATCACAAGATTATGGCATCTGA

GAATAGCAGCGCTATGGTGCAGGTTCACAGCCAAGGCAGAGAGGCCGCGGTTTCCCATCTCACAACAT

TG G CT ACT ATC AG C ACCTCTCCTC A AAG CCTG AC AAC A AAG CCTG GTCC AG ATA ATTCTACCC AT AATAC

TCCAGTGTATAAACTCGACATTTCTGAGGCTACACAAGTGGAGCAGCACCATAGAAGAACAGACAACG

ACTCAACTGCCTCCGACACCCCTTCAGCGACAACCGCCGCCGGCCCTCCTAAGGCGGAAAACACTAATA

CTTCGAAATCAACCGA 1 1 1 1 C 1 GGATCCAGCGACCACCACCTCGCCTCAGAATCATTCGGAAACGGCTG

GAAATAATAACACCCACCACCAGGACACCGGCGAAGAAAGTGCCAGCAGTGGCAAGCTCGGGCTGATC

ACCAATACTATCGCGGGGGTGGCCGGGCTAATTACAGGCGGCAGACGGACCAGGAGAGAGGCCATTG

TAAACGCCCAGCCTAAATGCAACCCCAACCTGCATTACTGGACCACCCAAGACGAGGGCGCCGCAATTG

GTTTGGCTTGGATCCCCTA 1 1 1 1 GGACCCGCCGCCGAAGGGATTTATATCGAAGGACTGATGCACAACC

AGGATGGCCTTATTTGTGGGCTTCGGCAGCTGGCCAATGAGACGACACAAGCGCTGCAGTTATTCTTGA

GAGCCACTACCGAGCTTAGGACCTTCAGCATTCTGAATCGCAAGGCCATCGACTTCCTGTTGCAGCGAT

GGGGGGGTACCTGCCATATTCTGGGCCCTGATTGCTGTATAGAACCCCACGACTGGACGAAGAATATA

ACCGATAAAATAGATCAGATCATCCACGACTTTGTCGACAAAACACTCCCAGATCAGGGCGATAACGAT

AATTGGTGGACCGGCTGGAGGCAGTGGATCCCAGCAGGAATCGGGGTGACAGGCGTTATTATCGCGG

TCATAGCACTG 1 1 1 1 G C ATCTG C AAGTTCGTCTTT (SEQ ID NO. 5)

A 1 GGGGG 1 GACAGGGA 1 A 1 1 GCAGC 1 GO. 1 CGGGA 1 CGC 1 1 CAAGCGCACC 1 1 1 1 1 1 1 C 1 1 GGG 1 C

ATC ATCCTCTTTC AG CG C AC ATTT AG C ATTCCG CTCG G CGTA ATTC AT AATTC AACTCTAC AAGTCTCTG A

CGTGGACAAATTGGTCTGCCGGGACAAGTTGTCTTCGACAAACCAATTGAGGTCTGTAGGGCTGAACCT

CGAGGGCAATGGTGTGGCGACGGATGTACCCTCGGCCACCAAGCGTTGGGGCTTTAGGTCTGGCGTCC

CCCCTAAAGTGGTGAACTATGAAGCTGGGGAATGGGCCGAAAATTGCTACAATCTGGAAATTAAGAAA

CCTGACGGAAGCGAGTGTCTCCCAGCTGCCCCAGACGGTATCCGCGGGTTCCCCAGATGCAGGTACGT

TCATAAAGTGAGTGGAACCGGCCCATGCGCCGGAGATTTCGCATTCCACAAGGAAGGTGCTTTCTTTCT

TTACGACAGGCTAGCCTCCACAGTGATCTATCGCGGCACCACCTTCGCAGAGGGCGTGGTTGCC 1 1 1 1 1

GATCCTGCCTCAGGCCAAAAAAGACTTCTTCAGTTCTCACCCTCTGAGGGAACCAGTGAACGCCACCGA

GGACCCTTCCAGCGGATACTACAGCACCACGATCCGCTATCAAGCGACCGGTTTCGGGACCAACGAGA

CTGAGTACCTCTTTGAAGTGGATAACCTGACATATGTGCAGCTGGAATCTCGGTTTACCCCGCAGTTCCT

TCTCCAGCTGAATGAAACTATATACACCTCAGGCAAGAGATCCAATACCACAGGTAAATTGATTTGGAA

AGTTAATCCCGAAATCGACACAACGATTGGGGAGTGGGCC I 1 1 1 GGGAGACCAAGAAAAATCTGACAC

GCAAGATCAGAAGTGAGGAGTTGAGTTTCACCGTGGTCAGCAATGGCGCGAAAAATATTTCCGGTCAA

TCTCCAGCCAGGACTAGCTCTGACCCTGGAACGAACACTACCACAGAAGACCATAAGATCATGGCAAGT

GAGAATTCTTCCGCTATGGTTCAGGTGCACTCCCAGGGCCGGGAAGCCGCCGTCTCACACCTGACAACC

CTCGCCACAATCTCCACTTCCCCACAGTCCCTGACAACCAAACCCGGGCCTGACAACTCAACACATAATA

CGCCAGTTTACAAGCTCGATATCTCTGAGGCTACCCAGGTGGAGCAACACCACAGGCGGACTGACAAC

GACAGCACCGCTAGTGATACCCCCTCAGCCACCACTGCCGCAGGTCCCCCCAAGGCCGAAAATACAAAT

ACCAGTAAAAGTACGGACTTCCTGGACCCCGCAACTACAACCTCCCCCCAAAACCACAGCGAAACGGCA

GGGAATAATAATACACACCATCAGGATACCGGAGAGGAGTCCGCTTCTTCCGGCAAGCTGGGACTGAT

AACCAACACAATTGCCGGAGTGGCAGGTCTGATTACGGGAGGGCGCAGGACTAGACGGGAAGCAATC

GTGAACGCACAGCCAAAGTGCAATCCTAACCTGCACTACTGGACTACCCAAGATGAAGGGGCAGCCAT

TGGGCTTGCATGGATCCCCTACTTTGGGCCTGCAGCGGAGGGGATTTATATCGAAGGCCTGATGCATAA

TCAGGATGGATTGATTTGCGGGCTCAGGCAGTTGGCGAATGAAACAACACAAGCCCTACAATTATTTCT

GAGGGCCACTACTGAGTTGCGAACATTTAGTATCCTCAATAGAAAGGCCATAGACTTCCTGCTCCAGCG

CTGGGGCGGGACTTGCCACATTCTAGGCCCGGACTGTTGTATCGAACCCCATGACTGGACCAAGAACAT

AACTGATAAGATAGACCAGATCATCCATGATTTCGTTGATAAAACACTGCCCGATCAGGGTGACAACGA

CAATTGGTGGACTGGCTGGAGGCAGTGGATCCCAGCTGGCATTGGGGTTACAGGTGTTATAATTGCCG

TG ATTG CTCTCTTCTG C ATCTG C AA ATTTGTCTTCG GT AA ACC AATACCC A ATCC ACTG CTG G G CCTG GAT AGCACG (SEQ I D NO. 6)

ATGGGAGTGACTGGCATTCTTCAGCTTCCTCGCGACAGGTTTAAGAGAACCTCGTTCTTCCTGTGGGTC

ATTATAC 1 1 1 1 CC AG CG C AC ATTC AG C ATCCC ACTTG G AGTG ATTC ATA ATTCT AC ACTTC AG GTTAG CG A

CGTGGACAAGCTGGTCTGCCGGGACAAACTGTCGTCAACAAATCAGCTTAGGTCCGTAGGCCTGAACCT

TGAAGGTAACGGCGTTGCAACAGATGTGCCTAGCGCAACGAAGCGGTGGGGATTTAGGTCTGGTGTTC

CACCAAAAGTGGTCAATTACGAGGCCGGAGAGTGGGCAGAGAACTGCTATAACCTGGAAATAAAAAA

GCCTGACGGTAGCGAATGCCTGCCAGCCGCGCCAGATGGCATTCGGGGGTTTCCCCGCTGTCGCTACG

TCCACAAAGTAAGTGGTACTGGGCCTTGTGCCGGTGACTTCGCCTTTCACAAAGAGGGCGCG 1 1 C I 1 1 1

TGTATGACCGTCTCGCCTCCACAGTAATTTACCGAGGCACTACATTTGCAGAAGGGGTGGTGGCATTCC

TTATATTACCCCAGGCTAAAAAGGACTTCTTCTCTAGCCACCCACTCCGGGAACCAGTGAATGCAACCGA

G G ATCC A AG CTCG G G GT ACT ATTCT ACC ACT ATC AG GTATC AG G C A AC AG G GTTTG GTAC AA ATG AG AC

TGAATATCTCTTCGAGGTTGACAACCTGACCTACGTGCAGCTGGAAAGTCGTTTCACTCCGCAGTTCCTC

TTGCAGCTCAATGAGACCATATATACCAGTGGGAAACGGTCTAACACAACCGGAAAACTGATTTGGAA

AGTTAACCCAGAAATTGATACTACGATTGGTGAGTGGGCG 1 1 1 1 GGGAGACAAAGAAGAATTTAACTA

GGAAGATCAGGTCCGAAGAACTGTCATTCACTGTGGTGTCTAACGGCGCGAAAAATATCTCTGGGCAG

TCTCCAGCCCGCACATCCTCCGATCCTGGAACGAATACAACCACCGAAGATCATAAGATCATGGCCAGC

GAAAACTCCTCCGCCATGGTCCAAGTCCATTCCCAAGGGAGGGAAGCTGCAGTCTCCCACCTCACTACT

TTGGCAACCATTAGCACTTCGCCACAAAGCCTGACCACCAAGCCCGGCCCGGATAATTCAACCCACAAC

ACCCCAGTTTATAAACTGGATATCAGTGAGGCGACTCAGGTGGAGCAGCATCACCGAAGAACTGATAA

TGATTCTACTGCCAGCGACACCCCAAGCGCGACCACCGCTGCCGGACCTCCCAAGGCCGAGAATACTAA

CACTTCGAAATCAACAGACTTTCTGGATCCCGCCACAACCACGTCCCCACAGAATCACTCTGAAACCGCC

GGTAACAATAATACACATCACCAGGATACCGGGGAGGAATCCGCTTCTAGCGGGAAGCTGGGCCTTAT

AACAAATACGATAGCAGGGGTGGCCGGACTCATCACAGGGGGTCGGAGGACACGGCGGGAGGCTATA

GTCAATGCTCAGCCTAAATGCAACCCGAATCTGCACTATTGGACCACACAGGACGAGGGAGCCGCCATC

GGGCTGGCATGGATTCCATATTTCGGCCCAGCCGCTGAGGGCATCTACATCGAGGGTCTTATGCACAAC

CAGGACGGGCTAATCTGCGGACTTAGGCAGCTGGCCAACGAGACAACACAGGCACTCCAGCTCTTCCTT

CG CG CTAC A AC AG AG CTACG G ACC 1 1 1 1 CC ATTCTC A AC AG G AAG G CC AT AG ACTTCCTCTTG C AG CG G

TGGGGAGGCACCTGTCATATTCTCGGGCCCGATTGTTGTATCGAACCTCATGATTGGACCAAAAATATT

ACAGATAAGATTGATCAAATCATCCACGATTTCGTAGATAAGACACTCCCCGACCAGGGAGACAATGAC

AACTGGTGGACGGGGTGGCGACAG (SEQ ID NO. 7)

ATGGGCGTGACTGGGATCCTCCAACTGCCTCGCGATAGATTCAAGCGAACTAGTTTCTTTCTATGGGTC

ATCATACTCTTCCAGCGAACCTTCTCAATTCCACTGGGAGTCATTCACAACTCCACCTTGCAAGTGTCAG

ATGTGGACAAGTTGGTATGCCGAGACAAACTCTCTTCTACTAATCAGCTCAGGTCTGTGGGGCTGAACC

TTGAAGGTAACGGCGTGGCCACAGATGTGCCTTCGGCTACTAAGCGATGGGGTTTCCGGTCCGGTGTG

CCCCCGAAGGTGGTAAATTATGAGGCTGGCGAGTGGGCCGAAAACTGCTATAACTTGGAGATTAAAAA

GCCTGACGGTAGCGAATGTTTACCAGCCGCTCCCGATGGAATTAGGGGCTTTCCCAGATGTCGGTATGT

CC AC A A AGTGTCTG G A ACTG G G CCTTGTG CTG G G G A 1 1 1 1 GCCTTCCATAAAGAAGGGGCCTTCTTTCT

ATACGATCGGCTGGCCAGTACCGTCATTTATCGAGGCACTACTTTCGCCGAAGGCGTTGTGGCG 1 1 1 1 1

GATTCTCCCACAGGCCAAGAAAGACTTC 1 1 1 1 CCTCACATCCTCTACGGGAGCCAGTAAATGCAACCGAA

GATCCTTCTAGTGGCTACTACTCCACCACTATTCGGTACCAGGCTACCGGATTCGGCACCAATGAGACA

GAATATTTATTTGAGGTGGACAACCTTACTTATGTGCAGTTGGAAAGCAGGTTCACCCCCCAGTTCCTCC

TGCAGTTGAACGAGACAATTTATACATCAGGTAAGCGGTCTAACACTACCGGTAAGTTAATCTGGAAAG

TGAACCCCGAGATCGACACGACCATCGGGGAATGGG I 1 1 1 1 GGGAGACCAAGAAAAACCTGACGCGC

AAGATTAGATCCGAGGAGCTCAGTTTCACTGTGGTGAGTAATGGCGCTAAAAATATCAGTGGGCAGAG

CCCCGCCCGGACAAGCAGCGACCCTGGGACAAACACTACAACCGAAGACCACAAAATAATGGCCTCAG

AAAACAGTAGCGCGATGGTGCAAGTGCATAGCCAAGGACGCGAAGCCGCTGTATCCCATCTTACCACC

CTGGCTACGATTAGCACCTCGCCCCAGTCTCTGACTACGAAGCCAGGGCCTGACAATTCTACACACAAC

ACACCCGTGTACAAACTTGACATCTCAGAAGCAACGCAGGTGGAACAGCATCACCGCCGGACGGATAA

CGACAGCACCGCAAGCGATACCCCCAGTGCTACTACTGCAGCCGGTCCCCCAAAAGCAGAAAATACGA

ATACCAGCAAGAGTACAGACTTCTTGGACCCGGCCACCACAACAAGCCCACAAAATCACAGCGAGACT GCAGGCAACAATAACACACATCACCAGGATACGGGGGAGGAATCTGCTTCGTCCGGCAAACTCGGACT

GATCACAAATACGATCGCCGGGGTGGCCGGCTTGATCACCGGCGGCAGGCGGACTCGGAGAGAAGCC

ATAGTGAACGCTCAGCCTAAGTGCAATCCGAATCTACACTATTGGACTACTCAGGACGAAGGCGCAGCT

ATTGGCTTGGCTTGGATTCCATACTTTGGGCCAGCAGCAGAGGGAATATACATCGAGGGACTGATGCA

TAACCAGGATGGCCTGATCTGTGGCCTCCGACAGTTGGCTAATGAAACTACCCAGGCCCTCCAATTGTT

CCTGCGCGCTACAACTGAACTTCGGACCTTTTCTA I 1 1 1 GAACAGGAAGGCCATAGATTTCCTGCTTCAG

CGTTGGGGCGGAACATGCCATATCCTGGGCCCCGACTGTTGTATCGAGCCTCATGATTGGACGAAGAA

TATAACTGATAAAATAGATCAAATAATCCACGACTTCGTCGACAAGACCCTGCCTGACCAGGGGGACAA

TGATAACTGGTGGACTGGATGGCGACAGGGTAAGCCAATCCCGAACCCTCTTCTCGGTCTGGATAGCA

CG (SEQ ID NO. 8)

25 ATGGACAGTAGGCCCCAGAAGATCTGGATGGCACCATCTCTCACTGAGAGTGATATGGACTATCATAA

GATCCTGACGGCAGGGCTGAGCGTGCAGCAAGGCATCGTCAGGCAGCGGGTGATCCCAGTATATCAG

GTCAACAACCTTGAGGAGATTTGTCAACTGATAATTCAGGCCTTTGAAGCCGGCGTCGACTTTCAAGAA

AGTGCAGATAGCTTTCTCCTGATGCTGTGTTTGCACCATGCGTACCAGGGCGACTATAAGCTGTTCCTG

GAGTCTGGGGCTGTTAAGTACCTGGAGGGGCACGGTTTCAGGTTTGAGGTGAAGAAGCGGGACGGCG

TCAAGAGGTTAGAGGAACTGCTGCCAGCAGTGAGCTCTGGTAAGAATATTAAAAGGACCCTCGCTGCA

ATGCCGGAGGAGGAAACCACAGAGGCAAACGCTGGCCAGTTCCTGAGCTTCGCATCTCTTTTTCTACCG

AAGCTAGTGGTAGGAGAAAAAGCTTGCCTCGAAAAGGTACAGAGACAGATCCAGGTTCACGCTGAACA

GGGGCTTATCCAGTACCCAACCGCTTGGCAGTCGGTGGGTCACATGATGGTGA I 1 1 1 CAGACTGATGCG

G AC AA ATTTTCTG ATTA AGTTCCTG CTTATTC ACC AG G G G ATG C AT ATG GTG G C AG G CC ACG ATG C A A A

TGACGCCGTAATCTCTAATAGCGTTGCTCAAGCGCGCTTCTCTGGGCTGCTGATCGTGAAAACAGTGCT

GGATCATATCTTACAAAAGACTGAGAGGGGTGTGAGGCTGCACCCCCTGGCTAGAACCGCAAAGGTCA

AGAATGAAGTGAATTCGTTTAAGGCTGCCCTCTCTTCACTCGCCAAACACGGGGAGTACGCGCCTTTCG

CAAGACTGCTGAACCTTTCAGGGGTGAACAATCTTGAGCATGGATTGTTTCCACAGTTGTCCGCCATTG

CACTGGGCGTGGCAACAGCACACGGGTCCACTCTGGCCGGTGTGAACGTTGGGGAGCAGTATCAACAG

CTCAGGGAAGCTGCCACTGAGGCCGAGAAACAGCTCCAGCAGTACGCCGAGTCCCGCGAATTGGATCA

CCTCGGCCTCGACGACCAGGAAAAGAAGATTCTGATGAA I 1 1 1 CATCAAAAAAAAAACGAGATTTCATT

TCAACAGACCAACGCAATGGTGACTCTTAGAAAAGAGCGCCTAGCCAAATTAACGGAGGCCATAACCG

CAGCCAGCCTACCAAAGACATCTGGGCACTACGACGACGACGACGACATCCCGTTTCCCGGTCCAATTA

ATGACGACGACAATCCCGGCCACCAAGACGACGACCCAACTGACTCGCAGGACACCACGATTCCCGAT

GTCGTGGTGGACCCCGATGACGGGAGCTACGGAGAATATCAAAGCTACTCTGAGAACGGCATGAATGC

ACCCGACGATCTGGTGCTGTTTGACTTGGACGAGGACGATGAAGATACTAAGCCCGTGCCCAATAGGT

CAACTAAAGGCGGCCAGCAGAAAAATTCCCAAAAGGGGCAGCACATCGAGGGCAGACAAACACAATC

CAGACCTATACAGAATGTGCCTGGACCCCACCGCACTATTCACCACGCAAGTGCTCCTCTGACCGACAAT

GATCGCAGGAATGAGCCATCTGGTTCCACATCTCCGAGAATGCTTACACCTATCAACGAAGAGGCCGAC

CCGCTCGACGATGCGGACGACGAGACCTCTAGTTTGCCTCCTCTTGAGTCAGATGACGAAGAACAGGA

CCGGGATGGAACATCGAATAGAACTCCTACAGTGGCTCCGCCCGCTCCAGTATACCGCGATCACAGTGA

GAAGAAGGAGCTGCCACAGGATGAACAACAGGACCAGGATCACACCCAAGAAGCACGTAACCAGGAC

TCGGATAACACTCAGTCAGAACACTCTTTTGAGGAGATGTATCGGCACATTCTTCGATCGCAAGGTCCAT

TTGACGCAGTCCTCTACTACCACATGATGAAGGATGAACCCGTCG 1 1 1 1 1 1 CCACAAGCGATGGGAAAG

AGTATACATACCCGGATTCCTTGGAGGAGGAGTACCCGCCCTGGCTGACAGAGAAAGAGGCGATGAAC

GAGGAGAACCGCTTTGTAACTCTGGACGGTCAGCAA I 1 1 1 ATTGGCCCGTTATG AATCACAAGAACAAG

TTC ATG GCTATCTTG C AAC ATC ACC AG (SEQ I D NO. 25)

Table 5

QATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAF

WETKKNLTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGR

EAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAG

PPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGR

RTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANET

TQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLP

DQGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCICKFVF (SEQ ID NO.9)

METPAQLLFLLLLWLPDTTGIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPS

ATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCA

GDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRY

QATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAF

WETKKNLTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGR

EAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAG

PPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGR

RTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANET

TQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLP

DQGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCICKFVFGKPIPNPLLGLDST (SEQ ID NO.10)

METPAQLLFLLLLWLPDTTGIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPS

ATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCA

GDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRY

QATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAF

WETKKNLTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGR

EAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAG

PPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGR

RTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANET

TQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLP

DQGDNDNWWTGWRQ (SEQ ID NO.11)

METPAQLLFLLLLWLPDTTGHHHHHHGKPIPNPLLGLDSTGDWKWDGGLVPRGSDEMLRELQETNA

ALQDVRELLRQQVRQITFLRCLLMGGRLLCRLEELERRLEELERRLEELERAINTVDELAALRRRLEELARG

SIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYE

AGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTV

IYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTY

VQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVS

NGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATISTSPQSLTTK

PGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPATTT

SPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYW

TTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRK

AIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQGDNDNWWTGWRQ (SEQ

ID NO.12)

MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLE

GNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYV

HKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATED

PSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPE

IDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAM

VQVHSQGREAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASD

TPSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAG

VAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLIC

GLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQII

HDFVDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCICKFVF (SEQ ID NO.13)

MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLE GNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYV HKVSGTGPCAGDFAFHKEGAFFLYD LASTVIY GTTFAEGVVAFLILPQAKKDFFSSHPL EPVNATED

PSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPE

IDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAM

VQVHSQGREAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASD

TPSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAG

VAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLIC

GLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQII

HDFVDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCICKFVFGKPIPNPLLGLDST (SEQ

ID NO.14)

15 MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLE

GNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYV

HKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATED

PSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPE

IDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAM

VQVHSQGREAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASD

TPSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAG

VAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLIC

GLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQII

HDFVDKTLPDQGDNDNWWTGWRQ (SEQ ID NO.15)

16 MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLE

GNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYV

HKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATED

PSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPE

IDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAM

VQVHSQGREAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASD

TPSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAG

VAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLIC

GLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQII

HDFVDKTLPDQGDNDNWWTGWRQGKPIPNPLLGLDST (SEQ ID NO.16)

26 MDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEICQLIIQAFEAGVDFQES

ADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVKRLEELLPAVSSGKNIKRTLAAMPE

EETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQVHAEQGLIQYPTAWQSVGHMMVIFRLMRT

NFLIKFLLIHQGMHMVAGHDANDAVISNSVAQARFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKN

EVNSFKAALSSLAKHGEYAPFARLLNLSGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLR

EAATEAEKQLQQYAESRELDHLGLDDQEKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAAS

LPKTSGHYDDDDDIPFPGPINDDDNPGHQDDDPTDSQDTTIPDVVVDPDDGSYGEYQSYSENGMNA

PDDLVLFDLDEDDEDTKPVPNRSTKGGQQKNSQKGQHIEGRQTQSRPIQNVPGPHRTIHHASAPLTD

NDRRNEPSGSTSPRMLTPINEEADPLDDADDETSSLPPLESDDEEQDRDGTSNRTPTVAPPAPVYRDHS

EKKELPQDEQQDQDHTQEARNQDSDNTQSEHSFEEMYRHILRSQGPFDAVLYYHMMKDEPVVFSTS

DGKEYTYPDSLEEEYPPWLTEKEAMNEENRFVTLDGQQFYWPVMNHKNKFMAILQHHQ (SEQ ID

NO.26)

Table 6

SEQ Nucleic Acid Sequence

ID

NO.

17 1 LAAGL 1111 GGALLL 1 L 1 ALAGAAGL 1 AA 1 ALGAL 1 LAL 1 A 1 AGGGAAA 1 AAGAGAGAAAAGAAGAG

TAAGAAGAAATATAAGAGCCACCATGGAGACTCCAGCCCAGCTGCTGTTCTTACTCTTACTCTGGCTGCC

TGATACCACGGGAATACCGTTAGGCGTTATTCATAATTCGACACTCCAGGTGTCAGACGTGGATAAGCTG

GTCTGCCGCGATAAGCTGAGTAGTACGAACCAGCTGAGATCCGTCGGTCTAAACCTTGAGGGAAATGGT

GTGGCCACTGACGTCCCCAGCGCGACCAAGCGGTGGGGGTTCAGGTCTGGTGTCCCACCTAAAGTAGTG AACTACGAAGCAGGTGAGTGGGCAGAAAACTGCTACAACTTAGAAATCAAAAAGCCAGATGGATCTGA

ATGCCTGCCCGCCGCGCCAGATGGGATCCGCGGCTTCCCAAGGTGCCGCTACGTACACAAGGTGAGCGG

AACCGGGCCTTGTGCCGGGGATTTCGC 1 1 1 1 LA I AAAGAGGGGGL 1 1 L I 1 1 1 1 GTATGATAGATTGGCG

AGCACCGTTATCTACCGCGGTACCACCTTCGCCGAAGGCGTGGTGGCCTTTCTTATCCTGCCTCAGGCCA

AAAAGGACTTCTTCTCCAGTCACCCGTTGCGCGAACCCGTCAATGCTACCGAGGACCCATCTTCAGGATA

CTATTCCACCACTATACGCTATCAGGCCACAGGCTTCGGGACCAATGAAACTGAGTACCTCTTTGAGGTC

GACAACCTTACATATGTCCAACTCGAATCACGCTTCACTCCTCAATTTCTGCTTCAGTTGAACGAGACAAT

ATATACATCAGGCAAGCGATCTAATACTACCGGCAAGCTGATATGGAAGGTGAATCCAGAGATCGATAC

CACAATCGGCGAGTGGGCL I 1 1 1 GGGAGACTAAGAAAAACTTGACTCGCAAAATCCGGAGTGAGGAGC

TGAGTTTCACTGTGGTGTCTAATGGTGCGAAGAATATAAGCGGCCAATCCCCTGCTAGGACCTCTTCGGA

TCCCGGTACGAACACAACAACTGAGGACCACAAGATTATGGCCAGCGAAAATTCTAGCGCAATGGTACA

GGTGCATTCACAGGGCCGGGAGGCGGCAGTCTCTCACCTAACGACACTGGCGACCATATCGACCTCTCC

ACAGTCACTGACAACTAAGCCTGGACCCGATAATTCCACACACAACACCCCCGTCTACAAGTTAGATATA

TCTGAGGCAACTCAAGTAGAACAACATCACCGGCGCACAGACAATGACTCCACCGCCTCTGACACCCCTA

GCGCCACTACGGCAGCAGGGCCACCTAAGGCAGAGAACACCAACACATCGAAATCAACCGACTTTCTGG

ACCCGGCAACTACCACATCTCCCCAGAATCATTCAGAAACAGCCGGAAACAATAATACACATCACCAAGA

TACAGGCGAGGAGAGTGCCAGCTCCGGAAAGTTGGGTCTCATAACCAACACAATTGCTGGCGTTGCCGG

CCTTATCACAGGTGGCAGACGGACAAGGAGAGAAGCTATTGTGAACGCCCAGCCTAAGTGCAACCCAAA

TCTCCACTACTGGACGACACAGGATGAAGGGGCAGCGATCGGGCTGGCCTGGATACCTTACTTTGGACC

CGCAGCAGAGGGCATCTATATCGAGGGCTTGATGCACAACCAAGACGGGCTGATCTGCGGCCTGCGCCA

GCTGGCCAACGAAACTACACAGGCCCTGCAGCTGTTTCTGCGGGCTACAACTGAGCTCCGGAC 1 1 1 I AGT

ATTCTGAATCGAAAGGCCATAGATTTCCTTCTGCAGAGATGGGGTGGCACATGTCACATCCTTGGCCCCG

ATTGCTGCATCGAACCCCACGATTGGACAAAAAACATTACCGATAAGATAGACCAGATCATACACGATTT

CGTTGACAAGACATTGCCCGACCAAGGAGATAATGACAATTGGTGGACAGGATGGAGGCAGTGGATAC

C AG CTG G CAT AG GCGTGACCGGG GTC ATC ATCG C AGTG ATTG CCTTG 1 1 1 1 G C ATTTG C A AGTTCGTGTT

CTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCT

TCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ I D NO. 17)

1 LAAGL 1 1 1 1 GGALLL 1 L 1 ALAGAAGL 1 AA 1 ALGAL 1 LAL 1 A 1 AGGGAAA 1 AAG AGAGAAAAGAAGAG

TAAGAAGAAATATAAGAGCCACCATGGAGACCCCCGCTCAGCTCCTGTTTCTGCTCCTGCTATGGTTACC

AGATACAACTGGTATTCCGCTCGGTGTTATACACAATTCCACACTCCAGGTCTCAGATGTAGATAAATTA

GTTTGTCGGGATAAACTGTCTAGTACCAATCAGCTTAGGTCTGTCGGTCTTAATCTCGAAGGAAACGGCG

TTGCCACCGATGTGCCTAGCGCTACGAAGAGATGGGGCTTTCGAAGCGGAGTGCCCCCAAAGGTGGTCA

ACTATGAAGCTGGAGAATGGGCCGAGAACTGCTATAACCTCGAAATAAAAAAGCCTGACGGCTCTGAGT

GTCTGCCAGCCGCCCCAGATGGAATTAGAGGGTTTCCACGATGCAGGTACGTACATAAAGTCAGCGGCA

CCGGTCCATGCGCGGGCGACTTTGCATTTCACAAAGAGGGAGCCTTCTTCCTATACGACAGGTTAGCCAG

CACAGTGATATACAGGGGGACTACGTTCGCTGAGGGCGTCGTTGCA I 1 1 1 1 GATTCTACCACAAGCAAAA

AAAGACTTTTTCAGTTCTCATCCTCTGCGAGAACCCGTAAATGCTACTGAGGACCCCTCCTCAGGCTACTA

TTCTACCACAATTCGTTATCAGGCGACAGGCTTTGGTACTAATGAAACCGAGTATL I 1 1 1 CGAAGTAGAC

AATCTGACGTACGTGCAGCTCGAATCACGTTTCACCCCACAATTCCTCCTGCAACTCAACGAGACAATCTA

TACATCCGGTAAACGCAGTAACACTACTGGGAAACTCATCTGGAAGGTGAATCCTGAGATAGACACTACT

ATCGGCGAGTGGGCA 1 1 1 1 GGGAAACCAAAAAAAATCTGACTCGTAAGATTAGATCAGAAGAGTTATCC

TTTACTGTCGTCAGTAATGGAGCCAAAAACATCAGTGGGCAGTCCCCGGCTCGGACGAGCTCCGACCCC

GGCACCAATACAACTACTGAAGATCATAAGATCATGGCATCCGAAAATAGTTCCGCCATGGTACAGGTG

C AC AG CC AG G G C AG AG AAGCCG CTGTCTC AC ACCTTACTACG CTG G CTACT AT AAG C ACCTCG CCTC AAT

CCCTGACCACAAAGCCTGGTCCCGACAACTCCACCCATAACACCCCTGTTTACAAGCTGGACATTAGCGA

AGCAACTCAGGTCGAACAGCATCACAGGCGTACAGATAACGATAGCACCGCAAGTGATACCCCATCTGC

CACGACCGCCGCCGGACCCCCTAAGGCAGAGAACACCAACACATCGAAGTCAACAGATTTTCTTGACCCA

GCCACTACCACATCACCCCAGAATCACTCTGAAACTGCTGGCAACAATAATACTCATCACCAGGATACTG

GCGAAGAGAGCGCCAGCTCTGGCAAACTGGGATTGATTACAAACACGATTGCAGGAGTTGCCGGTCTCA

TCACCGGAGGTAGAAGAACCCGCCGGGAGGCTATTGTGAACGCCCAGCCAAAGTGTAACCCTAACCTTC

ATTACTGGACCACTCAGGATGAAGGAGCTGCAATTGGCCTCGCCTGGATTCCCTACTTCGGACCAGCCGC CGAGGGTATTTACATCGAGGGCCTCATGCATAACCAGGATGGCCTGATCTGCGGCCTAAGGCAGTTGGC CAACGAAACGACGCAAGCGCTCCAGTTATTTCTTCGAGCAACTACAGAACTACGCAC I 1 1 1 1 CAATCCTGA ACCGAAAAGCCATCGACTTTCTCCTGCAGAGATGGGGCGGCACTTGTCACATTCTGGGGCCTGATTGTTG TATCGAGCCACATGACTGGACCAAAAATATCACGGACAAGATCGATCAGATCATTCATGACTTTGTGGAT AAAACACTGCCAGACCAAGGGGATAACGATAACTGGTGGACTGGCTGGCGGCAGTGGATCCCCGCAGG TATTG G G GTC ACTG G AGT AATTATCG CCGTG ATTG CTCTGTTTTG C ATTTG C A AGTTCGTTTTCG GT AA AC CCATTCCAAATCCCCTACTAGGCCTGGACAGCACATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCT TGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTC TGAGTGGGCGGC (SEQ ID NO. 18)

