CN117716036A - Temperature controllable self-replicating RNA vaccine for viral diseases - Google Patents

Temperature controllable self-replicating RNA vaccine for viral diseases Download PDF

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CN117716036A
CN117716036A CN202280043353.2A CN202280043353A CN117716036A CN 117716036 A CN117716036 A CN 117716036A CN 202280043353 A CN202280043353 A CN 202280043353A CN 117716036 A CN117716036 A CN 117716036A
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M·S·H·柯
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Iliksgen Treatment Co
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Iliksgen Treatment Co
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Abstract

The present disclosure relates to mRNA, self-replicating RNA, and temperature-sensitive self-replicating RNA encoding a coronavirus nucleocapsid protein or an influenza virus nucleocapsid protein operably combined with a mammalian signal peptide. The present disclosure relates to mRNA, self-replicating RNA, and temperature-sensitive self-replicating RNA encoding one or more other viral nucleocapsid proteins in operable combination with a mammalian signal peptide. The RNA construct is suitable for active immunization against a virus in a mammalian subject, such as a human subject.

Description

Temperature controllable self-replicating RNA vaccine for viral diseases
Cross Reference to Related Applications
The present application claims the benefits of U.S. provisional application No. 63/275,398, U.S. provisional application No. 63/240,278, and U.S. provisional application No. 63/211,974, filed on 3, 11, 2021, 9, 2, 2021, 6, 17, each of which are hereby incorporated by reference in their entirety.
Submission of ASCII text file sequence Listing
The contents of the following submitted ASCII text files are incorporated herein by reference in their entirety: a sequence listing in Computer Readable Form (CRF) (file name: 699442001440seqlist. Txt, date recorded: 2022, 6, 16 days, size: 126,113 bytes).
Technical Field
The present disclosure relates to mRNA, self-replicating RNA, and temperature-sensitive self-replicating RNA encoding a coronavirus nucleocapsid protein or an influenza virus nucleocapsid protein operably combined with a mammalian signal peptide. The present disclosure relates to mRNA, self-replicating RNA, and temperature-sensitive self-replicating RNA encoding one or more other viral nucleocapsid proteins in operable combination with a mammalian signal peptide. The RNA construct is suitable for active immunization against a virus in a mammalian subject, such as a human subject.
Background
The genus beta coronavirus encompasses Severe Acute Respiratory Syndrome (SARS) -CoV-2, which causes a pandemic in COVID-19, SARS-CoV-1, which causes a 2002-2004SARS outbreak, and Middle East Respiratory Syndrome (MERS) -CoV. The popularity of covd-19 makes vaccine design and production an urgent need for immunization of a large population worldwide.
The SARS-CoV-2 vaccine currently approved by the U.S. food and drug administration (U.S. food & Drug Administration) is designed to elicit neutralizing antibodies (nAb) against spike (S) protein or the Receptor Binding Domain (RBD) of S protein prior to infection. However, this approach presents a significant challenge because the S protein is not very conserved even between SARS-CoV-1 and SARS-CoV-2 strains. In particular, small amino acid changes that occur between variants often result in conformational changes in the S protein, which may significantly reduce the effectiveness of the nAb triggered by a particular S protein of the covd-19 vaccine.
Thus, continued vaccine development targeting only the β -coronavirus S protein is expected to follow the path of seasonal influenza vaccines. This means that the continued advent of varieties would likely require the periodic development and production of new vaccines. While the annual production of beta coronavirus vaccines may be technically feasible, global vaccination efforts involving annual administration of new vaccines are economically and logistically impractical. The problem with the annual administration of new vaccines places a particularly undue burden on low and medium income countries.
Thus, there is a need in the art for a beta coronavirus vaccine that safely induces a sustained immune response with broad responsiveness against SARS-CoV-2 variants. Preferably, the persistent immune response is broadly responsive to other beta coronaviruses causing human disease. There is also a need in the art for an influenza virus vaccine that is safe and effective in inducing a broad reactive immune response against influenza a virus and/or influenza b virus.
Disclosure of Invention
The present disclosure relates to the use of nucleoprotein (also referred to herein as nucleocapsid protein) from a beta coronavirus as a vaccine antigen to induce a cellular immune response with broad responsiveness to a beta coronavirus variant. In some embodiments, temperature-controlled self-replicating RNA (referred to herein as srRNAts and c-srrrna) vaccine platforms are utilized. The c-srRNA vaccine platform is advantageous for inducing an effective cellular immune response following intradermal administration. In some embodiments, the nucleoprotein from SARS-CoV-2 is expressed in the host cell to address the infection caused by both SARS-CoV-2 and SARS-CoV-1, as well as variants thereof. In some embodiments, a nucleoprotein from a coronavirus is fused to a signal peptide of a human CD5 antigen and expressed in a host cell to enhance a cellular immune response elicited against the coronavirus. In some embodiments, the nucleoprotein from a first coronavirus is fused to a nucleoprotein from a second coronavirus, the second coronavirus being different from the first coronavirus. In some embodiments, the fusion protein comprises a tandem array of two or three coronavirus nucleoproteins. In a subset of these embodiments, the fusion protein comprises SARS-CoV-2 nucleoprotein and MERS-CoV nucleoprotein. In some embodiments, the fusion protein further comprises a coronavirus spike protein or fragment thereof. In this way, a more broadly reactive coronavirus-specific immune response is stimulated.
The present disclosure also relates to the use of nucleoprotein (also referred to herein as nucleocapsid protein) from influenza virus as vaccine antigen to induce a cellular immune response with broad reactivity to influenza a virus and/or influenza b virus that changes rapidly over time due to antigen drift and antigen shift. In some embodiments, a temperature-controllable self-replicating RNA vaccine platform is utilized. The c-srRNA vaccine platform is advantageous for inducing an effective cellular immune response following intradermal administration. In some embodiments, a nucleoprotein from one subtype of influenza a (FluA) virus is expressed in a host cell to address infections caused by the same and different subtypes of FluA. In some embodiments, a nucleoprotein from one lineage of influenza b (FluB) virus is expressed in a host cell to address infections caused by the same and different lineages of FluB. In some embodiments, a nucleoprotein from influenza virus is fused to a signal peptide of human CD5 antigen and expressed in a host cell to enhance the cellular immune response elicited against influenza virus. In some embodiments, the nucleoprotein from the FluA virus is fused to the nucleoprotein from the FluB virus. In some embodiments, the fusion protein comprises a tandem array of two or three nuclear proteins from one or more strains of FluA and/or one or more lineages of FluB. In some embodiments, the fusion protein further comprises influenza hemagglutinin or a fragment thereof. In this way, a more broadly reactive influenza-specific immune response is stimulated.
The present disclosure also relates to the use of a nucleoprotein (also referred to herein as a nucleocapsid protein) from ebola virus as a vaccine antigen to induce a broadly reactive cellular immune response to two, three or four ebola virus species that infect humans. In some embodiments, a temperature-controllable self-replicating RNA vaccine platform is utilized. The c-srRNA vaccine platform is advantageous for inducing an effective cellular immune response following intradermal administration. In some embodiments, a nucleoprotein from ebola virus is fused to a signal peptide of human CD5 antigen and expressed in a host cell to enhance the cellular immune response elicited against the ebola virus. In some embodiments, a nucleoprotein from a first ebola virus species is fused to a nucleoprotein from a second ebola virus species, the nucleoprotein from the second ebola virus species is optionally fused to a nucleoprotein of a third ebola virus species, and the nucleoprotein of the third ebola virus species is optionally fused to a nucleoprotein of a fourth ebola virus species. In some embodiments, the fusion protein comprises a tandem array of two, three, or four nucleoproteins, or fragments thereof, from two or more ebola virus species. In some embodiments, the fusion protein further comprises ebola virus envelope glycoprotein or a fragment thereof. In this way, a more broadly reactive ebola virus-specific immune response was stimulated.
In other embodiments, the present disclosure provides compositions comprising an excipient and a temperature-controllable self-replicating RNA. In some embodiments, the composition comprises chitosan. In some embodiments, the chitosan is a low molecular weight (about 3-5 kDa) chitosan oligosaccharide, such as chitosan oligosaccharide lactic acid. In some embodiments, the composition does not comprise a liposome or lipid nanoparticle.
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FIG. 1 shows a schematic representation of the mechanism by which the immune response of cells (CD4+ and CD8+ T cells) is induced following intradermal injection of temperature controlled self-replicating RNA (herein referred to as srRNAts and c-srRNA) vaccines.
FIG. 2 shows a schematic representation of SARS-CoV-2 nucleocapsid (N) protein expressed by mRNA, self-replicating RNA or temperature-sensitive self-replicating RNA (srRNAts) delivered to mammalian host cells. In an exemplary embodiment, the coding region of the N protein is a gene of interest (GOI) inserted into srrrnats. The amino acid sequence of the G5004 antigen is shown as SEQ ID NO. 5. The G5004 antigen is SARS-CoV-2N protein lacking a signal peptide. The amino acid sequence of the G5005 antigen is shown as SEQ ID NO. 6. The G5005 antigen is a fusion protein comprising a signal peptide (CD 5-SP) sequence from the human CD5 antigen as shown in SEQ ID NO. 8 and the SARS-CoV-2N protein, wherein CD5-SP replaces the initiating methionine at position 1 of the N protein. The amino acid sequence of the G5006 antigen is shown as SEQ ID NO. 7. The G5006 antigen is a fusion protein comprising the signal peptide sequence of CD5-SP, the SARS-CoV-2N protein and the MERS-CoV N protein. The nucleotide sequence of the coding G5004 antigen is shown as SEQ ID NO. 1. The nucleotide sequence of the coding G5005 antigen is shown as SEQ ID NO. 2. The nucleotide sequence encoding the G5006 antigen is shown in SEQ ID NO. 3 and in codon optimized form in SEQ ID NO. 4.
Fig. 3 shows a schematic diagram of an exemplary method for stimulating an immune response against coronavirus in a human subject. Temperature sensitive agents (ts agents) such as srrrnats are functional at licensed temperatures but non-functional at unlicensed temperatures. The temperature at or below the body surface of a human subject (surface body temperature) is the permissible temperature, while the core body temperature of a human subject is the higher unlicensed temperature. Thus, ts agents administered intradermally to a human subject are functional at a permissible temperature below the body surface of the human subject.
FIGS. 4A and 4B show the frequency of cytokine secreting cells in spleen cell samples obtained from CD-1-outcrossing mice that have been immunized by a single intradermal injection with 100. Mu.L of a solution containing 5. Mu.g or 25. Mu.g of temperature controllable self-replicating RNA encoding the G5004 antigen (srRNA 1ts2[ PCT/US20/67506 ]) or placebo (PBO: buffer only). FIG. 4A shows the frequency of interferon-gamma (INF-gamma) Spot Forming Cells (SFCs) in 1x 10 x 6 spleen cells after restimulation by culturing the spleen cells in the presence or absence of SARS-CoV-2 nucleoprotein peptide pool, and FIG. 4B shows the frequency of interleukin-4 (IL-4) SFCs in the spleen cells. After subtracting the frequency obtained in the absence of peptide (background), the frequency obtained in the presence of peptide is plotted in the figure. Mean and standard deviation (error bars) of five mice per group (n=5) are shown. Splenocytes were isolated 14 days after intradermal injection.
FIGS. 5A and 5B show the frequency of cytokine secreting cells in spleen cell samples obtained from CD-1-outcrossing mice that have been immunized by a single intradermal injection with 100. Mu.L of a solution containing 5. Mu.g or 25. Mu.g of temperature controllable self-replicating RNA encoding the G5005 antigen (srRNA 1ts2[ PCT/US20/67506 ]) or placebo (PBO: buffer only). FIG. 5A shows the frequency of interferon-gamma (INF-gamma) Spot Forming Cells (SFCs) in 1x 10 x 6 spleen cells after restimulation by culturing the spleen cells in the presence or absence of SARS-CoV-2 nucleoprotein peptide pool, and FIG. 5B shows the frequency of interleukin-4 (IL-4) SFCs in the spleen cells. After subtracting the frequency obtained in the absence of peptide (background), the frequency obtained in the presence of peptide is plotted in the figure. Mean and standard deviation (error bars) of five mice per group (n=5) are shown. Splenocytes were isolated 14 days after intradermal injection.