1 LAAGL 1 1 1 1 GGALLL 1 L 1 ALAGAAGL 1 AA 1 ALGAL 1 LAL 1 A 1 AGGGAAA 1 AAGAGAGAAAAGAAGAG

TAAGAAGAAATATAAGAGCCACCATGGAAACACCAGCCCAGCTGCTGTTTCTCCTGCTGTTGTGGTTGCC

AGACACTACAGGAATCCCCCTTGGTGTGATTCATAACTCCACGCTTCAAGTGTCGGATGTAGACAAACTT

GTGTGTCGGGATAAGCTTTCGTCTACAAACCAATTAAGATCGGTGGGACTGAACCTGGAAGGAAATGGA

GTGGCCACTGACGTCCCCTCCGCGACCAAACGGTGGGGATTTCGGTCAGGCGTGCCCCCCAAAGTCGTC

AATTACGAGGCCGGAGAGTGGGCAGAAAACTGTTATAACCTCGAAATCAAGAAGCCCGATGGATCGGA

ATGCTTGCCGGCAGCCCCTGACGGGATCCGCGGGTTCCCGAGGTGCAGATACGTGCACAAAGTCTCCGG

CACCGGACCTTGCGCCGGCGACTTCGCGTTTCACAAAGAAGGCGCA I 1 1 1 1 1 L 1 GTACGATCGACTTGCA

AGCACTGTTATATATAGAGGGACGACATTCGCTGAAGGCGTTGTGGCATTTCTGATACTGCCCCAAGCTA

AAAAGGA I 1 1 1 1 1 1 AGCTCACATCCACTACGGGAACCGGTCAATGCTACTGAAGATCCATCAAGCGGCTA

TTATTC A ACC ACC ATA AG GTATC AG G CG AC AG G 1 1 1 1 GGCACTAATGAAACCGAGTACCTCTTTGAAGTA

GACAATCTCACTTACGTGCAACTTGAGTCCCGATTTACCCCTCAGTTTCTGCTGCAATTAAACGAGACTAT

TTACACTTCCGGCAAAAGGAGTAACACGACAGGCAAACTGATCTGGAAGGTCAACCCTGAGATCGATAC

CACGATTGGCGAGTGGGCATTCTGGGAGACTAAAAAAAACCTGACCAGGAAGATACGGTCCGAAGAGC

TGTCTTTCACAGTCGTTAGCAACGGTGCCAAGAACATTTCCGGCCAGTCCCCTGCCCGGACCTCGAGTGA

TCCCGGAACCAATACGACAACCGAGGATCATAAGATCATGGCCAGCGAAAATAGCAGCGCAATGGTACA

AGTTCATTCCCAAGGGCGAGAAGCCGCGGTGTCCCACCTGACTACTCTGGCCACTATTTCCACAAGCCCT

CAGTCGTTAACAACCAAACCTGGGCCTGACAATTCAACCCATAACACACCTGTATATAAGCTGGATATTTC

AGAGGCAACCCAAGTGGAACAGCACCATAGGAGAACGGACAATGACTCTACAGCCTCCGACACCCCCTC

TGCAACGACCGCTGCCGGACCACCCAAGGCTGAAAACACCAACACATCCAAGAGTACCGA I 1 1 1 1 I GGAC

CCGGCGACCACTACGAGTCCTCAGAATCACAGTGAAACGGCAGGGAATAACAACACCCACCATCAGGAT

ACGGGCGAAGAGTCTGCCAGTTCTGGCAAGTTGGGGCTCATCACGAACACCATTGCCGGGGTGGCTGGT

CTTATCACTGGAGGTCGCAGGACGAGACGTGAGGCTATAGTGAACGCACAGCCAAAGTGTAATCCCAAC

CTTCATTACTGGACCACACAGGACGAGGGAGCTGCAATTGGGTTGGCCTGGATCCCTTACTTTGGACCCG

CAGCCGAAGGCATCTACATCGAAGGCCTAATGCACAATCAGGACGGCCTTATCTGCGGATTGCGACAGC

TTGCCAATGAAACAACCCAGGCTCTTCAACTGTTTCTAAGAGCCACAACTGAGTTGAGGAL I 1 1 1 AG CAT

CTTGAATAGGAAAGCCATAGACTTCTTGTTACAGAGGTGGGGGGGCACATGTCACATCCTCGGACCCGA

CTGTTGTATAGAGCCTCATGACTGGACCAAGAACATCACAGATAAGATTGACCAGATTATCCATGA I 1 1 1

GTCGATAAAACACTTCCCGACCAAGGGGATAATGATAACTGGTGGACCGGCTGGCGCCAGTGATAATAG

GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCC

GTACCCCCGTG GTCTTTG AAT AA AGTCTG AGTG G G CG G C (SEQ ID NO. 19)

1 LAA L 1 1 1 1 GALLL 1 L 1 ALAGAAGL 1 AA 1 ALGAL 1 LAL 1 A 1 AGGGAAA 1 AAGAGAGAAAAGAAGAG

TAAGAAGAAATATAAGAGCCACCATGGAAACGCCAGCACAATTAC 1 1 1 1 I L I GL I 1 1 1 GCTGTGGCTCCCT

GACACAACTGGACATCACCACCATCATCATGGCAAACCTATACCTAACCCTCTGCTGGGTCTGGACTCTAC

CGGTGATTGGAAATGGGATGGAGGCCTGGTCCCTCGGGGCTCTGATGAGATGTTGCGAGAGCTGCAGG

AGACTAACGCAGCACTCCAGGACGTCCGCGAACTTCTCCGACAGCAGGTTAGACAGATCACCTTCCTTAG

ATGTTTACTTATGGGGGGGAGACTGCTCTGCAGATTGGAAGAGCTGGAGAGGCGTCTGGAAGAGCTGG

AGCGGCGCCTTGAGGAACTGGAACGGGCCATTAATACAGTTGATGAGTTGGCCGCCTTGAGGAGACGT

CTGGAGGAACTAGCACGCGGCTCTATACCTCTAGGGGTTATCCATAATTCCACGCTCCAGGTTAGTGACG

TTGACAAGCTGGTGTGCCGCGATAAATTATCTTCCACGAACCAGTTGAGATCCGTTGGACTAAATCTTGA

GGGAAACGGCGTTGCCACAGACGTGCCATCTGCCACCAAAAGGTGGGGTTTCCGCTCCGGTGTGCCCCC

CAAAGTGGTGAATTACGAGGCAGGGGAATGGGCGGAAAACTGTTACAATCTGGAAATTAAGAAGCCTG ACGGCAGTGAGTGTCTGCCGGCCGCTCCTGACGGAATAAGAGGGTTCCCAAGGTGTAGATACGTGCACA

AGGTCTCTGGAACCGGACCTTGCGCCGGGGACTTCGCCTTCCACAAAGAAGGTGCA I 1 1 1 1 CCTATATGA

CCG CTT AG CCTCG AC AGTG ATTT AC AG G G G C ACC ACTTTCG C AG AG G G G GT AGTCG CTTTCTTA A 1 I M G

CCTCAGGCGAAAAAGGAC I 1 1 1 1 1 1 C 1 AGTCACCCCCTCAGGGAGCCTGTAAACGCAACCGAGGACCCAA

GTAGCGGTTACTACTCCACAACGATCAGGTACCAGGCCACCGGATTTGGCACAAACGAAACGGAATACC

TATTTGAAGTCGACAACCTGACCTACGTCCAGCTGGAGTCCCGATTCACGCCCCAGTTCCTACTGCAGCT

GAATGAGACAATTTACACTTCTGGTAAAAGAAGTAACACCACTGGAAAACTCATCTGGAAGGTCAATCCA

GAAATTGATACAACAATCGGTGAGTGGGC I 1 1 1 1 GGGAGACGAAAAAAAACTTGACCAGAAAAATCAGG

TCCGAGGAGCTGAGTTTCACGGTAGTTAGCAACGGAGCGAAAAATATCTCAGGACAGAGTCCAGCCCGA

ACCTCTTCGGATCCAGGCACTAATACGACAACAGAAGACCATAAGATTATGGCTAGTGAGAACTCATCTG

CTATGGTCCAGGTGCACTCGCAAGGACGCGAAGCAGCTGTGTCACATCTGACCACCCTTGCAACTATCAG

CACAAGTCCTCAGAGCTTAACCACCAAGCCTGGTCCCGACAATTCTACACACAATACTCCTGTTTACAAAT

TAGACATCAGTGAAGCCACCCAGGTTGAACAACACCACCGGCGGACGGACAATGACTCAACGGCATCTG

ACACCCCATCCGCCACAACGGCTGCCGGGCCACCAAAAGCTGAGAATACCAACACATCAAAGTCAACCG

ACTTCTTAGACCCCGCTACAACCACCTCCCCCCAAAATCATTCCGAAACTGCCGGTAACAACAACACCCAC

CACCAGGATACCGGGGAGGAAAGCGCCAGCAGCGGCAAGCTAGGCCTGATCACAAACACAATCGCCGG

GGTCGCAGGCCTAATCACCGGAGGCAGACGTACACGTCGGGAGGCTATTGTTAATGCCCAGCCTAAATG

CAATCCGAATCTTCATTATTGGACCACCCAGGATGAAGGAGCTGCCATTGGACTGGCTTGGATCCCTTAC

TTTGGCCCCGCCGCTGAGGGAATTTATATAGAGGGGCTGATGCACAACCAAGATGGACTAATTTGTGGA

CTCCGCCAGCTGGCCAATGAAACCACCCAAGCTTTACAACTCTTTCTGAGAGCAACAACAGAACTTCGGA

CA I 1 1 1 C 1 ATATTGAATAGAAAGGCTATTGATTTCCTTCTCCAGAGATGGGGTGGTACCTGCCACATACTC

GGTCCAGATTGTTGCATAGAACCTCATGACTGGACCAAAAATATTACTGACAAAATCGATCAGATCATTC

ACGATTTCGTGGATAAGACATTACCCGACCAAGGCGACAACGACAACTGGTGGACTGGATGGAGGCAG

TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTT

CCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO. 20)

1 CAAGC 1 1 1 1 GGACCC 1 CG 1 ACAGAAGC 1 AA 1 ALGAL 1 LAL 1 A 1 AGGGAAA 1 AAGAGAGAAAAGAAGAG

TAAGAAGAAATATAAGAGCCACCATGGGAGTCACCGGCATTCTGCAGCTCCCCAGGGATCGTTTCAAAA

GAACTTCC 1 1 1 1 1 1 L 1 1 G 1 AA 1 LA 1 LL 1 1 1 LLAAAGGAL 1 1 1 1 CCATCCCCCTGGGAGTTATACAC

AACAGTACATTGCAGGTCTCTGACGTAGACAAACTCGTCTGCCGGGACAAACTTTCTAGCACTAATCAGC

TGCGGTCGGTAGGTCTGAACTTGGAAGGTAATGGTGTCGCCACAGATGTGCCATCCGCCACCAAACGCT

GGGGGTTCCGGTCAGGCGTGCCCCCAAAAGTCGTCAACTATGAAGCAGGCGAATGGGCGGAAAACTGT

TATAATCTCGAGATCAAAAAACCTGACGGGTCCGAGTGTTTGCCCGCCGCTCCTGACGGCATCAGAGGCT

TCCCTAGGTGCAGATATGTCCACAAGGTATCAGGTACCGGCCCATGTGCTGGGGACTTCGCTTTCCACAA

GGAAGGAGCTTTCTTTCTCTACGACAGGCTGGCCTCTACCGTCATATATAGGGGAACTACCTTCGCCGAA

GGCGTTGTTGCL 1 1 1 1 1 A 1 LL 1 LL 1 LA LLAAAAAA AL 1 I L I 1 1 I L I 1 CCCATCCCCTGCGGGAGCC

AGTTAACGCTACCGAGGACCCGAGCTCTGGCTATTACAGTACCACCATCCGGTATCAAGCTACCGGATTC

GGCACGAACGAAACCGAGTACCTATTCGAGGTGGACAACCTGACATATGTTCAACTGGAGTCCCGGTTC

ACCCCCCAGTTTCTGCTGCAGTTGAACGAGACCATCTACACAAGCGGTAAAAGGTCTAATACCACTGGAA

AACTGATCTGGAAAGTAAACCCGGAGATAGATACCACCATTGGAGAATGGGCATTCTGGGAGACCAAG

AAAAATTTGACCAGGAAAATACGCTCTGAAGAACTGTCCTTTACGGTGGTGTCTAATGGGGCGAAGAAC

ATCTCCGGACAGAGCCCTGCGCGCACATCCTCAGATCCAGGCACAAATACCACAACAGAGGATCACAAG

ATTATGGCATCTGAGAATAGCAGCGCTATGGTGCAGGTTCACAGCCAAGGCAGAGAGGCCGCGGTTTCC

CATCTCACAACATTGGCTACTATCAGCACCTCTCCTCAAAGCCTGACAACAAAGCCTGGTCCAGATAATTC

TACCCATAATACTCCAGTGTATAAACTCGACATTTCTGAGGCTACACAAGTGGAGCAGCACCATAGAAGA

ACAGACAACGACTCAACTGCCTCCGACACCCCTTCAGCGACAACCGCCGCCGGCCCTCCTAAGGCGGAA

AACACTAATACTTCGAAATCAACCGATTTTCTGGATCCAGCGACCACCACCTCGCCTCAGAATCATTCGGA

AACGGCTGGAAATAATAACACCCACCACCAGGACACCGGCGAAGAAAGTGCCAGCAGTGGCAAGCTCG

GGCTGATCACCAATACTATCGCGGGGGTGGCCGGGCTAATTACAGGCGGCAGACGGACCAGGAGAGAG

GCCATTGTAAACGCCCAGCCTAAATGCAACCCCAACCTGCATTACTGGACCACCCAAGACGAGGGCGCC

GCAATTGGTTTGGCTTGGATCCCCTA 1 1 1 1 GGACCCGCCGCCGAAGGGATTTATATCGAAGGACTGATGC

ACAACCAGGATGGCCTTATTTGTGGGCTTCGGCAGCTGGCCAATGAGACGACACAAGCGCTGCAGTTAT TCTTGAGAGCCACTACCGAGCTTAGGACCTTCAGCATTCTGAATCGCAAGGCCATCGACTTCCTGTTGCA

GCGATGGGGGGGTACCTGCCATATTCTGGGCCCTGATTGCTGTATAGAACCCCACGACTGGACGAAGAA

TATAACCGATAAAATAGATCAGATCATCCACGACTTTGTCGACAAAACACTCCCAGATCAGGGCGATAAC

GATAATTGGTGGACCGGCTGGAGGCAGTGGATCCCAGCAGGAATCGGGGTGACAGGCGTTATTATCGC

GGTCATAGCACTG 1 1 1 1 CA I C I GCAA 1 1 CG I C I 1 1 1 G AT A AT AG GCTGGAGCCTCGGTGGCCATG CTTC

TTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGT

CTGAGTGGGCGGC (SEQ ID NO. 21)

1 CAAGC 1 1 1 1 GACCC 1 C 1 ACAGAAGC 1 AA 1 ACGAC 1 CAC 1 A 1 AGGGAAA 1 AAGAGAGAAAAGAAGAG

TAAGAAGAAATATAAGAGCCACCATGGGGGTGACAGGGATATTGCAGCTGCCTCGGGATCGCTTCAAG

CGCACCTCC I 1 1 1 1 1 C 1 GTGGGTCATCATCCTCTTTCAGCGCACATTTAGCATTCCGCTCGGCGTAATTCAT

AATTCAACTCTACAAGTCTCTGACGTGGACAAATTGGTCTGCCGGGACAAGTTGTCTTCGACAAACCAAT

TGAGGTCTGTAGGGCTGAACCTCGAGGGCAATGGTGTGGCGACGGATGTACCCTCGGCCACCAAGCGTT

GGGGCTTTAGGTCTGGCGTCCCCCCTAAAGTGGTGAACTATGAAGCTGGGGAATGGGCCGAAAATTGCT

ACAATCTGGAAATTAAGAAACCTGACGGAAGCGAGTGTCTCCCAGCTGCCCCAGACGGTATCCGCGGGT

TCCCCAGATGCAGGTACGTTCATAAAGTGAGTGGAACCGGCCCATGCGCCGGAGATTTCGCATTCCACA

AGGAAGGTGCTTTCTTTCTTTACGACAGGCTAGCCTCCACAGTGATCTATCGCGGCACCACCTTCGCAGA

GGGCGTGGTTGCC 1 1 1 1 1 GATCCTGCCTCAGGCCAAAAAAGACTTCTTCAGTTCTCACCCTCTGAGGGAA

CCAGTGAACGCCACCGAGGACCCTTCCAGCGGATACTACAGCACCACGATCCGCTATCAAGCGACCGGT

TTCGGGACCAACGAGACTGAGTACCTCTTTGAAGTGGATAACCTGACATATGTGCAGCTGGAATCTCGGT

TTACCCCGCAGTTCCTTCTCCAGCTGAATGAAACTATATACACCTCAGGCAAGAGATCCAATACCACAGG

TAAATTGATTTGGAAAGTTAATCCCGAAATCGACACAACGATTGGGGAGTGGGCC I 1 1 1 GGGAGACCAA

GAAAAATCTGACACGCAAGATCAGAAGTGAGGAGTTGAGTTTCACCGTGGTCAGCAATGGCGCGAAAA

ATATTTCCGGTCAATCTCCAGCCAGGACTAGCTCTGACCCTGGAACGAACACTACCACAGAAGACCATAA

GATCATGGCAAGTGAGAATTCTTCCGCTATGGTTCAGGTGCACTCCCAGGGCCGGGAAGCCGCCGTCTC

ACACCTGACAACCCTCGCCACAATCTCCACTTCCCCACAGTCCCTGACAACCAAACCCGGGCCTGACAACT

CAACACATAATACGCCAGTTTACAAGCTCGATATCTCTGAGGCTACCCAGGTGGAGCAACACCACAGGC

GGACTGACAACGACAGCACCGCTAGTGATACCCCCTCAGCCACCACTGCCGCAGGTCCCCCCAAGGCCG

AAAATACAAATACCAGTAAAAGTACGGACTTCCTGGACCCCGCAACTACAACCTCCCCCCAAAACCACAG

CGAAACGGCAGGGAATAATAATACACACCATCAGGATACCGGAGAGGAGTCCGCTTCTTCCGGCAAGCT

GGGACTGATAACCAACACAATTGCCGGAGTGGCAGGTCTGATTACGGGAGGGCGCAGGACTAGACGGG

AAGCAATCGTGAACGCACAGCCAAAGTGCAATCCTAACCTGCACTACTGGACTACCCAAGATGAAGGGG

CAGCCATTGGGCTTGCATGGATCCCCTACTTTGGGCCTGCAGCGGAGGGGATTTATATCGAAGGCCTGA

TGCATAATCAGGATGGATTGATTTGCGGGCTCAGGCAGTTGGCGAATGAAACAACACAAGCCCTACAAT

TATTTCTGAGGGCCACTACTGAGTTGCGAACATTTAGTATCCTCAATAGAAAGGCCATAGACTTCCTGCTC

CAGCGCTGGGGCGGGACTTGCCACATTCTAGGCCCGGACTGTTGTATCGAACCCCATGACTGGACCAAG

AACATAACTGATAAGATAGACCAGATCATCCATGATTTCGTTGATAAAACACTGCCCGATCAGGGTGACA

ACGACAATTGGTGGACTGGCTGGAGGCAGTGGATCCCAGCTGGCATTGGGGTTACAGGTGTTATAATTG

CCGTGATTGCTCTCTTCTGCATCTGCAAATTTGTCTTCGGTAAACCAATACCCAATCCACTGCTGGGCCTG

GATAGCACGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCC

TCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO. 22)

1 CAAGC 1 1 1 1 GGACCC 1 CG 1 ACAGAAGC 1 AA 1 ACGAC 1 CAC 1 A 1 AGGGAAA 1 AAGAGAGAAAAGAAGAG

TAAGAAGAAATATAAGAGCCACCATGGGAGTGACTGGCATTCTTCAGCTTCCTCGCGACAGGTTTAAGA

G AACCTCGTTCTTCCTGTG G GTC ATTAT AC 1 1 1 1 CCAGCGCACATTCAGCATCCCACTTGGAGTGATTCAT

AATTCTACACTTCAGGTTAGCGACGTGGACAAGCTGGTCTGCCGGGACAAACTGTCGTCAACAAATCAG

CTTAGGTCCGTAGGCCTGAACCTTGAAGGTAACGGCGTTGCAACAGATGTGCCTAGCGCAACGAAGCGG

TGGGGATTTAGGTCTGGTGTTCCACCAAAAGTGGTCAATTACGAGGCCGGAGAGTGGGCAGAGAACTG

CTATAACCTGGAAATAAAAAAGCCTGACGGTAGCGAATGCCTGCCAGCCGCGCCAGATGGCATTCGGGG

GTTTCCCCGCTGTCGCTACGTCCACAAAGTAAGTGGTACTGGGCCTTGTGCCGGTGACTTCGCCTTTCAC

AAAGAGGGCGCGTTC 1 1 1 1 1 GTATGACCGTCTCGCCTCCACAGTAATTTACCGAGGCACTACATTTGCAG

AAGGGGTGGTGGCATTCCTTATATTACCCCAGGCTAAAAAGGACTTCTTCTCTAGCCACCCACTCCGGGA

ACCAGTGAATGCAACCGAGGATCCAAGCTCGGGGTACTATTCTACCACTATCAGGTATCAGGCAACAGG GTTTGGTACAAATGAGACTGAATATCTCTTCGAGGTTGACAACCTGACCTACGTGCAGCTGGAAAGTCGT

TTCACTCCGCAGTTCCTCTTGCAGCTCAATGAGACCATATATACCAGTGGGAAACGGTCTAACACAACCG

GAAAACTGATTTGGAAAGTTAACCCAGAAATTGATACTACGATTGGTGAGTGGGCG 1 1 1 1 GGGAGACAA

AGAAGAATTTAACTAGGAAGATCAGGTCCGAAGAACTGTCATTCACTGTGGTGTCTAACGGCGCGAAAA

ATATCTCTGGGCAGTCTCCAGCCCGCACATCCTCCGATCCTGGAACGAATACAACCACCGAAGATCATAA

GATCATGGCCAGCGAAAACTCCTCCGCCATGGTCCAAGTCCATTCCCAAGGGAGGGAAGCTGCAGTCTC

CCACCTCACTACTTTGGCAACCATTAGCACTTCGCCACAAAGCCTGACCACCAAGCCCGGCCCGGATAAT

TCAACCCACAACACCCCAGTTTATAAACTGGATATCAGTGAGGCGACTCAGGTGGAGCAGCATCACCGA

AGAACTGATAATGATTCTACTGCCAGCGACACCCCAAGCGCGACCACCGCTGCCGGACCTCCCAAGGCC

GAGAATACTAACACTTCGAAATCAACAGACTTTCTGGATCCCGCCACAACCACGTCCCCACAGAATCACT

CTGAAACCGCCGGTAACAATAATACACATCACCAGGATACCGGGGAGGAATCCGCTTCTAGCGGGAAGC

TGGGCCTTATAACAAATACGATAGCAGGGGTGGCCGGACTCATCACAGGGGGTCGGAGGACACGGCGG

GAGGCTATAGTCAATGCTCAGCCTAAATGCAACCCGAATCTGCACTATTGGACCACACAGGACGAGGGA

GCCGCCATCGGGCTGGCATGGATTCCATATTTCGGCCCAGCCGCTGAGGGCATCTACATCGAGGGTCTTA

TGCACAACCAGGACGGGCTAATCTGCGGACTTAGGCAGCTGGCCAACGAGACAACACAGGCACTCCAGC

TCTTCCTTCG CG CTAC A AC AG AG CTACG G ACL 1 1 1 1 CCATTCTCAACAGGAAGGCCATAGACTTCCTCTTG

CAGCGGTGGGGAGGCACCTGTCATATTCTCGGGCCCGATTGTTGTATCGAACCTCATGATTGGACCAAA

AATATTACAGATAAGATTGATCAAATCATCCACGATTTCGTAGATAAGACACTCCCCGACCAGGGAGACA

ATGACAACTGGTGGACGGGGTGGCGACAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCC

CTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAG

TGGGCGGC (SEQ I D NO. 23)

1 CAAGC 1 1 1 1 GGACCC 1 CG 1 ACAGAAGC 1 AA 1 ACGAC 1 CAC 1 A 1 AGGGAAA 1 AAGAGAGAAAAGAAGAG

TAAGAAGAAATATAAGAGCCACCATGGGCGTGACTGGGATCCTCCAACTGCCTCGCGATAGATTCAAGC

GAACTAGTTTCTTTCTATGGGTCATCATACTCTTCCAGCGAACCTTCTCAATTCCACTGGGAGTCATTCACA

ACTCCACCTTGCAAGTGTCAGATGTGGACAAGTTGGTATGCCGAGACAAACTCTCTTCTACTAATCAGCT

CAGGTCTGTGGGGCTGAACCTTGAAGGTAACGGCGTGGCCACAGATGTGCCTTCGGCTACTAAGCGATG

GGGTTTCCGGTCCGGTGTGCCCCCGAAGGTGGTAAATTATGAGGCTGGCGAGTGGGCCGAAAACTGCT

ATAACTTGGAGATTAAAAAGCCTGACGGTAGCGAATGTTTACCAGCCGCTCCCGATGGAATTAGGGGCT

TTCCCAGATGTCGGTATGTCCACAAAGTGTCTGGAACTGGGCCTTGTGCTGGGGA I 1 1 1 GCCTTCCATAA

AGAAGGGGCCTTCTTTCTATACGATCGGCTGGCCAGTACCGTCATTTATCGAGGCACTACTTTCGCCGAA

GGCGTTGTGGCG 1 1 1 1 I GA I 1 C 1 CCCACAGGCCAAGAAAGAC 1 I C I 1 1 1 CCTCACATCCTCTACGGGAGCC

AGTAAATGCAACCGAAGATCCTTCTAGTGGCTACTACTCCACCACTATTCGGTACCAGGCTACCGGATTC

GGCACCAATGAGACAGAATATTTATTTGAGGTGGACAACCTTACTTATGTGCAGTTGGAAAGCAGGTTC

ACCCCCCAGTTCCTCCTGCAGTTGAACGAGACAATTTATACATCAGGTAAGCGGTCTAACACTACCGGTA

AGTTAATCTGGAAAGTGAACCCCGAGATCGACACGACCATCGGGGAATGGGC I 1 1 1 1 GGGAGACCAAG

AAAAACCTGACGCGCAAGATTAGATCCGAGGAGCTCAGTTTCACTGTGGTGAGTAATGGCGCTAAAAAT

ATCAGTGGGCAGAGCCCCGCCCGGACAAGCAGCGACCCTGGGACAAACACTACAACCGAAGACCACAA

AATAATGGCCTCAGAAAACAGTAGCGCGATGGTGCAAGTGCATAGCCAAGGACGCGAAGCCGCTGTAT

CCCATCTTACCACCCTGGCTACGATTAGCACCTCGCCCCAGTCTCTGACTACGAAGCCAGGGCCTGACAA

TTCTACACACAACACACCCGTGTACAAACTTGACATCTCAGAAGCAACGCAGGTGGAACAGCATCACCGC

CGGACGGATAACGACAGCACCGCAAGCGATACCCCCAGTGCTACTACTGCAGCCGGTCCCCCAAAAGCA

GAAAATACGAATACCAGCAAGAGTACAGACTTCTTGGACCCGGCCACCACAACAAGCCCACAAAATCAC

AGCGAGACTGCAGGCAACAATAACACACATCACCAGGATACGGGGGAGGAATCTGCTTCGTCCGGCAA

ACTCGGACTGATCACAAATACGATCGCCGGGGTGGCCGGCTTGATCACCGGCGGCAGGCGGACTCGGA

GAGAAGCCATAGTGAACGCTCAGCCTAAGTGCAATCCGAATCTACACTATTGGACTACTCAGGACGAAG

GCGCAGCTATTGGCTTGGCTTGGATTCCATACTTTGGGCCAGCAGCAGAGGGAATATACATCGAGGGAC

TGATGCATAACCAGGATGGCCTGATCTGTGGCCTCCGACAGTTGGCTAATGAAACTACCCAGGCCCTCCA

ATTGTTCCTG CG CG CTAC A ACTG AACTTCG G ACC 1 1 1 I C I A l 1 1 1 GAACAGGAAGGCCATAGATTTCCTGC

TTCAGCGTTGGGGCGGAACATGCCATATCCTGGGCCCCGACTGTTGTATCGAGCCTCATGATTGGACGA

AGAATATAACTGATAAAATAGATCAAATAATCCACGACTTCGTCGACAAGACCCTGCCTGACCAGGGGG

ACAATGATAACTGGTGGACTGGATGGCGACAGGGTAAGCCAATCCCGAACCCTCTTCTCGGTCTGGATA GCACGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCT CCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO. 24)

27 1 LAAGL 1 1 1 1 GGALLL 1 L 1 ALAGAAGL 1 AA 1 ALGAL 1 LAL 1 A 1 AGGGAAA 1 AAGAGAGAAAAGAAGAG

TAAGAAGAAATATAAGAGCCACCATGGACAGTAGGCCCCAGAAGATCTGGATGGCACCATCTCTCACTG

AGAGTGATATGGACTATCATAAGATCCTGACGGCAGGGCTGAGCGTGCAGCAAGGCATCGTCAGGCAG

CGGGTGATCCCAGTATATCAGGTCAACAACCTTGAGGAGATTTGTCAACTGATAATTCAGGCCTTTGAAG

CCGGCGTCGACTTTCAAGAAAGTGCAGATAGCTTTCTCCTGATGCTGTGTTTGCACCATGCGTACCAGGG

CG ACT AT A AG CTGTTCCTG G AGTCTG G G G CTGTTA AGT ACCTG G AG G GG C ACG GTTTC AG GTTTG AG GT

GAAGAAGCGGGACGGCGTCAAGAGGTTAGAGGAACTGCTGCCAGCAGTGAGCTCTGGTAAGAATATTA

AAAGGACCCTCGCTGCAATGCCGGAGGAGGAAACCACAGAGGCAAACGCTGGCCAGTTCCTGAGCTTC

GCATCTCTTTTTCTACCGAAGCTAGTGGTAGGAGAAAAAGCTTGCCTCGAAAAGGTACAGAGACAGATC

CAGGTTCACGCTGAACAGGGGCTTATCCAGTACCCAACCGCTTGGCAGTCGGTGGGTCACATGATGGTG

A l 1 1 1 LAGAL 1 A 1 LGGALAAA 1 1 1 1 L 1 GATTAAGTTCCTGCTTATTCACCAGGGGATGCATATGGTGGC

AGGCCACGATGCAAATGACGCCGTAATCTCTAATAGCGTTGCTCAAGCGCGCTTCTCTGGGCTGCTGATC

GTGAAAACAGTGCTGGATCATATCTTACAAAAGACTGAGAGGGGTGTGAGGCTGCACCCCCTGGCTAGA

ACCGCAAAGGTCAAGAATGAAGTGAATTCGTTTAAGGCTGCCCTCTCTTCACTCGCCAAACACGGGGAG

TACGCGCCTTTCGCAAGACTGCTGAACCTTTCAGGGGTGAACAATCTTGAGCATGGATTGTTTCCACAGT

TGTCCGCCATTGCACTGGGCGTGGCAACAGCACACGGGTCCACTCTGGCCGGTGTGAACGTTGGGGAGC

AGTATCAACAGCTCAGGGAAGCTGCCACTGAGGCCGAGAAACAGCTCCAGCAGTACGCCGAGTCCCGC

GAATTGGATCACCTCGGCCTCGACGACCAGGAAAAGAAGATTCTGATGAA I 1 1 1 CATCAAAAAAAAAAC

GAGATTTCATTTCAACAGACCAACGCAATGGTGACTCTTAGAAAAGAGCGCCTAGCCAAATTAACGGAG

GCCATAACCGCAGCCAGCCTACCAAAGACATCTGGGCACTACGACGACGACGACGACATCCCGTTTCCC

GGTCCAATTAATGACGACGACAATCCCGGCCACCAAGACGACGACCCAACTGACTCGCAGGACACCACG

ATTCCCGATGTCGTGGTGGACCCCGATGACGGGAGCTACGGAGAATATCAAAGCTACTCTGAGAACGGC

ATGAATGCACCCGACGATCTGGTGCTGTTTGACTTGGACGAGGACGATGAAGATACTAAGCCCGTGCCC

AATAG GTC AACT AA AG G CG G CC AG C AG A AA AATTCCC AAA AG G G G C AG C AC ATCG AG G G C AG AC AA AC

ACAATCCAGACCTATACAGAATGTGCCTGGACCCCACCGCACTATTCACCACGCAAGTGCTCCTCTGACC

GACAATGATCGCAGGAATGAGCCATCTGGTTCCACATCTCCGAGAATGCTTACACCTATCAACGAAGAG

GCCGACCCGCTCGACGATGCGGACGACGAGACCTCTAGTTTGCCTCCTCTTGAGTCAGATGACGAAGAA

CAGGACCGGGATGGAACATCGAATAGAACTCCTACAGTGGCTCCGCCCGCTCCAGTATACCGCGATCAC

AGTGAGAAGAAGGAGCTGCCACAGGATGAACAACAGGACCAGGATCACACCCAAGAAGCACGTAACCA

GGACTCGGATAACACTCAGTCAGAACACTCTTTTGAGGAGATGTATCGGCACATTCTTCGATCGCAAGGT

CCATTTGACGCAGTCCTCTACTACCACATGATGAAGGATGAACCCGTCG 1 1 1 1 1 1 CCACAAGCGATGGGA

AAGAGTATACATACCCGGATTCCTTGGAGGAGGAGTACCCGCCCTGGCTGACAGAGAAAGAGGCGATG

AACGAGGAGAACCGCTTTGTAACTCTGGACGGTCAGCAA I 1 1 1 ATTGGCCCGTTATGAATCACAAGAACA

AGTTCATGGCTATCTTGCAACATCACCAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCC

TTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGT

GGGCGGC (SEQ ID NO. 27)

Table 7. Full-length Ebola GP-Small Amino Acid Sequences {Homo sapiens strains)

AER59711 Democratic 12/31/2008 11/7/2011 Zaire ebolavirus isolate 034- KS, partial genome Republic of

the Congo

AKU75160 Sierra 2/19/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML12033/SLe/WesternUrban 20150219, complete genome

AKU75169 Sierra 2/21/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML1205 l/SLe/WesternUrban 20150221 , complete genome

AKU75178 Sierra 2/26/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML12116/SLe/WesternUrban 20150226, complete genome

AKU75187 Sierra 2/26/2015 8/11/2015 Zaire ebolavirus isolate

Leone EB OV/DML 12117/SLe/WesternUrban 20150226 , complete genome

AKU75196 Sierra 2/27/2015 8/11/2015 Zaire ebolavirus isolate

Leone EB OV/DML 12120/SLe/WesternUrban 20150227 , complete genome

AKU75214 Sierra 2/28/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML 12137/SLe/WesternUrban 20150228 , complete genome

AKU75205 Sierra 3/4/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML 12194/SLe/WesternUrban 20150304, complete genome

AKU75223 Sierra 3/7/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML12239/SLe/WesternUrban 20150309, complete genome

AKU75232 Sierra 3/9/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML12260/SLe/WesternUrban 20150309, complete genome

AKU75241 Sierra 3/10/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML12268/SLe/WesternUrban 20150310, complete genome

AKU75250 Sierra 3/28/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML 12458/SLe/WesternUrban 20150328 , complete genome

AKU75259 Sierra 3/31/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML12485/SLe/WesternUrban 20150331 , partial genome

AKU75583 Sierra 6/30/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML14077/SLe/WesternUrban 20150630, complete genome

AKU75565 Sierra 7/3/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML 14163/SLe/WesternUrban 20150703 , complete genome

AKU75574 Sierra 7/11/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML14366/SLe/WesternUrban 20150711, complete genome

AKU75268 Sierra 1/13/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24502/SLe/Kono/20150113, partial genome

AKU75277 Sierra 1/13/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24504/SLe/Kono/20150113,

complete genome AKU75286 Sierra 1/14/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24506/SLe/Kono/20150114,

complete genome

AKU75295 Sierra 1/14/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML2451 l/SLe/Kono/20150114,

complete genome

AKU75304 Sierra 1/17/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24552/SLe/Kono/20150117,

complete genome

AKU75313 Sierra 1/17/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24553/SLe/Kono/20150117,

complete genome

AKU75326 Sierra 1/18/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24573/SLe/Kono/20150118,

complete genome

AKU75331 Sierra 1/19/2015 8/11/2015 Zaire ebolavirus isolate

Leone EB O V/DML24581 /SLe/Kono/20150119,

complete genome

AKU75340 Sierra 1/20/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24592/SLe/Kono/20150120,

complete genome

AKU75351 Sierra 1/20/2015 8/11/2015 Zaire ebolavirus isolate

Leone EB O V/DML24601 /SLe/Kono/20150120,

complete genome

AKU75358 Sierra 1/20/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24604/SLe/Kono/20150120,

complete genome

AKU75367 Sierra 1/20/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24605/SLe/Kono/20150120,

complete genome

AKU75376 Sierra 1/20/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24606/SLe/Kono/20150120,

complete genome

AKU75385 Sierra 1/20/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24608/SLe/Kono/20150120,

complete genome

AKU75394 Sierra 1/21/2015 8/11/2015 Zaire ebolavirus isolate

Leone EB O V/DML24611 /SLe/Kono/20150121,

complete genome

AKU75403 Sierra 1/21/2015 8/11/2015 Zaire ebolavirus isolate

Leone EB O V/DML24620/SLe/Kono/20150121, partial genome

AKU75412 Sierra 1/25/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24669/SLe/Kono/20150125 ,

complete genome

AKU75421 Sierra 1/25/2015 8/11/2015 Zaire ebolavirus isolate

Leone EB O V/DML24677/SLe/Kono/20150125,

complete genome

AKU75430 Sierra 1/26/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24683/SLe/Kono/20150126,

complete genome

AKU75439 Sierra 1/28/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24706/SLe/Kono/20150128,

complete genome AKU75448 Sierra 1/28/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24708/SLe/Kono/20150128,

complete genome

AKU75457 Sierra 1/29/2015 8/11/2015 Zaire ebolavirus isolate

Leone EB O V/DML24720/SLe/Kono/20150129 ,

complete genome

AKU75466 Sierra 1/30/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24758/SLe/Kono/20150130,

complete genome

AKU75475 Sierra 2/3/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24818/SLe/Kono/20150203 ,

complete genome

AKU75484 Sierra 2/4/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24825/SLe/Tonkolili/20150204, complete genome

AKU75493 Sierra 2/6/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24853/SLe/Kono/20150206,

complete genome

AKU75502 Sierra 2/6/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML24854/SLe/Kono/20150206,

complete genome

AKU75511 Sierra 2/18/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML25083/SLe/Kono/20150218 ,

complete genome

AKU75520 Sierra 2/19/2015 8/11/2015 Zaire ebolavirus isolate

Leone EB O V/DML25103/SLe/Kono/20150219 ,

complete genome

AKU75529 Sierra 2/18/2015 8/11/2015 Zaire ebolavirus isolate

Leone EB O V/DML25123/SLe/Kenema/20150218 , partial genome

AKU75538 Sierra 2/23/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML25180/SLe/Kono/20150223 ,

complete genome

AKU75547 Sierra 3/6/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML25344/SLe/Kono/20150306,

complete genome

AKU75556 Sierra 3/10/2015 8/11/2015 Zaire ebolavirus isolate

Leone EBOV/DML2541 l/SLe/Kono/20150310, partial genome

AGB56840 Democratic 1976 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- Republic of tc/COD/1976/deRoover, complete genome the Congo

AGB56750 Democratic 1977 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- Republic of tc/COD/1977/Bonduni, complete genome the Congo

AGB56795 Democratic 1995 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- Republic of tc/COD/1995/13625 Kikwit, complete genome the Congo

AGB56822 Democratic 1995 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- Republic of tc/COD/1995/13709 Kikwit, complete genome the Congo

AGB56696 Democratic 2007 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- Republic of tc/COD/2007/0 Luebo, complete genome the Congo AGB56705 Democratic 2007 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- Republic of tc/COD/2007/1 Luebo, complete genome the Congo

AGB56714 Democratic 2007 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- Republic of tc/COD/2007/23 Luebo, complete genome the Congo

AGB56732 Democratic 2007 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- Republic of tc/COD/2007/4 Luebo, complete genome the Congo

AGB56723 Democratic 2007 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- Republic of tc/COD/2007/43 Luebo, complete genome the Congo

AGB56741 Democratic 2007 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- Republic of tc/COD/2007/5 Luebo, complete genome the Congo

AGB56687 Democratic 2007 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- Republic of tc/COD/2007/9 Luebo, complete genome the Congo

AGB56759 Gabon 1994 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- tc/GAB/1994/Gabon, complete genome

AGB56768 Gabon 1996 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- tc/GAB/1996/lEko, complete genome

AGB56813 Gabon 1996 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- tc/GAB/1996/lIkot, complete genome

AGB56786 Gabon 1996 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- tc/GAB/1996/lMbie, complete genome

AGB56804 Gabon 1996 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- tc/GAB/1996/10ba, complete genome

AGB56777 Gabon 1996 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- tc/GAB/1996/2Nza, complete genome

AGB56831 Gabon 2002 1/7/2013 Zaire ebolavirus isolate EBOV/H.sapiens- tc/GAB/2002/Ilembe, complete genome

AKA59361 United 3/12/2015 4/4/2015 Zaire ebolavirus isolate Ebola virus H.sapiens- Kingdom wt/GBR/2015/Makona-UK3, complete genome

AKC01436 Liberia 2014 4/14/2015 Zaire ebolavirus isolate Ebola virus H.sapiens- wt/LBR/2014/Makona-Liberia-DQEl , complete genome

AKC01476 Liberia 2014 4/14/2015 Zaire ebolavirus isolate Ebola virus H.sapiens- wt/LBR/2014/Makona-Liberia-DQE 12, complete genome

AKC01484 Liberia 2014 4/14/2015 Zaire ebolavirus isolate Ebola virus H.sapiens- wt/LBR/2014/Makona-Liberia-DQE 13 , complete genome

AKC01492 Liberia 2014 4/14/2015 Zaire ebolavirus isolate Ebola virus H.sapiens- wt/LBR/2014/Makona-Liberia-DQE 14, complete genome

AKC01444 Liberia 2015 4/14/2015 Zaire ebolavirus isolate Ebola virus H.sapiens- wt/LBR/2015/Makona-Liberia-DQE3, complete genome

AKC01452 Liberia 2015 4/14/2015 Zaire ebolavirus isolate Ebola virus H.sapiens- wt/LBR/2015/Makona-Liberia-DQE4, complete genome

AKC01460 Liberia 2015 4/14/2015 Zaire ebolavirus isolate Ebola virus H.sapiens- wt/LBR/2015/Makona-Liberia-DQE5, complete genome

AKC01468 Liberia 2015 4/14/2015 Zaire ebolavirus isolate Ebola virus H.sapiens- wt/LBR/2015/Makona-Liberia-DQE6, complete genome

AKT08841 Sierra 2/19/2015 8/4/2015 Zaire ebolavirus isolate Ebola virus H.sapiens- Leone wt/SLE/2015/Makona-Goderich 1 , complete genome

AI011752 Democratic 2014 10/17/2014 Zaire ebolavirus isolate Ebola virus/H.sap- Republic of wt/COD/2014/Boende-Lokolia, partial genome the Congo

AIR94007 Democratic 1976 10/3/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Republic of tc/COD/1976/Yambuku-Ecran, complete genome the Congo

AIY29183 United 8/25/2014 11/26/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Kingdom tc/GBR/2014/Makona-UKl.l, complete genome

AJF38896 Italy 11/25/2014 2/1/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- tc/SLE/2014/Makona-Italy-INMI 1 , complete genome

AJG44193 Switzerland 11/21/2014 2/9/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/CHE/2014/Makona-GEl, complete genome

AKI84248 Democratic 8/16/2014 7/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Republic of wt/COD/2014/Lomela-Lokolia-Bl 1, partial the Congo genome

AJA04385 Democratic 8/20/2014 12/22/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Republic of wt/COD/2014/Lomela-Lokolial6, complete the Congo genome

AJA04394 Democratic 8/20/2014 12/22/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Republic of wt/COD/2014/Lomela-Lokolial7, partial genome the Congo

AJA04403 Democratic 8/20/2014 12/22/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Republic of wt/COD/2014/Lomela-Lokolia 19 , complete the Congo genome

AIW65951 United 8/25/2014 11/15/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Kingdom wt/GBR/2014/Makona-UKl, complete genome

AJE60745 United 12/29/2014 1/21/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Kingdom wt/GBR/2014/Makona-UK2, partial genome

AKL91083 Guinea 3/19/2014 6/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-C05, partial genome

AKL91092 Guinea 3/19/2014 6/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-C05, partial genome

AKL91101 Guinea 3/20/2014 6/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-C07, partial genome

AKL91110 Guinea 3/20/2014 6/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-C07, partial genome

AKL91119 Guinea 3/17/2014 6/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-C15, partial genome

AKL91128 Guinea 3/17/2014 6/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-C15, partial genome

AKG65728 Guinea 9/18/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1027 , partial genome

AKG65737 Guinea 9/19/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1039, partial genome AKG65278 Guinea 9/19/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1043 , partial genome

AKG65296 Guinea 9/21/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1059, partial genome

AKG65323 Guinea 9/22/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1081, partial genome

AKG65746 Guinea 9/24/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1105, partial genome

AKG65350 Guinea 9/25/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1120, partial genome

AKG65359 Guinea 9/25/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1121, partial genome

AKG65755 Guinea 9/25/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1128, partial genome

AKG65764 Guinea 9/25/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1129, partial genome

AKG65773 Guinea 9/27/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1149, partial genome

AKG65368 Guinea 9/28/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1193, partial genome

AKG65377 Guinea 10/2/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1205 , partial genome

AKG65386 Guinea 10/2/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1210, partial genome

AKG65782 Guinea 10/2/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1213, partial genome

AKG65791 Guinea 10/2/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1215, partial genome

AKG65404 Guinea 10/4/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1249 , partial genome

AKG65800 Guinea 10/4/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1250, partial genome

AKG65440 Guinea 10/6/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1298 , partial genome

AKG65494 Guinea 10/8/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1340, partial genome AKG65503 Guinea 10/8/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1342, partial genome

AKG65530 Guinea 10/10/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1371, partial genome

AKG65566 Guinea 10/14/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1445 , partial genome

AKG65575 Guinea 10/14/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1454, partial genome

AKG65584 Guinea 10/15/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1480, partial genome

AKG65710 Guinea 10/15/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1481, partial genome

AKG65719 Guinea 10/16/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1491 , partial genome

AKG65593 Guinea 10/18/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1551, partial genome

AKG65602 Guinea 10/19/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1561, partial genome

AKG65665 Guinea 10/24/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry- 1651, partial genome

ALF04602 Guinea 10/13/2014 9/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-211, partial genome

ALF04596 Guinea 10/14/2014 9/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-223 , partial genome

AKG65097 Guinea 7/24/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-505 , partial genome

AKG65809 Guinea 7/24/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-507, partial genome

AKG65107 Guinea 7/24/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-509, partial genome

AKG65134 Guinea 8/2/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-573 , partial genome

AKG65161 Guinea 8/15/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-653 , partial genome

AKG65170 Guinea 8/16/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-657, partial genome AKG65179 Guinea 8/19/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-678 , partial genome

AKG65188 Guinea 8/20/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-684, partial genome

AKG65197 Guinea 8/21/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-691 , partial genome

AKG65206 Guinea 8/22/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-701 , partial genome

AKG65215 Guinea 8/26/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-740, partial genome

AKG65818 Guinea 8/26/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-742, partial genome

AKG65827 Guinea 8/27/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-768 , partial genome

AKG65224 Guinea 8/28/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-786, partial genome

AKG65233 Guinea 8/28/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-787, partial genome

AKG65260 Guinea 9/14/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Conakry-976 , partial genome

AKG65305 Guinea 9/21/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coyah- 1063 , partial genome

AKG65413 Guinea 10/4/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coyah- 1274, partial genome

AKG65422 Guinea 10/4/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coyah- 1277 , partial genome

AKG65431 Guinea 10/4/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coyah- 1278 , partial genome

AKG65836 Guinea 10/4/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coyah- 1279 , partial genome

AKG65449 Guinea 10/7/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coyah- 1316 , partial genome

AKG65845 Guinea 10/7/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coyah- 1320, partial genome

AKG65458 Guinea 10/7/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coyah- 1321, partial genome AKG65854 Guinea 10/7/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coy ah- 1327 , partial genome

AKG65476 Guinea 10/7/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coy ah- 1333 , partial genome

AKG65485 Guinea 10/8/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coyah-1339, partial genome

AKG65512 Guinea 10/9/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coy ah- 1355 , partial genome

AKG65539 Guinea 10/10/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coy ah- 1374, partial genome

AKG65548 Guinea 10/11/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coy ah- 1394, partial genome

AKG65557 Guinea 10/13/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coy ah- 1436 , partial genome

AKG65674 Guinea 10/24/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coyah-1652, partial genome

AKG65683 Guinea 10/24/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coyah-1686, partial genome

AKG65692 Guinea 10/25/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coyah-1689, partial genome

AKG65701 Guinea 10/25/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coy ah- 1690, partial genome

AKG65251 Guinea 9/11/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Coyah-955, partial genome

AKG65332 Guinea 9/24/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Dalaba-l 104, partial genome

AKG65341 Guinea 9/24/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Dalaba-l 116, partial genome

AKG65395 Guinea 10/2/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Dalaba-1211, partial genome

AKG65242 Guinea 8/29/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Dubreka-789, partial genome

AKI82636 Guinea 9/1/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000015 , partial genome

AKI82645 Guinea 9/1/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000027 , partial genome

AKI82654 Guinea 9/1/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000028 , partial genome

AKI82663 Guinea 9/4/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000127 , partial genome

AKI82672 Guinea 9/4/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000128, partial genome

AKI82681 Guinea 9/7/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000218 , partial genome

AKI82690 Guinea 9/7/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000219 , partial genome

AKI82699 Guinea 9/9/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000321 , partial genome

AKI82708 Guinea 9/12/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000457 , partial genome

AKI82717 Guinea 9/13/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000500, partial genome

AKI82726 Guinea 9/13/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000501 , partial genome

AKI82735 Guinea 9/13/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000502, partial genome

AKI82744 Guinea 9/21/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000706 , partial genome

AKI82753 Guinea 9/22/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000707 , partial genome

AKI82762 Guinea 9/29/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000921 , partial genome

AKI82771 Guinea 9/29/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000925 , partial genome

AKI82780 Guinea 9/29/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000934, partial genome

AKI82789 Guinea 9/30/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000958 , partial genome

AKI82798 Guinea 10/1/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000968 , partial genome

AKI82807 Guinea 10/1/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000982, partial genome

AKI82816 Guinea 10/1/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_000983 , partial genome

AKI82825 Guinea 10/5/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_001101 , partial genome

AKI82834 Guinea 10/5/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_001102, partial genome

AKI82843 Guinea 12/20/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_004059 , partial genome

AKI82852 Guinea 12/19/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_004085 , partial genome

AKI82861 Guinea 12/22/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_004192, partial genome

AKI82870 Guinea 12/24/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_004201 , partial genome

AKI82879 Guinea 12/26/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_004259 , partial genome

AKI82888 Guinea 12/27/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_004290, partial genome

AKI82996 Guinea 7/18/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_074335 , partial genome

AKI83050 Guinea 7/22/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_074354, partial genome

AKI83077 Guinea 8/1/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_074436, partial genome

AKI83086 Guinea 8/1/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_074437 , partial genome

AKI83095 Guinea 8/1/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_074438 , partial genome

AKI83104 Guinea 8/1/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_074439 , partial genome

AKI83113 Guinea 8/4/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_074461 , partial genome

AKI83122 Guinea 8/4/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_074462, partial genome

AKI83131 Guinea 8/8/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_074531 , partial genome

AKI83149 Guinea 8/14/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_074684, partial genome

AKI83167 Guinea 8/16/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_074785 , partial genome

AKI83203 Guinea 8/23/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_075076, partial genome

AKI83212 Guinea 8/29/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_075368, partial genome

AKI83221 Guinea 8/30/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_075373 , partial genome

AKI83230 Guinea 8/30/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_075435 , partial genome

AKI83239 Guinea 8/31/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_075447, partial genome

AKI83248 Guinea 10/10/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_075928 , partial genome

AKI83257 Guinea 10/10/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_075929, partial genome

AKI83266 Guinea 10/10/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_075930, partial genome

AKI83275 Guinea 10/10/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_075931 , partial genome

AKI83284 Guinea 10/10/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_075932, partial genome

AKI83293 Guinea 10/17/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076138, partial genome

AKI83302 Guinea 10/18/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076191, partial genome

AKI83311 Guinea 10/18/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076192, partial genome

AKI83320 Guinea 10/18/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076193, partial genome

AKI83329 Guinea 10/19/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076217 , partial genome

AKI83338 Guinea 10/22/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076322, partial genome

AKI83347 Guinea 10/23/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076334, partial genome

AKI83356 Guinea 10/23/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076335 , partial genome

AKI83365 Guinea 10/26/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076383 , partial genome

AKI83374 Guinea 10/27/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076403 , partial genome

AKI83383 Guinea 10/29/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076472, partial genome

AKI83392 Guinea 11/1/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076533 , partial genome

AKI83401 Guinea 11/1/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076534, partial genome

AKI83410 Guinea 11/3/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076610, partial genome

AKI83419 Guinea 11/3/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076615 , partial genome

AKI83428 Guinea 11/8/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076769, partial genome

AKI83437 Guinea 11/8/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076770, partial genome

AKI83446 Guinea 11/13/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076948 , partial genome

AKI83455 Guinea 11/13/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076949 , partial genome

AKI83464 Guinea 11/13/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_076951 , partial genome

AKI83473 Guinea 11/24/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078415 , partial genome

AKI83482 Guinea 11/24/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078416 , partial genome

AKI83491 Guinea 12/1/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078555, partial genome

AKI83500 Guinea 12/1/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078556, partial genome

AKI83509 Guinea 12/4/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078608 , partial genome

AKI83518 Guinea 12/3/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078638 , partial genome

AKI83527 Guinea 12/4/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078639, partial genome

AKI83536 Guinea 12/9/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078654, partial genome

AKI83545 Guinea 12/9/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078656, partial genome

AKI83554 Guinea 12/11/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078670, partial genome

AKI83563 Guinea 12/11/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078683, partial genome

AKI83572 Guinea 12/14/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078694, partial genome

AKI83581 Guinea 12/14/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078697, partial genome

AKI83590 Guinea 12/15/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078706, partial genome

AKI83599 Guinea 12/14/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078709, partial genome

AKI83608 Guinea 12/16/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078722, partial genome

AKI83617 Guinea 12/17/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078763 , partial genome

AKI83626 Guinea 12/18/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_078779, partial genome

AKI83635 Guinea 7/30/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079388 , partial genome

AKI83644 Guinea 3/28/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079404, partial genome

AKI83653 Guinea 3/28/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079405 , partial genome

AKI83662 Guinea 3/31/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079408 , partial genome

AKI83671 Guinea 3/31/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079410, partial genome

AKI83680 Guinea 3/31/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079412, partial genome

AKI83689 Guinea 3/31/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079413 , partial genome

AKI83698 Guinea 3/31/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079414, partial genome

AKI83707 Guinea 3/30/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079421 , partial genome

AKI83716 Guinea 3/27/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079422, partial genome

AKI83725 Guinea 3/27/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079423 , partial genome

AKI83734 Guinea 3/27/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079424, partial genome

AKI83743 Guinea 4/2/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079429 , partial genome

AKI83752 Guinea 4/2/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079434, partial genome

AKI83761 Guinea 4/3/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079442, partial genome

AKI83770 Guinea 4/2/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079444, partial genome

AKI83788 Guinea 4/4/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079464, partial genome

AKI83797 Guinea 4/7/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079497 , partial genome

AKI83806 Guinea 4/10/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079514, partial genome

AKI83815 Guinea 4/11/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079517, partial genome

AKI83824 Guinea 4/12/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079542, partial genome

AKI83833 Guinea 4/13/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079549, partial genome

AKI83842 Guinea 4/18/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079578 , partial genome

AKI83851 Guinea 4/22/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079587, partial genome

AKI83860 Guinea 4/28/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079622, partial genome

AKI83869 Guinea 5/1/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079630, partial genome

AKI83878 Guinea 5/7/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079657, partial genome

AKI83887 Guinea 5/7/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079659, partial genome

AKI83896 Guinea 5/10/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079677 , partial genome

AKI83905 Guinea 5/11/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079681 , partial genome

AKI83914 Guinea 5/11/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079685 , partial genome

AKI83923 Guinea 5/14/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079702, partial genome

AKI83932 Guinea 5/18/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079731 , partial genome

AKI83941 Guinea 5/21/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079749 , partial genome

AKI83950 Guinea 5/21/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079750, partial genome

AKI83959 Guinea 5/22/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079753 , partial genome

AKI83968 Guinea 5/24/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079772, partial genome

AKI83977 Guinea 5/24/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079775 , partial genome

AKI83986 Guinea 5/28/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079815 , partial genome

AKI83995 Guinea 6/1/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079859, partial genome

AKI84004 Guinea 6/5/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079876, partial genome

AKI84013 Guinea 6/5/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079880, partial genome

AKI84022 Guinea 6/9/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079910, partial genome

AKI84031 Guinea 6/9/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079911, partial genome

AKI84040 Guinea 6/9/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079912, partial genome

AKI84049 Guinea 6/9/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079913 , partial genome

AKI84058 Guinea 6/10/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079914, partial genome

AKI84067 Guinea 6/10/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_079915 , partial genome

AKI84103 Guinea 6/20/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_080063 , partial genome

AKI84148 Guinea 6/22/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_080076, partial genome

AKI84166 Guinea 6/25/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_080141 , partial genome

AKI84211 Guinea 7/10/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-EM_080253 , partial genome

AKG65314 Guinea 9/21/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Forecariah- 1069, partial genome

AKG65521 Guinea 10/9/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Forecariah- 1365, partial genome

AKG65611 Guinea 10/18/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Forecariah- 1567, partial genome

AKG65620 Guinea 10/18/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Forecariah- 1568, partial genome

AKG65629 Guinea 10/20/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Forecariah- 1571, partial genome

AKG65647 Guinea 10/24/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Forecariah- 1623 , partial genome

AKG65269 Guinea 9/15/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Forecariah-989, partial genome

AKG65143 Guinea 8/12/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Gueckedou-633 , partial genome

AKG65467 Guinea 10/7/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Kerouane- 1331 , partial genome

AKG65287 Guinea 9/20/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Kindia- 1047 , partial genome

AKG65656 Guinea 10/23/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Kindia- 1648 , partial genome

ALF04584 Guinea 12/18/2014 9/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Kindia-802, partial genome

AKG65125 Guinea 7/27/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Kouroussa-531 , partial genome

AKG65152 Guinea 8/14/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Macenta-645 , partial genome

AKG65638 Guinea 10/22/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Nzeerekore- 1622, partial genome

AKG65116 Guinea 7/24/2014 6/26/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2014/Makona-Siguiri-517, partial genome

AKI82897 Guinea 1/2/2015 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2015/Makona-EM_004414, partial genome

AKI82906 Guinea 1/2/2015 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2015/Makona-EM_004422, partial genome

AKI82915 Guinea 1/4/2015 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2015/Makona-EM_004437, partial genome

AKI82924 Guinea 1/4/2015 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2015/Makona-EM_004438 , partial genome

AKI82933 Guinea 1/11/2015 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2015/Makona-EM_004481 , partial genome

AKI82942 Guinea 1/14/2015 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2015/Makona-EM_004494, partial genome

AKI82951 Guinea 1/15/2015 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2015/Makona-EM_004503 , partial genome

AKI82960 Guinea 1/22/2015 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2015/Makona-EM_004555, partial genome

AKI82969 Guinea 1/25/2015 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2015/Makona-EM_004563 , partial genome AKI82978 Guinea 1/27/2015 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2015/Makona-EM_004580, partial genome

AKI82987 Guinea 1/31/2015 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/GIN/2015/Makona-EM_004589 , partial genome

AJZ74730 Liberia 9/23/2014 3/30/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-10054, partial genome

AIY27577 Liberia 8/3/2014 11/26/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-201403007 , complete genome

AKI83005 Liberia 7/22/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_074349, partial genome

AKI83014 Liberia 7/22/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_074350, partial genome

AKI83023 Liberia 7/22/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_074351 , partial genome

AKI83032 Liberia 7/22/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_074352, partial genome

AKI83041 Liberia 7/22/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_074353, partial genome

AKI83059 Liberia 7/26/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_074391, partial genome

AKI83068 Liberia 7/25/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_074392, partial genome

AKI83140 Liberia 8/8/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_074548 , partial genome

AKI83158 Liberia 8/14/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_074720, partial genome

AKI83176 Liberia 8/17/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_074821 , partial genome

AKI83185 Liberia 8/17/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_074822, partial genome

AKI83194 Liberia 8/22/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_075043 , partial genome

AKI83779 Liberia 4/1/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_079450, partial genome

AKI84112 Liberia 6/20/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_080064, partial genome

AKI84121 Liberia 6/20/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_080065 , partial genome

AKI84130 Liberia 6/20/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_080066, partial genome

AKI84139 Liberia 6/20/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_080067, partial genome

AKI84184 Liberia 6/29/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_080193 , partial genome

AKI84193 Liberia 7/3/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_080213 , partial genome

AKI84202 Liberia 7/4/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_080223 , partial genome

AKI84220 Liberia 7/12/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_080261, partial genome

AKI84238 Liberia 7/12/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-EM_080269, partial genome

AJZ74520 Liberia 11/5/2014 3/30/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-LIBR0058, partial genome

AJZ74547 Liberia 11/6/2014 3/30/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-LIBR0067, partial genome

AJZ74556 Liberia 11/6/2014 3/30/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-LIBR0073, partial genome

AJZ74565 Liberia 11/8/2014 3/30/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-LIBR0090, partial genome

AJZ74574 Liberia 11/8/2014 3/30/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-LIBR0092, partial genome

AJZ74583 Liberia 11/8/2014 3/30/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-LIBR0093, partial genome

AJZ74592 Liberia 11/10/2014 3/30/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-LIBR0116, partial genome

AJZ74601 Liberia 11/13/2014 3/30/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-LIBR0168, partial genome

AJZ74626 Liberia 11/22/2014 3/30/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-LIBR0286, partial genome

AJZ74635 Liberia 11/25/2014 3/30/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-LIBR0333, partial genome

AJZ74644 Liberia 12/3/2014 3/30/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-LIBR0423, partial genome

AJZ74660 Liberia 12/10/2014 3/30/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-LIBR0503, partial genome

AJZ74669 Liberia 12/10/2014 3/30/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-LIBR0505, partial genome

AJZ74721 Liberia 10/1/2014 3/30/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2014/Makona-LIBR10053, partial genome

AJZ74695 Liberia 1/20/2015 3/30/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2015/Makona-LIBR0993, partial genome AJZ74712 Liberia 2/14/2015 3/30/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/LBR/2015/Makona-LIBR1413, partial genome

AIZ68608 Mali 10/23/2014 12/12/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/MLI/2014/Makona-Mali-DPRl , complete genome

AIZ68616 Mali 11/12/2014 12/12/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/MLI/2014/Makona-Mali-DPR2, complete genome

AIZ68624 Mali 11/21/2014 12/12/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/MLI/2014/Makona-Mali-DPR3 , complete genome

AIZ68632 Mali 11/12/2014 12/12/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- wt/MLI/2014/Makona-Mali-DPR4, complete genome

AKG95786 Sierra 8/22/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140008, partial genome

AKG95795 Sierra 8/20/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140024, partial genome

AKG95930 Sierra 8/23/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140038, partial genome

AKG95678 Sierra 8/22/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140091, partial genome

AKG95696 Sierra 8/24/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140100, partial genome

AKG95570 Sierra 8/26/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140134, partial genome

AKG95912 Sierra 8/27/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140161, partial genome

AKG96173 Sierra 8/27/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140174, partial genome

AKG96191 Sierra 8/29/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140254, partial genome

AKG96038 Sierra 9/2/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140395, partial genome

AKG95741 Sierra 9/3/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140433, partial genome

AKG96110 Sierra 9/3/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140436, partial genome

AKG95642 Sierra 9/4/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140489, partial genome

AKG95894 Sierra 9/5/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140517, partial genome

AKG96029 Sierra 9/7/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140590, partial genome

AKG96101 Sierra 9/10/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140729, partial genome

AKG96200 Sierra 9/15/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140872, partial genome

AKG95948 Sierra 9/18/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140910, partial genome

AKG96047 Sierra 9/16/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20140933, partial genome

AKG95723 Sierra 9/21/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141012, partial genome AKG96272 Sierra 9/21/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141043, partial genome

AKG95777 Sierra 9/21/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141061, partial genome

AKG96146 Sierra 9/22/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141123, partial genome

AKG96254 Sierra 9/26/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141227, partial genome

AKG95921 Sierra 9/25/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141232, partial genome

AKG96227 Sierra 9/24/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141241 , partial genome

AKG95876 Sierra 9/24/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141271, partial genome

AKG96011 Sierra 9/23/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141280, partial genome

AKG95588 Sierra 9/23/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141282, partial genome

AKG95615 Sierra 9/23/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141288, partial genome

AKG96083 Sierra 9/26/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141352, partial genome

AKG96119 Sierra 9/28/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141397, partial genome

AKG95867 Sierra 9/28/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141429, partial genome

AKG96128 Sierra 10/1/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141491, partial genome

AKG95984 Sierra 10/1/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141497, partial genome

AKG95597 Sierra 10/3/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141582, partial genome

AKG95624 Sierra 10/4/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141643, partial genome

AKG95552 Sierra 10/5/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141650, partial genome

AKG96074 Sierra 10/9/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141960, partial genome

AKG96164 Sierra 10/10/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20141997, partial genome

AKG95993 Sierra 10/11/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20142065, partial genome

AKG95633 Sierra 10/12/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20142127, partial genome

AKG95885 Sierra 10/14/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20142260, partial genome

AKG95768 Sierra 10/16/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20142407, partial genome

AKG95813 Sierra 10/18/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20142417, partial genome

AKG95822 Sierra 10/19/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20142477, partial genome

AKG95561 Sierra 10/18/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20142551, partial genome AKG96236 Sierra 10/23/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20142843, partial genome

AKG96245 Sierra 10/23/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20142856, partial genome

AKG95939 Sierra 10/24/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20142895, partial genome

AKG95975 Sierra 10/26/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143018, partial genome

AKG96002 Sierra 10/25/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143031, partial genome

AKG96092 Sierra 10/24/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143036, partial genome

AKG95750 Sierra 10/25/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143107, partial genome

AKG96020 Sierra 10/27/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143164, partial genome

AKG96209 Sierra 10/28/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143187, partial genome

AKG96065 Sierra 10/29/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143317, partial genome

AKG95849 Sierra 10/30/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143360, partial genome

AKG95759 Sierra 10/31/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143415, partial genome

AKG95732 Sierra 11/1/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143458, partial genome

AKG96263 Sierra 11/1/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143466, partial genome