FIGS. 6A and 6B show the frequency of cytokine secreting cells in spleen cell samples obtained from BALB/c mice that have been immunized by a single intradermal injection of 100. Mu.L solution containing 5. Mu.g or 25. Mu.g of temperature controllable self replicating RNA encoding G5005 antigen (srRNA 1ts2[ PCT/US20/67506 ]) or placebo (PBO: buffer only). FIG. 6A shows the frequency of interferon-gamma (INF-gamma) Spot Forming Cells (SFCs) in 1x 10 x 6 spleen cells after restimulation by culturing the spleen cells in the presence or absence of SARS-CoV-2 nucleoprotein peptide pool, and FIG. 6B shows the frequency of interleukin-4 (IL-4) SFCs in the spleen cells. After subtracting the frequency obtained in the absence of peptide (background), the frequency obtained in the presence of peptide is plotted in the figure. Mean and standard deviation (error bars) of five mice per group (n=5) are shown. Splenocytes were isolated 30 days after vaccination.
FIG. 7 shows the level of SARS-CoV-2 antigen reactive immunoglobulin G (IgG) in serum of BALB/c mice that have been immunized by a single intradermal injection with 100. Mu.L of a solution containing 5. Mu.g or 25. Mu.g of temperature controllable self-replicating RNA encoding G5005 antigen (srRNA 1ts2[ PCT/US20/67506 ]) or placebo (PBO: buffer only). IgG levels are represented by OD450 in ELISA. IgG levels before (day-1) and after (day 30) vaccination are shown. Mean and standard deviation (error bars) of five mice per group (n=5) are shown.
FIG. 8 shows the frequency of interferon-gamma (INF-gamma) secreting cells in spleen cell samples obtained from BALB/c mice that have been immunized by a single intradermal injection of 100. Mu.L solution containing 5. Mu.g or 25. Mu.g of temperature controlled self-replicating RNA encoding G5006 antigen (srRNA 1ts2[ PCT/US20/67506 ]) or placebo (PBO: buffer only). Specifically, FIG. 8 shows the frequency of INF-gamma Spot Forming Cells (SFC) in 1x 10≡6 spleen cells after restimulation by culturing the spleen cells in the presence or absence of SARS-CoV-2 nucleoprotein peptide pool. After subtracting the frequency obtained in the absence of peptide (background), the frequency obtained in the presence of peptide is plotted in the figure. Mean and standard deviation (error bars) of five mice per group (n=5) are shown. Splenocytes were isolated 14 days after vaccination.
Fig. 9 shows a schematic diagram of an exemplary pandemic influenza vaccine. Briefly, fusion proteins comprising a nucleoprotein from influenza a virus (FluA) and a nucleoprotein from influenza b virus (FluB) are expressed by mRNA, self-replicating RNA, or temperature-sensitive self-replicating RNA (srRNAts) delivered to mammalian host cells. In an exemplary embodiment, the coding region of the fusion protein is a gene of interest (GOI) inserted into srRNAts. Specifically, G5010 is a fusion protein comprising: the signal peptide sequence (CD 5-SP) from the human CD5 antigen, fluA nucleoprotein (influenza A, subtype H5N8 [ A/b.sub.der dock/Korea/Gochang 1/2014], genBank number KJ413835.1, proteinID number AHL 21420.1) and FluB nucleoprotein (influenza B [ B/Florida/4/2006], genBank number CY033879.1, proteinID number ACF 54251.1) as shown in SEQ ID No. 8. In G5010, CD5-SP replaces the initiator methionine of the FluA nucleoprotein and the FluA nucleoprotein was fused to the methionine of the initiation codon of the FluB nucleoprotein.
FIG. 10 shows an alignment of the nucleoprotein (SEQ ID NO: 13) of influenza A (H5N 8 strain; proteinID AHL 21420.1) used as vaccine antigen in G5010 and the nucleoprotein (SEQ ID NO: 17) of influenza A (NP/Annarbor H2N2; proteinID P21433) used as a source of the peptide pool for the ELISPot assay.
FIGS. 11A and 11B show the frequency of cytokine secreting cells in spleen cell samples obtained from BALB/c mice that have been immunized by a single intradermal injection of 100. Mu.L solution containing 5. Mu.g or 25. Mu.g of temperature controllable self replicating RNA encoding G5010 antigen (srRNA 1ts2[ PCT/US20/67506 ]) or placebo (PBO: buffer only). FIG. 11A shows the frequency of interferon-gamma (INF-gamma) Spot Forming Cells (SFC) in 1x 10≡6 spleen cells after restimulation by culturing the spleen cells in the presence or absence of influenza A (H2N 2) nucleoprotein peptide pool, and FIG. 11B shows the frequency of interleukin-4 (IL-4) SFC in the spleen cells. After subtracting the frequency obtained in the absence of peptide (background), the frequency obtained in the presence of peptide is plotted in the figure. Mean and standard deviation (error bars) of five mice per group (n=5) are shown. Splenocytes were isolated 14 days after vaccination.
Fig. 12 shows a schematic diagram of an exemplary generalized ebola virus vaccine. Briefly, fusion proteins comprising the nucleoproteins of four different ebola strains are expressed from mRNA, self-replicating RNA, or temperature-sensitive self-replicating RNA (srrrnats) delivered to mammalian host cells. In an exemplary embodiment, the coding region of the fusion protein is a gene of interest (GOI) inserted into srRNAts. Specifically, exemplary pan-ebola antigens are fusion proteins comprising: the signal peptide sequence from human CD5 antigen (CD 5-SP) as shown in SEQ ID NO:8, the portion of the nucleoprotein of the Zaeoloma type Ebola virus as shown in SEQ ID NO:18 (residues 2-739; total 738aa;GenBank ID:AF272001), the portion of the nucleoprotein of the Sudan type Ebola virus as shown in SEQ ID NO:19 (residues 403-738; total 336aa;GenBank ID:AF173836), the portion of the nucleoprotein of the Bendibu Jiao Xingai Bola virus as shown in SEQ ID NO:20 (residues 403-739; total 337aa;GenBank ID:FJ217161), and the portion of the nucleoprotein of the Taaeoloma type Ebola virus as shown in SEQ ID NO:21 (residues 483-651; total 169aa;GenBank ID:FJ217162). The amino acid sequence of the ubiquitin Ebola antigen is shown as SEQ ID NO. 22, and the nucleic acid sequence of the coding ubiquitin Ebola antigen is shown as SEQ ID NO. 23.
Figure 13 shows amino acid sequence similarity as a percentage of identity between four ebola virus species. Amino acid sequences of zaire type ebola virus NP (GenBank ID: AF 272001), sudan type ebola virus NP (GenBank ID: AF 173836), bund Jiao Xingai ebola virus NP (GenBank ID: FJ 217161), tay forest type ebola virus NP (GenBank ID: FJ 217162) were compared with each other by using NCBI BlastP algorithm. Based on the sequence alignment, proteins are divided into well-conserved regions (a) and less-conserved regions (B). For region a, the amino acid sequence identity between zaire-type ebola virus NP and sudan-type ebola virus NP is 88%, while for region B, the amino acid sequence identity is 42%. For region a, the amino acid sequence identity between zaire ebola virus NP and bundi Jiao Xingai bola virus NP is 92%, while for region B, the amino acid sequence identity is 53%. For region a, the amino acid sequence identity between zaire ebola virus NP and tailin ebola virus NP is 92%, while for region B the amino acid sequence identity is 54%. For region B, the present dibuch (B) and tazicar (B) sequences have a relatively high level of sequence similarity. Proteins are divided into well conserved regions (80% and 86% similarity; no tag) and less conserved regions (40% identity; referred to herein as region C) based on sequence alignment of region B.
FIGS. 14A and 14B show the frequency of cytokine secreting cells in spleen cell samples obtained from BALB/c mice that have been immunized by a single intradermal injection with 25. Mu.g of a 100. Mu.L solution of temperature controllable self-replicating RNA (also called c-srRNA) 1ts2 as described in WO 2021/138447 A1, or placebo (PBO: buffer only) encoding pan-type ebola antigen (srRNA 1ts 2-pan-type ebola, also called G5011). Fig. 14A shows the results obtained by treating the cells with a virus selected from the group consisting of the cross-tahlamys forest type ebola virus nucleoprotein (Swiss-Prot ID: b8XCN 6) [ JPT peptide; pepnix takie forest type ebola virus (NP); JPT product code: peptide scan of PM-TEBOV-NP ] the frequency of interferon-gamma (INF-gamma) Spot Forming Cells (SFC) in 1x10 x 6 spleen cells after restimulation was cultured in the case of pool of 182 peptides (15 mer with 11 amino acid overlap), and FIG. 14B shows the frequency of interleukin-4 (IL-4) SFC in the spleen cells. After subtracting the frequency obtained in the absence of peptide (background), the frequency obtained in the presence of peptide is plotted in the figure. Mean and standard deviation (error bars) of five mice per group (n=5) are shown. Splenocytes were isolated 14 days after vaccination.
FIG. 15 depicts a schematic diagram showing an exemplary srRNA1ts2 construct encoding the Receptor Binding Domain (RBD) of the spike protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). G5003 is the same antigen as "srRNA1ts2-2019-nCoV-RBD1" presented in FIG. 21 of WO 2021/138447 A1; and G5003 encodes a fusion protein comprising the signal peptide of CD5 (residues 1-24) and the RBD of the spike protein of SARS-CoV-2 (original strain). G5003o encodes a fusion protein (SEQ ID NO: 25) comprising the signal peptide of CD5 (residues 1-24) and the RBD of the spike protein of SARS-CoV-2 (Ornidus strain B.1.1.529: science Brief: ornidus (B.1.1.529) variant |CDC). The nucleotide sequence of the G5003o open reading frame is shown as SEQ ID NO. 24.
FIGS. 16A and 16B show the frequency of cytokine secreting cells in spleen cell samples obtained from C57BL/6 mice that have been immunized by a single intradermal injection with 100. Mu.L of a solution containing placebo (PBO: buffer only) or 25. Mu.g of temperature controllable self-replicating RNA encoding G5003o antigen (srRNA 1ts2 as described in WO 2021/138447 A1). FIG. 16A shows the frequency of interferon-gamma (INF-gamma) Spot Forming Cells (SFC) in 1x 10≡6 splenocytes from immunized mice re-stimulated by the following manner and FIG. 16B shows the frequency of interleukin-4 (IL-4) SFC in the splenocytes: in the presence or absence of the RBD [ JPT peptide product code from Rong Bianchong (b.1.1.529) by SARS-CoV-2 obreck: spleen cells were cultured in the presence of pools of 53 peptides (15 mer with 11 amino acid overlap) obtained by peptide scanning of PM-SARS2-RBDMUT 08-1. The assay was performed by ELISpot assay. After subtracting the frequency obtained in the absence of peptide (background), the frequency obtained in the presence of peptide is plotted in the figure. Mean and standard deviation (error bars) of five mice per group (n=5) are shown. Splenocytes were isolated 14 days after vaccination.
Figures 17A-17C show induction of both cellular and humoral immunity in mice as a result of administration of a composition comprising C-srRNA encoding an antigen, followed by administration of a composition comprising a protein antigen. Fig. 17A depicts a schematic of the experimental procedure. On day-40, blood was drawn from female BALB/c mice for Plaque Reduction Neutralization Test (PRNT). On day-36, these mice were treated with c-srRNA encoding the G5003 antigen. The c-srRNA was injected intradermally as naked RNA into the skin of mice without any nanoparticles or transfection reagents. On day-22 (14 days after c-srRNA-G5003 vaccination), half of the mice were sacrificed to obtain spleen cells for ELISPot assay. On day 0, the remaining mice were injected intradermally with adjuvant (adavax, commercially available from invitrogen TM Adjuvant) spike protein of the mixed SARS-CoV-2 delta variant (b.1.617.2). On day 7 (7 days after spike protein injection),blood was drawn for PRNT assay. FIG. 17B shows induction of cellular immunity against RBD proteins by single intradermal vaccination with the c-srRNA-G5003 vaccine. The graph shows the frequency of interferon-gamma (INF-gamma) Spot Forming Cells (SFC) in 1x 10≡6 splenocytes from immunized mice re-stimulated by: spleen cells were cultured in the presence or absence of a pool of 53 peptides (15 mers with 11 amino acid overlaps) covering SARS-CoV-2RBD (original strain). The assay was performed by ELISpot assay. After subtracting the frequency obtained in the absence of peptide (background), the frequency obtained in the presence of peptide is plotted in the figure. Mean and standard deviation (error bars) of five mice per group (n=5) are shown. Spleen cells were isolated on day-22 (14 days post vaccination). FIG. 17C shows the titers of serum antibodies that can neutralize (50%) of SARS-CoV-2 virus (delta variant B.1.617.2) as measured by a plaque reduction neutralization assay (PRNT). Exposure to the spike protein of the SARS-CoV-2 virus (delta variant B.1.617.2) induced neutralizing antibodies specific for the delta variant of the SARS-CoV-2 virus only in mice vaccinated with vaccine c-srRNA-G5003 (RBD encoding SARS-CoV-2 (original strain)).
Fig. 18A-18C show induction of cellular immunity in mice as a result of administration of a composition comprising a protein antigen followed by administration of a composition comprising C-srrrna encoding the antigen. Fig. 18A depicts a schematic of the experimental procedure. On day 0 (treatment 1), female C57BL/6 mice were injected intradermally with 10. Mu.g RBD protein (Sino Biological SARS-CoV-2[2019-nCoV]) +adjuvant (AddaVax commercially available from Invivogen TM Adjuvant). On day 14 (treatment 2), mice were treated with intradermal placebo (PBO: buffer only), 25. Mu.g of c-srRNA encoding G5003 antigen, 25. Mu.g of c-srRNA encoding G5003o antigen, or 10. Mu.g of RBD protein (Sino Biological SARS-CoV-2[ 2019-nCoV)]) +adjuvant (AddaVax) TM Adjuvant). On day 28, mice were sacrificed and spleen cells and serum were collected. FIG. 18B shows the frequency of interferon-gamma (INF-gamma) Spot Forming Cells (SFC) in 1X 10≡6 splenocytes re-stimulated by culture in the presence or absence of a pool of 53 peptides (15 mers with 11 amino acid overlaps) covering SARS-CoV-2RBD (original strain)And FIG. 18C shows the frequency of interleukin 4 (IL-4) SFC in the spleen cells. The assay was performed by ELISpot assay. After subtracting the frequency obtained in the absence of peptide (background), the frequency obtained in the presence of peptide is plotted in the figure.
FIG. 19 shows serum antibody levels (represented by OD450 measurements) of RBD against SARS-CoV-2 virus (original strain) as determined by ELISA assay. Mean and standard deviation (error bars) of five mice per group (n=5) are shown. Data on day-1 (before treatment 1) and day 28 (after treatment 2) of each group are shown.
FIGS. 20A-20D show the frequency of interferon-gamma (INF-gamma) secreting cells or interleukin 4 (IL-4) secreting cells in spleen cell samples obtained from BALB/c mice that have been immunized by a single intradermal injection of 100. Mu.L solution containing 5. Mu.g (n=1) or 25. Mu.g (n=4) of temperature controllable self-replicating RNA encoding G5006 antigen (srRNA 1ts2 as described in WO 2021/138447 A1) (FIG. 2) or placebo (PBO: buffer: n=5 only). After subtracting the frequency obtained in the absence of peptide (background), the frequency obtained in the presence of peptide is plotted in the figure. The mean and standard deviation (error bars) of one mouse (n=1) or four mice (n=4) per group are shown. Splenocytes were isolated 14 days after vaccination. FIGS. 20A and 20B show the results after restimulation by culturing spleen cells in the presence or absence of a SARS-CoV-2 nucleoprotein peptide pool. FIGS. 20C and 20D show the results after restimulation by culturing spleen cells in the presence or absence of MERS-CoV-2 nucleoprotein peptide pool.
FIG. 21 shows survival (%) of female BALB/c mice vaccinated with c-srRNA-G5006, followed by injection of tumor cells expressing the G5006 antigen.
FIG. 22 depicts a schematic diagram showing an exemplary srRNA1ts2 construct encoding a fusion protein (designated herein as G5006 d) that is a signal peptide of CD5 (residues 1-24), the Receptor Binding Domain (RBD) of the spike protein of Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the nucleoprotein of SARS-CoV-2, the nucleoprotein of MERS-CoV, and the fusion protein of the RBD of MERS-CoV. The amino acid sequence of the pan-type coronavirus antigen (G5006 d) is shown as SEQ ID NO. 27, and the nucleotide sequence of the open reading frame is shown as SEQ ID NO. 26.
FIGS. 23A-23B show the frequency of cytokine secreting cells in spleen cell samples obtained from female C57BL/6 mice that have been immunized by a single intradermal injection with 100. Mu.L solution of placebo (PBO: buffer only), 25. Mu.g of temperature controllable self-replicating RNA encoding G5006 antigen (srRNA 1ts2 as described in WO 2021/138447 A1), or 25. Mu.g of temperature controllable self-replicating RNA encoding G5006d antigen (srRNA 1ts2 as described in WO 2021/138447 A1). FIG. 23A shows the frequency of interferon-gamma (INF-gamma) Spot Forming Cells (SFC) in 1x 10≡6 splenocytes from immunized mice re-stimulated by the following manner, and FIG. 23B shows the frequency of interleukin-4 (IL-4) SFC in the splenocytes: in the presence or absence of the RBD [ JPT peptide product code from spike protein passing through (A) SARS-CoV-2: PM-WCPV-S-RBD-2]; (B) nucleoprotein of SARS-CoV-2 [ JPT peptide product code: PM-WCPV-NCAP ]; (C) nuclear protein of MERS-CoV [ JPT peptide, custom ]; and (D) MERS-CoV spike protein [ JPT peptide product code: peptide scan of PM-MERS-CoV-S-1] spleen cells were cultured with a pool of peptides (15 mer with 11 amino acid overlap). The assay was performed by ELISpot assay. After subtracting the frequency obtained in the absence of peptide (background), the frequency obtained in the presence of peptide is plotted in the figure. The mean and standard deviation (error bars) of five mice per group for PBO (n=5), four mice for G5006 (n=4) and five mice for G5006d (n=5) are shown. Splenocytes were isolated 14 days after vaccination.
FIG. 24 depicts a schematic diagram showing an exemplary srRNA1ts2 construct encoding a fusion protein (G5012) that is a fusion protein of the signal peptide of CD5 (residues 1-24), a portion of the Hemagglutinin (HA) of influenza A (A/New Caledonia/20/1999 (H1N 1)) (residues 25-165), a nucleoprotein of influenza A (A/breeder stack/Korea/Gochang 1/2014 (H5N 8)) (residues 166-662), a nucleoprotein of influenza B (B/Florida/4/2006) (residues 663-1222), and a portion of the Hemagglutinin (HA) of influenza B (B/Florida/4/2006) (residues 1223-1365). The amino acid sequence of the pan-type influenza virus antigen (G5012) is shown as SEQ ID NO. 29, and the nucleotide sequence of the open reading frame is shown as SEQ ID NO. 28.
FIG. 25 shows the effect of chitosan oligomer on gene (luciferase) expression from srRNA1ts2 (exemplary c-srRNA) in mice. c-srRNA encoding luciferase was injected intradermally into mice under the following conditions: 1, control-c-srRNA only; 2, c-srRNA mixed with chitosan oligosaccharide (0.001. Mu.g/mL); 3, c-srRNA mixed with chitosan oligosaccharide (0.01. Mu.g/mL); 4, c-srRNA mixed with chitosan oligosaccharide (0.5. Mu.g/mL); and 5, c-srRNA mixed with chitosan oligosaccharide lactic acid (0.1. Mu.g/mL).
Detailed Description
The broader, more durable protection against SARS-CoV-1, SARS-CoV-2, MERS-CoV and variants thereof is best achieved by a vaccine that induces cellular immunity (i.e., a T cell induction vaccine that involves CD8+ killer T cells and CD4+ helper T cells). This is in contrast to the current covd-19 vaccine paradigm that focuses on neutralizing antibodies as discussed in the background section. The critical importance of cellular immunity in combating coronaviruses has been demonstrated through experimentation and is widely discussed [ Sette and Crotty 2021]. Cellular immunity alone can provide protection via cd8+ killer T cells [ Matchett et al, 2021]. Furthermore, cellular immunity is dependent on linear T cell epitopes, whereas humoral immunity is dependent on conformational (as well as linear) B cell epitopes. Thus, cellular immunity is much more robust to variation than humoral immunity. Furthermore, memory T cells last longer than memory B cells, and thus may provide life-long immunity. This requires both a suitable antigen and a cell immunity based vaccine platform.
mRNA vaccine platform based on cellular immunity
Vaccine platforms are described in the earlier patent application by Elixirgen [ PCT/US20/67506, now published as WO 2021/138447A1 ]. This vaccine platform was optimized to induce cellular immunity, which was made possible by combining prior knowledge of vaccine biology with temperature-controlled self-replicating mRNA (srRNAts) based on alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV). The terms c-srRNA and srRNA are used interchangeably throughout this disclosure, with srRNA1ts2 (described in WO 2021/138447 A1) being an exemplary embodiment. srrrnats are based on srrrna, also known as self-amplified mRNA (saRNA or SAM), providing temperature sensitivity by incorporating small amino acid changes in the alphavirus replicase. Elixirgen Therapeutic the srRNAts function at 30-35 ℃, but not at or above 37 ℃ + -0.5 ℃. It has all the advantages of the mRNA platform: no genome integration, rapid development and deployment, and simple superior manufacturing process (GMP) and other advantages of the srRNA platform compared to the mRNA platform, in particular longer expression [ Johanning et al, 1995] and higher immunogenicity at lower doses [ Brito et al, 2014]. However, this simple temperature-controllable feature makes it possible to combine many of the desirable features of the T cell-inducing vaccines described herein.
Briefly, srRNA1ts2 is a temperature-sensitive self-replicating VEEV-based RNA replicon developed for transient expression of heterologous proteins. Temperature sensitivity was conferred by inserting five amino acid residues into the nonstructural protein 2 (nsP 2) of VEEV. The nsP2 protein is a helicase/protease that, together with nsP1, nsP3 and nsP4, constitutes a VEEV replicase. srRNA1ts2 does not contain VEEV structural proteins (capsids, E1, E2 and E3). The disclosure of WO 2021/138447 A1 of Elixirgen Therapeutics, inc. In particular, example 3 of WO 2021/138447 A1, FIG. 12 and SEQ ID NO.29-49 are hereby incorporated by reference.
Overall, the convincing potential of the srrrnats platform for immunogenicity (dose throttling) and safety benefits (temperature control and naked delivery), the ability to provide long-lasting baseline cellular immunity, and to provide rapid humoral responses across variants, makes the platform a strong candidate for large-scale deployment that can meet the global needs for inexpensive, safe, vaccine to handle variants that provide long-term immunity.
General techniques and definitions
Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.
As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "an" excipient includes one or more excipients.
The phrase "comprising" as used herein is open ended, meaning that embodiments may include additional elements. In contrast, the phrase "consisting of … …" is closed, indicating that such embodiments do not include additional elements (other than trace impurities). The phrase "consisting essentially of … …" is partially enclosed, meaning that the embodiments may further include elements that do not substantially alter the essential characteristics of the embodiments.
As used herein, the term "about" with respect to a value encompasses 90% to 110% of the value (e.g., when used with respect to chitosan oligosaccharides, a molecular weight of about 5,000 daltons refers to 4,500 daltons to 5,500 daltons).
The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or T cell antigen receptor. Antigens may include peptides, polypeptides, proteins, glycoproteins, polysaccharides, complex carbohydrates, sugars, gangliosides, lipids, and phospholipids; portions thereof and combinations thereof. In the context of the present disclosure, the term "antigen" typically refers to a polypeptide or protein antigen of at least eight amino acid residues in length, which may comprise one or more post-translational modifications.
Unless otherwise specified, the terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues, and are not limited to certain lengths. The polypeptide may comprise natural amino acid residues or a combination of natural and unnatural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptide may contain modifications with respect to native or native sequences so long as the protein retains the desired activity (e.g., antigenicity).
As used herein, the terms "isolated" and "purified" refer to materials that are removed from at least one component with which they are naturally associated (e.g., from their original environment). The term "isolated" when used in reference to a recombinant protein refers to a protein that has been removed from the medium of the host cell that produced the protein. In some embodiments, the isolated protein (e.g., SARS-CoV-2 spike protein) is at least 75%, 90%, 95%, 96%, 97%, 98% or 99% pure as determined by HPLC.
An "effective amount" or "sufficient amount" of a substance is an amount sufficient to achieve a beneficial or desired result, including a clinical result, and thus, the "effective amount" depends on the context in which it is used. In the context of administering a composition of the present disclosure comprising an mRNA encoding an antigen, an effective amount contains sufficient mRNA to stimulate an immune response (preferably a cellular immune response to the antigen).