AKG95579 Sierra 11/1/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143550, partial genome

AKG95804 Sierra 11/3/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143648, partial genome

AKG95831 Sierra 11/3/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143659, partial genome

AKG96155 Sierra 11/4/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143716, partial genome

AKG95651 Sierra 11/5/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143753, partial genome

AKG95840 Sierra 11/5/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143796, partial genome

AKG95687 Sierra 11/6/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143918, partial genome

AKG95903 Sierra 11/7/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143938, partial genome

AKG95705 Sierra 11/7/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20143964, partial genome

AKG95669 Sierra 11/8/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20144192, partial genome

AKG95966 Sierra 11/12/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20144521, partial genome

AKG95957 Sierra 11/12/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20144610, partial genome

AKG96218 Sierra 11/15/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20144819, partial genome AKG95714 Sierra 11/15/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20144820, partial genome

AKG95858 Sierra 11/14/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20144837, partial genome

AKG95606 Sierra 11/13/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20144865, partial genome

AKG96182 Sierra 11/25/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20145835, partial genome

AKG96137 Sierra 11/26/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20145853, partial genome

AKG95660 Sierra 11/24/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20146001, partial genome

AKG95543 Sierra 12/22/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20146553, partial genome

AKG96056 Sierra 12/26/2014 5/17/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-20146578, partial genome

AIE11810 Sierra 5/25/2014 6/30/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM095, complete genome

AIE11801 Sierra 5/25/2014 6/30/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM095B , complete

genome

AIE11819 Sierra 5/26/2014 6/30/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM096, complete genome

AIE 11828 Sierra 5/26/2014 6/30/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM098, complete genome

AIG95888 Sierra 6/2/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM104, complete genome

AIG95897 Sierra 6/2/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM106, complete genome

AIG95906 Sierra 6/3/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM110, complete genome

AIG95915 Sierra 6/3/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EMl l l, complete genome

AIG95924 Sierra 6/3/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM112, complete genome

AIG95933 Sierra 6/3/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM113, complete genome

AIG95942 Sierra 6/3/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM115, complete genome

AIG95951 Sierra 6/3/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM119, complete genome

AIG95960 Sierra 6/3/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM120, complete genome

AIG95969 Sierra 6/4/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM121, complete genome

AIG95978 Sierra 6/4/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM 124.1 , complete

genome

AIG95987 Sierra 6/6/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM 124.2, complete

genome

AIG95996 Sierra 6/8/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM 124.3 , complete

genome AIG96005 Sierra 6/9/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM 124.4, complete genome

AKI84076 Sierra 6/13/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM_079983 , partial genome

AKI84085 Sierra 6/14/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM_080003 , partial genome

AKI84094 Sierra 6/15/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM_080011, partial genome

AKI84157 Sierra 6/24/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM_080132, partial genome

AKI84175 Sierra 6/26/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM_080165 , partial genome

AKI84229 Sierra 7/12/2014 6/12/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-EM_080265 , partial genome

AIE11837 Sierra 5/27/2014 6/30/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3670.1 , complete genome

AIE 11846 Sierra 5/27/2014 6/30/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3676.1 , complete genome

AIE11855 Sierra 6/6/2014 6/30/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3676.2, complete genome

AIE11864 Sierra 5/26/2014 6/30/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3677.1 , complete genome

AIE 11873 Sierra 5/27/2014 6/30/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3677.2, complete genome

AIE11882 Sierra 5/28/2014 6/30/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3679.1 , complete genome

AIE11891 Sierra 5/28/2014 6/30/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3680.1 , complete genome

AIE 11900 Sierra 5/28/2014 6/30/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3682.1 , complete genome

AIE 11909 Sierra 5/28/2014 6/30/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3683.1 , complete genome

AIE11918 Sierra 5/28/2014 6/30/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3686.1 , complete genome

AIE 11927 Sierra 5/28/2014 6/30/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3687.1 , complete genome AIG96014 Sierra 5/31/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3707, complete genome

AIG96023 Sierra 6/9/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3713.2, complete

genome

AIG96032 Sierra 6/11/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3713.3, complete

genome

AIG96041 Sierra 6/13/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3713.4, complete

genome

AIG96050 Sierra 6/5/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3724, complete genome

AIG96059 Sierra 6/7/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3729, complete genome

AIG96068 Sierra 6/7/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3734.1 , complete

genome

AIG96077 Sierra 6/7/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3735.1 , complete

genome

AIG96086 Sierra 6/9/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3735.2, complete

genome

AIG96095 Sierra 6/10/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3750.1 , complete

genome

AIG96104 Sierra 6/12/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3750.2, complete

genome

AIG96113 Sierra 6/14/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3750.3 , complete

genome

AIG96122 Sierra 6/10/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3752, complete genome

AIG96131 Sierra 6/11/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3758, complete genome

AIG96140 Sierra 6/12/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3764, complete genome

AIG96149 Sierra 6/14/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3765.2, complete

genome

AIG96158 Sierra 6/12/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3769.1 , complete

genome

AIG96167 Sierra 6/13/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3769.2, complete

genome

AIG96176 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3769.3 , complete

genome

AIG96185 Sierra 6/16/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3769.4, complete

genome AIG96194 Sierra 6/12/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3770.1 , complete

genome

AIG96203 Sierra 6/14/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3770.2, complete

genome

AIG96212 Sierra 6/12/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3771, complete genome

AIG96221 Sierra 6/14/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3782, complete genome

AIG96230 Sierra 6/14/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3786, complete genome

AIG96239 Sierra 6/14/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3787, complete genome

AIG96248 Sierra 6/14/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3788, complete genome

AIG96257 Sierra 6/14/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3789.1 , complete

genome

AIG96266 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3795, complete genome

AIG96275 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3796, complete genome

AIG96284 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3798, complete genome

AIG96293 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3799, complete genome

AIG96302 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3800, complete genome

AIG96311 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3805.1 , complete

genome

AIG96320 Sierra 6/20/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3805.2, complete

genome

AIG96329 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3807, complete genome

AIG96338 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3808, complete genome

AIG96347 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3809, complete genome

AIG96356 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3810.1, complete

genome

AIG96365 Sierra 6/17/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3810.2, complete

genome

AIG96374 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3814, complete genome

AIG96383 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3816, complete genome

AIG96392 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3817, complete genome AIG96401 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3818, complete genome

AIG96410 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3819, complete genome

AIG96419 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3820, complete genome

AIG96428 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3821, complete genome

AIG96437 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3822, complete genome

AIG96446 Sierra 6/15/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3823, complete genome

AIG96455 Sierra 6/16/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3825.1 , complete

genome

AIG96464 Sierra 6/17/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3825.2, complete

genome

AIG96473 Sierra 6/16/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3826, complete genome

AIG96482 Sierra 6/16/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3827, complete genome

AIG96491 Sierra 6/16/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3829, complete genome

AIG96500 Sierra 6/16/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3831, complete genome

AIG96509 Sierra 6/17/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3834, complete genome

AIG96518 Sierra 6/17/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3838, complete genome

AKC35925 Sierra 6/16/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3838.2, partial genome

AIG96527 Sierra 6/17/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3840, complete genome

AIG96536 Sierra 6/17/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3841, complete genome

AIG96545 Sierra 6/18/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3845, complete genome

AKC35934 Sierra 6/19/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3845.2, partial genome

AIG96554 Sierra 6/18/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3846, complete genome

AIG96563 Sierra 6/18/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3848, complete genome

AIG96572 Sierra 6/18/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3850, complete genome

AIG96581 Sierra 6/18/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3851, complete genome

AKC35943 Sierra 6/18/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3851.2, partial genome

AKC35952 Sierra 6/17/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3855.2, partial genome

AIG96590 Sierra 6/18/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3856.1 , complete genome

AIG96599 Sierra 6/20/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3856.3, complete

genome

AIG96608 Sierra 6/18/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3857, complete genome

AKA43781 Sierra 4/1/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3864.1 , complete

genome

AKC35961 Sierra 6/19/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3886.1, partial genome

AKC35970 Sierra 6/19/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3889.1, partial genome

AKC35979 Sierra 6/21/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3913.1, partial genome

AKC35988 Sierra 6/21/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3917.1, partial genome

AKC35997 Sierra 6/22/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3926.2, partial genome

AKC36006 Sierra 6/24/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3949.1, partial genome

AKC36015 Sierra 6/24/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3950.1, partial genome

AKC36024 Sierra 6/25/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3952.1, partial genome

AKC36033 Sierra 6/24/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G3972.1, partial genome

AKC36042 Sierra 7/5/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4132.1, partial genome

AKC36051 Sierra 7/4/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4133.1, partial genome

AKC36060 Sierra 7/6/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4151.1, partial genome

AKC36069 Sierra 7/7/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4190.1, partial genome

AKC36078 Sierra 7/8/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4200.1, partial genome

AKC36087 Sierra 7/8/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4217.1, partial genome

AKC36096 Sierra 7/9/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4221.1, partial genome

AKC36105 Sierra 7/11/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4235.1, partial genome

AKC36114 Sierra 7/10/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4236.1, partial genome

AKC36123 Sierra 7/11/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4250.1, partial genome

AKC36132 Sierra 7/11/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4251.1, partial genome

AKC36141 Sierra 7/11/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4252.1, partial genome

AKC36150 Sierra 7/11/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4254.1, partial genome AKC36159 Sierra 7/11/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4255.1 , partial genome

AKC36168 Sierra 7/11/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4263.1 , partial genome

AKC36177 Sierra 7/11/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4264.1 , partial genome

AKC36186 Sierra 7/12/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4299.1 , partial genome

AKC36195 Sierra 7/12/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4312.2, partial genome

AKC36204 Sierra 7/13/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4316.1 , partial genome

AKC36213 Sierra 7/14/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4323.2, partial genome

AKC36222 Sierra 7/14/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4324.1 , partial genome

AKC36231 Sierra 7/14/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4325.1 , partial genome

AKC36240 Sierra 7/14/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4329.1 , partial genome

AKC36249 Sierra 7/13/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4333.1 , partial genome

AKC36258 Sierra 7/14/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4334.1 , partial genome

AKC36267 Sierra 7/14/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4337.1 , partial genome

AKC36276 Sierra 7/18/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4345.1 , partial genome

AKC36285 Sierra 7/15/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4347.1 , partial genome

AKC36294 Sierra 7/9/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4348.1 , partial genome

AKC36303 Sierra 7/14/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4350.1 , partial genome

AKC36312 Sierra 7/15/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4380.1 , partial genome

AKC36321 Sierra 7/16/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4382.1 , partial genome

AKC36330 Sierra 7/16/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4389.1 , partial genome

AKC36339 Sierra 7/18/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4406.1 , partial genome

AKC36348 Sierra 7/18/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4415.1 , partial genome

AKC36357 Sierra 7/18/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4416.1 , partial genome

AKC36366 Sierra 7/19/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4419.1 , partial genome

AKC36375 Sierra 7/19/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4422.1 , partial genome

AKC36384 Sierra 7/19/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4423.1 , partial genome

AKC36393 Sierra 7/19/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4424.1 , partial genome AKC36402 Sierra 7/20/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4431.1, partial genome

AKC36411 Sierra 7/20/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4433.1, partial genome

AKC36420 Sierra 7/20/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4437.1, partial genome

AKC36429 Sierra 7/21/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4445.1, partial genome

AKC36438 Sierra 7/21/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4450.1, partial genome

AKC36447 Sierra 7/21/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4454.1, partial genome

AKC36456 Sierra 8/5/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4465.1, partial genome

AKC36465 Sierra 7/22/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4466.1, partial genome

AKC36474 Sierra 7/26/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4527.2, partial genome

AKC36483 Sierra 8/1/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4683.1, partial genome

AKC36492 Sierra 8/3/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4698.1, partial genome

AKC36501 Sierra 8/3/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4701.1, partial genome

AKC36510 Sierra 8/3/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4702.1, partial genome

AKC36519 Sierra 8/4/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4717.1, partial genome

AKC36528 Sierra 8/4/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4725.1, partial genome

AKC36537 Sierra ¾£§ 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone 5/2014 wt/SLE/2014/Makona-G4730.1, partial genome

AKC36546 Sierra 8/5/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4736.1, partial genome

AKC36555 Sierra 8/5/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4748.1, partial genome

AKC36564 Sierra 8/5/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4751.1, partial genome

AKC36572 Sierra 8/9/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4837.1, partial genome

AKC36581 Sierra 8/10/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4856.1, partial genome

AKC36590 Sierra 8/10/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4861.1, partial genome

AKC36599 Sierra 8/10/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4868.1, partial genome

AKC36608 Sierra 8/11/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4886.1, partial genome

AKC36617 Sierra 8/12/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4907.1, partial genome

AKC36626 Sierra 8/13/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4937.1, partial genome

AKC36635 Sierra 8/12/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4942.1, partial genome AKC36644 Sierra 8/13/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4946.1, partial genome

AKC36653 Sierra 8/13/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4955.1, partial genome

AKC36662 Sierra 8/13/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4956.1, partial genome

AKC36671 Sierra 8/14/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4960.1, partial genome

AKC36680 Sierra 8/14/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4971.1, partial genome

AKC36689 Sierra 8/14/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4972.1, partial genome

AKC36698 Sierra 8/12/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4973.1, partial genome

AKC36707 Sierra 8/14/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4981.1, partial genome

AKC36716 Sierra 8/14/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4982.1, partial genome

AKC36725 Sierra 8/15/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4994.1, partial genome

AKC36734 Sierra 8/15/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4996.1, partial genome

AKC36743 Sierra 8/15/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G4999.1, partial genome

AKC36752 Sierra 8/15/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5012.3, partial genome

AKC36761 Sierra 8/16/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5016.1, partial genome

AKC36770 Sierra 8/16/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5019.1, partial genome

AKC36779 Sierra 8/17/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5039.1, partial genome

AKC36788 Sierra 8/18/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5059.1, partial genome

AKC36797 Sierra 8/17/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5064.1, partial genome

AKC36806 Sierra 8/19/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5112.1, partial genome

AKC36815 Sierra 8/18/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5114.1, partial genome

AKC36824 Sierra 8/24/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5119.1, partial genome

AKC36833 Sierra 8/22/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5244.1, partial genome

AKC36842 Sierra 8/25/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5295.1, partial genome

AKC36851 Sierra 8/25/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5296.1, partial genome

AKC36860 Sierra 8/28/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5304.1, partial genome

AKC36869 Sierra 8/28/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5364.1, partial genome

AKC36878 Sierra 8/28/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5370.1, partial genome AKC36887 Sierra 9/4/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5516.1, partial genome

AKC36896 Sierra 9/5/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5520.1, partial genome

AKC36905 Sierra 9/5/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5529.1, partial genome

AKC36914 Sierra 9/8/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5570.1, partial genome

AKC36923 Sierra 9/8/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5571.1, partial genome

AKC36932 Sierra 9/10/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5617.1, partial genome

AKC36941 Sierra 9/11/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5640.1, partial genome

AKC36950 Sierra 9/11/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5644.1, partial genome

AKC36959 Sierra 9/11/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5647.1, partial genome

AKC36968 Sierra 9/13/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5684.1, partial genome

AKC36977 Sierra 9/13/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5685.1, partial genome

AKC36986 Sierra 9/14/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5691.1, partial genome

AKC36995 Sierra 9/15/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5723.1, partial genome

AKC37004 Sierra 9/16/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5731.1, partial genome

AKC37013 Sierra 9/16/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5737.1, partial genome

AKC37022 Sierra 9/15/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5738.1, partial genome

AKC37031 Sierra 9/17/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5743.1, partial genome

AKC37040 Sierra 9/18/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5756.1, partial genome

AKC37049 Sierra 9/16/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5763.1, partial genome

AKC37058 Sierra 9/16/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5765.1, partial genome

AKC37067 Sierra 9/16/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5767.1, partial genome

AKC37076 Sierra 9/21/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5844.1, partial genome

AKC37085 Sierra 9/21/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5853.1, partial genome

AKC37094 Sierra 9/22/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5879.1, partial genome

AKC37103 Sierra 9/22/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5898.1, partial genome

AKC37112 Sierra 9/25/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5982.1, partial genome

AKC37121 Sierra 9/25/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5983.1, partial genome AKC37139 Sierra 9/25/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5986.1, partial genome

AKC37148 Sierra 9/25/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5988.1, partial genome

AKC37157 Sierra 9/25/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5996.1, partial genome

AKC37166 Sierra 9/25/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5997.1, partial genome

AKC37175 Sierra 9/25/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G5998.1, partial genome

AKC37184 Sierra 9/25/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G6012.1, partial genome

AKC37193 Sierra 9/25/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G6020.1, partial genome

AKC37202 Sierra 9/25/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G6060.1, partial genome

AKC37211 Sierra 9/25/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G6062.1, partial genome

AKC37220 Sierra 9/25/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G6069.1, partial genome

AKC37229 Sierra 9/27/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G6089.1, partial genome

AKC37238 Sierra 9/27/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G6091.1, partial genome

AKC37247 Sierra 9/27/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G6095.1, partial genome

AKC37256 Sierra 9/28/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G6103.1, partial genome

AKC37265 Sierra 9/28/2014 4/19/2015 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-G6104.1, partial genome

AIG96617 Sierra 6/4/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-NM042.1 , complete genome

AIG96626 Sierra 6/9/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-NM042.2, complete genome

AIG96635 Sierra 6/12/2014 7/25/2014 Zaire ebolavirus isolate Ebola virus/H.sapiens- Leone wt/SLE/2014/Makona-NM042.3 , complete genome

AJP14319 Sierra 9/27/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H.sapiens- Leone wt/SLE/2014/Makona-J0001, partial genome

AJP14355 Sierra 9/27/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H.sapiens- Leone wt/SLE/2014/Makona-J0002, partial genome

AJP14453 Sierra 9/28/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H.sapiens- Leone wt/SLE/2014/Makona-J0003, partial genome

AJP 14606 Sierra 9/28/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H.sapiens- Leone wt/SLE/2014/Makona-J0004, partial genome

AJP 14247 Sierra 9/26/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H.sapiens- Leone wt/SLE/2014/Makona-J0005, partial genome

AJP 14265 Sierra 9/28/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H.sapiens- Leone wt/SLE/2014/Makona-J0006, partial genome

AJP 14274 Sierra 9/27/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H.sapiens- Leone wt/SLE/2014/Makona-J0007, partial genome AJP15056 Sierra 9/29/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0008, partial genome

AJP 15200 Sierra 9/28/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0009, partial genome

AJP 14346 Sierra 9/28/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0010, partial genome

AJP14364 Sierra 9/29/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0011, partial genome

AJP 14373 Sierra 9/29/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0012, partial genome

AJP 14382 Sierra 9/28/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0013, partial genome

AJP 15254 Sierra 9/29/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0014, partial genome

AJP 15263 Sierra 9/26/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0015, partial genome

AJP 15272 Sierra 9/30/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0016, partial genome

AJP15317 Sierra 9/26/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0017, partial genome

AJP 14391 Sierra 9/30/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0018, partial genome

AJP 15380 Sierra 9/25/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0019, partial genome

AJP 15389 Sierra 9/25/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0020, partial genome

AJP15398 Sierra 9/29/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0021, partial genome

AJP 15407 Sierra 9/25/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0022, partial genome

AJP 14462 Sierra 10/1/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0023, partial genome

AJP14561 Sierra 9/29/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0024, partial genome

AJP14588 Sierra 10/2/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0025, partial genome

AJP 14624 Sierra 10/2/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0026, partial genome

AJP 14696 Sierra 10/2/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0027, partial genome

AJP 14741 Sierra 10/2/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0028, partial genome

AJP14786 Sierra 9/29/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0029, partial genome

AJP 14049 Sierra 10/3/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0030, partial genome

AJP 14058 Sierra 10/3/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0031, partial genome

AJP14813 Sierra 9/30/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0032, partial genome

AJP 14822 Sierra 10/3/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0033, partial genome

AJP 14067 Sierra 9/30/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0034, partial genome AJP 14076 Sierra 10/1/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0035, partial genome

AJP 14840 Sierra 10/3/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0036, partial genome

AJP14130 Sierra 10/3/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0037, partial genome

AJP14157 Sierra 9/30/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0038, partial genome

AJP14175 Sierra 10/3/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0039, partial genome

AJP14256 Sierra 10/4/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0040, partial genome

AJP14984 Sierra 10/4/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0041, partial genome

AJP 14993 Sierra 10/4/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0042, partial genome

AJP 15002 Sierra 10/4/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0043, partial genome

AJP15011 Sierra 10/5/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0044, partial genome

AJP 15020 Sierra 10/5/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0045, partial genome

AJP 15029 Sierra 10/7/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0046, partial genome

AJP14283 Sierra 10/5/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0047, partial genome

AJP 15038 Sierra 10/5/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0048, partial genome

AJP 15047 Sierra 10/7/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0049, partial genome

AJP 15065 Sierra 10/5/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0050, partial genome

AJP 14292 Sierra 10/7/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0051, partial genome

AJP 15074 Sierra 10/8/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0052, partial genome

AJP15083 Sierra 10/6/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0053, partial genome

AJP 15092 Sierra 10/6/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0054, partial genome

AJP15101 Sierra 10/6/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0055, partial genome

AJP 14301 Sierra 10/8/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0056, partial genome

AJP14310 Sierra 10/5/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0057, partial genome

AJP15110 Sierra 10/8/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0058, partial genome

AJP15119 Sierra 10/8/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0059, partial genome

AJP15128 Sierra 10/6/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0060, partial genome

AJP 14328 Sierra 10/10/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0061, partial genome AJP15137 Sierra 10/10/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0062, partial genome

AJP14337 Sierra 10/10/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0063, partial genome

AJP15146 Sierra 10/9/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0064, partial genome

AJP15155 Sierra 10/9/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0065, partial genome

AJP15164 Sierra 10/10/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0066, partial genome

AJP15173 Sierra 10/9/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0067, partial genome

AJP15182 Sierra 10/9/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0068, partial genome

AJP15191 Sierra 10/9/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0069, partial genome

AJP 15209 Sierra 10/9/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0070, partial genome

AJP15218 Sierra 10/9/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0071, partial genome

AJP 15227 Sierra 10/9/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0072, partial genome

AJP15236 Sierra 10/12/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0073, partial genome

AJP 15245 Sierra 10/13/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0074, partial genome

AJP 15281 Sierra 10/17/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0075, partial genome

AJP 15290 Sierra 10/17/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0076, partial genome

AJP 15299 Sierra 10/17/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0077, partial genome

AJP 15308 Sierra 10/18/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0078, partial genome

AJP 15326 Sierra 10/16/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0079, partial genome

AJP15335 Sierra 10/18/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0080, partial genome

AJP 15344 Sierra 10/16/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0081, partial genome

AJP15353 Sierra 10/16/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0082, partial genome

AJP15362 Sierra 10/18/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0083, partial genome

AJP 15371 Sierra 10/16/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0084, partial genome

AJP 14400 Sierra 10/17/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0085, partial genome

AJP14417 Sierra 10/23/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0087, partial genome

AJP 14426 Sierra 10/20/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0088, partial genome

AJP15416 Sierra 10/23/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0089, partial genome AJP14435 Sierra 10/23/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0090, partial genome

AJP 15425 Sierra 10/25/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0091, partial genome

AJP 14444 Sierra 10/25/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0092, partial genome

AJP 15434 Sierra 10/24/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0093, partial genome

AJP 15443 Sierra 10/27/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0094, partial genome

AJP 14471 Sierra 10/27/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0095, partial genome

AJP 14480 Sierra 10/27/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0096, partial genome

AJP 15460 Sierra 10/28/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0098, partial genome

AJP 15469 Sierra 10/28/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0099, partial genome

AJP 14489 Sierra 10/26/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0100, partial genome

AJP14498 Sierra 10/29/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0101, partial genome

AJP 14507 Sierra 10/27/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0102, partial genome

AJP14516 Sierra 10/29/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0103, partial genome

AJP15478 Sierra 10/29/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0104, partial genome

AJP 15487 Sierra 10/29/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0105, partial genome

AJP 15496 Sierra 10/28/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0106, partial genome

AJP 15505 Sierra 10/29/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0107, partial genome

AJP 14525 Sierra 10/28/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0108, partial genome

AJP 13941 Sierra 10/31/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0109, partial genome

AJP14534 Sierra 10/31/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0110, partial genome

AJP 13950 Sierra 10/30/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0111, partial genome

AJP 14543 Sierra 10/31/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0112, partial genome

AJP14552 Sierra 10/30/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0113, partial genome

AJP 13959 Sierra 10/29/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0114, partial genome

AJP13968 Sierra 10/29/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0115, partial genome

AJP 14570 Sierra 10/30/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0116, partial genome

AJP 14579 Sierra 10/31/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0117, partial genome AJP 13977 Sierra 10/31/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0118, partial genome

AJP 14597 Sierra 10/30/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0119, partial genome

AJP13986 Sierra 10/30/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0120, partial genome

AJP14615 Sierra 10/29/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0121, partial genome

AJP14633 Sierra 10/29/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0122, partial genome

AJP 14642 Sierra 11/1/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0123, partial genome

AJP 14651 Sierra 11/2/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0124, partial genome

AJP 14660 Sierra 10/31/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0125, partial genome

AJP 14669 Sierra 10/31/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0126, partial genome

AJP14678 Sierra 10/30/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0127, partial genome

AJP14687 Sierra 10/31/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0128, partial genome

AJP 13995 Sierra 11/2/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0129, partial genome

AJP 14705 Sierra 11/2/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0130, partial genome

AJP14714 Sierra 10/30/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0131, partial genome

AJP 14004 Sierra 11/2/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0132, partial genome

AJP 14723 Sierra 10/31/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0133, partial genome

AJP 14732 Sierra 10/31/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0134, partial genome

AJP14013 Sierra 11/3/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0135, partial genome

AJP 14750 Sierra 11/1/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0136, partial genome

AJP 14759 Sierra 11/1/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0137, partial genome

AJP14768 Sierra 11/1/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0138, partial genome

AJP 14777 Sierra 10/30/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0139, partial genome

AJP 14022 Sierra 10/30/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0140, partial genome

AJP 14795 Sierra 11/4/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0141, partial genome

AJP 14804 Sierra 11/3/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0142, partial genome

AJP 14031 Sierra 11/4/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0143, partial genome

AJP 14040 Sierra 11/4/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0144, partial genome AJP14831 Sierra 11/6/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0145, partial genome

AJP14085 Sierra 11/7/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0146, partial genome

AJP 14094 Sierra 11/7/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0147, partial genome

AJP14103 Sierra 11/6/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0148, partial genome

AJP 14849 Sierra 11/7/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0149, partial genome

AJP14858 Sierra 11/6/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0150, partial genome

AJP14112 Sierra 11/7/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0151, partial genome

AJP14121 Sierra 11/7/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0152, partial genome

AJP14139 Sierra 11/7/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0153, partial genome

AJP14148 Sierra 11/7/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0154, partial genome

AJP14867 Sierra 11/7/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0155, partial genome

AJP 14876 Sierra 11/7/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0156, partial genome

AJP14885 Sierra 11/6/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0157, partial genome

AJP 14894 Sierra 11/8/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0158, partial genome

AJP14166 Sierra 11/5/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0159, partial genome

AJP14184 Sierra 11/7/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0160, partial genome

AJP 14903 Sierra 11/8/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0161, partial genome

AJP14193 Sierra 11/9/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0162, partial genome

AJP14912 Sierra 11/8/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0163, partial genome

AJP 14202 Sierra 11/9/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0164, partial genome

AJP 14921 Sierra 11/8/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0165, partial genome

AJP 14930 Sierra 11/8/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0166, partial genome

AJP14211 Sierra 11/8/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0167, partial genome

AJP 14939 Sierra 11/11/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0168, partial genome

AJP 14948 Sierra 11/9/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0169, partial genome

AJP 14220 Sierra 11/10/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0170, partial genome

AJP 14957 Sierra 11/10/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H. sapiens- Leone wt/SLE/2014/Makona-J0171, partial genome AJP 14229 Sierra 11/11/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H.sapiens- Leone wt/SLE/2014/Makona-J0172, partial genome

AJP14238 Sierra 11/11/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H.sapiens- Leone wt/SLE/2014/Makona-J0173, partial genome

AJP 14966 Sierra 11/11/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H.sapiens- Leone wt/SLE/2014/Makona-J0174, partial genome

AJP 14975 Sierra 11/11/2014 3/1/2015 Zaire ebolavirus isolate Ebolavirus/H.sapiens- Leone wt/SLE/2014/Makona-J0175, partial genome

AIW47453 Guinea 2014/03 11/10/2014 Zaire ebolavirus isolate H.sapiens- tc/GIN/14/WPG-C05, complete genome

AIW47461 Guinea 2014/03 11/10/2014 Zaire ebolavirus isolate H.sapiens- tc/GIN/14/WPG-C07, complete genome

AIW47469 Guinea 2014/03 11/10/2014 Zaire ebolavirus isolate H.sapiens- tc/GIN/14/WPG-C15, complete genome

ALH21455 Guinea 2014 10/10/2015 Zaire ebolavirus isolate H.sapiens- wt/GIN/2014/Makona-Conakry-CREMS - 1022, complete genome

ALH21464 Guinea 2014 10/10/2015 Zaire ebolavirus isolate H.sapiens- wt/GIN/2014/Makona-Conakry-CREMS -2214, complete genome

AHX24668 Guinea 2014 4/18/2014 Zaire ebolavirus isolate H.sapiens- wt/GIN/2014/Makona-Gueckedou-C05 , complete genome

AHX24659 Guinea 2014 4/18/2014 Zaire ebolavirus isolate H.sapiens- wt/GIN/2014/Makona-Gueckedou-C07, complete genome

AHX24650 Guinea 2014 4/18/2014 Zaire ebolavirus isolate H.sapiens- wt/GIN/2014/Makona-Kissidougou-C 15 , complete genome

AJT59735 Democratic 2003 3/17/2015 Zaire ebolavirus isolate Kelle 1 NP protein (NP),

Republic of VP35 protein (VP35), VP40 protein (VP40), GP the Congo protein (GP), VP30 protein (VP30), VP24 protein

(VP24), and L protein (L) genes, complete cds

AER59717 Democratic 8/31/2007 11/7/2011 Zaire ebolavirus isolate M-M, partial genome

Republic of

the Congo

AKI84266 Democratic 1995 6/2/2015 Zaire ebolavirus isolate Zaire ebolavirus H.

Republic of sapiens-tc/ZAI/1995/Zaire- 199510621 , partial the Congo genome

AKI84257 Sierra 2014 6/2/2015 Zaire ebolavirus isolate Zaire

Leone ebolavirus/H.sapiens-tc/SL/2014/Makona- SL3864.1, partial genome

Existing Ebola vaccines include conventional inactivated (by heat, formalin, or γ- irradiation) viral vaccines, sub-unit Ebola virus genes inserted into a DNA plasmid and

Virus-like-particles (VLP) based on VP40 alone or with GP. Studies using conventional inactivated and DNA vaccines have shown consistently low survival rates in non-human primates. The mRNA Ebola vaccines of the invention have unique advantages over conventional vaccines. As shown in the data presented in the Examples the constructs of the invention provided 100% protection against infection in an animal model of Ebola.

The fourth gene of the Ebola genome encodes a 160-kDa envelope-attached glycoprotein (GP) and a 110 kDa secreted glycoprotein (sGP). Both GP and sGP have an identical 295-residue N-terminus, however, they have different C-terminal sequences. GP is a class I fusion protein which assembles as trimers on viral surface and plays an important role in virus entry and attachment. Mature GP is a disulfide-linked heterodimer formed by two subunits, GP1 and GP2, which are generated from the proteolytic process of GP precursor (pre-GP) by cellular furin during virus assembly. The GP1 subunit contains a mucin domain (Muc) and a receptor-binding domain (RBD); the GP2 subunit has a fusion peptide, a helical heptad-repeat (HR) region, a transmembrane (TM) domain, and a 4-residue cytoplasmic tail. The RBD of GP1 mediates the interaction of Ebola virus with cellular receptors such as DC- SIGN/LSIGN, TIM-1, hMGL, NPC1, β-integrins, folate receptor-a, and Tyro3 family receptors. The mucin domain has N- and O-linked glycans and enhances the viral attachment to cellular hMGL, and participates in shielding key neutralization epitopes, which helps the virus evade host immune responses.

The entire contents of International Application No. PCT/US2015/02740 is incorporated herein by reference. Nucleic Acids/Polynucleotides

Ebola virus vaccines, as provided herein, comprise at least one (one or more) RNA {e.g., mRNA) polynucleotide having an open reading frame encoding at least one Ebola virus antigenic polypeptide. 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.

Nucleic acids (also referred to as polynucleotides) may be or may include, for example, 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, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino- a- 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 function as messenger RNA (mRNA). "Messenger RNA" (mRNA) refers to any polynucleotide that encodes a (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.

The basic components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly- A tail. Polynucleotides of the present disclosure may function as mRNA but can be distinguished from wild-type mRNA in their functional and/or structural design features which serve to overcome existing problems of effective polypeptide expression using nucleic-acid based therapeutics.

In some embodiments, a RNA polynucleotide of an Ebola virus 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 an Ebola virus vaccine encodes at least 10, 20, 30, 40, 50 , 60, 70, 80, 90 or 100 antigenic polypeptides. In some embodiments, a RNA polynucleotide of an Ebola virus vaccine encodes at least 100 or at least 200 antigenic polypeptides. In some embodiments, a RNA polynucleotide of an Ebola virus 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.

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 CA) 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 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, an Ebola virus 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 term "polypeptide variant" refers to molecules which differ in their amino acid sequence from a native 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 or reference sequence. Ordinarily, variants possess at least 50% identity to a native or reference sequence. In some embodiments, variants share at least 80%, or at least 90% identity with a native or reference sequence.

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.

"Orthologs" refers to genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. Identification of orthologs is critical for reliable prediction of gene function in newly sequenced genomes.

"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 polynucleotide or 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. For example, sequence tags or amino acids, such as one or more lysines, can be added to peptide sequences (e.g. , at the N-terminal or C-terminal ends).

Sequence tags can be used for peptide detection, purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. 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.

"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. In another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 amino acids that are greater than 80%, 90%, 95%, or 100% identical to any of the sequences described herein, wherein the protein has a stretch of 5, 10, 15, 20, 25, or 30 amino acids that are less than 80%, 75%, 70%, 65% or 60% identical to any of the sequences described herein can be utilized in accordance with the disclosure.

Polypeptide or polynucleotide molecules of the present disclosure may share a certain degree of sequence similarity or identity with the reference molecules (e.g. , reference polypeptides or reference polynucleotides), for example, with art-described molecules (e.g. , engineered or designed molecules or wild-type molecules). The term "identity" as known in the art, refers to a relationship between the sequences of two or more 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 nucleic acid residues. 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. "% identity" as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. It is understood that identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. 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). Another popular local alignment technique is based on the Smith- Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981) "Identification of common molecular subsequences." J. Mol. Biol. 147: 195-197). A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, CD. (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48:443-453.). 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. Polymeric molecules (e.g. nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or polypeptide molecules) that share a threshold level of similarity or identity determined by alignment of matching residues are termed homologous. Homology is a qualitative term that describes a relationship between molecules and can be based upon the quantitative similarity or identity. Similarity or identity is a quantitative term that defines the degree of sequence match between two compared sequences. 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.

Homology implies that the compared sequences diverged in evolution from a common origin. The term "homolog" refers to a first amino acid sequence or nucleic acid sequence (e.g. , gene (DNA or RNA) or protein sequence) that is related to a second amino acid sequence or nucleic acid sequence by descent from a common ancestral sequence. The term "homolog" may apply to the relationship between genes and/or proteins separated by the event of speciation or to the relationship between genes and/or proteins separated by the event of genetic duplication. "Orthologs" are genes (or proteins) in different species that evolved from a common ancestral gene (or protein) by speciation. Typically, orthologs retain the same function in the course of evolution. "Paralogs" are genes (or proteins) related by duplication within a genome. Orthologs retain the same function in the course of evolution, whereas paralogs evolve new functions, even if these are related to the original one.

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 polynucleic acid 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 nucleic acid 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 nucleic acid 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 nucleic acid 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 ah, Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al, J. Molec. Biol, 215, 403 (1990)).

Chemical Modifications

RNA {e.g., mRNA) vaccines of the present disclosure comprise at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one Ebola virus 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.

Polypeptides, 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 intemucleotide 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 phosphdioester 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 "LP'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- threonylcarbamoyladenosine; l,2'-0-dimethyladenosine; 1-methyladenosine; 2'-0- methyladenosine; 2'-0-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine; 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine; 2'-0- methyladenosine; 2'-0-ribosyladenosine (phosphate); Isopentenyladenosine; N6-(cis- hydroxyisopentenyl)adenosine; N6,2'-0-dimethyladenosine; N6,2'-0-dimethyladenosine; N6,N6,2'-0-trimethyladenosine; N6,N6-dimethyladenosine; N6-acetyladenosine; N6- hydroxynorvalylcarbamoyladenosine; N6-methyl-N6-threonylcarbamoyladenosine; 2- methyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine; N 1-methyladenosine; N6, N6 (dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; a-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; 2'0-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'-0-methylcytidine; 2'-0- methylcytidine; 5,2'-0-dimethylcytidine; 5-formyl-2'-0-methylcytidine; Lysidine; N4,2'-0- dimethylcytidine; N4-acetyl-2'-0-methylcytidine; N4-methylcytidine; N4,N4-Dimethyl-2'- OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine; Pseudo-iso-cytidine; pyrrolo-cytidine; a-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'-0- 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-l- methyl-pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-thio-l -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'-0-Methyl-N4-Acetyl-cytidine TP; 2'0-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'-0-Methyl-5-(l- propynyl)cytidine TP; 3'-Ethynylcytidine TP; 4'-Azidocytidine TP; 4'-Carbocyclic cytidine TP; 4'-Ethynylcytidine TP; 5-(l-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'-0-dimethylguanosine; N2- methylguanosine; Wyosine; l,2'-0-dimethylguanosine; 1-methylguanosine; 2'-0- methylguanosine; 2'-0-ribosylguanosine (phosphate); 2'-0-methylguanosine; 2'-0- ribosylguanosine (phosphate); 7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine; Methylwyosine; N2,7-dimethylguanosine; N2,N2,2'-0-trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine; N2,7,2'-0-trimethylguanosine; 6- thio-guanosine; 7-deaza-guanosine; 8-oxo-guanosine; Nl-methyl-guanosine; a-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; l-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; 2'0-methyl-N2-isobutyl-guanosine TP; 2'-a-Ethynylguanosine TP; 2'-a- Trifluoromethylguanosine TP; 2'-b-Ethynylguanosine 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; l,2'-0- dimethylinosine; 2'-0-methylinosine; 7-methylinosine; 2'-0-methylinosine; Epoxyqueuosine; galactosyl-queuosine; Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deaza thymidine; deoxy-thymidine; 2'-0-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; l-methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1-methylpseduouridine; 1-methyl- pseudouridine; 2'-0-methyluridine; 2'-0-methylpseudouridine; 2'-0-methyluridine; 2-thio-2'- O-methyluridine; 3-(3-amino-3-carboxypropyl)uridine; 3,2'-0-dimethyluridine; 3-Methyl- pseudo-Uridine TP; 4-thiouridine; 5-(carboxyhydroxymethyl)uridine; 5- (carboxyhydroxymethyl)uridine methyl ester; 5,2'-0-dimethyluridine; 5,6-dihydro-uridine; 5- aminomethyl-2-thiouridine; 5-carbamoylmethyl-2'-0-methyluridine; 5- carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine methyl ester; 5-carboxymethylaminomethyl-2'-0-methyluridine; 5- carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyl-2-thiouridine; 5- carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5- Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2'-0-methyluridine; 5- methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5-methyluridine), 5-methoxyuridine; 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; Nl-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'-0-methyluridine TP; 5-(iso-Pentenylaminomethyl)uridine TP; 5- propynyl uracil; a-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; l-(aminoalkylamino-carbonylethylenyl)-2- (thio)-pseudouracil; l-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP; l-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-2'-a-azidouridine TP; 2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4 (thio)pseudouracil; 4-(thio )pseudouracil; 4-(thio)uracil; 4-thiouracil; 5 (l,3-diazole-l-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-(l,3-diazole-l-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; P seudo-UTP-l-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-l -methyl- 1-deaza-pseudouridine; 2- thio-l-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-l-methyl-pseudouridine; 4-thio-pseudouridine; 5-aza- uridine;

Dihydropseudouridine; (+)l-(2-Hydroxypropyl)pseudouridine TP; (2R)-l-(2- Hydroxypropyl)pseudouridine TP; (2S)-l-(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; l-(2,2,2-Trifluoroethyl)-pseudo-UTP; 1- (2,2,3, 3,3-Pentafluoropropyl)pseudouridine TP; l-(2,2-Diethoxyethyl)pseudouridine TP; 1- (2,4,6-Trimethylbenzyl)pseudouridine TP; l-(2,4,6-Trimethyl-benzyl)pseudo-UTP; l-(2,4,6- Trimethyl-phenyl)pseudo-UTP; l-(2-Amino-2-carboxyethyl)pseudo-UTP; l-(2-Amino- ethyl)pseudo-UTP; l-(2-Hydroxyethyl)pseudouridine TP; l-(2-Methoxyethyl)pseudouridine TP; l-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP; l-(3,4- Dimethoxybenzyl)pseudouridine TP; l-(3-Amino-3-carboxypropyl)pseudo-UTP; l-(3- Amino-propyl)pseudo-UTP; l-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP; l-(4-Amino- 4-carboxybutyl)pseudo-UTP; l-(4-Amino-benzyl)pseudo-UTP; l-(4-Amino-butyl)pseudo- UTP; l-(4-Amino-phenyl)pseudo-UTP; l-(4-Azidobenzyl)pseudouridine TP; l-(4- Bromobenzyl)pseudouridine TP; l-(4-Chlorobenzyl)pseudouridine TP; l-(4- Fluorobenzyl)pseudouridine TP; l-(4-Iodobenzyl)pseudouridine TP; l-(4- Methanesulfonylbenzyl)pseudouridine TP; l-(4-Methoxybenzyl)pseudouridine TP; l-(4- Methoxy-benzyl)pseudo-UTP; l-(4-Methoxy-phenyl)pseudo-UTP; l-(4- Methylbenzyl)pseudouridine TP; l-(4-Methyl-benzyl)pseudo-UTP; l-(4- Nitrobenzyl)pseudouridine TP; l-(4-Nitro-benzyl)pseudo-UTP; l(4-Nitro-phenyl)pseudo- UTP; l-(4-Thiomethoxybenzyl)pseudouridine TP; l-(4- Trifluoromethoxybenzyl)pseudouridine TP; l-(4-Trifluoromethylbenzyl)pseudouridine TP; l-(5-Amino-pentyl)pseudo-UTP; l-(6-Amino-hexyl)pseudo-UTP; 1,6-Dimethyl-pseudo- UTP; l-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridine TP; l-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl } pseudouridine TP; 1-Acetylpseudouridine TP; l-Alkyl-6-(l-propynyl)-pseudo-UTP; l-Alkyl-6-(2-propynyl)-pseudo-UTP; l-Alkyl-6-allyl- pseudo-UTP; l-Alkyl-6-ethynyl-pseudo-UTP; l-Alkyl-6-homoallyl-pseudo-UTP; l-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; l-Me-2-thio-pseudo-UTP; l-Me-4-thio-pseudo-UTP; 1-Me-alpha- thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine TP; 1-

Methoxymethylpseudouridine TP; l-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP; 1-Methyl- 6-(4-morpholino)-pseudo-UTP; l-Methyl-6-(4-thiomorpholino)-pseudo-UTP; l-Methyl-6- (substituted phenyl)pseudo-UTP; l-Methyl-6-amino-pseudo-UTP; l-Methyl-6-azido-pseudo- UTP; l-Methyl-6-bromo-pseudo-UTP; l-Methyl-6-butyl-pseudo-UTP; l-Methyl-6-chloro- pseudo-UTP; l-Methyl-6-cyano-pseudo-UTP; l-Methyl-6-dimethylamino-pseudo-UTP; 1- Methyl-6-ethoxy-pseudo-UTP; l-Methyl-6-ethylcarboxylate-pseudo-UTP; l-Methyl-6-ethyl- pseudo-UTP; l-Methyl-6-fluoro-pseudo-UTP; l-Methyl-6-formyl-pseudo-UTP; l-Methyl-6- hydroxyamino-pseudo-UTP; l-Methyl-6-hydroxy-pseudo-UTP; l-Methyl-6-iodo-pseudo- UTP; l-Methyl-6-iso-propyl-pseudo-UTP; l-Methyl-6-methoxy-pseudo-UTP; l-Methyl-6- methylamino-pseudo-UTP; l-Methyl-6-phenyl -pseudo-UTP; l-Methyl-6-propyl-pseudo- UTP; l-Methyl-6-tert-butyl-pseudo-UTP; l-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; l-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-2'-a-mercaptouridine TP; 2'-Deoxy-2'-a-thiomethoxyuridine TP; 2'-Deoxy-2'-b-aminouridine TP; 2'-Deoxy-2'-b- azidouridine TP; 2'-Deoxy-2'-b-bromouridine TP; 2'-Deoxy-2'-b-chlorouridine TP; 2'-Deoxy- 2'-b-fluorouridine TP; 2'-Deoxy-2'-b-iodouridine TP; 2'-Deoxy-2'-b-mercaptouridine TP; 2'- Deoxy-2'-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine; 2-methoxy uridine; 2'-0- Methyl-5-(l-propynyl)uridine TP; 3-Alkyl-pseudo-UTP; 4'-Azidouridine TP; 4'-Carbocyclic uridine TP; 4'-Ethynyluridine TP; 5-(l-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 l-(4- methylbenzenesulfonic acid) TP; Pseudouridine l-(4-methylbenzoic acid) TP; Pseudouridine TP l-[3-(2-ethoxy)]propionic acid; Pseudouridine TP l-[3-{2-(2-[2-(2-ethoxy )-ethoxy]- ethoxy )-ethoxy}] propionic acid; Pseudouridine TP l-[3-{2-(2-[2-{2(2-ethoxy )-ethoxy}- ethoxy]-ethoxy )-ethoxy}]propionic acid; Pseudouridine TP l-[3-{2-(2-[2-ethoxy ]-ethoxy)- ethoxy}] ropionic acid; Pseudouridine TP l-[3-{2-(2-ethoxy)-ethoxy}] propionic acid;

Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP 1-methylphosphonic acid diethyl ester; Pseudo-UTP-Nl-3-propionic acid; Pseudo-UTP-Nl-4-butanoic acid; Pseudo- UTP-Nl-5-pentanoic acid; Pseudo-UTP-Nl-6-hexanoic acid; Pseudo-UTP-Nl-7-heptanoic acid; Pseudo-UTP-Nl-methyl-p-benzoic acid; Pseudo-UTP-Nl-p-benzoic acid; Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine; undermodified hydroxywybutosine; 4- demethylwyosine; 2,6-(diamino)purine;l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl: l,3-( diaza)-2-( oxo )-phenthiazin-l-yl;l,3-(diaza)-2-(oxo)-phenoxazin-l-yl;l,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'iluro-uridine;2'-amino-2'-deoxyribose; 2-amino-6-Chloro-purine; 2-aza-inosinyl; 2'-azido- 2'-deoxyribose; 2'iluoro-2'-deoxyribose; 2'-fluoro-modified bases; 2'-0-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-(iluoro)-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)- l-(aza)-2-(thio )-3-(aza)-phenthiazin-l- yl; 7-(aminoalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl; 7-(aminoalkylhydroxy)- l,3-(diaza)-2-(oxo)-phenoxazin-l-yl; 7-(aminoalkylhydroxy)-l,3-( diaza)-2-( oxo )- phenthiazin-l-yl; 7-(aminoalkylhydroxy)-l,3-( diaza)-2-(oxo)-phenoxazin-l-yl; 7-(aza)indolyl; 7-(guanidiniumalkylhydroxy)-l-(aza)-2-(thio )-3-(aza)-phenoxazinl-yl; 7- (guanidiniumalkylhydroxy)- l-(aza)-2-(thio )-3-(aza)-phenthiazin-l-yl; 7- (guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl; 7- (guanidiniumalkylhydroxy)-l,3-(diaza)-2-(oxo)-phenoxazin-l-yl; 7-(guanidiniumalkyl- hydroxy)-l,3-( diaza)-2-( oxo )-phenthiazin-l-yl; 7-(guanidiniumalkylhydroxy)-l,3-(diaza)-2-( oxo )-phenoxazin-l-yl; 7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl, propynyl- 7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl; 7- substituted l,3-(diaza)-2-(oxo)-phenoxazin-l-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; Diiluorotolyl; 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, polynucleotides (e.g. , RNA polynucleotides, such as mRNA polynucleotides) include a combination of at least two (e.g. , 2, 3, 4 or more) of the aforementioned modified nucleobases.

In some embodiments, modified nucleobases in polynucleotides (e.g. , RNA polynucleotides, such as mRNA polynucleotides) are selected from the group consisting of pseudouridine (ψ), Nl-methylpseudouridine (m¹!)/)_* 2-thiouridine, 4'-thiouridine, 5- methylcytosine, 2-thio- l -methyl- 1-deaza-pseudouridine, 2-thio-l -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-l -methyl - pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine), 5-methoxyuridine and 2'-0-methyl uridine. In some embodiments, polynucleotides (e.g. , RNA polynucleotides, such as mRNA polynucleotides) include a combination of at least two (e.g. , 2, 3, 4 or more) of the aforementioned modified nucleobases.

In some embodiments, modified nucleobases in polynucleotides (e.g. , RNA polynucleotides, such as mRNA polynucleotides) are selected from the group consisting of 1- methyl-pseudouridine (m¹!)/)_* 5-methoxy-uridine (mo⁵U), 5-methyl-cytidine (m⁵C), pseudouridine (ψ), α-thio-guanosine and a-thio-adenosine. In some embodiments, polynucleotides includes a combination of at least two (e.g. , 2, 3, 4 or more) of the aforementioned modified nucleobases.

In some embodiments, polynucleotides (e.g. , RNA polynucleotides, such as mRNA polynucleotides) comprise pseudouridine (ψ) and 5-methyl-cytidine (m⁵C). In some embodiments, polynucleotides (e.g. , RNA polynucleotides, such as mRNA polynucleotides) comprise 1-methyl-pseudouridine (m ψ). In some embodiments, polynucleotides (e.g. , RNA polynucleotides, such as mRNA polynucleotides) comprise 1-methyl-pseudouridine (m^) and 5-methyl-cytidine (m⁵C). In some embodiments, polynucleotides (e.g. , RNA

polynucleotides, such as mRNA polynucleotides) comprise 2-thiouridine (s U). In some embodiments, polynucleotides (e.g. , RNA polynucleotides, such as mRNA polynucleotides) comprise 2-thiouridine and 5-methyl-cytidine (m⁵C). In some embodiments, polynucleotides (e.g. , RNA polynucleotides, such as mRNA polynucleotides) comprise methoxy-uridine (mo⁵U). In some embodiments, polynucleotides (e.g. , RNA polynucleotides, such as mRNA polynucleotides) comprise 5-methoxy-uridine (mo⁵U) and 5-methyl-cytidine (m⁵C). In some embodiments, polynucleotides (e.g. , RNA polynucleotides, such as mRNA polynucleotides) comprise 2'-0-methyl uridine. In some embodiments polynucleotides (e.g. , RNA

polynucleotides, such as mRNA polynucleotides) comprise 2'-0-methyl uridine and 5- methyl-cytidine (m⁵C). In some embodiments, polynucleotides (e.g. , RNA polynucleotides, such as mRNA polynucleotides) comprise N6-methyl-adenosine (m⁶A). In some embodiments, polynucleotides (e.g. , RNA polynucleotides, such as mRNA polynucleotides) comprise N6-methyl-adenosine (m⁶A) and 5-methyl-cytidine (m⁵C).

In some embodiments, polynucleotides (e.g. , RNA polynucleotides, such as mRNA polynucleotides) are 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 those set forth above.

Exemplary 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), and 2- thio-5-methyl-cytidine.

In some embodiments, a modified nucleobase is a modified uridine. Exemplary nucleobases and In some embodiments, a modified nucleobase is a modified cytosine.

nucleosides having a modified uridine include 5-cyano uridine, and 4'-thio uridine.

In some embodiments, a modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 7-deaza- adenine, 1-methyl- adenosine (mlA), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A). In some embodiments, a modified nucleobase is a modified guanine. Exemplary 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 (preQO), 7-aminomethyl-7-deaza-guanosine (preQl), 7-methyl-guanosine (m7G), 1-methyl-guanosine (mlG), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

The polynucleotides of the present disclosure may be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g. , purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in a polynucleotide of the invention, or in a given predetermined sequence region thereof (e.g. , in the mRNA including or excluding the polyA tail). In some embodiments, all nucleotides X in a polynucleotide of the present disclosure (or in a given sequence region thereof) are modified nucleotides, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

The polynucleotide may contain from about 1% to about 100% modified nucleotides

(either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g. , from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.

The polynucleotides may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the polynucleotides may contain a modified pyrimidine such as a modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the polynucleotide is replaced with a modified uracil (e.g. , a 5-substituted uracil). The modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g. , 2, 3, 4 or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the polynucleotide is replaced with a modified cytosine (e.g. , a 5-substituted cytosine). The modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g. , 2, 3, 4 or more unique structures).

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 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 (s U), 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

3 5 5

(m U), 5-methoxy-uridine (mo U), uridine 5-oxyacetic acid (cmo U), 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-

5 2 5 2 methoxycarbonylmethyl-2-thio-uridine (mem s U), 5-aminomethyl-2-thio-uridine (nm s U),

5 5 2

5-methylaminomethyl-uridine (mnm U), 5-methylaminomethyl-2-thio-uridine (mnm s U), 5-

5 2 5 methylaminomethyl-2-seleno-uridine (mnm se U), 5-carbamoylmethyl-uridine (ncm U), 5- carboxymethylaminomethyl-uridine (cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine

5 2 5 (cmnm s U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (xm U),

5 2

l-taurinomethyl -pseudouridine, 5-taurinomethyl-2-thio-uridine(xm s U), l-taurinomethyl-4- thio-pseudouridine, 5-methyl-uridine (m⁵U, i.e., having the nucleobase deoxy thymine), 1-

1 5 2

methyl-pseudouridine (m ψ), 5-methyl-2-thio-uridine (m s U), l-methyl-4-thio-

1 4 3 pseudouridine (m s ψ), 4-thio- l -methyl-pseudouridine, 3-methyl-pseudouridine (m ψ), 2- thio-1 -methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouridine, 2-thio-l -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, Nl -methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp U), 1- methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp ψ), 5-

(isopentenylaminomethyl)uridine (inm⁵U), 5-(isopentenylaminomethyl)-2-thio-uridine

5 2 5

(inm s U), a-thio-uridine, 2'-0-methyl-uridine (Um), 5,2'-0-dimethyl-uridine (m Um), 2'-0- methyl-pseudouridine (fm), 2-thio-2'-0-methyl-uridine (s Um), 5-methoxycarbonylmethyl- 2'-0-methyl-uridine (mcm⁵Um), 5-carbamoylmethyl-2'-0-methyl-uridine (ncm⁵Um), 5- carboxymethylaminomethyl-2'-0-methyl-uridine (cmnm⁵Um), 3,2'-0-dimethyl-uridine

3 5

(m Um), and 5-(isopentenylaminomethyl)-2'-0-methyl-uridine (inm Um), 1-thio-uridine, deoxythymidine, 2' -F-ara-uridine, 2' -F-uridine, 2' -OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(l -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-formyl- cytidine (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 (s C), 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1-methyl-pseudoisocytidine, 4-thio-l -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), a-thio-cytidine, 2'-0-methyl-cytidine (Cm), 5,2'-0- dimethyl-cytidine (m⁵Cm), N4-acetyl-2'-0-methyl-cytidine (ac⁴Cm), N4,2'-0-dimethyl- cytidine (m⁴Cm), 5-formyl-2'-0-methyl-cytidine (f⁵Cm), N4,N4,2'-0-trimethyl-cytidine (m⁴ ₂Cm), 1-thio-cytidine, 2' -F-ara-cytidine, 2' -F-cytidine, and 2' -OH-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 ( ^lA), 2-methyl- adenine (m²A), N6-methyl-adenosine (m⁶A), 2-methylthio-N6-methyl-adenosine (ms²m⁶A), 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 (m⁶2A), N6-hydroxynorvalylcarbamoyl- adenosine (hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A), N6- acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a- thio-adenosine, 2'-0-methyl-adenosine (Am), N6,2'-0-dimethyl-adenosine (m⁶Am),

N6,N6,2'-0-trimethyl-adenosine (m⁶ ₂Am), l,2'-0-dimethyl-adenosine (n^Am), 2'-0- ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido- adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2' -OH-ara-adenosine, and N6-(19-amino- pentaoxanonadecyl) - adeno sine .

In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m¹!), 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 (preQo), 7-aminomethyl-7-deaza-guanosine (preQi), 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 (m G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6- methoxy-guanosine, 1-methyl-guanosine (m 1 G), N2-methyl-guanosine (m 2 G), N2,N2- dimethyl-guanosine (m 2₂G), N2,7-dimethyl-guanosine (m 2 ' 7 G), N2, N2,7-dimethyl-guanosine

(m 2 ' 2 ' 7 G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2- methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-0-methyl- guanosine (Gm), N2-methyl-2'-0-methyl-guanosine (m²Gm), N2,N2-dimethyl-2'-0-methyl- guanosine (m 2₂Gm), l-methyl-2'-0-methyl-guanosine (m 1 Gm), N2,7-dimethyl-2'-0-methyl- guanosine (m 2 ' 7 Gm), 2'-0-methyl-i ·nosine (Im), l,2'-0-dimethyl-inosine (m 1'lm), 2'-0- ribosylguanosine (phosphate) (Gr(p)) , 1-thio-guanosine, 06-methyl-guanosine, 2'-F-ara- guanosine, and 2'-F-guanosine.

In Vitro Transcription ofRNA (e.g., mRNA)

Ebola virus vaccines 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 nucleic acid 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 DNA beginning with a start codon

(e.g. , methionine (ATG)), and ending with a stop codon (e.g. , TAA, TAG or TGA) and 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 polynucleotide includes 200 to 3,000 nucleotides. For example, a polynucleotide may include 200 to 500, 200 to 1000, 200 to 1500, 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, or 2000 to 3000 nucleotides).

Methods of Treatment

Provided herein are compositions (e.g. , pharmaceutical compositions), methods, kits and reagents for prevention and/or treatment of Ebola virus in humans and other mammals. Ebola virus 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 Ebola virus RNA vaccines of the present disclosure are used to provide prophylactic protection from Ebola virus. Prophylactic protection from Ebola virus can be achieved following administration of a Ebola virus RNA vaccine of the present disclosure. 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.

It is envisioned that there may be situations where persons are at risk for infection with more than one strain of Ebola virus. RNA (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 Ebola virus, a combination vaccine can be administered that includes RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a first Ebola virus antigen and further includes RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a second Ebola virus antigen. Additionally, or alternatively an epitope may be selected that has cross-strain homology and thus produces an immune response against more than one strain. RNAs (mRNAs) can be co-formulated, 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 Ebola virus is provided in aspects of the invention. The method involves administering to the subject a Ebola virus RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one Ebola virus antigenic polypeptide or an immunogenic fragment thereof, thereby inducing in the subject an immune response specific to Ebola virus 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 Ebola virus. 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 exemplary embodiments, a traditional vaccine is a vaccine that has achieved regulatory approval and/or is registered by a national drug regulatory body, for example the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EM A).

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 prophylactic ally effective dose of a traditional vaccine against the Ebola virus.

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 Ebola virus.

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 Ebola virus.

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 Ebola virus.

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 Ebola virus.

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 Ebola virus.

A method of eliciting an immune response in a subject against a Ebola virus is provided in other aspects of the invention. The method involves administering to the subject a Ebola virus RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one Ebola virus antigenic polypeptide or an immunogenic fragment thereof, thereby inducing in the subject an immune response specific to Ebola virus 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 Ebola virus 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 Ebola virus 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 Ebola virus 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 Ebola virus 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 5 times the dosage level relative to the Ebola virus 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 Ebola virus 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 Ebola virus 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 Ebola virus 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 Ebola virus 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 Ebola virus RNA vaccine.

In other embodiments the immune response is assessed by determining anti-antigenic polypeptide antibody titer in the subject. In other aspects the invention is a method of eliciting an immune response in a subject against a Ebola virus by administering to the subject a Ebola virus RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one Ebola virus antigenic polypeptide or an immunogenic fragment thereof, thereby inducing in the subject an immune response specific to Ebola virus 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 prophylactic ally effective dose of a traditional vaccine against the Ebola virus. 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.

A method of v eliciting an immune response in a subject against a Ebola virus by administering to the subject a Ebola virus 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 {e.g., pharmaceutical compositions), methods, kits and reagents for prevention, treatment or diagnosis of Ebola virus in humans and other mammals, for example. Ebola virus 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 some embodiments, the Ebola virus vaccines of the invention can be envisioned for use in 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 exemplary embodiments, an Ebola virus vaccine containing RNA polynucleotides 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 Ebola virus RNA vaccines may be induced for translation of a polypeptide (e.g., antigen or immunogen) in a cell, tissue or organism. In exemplary embodiments, such translation occurs in vivo, although there can be envisioned embodiments where such translation occurs ex vivo, in culture or in vitro. In exemplary embodiments, the cell, tissue or organism is contacted with an effective amount of a composition containing an Ebola virus RNA vaccine that contains a polynucleotide that has at least one a translatable region encoding an antigenic polypeptide.

An "effective amount" of an Ebola virus RNA vaccine 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 Ebola virus RNA vaccine, and other determinants. In general, an effective amount of the Ebola virus RNA vaccine composition 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 their encoded polypeptides) in accordance with the present disclosure may be used for treatment of Ebola virus.

Ebola virus 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 vaccines of the present disclosure provided to a cell, a tissue or a subject may be an amount effective for immune prophylaxis.

Ebola virus RNA vaccines may be administrated 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, 18 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. In exemplary embodiments, the time of administration between the initial administration of the

prophylactic composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months or 1 year.

In some embodiments, Ebola virus RNA vaccines may be administered

intramuscularly or intradermally, similarly to the administration of inactivated vaccines known in the art. Ebola virus 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 a variety of infectious disease. 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 Ebola virus RNA vaccines and RNA vaccine compositions and/or complexes optionally in combination with one or more pharmaceutically acceptable excipients.

Ebola virus RNA vaccines may be formulated or administered alone or in conjunction with one or more other components. In some embodiments, Ebola virus RNA vaccines do not include an adjuvant (they are adjuvant free).

Ebola virus 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 substances, such as, for example, a therapeutic ally- 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, Ebola virus 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 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.

Ebola virus 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 Ebola virus 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, Ebola virus RNA {e.g., mRNA) vaccines are formulated in a nanoparticle. In some embodiments, Ebola virus RNA vaccines are formulated in a lipid nanoparticle. In some embodiments, Ebola virus 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 polycation 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, Ebola virus 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. In one example by Semple et al. {Nature Biotech. 2010 28: 172-176; herein incorporated by reference in its entirety), the lipid nanoparticle formulation is composed of 57.1 % cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3 % cholesterol, and 1.4% PEG-c-DMA. As another example, changing the composition of the cationic lipid can more effectively deliver siRNA 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-[(co-methoxy- poly(ethyleneglycol)2000)carbamoyl)]-l,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, D Lin-DMA, C 12-200 and DLin-KC2- DMA.

In some embodiments, an Ebola virus 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 -DM A, 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-l- yloxy]-2-{ [(9Z,2Z)-octadeca-9,12-dien-l-yloxy]methyl}propan-l-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en- 1 -yloxy] -2- { [(9Z)-octadec-9-en- 1 - yloxy]methyl}propan-l-ol (Compound 2 in US20130150625); 2-amino-3-[(9Z,12Z)- octadeca-9,12-dien-l-yloxy]-2-[(octyloxy)methyl]propan-l-ol (Compound 3 in

US20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-2- { [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]methyl}propan-l-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-[l,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- [l,3]-dioxolane (DLin-KC2-DMA) , dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3- DMA), and di((Z)-non-2-en-l-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-[l,3]- dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3- DMA), and di((Z)-non-2-en-l-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- CM 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-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3 -DMA) , and di((Z)-non-2-en- l-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-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-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-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-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-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-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-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-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-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-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-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-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-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-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 .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 Ebola 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. Liposomes, Lipoplexes, and Lipid Nanoparticles

The RNA vaccines of the invention can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In one embodiment, pharmaceutical compositions of RNA vaccines include liposomes. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.

The formation of liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to- batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.

As a non-limiting example, liposomes such as synthetic membrane vesicles may be prepared by the methods, apparatus and devices described in US Patent Publication No. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373 and US20130183372, the contents of each of which are herein incorporated by reference in its entirety.

In one embodiment, pharmaceutical compositions described herein may include, without limitation, liposomes such as those formed from l,2-dioleyloxy-N,N- dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech

(Bothell, WA), l,2-dilinoleyloxy-3 -dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), and MC3 (US 20100324120; herein incorporated by reference in its entirety) and liposomes which may deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, PA).

In one embodiment, pharmaceutical compositions described herein may include, without limitation, liposomes such as those formed from the synthesis of stabilized plasmid- lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. Gene Therapy. 1999 6: 1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2: 1002-1007; Zimmermann et al., Nature. 2006 441: 111-114; Heyes et al. J Contr Rel. 2005 107:276-287; Semple et al. Nature Biotech. 2010 28: 172-176; Judge et al. J Clin Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19: 125-132; U.S. Patent Publication No US20130122104; all of which are incorporated herein in their entireties). The original manufacture method by Wheeler et al. was a detergent dialysis method, which was later improved by Jeffs et al. and is referred to as the spontaneous vesicle formation method. The liposome formulations are composed of 3 to 4 lipid components in addition to the

polynucleotide. As an example a liposome can contain, but is not limited to, 55%

cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15% 1,2- dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al. As another example, certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be 1,2- distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2- dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described by Heyes et al.

In some embodiments, liposome formulations may comprise from about about 25.0% cholesterol to about 40.0% cholesterol, from about 30.0% cholesterol to about 45.0% cholesterol, from about 35.0% cholesterol to about 50.0% cholesterol and/or from about 48.5% cholesterol to about 60% cholesterol. In a preferred embodiment, formulations may comprise a percentage of cholesterol selected from the group consisting of 28.5%, 31.5%, 33.5%, 36.5%, 37.0%, 38.5%, 39.0% and 43.5%. In some embodiments, formulations may comprise from about 5.0% to about 10.0% DSPC and/or from about 7.0% to about 15.0% DSPC.

In one embodiment, pharmaceutical compositions may include liposomes which may be formed to deliver polynucleotides which may encode at least one immunogen (antigen) or any other polypeptide of interest. The RNA vaccine may be encapsulated by the liposome and/or it may be contained in an aqueous core which may then be encapsulated by the liposome (see International Pub. Nos. WO2012031046, WO2012031043, WO2012030901 and WO2012006378 and US Patent Publication No. US20130189351, US20130195969 and US20130202684; the contents of each of which are herein incorporated by reference in their entirety).