In the present disclosure, the terms "individual" and "subject" refer to mammals. "mammal" includes, but is not limited to, humans, non-human primates (e.g., monkeys), farm animals, sports animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats). In some preferred embodiments, the subject is a human subject.
The term "dose" as used herein with respect to a composition comprising an mRNA encoding an antigen refers to the measured portion of the subject taken (administered or received) at any one time. Administering a composition of the present disclosure to a subject in need thereof includes administering an effective amount of a composition comprising mRNA encoding an antigen to stimulate an immune response to the antigen in the subject.
"stimulation" of a response or parameter includes eliciting and/or enhancing the response or parameter when compared to a condition that is otherwise identical except for the parameter of interest, or alternatively, as compared to another condition (e.g., increased secretion of an antigen-specific cytokine upon administration of a composition comprising or encoding the antigen, as compared to administration of a control composition that does not comprise or encode the antigen). For example, "stimulation" of an immune response (e.g., a Th1 response) means an increase in the response. Depending on the measured parameters, the increase may be 2-fold to 200-fold or more, 5-fold to 500-fold or more, 10-fold to 1000-fold or more, or 2, 5, 10, 50 or 100-fold to 200, 500, 1,000, 5,000 or 10,000-fold.
Conversely, "suppressing" a response or parameter includes reducing and/or suppressing the response or parameter when compared to a condition that is otherwise identical except for the parameter of interest, or alternatively, as compared to another condition. For example, "inhibition" of an immune response (e.g., a Th2 response) means a decrease in the response. Depending on the measured parameters, the decrease may be 2-fold to 200-fold, 5-fold to 500-fold or more, 10-fold to 1000-fold or more, or 2, 5, 10, 50 or 100-fold to 200, 500, 1,000, 2,000, 5,000 or 10,000-fold.
The relative terms "higher" and "lower" refer to a measurable increase or decrease in response or parameter, respectively, when compared to a condition that is otherwise identical except for the parameter of interest, or alternatively, as compared to another condition. For example, "higher antibody titer" refers to an antigen-reactive antibody titer that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold higher as a result of administering a composition of the present disclosure comprising an mRNA encoding an antigen than an antigen-reactive antibody titer as a result of a control condition (e.g., administering a comparative composition comprising no mRNA or no control mRNA encoding an antigen). Likewise, "lower antibody titer" refers to an antigen-reactive antibody titer that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold lower as a result of the control conditions (e.g., administration of a comparative composition comprising no mRNA or comprising a control mRNA that does not encode an antigen) than an antigen-reactive antibody titer that is a result of the administration of a composition of the present disclosure comprising an mRNA that encodes an antigen.
As used herein, the term "immunization" refers to a process of increasing the response of a mammalian subject to an antigen, and thus improving its ability to resist or overcome an infection and/or to resist a disease.
As used herein, the term "vaccination" refers to the introduction of a vaccine into the body of a mammalian subject.
As used herein, "percent amino acid sequence identity (%)" and "percent identity" and "sequence identity" when used with respect to an amino acid sequence (reference polypeptide sequence) are defined as the percentage of amino acid residues in a candidate sequence (e.g., a subject antigen) that are identical to amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity. The alignment used to determine the percent amino acid sequence identity can be accomplished in a variety of ways well known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the compared sequences.
Amino acid substitutions may include substitution of one amino acid in the polypeptide with another amino acid. Amino acid substitutions may be introduced into the antigen of interest and the product screened for desired activity (e.g., increased stability and/or immunogenicity).
Amino acids can generally be grouped according to the following common side chain characteristics:
(1) Hydrophobicity: norleucine, met, ala, val, leu, ile;
(2) Neutral hydrophilicity: cys, ser, thr, asn, gln;
(3) Acid: asp, glu;
(4) Alkaline: his, lys, arg;
(5) Residues that affect chain orientation: gly, pro; and
(6) Aromatic: trp, tyr, phe.
Conservative amino acid substitutions will involve the exchange of a member of one of these classes with another member of the same class. Non-conservative amino acid substitutions will involve exchanging members of one of these classes with members of the other class.
As used herein, the term "excipient" refers to a compound that is present in a composition comprising an active ingredient (e.g., mRNA encoding an antigen). Pharmaceutically acceptable excipients are inert pharmaceutical compounds and may include, for example, solvents, fillers, buffers, tonicity adjusting agents and preservatives (Pramantick et al, pharma Times,45:65-77,2013). In some embodiments, the compositions of the present disclosure include excipients that function as one or more of solvents, fillers, buffers, and tonicity adjusting agents (e.g., sodium chloride in saline may act as both an aqueous vehicle and tonicity adjusting agent).
Optimizing for intradermal delivery
Intradermal vaccination results in durable cellular immunity and increased immunogenicity [ hicking and Jones,2009]. Human skin (epidermis and dermis) is rich in Antigen Presenting Cells (APCs), including langerhans cells and dermal Dendritic Cells (DCs). Intradermal vaccination is known to be 5 to 10 times more effective than subcutaneous or intramuscular vaccination because it targets APCs present in the skin [ hicking and Jones,2009], thereby activating the T cell immune pathway to obtain durable immunity. By intradermal injection, srRNAts are taken up mainly by skin APCs, where they are replicated, antigen is produced, the antigen is digested into peptides, and the peptides are presented to T cells (fig. 1). Peptides presented by this pathway stimulate MHC-I restricted cd8+ killer T cells. In another approach, APCs also absorb antigens produced by nearby skin cells. Peptides presented by this pathway stimulate MHC-II restricted cd4+ helper T cells, which help B cells produce neutralizing antibodies (nAb) to combat viral infection.
Problems and solutions for intradermal injection
Here is the solution that we have identified as a potential problem and that provided by the srrrnats platform.
(1) One key unrecognized obstacle to the use of srRNA as an intradermal vaccine platform is that both mRNA and srRNA do not express antigen well at skin temperature [ PCT/US20/67506]. Non-intuitively, the temperature of human skin (about 30 ℃ -35 ℃) is lower than the human core body temperature (about 37 ℃); this means that the vectors and platforms developed at 37 ℃ are not optimal for intradermal injection. One innovation of the srRNAts platform is that it strongly expresses an antigen at skin temperature [ PCT/US20/67506]. In addition, such temperature control also minimizes safety risks caused by undesired systemic distribution of srRNAts, since srRNAts are deactivated once the temperature of the srRNAts rises above its allowable threshold (as it gets closer to the body core). In other words, the srrrNAts platform expresses the antigen optimally in case of intradermal injection compared to mRNA and srRNA, and it additionally has safety features: the ability of the vector to spread and produce is limited in other areas of the subject's body or the vector is inactive.
(2) Another challenge of intradermal vaccination is the lack of suitable additives. Because adjuvants (such as aluminum salts and oil-in-water) are too locally reactive for delivery by the intradermal route, no adjuvant is incorporated into clinically approved intradermal vaccines, resulting in lower immunogenicity [ Hickling and Jones,2009]. Lipid Nanoparticles (LNPs) for intramuscularly administered mRNA and srRNA vaccines are also oil-in-water, which may cause skin reactivities and increase the risk of allergic reactions to LNP components (such as PEG). The c-srRNA platform is a solution to this problem because the platform is injected with naked c-srRNA (LNP free, adjuvant free). First, the self-replication of RNA within cells (especially APCs) induces strong innate immunity, which replaces the primary function of adjuvants. Second, the data in the literature and obtained during the development of the present disclosure indicate that, particularly for intradermal injection, naked mRNA/srrrna is equally effective in producing antigen compared to electroporation of mRNA/srrrna [ Johansson et al 2012] and mRNA/srrrnas combined with LNP [ Golombek et al 2018 ].
(3) A third challenge is the limited number of precedents for intradermal vaccines. Only BCG vaccine is routinely administered intradermally, and currently available covd-19 vaccines are all administered intramuscularly. One way we have reduced the obstacles to using intradermal injections is to use specialized devices such as MicronJet600 (NanoPass) and Immucise (Terumo), which now allow for simple, consistent intradermal injections. These devices are also good candidates for mass production and deployment. However, intradermal injection by Mantoux technology using standard needles and syringes is also an option due to the relatively high cost of these special devices.
Design of suitable antigens
Methods focused on cellular immunity allow reconsidering all proteins encoded on the viral genome as antigen candidates, as humoral immunity (i.e. induction of neutralizing antibodies) is not a major consideration.
When selecting antigens that will provide broader protection against SARS-CoV-1, SARS-CoV-2, MERS-CoV and variants thereof, it is determined that nucleoprotein (N) is most suitable because (1) N is the most abundant protein, followed by membrane (M) and spike (S) in the viral particle [ Finkel et al, 2021], (2) N is generally the most conserved protein in the above mentioned beta coronaviruses [ Grifoni et al, 2020], and (3) epitopes of B and T cells are the most abundant in S and N [ Grifoni et al, 2020]. This is consistent with previous recommendations (N is the best antigen for a vaccine) [ Dutta et al 2020]. Notably, recent reports clearly indicate that vaccines using N alone as antigen can provide S-independent protective immunity in both hamsters and mice [ matrichett et al 2021]. Although disease enhancement of N-vaccine as well as S-vaccine was previously observed [ Lambert et al 2020], these data were obtained by using different vectors with unfavorable Th2> Th1 characteristics.
An exemplary vaccine candidate, srRNA1ts2-G5005, was designed to express the N protein of SARS-CoV-2 (SARS 2-N). However, MERS-N forms a distinct group and shows only 48% identity [ Tilocca et al 2020]. In this regard, another exemplary vaccine candidate, srRNA1ts2-G5006, was designed to express a fusion protein of SARS2-N and MERS-N. The G5005 and G5006 antigens are schematically shown in figure 2. srRNA1ts2-G5005 is suitable for inducing an immune response against SARS-CoV-1, SARS-CoV-2 and variants thereof. In contrast, srRNA1ts2-G5006 is suitable for inducing an immune response against the pan-type coronavirus (e.g., against SARS-CoV-1, SARS-CoV-2, MERS-CoV, and variants thereof).
To cope with the emergence of the variant (mutant) form of SARS-CoV-2 virus, C-srRNA encoding RBD of SARS-CoV-2 Ormi Rong Bianchong (G5003 o) was generated and administered intradermally to C57BL/6 mice (example 8 and FIG. 15). Cellular immunity was assessed 14 days after vaccination. The results clearly show that c-srRNA can induce an obreck Rong Bianchong-specific cellular immunity when it contains the open reading frame of the Receptor Binding Domain (RBD) of the obreck variety. Importantly, c-srRNA encoding G5003o antigen was found to induce a Th 1-biased response, as shown in FIGS. 16A-16B [ Th1 (INF-gamma) > Th2 (IL-4) ], which is advantageous for vaccines.
Inclusion of prime boost regimens
One of the unique features of the intradermally administered c-srRNA vaccine is its ability to induce cellular immunity but not significantly induce humoral immunity (i.e., antibodies). As determined during the development of the present disclosure, the c-srRNA vaccine was able to elicit a humoral immune response against the subsequently encountered protein antigen. Briefly, mice were first treated with c-srRNA encoding antigen (i.e., RBD of SARS-CoV-2 original strain), followed by adjuvanted variant RBD protein (i.e., RBD of SARS-CoV-2 delta variant), as described in example 9 and shown in FIG. 17A.
Cellular immunity (assessed by measuring the presence of antigen-specific IFN-gamma secreting T cells) has been induced on day 14 after primary vaccination (priming), as shown in FIG. 17B. No antigen-specific antibodies were detected at this time. Antibodies were induced after treatment with adjuvanted protein antigen as early as day 7 after the second vaccination (boost), as shown in figure 17C. Early induction of this antibody was consistent with a secondary immune response, indicating that c-srRNA has elicited humoral immunity. Importantly, antibodies raised against protein antigens are capable of neutralizing viral variants having RBD sequences that differ from the RBD antigens encoded by the c-srRNA vaccine. This unexpected finding suggests that the c-srRNA vaccine can induce a protective immune response against pathogens having an antigen sequence that is different from the antigen sequence encoded by the c-srRNA vaccine. Thus, the c-srRNA vaccine is expected to induce a broadly reactive immune response, which is crucial for providing protection against variant pathogens.