In another embodiment, liposomes may be formulated for targeted delivery. As a non-limiting example, the liposome may be formulated for targeted delivery to the liver. The liposome used for targeted delivery may include, but is not limited to, the liposomes described in and methods of making liposomes described in US Patent Publication No.

US20130195967, the contents of which are herein incorporated by reference in its entirety.

In another embodiment, the polynucleotide which may encode an immunogen (antigen) may be formulated in a cationic oil-in-water emulsion where the emulsion particle comprises an oil core and a cationic lipid which can interact with the polynucleotide anchoring the molecule to the emulsion particle (see International Pub. No. WO2012006380; herein incorporated by reference in its entirety).

In one embodiment, the RNA vaccines may be formulated in a water-in-oil emulsion comprising a continuous hydrophobic phase in which the hydrophilic phase is dispersed. As a non-limiting example, the emulsion may be made by the methods described in International Publication No. WO201087791, the contents of which are herein incorporated by reference in its entirety.

In another embodiment, the lipid formulation may include at least cationic lipid, a lipid which may enhance transfection and a least one lipid which contains a hydrophilic head group linked to a lipid moiety (International Pub. No. WO2011076807 and U.S. Pub. No. 20110200582; the contents of each of which is herein incorporated by reference in their entirety). In another embodiment, the polynucleotides encoding an immunogen may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers (see U.S. Pub. No. 20120177724, the contents of which is herein incorporated by reference in its entirety). In one embodiment, the polynucleotides may be formulated in a lipsome as described in International Patent Publication No. WO2013086526, the contents of which is herein incorporated by reference in its entirety. The RNA vaccines may be encapsulated in a liposome using reverse pH gradients and/or optimized internal buffer compositions as described in International Patent Publication No. WO2013086526, the contents of which is herein incorporated by reference in its entirety.

In one embodiment, the RNA vaccine pharmaceutical compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, WA), SMARTICLES® (Marina Biotech, Bothell, WA), neutral DOPC (1,2- dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713); herein incorporated by reference in its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).

In one embodiment, the cationic lipid may be a low molecular weight cationic lipid such as those described in US Patent Application No. 20130090372, the contents of which are herein incorporated by reference in its entirety.

In one embodiment, the RNA vaccines may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers.

In one embodiment, the RNA vaccines may be formulated in a liposome comprising a cationic lipid. The liposome may have a molar ratio of nitrogen atoms in the cationic lipid to the phophates in the RNA (N:P ratio) of between 1: 1 and 20: 1 as described in International Publication No. WO2013006825, herein incorporated by reference in its entirety. In another embodiment, the liposome may have a N:P ratio of greater than 20: 1 or less than 1: 1.

In one embodiment, the RNA vaccines may be formulated in a lipid-polycation complex. The formation of the lipid-polycation complex 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 polycation 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 another embodiment, the RNA vaccines may be formulated in a lipid-polycation complex which may further include a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

In one embodiment, the RNA vaccines may be formulated in an aminoalcohol lipidoid. Aminoalcohol lipidoids which may be used in the present invention may be prepared by the methods described in U.S. Patent No. 8,450,298, herein incorporated by reference in its entirety.

The liposome 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. In one example by Semple et al. (Semple et al. Nature Biotech. 2010 28: 172-176; herein incorporated by reference in its entirety), the liposome formulation was composed of 57.1 % cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3 % cholesterol, and 1.4% PEG-c-DMA. As another example, changing the composition of the cationic lipid could more effectively deliver siRNA to various antigen presenting cells (Basha et al. Mol Ther. 2011 19:2186- 2200; herein incorporated by reference in its entirety). In some embodiments, liposome formulations may comprise from about 35 to about 45% cationic lipid, from about 40% to about 50% cationic lipid, from about 50% to about 60% cationic lipid and/or from about 55% to about 65% cationic lipid. In some embodiments, the ratio of lipid to mRNA in liposomes may be from about about 5: 1 to about 20: 1, from about 10: 1 to about 25: 1, from about 15: 1 to about 30: 1 and/or at least 30: 1.

In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP) formulations may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified from C 14 to C18 to alter the pharmacokinetics and/or biodistribution of the LNP formulations. As a non-limiting example, LNP formulations may contain from about 0.5% to about 3.0%, from about 1.0% to about 3.5%, from about 1.5% to about 4.0%, from about 2.0% to about 4.5%, from about 2.5% to about 5.0% and/or from about 3.0% to about 6.0% of the lipid molar ratio of PEG-c-DOMG (R-3-[(co-methoxy- poly(ethyleneglycol)2000)carbamoyl)]-l,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) as compared to the cationic lipid, DSPC and cholesterol. In another embodiment 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, D Lin-DMA, C 12-200 and DLin-KC2- DMA.

In one embodiment, the RNA vaccines may be formulated in a lipid nanoparticle such as those described in International Publication No. WO2012170930, the contents of which is herein incorporated by reference in its entirety. In one embodiment, the RNA vaccine formulation comprising the polynucleotide is a nanoparticle which may comprise at least one lipid. The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C 12-200, DLin-MC3-DMA, DLin- KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. In another aspect, 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-l-yloxy]-2-{ [(9Z,2Z)-octadeca-9,12-dien-l-yloxy]methyl}propan-l-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-l-yloxy]-2-{ [(9Z)- octadec-9-en-l-yloxy]methyl}propan-l-ol (Compound 2 in US20130150625); 2-amino-3- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-2-[(octyloxy)methyl]propan-l-ol (Compound 3 in US20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-2- { [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]methyl}propan-l-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-[l,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 one embodiment, the lipid nanoparticle formulation consists essentially of (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl- [l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3- DMA), and di((Z)-non-2-en-l-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 about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15% PEG-lipid.

In one embodiment, the formulation includes from about 25% to about 75% on a molar basis of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3- DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., from about 35 to about 65%, from about 45 to about 65%, about 60%, about 57.5%, about 50% or about 40% on a molar basis.

In one embodiment, the formulation includes from about 0.5% to about 15% on a molar basis of the neutral lipid e.g., from about 3 to about 12%, from about 5 to about 10% or about 15%, about 10%, or about 7.5% on a molar basis. Exemplary neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE and SM. In one embodiment, the formulation includes from about 5% to about 50% on a molar basis of the sterol (e.g., about 15 to about 45%, about 20 to about 40%, about 40%, about 38.5%, about 35%, or about 31% on a molar basis. An exemplary sterol is cholesterol. In one embodiment, the formulation includes from about 0.5% to about 20% on a molar basis of the PEG or PEG-modified lipid (e.g., about 0.5 to about 10%, about 0.5 to about 5%, about 1.5%, about 0.5%, about 1.5%, about 3.5%, or about 5% on a molar basis. In one embodiment, the PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of 2,000 Da. In other embodiments, the 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. Exemplary PEG-modified lipids include, but are not limited to, 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 one embodiment, the formulations of the inventions include 25-75% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-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 one embodiment, the formulations of the inventions include 35-65% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-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 one embodiment, the formulations of the inventions include 45-65% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-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 one embodiment, the formulations of the inventions include about 60% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,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), about 7.5% of the neutral lipid, about 31 % of the sterol, and about 1.5% of the PEG or PEG-modified lipid on a molar basis.

In one embodiment, the formulations of the inventions include about 50% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,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), about 10% of the neutral lipid, about 38.5 % of the sterol, and about 1.5% of the PEG or PEG-modified lipid on a molar basis.

In one embodiment, the formulations of the inventions include about 50% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,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), about 10% of the neutral lipid, about 35 % of the sterol, about 4.5% or about 5% of the PEG or PEG-modified lipid, and about 0.5% of the targeting lipid on a molar basis.

In one embodiment, the formulations of the inventions include about 40% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,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), about 15% of the neutral lipid, about 40% of the sterol, and about 5% of the PEG or PEG-modified lipid on a molar basis.

In one embodiment, the formulations of the inventions include about 57.2% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,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), about 7.1% of the neutral lipid, about 34.3% of the sterol, and about 1.4% of the PEG or PEG-modified lipid on a molar basis.

In one embodiment, the formulations of the inventions include about 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), about 7.5% of the neutral lipid, about 31.5 % of the sterol, and about 3.5% of the PEG or PEG-modified lipid on a molar basis.

In preferred embodiments, lipid nanoparticle formulation consists essentially of a lipid mixture in molar ratios of about 20-70% cationic lipid: 5-45% neutral lipid: 20-55% cholesterol: 0.5-15% PEG-modified lipid; more preferably in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% cholesterol: 0.5-15% PEG-modified lipid.

In particular embodiments, the molar lipid ratio is approximately 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).

Exemplary lipid nanoparticle compositions and methods of making same 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 one embodiment, the lipid nanoparticle formulations described herein may comprise a cationic lipid, a PEG lipid and a structural lipid and optionally comprise a non- cationic lipid. As a non-limiting example, the lipid nanoparticle may comprise about 40-60% of cationic lipid, about 5-15% of a non-cationic lipid, about 1-2% of a PEG lipid and about 30-50% of a structural lipid. As another non-limiting example, the lipid nanoparticle may comprise about 50% cationic lipid, about 10% non-cationic lipid, about 1.5% PEG lipid and about 38.5% structural lipid. As yet another non-limiting example, the lipid nanoparticle may comprise about 55% cationic lipid, about 10% non-cationic lipid, about 2.5% PEG lipid and about 32.5% structural lipid. In one embodiment, 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 one embodiment, 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 about 40-60% of cationic lipid, about 5-15% of a non-cationic lipid, about 1-2% of a PEG lipid and about 30-50% of a structural lipid. As another non- limiting example, the lipid nanoparticle may comprise about 50% cationic lipid, about 10% non-cationic lipid, about 1.5% PEG lipid and about 38.5% structural lipid. As yet another non-limiting example, the lipid nanoparticle may comprise about 55% cationic lipid, about 10% non-cationic lipid, about 2.5% PEG lipid and about 32.5% structural lipid. In one embodiment, 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 one embodiment, 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 about 50% of the cationic lipid DLin-KC2- DMA, about 10% of the non-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DOMG and about 38.5% of the structural lipid cholesterol. As a non-limiting example, the lipid nanoparticle comprise about 50% of the cationic lipid DLin-MC3-DMA, about 10% of the non-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DOMG and about 38.5% of the structural lipid cholesterol. As a non-limiting example, the lipid nanoparticle comprise about 50% of the cationic lipid DLin-MC3 -DM A, about 10% of the non-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DMG and about 38.5% of the structural lipid cholesterol. As yet another non-limiting example, the lipid nanoparticle comprise about 55% of the cationic lipid L319, about 10% of the non-cationic lipid DSPC, about 2.5% of the PEG lipid PEG-DMG and about 32.5% of the structural lipid cholesterol.

In one embodiment, the cationic lipid may be selected from, but not limited to, a cationic lipid described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865, WO2008103276, WO2013086373 and WO2013086354, US Patent Nos. 7,893,302, 7,404,969, 8,283,333, and 8,466,122 and US Patent Publication No. US20100036115, US20120202871,

US20130064894, US20130129785, US20130150625, US20130178541 and US20130225836; the contents of each of which are herein incorporated by reference in their entirety. In another embodiment, the cationic lipid may be selected from, but not limited to, formula A described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638 and WO2013116126 or US Patent Publication No.

US20130178541 and US20130225836; the contents of each of which is herein incorporated by reference in their entirety. In yet another embodiment, the cationic lipid may be selected from, but not limited to, formula CLI-CLXXIX of International Publication No.

WO2008103276, formula CLI-CLXXIX of US Patent No. 7,893,302, formula CLI- CLXXXXII of US Patent No. 7,404,969 and formula I- VI of US Patent Publication No. US20100036115, formula I of US Patent Publication No US20130123338; each of which is herein incorporated by reference in their entirety. As a non-limiting example, the cationic lipid may be selected from (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine,

(17Z,20Z)-N,N-dimemylhexacosa-17,20-dien-9-amine, (lZ,19Z)-N5N-dimethylpentacosa-l 6, 19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)-N,N- dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine, ( 15Z, 18Z)-N,N-dimethyltetracosa- 15,18-dien-7-amine, ( 18Z,2 lZ)-N,N-dimethylheptacosa- 18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z.17Z)- N,N-dimethyltricosa-14,17-dien-4-amine, (19Z,22Z)-N,N-dimeihyloctacosa-19,22-dien-9- amine, (18Z,21 Z)-N,N-dimethylheptacosa- 18 ,21 -dien-8 -amine, (17Z,20Z)-N,N- dimethylhexacosa- 17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-6- amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, (21 Z ,24Z)-N,N- dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimetylheptacos-18-en-10-amine, (17Z)- N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosan-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien-10- amine, l-[(HZ,14Z)-l-nonylicosa-l l,14-dien-l-yl] pyrrolidine, (20Z)-N,N-dimethylheptacos- 20-en-l 0-amine, (15Z)-N,N-dimethyl eptacos-15-en-l 0-amine, (14Z)-N,N-dimethylnonacos- 14-en-10-amine, (17Z)-N,N-dimethylnonacos-17-en-10-amine, (24Z)-N,N-dimethyltritriacont- 24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-l 0-amine, (22Z)-N,N- dimethylhentriacont-22-en-10-amine, (16Z)-N,N-dimethylpentacos-16-en-8-amine,

(12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-l-amine, (13Z,16Z)-N,N-dimethyl-3- nonyldocosa-13 , 16-dien-l-amine, N,N-dimethyl-l- [(IS ,2R)-2-octylcyclopropyl] eptadecan-8- amine, l-[(lS,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethyl-l- [( 1 S ,2R)-2-octylcyclopropyl] nonadecan- 10-amine, N,N-dimethyl-21 - [(IS ,2R)-2- octylcyclopropyl]henicosan-10-amine,N,N-dimethyl- 1 - [( 1 S ,2S)-2- { [(lR,2R)-2- pentylcyclopropyl] methyl } cyclopropyl] nonadecan- 10-amine ,Ν,Ν-dimethyl- 1 - [( 1 S ,2R)-2- octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(lR,2S)-2- undecyIcyclopropyl]tetradecan-5-amine, N,N-dimethyl-3-{7-[(lS,2R)-2- octylcyclopropyl]heptyl} dodecan-l-amine, l-[(lR,2S)-2-hepty lcyclopropyl]-N,N- dimethyloctadecan-9-amine, 1 - [( 1 S ,2R)-2-decylcyclopropyl] -N,N-dimethylpentadecan-6- amine, N,N-dimethyl-l-[(lS,2R)-2-octylcyclopropyl]pentadecan-8-amine, R-N,N-dimethyl- 1- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-(octyloxy)propan-2-amine, S-N,N-dimethyl-l- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-(octyloxy)propan-2-amine, l-{2-[(9Z,12Z)- octadeca-9, 12-dien- 1 -yloxy] - 1 - [(octyloxy)methyl] ethyl jpyrrolidine, (2S )-N,N-dimethyl- 1 - [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-[(5Z)-oct-5-en-l-yloxy]propan-2-amine, l-{2- [(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy] - 1 - [(octyloxy)methyl] ethyl } azetidine, (2S )- 1 - (hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, (2S)-1- (heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, N,N- dimethyl-l-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, N,N- dimethyl-l-[(9Z)-octadec-9-en-l-yloxy]-3-(octyloxy)propan-2-amine; (2S)-N,N-dimethyl-l- [(6Z,9Z,12Z)-octadeca-6,9,12-trien-l-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1- [(l lZ,14Z)-icosa-l l,14-dien-l-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1- (hexyloxy)-3-[(l lZ,14Z)-icosa-l l,14-dien-l-yloxy]-N,N-dimethylpropan-2-amine, 1- [(1 lZ,14Z)-icosa-l l,14-dien-l-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1- [( 13Z, 16Z)-docosa-13 , 16-dien-l-yloxy] -N,N-dimethyl-3-(octyloxy)propan-2- amine, (2S)- 1 - [( 13Z, 16Z)-docosa- 13 , 16-dien- 1 -yloxy] -3-(hexyloxy)-N,N-dimethylpropan-2- amine, (2S)- 1 - [(13Z)-docos-13-en-l-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, l-[(13Z)-docos- 13-en- 1 -yloxy] -N,N-dimethyl-3 -(octyloxy)propan-2-amine, 1 - [(9Z)-hexadec-9-en- 1 -yloxy] - N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(l-metoylo ctyl)oxy]-3- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, (2R)-l-[(3,7-dimethyloctyl)oxy]- N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, N,N-dimethyl-l- (octyloxy)-3-({ 8-[(lS,2S)-2-{ [(lR,2R)-2- pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, Ν,Ν-dimethyl- 1-{ [8-(2- oc 1 ylcyclopropyl)octyl] oxy } -3 -(octyloxy)propan-2-amine and ( 11E,20Z,23Z) -N,N- dimethylnonacosa-ll,20,2-trien-10-amine or a pharmaceutically acceptable salt or stereoisomer thereof.

In one embodiment, the lipid may be a cleavable lipid such as those described in

International Publication No. WO2012170889, herein incorporated by reference in its entirety. In another embodiment, the lipid may be a cationic lipid such as, but not limited to, Formula (I) of U.S. Patent Application No. US20130064894, the contents of which are herein incorporated by reference in its entirety.

In one embodiment, the cationic lipid may be synthesized by methods known in the art and/or as described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865, WO2013086373 and WO2013086354; the contents of each of which are herein incorporated by reference in their entirety.

In another embodiment, the cationic lipid may be a trialkyl cationic lipid. Non- limiting examples of trialkyl cationic lipids and methods of making and using the trialkyl cationic lipids are described in International Patent Publication No. WO2013126803, the contents of which are herein incorporated by reference in its entirety.

In one embodiment, the LNP formulations of the RNA vaccines may contain PEG-c- DOMG at 3% lipid molar ratio. In another embodiment, the LNP formulations RRNA vaccines may contain PEG-c-DOMG at 1.5% lipid molar ratio.

In one embodiment, the pharmaceutical compositions of the RNA vaccines may include at least one of the PEGylated lipids described in International Publication No.

WO2012099755, the contents of which is herein incorporated by reference in its entirety.

In one embodiment, the LNP formulation may contain PEG-DMG 2000 (1 ,2- dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethylene glycol)-2000). In one embodiment, the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art and at least one other component. In another embodiment, the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art, DSPC and cholesterol. As a non-limiting example, the LNP formulation may contain PEG-DMG 2000, DLin-DMA,

DSPC and cholesterol. As another non-limiting example the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol in a molar ratio of 2:40: 10:48 (see e.g., Geall et al., Nonviral delivery of self- amplifying RNA vaccines, PNAS 2012; PMID:

22908294; herein incorporated by reference in its entirety).

In one embodiment, the LNP formulation may be formulated by the methods described in International Publication Nos. WO2011127255 or

WO2008103276, the contents of each of which is herein incorporated by reference in their entirety. As a non-limiting example, the RNA vaccines described herein may be encapsulated in LNP formulations as described in WO2011127255 and/or WO2008103276; each of which is herein incorporated by reference in their entirety.

In one embodiment, the RNA vaccines described herein may be formulated in a nanoparticle to be delivered by a parenteral route as described in U.S. Pub. No.

US20120207845; the contents of which are herein incorporated by reference in its entirety.

In one embodiment, the RNA vaccines may be formulated in a lipid nanoparticle made by the methods described in US Patent Publication No US20130156845 or

International Publication No WO2013093648 or WO2012024526, each of which is herein incorporated by reference in its entirety.

The lipid nanoparticles described herein may be made in a sterile environment by the system and/or methods described in US Patent Publication No. US20130164400, herein incorporated by reference in its entirety.

In one embodiment, the LNP formulation may be formulated in a nanoparticle such as a nucleic acid-lipid particle described in US Patent No. 8,492,359, the contents of which are herein incorporated by reference in its entirety. As a non-limiting example, the lipid particle may comprise one or more active agents or therapeutic agents; one or more cationic lipids comprising from about 50 mol % to about 85 mol % of the total lipid present in the particle; one or more non-cationic lipids comprising from about 13 mol % to about 49.5 mol % of the total lipid present in the particle; and one or more conjugated lipids that inhibit aggregation of particles comprising from about 0.5 mol % to about 2 mol % of the total lipid present in the particle. The nucleic acid in the nanoparticle may be the polynucleotides described herein and/or are known in the art.

In one embodiment, the LNP formulation may be formulated by the methods described in International Publication Nos. WO2011127255 or WO2008103276, the contents of each of which are herein incorporated by reference in their entirety. As a non-limiting example, modified RNA described herein may be encapsulated in LNP formulations as described in WO2011127255 and/or WO2008103276; the contents of each of which are herein incorporated by reference in their entirety.

In one embodiment, LNP formulations described herein may comprise a polycationic composition. As a non-limiting example, the polycationic composition may be selected from formula 1-60 of US Patent Publication No. US20050222064; the content of which is herein incorporated by reference in its entirety. In another embodiment, the LNP formulations comprising a polycationic composition may be used for the delivery of the modified RNA described herein in vivo and/or in vitro. In one embodiment, the LNP formulations described herein may additionally comprise a permeability enhancer molecule. Non-limiting permeability enhancer molecules are described in US Patent Publication No. US20050222064; the content of which is herein incorporated by reference in its entirety.

In one embodiment, the RNA vaccine pharmaceutical compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, WA), SMARTICLES® (Marina Biotech, Bothell, WA), neutral DOPC (1,2- dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713); herein incorporated by reference in its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).

In one embodiment, the RNA vaccines may be formulated in a lyophilized gel-phase liposomal composition as described in US Publication No. US2012060293, herein incorporated by reference in its entirety.

The nanoparticle formulations may comprise a phosphate conjugate. The phosphate conjugate may increase in vivo circulation times and/or increase the targeted delivery of the nanoparticle. Phosphate conjugates for use with the present invention may be made by the methods described in International Application No. WO2013033438 or US Patent

Publication No. US20130196948, the contents of each of which are herein incorporated by reference in its entirety. As a non-limiting example, the phosphate conjugates may include a compound of any one of the formulas described in International Application No.

WO2013033438, herein incorporated by reference in its entirety.

The nanoparticle formulation may comprise a polymer conjugate. The polymer conjugate may be a water soluble conjugate. The polymer conjugate may have a structure as described in U.S. Patent Application No. 20130059360, the contents of which are herein incorporated by reference in its entirety. In one aspect, polymer conjugates with the polynucleotides of the present invention may be made using the methods and/or segmented polymeric reagents described in U.S. Patent Application No. 20130072709, herein incorporated by reference in its entirety. In another aspect, the polymer conjugate may have pendant side groups comprising ring moieties such as, but not limited to, the polymer conjugates described in US Patent Publication No. US20130196948, the contents of which is herein incorporated by reference in its entirety.

The nanoparticle formulations may comprise a conjugate to enhance the delivery of nanoparticles of the present invention in a subject. Further, the conjugate may inhibit phagocytic clearance of the nanoparticles in a subject. In one aspect, the conjugate may be a "self peptide designed from the human membrane protein CD47 (e.g., the "self particles described by Rodriguez et al (Science 2013 339, 971-975), herein incorporated by reference in its entirety). As shown by Rodriguez et al. the self peptides delayed macrophage-mediated clearance of nanoparticles which enhanced delivery of the nanoparticles. In another aspect, the conjugate may be the membrane protein CD47 (e.g., see Rodriguez et al. Science 2013 339, 971-975, herein incorporated by reference in its entirety). Rodriguez et al. showed that, similarly to "self peptides, CD47 can increase the circulating particle ratio in a subject as compared to scrambled peptides and PEG coated nanoparticles.

In one embodiment, the RNA vaccines of the present invention are formulated in nanoparticles which comprise a conjugate to enhance the delivery of the nanoparticles of the present invention in a subject. The conjugate may be the CD47 membrane or the conjugate may be derived from the CD47 membrane protein, such as the "self peptide described previously. In another aspect the nanoparticle may comprise PEG and a conjugate of CD47 or a derivative thereof. In yet another aspect, the nanoparticle may comprise both the "self peptide described above and the membrane protein CD47.

In another aspect, a "self peptide and/or CD47 protein may be conjugated to a viruslike particle or pseudovirion, as described herein for delivery of the RNA vaccines of the present invention.

In another embodiment, RNA vaccine pharmaceutical compositions comprising the polynucleotides of the present invention and a conjugate which may have a degradable linkage. Non-limiting examples of conjugates include an aromatic moiety comprising an ionizable hydrogen atom, a spacer moiety, and a water-soluble polymer. As a non-limiting example, pharmaceutical compositions comprising a conjugate with a degradable linkage and methods for delivering such pharmaceutical compositions are described in US Patent Publication No. US20130184443, the contents of which are herein incorporated by reference in its entirety.

The nanoparticle formulations may be a carbohydrate nanoparticle comprising a carbohydrate carrier and a RNA vaccine. As a non-limiting example, the carbohydrate carrier may include, but is not limited to, an anhydride-modified phytoglycogen or glycogen- type material, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride- modified phytoglycogen beta-dextrin. (See e.g., International Publication No.

WO2012109121; the contents of which are herein incorporated by reference in its entirety).

Nanoparticle formulations of the present invention may be coated with a surfactant or polymer in order to improve the delivery of the particle. In one embodiment, the nanoparticle may be coated with a hydrophilic coating such as, but not limited to, PEG coatings and/or coatings that have a neutral surface charge. The hydrophilic coatings may help to deliver nanoparticles with larger payloads such as, but not limited to, RNA vaccines within the central nervous system. As a non-limiting example nanoparticles comprising a hydrophilic coating and methods of making such nanoparticles are described in US Patent Publication No. US20130183244, the contents of which are herein incorporated by reference in its entirety.

In one embodiment, the lipid nanoparticles of the present invention may be hydrophilic polymer particles. Non-limiting examples of hydrophilic polymer particles and methods of making hydrophilic polymer particles are described in US Patent Publication No. US20130210991, the contents of which are herein incorporated by reference in its entirety.

In another embodiment, the lipid nanoparticles of the present invention may be hydrophobic polymer particles.

Lipid nanoparticle formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle

(reLNP). lonizable cationic lipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in plasma and tissues over time and may be a potential source of toxicity. The rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of the lipid nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of an enzymatically degraded ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation. The ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain. The internal ester linkage may replace any carbon in the lipid chain.

In one embodiment, the internal ester linkage may be located on either side of the saturated carbon.

In one embodiment, an immune response may be elicited by delivering a lipid nanoparticle which may include a nanospecies, a polymer and an immunogen. (U.S.

Publication No. 20120189700 and International Publication No. WO2012099805; each of which is herein incorporated by reference in their entirety). The polymer may encapsulate the nanospecies or partially encapsulate the nanospecies. The immunogen may be a recombinant protein, a modified RNA and/or a polynucleotide described herein. In one embodiment, the lipid nanoparticle may be formulated for use in a vaccine such as, but not limited to, against a pathogen. Lipid nanoparticles may be engineered to alter the surface properties of particles so the lipid nanoparticles may penetrate the mucosal barrier. Mucus is located on mucosal tissue such as, but not limted to, oral (e.g., the buccal and esophageal membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal, tracheal and bronchial membranes), genital (e.g., vaginal, cervical and urethral membranes). Nanoparticles larger than 10-200 nm which are preferred for higher drug encapsulation efficiency and the ability to provide the sustained delivery of a wide array of drugs have been thought to be too large to rapidly diffuse through mucosal barriers. Mucus is continuously secreted, shed, discarded or digested and recycled so most of the trapped particles may be removed from the mucosla tissue within seconds or within a few hours. Large polymeric nanoparticles (200nm -500nm in diameter) which have been coated densely with a low molecular weight polyethylene glycol (PEG) diffused through mucus only 4 to 6-fold lower than the same particles diffusing in water (Lai et al. PNAS 2007 104(5): 1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2): 158-171; each of which is herein incorporated by reference in their entirety). The transport of nanoparticles may be determined using rates of permeation and/or fluorescent microscopy techniques including, but not limited to, fluorescence recovery after photobleaching (FRAP) and high resolution multiple particle tracking (MPT). As a non-limiting example, compositions which can penetrate a mucosal barrier may be made as described in U.S. Pat. No. 8,241,670 or International Patent Publication No. WO2013110028, the contents of each of which are herein incorporated by reference in its entirety.

The lipid nanoparticle engineered to penetrate mucus may comprise a polymeric material (i.e. a polymeric core) and/or a polymer-vitamin conjugate and/or a tri-block copolymer. The polymeric material may include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The polymeric material may be biodegradable and/or biocompatible. Non-limiting examples of biocompatible polymers are described in International Patent Publication No.

WO2013116804, the contents of which are herein incorporated by reference in its entirety. The polymeric material may additionally be irradiated. As a non-limiting example, the polymeric material may be gamma irradiated (See e.g., International App. No.

WO201282165, herein incorporated by reference in its entirety). Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L- lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L- lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L- lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol, poly-L-glutamic acid,

poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose,

carboxymethylcellulose, polymers of acrylic acids, such as poly(methyl(meth)acrylate)

(PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co- caprolactone), PEG-PLGA-PEG and trimethylene carbonate, polyvinylpyrrolidone. The lipid nanoparticle may be coated or associated with a co-polymer such as, but not limited to, a block co-polymer (such as a branched polyether-polyamide block copolymer described in International Publication No. WO2013012476, herein incorporated by reference in its entirety), and (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymer (see e.g., US Publication 20120121718 and US Publication 20100003337 and U.S. Pat. No. 8,263,665; each of which is herein incorporated by reference in their entirety). The co-polymer may be a polymer that is generally regarded as safe (GRAS) and the formation of the lipid nanoparticle may be in such a way that no new chemical entities are created. For example, the lipid nanoparticle may comprise poloxamers coating PLGA nanoparticles without forming new chemical entities which are still able to rapidly penetrate human mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; the contents of which are herein incorporated by reference in its entirety). A non-limiting scalable method to produce nanoparticles which can penetrate human mucus is described by Xu et al. (See e.g., J Control Release 2013, 170(2):279-86; the contents of which are herein incorporated by reference in its entirety).

The vitamin of the polymer-vitamin conjugate may be vitamin E. The vitamin portion of the conjugate may be substituted with other suitable components such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a hydrophobic component of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains and alkylene oxide chains).

The lipid nanoparticle engineered to penetrate mucus may include surface altering agents such as, but not limited to, polynucleotides, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as for example dimethyldioctadecyl- ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4 dornase alfa, neltenexine, erdosteine) and various DNases including rhDNase. The surface altering agent may be embedded or enmeshed in the particle's surface or disposed (e.g., by coating, adsorption, covalent linkage, or other process) on the surface of the lipid nanoparticle. (see e.g., US Publication 20100215580 and US Publication

20080166414 and US20130164343; the contents of each of which is herein incorporated by reference in their entirety).

In one embodiment, the mucus penetrating lipid nanoparticles may comprise at least one polynucleotide described herein. The polynucleotide may be encapsulated in the lipid nanoparticle and/or disposed on the surface of the paricle. The polynucleotide may be covalently coupled to the lipid nanoparticle. Formulations of mucus penetrating lipid nanoparticles may comprise a plurality of nanoparticles. Further, the formulations may contain particles which may interact with the mucus and alter the structural and/or adhesive properties of the surrounding mucus to decrease mucoadhesion which may increase the delivery of the mucus penetrating lipid nanoparticles to the mucosal tissue.

In another embodiment, the mucus penetrating lipid nanoparticles may be a hypotonic formulation comprising a mucosal penetration enhancing coating. The formulation may be hypotonice for the epithelium to which it is being delivered. Non-limiting examples of hypotonic formulations may be found in International Patent Publication No.

WO2013110028, the contents of which are herein incorporated by reference in its entirety. In one embodiment, in order to enhance the delivery through the mucosal barrier the RNA vaccine formulation may comprise or be a hypotonic solution. Hypotonic solutions were found to increase the rate at which mucoinert particles such as, but not limited to, mucus-penetrating particles, were able to reach the vaginal epithelial surface (See e.g., Ensign et al. Biomaterials 2013 34(28):6922-9; the contents of which is herein incorporated by reference in its entirety).

In one embodiment, the RNA vaccine is formulated as a lipoplex, such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom),

STEMFECTTM from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids acids (Aleku et al.

Cancer Res. 2008 68:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene Ther 2006 13: 1222-1234; Santel et al., Gene Ther 2006 13: 1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et al. Microvasc Res 2010 80:286-293 Weide et al. J Immunother. 2009 32:498-507; Weide et al. J Immunother. 2008 31: 180-188; Pascolo Expert Opin. Biol. Ther. 4: 1285-1294; Fotin-Mleczek et al., 2011 J. Immunother. 34: 1-15; Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc Natl Acad Sci U S A. 2007 6; 104:4095-4100; deFougerolles Hum Gene Ther. 2008 19: 125-132; all of which are incorporated herein by reference in its entirety).

In one embodiment such formulations may also be constructed or compositions altered such that they passively or actively are directed to different cell types in vivo, including but not limited to hepatocytes, immune cells, tumor cells, endothelial cells, antigen presenting cells, and leukocytes (Akinc et al. Mol Ther. 2010 18: 1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge et al., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res 2010 80:286-293; Santel et al., Gene Ther 2006 13: 1222-1234; Santel et al., Gene Ther 2006 13: 1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske and Cullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 2011 18: 1127-1133; all of which are incorporated herein by reference in its entirety). One example of passive targeting of formulations to liver cells includes the DLin-DMA, DLin-KC2-DMA and DLin-MC3 -DMA-based lipid nanoparticle formulations which have been shown to bind to apolipoprotein E and promote binding and uptake of these formulations into hepatocytes in vivo (Akinc et al. Mol Ther. 2010 18: 1357-1364; herein incorporated by reference in its entirety). Formulations can also be selectively targeted through expression of different ligands on their surface as exemplified by, but not limited by, folate, transferrin, N- acetylgalactosamine (GalNAc), and antibody targeted approaches (Kolhatkar et al., Curr Drug Discov Technol. 2011 8: 197-206; Musacchio and Torchilin, Front Biosci. 2011 16: 1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008 25: 1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther. 2010 18: 1357-1364; Srinivasan et al., Methods Mol Biol. 2012 820: 105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control Release. 20:63-68; Peer et al., Proc Natl Acad Sci U S A. 2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., Nat Biotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 2011 18: 1127-1133; all of which are incorporated herein by reference in its entirety).