Subunit vaccines against pathogens typically do not provide long-lasting humoral immunity (i.e., pathogen-specific antibodies), and thus one or more booster vaccines are needed. As determined during development of the present disclosure, the c-srRNA vaccine is suitable for use as a booster vaccine when the adjuvanted protein is administered as a primary vaccine. Briefly, mice were first treated with adjuvanted protein (i.e., RBD of SARS-CoV-2 original strain), followed by placebo (PBO: buffer only), c-srRNA encoding G5003 antigen (original RBD), c-srRNA encoding G5003o antigen (obrong RBD), or adjuvanted protein antigen (original RBD), as described in example 10 and FIG. 18A.
As shown in fig. 17C, the C-srRNA vaccine alone did not induce humoral immunity in the form of a neutralizing antibody response (see, PBO day 7). However, when humoral immunity was elicited by adjuvanted proteins (as a model for primary vaccination), the C-srRNA boost vaccine was able to induce both antigen-specific cytokine responses (FIGS. 18B-18C) and antigen-specific antibody responses (FIG. 19). Notably, under current experimental conditions, shan Jizuo agent proteins do not induce RBD-specific antibodies. Clearly, cellular immunity induced by c-srRNA is able to stimulate antibody production against the protein antigens encountered earlier. This observation suggests that an important interaction between the cellular immune response and the humoral immune response occurs.
Elimination of antigen expressing cells in vivo
The c-srRNA vaccine is capable of inducing a strong cellular immune response (i.e., antigen specific CD8+ cytotoxic T lymphocytes and CD4+ helper T lymphocytes). Antigen-specific cd8+ CTLs lyse cells expressing the antigen. Antigen recognition by cd8+ CTLs is based on the presentation of short peptide fragments (T cell epitopes) by MHC class I molecules, so that the antigen need not be expressed on the surface of the target cells. For vaccines against pathogens, the vaccine is expected to lyse cells infected with the pathogen. For vaccines against cancer, the vaccine is expected to lyse cancer cells.
A c-srRNA vaccine was produced that encodes a fusion protein of SARS-CoV-2 nucleoprotein and MERS-CoV nucleoprotein (referred to as SMN protein or G5006) as antigens. To model virus-infected cells, a 4T1 breast cancer cell line was selected, which was derived from BALB/c mice and is known as a model for triple negative stage IV human breast cancer. When injected into BALB/c mice, 4T1 cells grew rapidly and formed tumors. This syngeneic mouse model was used to model the rapid increase in infected cells. 4T1 cells expressing SMN protein (designated 4T 1-SMN) were established by transfecting a plasmid vector encoding the SMN protein under the CMV promoter such that the protein was constitutively expressed in the 4T1 cells. The fusion protein is identical to G5006 except that the CD5 signal peptide is removed from the N-terminus of SMN protein expressed in 4T1 cells.
BALB/C mice were vaccinated with C-srRNA-G5006 and induction of cellular immunity was demonstrated by the presence of T cells that responded to both SARS-CoV-2 nucleoprotein (FIGS. 20A-20B) and MERS-CoV nucleoprotein (FIGS. 20C-20D). Subsequently, on day 24 (24 days after vaccination), 4T1-SMN cells were injected into BALB/c mice vaccinated with c-srRNA-G5006. As expected, 4T1-SMN cells grew rapidly in mice receiving placebo (no-vaccine group). In contrast, in c-srRNA-G5006 vaccinated mice, the growth of 4T1-SMN tumors was inhibited. In two mice receiving the 25 μ G c-srRNA-G5006 vaccine, even though the tumor initially grew, the mice eventually became tumor-free and survived for a long time after death of the placebo recipients. Furthermore, there was no tumor growth even after the second round of 4T1-SMN tumor injection at day 143 post-vaccination, and mice remained viable and tumor-free for the duration of the study (fig. 21). This result suggests that the c-srRNA vaccine encoding the G5006 antigen (i.e., SMN protein) can induce a protective immune response by eliminating cells infected with SARS-CoV-2 or MERS-CoV.
Ubiquitin-type coronavirus-enhanced vaccine
For infectious diseases, such as covd-19, world health organization (World Health Organization) guidelines require licensed vaccines capable of inducing neutralizing antibodies (nabs). This requirement is significant because nabs can prevent cells from being infected, and thus nabs can effectively control the spread of infection. However, the na b levels typically drop rapidly, so periodic (e.g., once or twice a year) boosting of the vaccine is required after the primary vaccination series (1 st and 2 nd vaccinations) are completed to maintain adequate na b levels. The high mutation rate of SARS-CoV-2 (particularly within the RBD of spike protein that is the target of nAb) is a major problem associated with the use of the first generation of COVID-19 vaccines that typically target SARS-CoV-2 spike protein.
To solve these problems, a new booster vaccine, c-srRNA-G5006d, was developed, which encodes a fusion protein comprising: CD5 signal peptide, spike-RBD of SARS-CoV-2, nucleoprotein of MERS-CoV, and spike-RBD of MERS-CoV (example 12 and FIG. 22). The amino acid sequence of the pan-type coronavirus antigen (G5006 d) is shown as SEQ ID NO. 27, and the nucleotide sequence of the open reading frame is shown as SEQ ID NO. 26. The order of each sequence segment of the fusion protein (RBD of SARS-CoV-2; nucleoprotein of MERS-CoV; RBD of MERS-CoV) can be altered and the amino acid sequence of each segment need not be 100% identical to the exemplary sequences provided herein.
After receiving a primary vaccine series (either vaccination 1 or vaccination 1 and vaccination 2) against the spike antigen or fragment thereof (RBD), the c-srRNA-G5006d vaccine is intended to be used as a booster vaccine. However, the c-srRNA-G5006d vaccine can also be used as part of the primary vaccine family.
The c-srRNA-G5006d vaccine boosts nAb levels and provides cellular immunity against human-infected beta coronaviruses. Cellular immunity is important to provide long-term protection against severe disease, hospitalization, and death.
When a c-srRNA vaccine encoding spike-RBD is used as a booster vaccine, the c-srRNA vaccine can increase the level of antibodies or nAbs against spike-RBD after administration of a vaccine that can elicit or induce humoral immunity, as described in example 10.
c-srRNA-G5006d encodes both the spike-RBD protein of SARS-CoV-2 and the spike-RBD protein of MERS-CoV. Thus, c-srRNA-G5006d can be used as a booster vaccine for both SARS-CoV-2 and MERS-CoV.
The spike proteins of SARS-CoV-2 and SARS-CoV are similar (about 76% identity) (Grifoni et al 2020). Thus, c-srRNA-G5006d is effective as a potentiator against SARS-CoV-2, SARS-CoV and its variants. In another aspect, the spike proteins of SARS-CoV-2 and MERS-CoV are different (about 35% identity) (Grifoni et al 2020). However, c-srRNA-G5006d also encodes spike-RBD of MERS-CoV. Thus, c-srRNA-G5006d is effective as a booster for MERS-CoV and its variants. In summary, c-srRNA-G5006d is effective as a potentiator against SARS-CoV-2, SARS-CoV, MERS-CoV and variants thereof.
c-srRNA-G5006d also encodes the nucleoprotein of SARS-CoV-2 and MERS-CoV. Thus, c-srRNA-G5006d is capable of inducing strong cellular immunity against SARS-CoV-2 and MERS-CoV. The nucleoproteins of SARS-CoV-2 and SARS-CoV are very similar to each other (about 90% identity) (Grifoni et al 2020). Thus, c-srRNA-G5006d provides strong cellular immunity against SARS-CoV-2, SARS-CoV and its variants. In contrast, the nucleoproteins of SARS-CoV-2 and MERS-CoV are different (about 48% identity) (Grifoni et al 2020). However, c-srRNA-G5006d also encodes the nucleoprotein of MERS-CoV. Thus, c-srRNA-G5006d is expected to provide strong cellular immunity against MERS-CoV and its variants. In summary, c-srRNA-G5006 induced potent immune responses against SARS-CoV-2, SARS-CoV, MERS-CoV and variants thereof.
As described in example 9 and example 10, the c-srRNA vaccine has a significant mode of action. That is, the encoded antigen does not appear to directly stimulate B cells, and thus the three-dimensional structure of the encoded antigen need not be considered. This is in contrast to conventional vaccines designed to directly stimulate B cells to produce antibodies directed against conformational epitopes (the three-dimensional structure of the antigen). That is why it is appropriate to use a fusion protein for the c-srRNA vaccine, whereas it is complicated to use a fusion protein for the conventional subunit vaccine because the natural three-dimensional structure of each antigen may be destroyed when expressed as a fusion protein. The c-srRNA boost vaccine stimulates antibody production by activation of CD4+ helper T cells, and thus it relies on a short peptide epitope (about 15 mer). Thus, two or more different antigens can simply be put together to form a single fusion protein to the antigen encoded by the c-srRNA vaccine, and this mechanism can be problematic for subunit vaccine design.
The fact that c-srRNA relies on short peptide epitopes for inducing cellular and humoral immune responses also provides advantages for vaccines that elicit broader reactivity against protection of variant pathogens. Many T cell epitopes are present in a single protein and thus any single mutation is unlikely to cause loss of immunogenicity. On the other hand, conventional subunit vaccines rely on the three-dimensional structure of protein antigens, and thus even a single mutation may alter the conformation of the protein, which may lead to loss of immunogenicity.
As shown in fig. 23A-23B, c-srRNA-G5006d can stimulate cellular immunity against all of the following proteins encoded by this vaccine: spike-RBD of SARS-CoV-2, nucleoprotein of MERS-CoV and spike-RBD of MERS-CoV.
Pandemic influenza booster vaccine
As determined during development of the present disclosure (see, example 6), when fusion proteins comprising nucleoproteins from representative influenza a and influenza b strains are expressed by intradermally injected temperature controllable self-replicating RNAs, the fusion proteins are capable of inducing a strong antigen-specific cellular immune response. Protection is generally thought to be mediated primarily by neutralizing antibodies directed against Hemagglutinin (HA), one of the surface proteins of influenza virus. Thus, FDA approved influenza vaccines include HA as an antigen alone or in combination with other influenza antigens. Since the c-srRNA based boost vaccine only requires CD4+ T cell epitopes on the HA protein to enhance Ab production, the three dimensional structure of the HA protein need not be considered. It is known that only some portion of the HA protein of H1N1 influenza virus can act as a CD4+ T cell epitope (Knowlden et al, pathogenens.8 (4): 220,2019). B cell epitopes and CD4+ T cell epitopes have been identified in both influenza A and influenza B (Terajima et al Virol J,10:244, 2013). The sequences of the HA proteins of representative H1N1 influenza viruses were aligned (Darricurrre et al, J Virol,92 (22): e01349-18, 2018) and regions with well conserved sequences were identified. Based on these considerations, the HA protein fragment (residues 316-456) of influenza A virus (A/New Caledonia/20/1999 (H1N 1)) [ GenBank accession number EU103824] and the HA protein fragment (residues 332-474) of influenza B virus (B/Florida/4/2006) [ GenBank accession number CY033876] were selected. Also included are nucleoproteins from influenza a and influenza b, which have been described in example 6 and are represented as G5010 antigens.
Figure 24 shows the design of a pan-type influenza booster vaccine. c-srRNA-G5012 encodes a fusion protein (G5012) comprising a signal peptide (residues 1-24) of CD5, a portion of Hemagglutinin (HA) of influenza A, a nucleoprotein of influenza B, and a portion of Hemagglutinin (HA) of influenza B. The amino acid sequence of the pan-type influenza virus antigen (G5012) is shown as SEQ ID NO. 29, and the nucleotide sequence of the open reading frame is shown as SEQ ID NO. 28. The order of each sequence segment of the fusion protein (part of HA for influenza a; nucleoprotein for influenza b; part of HA for influenza b) may be altered and the amino acid sequence of each segment need not be 100% identical to the exemplary sequences provided herein.
This c-srRNA-G5012 influenza vaccine potentiates nAb levels by enhancing HA-specific CD4+ helper T cells. It also provides cellular immunity against substantially all influenza viruses through evolutionarily conserved nucleoproteins. Cellular immunity is known to provide long-term protection against severe disease, hospitalization, and death.
Chitosan enhancement of in vivo Gene expression
Rnase inhibitors (proteins purified from human placenta) slightly enhance immunogenicity against antigens encoded on C-srRNA, most likely by enhancing expression of antigens from C-srRNA in vivo when injected intradermally into mice (see, e.g., figure 25C of WO 2021/138447 A1). RNase inhibitors can protect c-srRNA from RNase-mediated degradation in vivo. However, it is desirable to find alternative agents that can enhance the expression of a gene of interest (GOI) in vivo for therapeutic purposes, because it is difficult to use protein-based rnase inhibitors as excipients in injectable products.