In one embodiment, the RNA vaccine is formulated as a solid lipid nanoparticle. A solid lipid nanoparticle (SLN) may be spherical with an average diameter between 10 to 1000 nm. SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and may be stabilized with surfactants and/or emulsifiers. In a further embodiment, the lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702; the contents of which are herein incorporated by reference in its entirety). As a non-limiting example, the SLN may be the SLN described in International Patent

Publication No. WO2013105101, the contents of which are herein incorporated by reference in its entirety. As another non-limiting example, the SLN may be made by the methods or processes described in International Patent Publication No. WO2013105101, the contents of which are herein incorporated by reference in its entirety.

Liposomes, lipoplexes, or lipid nanoparticles may be used to improve the efficacy of polynucleotides directed protein production as these formulations may be able to increase cell transfection by the RNA vaccine; and/or increase the translation of encoded protein. One such example involves the use of lipid encapsulation to enable the effective systemic delivery of polyplex plasmid DNA (Heyes et al., Mol Ther. 2007 15:713-720; herein incorporated by reference in its entirety). The liposomes, lipoplexes, or lipid nanoparticles may also be used to increase the stability of the polynucleotide.

In one embodiment, the RNA vaccines of the present invention can be formulated for controlled release and/or targeted delivery. As used herein, "controlled release" refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome. In one embodiment, the RRNA vaccines may be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery. As used herein, the term "encapsulate" means to enclose, surround or encase. As it relates to the formulation of the compounds of the invention, encapsulation may be substantial, complete or partial. The term "substantially encapsulated" means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of the pharmaceutical composition or compound of the invention may be enclosed, surrounded or encased within the delivery agent. "Partially encapsulation" means that less than 10, 10, 20, 30, 40 50 or less of the pharmaceutical composition or compound of the invention may be enclosed, surrounded or encased within the delivery agent.

Advantageously, encapsulation may be determined by measuring the escape or the activity of the pharmaceutical composition or compound of the invention using fluorescence and/or electron micrograph. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical composition or compound of the invention are encapsulated in the delivery agent.

In one embodiment, the controlled release formulation may include, but is not limited to, tri-block co-polymers. As a non-limiting example, the formulation may include two different types of tri-block co-polymers (International Pub. No. WO2012131104 and

WO2012131106; the contents of each of which is herein incorporated by reference in its entirety).

In another embodiment, the RNA vaccines may be encapsulated into a lipid nanoparticle or a rapidly eliminated lipid nanoparticle and the lipid nanoparticles or a rapidly eliminated lipid nanoparticle may then be encapsulated into a polymer, hydrogel and/or surgical sealant described herein and/or known in the art. As a non-limiting example, the polymer, hydrogel or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, IL).

In another embodiment, the lipid nanoparticle may be encapsulated into any polymer known in the art which may form a gel when injected into a subject. As another non-limiting example, the lipid nanoparticle may be encapsulated into a polymer matrix which may be biodegradable.

In one embodiment, the the RNA vaccine formulation for controlled release and/or targeted delivery may also include at least one controlled release coating. Controlled release coatings include, but are not limited to, OPADRY®, polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such as ethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®).

In one embodiment, the RNA vaccine controlled release and/or targeted delivery formulation may comprise at least one degradable polyester which may contain polycationic side chains. Degradeable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In another embodiment, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.

In one embodiment, the RNA vaccine controlled release and/or targeted delivery formulation comprising at least one polynucleotide may comprise at least one PEG and/or PEG related polymer derivatives as described in US Patent No. 8,404,222, herein

incorporated by reference in its entirety.

In another embodiment, the RNA vaccine controlled release delivery formulation comprising at least one polynucleotide may be the controlled release polymer system described in US20130130348, herein incorporated by reference in its entirety.

In one embodiment, the the RNA vaccines of the present invention may be encapsulated in a therapeutic nanoparticle, referred to herein as "therapeutic nanoparticle RRNA vaccines." Therapeutic nanoparticles may be formulated by methods described herein and known in the art such as, but not limited to, International Pub Nos. WO2010005740, WO2010030763, WO2010005721, WO2010005723, WO2012054923, US Pub. Nos.

US20110262491, US20100104645, US20100087337, US20100068285, US20110274759, US20100068286, US20120288541, US20130123351 and US20130230567 and US Pat No. 8,206,747, 8,293,276, 8,318,208 and 8,318,211; the contents of each of which are herein incorporated by reference in their entirety. In another embodiment, therapeutic polymer nanoparticles may be identified by the methods described in US Pub No. US20120140790, the contents of which is herein incorporated by reference in its entirety.

In one embodiment, the therapeutic nanoparticle RNA vaccine may be formulated for sustained release. As used herein, "sustained release" refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time may include, but is not limited to, hours, days, weeks, months and years. As a non- limiting example, the sustained release nanoparticle may comprise a polymer and a therapeutic agent such as, but not limited to, the the polynucleotides of the present invention (see International Pub No. 2010075072 and US Pub No. US20100216804, US20110217377 and US20120201859, each of which is herein incorporated by reference in their entirety). In another non-limiting example, the sustained release formulation may comprise agents which permit persistent bioavailability such as, but not limited to, crystals, macromolecular gels and/or particulate suspensions (see US Patent Publication No US20130150295, the contents of which is herein incorporated by reference in its entirety).

In one embodiment, the therapeutic nanoparticle RNA vaccines may be formulated to be target specific. As a non-limiting example, the thereapeutic nanoparticles may include a corticosteroid (see International Pub. No. WO2011084518; herein incorporated by reference in its entirety). As a non-limiting example, the therapeutic nanoparticles may be formulated in nanoparticles described in International Pub No. WO2008121949, WO2010005726, WO2010005725, WO2011084521 and US Pub No. US20100069426, US20120004293 and US20100104655, each of which is herein incorporated by reference in their entirety.

In one embodiment, the nanoparticles of the present invention may comprise a polymeric matrix. As a non-limiting example, the nanoparticle may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,

polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.

In one embodiment, the therapeutic nanoparticle comprises a diblock copolymer. In one embodiment, the diblock copolymer may include PEG in combination with a polymer such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4- hydroxy-L-proline ester) or combinations thereof. In another embodiment, the diblock copolymer may comprise the diblock copolymers described in European Patent Publication No. the contents of which are herein incorporated by reference in its entirety. In yet another embodiment, the diblock copolymer may be a high-X diblock copolymer such as those described in International Patent Publication No. WO2013120052, the contents of which are herein incorporated by reference in its entirety. As a non-limiting example the therapeutic nanoparticle comprises a PLGA-PEG block copolymer (see US Pub. No. US20120004293 and US Pat No. 8,236,330, each of which is herein incorporated by reference in their entirety). In another non-limiting example, the therapeutic nanoparticle is a stealth nanoparticle comprising a diblock copolymer of PEG and PLA or PEG and PLGA (see US Pat No 8,246,968 and International Publication No.

WO2012166923, the contents of each of which are herein incorporated by reference in its entirety). In yet another non-limiting example, the therapeutic nanoparticle is a stealth nanoparticle or a target- specific stealth nanoparticle as described in US Patent Publication No. US20130172406, the contents of which are herein incorporated by reference in its entirety.

In one embodiment, the therapeutic nanoparticle may comprise a multiblock copolymer (See e.g., U.S. Pat. No. 8,263,665 and 8,287,910 and US Patent Pub. No.

US20130195987; the contents of each of which are herein incorporated by reference in its entirety).

In yet another non-limiting example, the lipid nanoparticle comprises the block copolymer PEG-PLGA-PEG (see e.g., the thermosensitive hydrogel (PEG-PLGA-PEG) was used as a TGF-betal gene delivery vehicle in Lee et al. Thermosensitive Hydrogel as a Tgf- βΐ Gene Delivery Vehicle Enhances Diabetic Wound Healing. Pharmaceutical Research, 2003 20(12): 1995-2000; as a controlled gene delivery system in Li et al. Controlled Gene Delivery System Based on Thermosensitive Biodegradable Hydrogel. Pharmaceutical

Research 2003 20(6):884-888; and Chang et al., Non-ionic amphiphilic biodegradable PEG- PLGA-PEG copolymer enhances gene delivery efficiency in rat skeletal muscle. J Controlled Release. 2007 118:245-253; each of which is herein incorporated by reference in its entirety). The RNA vaccines of the present invention may be formulated in lipid nanoparticles comprising the PEG-PLGA-PEG block copolymer.

In one embodiment, the therapeutic nanoparticle may comprise a multiblock copolymer (See e.g., U.S. Pat. No. 8,263,665 and 8,287,910 and US Patent Pub. No.

US20130195987; the contents of each of which are herein incorporated by reference in its entirety).

In one embodiment, the block copolymers described herein may be included in a polyion complex comprising a non-polymeric micelle and the block copolymer. (See e.g., U.S. Pub. No. 20120076836; herein incorporated by reference in its entirety).

In one embodiment, the therapeutic nanoparticle may comprise at least one acrylic polymer. Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates and combinations thereof.

In one embodiment, the therapeutic nanoparticles may comprise at least one poly(vinyl ester) polymer. The poly(vinyl ester) polymer may be a copolymer such as a random copolymer. As a non-limiting example, the random copolymer may have a structure such as those described in International Application No. WO2013032829 or US Patent Publication No US20130121954, the contents of which are herein incorporated by reference in its entirety. In one aspect, the poly(vinyl ester) polymers may be conjugated to the polynucleotides described herein. In another aspect, the poly(vinyl ester) polymer which may be used in the present invention may be those described in, herein incorporated by reference in its entirety.

In one embodiment, the therapeutic nanoparticle may comprise at least one diblock copolymer. The diblock copolymer may be, but it not limited to, a poly(lactic) acid- poly(ethylene)glycol copolymer (see e.g., International Patent Publication No.

WO2013044219; herein incorporated by reference in its entirety). As a non-limiting example, the therapeutic nanoparticle may be used to treat cancer (see International publication No. WO2013044219; herein incorporated by reference in its entirety).

In one embodiment, the therapeutic nanoparticles may comprise at least one cationic polymer described herein and/or known in the art.

In one embodiment, the therapeutic nanoparticles may comprise at least one amine- containing polymer such as, but not limited to polylysine, polyethylene imine,

poly(amidoamine) dendrimers, poly (beta- amino esters) (See e.g., U.S. Pat. No. 8,287,849; herein incorporated by reference in its entirety) and combinations thereof.

In another embodiment, the nanoparticles described herein may comprise an amine cationic lipid such as those described in International Patent Application No.

WO2013059496, the contents of which are herein incorporated by reference in its entirety. In one aspect the cationic lipids may have an amino-amine or an amino-amide moiety.

In one embodiment, the therapeutic nanoparticles may comprise at least one degradable polyester which may contain polycationic side chains. Degradeable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4- hydroxy-L-proline ester), and combinations thereof. In another embodiment, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer. In another embodiment, the therapeutic nanoparticle may include a conjugation of at least one targeting ligand. The targeting ligand may be any ligand known in the art such as, but not limited to, a monoclonal antibody. (Kirpotin et al, Cancer Res. 2006 66:6732-6740; herein incorporated by reference in its entirety).

In one embodiment, the therapeutic nanoparticle may be formulated in an aqueous solution which may be used to target cancer (see International Pub No. WO2011084513 and US Pub No. US20110294717, each of which is herein incorporated by reference in their entirety).

In one embodiment, the therapeutic nanoparticle RNA vaccines, e.g., therapeutic nanoparticles comprising at least one RNA vaccine may be formulated using the methods described by Podobinski et al in US Patent No. 8,404,799, the contents of which are herein incorporated by reference in its entirety.

In one embodiment, the RNA vaccines may be encapsulated in, linked to and/or associated with synthetic nanocarriers. Synthetic nanocarriers include, but are not limited to, those described in International Pub. Nos. WO2010005740, WO2010030763,

WO201213501, WO2012149252, WO2012149255, WO2012149259, WO2012149265, WO2012149268, WO2012149282, WO2012149301, WO2012149393, WO2012149405, WO2012149411, WO2012149454 and WO2013019669, and US Pub. Nos. US20110262491, US20100104645, US20100087337 and US20120244222, each of which is herein

incorporated by reference in their entirety. The synthetic nanocarriers may be formulated using methods known in the art and/or described herein. As a non-limiting example, the synthetic nanocarriers may be formulated by the methods described in International Pub Nos. WO2010005740, WO2010030763 and WO201213501and US Pub. Nos. US20110262491, US20100104645, US20100087337 and US2012024422, each of which is herein incorporated by reference in their entirety. In another embodiment, the synthetic nanocarrier formulations may be lyophilized by methods described in International Pub. No. WO2011072218 and US Pat No. 8,211,473; the content of each of which is herein incorporated by reference in their entirety. In yet another embodiment, formulations of the present invention, including, but not limited to, synthetic nanocarriers, may be lyophilized or reconstituted by the methods described in US Patent Publication No. US20130230568, the contents of which are herein incorporated by reference in its entirety.

In one embodiment, the synthetic nanocarriers may contain reactive groups to release the polynucleotides described herein (see International Pub. No. WO20120952552 and US Pub No. US20120171229, each of which is herein incorporated by reference in their entirety). In one embodiment, the synthetic nanocarriers may contain an immuno stimulatory agent to enhance the immune response from delivery of the synthetic nanocarrier. As a non- limiting example, the synthetic nanocarrier may comprise a Thl immunostimulatory agent which may enhance a Thl-based response of the immune system (see International Pub No. WO2010123569 and US Pub. No. US20110223201, each of which is herein incorporated by reference in its entirety).

In one embodiment, the synthetic nanocarriers may be formulated for targeted release. In one embodiment, the synthetic nanocarrier is formulated to release the polynucleotides at a specified pH and/or after a desired time interval. As a non-limiting example, the synthetic nanoparticle may be formulated to release the RNA vaccines after 24 hours and/or at a pH of 4.5 (see International Pub. Nos. WO2010138193 and WO2010138194 and US Pub Nos. US20110020388 and US20110027217, each of which is herein incorporated by reference in their entireties).

In one embodiment, the synthetic nanocarriers may be formulated for controlled and/or sustained release of the polynucleotides described herein. As a non-limiting example, the synthetic nanocarriers for sustained release may be formulated by methods known in the art, described herein and/or as described in International Pub No. WO2010138192 and US Pub No. 20100303850, each of which is herein incorporated by reference in their entirety.

In one embodiment, the RNA vaccine may be formulated for controlled and/or sustained release wherein the formulation comprises at least one polymer that is a crystalline side chain (CYSC) polymer. CYSC polymers are described in U.S. Patent No. 8,399,007, herein incorporated by reference in its entirety.

In one embodiment, the synthetic nanocarrier may be formulated for use as a vaccine. In one embodiment, the synthetic nanocarrier may encapsulate at least one polynucleotide which encode at least one antigen. As a non-limiting example, the synthetic nanocarrier may include at least one antigen and an excipient for a vaccine dosage form (see International Pub No. WO2011150264 and US Pub No. US20110293723, each of which is herein incorporated by reference in their entirety). As another non-limiting example, a vaccine dosage form may include at least two synthetic nanocarriers with the same or different antigens and an excipient (see International Pub No. WO2011150249 and US Pub No. US20110293701, each of which is herein incorporated by reference in their entirety). The vaccine dosage form may be selected by methods described herein, known in the art and/or described in International Pub No. WO2011150258 and US Pub No. US20120027806, each of which is herein incorporated by reference in their entirety). In one embodiment, the synthetic nanocarrier may comprise at least one polynucleotide which encodes at least one adjuvant. As non-limiting example, the adjuvant may comprise dimethyldioctadecylammonium-bromide, dimethyldioctadecylammonium- chloride, dimethyldioctadecylammonium-phosphate or dimethyldioctadecylammonium- acetate (DDA) and an apolar fraction or part of said apolar fraction of a total lipid extract of a mycobacterium (See e.g, U.S. Pat. No. 8,241,610; herein incorporated by reference in its entirety). In another embodiment, the synthetic nanocarrier may comprise at least one polynucleotide and an adjuvant. As a non-limiting example, the synthetic nanocarrier comprising and adjuvant may be formulated by the methods described in International Pub No. WO2011150240 and US Pub No. US20110293700, each of which is herein incorporated by reference in its entirety.

In one embodiment, the synthetic nanocarrier may encapsulate at least one

polynucleotide which encodes a peptide, fragment or region from a virus. As a non-limiting example, the synthetic nanocarrier may include, but is not limited to, the nanocarriers described in International Pub No. WO2012024621, WO201202629, WO2012024632 and

US Pub No. US20120064110, US20120058153 and US20120058154, each of which is herein incorporated by reference in their entirety.

In one embodiment, the synthetic nanocarrier may be coupled to a polynucleotide which may be able to trigger a humoral and/or cytotoxic T lymphocyte (CTL) response (See e.g., International Publication No. WO2013019669, herein incorporated by reference in its entirety).

In one embodiment, the RNA vaccine may be encapsulated in, linked to and/or associated with zwitterionic lipids. Non-limiting examples of zwitterionic lipids and methods of using zwitterionic lipids are described in US Patent Publication No. US20130216607, the contents of which are herein incorporated by reference in its entirety. In one aspect, the zwitterionic lipids may be used in the liposomes and lipid nanoparticles described herein.

In one embodiment, the RNA vaccine may be formulated in colloid nanocarriers as described in US Patent Publication No. US20130197100, the contents of which are herein incorporated by reference in its entirety.

In one embodiment, the nanoparticle may be optimized for oral administration. The nanoparticle may comprise at least one cationic biopolymer such as, but not limited to, chitosan or a derivative thereof. As a non-limiting example, the nanoparticle may be formulated by the methods described in U.S. Pub. No. 20120282343; herein incorporated by reference in its entirety. In some embodiments, LNPs comprise the lipid KL52 (an amino-lipid disclosed in U.S. Application Publication No. 2012/0295832 expressly incorporated herein by reference in its entirety). Activity and/or safety (as measured by examining one or more of ALT/AST, white blood cell count and cytokine induction) of LNP administration may be improved by incorporation of such lipids. LNPs comprising KL52 may be administered intravenously and/or in one or more doses. In some embodiments, administration of LNPs comprising KL52 results in equal or improved mRNA and/or protein expression as compared to LNPs comprising MC3.

In some embodiments, RNA vaccine may be delivered using smaller LNPs. Such particles may comprise a diameter from below 0.1 um up to 100 nm such as, but not limited to, less than 0.1 um, less than 1.0 um, less than 5 um, less than 10 um, less than 15 um, less than 20 um, less than 25 um, less than 30 um, less than 35 um, less than 40 um, less than 50 um, less than 55 um, less than 60 um, less than 65 um, less than 70 um, less than 75 um, less than 80 um, less than 85 um, less than 90 um, less than 95 um, less than 100 um, less than 125 um, less than 150 um, less than 175 um, less than 200 um, less than 225 um, less than 250 um, less than 275 um, less than 300 um, less than 325 um, less than 350 um, less than 375 um, less than 400 um, less than 425 um, less than 450 um, less than 475 um, less than 500 um, less than 525 um, less than 550 um, less than 575 um, less than 600 um, less than 625 um, less than 650 um, less than 675 um, less than 700 um, less than 725 um, less than 750 um, less than 775 um, less than 800 um, less than 825 um, less than 850 um, less than 875 um, less than 900 um, less than 925 um, less than 950 um, less than 975 um.

In another embodiment, RNA vaccines may be delivered using smaller LNPs which may comprise a diameter from about 1 nm to about 100 nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm to about 90 nm, about 10 to about 50 nM, from about 20 to about 50 nm, from about 30 to about 50 nm, from about 40 to about 50 nm, from about 20 to about 60 nm, from about 30 to about 60 nm, from about 40 to about 60 nm, from about 20 to about 70 nm, from about 30 to about 70 nm, from about 40 to about 70 nm, from about 50 to about 70 nm, from about 60 to about 70 nm, from about 20 to about 80 nm, from about 30 to about 80 nm, from about 40 to about 80 nm, from about 50 to about 80 nm, from about 60 to about 80 nm, from about 20 to about 90 nm, from about 30 to about 90 nm, from about 40 to about 90 nm, from about 50 to about 90 nm, from about 60 to about 90 nm and/or from about 70 to about 90 nm.

In some embodiments, such LNPs are synthesized using methods comprising microfluidic mixers. Exemplary microfluidic mixers may include, but are not limited to a slit interdigitial micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I.V. et al., Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing have been published (Langmuir. 2012. 28:3633-40; Belliveau, N.M. et al., Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA. Molecular

Therapy-Nucleic Acids. 2012. I:e37; Chen, D. et al., Rapid discovery of potent siRNA- containing lipid nanoparticles enabled by controlled microfluidic formulation. J Am Chem Soc. 2012. 134(16):6948-51; each of which is herein incorporated by reference in its entirety). In some embodiments, methods of LNP generation comprising SHM, further comprise the mixing of at least two input streams wherein mixing occurs by microstructure- induced chaotic advection (MICA). According to this method, fluid streams flow through channels present in a herringbone pattern causing rotational flow and folding the fluids around each other. This method may also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling. Methods of generating LNPs using SHM include those disclosed in U.S. Application Publication Nos. 2004/0262223 and

2012/0276209, each of which is expressly incorporated herein by reference in their entirety.

In one embodiment, the RNA vaccine of the present invention may be formulated in lipid nanoparticles created using a micromixer such as, but not limited to, a Slit Interdigital Micro structured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM)from the Institut fiir Mikrotechnik Mainz GmbH, Mainz Germany).

In one embodiment, the RNA vaccines of the present invention may be formulated in lipid nanoparticles created using microfluidic technology (see Whitesides, George M. The Origins and the Future of Microfluidic s. Nature, 2006 442: 368-373; and Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651; each of which is herein incorporated by reference in its entirety). As a non-limiting example, controlled microfluidic formulation includes a passive method for mixing streams of steady pressure-driven flows in micro channels at a low Reynolds number (See e.g., Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651; which is herein incorporated by reference in its entirety).

In one embodiment, the RNA vaccines of the present invention may be formulated in lipid nanoparticles created using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, MA) or Dolomite Microfluidics (Royston, UK). A

micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism.

In one embodiment, the RNA vaccines of the invention may be formulated for delivery using the drug encapsulating microspheres described in International Patent Publication No. WO2013063468 or U.S. Patent No. 8,440,614, each of which is herein incorporated by reference in its entirety. The microspheres may comprise a compound of the formula (I), (II), (III), (IV), (V) or (VI) as described in International Patent Publication No. WO2013063468, the contents of which are herein incorporated by reference in its entirety. In another aspect, the amino acid, peptide, polypeptide, lipids (APPL) are useful in delivering the RNA vaccines of the invention to cells (see International Patent Publication No.

WO2013063468, the contents of which is herein incorporated by reference in its entirety).

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.

In one aspect, the lipid nanoparticle may be a limit size lipid nanoparticle described in International Patent Publication No. WO2013059922, the contents of which are herein incorporated by reference in its entirety. The limit size lipid nanoparticle may comprise a lipid bilayer surrounding an aqueous core or a hydrophobic core; where the lipid bilayer may comprise a phospholipid such as, but not limited to, diacylphosphatidylcholine, a

diacylphosphatidylethanolamine, a ceramide, a sphingomyelin, a dihydrosphingomyelin, a cephalin, a cerebroside, a C8-C20 fatty acid diacylphophatidylcholine, and l-palmitoyl-2- oleoyl phosphatidylcholine (POPC). In another aspect the limit size lipid nanoparticle may comprise a polyethylene glycol-lipid such as, but not limited to, DLPE-PEG, DMPE-PEG, DPPC-PEG and DSPE-PEG.

In one embodiment, the RNA vaccines may be delivered, localized and/or

concentrated in a specific location using the delivery methods described in International Patent Publication No. WO2013063530, the contents of which are herein incorporated by reference in its entirety. As a non-limiting example, a subject may be administered an empty polymeric particle prior to, simultaneously with or after delivering the RNA vaccines to the subject. The empty polymeric particle undergoes a change in volume once in contact with the subject and becomes lodged, embedded, immobilized or entrapped at a specific location in the subject.

In one embodiment, the RNA vaccines may be formulated in an active substance release system (See e.g., US Patent Publication No. US20130102545, the contents of which is herein incorporated by reference in its entirety). The active substance release system may comprise 1) at least one nanoparticle bonded to an oligonucleotide inhibitor strand which is hybridized with a catalytically active nucleic acid and 2) a compound bonded to at least one substrate molecule bonded to a therapeutically active substance (e.g., polynucleotides described herein), where the therapeutically active substance is released by the cleavage of the substrate molecule by the catalytically active nucleic acid.

In one embodiment, the RNA vaccines may be formulated in a nanoparticle comprising an inner core comprising a non-cellular material and an outer surface comprising a cellular membrane. The cellular membrane may be derived from a cell or a membrane derived from a virus. As a non-limiting example, the nanoparticle may be made by the methods described in International Patent Publication No. WO2013052167, herein incorporated by reference in its entirety. As another non-limiting example, the nanoparticle described in International Patent Publication No. WO2013052167, herein incorporated by reference in its entirety, may be used to deliver the RNA vaccines described herein.

In one embodiment, the RNA vaccines may be formulated in porous nanoparticle- supported lipid bilayers (protocells). Protocells are described in International Patent

Publication No. WO2013056132, the contents of which are herein incorporated by reference in its entirety.

In one embodiment, the RNA vaccines described herein may be formulated in polymeric nanoparticles as described in or made by the methods described in US Patent No. 8,420,123 and 8,518,963 and European Patent No. EP2073848B 1, the contents of each of which are herein incorporated by reference in their entirety. As a non-limiting example, the polymeric nanoparticle may have a high glass transition temperature such as the nanoparticles described in or nanoparticles made by the methods described in US Patent No. 8,518,963, the contents of which are herein incorporated by reference in its entirety. As another non- limiting example, the polymer nanoparticle for oral and parenteral formulations may be made by the methods described in European Patent No. EP2073848B 1, the contents of which are herein incorporated by reference in its entirety.

In another embodiment, the RNA vaccines described herein may be formulated in nanoparticles used in imaging. The nanoparticles may be liposome nanoparticles such as those described in US Patent Publication No US20130129636, herein incorporated by reference in its entirety. As a non-limiting example, the liposome may comprise

gadolinium(III)2-{4,7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N'-amido-methyl]- 1,4,7, 10-tetra-azacyclododec-l-yl}-acetic acid and a neutral, fully saturated phospholipid component (see e.g., US Patent Publication No US20130129636, the contents of which is herein incorporated by reference in its entirety).

In one embodiment, the nanoparticles which may be used in the present invention are formed by the methods described in U.S. Patent Application No. US20130130348, the contents of which is herein incorporated by reference in its entirety.

The nanoparticles of the present invention may further include nutrients such as, but not limited to, those which deficiencies can lead to health hazards from anemia to neural tube defects (see e.g, the nanoparticles described in International Patent Publication No WO2013072929, the contents of which is herein incorporated by reference in its entirety). As a non-limiting example, the nutrient may be iron in the form of ferrous, ferric salts or elemental iron, iodine, folic acid, vitamins or micronutrients.

In one embodiment, the RNA vaccines of the present invention may be formulated in a swellable nanoparticle. The swellable nanoparticle may be, but is not limited to, those described in U.S. Patent No. 8,440,231, the contents of which is herein incorporated by reference in its entirety. As a non-limiting embodiment, the swellable nanoparticle may be used for delivery of the RNA vaccines of the present invention to the pulmonary system (see e.g., U.S. Patent No. 8,440,231, the contents of which is herein incorporated by reference in its entirety).

The RNA vaccines of the present invention may be formulated in polyanhydride nanoparticles such as, but not limited to, those described in U.S. Patent No. 8,449,916, the contents of which is herein incorporated by reference in its entirety.

The nanoparticles and microparticles of the present invention may be geometrically engineered to modulate macrophage and/or the immune response. In one aspect, the geometrically engineered particles may have varied shapes, sizes and/or surface charges in order to incorporated the polynucleotides of the present invention for targeted delivery such as, but not limited to, pulmonary delivery (see e.g., International Publication No

WO2013082111, the contents of which is herein incorporated by reference in its entirety). Other physical features the geometrically engineering particles may have include, but are not limited to, fenestrations, angled arms, asymmetry and surface roughness, charge which can alter the interactions with cells and tissues. As a non-limiting example, nanoparticles of the present invention may be made by the methods described in International Publication No WO2013082111, the contents of which is herein incorporated by reference in its entirety.

In one embodiment, the nanoparticles of the present invention may be water soluble nanoparticles such as, but not limited to, those described in International Publication No. WO2013090601, the contents of which is herein incorporated by reference in its entirety. The nanoparticles may be inorganic nanoparticles which have a compact and zwitterionic ligand in order to exhibit good water solubility. The nanoparticles may also have small hydrodynamic diameters (HD), stability with respect to time, pH, and salinity and a low level of non-specific protein binding.

In one embodiment the nanoparticles of the present invention may be developed by the methods described in US Patent Publication No. US20130172406, the contents of which are herein incorporated by reference in its entirety. In one embodiment, the nanoparticles of the present invention are stealth nanoparticles or target- specific stealth nanoparticles such as, but not limited to, those described in US Patent Publication No. US20130172406; the contents of which is herein incorporated by reference in its entirety. The nanoparticles of the present invention may be made by the methods described in US Patent Publication No. US20130172406, the contents of which are herein incorporated by reference in its entirety.

In another embodiment, the stealth or target- specific stealth nanoparticles may comprise a polymeric matrix. The polymeric matrix may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polyesters, polyanhydrides, polyethers, polyurethanes, polymethacrylates, polyacrylates, polycyanoacrylates or combinations thereof.

In one embodiment, the nanoparticle may be a nanoparticle-nucleic acid hybrid structure having a high density nucleic acid layer. As a non-limiting example, the

nanoparticle-nucleic acid hybrid structure may made by the methods described in US Patent Publication No. US20130171646, the contents of which are herein incorporated by reference in its entirety. The nanoparticle may comprise a nucleic acid such as, but not limited to, polynucleotides described herein and/or known in the art.

At least one of the nanoparticles of the present invention may be embedded in in the core a nanostructure or coated with a low density porous 3-D structure or coating which is capable of carrying or associating with at least one pay load within or on the surface of the nanostructure. Non-limiting examples of the nanostructures comprising at least one nanoparticle are described in International Patent Publication No. WO2013123523, the contents of which are herein incorporated by reference in its entirety.

In some embodiments the RNA vaccine may be associated with a cationic or polycationic compounds, including protamine, nucleoline, spermine or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), polyarginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV-binding peptides, HIV-1 Tat (fflV), Tat- derived peptides, Penetratin, VP 22 derived or analog peptides, Pestivirus Erns, HSV, VP 22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs), PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides (particularly from Drosophila antennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(l), pVEC, hCT-derived peptides, SAP, histones, cationic polysaccharides, for example chitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g. DOTMA: [l-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14- amidine, DOTIM, SAINT, DC-Choi, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS:

Dioctadecylamidoglicylspermin, DIMRI: Dimyristooxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: 0,0- ditetradecanoyl-N-.alpha.-trimethylammonioacetyl)diethanolamine chloride, CLIP 1: rac- [(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac- [2(2,3-dihexadecyloxypropyloxymethyloxy)ethyl]-trimethylammonium, CLIP9: rac-[2(2,3- dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammo- nium, oligofectamine, or cationic or polycationic polymers, e.g. modified polyaminoacids, such as beta-aminoacid- polymers or reversed polyamides, etc., modified polyethylenes, such as PVP (poly(N-ethyl-4- vinylpyridinium bromide)), etc., modified acrylates, such as pDMAEMA

(poly(dimethylaminoethyl methylacrylate)), etc., modified amidoamines such as pAMAM (poly(amidoamine)), etc., modified polybetaminoester (PBAE), such as diamine end modified 1,4 butanediol diacrylate-co-5 -amino- 1-pentanol polymers, etc., dendrimers, such as polypropylamine dendrimers or pAMAM based dendrimers, etc., polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine), etc., polyallylamine, sugar backbone based polymers, such as cyclodextrin based polymers, dextran based polymers, chitosan, etc., silan backbone based polymers, such as PMOXA-PDMS copolymers, etc., blockpolymers consisting of a combination of one or more cationic blocks (e.g. selected from a cationic polymer as mentioned above) and of one or more hydrophilic or hydrophobic blocks (e.g. polyethyleneglycole); etc.

In other embodiments the RNA vaccine is not associated with a cationic or polycationic compounds.

Modes of Vaccine Administration

Ebola virus 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. Ebola virus 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 Ebola virus 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 some embodiments, Ebola virus RNA vaccines compositions 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, Ebola virus RNA vaccines compositions 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, Ebola virus RNA vaccine compositions 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, Ebola virus RNA vaccine compositions 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 Ebola virus RNA vaccine composition may be administered three or four times.

In some embodiments, Ebola virus RNA vaccine compositions 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).

Some aspects of the present disclosure provide formulations of the Ebola virus RNA (e.g. , mRNA) vaccine, wherein the Ebola virus 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 an anti-Ebola virus antigenic polypeptide). "An effective amount" is a dose of an Ebola virus 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-Ebola virus antigenic polypeptide antibody titer produced in a subject administered a Ebola virus 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 (e.g. , an anti-Ebola virus antigenic polypeptide) 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 embodiemnts, an antibody titer is used to determine the strength of an autoimmune response, to determine whether a booster immunization is needed, to derermine 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 Ebola virus RNA vaccine.