Low molecular weight chitosan (molecular weight about 6 kDa) has been shown to inhibit RNase activity with an inhibition constant ranging from 30-220nM (Yakovlev et al Biochem Biophys Res Commun,357 (3): 584-8, 2007). Although this was only demonstrated in vitro and also for artificially prepared polynucleotides like poly (a)/poly (U), it was necessary to test in mice by intradermal injection of c-srRNA whether chitosan oligosaccharides could enhance expression of GOI from c-srRNA in vivo. As shown in example 14, the following two different chitosan oligomers were tested: chitosan oligomer (molecular weight. Ltoreq.5 kDa,. Gtoreq.75% deacetylation: heppe Medical Chitosan GmbH: product number 44009) and chitosan oligosaccharide lactic acid (molecular weight about 5kDa, >90% deacetylation: sigma-Aldrich: product number 523682). Unexpectedly, it was found that even very low levels of chitosan oligomer, as low as 0.001. Mu.g/mL (about 0.2nM: about 1/100 of the inhibition constant found by Yakovlev et al, supra, 2007) was able to enhance the expression of the luciferase encoded on c-srRNA by about 10-fold (FIG. 25). Similar enhancement of GOI expression was achieved by up to 0.5. Mu.g/mL of chitosan oligomer and 0.1. Mu.g/mL of chitosan oligosaccharide lactic acid.
Chitosan has been used as a nucleotide (DNA and RNA) delivery vehicle because it can form complexes or nanoparticles (reviewed in bussmann et al Adv Drug Deliv Rev,65 (9): 1234-70,2013; and Cao et al Drugs,17:381, 2019). However, it is notable that enhancement of GOI expression by chitosan oligomers is unlikely to be mediated by nanoparticle or complex formation of c-srrrna and chitosan oligomers. First, such low concentrations of chitosan oligomers do not allow for complex formation with RNA. Second, chitosan oligomer was added to the c-srRNA immediately prior to intradermal injection, so there was insufficient time to form a complex.
Since chitosan oligomers enhance GOI expression in vivo at much lower concentrations than are effective in vitro as rnase inhibitors (Yakovlev et al, supra, 2007), it is conceivable that such enhanced GOI expression by chitosan oligomers may not be mediated by its rnase inhibition mechanism. For example, chitosan oligomers may promote the incorporation of c-srRNA into cells, and thus may enhance the expression of GOI from c-srRNA. Nevertheless, this unexpected finding should provide an effective means of enhancing the in vivo therapeutic expression of the GOI encoded on c-srRNA.
Detailed description of the illustrated embodiments
1. A composition for stimulating an immune response against a coronavirus in a mammalian subject, the composition comprising an excipient and a messenger RNA (mRNA) comprising an Open Reading Frame (ORF) encoding a fusion protein, wherein the ORF comprises from 5 'to 3':
(i) A nucleotide sequence encoding a mammalian signal peptide; and
(ii) Nucleotide sequence encoding a nucleocapsid protein of a coronavirus.
2. The composition of embodiment 1, wherein the coronavirus is a beta coronavirus, optionally wherein the beta coronavirus is a human beta coronavirus.
3. The composition of embodiment 2, wherein the beta coronavirus comprises severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), middle east respiratory syndrome related coronavirus (MERS-CoV), or a combination thereof.
4. The composition of embodiment 3, wherein the beta coronavirus comprises severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).
5. The composition of embodiment 4, wherein the coronavirus nucleocapsid protein comprises a first nucleocapsid protein and a second nucleocapsid protein, wherein the first nucleocapsid protein is a SARS-CoV-2 nucleocapsid protein from a first variant of a first clade and the second nucleocapsid protein is a SARS-CoV-2 nucleocapsid protein from a second variant of a second clade, and wherein the first clade and the second clade are different clades defined by one or more of the world health organization, pango, GISAID, and Nextstrain.
6. A composition for stimulating an immune response against a coronavirus in a mammalian subject, the composition comprising an excipient and a messenger RNA (mRNA) comprising an Open Reading Frame (ORF) encoding a fusion protein, wherein the ORF comprises from 5 'to 3':
(i) A nucleotide sequence encoding a mammalian signal peptide; and
(ii) Nucleotide sequences encoding two or more coronavirus nucleocapsid proteins.
7. The composition of embodiment 6, wherein the coronavirus is a beta coronavirus, optionally wherein the beta coronavirus is a human beta coronavirus.
8. The composition of embodiment 7, wherein the beta coronavirus comprises severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), middle east respiratory syndrome related coronavirus (MERS-CoV), or a combination thereof.
9. The composition of embodiment 8, wherein the beta coronavirus comprises severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).
10. The composition of embodiment 9, wherein the two or more coronavirus nucleocapsid proteins comprise a SARS-CoV-2 nucleocapsid protein and a MERS nucleocapsid protein.
11. The composition of embodiment 9, wherein the two or more coronavirus nucleocapsid proteins comprise a SARS-CoV-2 nucleocapsid protein, a SARS-CoV-1 nucleocapsid protein, and a MERS nucleocapsid protein.
12. The composition of any of embodiments 6-11, wherein the two or more coronavirus nucleocapsid proteins are separated by a linker of one to ten residues in length.
13. The composition of any of embodiments 1-12, wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in mammalian antigen presenting cells.
14. The composition of embodiment 13, wherein the mammalian signal peptide is a CD5 signal peptide and the amino acid sequence of the CD5 signal peptide comprises SEQ ID No. 8 or an amino acid sequence at least 90% or 95% identical to SEQ ID No. 8.
15. The composition of any one of embodiments 1-14, wherein the amino acid sequence of the nucleocapsid protein comprises residues 2-419 of SEQ ID No. 5 or an amino acid sequence that is at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to residues 2-419 of SEQ ID No. 5.
16. The composition of any of embodiments 1-14, wherein the amino acid sequence of the fusion protein comprises SEQ ID No. 6 or an amino acid sequence at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 6.
17. The composition of any of embodiments 6-14, wherein the amino acid sequence of the fusion protein comprises SEQ ID No. 7 or an amino acid sequence at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 7.
18. The composition of embodiment 16, wherein the open reading frame comprises the nucleotide sequence of SEQ ID No. 2.
19. The composition of embodiment 17, wherein the open reading frame comprises the nucleotide sequence of SEQ ID NO. 3 or SEQ ID NO. 4.
20. The composition of any of embodiments 1-14, wherein the amino acid sequence of the fusion protein comprises residues 2-413 of SEQ ID No. 9 or an amino acid sequence at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to residues 2-413 of SEQ ID No. 9.
21. The composition of any of embodiments 1-14, wherein the amino acid sequence of the fusion protein comprises residues 2-422 of SEQ ID No. 10 or an amino acid sequence that is at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to residues 2-422 of SEQ ID No. 10.
22. The composition of any one of embodiments 1-21, wherein the composition does not comprise a liposome or a lipid nanoparticle.
23. The composition of any one of embodiments 1-22, wherein the mRNA is a self-replicating mRNA.
24. The composition of embodiment 23, wherein the self-replicating RNA comprises an alphavirus replicon lacking viral structural protein coding regions.
25. The composition of embodiment 24, wherein the alphavirus is selected from the group consisting of venezuelan equine encephalitis virus, sindbis virus, and semliki forest virus.
26. The composition of embodiment 25, wherein the alphavirus is venezuelan equine encephalitis virus.
27. The composition of any one of embodiments 23-26, wherein the alphavirus replicon comprises a non-structural protein coding region in which 12-18 nucleotides are inserted resulting in expression of nsP2 comprising 4 to 6 additional amino acids between β -sheet 4 and β -sheet 6 of non-structural protein 2 (nsP 2).
28. The composition of any one of embodiments 1-27, wherein the self-replicating mRNA is a temperature-sensitive agent (ts agent) that is capable of expressing the fusion at a permissive temperature and not expressing the fusion at an non-permissive temperature.
29. The composition of embodiment 28, wherein the permissible temperature is 31 ℃ to 35 ℃ and the impermissible temperature is at least 37 ℃ ± 0.5 ℃.
30. A method for stimulating an immune response against a coronavirus in a mammalian subject, the method comprising administering to a mammalian subject the composition of any one of embodiments 1-29 to stimulate an immune response against the coronavirus nucleocapsid protein in the mammalian subject.
31. The method of embodiment 30, wherein the composition is administered intradermally.
32. The method of embodiment 30 or embodiment 31, wherein the immune response comprises a coronavirus-reactive cellular immune response.
33. The method of embodiment 32, wherein the immune response further comprises a coronavirus-reactive humoral immune response.
34. The method of any one of embodiments 30-33, wherein the mammalian subject is a human subject.
35. A kit, the kit comprising:
the composition according to any one of embodiments 1-29 or any one of embodiments 37-62; and
a device for intradermal delivery of the composition to a mammalian subject.
36. The kit of embodiment 35, wherein the device comprises a syringe and a needle.
37. A composition for stimulating an immune response against two or more viruses in a mammalian subject, the composition comprising an excipient and a messenger RNA (mRNA) comprising an Open Reading Frame (ORF) encoding a fusion protein, wherein the ORF comprises from 5 'to 3':
(i) A nucleotide sequence encoding a mammalian signal peptide; and
(ii) Nucleotide sequences encoding a first nucleocapsid protein of a first virus and a second nucleocapsid protein of a second virus.
38. The composition of embodiment 37, wherein the first virus and the second virus are capable of causing a disease upon infection of a human subject.
39. The composition of embodiment 38, wherein the first virus and the second virus are different varieties, subtypes or lineages of the same species.
40. The composition of embodiment 38, wherein the first virus and the second virus are different species of the same genus.
41. The composition of embodiment 40, wherein both the first virus and the second virus are members of the genus betacoronavirus.
42. The composition of embodiment 41, wherein the first virus and the second virus comprise severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and middle east respiratory syndrome related coronavirus (MERS-CoV).
43. The composition of embodiment 38, wherein the first virus and the second virus are members of different families, orders, classes, or gates of the same kingdom.
44. The composition of embodiment 43, wherein both the first virus and the second virus are members of the orthomyxoviridae family.
45. The composition of embodiment 44, wherein the first virus and the second virus comprise influenza a virus and influenza b virus.
46. The composition of embodiment 45, wherein the amino acid sequence of the fusion protein comprises SEQ ID NO 16 or an amino acid sequence that is at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 16.
47. The composition of embodiment 38, wherein both the first virus and the second virus are members of the kingdom orthoribo virus, optionally wherein the first virus and the second virus comprise: (a) Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1) or middle east respiratory syndrome related coronavirus (MERS-CoV); and (b) influenza a virus or influenza b virus.
48. The composition of embodiment 40, wherein both the first virus and the second virus are members of the genus ebola, optionally wherein the first virus and the second virus are selected from the group consisting of zaire-type ebola virus, sudan-type ebola virus, bund Jiao Xingai ebola virus, and tay forest-type ebola virus.
49. The composition of embodiment 48, wherein the nucleotide sequence further encodes a third nucleocapsid protein of a third virus and a fourth nucleocapsid protein of a fourth virus, and the first virus, the second virus, the third virus, and the fourth virus are zaire-type ebola virus, sudan-type ebola virus, bundi Jiao Xingai bola virus, and tay forest-type ebola virus.
50. The composition of embodiment 49, wherein the amino acid sequence of the fusion protein comprises SEQ ID NO. 22 or an amino acid sequence that is at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 22.
51. The composition of embodiment 49, wherein the nucleotide sequence (ii) encodes a shared portion of a first nucleocapsid protein of the first virus for stimulating an immune response against all of the first virus, the second virus, the third virus, and the fourth virus.
52. The composition of embodiment 51, wherein the nucleotide sequence (ii) encodes a separate portion of the first nucleocapsid protein, the second nucleocapsid protein, the third nucleocapsid protein, and the fourth nucleocapsid protein each for stimulating an immune response against all of the first virus, the second virus, the third virus, and the fourth virus.
53. The composition of embodiment 52, wherein the nucleotide sequence (ii) encodes a fragment of a separate portion of a second nucleocapsid protein of the second virus for stimulating an immune response against the second virus and the third virus.
54. The composition of embodiment 37, wherein the nucleotide sequence (ii) encodes a shared portion of a first nucleocapsid protein of the first virus for stimulating an immune response against both the first virus and the second virus.
55. The composition of embodiment 54, wherein the nucleotide sequence (ii) encodes a separate portion of the first nucleocapsid protein and the second nucleocapsid protein each for stimulating an immune response against both the first virus and the second virus.
56. The composition of any one of embodiments 37-48, wherein the nucleotide sequence (ii) further encodes at least one other nucleocapsid protein of at least one other virus, and wherein the at least one other virus is different from the first virus and the second virus.
57. The composition of any of embodiments 37-56, wherein the first nucleocapsid protein and the second nucleocapsid protein, or the first nucleocapsid protein, the second nucleocapsid protein and the other nucleocapsid proteins, are separated by a linker of one to ten residues in length.
58. The composition of any one of embodiments 37-57, wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in mammalian antigen presenting cells.
59. The composition of any one of embodiments 37-58, wherein the mRNA is a self-replicating mRNA.
60. The composition of embodiment 59, wherein the self-replicating mRNA is a temperature-sensitive agent (ts agent) that is capable of expressing the fusion protein at a permissive temperature and not expressing the fusion protein at an non-permissive temperature.
61. The composition of embodiment 60, wherein the permissible temperature is 31 ℃ to 35 ℃ and the impermissible temperature is at least 37 ℃ ± 0.5 ℃.
62. The composition of any one of embodiments 1-29 or any one of embodiments 37-61, wherein the composition further comprises chitosan.
63. A method for stimulating an immune response against two or more viruses in a mammalian subject, the method comprising administering to a mammalian subject a composition according to any one of embodiments 37-62 to stimulate an immune response against nucleocapsid proteins of the two or more viruses in the mammalian subject.
64. The method of embodiment 63, wherein the composition is administered intradermally.
65. The method of embodiment 63 or embodiment 64, wherein the immune response comprises a cellular immune response reactive to the two or more viruses.
66. The method of embodiment 65, wherein the cellular immune response comprises a nucleocapsid protein specific helper T lymphocyte (Th) response comprising nucleocapsid protein specific cytokine secretion.
67. The method of embodiment 66, wherein the nucleocapsid protein specific cytokine secretion comprises secretion of one or both of interferon-gamma and interleukin-4.
68. The method of embodiment 65, wherein the cellular immune response comprises a nucleocapsid protein specific Cytotoxic T Lymphocyte (CTL) response.
69. The method of any one of embodiments 65-68, wherein the immune response further comprises a humoral immune response reactive against the two or more viruses.
70. The method of any one of embodiments 63-69, wherein the mammalian subject is a human subject.
71. A composition for stimulating an immune response against a virus in a mammalian subject, the composition comprising an excipient and a messenger RNA (mRNA) comprising an Open Reading Frame (ORF) encoding a fusion protein, wherein the ORF comprises from 5 'to 3':
(i) A nucleotide sequence encoding a mammalian signal peptide;
(ii) A nucleotide sequence encoding a first viral antigen of a first virus or a fragment thereof; and
(iii) A nucleotide sequence encoding a second viral antigen or fragment thereof of the first virus or second virus, wherein the first viral antigen is a nucleocapsid protein and the second viral antigen is a surface protein, or the first viral antigen is a surface protein and the second viral antigen is a nucleocapsid protein.
72. A composition for stimulating an immune response against two or more viruses in a mammalian subject, the composition comprising an excipient and a messenger RNA (mRNA) comprising an Open Reading Frame (ORF) encoding a fusion protein, wherein the ORF comprises from 5 'to 3':
(i) A nucleotide sequence encoding a mammalian signal peptide;
(ii) A nucleotide sequence encoding a first viral antigen of a first virus or a fragment thereof;
(iii) A nucleotide sequence encoding a second viral antigen of the first virus or a fragment thereof;
(iv) A nucleotide sequence encoding a third viral antigen of a second virus or a fragment thereof;
(iii) A nucleotide sequence encoding a fourth viral antigen of said second virus or a fragment thereof,
wherein the first viral antigen is a first nucleocapsid protein and the second viral antigen is a first surface protein, or the first viral antigen is a first surface protein and the second viral antigen is a first nucleocapsid protein, and wherein the third viral antigen is a second nucleocapsid protein and the fourth viral antigen is a second surface protein, or the third viral antigen is a second surface protein and the fourth viral antigen is a second nucleocapsid protein.
73. The composition of embodiment 71 or embodiment 72, wherein the mRNA is a self-replicating mRNA.
74. The composition of embodiment 73, wherein the self-replicating RNA comprises an alphavirus replicon that lacks viral structural protein coding regions.
75. The composition of embodiment 74, wherein the alphavirus is selected from the group consisting of venezuelan equine encephalitis virus, sindbis virus, and semliki forest virus.
76. The composition of embodiment 74, wherein the alphavirus is venezuelan equine encephalitis virus.
77. The composition of any one of embodiments 73-76, wherein the self-replicating mRNA is a temperature-sensitive agent (ts agent) that is capable of expressing the fusion protein at a permissive temperature and not expressing the fusion protein at an non-permissive temperature.
78. The composition of embodiment 77, wherein said permissible temperature is 31 ℃ to 35 ℃ and said impermissible temperature is at least 37 ℃ ± 0.5 ℃.
79. The composition of any one of embodiments 74-78, wherein said alphavirus replicon comprises a non-structural protein coding region in which 12-18 nucleotides are inserted resulting in expression of nsP2 comprising 4 to 6 additional amino acids between β -sheet 4 and β -sheet 6 of non-structural protein 2 (nsP 2).
80. The composition of any one of embodiments 71-79, wherein the first virus and/or the second virus is a coronavirus, optionally wherein the coronavirus is a beta coronavirus, optionally wherein the beta coronavirus is a human beta coronavirus.
81. The composition of embodiment 80, wherein the first virus and/or the second virus is a beta coronavirus independently selected from the group consisting of: severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), and middle east respiratory syndrome related coronavirus (MERS-CoV).
82. The composition of embodiment 80, wherein the first virus is SARS-CoV-2 and the second virus is MERS-CoV.
83. The composition of any one of embodiments 80-82, wherein each of said surface protein, said first surface protein, and/or said second surface protein comprises a Receptor Binding Domain (RBD) of a coronavirus spike protein.
84. The composition of embodiment 83, wherein the amino acid sequence of the fusion protein comprises SEQ ID NO 27 or an amino acid sequence that is at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 27.
85. The composition of any one of embodiments 71-79, wherein said first virus and/or said second virus is a member of the orthomyxoviridae family.
86. The composition of embodiment 85, wherein said first virus and/or said second virus is independently selected from the group consisting of Influenza A Virus (IAV) and Influenza B Virus (IBV).
87. The composition of embodiment 86, wherein the first virus is an IAV and the second virus is an IBV.
88. The composition of any one of embodiments 85-87, wherein the surface protein, the first surface protein, and/or the second surface protein each comprise a portion of influenza hemagglutinin.
89. The composition of embodiment 88, wherein the amino acid sequence of the fusion protein comprises SEQ ID NO 29 or an amino acid sequence that is at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 29.
90. The composition of any one of embodiments 71-89, wherein the composition further comprises chitosan.
91. A kit, the kit comprising:
(i) The composition of any one of embodiments 71-90; and
(ii) A device for intradermal delivery of the composition to a mammalian subject.
92. The kit of embodiment 91, wherein the device comprises a syringe and a needle.
93. The kit of embodiment 91 or embodiment 92, further comprising instructions for using the device to administer the composition to a mammalian subject to stimulate an immune response against one or more of the first viral antigen, the second viral antigen, the third viral antigen, and the fourth viral antigen.
94. A method of stimulating an immune response in a mammalian subject, the method comprising administering to a mammalian subject the composition of any one of embodiments 71-90 to stimulate an immune response in the mammalian subject against one or more of the first viral antigen, the second viral antigen, the third viral antigen, and the fourth viral antigen.
95. The method of embodiment 94, wherein the composition is administered intradermally.
96. The method of embodiment 95, wherein the immune response comprises a cellular immune response reactive against one or more of the first viral antigen, the second viral antigen, the third viral antigen, and the fourth viral antigen.
97. The method of embodiment 96, wherein the immune response further comprises a humoral immune response reactive against one or more of the first viral antigen, the second viral antigen, the third viral antigen, and the fourth viral antigen.
98. The method of any one of embodiments 94-97, wherein the mammalian subject is a human subject.
99. A method for active boosting against at least one virus, the method comprising intradermally administering the composition according to any one of embodiments 1-29, embodiments 37-62, or embodiments 71-90 to a mammalian subject in need thereof to stimulate a secondary immune response against the virus, wherein the mammalian subject has undergone a primary immunization regimen against the virus.
100. The method of embodiment 99, wherein the primary immunization regimen comprises administration of at least one dose of a different vaccine against the virus.
101. The method of embodiment 100, wherein the different vaccine comprises a protein antigen of the at least one virus or an inactivated virus, optionally wherein the protein antigen is a recombinant protein or fragment thereof.
102. A method for active boosting immunity against at least one virus, the method comprising:
(i) Administering intradermally the composition of any one of embodiments 1-29, any one of embodiments 37-62, or any one of embodiments 71-90 to a mammalian subject in need thereof to stimulate a primary immune response against the virus; and
(ii) At least one dose of a different vaccine against the virus is administered to the mammalian subject to stimulate a secondary immune response against the virus.
103. The method of embodiment 102, wherein the different vaccine comprises a protein antigen of the at least one virus or an inactivated virus, optionally wherein the protein antigen is a recombinant protein or fragment thereof.
104. A method for active primary immunization against at least one virus, the method comprising:
(i) Administering intradermally to a mammalian subject in need thereof the composition according to any one of embodiments 1-29, any one of embodiments 37-62, or any one of embodiments 71-90, to stimulate a primary immune response against the virus, wherein the mammalian subject has not undergone a primary immune regimen against the virus.
105. The method of embodiment 104, the method further comprising:
(ii) At least one dose of a different vaccine against the virus is administered to the mammalian subject to stimulate a secondary immune response against the virus.
106. The method of embodiment 105, wherein the different vaccine comprises a protein antigen of the at least one virus or an inactivated virus, optionally wherein the protein antigen is a recombinant protein or fragment thereof.
107. The method of any one of embodiments 94-106, wherein the mammalian subject is a human subject. 108. An expression vector comprising the mRNA of any one of the preceding claims in operable combination with a promoter.
109. The expression vector of embodiment 108, wherein the promoter is a T7 promoter or an SP6 promoter.
110. The expression vector of embodiment 108, wherein the vector is a plasmid.
111. The expression vector according to any one of embodiments 108-110, further comprising a selectable marker.
Examples
Abbreviations: ab (antibody); APC (antigen presenting cell); coV (coronavirus); c-srRNA (temperature controlled self-replicating RNA); CTL (cytotoxic T lymphocytes); fluA or IAV (influenza A virus); fluB or IBV (influenza B virus); IL-4 (interleukin-4); INF-gamma (interferon gamma); GOI (gene of interest); HA (hemagglutinin); MERS (middle east respiratory syndrome associated); nAb (neutralizing antibody); n or NP (nucleocapsid or nucleoprotein); nsP (non-structural protein); ORF (open reading frame); PBO (placebo); RBD (receptor binding domain); s (spike); PRNT (plaque reduction neutralization test); SARS (severe acute respiratory syndrome); SFC (spot forming cells); SFU (spot forming unit); srRNAts (temperature sensitive self-replicating RNA); th (helper T lymphocytes); and Tx (processing). The terms c-srRNA and srRNA are used interchangeably throughout the disclosure, with srRNA1ts2 (described in WO 2021/138447 A1) being an exemplary embodiment.
EXAMPLE 1 cellular immunity induced by srRNA1ts2-G5004
This example describes the findings that: when SARS-CoV-2 nucleoprotein alone (G5004 antigen, without signal peptide) was expressed from intradermally injected temperature-controllable self-replicating RNA, the protein did not induce an effective cellular immune response.
Materials and methods.
CD-1 inbred female mice.
srRNA1ts2-G5004 mRNA was produced by in vitro transcription of a temperature controlled self-replicating RNA vector encoding the G5004 antigen (srRNA 1ts2 as described in PCT/US 2020/067506) (FIG. 2).
From nucleoprotein (UniProt: P0DTC 9) [ JPT peptide product code) by SARS-CoV-2: PM-WCPV-NCAP ] peptides were scanned to obtain 102 peptide (15 mer with 11 amino acid overlap) pools.