In some embodiments, an anti-Ebola virus antigenic polypeptide antibody titer produced in a subject is increased by at least 1 log relative to a control. For example, anti- Ebola virus antigenic polypeptide 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 anti-Ebola virus antigenic polypeptide 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 anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is increased by 1-3 log relative to a control. For example, the anti-Ebola virus antigenic polypeptide 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 anti-Ebola virus antigenic polypeptide antibody titer produced in a subject is increased at least 2 times relative to a control. For example, the anti- Ebola virus antigenic polypeptide 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 anti- Ebola virus antigenic polypeptide 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-Ebola virus antigenic polypeptide antibody titer produced in a subject is increased 2- 10 times relative to a control. For example, the anti-Ebola virus antigenic polypeptide 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 the anti-Ebola virus antigenic polypeptide antibody titer produced in a subject who has not been administered a Ebola virus RNA (e.g. , mRNA) vaccine. In some embodiments, a control is an anti-Ebola virus antigenic polypeptide antibody titer produced in a subject who has been administered a live attenuated Ebola virus 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-Ebola virus antigenic polypeptide antibody titer produced in a subject administered inactivated Ebola virus vaccine. In some embodiments, a control is an anti-Ebola virus antigenic polypeptide antibody titer produced in a subject administered a recombinant or purified Ebola virus 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 other embodiments the control is ChAd3-EBO-Z, Chimpanzee adenovirus vector for single EVI dose by GSK. In other embodiments the control is VSV-EBOV, a recombinant, replication-competent vaccine, consisting of a vesicular stomatitis virus, which has been genetically engineered to express Ebola glycoproteins so as to provoke an immune response against the complete Ebola virus.

In some embodiments the vaccination protocol is a ring vaccination. A ring vaccination vaccinates all suspected individuals in an area around an outbreak (e.g., family members of those infected).

In some embodiments, an effective amount of a Ebola virus RNA (e.g. , mRNA) vaccine is a dose that is reduced compared to the standard of care dose of a recombinant Ebola virus 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 Ebola virus protein vaccine, or a live attenuated or inactivated Ebola virus vaccine, that a physician/clinician or other medical professional would administer to a subject to treat or prevent Ebola virus, or a Ebola virus-related condition, while following the standard of care guideline for treating or preventing Ebola virus, or a Ebola virus-related condition.

In some embodiments, the anti-Ebola virus antigenic polypeptide antibody titer produced in a subject administered an effective amount of a Ebola virus RNA vaccine is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered a standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus vaccine.

In some embodiments, an effective amount of a Ebola virus 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 Ebola virus protein vaccine. For example, an effective amount of a Ebola virus 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 Ebola virus protein vaccine. In some embodiments, an effective amount of a Ebola virus 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 Ebola virus protein vaccine. In some embodiments, an effective amount of a Ebola virus 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 Ebola virus protein vaccine. In some embodiments, the anti-Ebola virus antigenic polypeptide antibody titer produced in a subject administered an effective amount of a Ebola virus RNA vaccine is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or protein Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus vaccine. In some embodiments, an effective amount of a Ebola virus 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 Ebola virus protein vaccine, wherein the anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus vaccine. In some embodiments, the effective amount of a Ebola virus 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 Ebola virus protein vaccine. In some embodiments, such as the foregoing, the anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus 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 Ebola virus protein vaccine. In some embodiments, such as the foregoing, an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus vaccine.

In some embodiments, the effective amount of a Ebola virus RNA (e.g. , mRNA) vaccine is a total dose of 50- 1000 μg. In some embodiments, the effective amount of a Ebola virus RNA (e.g. , mRNA) vaccine is a total dose of 50- 1000, 50- 900, 50-800, 50-700, SO- 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 Ebola virus 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 Ebola virus 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 Ebola virus 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 3ΌΗ (SEG. 1)

(b) 5' triphosphate segment which may include the coding region of a polypeptide and comprising a normal 3ΌΗ (SEG. 2)

(c) 5' monophosphate segment for the 3' end of the chimeric polynucleotide (e.g., the tail) comprising cordycepin or no 3ΌΗ (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 2x KAPA HIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, MA). This system includes 2x KAPA ReadyMix 12.5 μΐ; Forward Primer (10 μΜ) 0.75 μΐ; Reverse Primer (10 μΜ) 0.75 μΐ;

Template cDNA -100 ng; and dH₂0 diluted to 25.0 μΐ. 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, CA) 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 μ§

2 lOx transcription buffer (400 mM Tris-HCl pH 8.0, 190 mM MgCl₂, 50 mM DTT, 10 mM Spermidine) 2.0 μΐ

3 Custom NTPs (25mM each) 7.2 μΐ

4 RNase Inhibitor 20 U

5 T7 RNA polymerase 3000 U

6 dH₂0 Up to 20.0 μΐ. 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, TX) following the manufacturer's instructions. This kit can purify up to 500 μg 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: Enzymatic Capping

Capping of a polynucleotide is performed as follows where the mixture includes: rVT RNA 60 μg-180μg and dH₂0 up to 72 μΐ. The mixture is incubated at 65° C for 5 minutes to denature RNA, and then is transferred immediately to ice.

The protocol then involves the mixing of lOx Capping Buffer (0.5 M Tris-HCl (pH 8.0), 60 mM KC1, 12.5 mM MgCl₂) (10.0 μΐ); 20 mM GTP (5.0 μΐ); 20 mM S-Adenosyl Methionine (2.5 μΐ); RNase Inhibitor (100 U); 2'-0-Methyltransferase (400U); Vaccinia capping enzyme (Guanylyl transferase) (40 U); dH₂0 (Up to 28 μΐ); and incubation at 37° C for 30 minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.

The polynucleotide is then purified using Ambion's MEGACLEAR™ Kit (Austin, TX) following the manufacturer's instructions. Following the cleanup, the RNA is quantified using the NANODROP™ (ThermoFisher, Waltham, MA) and analyzed by agarose gel electrophoresis to confirm the RNA is the proper size and that no degradation of the RNA has occurred. The RNA product may also be sequenced by running a reverse-transcription-PCR to generate the cDNA for sequencing. Example 6: PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must be performed before cleaning the final product. This is done by mixing Capped IVT RNA (100 μΐ); RNase Inhibitor (20 U); lOx Tailing Buffer (0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM

MgCl₂)(12.0 μΐ); 20 mM ATP (6.0 μΐ); Poly-A Polymerase (20 U); d¾0 up to 123.5 μΐ and incubation at 37° C for 30 min. If the poly-A tail is already in the transcript, then the tailing reaction may be skipped and proceed directly to cleanup with Ambion' s MEGACLEAR™ kit (Austin, TX) (up to 500 μg). Poly-A Polymerase is preferably a recombinant enzyme expressed in yeast.

It should be understood that the processivity or integrity of the polyA tailing reaction may not always result in an exact size polyA tail. Hence polyA tails of approximately between 40-200 nucleotides, e.g., about 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 150-165, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scope of the invention. Example 7: Natural 5^f Caps and 5^f Cap Analogues

5 '-capping of polynucleotides may be completed concomitantly during the in vitro- transcription reaction using the following chemical RNA cap analogs to generate the 5'- guanosine cap structure according to manufacturer protocols: 3'-0-Me-m7G(5')ppp(5') G [the ARCA cap];G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New

England BioLabs, Ipswich, MA). 5'-capping of modified RNA may be completed post- transcriptionally using a Vaccinia Virus Capping Enzyme to generate the "Cap 0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Cap 1 structure may be generated using both Vaccinia Virus Capping Enzyme and a 2'-0 methyl-transferase to generate:

m7G(5')ppp(5')G-2'-0-methyl. Cap 2 structure may be generated from the Cap 1 structure followed by the 2'-0-methylation of the 5 '-antepenultimate nucleotide using a 2'-0 methyl- transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2'-0- methylation of the 5'-preantepenultimate nucleotide using a 2'-0 methyl-transferase.

Enzymes are preferably derived from a recombinant source. When transfected into mammalian cells, the modified mRNAs have a stability of between 12- 18 hours or more than 18 hours, e.g. , 24, 36, 48, 60, 72 or greater than 72 hours.

Example 8: Capping Assays

A. Protein Expression Assay

Polynucleotides encoding a polypeptide, containing any of the caps taught herein can be transfected into cells at equal concentrations. 6, 12, 24 and 36 hours post-transfection the amount of protein secreted into the culture medium can be assayed by ELISA. Synthetic polynucleotides that secrete higher levels of protein into the medium would correspond to a synthetic polynucleotide with a higher translationally-competent Cap structure.

B. Purity Analysis Synthesis

Polynucleotides encoding a polypeptide, containing any of the caps taught herein can be compared for purity using denaturing Agarose-Urea gel electrophoresis or HPLC analysis. Polynucleotides with a single, consolidated band by electrophoresis correspond to the higher purity product compared to polynucleotides with multiple bands or streaking bands.

Synthetic polynucleotides with a single HPLC peak would also correspond to a higher purity product. The capping reaction with a higher efficiency would provide a more pure polynucleotide population.

C. Cytokine Analysis

Polynucleotides encoding a polypeptide, containing any of the caps taught herein can be transfected into cells at multiple concentrations. 6, 12, 24 and 36 hours post-transfection the amount of pro-inflammatory cytokines such as TNF-alpha and IFN-beta secreted into the culture medium can be assayed by ELISA. Polynucleotides resulting in the secretion of higher levels of pro -inflammatory cytokines into the medium would correspond to a polynucleotides containing an immune-activating cap structure.

D. Capping Reaction Efficiency

Polynucleotides encoding a polypeptide, containing any of the caps taught herein can be analyzed for capping reaction efficiency by LC-MS after nuclease treatment. Nuclease treatment of capped polynucleotides would yield a mixture of free nucleotides and the capped 5 '-5 -triphosphate cap structure detectable by LC-MS. The amount of capped product on the LC-MS spectra can be expressed as a percent of total polynucleotide from the reaction and would correspond to capping reaction efficiency. The cap structure with higher capping reaction efficiency would have a higher amount of capped product by LC-MS. Example 9: Agarose Gel Electrophoresis of Modified RNA or RT PCR Products

Individual polynucleotides (200-400 ng in a 20 μΐ volume) or reverse transcribed PCR products (200-400 ng) are loaded into a well on a non-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, CA) and run for 12-15 minutes according to the manufacturer protocol.

Example 10: Nanodrop Modified RNA Quantification and UV Spectral Data

Modified polynucleotides in TE buffer (1 μΐ) are used for Nanodrop UV absorbance readings to quantitate the yield of each polynucleotide from an chemical synthesis or in vitro transcription reaction.

Example 11; Formulation of Modified mRNA Using Lipidoids

Polynucleotides are formulated for in vitro experiments by mixing the polynucleotides with the lipidoid at a set ratio prior to addition to cells. In vivo formulation may require the addition of extra ingredients to facilitate circulation throughout the body. To test the ability of these lipidoids to form particles suitable for in vivo work, a standard formulation process used for siRNA-lipidoid formulations may used as a starting point. After formation of the particle, polynucleotide is added and allowed to integrate with the complex. The

encapsulation efficiency is determined using a standard dye exclusion assays.

Example 12; Evaluation of immunogenicity of Ebola GP antigens

Introduction

The Ebola glycoprotein (GP) is the only virally expressed protein on the virion surface, where it is essential for the attachment to host cells and catalyzes membrane fusion. Therefore, the Ebola GP is a critical component of vaccines, as well as a target of neutralizing antibodies and inhibitors of attachment and fusion. Pre-GP is cleaved by furin at a multi- basic motif into two subunits, GPl and GP2, which remain associated through a disulfide linkage between Cys53 of GPl and Cys609 of GP2. The heterodimer (GPl and GP2) then assembles into a 450-kDa trimer (3 GPl and 3 GP2) at the surface of nascent virions, where it exerts its functions. The structure of Ebola GP and antigen constructs tested herein are shown in FIG. 1 and Table 1.

Breifly, "Matured" EBOV GP has been engineered to include a human signal peptide. "Secreted" EBOV GP has been engineered to remove the transmembrane domain, i.e., residues 651-676 as depicted in the schematic. The "peptide scaffold" is as described in Schroder et al 2008. This short peptide sequence is described in the art as being capable of facilitating nanostructure formation. In preliminary experiments, the scaffold did not enhance antigenicity in the constructs tested. Without being bound in theory, it is believed that the scaffold peptide is not able to facilitate nanostructure formation under the physiologic conditions exemplified.

Table 1: Ebola glycoprotein constructs

Immunogenicity Evaluation: Study Design

In order to evaluate the antigenicity of seven Ebola antigen constructs, the following protocol was developed. As shown in FIG. 2, the mice were vaccinated with MC3- formulated, mRNA-encoded Ebola GP on days 0 (primary) and 14 (first booster). The doses were 0.4mg/kg. Samples were collected on days 0, 10, 21, 33, 52, and 77. Mice were euthanized on Day 77. Recombinant EBOV GP and PBS were used as the positive and negative experimental controls, respectively.

Results

The initial anti-Ebola GP response at Day 10 after a single primary challenge was generally within the range of lU/mL of anti-Ebola GP mouse antibody, as measured by ELISA (FIG. 3). Serum samples were diluted 1: 100 for the assay. For the positive control, the colored bars represent the units depicted. For the various constructs tested, the colored bars represent antibody titers for individual mice tested. As compared to PBS control, essentially all constructs had detectable antibody titers at 10 days following immunization.

The anti-Ebola GP antibody titer of selected antigens on Day 21 and Day 23 was also examined (n=3 per group) (FIG. 4).

The antibody response to the Ebola GP antigen at Day 21 post-vaccination was also quantified in each group (FIG. 5). For the positive control, the colored bars represent the units depicted. For the various constructs tested, the colored bars represent antibody titers for individual mice tested. As compared to PBS control, essentially all constructs had significant antibody titers at 10 days following immunization.

The in vitro neutralization activity of serum samples in the Delta Vp30 Ebola virus system has been examined (Halfman et al., PNAS 105: 1129) (FIG. 6). Naive mouse serum was found to have blocking activity in the assay, which was particularly evident at the 1:20 dilution and is believed to have masked the potential specific neutralizing activity of serum samples from animals at higher concentrations. The 1:980 dilution was found to be the best with respect to the evaluating the virus neutralizing capability of the samples. The background at this dilution was typically less than 10% on the Day 0 time point.

Example 13: In Vivo Vaccination of Guinea Pigs

Introduction

Guinea Pigs were vaccinated with Ebola GP (either pre -protein GP or mature GP) according to the vaccination schedule shown in FIG. 7 and Table 2.

Table 2

The guinea pigs were primed with 20 ug of vaccine on day 0 and boosted with 20ug of vaccine on day 21 (both IM). Animals were challenged with 1,000 pfu of guinea pig- adapted Ebola virus on day 42. Blood was collected on days 42, 45, 48, 51, 54, 63, and 70, followed by euthanization of the animals on day 70. Two mRNA vaccine constructs were tested (EH_EB LA . matGP . IgKsp(mem) SEQ ID NO. 17 and EH_EB LA . wtGP(mem) SEQ ID NO. 21) and a control unvaccinated group received PBS. Dosing is IM and there are 2 doses/animal ( 2 and 10 ug). The mRNA vaccines include pseudo uridine modifications.

Quite surprisingly, vaccination with the mRNA vaccine conferred 100% protection against 10E3 PFUs of gp-adapted Ebola (Zaire species, Mayinga strain). Untreated animals succumbed to the infection completely by day 10 post infection. The data is shown in FIG. 8.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.

All references, including patent documents, disclosed herein are incorporated by reference in their entirety.

Claims

What is claimed is: CLAIMS

1. An Ebola virus vaccine, comprising:

at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding an Ebola virus antigenic polypeptide or an immunogenic fragment thereof, wherein the at least one RNA polynucleotide comprises at least one chemical modification.

2. The Ebola vaccine of claim 1, wherein the Ebola virus antigenic polypeptide is EBOV glycoprotein (GP), surface EBOV GP, wild type EBOV pro-GP, mature EBOV GP, secreted wild type EBOV pro-GP, secreted mature EBOV GP, or a combination of any two or more of the foregoing.

3. The Ebola vaccine of claim 1, wherein the Ebola virus antigenic polypeptide is EBOV nucleoprotein (NP).

4. The Ebola vaccine of claim 3, wherein the Ebola virus antigenic polypeptide is EBOV matrix protein selected from VP35, VP40, VP24, and VP30.

5. The Ebola vaccine of claim 4, wherein the Ebola virus antigenic polypeptide is EBOV RNA polymerase L.

6. The Ebola vaccine of any one of claims 1-5, wherein the Ebola virus antigenic polypeptide is from Ebola virus strain subtype Zaire, strain H.sapiens- wt/GIN/2014/Kissidougou-C15; subtype Bundibugyo, strain Uganda 2007; subtype Zaire, strain Mayinga 1976; subtype Sudan, strain Gulu or a combination thereof.

7. The Ebola virus vaccine of any one of claims 1-6, further comprising an adjuvant.

8. The Ebola virus vaccine of any one of claims 1-7, wherein the open reading from is codon-optimized .

9. The Ebola virus vaccine of any one of claims 1-8, wherein the vaccine is multivalent.

10. The Ebola virus vaccine of claim 9, wherein the at least one RNA polynucleotide encodes 1 -10 antigenic polypeptides.

11. The Ebola virus vaccine of claim 10, wherein the at least one RNA polynucleotide encodes 1 - 100 antigenic polypeptides.

12. The Ebola virus vaccine of any one of claims 1-8, wherein the at least one RNA polynucleotide encodes 10-100 antigenic polypeptides.

13. The Ebola virus vaccine of any one of claims 1-12, wherein the at least one RNA polynucleotide comprises at least two chemical modifications.

14. The Ebola virus vaccine of claim 13, wherein the chemical modification is selected from pseudouridine, Nl-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5- methylcytosine, 2-thio-l -methyl- 1-deaza-pseudouridine, 2-thio-l -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-l -methyl - pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine), 5-methoxyuridine and 2'-0-methyl uridine.

15. The Ebola virus vaccine of any one of claims 1-14 formulated in a nanoparticle.

16. The Ebola virus vaccine of claim 15, wherein the nanoparticle has a mean diameter of 50-200 nm.

17. The Ebola virus vaccine of claim 15 or 16, wherein the nanoparticle is a lipid nanoparticle.

18. The Ebola virus vaccine of claim 17, wherein the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid.

19. The Ebola virus vaccine of claim 18, wherein the cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol.

20. The Ebola virus vaccine of claim 19, wherein the cationic lipid is selected from 2,2- dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

21. The Ebola virus vaccine of any one of claims 15-20, wherein the nanoparticle has a polydiversity value of less than 0.4.

22. The Ebola virus vaccine of any one of claims 15-20, wherein the nanoparticle has a net neutral charge at a neutral pH value.

23. An Ebola virus vaccine, comprising:

at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding an Ebola virus antigenic polypeptide, at least one 5' terminal cap and at least one chemical modification, formulated within a lipid nanoparticle.

24. The Ebola virus vaccine of claim 23, wherein the 5' terminal cap is

7mG(5')ppp(5')NlmpNp.

25. The Ebola virus vaccine of claim 23 or 24, wherein the at least one chemical modification is selected from pseudouridine, Nl-methylpseudouridine, 2-thiouridine, 4'- thiouridine, 5-methylcytosine, 2-thio-l -methyl- 1-deaza-pseudouridine, 2-thio-l-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-l- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5- methyluridine), 5-methoxyuridine and 2'-0-methyl uridine.

26. The Ebola virus vaccine of any one of claims 15-25, wherein the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid.

27. The Ebola virus vaccine of claim 26, wherein the cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol.

28. The Ebola virus vaccine of claim 27, wherein the cationic lipid is selected from 2,2- dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

29. An Ebola virus vaccine, comprising:

at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding an Ebola virus antigenic polypeptide, wherein at least 80% of the uracil in the open reading frame have a chemical modification.

30. The Ebola virus vaccine of claim 29, wherein 100% of the uracil in the open reading frame have a chemical modification.

31. The Ebola virus vaccine of claim 29 or 30, wherein the chemical modification is in the 5 -position of the uracil.

32. The Ebola virus vaccine of any one of claims 29-31, wherein the chemical modification is a Nl -methyl pseudouridine.

33. The Ebola virus vaccine of any one of claims 29-32, wherein the vaccine is formulated in a lipid nanoparticle.

34. The Ebola virus vaccine of any one of claims 1-33, wherein the at least one RNA polynucleotide encodes an antigenic polypeptide having greater than 90% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and having membrane fusion activity.

35. The Ebola virus vaccine of claim 34, wherein the at least one RNA polynucleotide encodes an antigenic polypeptide having greater than 95% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and having membrane fusion activity.

36. The Ebola virus vaccine of claim 35, wherein the at least one RNA polynucleotide encodes an antigenic polypeptide having greater than 96% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and having membrane fusion activity.

37. The Ebola virus vaccine of claim 36, wherein the at least one RNA polynucleotide encodes an antigenic polypeptide having greater than 97% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and having membrane fusion activity.

38. The Ebola virus vaccine of claim 37, wherein the at least one RNA polynucleotide encodes an antigenic polypeptide having greater than 98% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and having membrane fusion activity.

39. The Ebola virus vaccine of claim 38, wherein the at least one RNA polynucleotide encodes an antigenic polypeptide having greater than 99% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and having membrane fusion activity.

40. The Ebola virus vaccine of claim 38, wherein the at least one RNA polynucleotide encodes an antigenic polypeptide having 95-99% identity to an amino acid sequence of any one of Tables 3, 5 and 7 and having membrane fusion activity.

41. The Ebola virus of any one of claims 1-33, wherein the at least one RNA

polynucleotide encodes an antigenic polypeptide having an amino acid sequence of any one of Tables 3, 5 and 7, and wherein the RNA polynucleotide is codon optimized mRNA.

42. The Ebola virus of any one of claims 1-33, wherein the at least one RNA

polynucleotide encodes an antigenic polypeptide having membrane fusion activity and an amino acid sequence of any one of Tables 3, 5 and 7, and wherein the RNA polynucleotide has less than 80% identity to wild-type mRNA sequence.

43. The Ebola virus of any one of claims 1-33, wherein the at least one RNA

polynucleotide encodes an antigenic polypeptide having membrane fusion activity and an amino acid sequence of any one of Tables 3, 5 and 7, and wherein the RNA polynucleotide has greater than 80% identity to wild-type mRNA sequence, but does not include wild-type mRNA sequence.

44. The Ebola virus of any one of claims 1-33, wherein the at least one RNA

polynucleotide encodes an antigenic nucleoprotein polypeptide which mediates viral attachment and having an amino acid sequence of any one of Tables 3, 5 and 7, and wherein the RNA polynucleotide has less than 80% identity to wild-type mRNA sequence.

45. The Ebola virus of any one of claims 1-33, wherein the at least one RNA

polynucleotide encodes an antigenic nucleoprotein polypeptide which mediates viral attachment and having an amino acid sequence of any one of Tables 3, 5 and 7, and wherein the RNA polynucleotide has greater than 80% identity to wild-type mRNA sequence, but does not include wild-type nucleoprotein mRNA sequence.

46. The Ebola virus of any one of claims 1-33, wherein the at least one RNA

polynucleotide encodes an antigenic matrix polypeptide which mediates viral assembly and budding and having an amino acid sequence of any one of Tables 3, 5 and 7, and wherein the RNA polynucleotide has less than 80% identity to wild-type mRNA sequence.

47. The Ebola virus of any one of claims 1-33, wherein the at least one RNA

polynucleotide encodes an antigenic matrix polypeptide which mediates viral assembly and budding and having an amino acid sequence of any one of Tables 3, 5 and 7, and wherein the RNA polynucleotide has greater than 80% identity to wild-type mRNA sequence, but does not include wild-type matrix protein mRNA sequence.

48. The Ebola virus of any one of claims 1-33, wherein the at least one RNA

polynucleotide encodes an antigenic polypeptide that attaches to cell receptors.

49. The Ebola virus of any one of claims 1-33, wherein the at least one RNA

polynucleotide encodes an antigenic polypeptide that mediates viral assembly and budding.

50. The Ebola virus of any one of claims 1-33, wherein the at least one RNA

polynucleotide encodes an antigenic polypeptide that mediates viral attachment.

51. The Ebola virus of any one of claims 1-33, wherein the at least one RNA

polynucleotide encodes an antigenic polypeptide that is responsible for binding of the Ebola virus to a cell being infected.

52. A method of inducing an antigen specific immune response in a subject, comprising administering to the subject the Ebola virus vaccine of any one of claims 1-35 in an amount effective to produce an antigen specific immune response.

53. The method of claim 52, wherein the antigen specific immune response comprises a T cell response.

54. The method of claim 52, wherein the antigen specific immune response comprises a B cell response.

55. The method of any one of claims 52-54, wherein the method of inducing an antigen specific immune response involves a single administration of the Ebola virus vaccine.

56. The method of any one of claims 52-54, further comprising administering a booster dose of the vaccine.

57. The method of any one of claims 52-56, wherein the vaccine is administered to the subject by intradermal or intramuscular injection.

58. The Ebola virus vaccine of any one of claims 1-33 for use in a method of inducing an antigen specific immune response in a subject, the method comprising administering to the subject the Ebola virus vaccine in an amount effective to produce an antigen specific immune response.

59. Use of the Ebola virus vaccine of any one of claims 1-33 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 to the subject the Ebola virus vaccine in an amount effective to produce an antigen specific immune response.

60. An Ebola virus vaccine, comprising:

at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding an Ebola virus antigenic polypeptide, wherein the RNA polynucleotide comprises a nucleic acid having at least 80% sequence identity to SEQ ID NOs 1-8.

61. The Ebola virus vaccine of claim 60, wherein the RNA polynucleotide comprises a polynucleotide having at least 90% sequence identity to SEQ ID NOs 1-8.

62. The Ebola virus vaccine of claim 60, wherein the RNA polynucleotide comprises a polynucleotide of any of SEQ ID NOs 1-8.

63. The Ebola virus vaccine of claim 60, wherein the RNA polynucleotide comprises a polynucleotide sequence derived from Zaire ebolavirus.

64. The Ebola virus vaccine of claim 60, wherein the Ebola virus antigenic polypeptide is a full length antigenic protein.

65. The Ebola virus vaccine of claim 60, wherein the Ebola virus antigenic polypeptide is an epitope.

66. The Ebola virus vaccine of claim 60, wherein the at least one RNA polynucleotide includes at least one chemical modification.

67. The Ebola virus vaccine of claim 60, wherein the at least one RNA polynucleotide encode a further antigenic polypeptide.

68. The Ebola virus vaccine of any one of claims 1-51, wherein the open reading frame is codon-optimized .

69. The Ebola virus vaccine of any one of claims 1-51, wherein the vaccine is multivalent.

70. The Ebola virus vaccine of any one of claims 1-51, formulated in an effective amount to produce an antigen- specific immune response.

71. A method of inducing an immune response in a subject, the method comprising administering to the subject the Ebola virus vaccine of any one of claims 1-51 in an amount effective to produce an antigen- specific immune response in the subject.

72. The method of claim 71, wherein the antigen specific immune response comprises a T cell response or a B cell response.

73. The method of claim 71 or 72, wherein the subject is administered a single dose of the Ebola virus vaccine.

74. The method of claim 72 or 73, wherein the subject is administered a booster dose of the Ebola virus vaccine.

75. The method of any one of claims 71-74, wherein the vaccine is administered to the subject by intradermal injection or intramuscular injection.

76. The method of any one of claims 71-75, wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is increased by at least 1 log relative to a control.

77. The method of claim 76, wherein the anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is increased by 1-3 log relative to a control.

78. The method of any one of claims 71-74, wherein the anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is increased at least 2 times relative to a control.

79. The method of claim 78, wherein the anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is increased at least 5 times relative to a control.

80. The method of claim 79, wherein the anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is increased at least 10 times relative to a control.

81. The method of any one of claims 71-74, wherein the anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is increased 2-10 times relative to a control.

82. The method of any one of claims 71-74, wherein the control is an anti-Ebola virus antigenic polypeptide antibody titer produced in a subject who has not been administered Ebola virus vaccine.

83. The method of any one of claims 71-74, wherein the control is an anti-Ebola virus antigenic polypeptide antibody titer produced in a subject who has been administered a live attenuated or inactivated Ebola virus vaccine.

84. The method of any one of claims 71-74, wherein the control is an anti-Ebola virus antigenic polypeptide antibody titer produced in a subject who has been administered a recombinant or purified Ebola virus protein vaccine.

85. The method of any one of claims 71-74, wherein the effective amount is a dose equivalent to an at least 2-fold reduction in the standard of care dose of a recombinant Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus vaccine.

86. The method of claim 85, wherein the effective amount is a dose equivalent to an at least 4-fold reduction in the standard of care dose of a recombinant Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus vaccine.

87. The method of claim 86, wherein the effective amount is a dose equivalent to an at least 10-fold reduction in the standard of care dose of a recombinant Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus vaccine.

88. The method of claim 87, wherein the effective amount is a dose equivalent to an at least 100-fold reduction in the standard of care dose of a recombinant Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus vaccine.

89. The method of claim 88, wherein the effective amount is a dose equivalent to an at least 1000-fold reduction in the standard of care dose of a recombinant Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus vaccine.

90. The method of any one of claims 71-89, wherein the effective amount is a dose equivalent to a 2-1000-fold reduction in the standard of care dose of a recombinant Ebola virus protein vaccine, and wherein an anti-Ebola virus antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-Ebola virus antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified Ebola virus protein vaccine or a live attenuated or inactivated Ebola virus vaccine.

91. The method of any one of claims 76-89, wherein the control is an anti-Ebola virus virus antigenic polypeptide antibody titer produced in a subject who has been administered a virus-like particle (VLP) vaccine comprising structural proteins of the Ebola virus.

92. The method of any one of claims 71-91, wherein the effective amount is a total dose of 30 μg-1000 μg.

93. The method of claim 92, wherein the effective amount is a total dose of 40 μg.

94. The method of claim 92, wherein the effective amount is a total dose of 100 μg.

95. The method of claim 92, wherein the total dose is 160 μg.

96. The method of any one of claims 71-90, wherein the total dose is 0.1-0.4 mg/kg for an average human.

97. The method of claim 92, wherein the effective amount is a dose of 25 μg administered to the subject a total of two times.

98. The method of claim 92, wherein the effective amount is a dose of 100 μg

administered to the subject a total of two times.

99. The method of claim 92, wherein the effective amount is a dose of 400 μg

administered to the subject a total of two times.

100. The method of claim 92, wherein the effective amount is a dose of 500 μg

administered to the subject a total of two times.

101. The method of any one of claims 71-100, wherein the efficacy of the vaccine against Ebola virus is greater than 60%.

102. The method of claim 101, wherein the efficacy of the vaccine against Ebola virus is greater than 65%.

103. The method of claim 101, wherein the efficacy of the vaccine against Ebola virus is greater than 70%.

104. The method of claim 101, wherein the efficacy of the vaccine against Ebola virus is greater than 75%.

105. The method of claim 101, wherein the efficacy of the vaccine against Ebola virus is greater than 80%.

106. The method of claim 105, wherein the efficacy of the vaccine against Ebola virus is greater than 85%.

107. The method of claim 106, wherein the efficacy of the vaccine against Ebola virus is greater than 90%.

108. The method of any one of claims 71-107, wherein the vaccine immunizes the subject against Ebola virus for up to 2 years.

109. The method of any one of claims 71-107, wherein the vaccine immunizes the subject against Ebola virus for more than 2 years.

110. The method of claim 109, wherein the vaccine immunizes the subject against Ebola virus for more than 3 years.

111. The method of claim 109, wherein the vaccine immunizes the subject against Ebola virus for more than 4 years.

112. The method of claim 109, wherein the vaccine immunizes the subject against Ebola virus for 5-10 years.

113. The method of any one of claims 71-107, wherein the subject is older than 45 years.

114. The method of claim 113, wherein the subject is older than 60 years.

115. The method of any one of claims 71-107, wherein the subject is younger than 9 years.

116. The method of claim 115, wherein the subject is younger than 5 years.

117. The method of claim 116, wherein the subject is younger than 1 year.

118. The method of any one of claims 71-117, wherein the subject is immunosuppressed.

119. A method of inducing an antigen specific immune response in a subject, the method comprising administering to a subject the Ebola virus vaccine of any one of claims 1-51, in an effective amount to produce an antigen specific immune response in a subject, wherein the subject is administered the vaccine within 3 days to 1 year before travel to an area having at least one reported case of Ebola virus infection.

120. The method of claim 119, wherein the subject is administered the vaccine within 3 days to 30 days before travel to an area having at least one reported case of Ebola virus infection.

120. The method of claim 119, wherein the subject is older than 6 months.

121. A method of inducing an antigen specific immune response in a subject, the method comprising administering to a subject the Ebola virus vaccine of any one of claims 1-51 in a single dose, wherein the subject does not receive a booster dose within 5 years following the single dose.

122. The Ebola virus vaccine of any one of claims 1-51 for use in a method of inducing an antigen specific immune response in a subject, the method comprising administering to the subject the Ebola virus vaccine in an amount effective to produce an antigen specific immune response in the subject.

123. Use of the Ebola virus vaccine of any one of claims 1-51 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 to the subject the Ebola virus vaccine in an amount effective to produce an antigen specific immune response in the subject.

124. A Ebola virus vaccine, comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one Ebola virus antigenic polypeptide or an immunogenic fragment thereof, at least one 5' terminal cap, at least one chemical

modification, wherein the vaccine is formulated within a lipid nanoparticle.

125. The Ebola virus vaccine of claim 124, wherein at least 80%, at least 90% or 100% of the uracil in the open reading frame have a chemical modification.