ELISPot assay plates and reagents for interferon gamma (INF-gamma) and interleukin-4 (IL-4) (Cellular Technology Limited, ohio).
Immunospot S6 Entry Analyzer (Cellular Technology Limited, ohio, U.S.A.).
Results
Recently, vaccination with nucleoprotein (N) alone has been shown to elicit cellular immunity and spike-independent protective immunity of SARS-CoV-2 in mice and hamsters (Machett et al, biorxiv.2021.04.26.441518.2021). Vaccination involves intravenous administration of a human adenovirus serotype 5 (Ad 5) vector (Ad 5-N) expressing an N sequence derived from the USA-WA1/2021 strain.
To test whether nuclear protein (N) alone (without signal peptide) could induce cellular immunity, ELISPot assays were performed 14 days after CD-1-outcrossing mice were vaccinated by a single intradermal injection of 5 μg or 25 μg of srRNA1ts2-G5004 (FIG. 2) or placebo (PBO: buffer only). Only weak induction of interferon-gamma (INF-gamma) secreting T cells (fig. 4A) and IL-4 secreting T cells (fig. 4B) was observed. Interestingly, no dose-dependent INF-gamma response was observed (5. Mu.g compared to 25. Mu.g).
It was concluded that nucleoprotein (N) alone did not induce an effective cellular immune response upon expression of temperature-controlled self-replicating RNA by intradermal injection.
EXAMPLE 2 cellular immunity induced by srRNA1ts2-G5005
This example describes the following findings: addition of the CD5 signal peptide to SARS-CoV-2 nucleoprotein induces an effective cellular immune response in CD-1 mice upon expression of temperature-controlled self-replicating RNA by intradermal injection.
Materials and methods
CD-1 inbred female mice.
srRNA1ts2-G5005 mRNA was produced by in vitro transcription of a temperature controlled self-replicating RNA vector encoding the G5005 antigen (srRNA 1ts2 as disclosed in PCT/US 2020/067506) (FIG. 2).
From nucleoprotein (UniProt: P0DTC 9) [ JPT peptide product code) by SARS-CoV-2: PM-WCPV-NCAP ] peptides were scanned to obtain 102 peptide (15 mer with 11 amino acid overlap) pools.
ELISPot assay plates and reagents for interferon gamma (INF-gamma) and interleukin-4 (IL-4) (Cellular Technology Limited, ohio).
Immunospot S6 Entry Analyzer (Cellular Technology Limited, ohio, U.S.A.).
Results
Wild-type nucleoprotein contains no signal peptide or transmembrane domain and is therefore not expected to be directed against the secretory pathway of mammalian host cells. The inventors speculate that the lack of signal peptide may be responsible for the lack of induction of an effective cellular immune response by wild-type nucleoprotein (expressed by srRNA1ts2-G5004 of example 1). With this in mind, the coding region of the signal peptide sequence from the human CD5 gene was added to the nucleoprotein coding region to replace the initiation codon (ATG) of nucleoprotein in srRNA1ts2-G5005 (FIG. 2). The amino acid sequence of the CD5 signal peptide is MPMGSLQPLATLYLLGMLVASCLG (shown as SEQ ID NO: 8).
Cellular immunity was assessed by ELISpot assay 14 days after CD-1-outcrossing mice were vaccinated by single intradermal injection of 5 μg or 25 μg of srRNA1ts2-G5005 (FIG. 2) or placebo (PBO: buffer only).
As shown in fig. 5A, antigen-specific INF- γ secreting T cells were strongly induced in a dose-dependent manner (5 μg compared to 25 μg). In contrast, antigen-specific IL-4 secreting T cells were hardly induced (FIG. 5B). Th1 cells secrete INF-gamma, whereas Th2 cells secrete IL-4. Th1> Th2 immune responses are generally considered to be an advantageous feature of vaccines.
In summary, the addition of a signal peptide derived from human CD5 to the N-terminus of nucleoprotein (N) results in the induction of a strong antigen-specific cellular immune response when the protein is expressed by intradermally injected temperature-controllable self-replicating RNA. The srRNA1ts2-G5005 vaccine also showed good Th1 skewed (Th 1> Th 2) immune response.
EXAMPLE 3 cellular immunity induced by srRNA1ts2-G5005
This example describes the following findings: addition of CD5 signal peptide to SARS-CoV-2 nucleoprotein induces potent cellular immune responses in BALB/c mice upon expression of temperature-controlled self-replicating RNA by intradermal injection.
Materials and methods
BALB/c female mice.
srRNA1ts2-G5005 mRNA was produced by in vitro transcription of a temperature controlled self-replicating RNA vector encoding the G5005 antigen (srRNA 1ts2 as described in PCT/US 2020/067506) (FIG. 2).
From nucleoprotein (UniProt: P0DTC 9) [ JPT peptide product code) by SARS-CoV-2: PM-WCPV-NCAP ] peptides were scanned to obtain 102 peptide (15 mer with 11 amino acid overlap) pools.
ELISPot assay plates and reagents for interferon gamma (INF-gamma) and interleukin-4 (IL-4) (Cellular Technology Limited, ohio).
Immunospot S6 Entry Analyzer (Cellular Technology Limited, ohio, U.S.A.).
Results
To test whether srRNA1ts2-G5005 could induce a strong cellular immune response in another mouse strain, immunogenicity studies were also performed in BALB/c mice. Cellular immunity was assessed by ELISpot assay 30 days after BALB/c mice were vaccinated by single intradermal injection of 5 μg or 25 μg of srRNA1ts2-G5005 (FIG. 2) or placebo (PBO: buffer only).
As shown in fig. 6A, antigen-specific INF- γ secreting T cells were strongly induced in a dose-dependent manner (5 μg compared to 25 μg). In contrast, antigen-specific IL-4 secreting T cells were not induced (FIG. 6B). Thus, favorable Th1> Th2 cellular responses were also observed in BALB/c mice.
In summary, the addition of a signal peptide derived from human CD5 to the N-terminus of nucleoprotein (N) significantly enhances the antigen-specific cellular immune response when the protein is expressed by intradermally injected temperature-controllable self-replicating RNA. As in CD-1 mice, the srRNA1ts2-G5005 vaccine showed an advantageous Th1 skewed (Th 1> Th 2) immune response in BALB/c mice.
EXAMPLE 4 humoral immunity induced by srRNA1ts2-G5005
This example describes the following findings: when SARS-CoV-2 nucleoprotein is expressed from intradermally injected temperature-controllable self-replicating RNA, the SARS-CoV-2 nucleoprotein induces an effective humoral immune response upon ligation to the human CD5 signal peptide.
Materials and methods
BALB/c female mice.
srRNA1ts2-G5005 mRNA was produced by in vitro transcription of a temperature controlled self-replicating RNA vector encoding the G5005 antigen (srRNA 1ts2 as described in PCT/US 20/67506) (FIG. 2).
SARS-CoV-2 nucleocapsid IgG ELISA KIT (ENZO: ENZ-KIT 193-0001).
Results
To test whether srRNA1ts2-G5005 could induce humoral immunity, nuclear protein-specific IgG levels in serum were measured by ELISA after 30 days of BALB/c mice were vaccinated by a single intradermal injection of 5 μg or 25 μg of srRNA1ts2-G5005 (FIG. 2) or placebo (PBO: buffer only). IgG levels are represented by OD450 in ELISA. IgG levels were measured before (day-1) and after (day 30) vaccination (day 0).
As shown in fig. 7, nucleoprotein-specific serum IgG (5 μg compared to 25 μg) was strongly induced in a dose-dependent manner.
In summary, the addition of a signal peptide derived from human CD5 to the N-terminus of nucleoprotein (N) induces an antigen-specific humoral immune response when the protein is expressed by intradermally injected temperature-controllable self-replicating RNA.
EXAMPLE 5 cellular immunity induced by srRNA1ts2-G5006
This example describes the following findings: when a fusion protein comprising SARS-CoV-2 nucleoprotein and MERS-CoV nucleoprotein is expressed by intradermally injected temperature-controllable self-replicating RNA, the fusion protein can induce strong cellular immunity against SARS-CoV-2 and MERS-CoV.
Materials and methods
BALB/c female mice.
srRNA1ts2-G5006 mRNA was produced by in vitro transcription of a temperature controlled self-replicating RNA vector encoding the G5006 antigen (srRNA 1ts2 as described in PCT/US 2020/067506) (FIG. C).
From nucleoprotein (UniProt: P0DTC 9) [ JPT peptide product code) by SARS-CoV-2: PM-WCPV-NCAP ] peptides the resulting pool of peptides (15 mer with 11 amino acid overlap) was scanned.
Peptide pools derived from peptide scans (15 MERS with 11 amino acid overlap) of nucleoprotein by MERS-CoV.
ELISPot assay plates and reagents for interferon gamma (INF-gamma) and interleukin-4 (IL-4) (Cellular Technology Limited, ohio).
Immunospot S6 Entry Analyzer (Cellular Technology Limited, ohio, U.S.A.).
Results
T cell epitopes are present in short linear peptides (typically in the size range of 8-11 residues for MHC class I and 10-30 residues for MHC class II). Unlike many B cell epitopes, the 3-D conformation of T cell epitopes is not important for recognition of immune cell receptors. Thus, the inventors speculate that nucleoproteins from different strains of β -coronavirus may be fused together in the absence of long linkers (greater than 10 amino acids in length) and used as vaccine antigens to elicit immune responses against different β -coronaviruses (e.g., SARS-CoV-1 and its variants, SARS-CoV-2 and its variants, and MERS-CoV and its variants).
To test this concept, a fusion protein comprising human CD5 signal peptide, SARS-CoV-2 nucleoprotein and MERS-CoV nucleoprotein was designed (see G5006 in FIG. 2C). Mice were vaccinated with srRNA1ts2-G5006 by intradermal injection and antigen-specific cellular immune responses were measured by the ELISPot assay. As expected, the srRNA1ts2-G5006 vaccine induced a strong INF-gamma secreting T cell response against both SARS-CoV-2 nucleoprotein (FIG. 8) and MERS-CoV nucleoprotein. Furthermore, cellular immune responses are expected to have a Th1> Th2 balance.
In summary, when fusion proteins comprising nucleoproteins from different beta coronaviruses are expressed by intradermally injected temperature-controllable self-replicating RNAs, the fusion proteins induce a strong antigen-specific cellular immune response.
Example 6 cellular immunity induced by srRNA1ts2-G5010 (influenza A vaccine)
This example describes an assessment of the immune response induced by a fusion protein comprising influenza a virus (FluA) nucleoprotein and influenza b virus (FluB) nucleoprotein when said fusion protein is expressed by intradermally injected temperature-controllable self-replicating RNA.
Materials and methods
BALB/c female mice.
srRNA1ts2-G5010 mRNA was produced by in vitro transcription of a temperature controllable self-replicating RNA vector (srRNA 1ts2[ PCT/US20/67506 ]) encoding the G5010 antigen (FIG. 9). The amino acid sequence of the G5010 fusion protein is shown as SEQ ID NO. 16. The nucleic acid sequence encoding the G5010 fusion protein was codon optimized for expression in human cells and is shown in SEQ ID NO. 15.
From Nucleoprotein (NP) through influenza a (H2N 2) (Swiss-Prot ID P21433) [ JPT peptide product code: peptide scan of PM-INFA-NPH2N2] a pool of 122 overlapping peptides (15 mer with 11 amino acid overlap) was obtained. The amino acid sequence of H2N2 nucleoprotein is shown in SEQ ID NO. 17.
ELISPot assay plates and reagents for interferon gamma (INF-gamma) and interleukin-4 (IL-4) (Cellular Technology Limited, ohio).
Immunospot S6 Entry Analyzer (Cellular Technology Limited, ohio, U.S.A.).
Results
Influenza a and b can infect humans and cause seasonal epidemics or pandemics (see "influenza virus type (Types of Influenza Viruses)" from CDC website www.cdc.gov/flu/abaout/viruses/types.htm). Nucleoprotein antigens are more conserved among different influenza strains than Hemagglutinin (HA) and Neuraminidase (NA) antigens conventionally included in influenza vaccines. For example, the amino acid sequences of the nucleoproteins of representative influenza a strains (H1N 1, H3N2, H5N8, H7N7, H7N9, H9N2, H10N 8) are very similar. Likewise, the amino acid sequences of the nucleoproteins of representative influenza b strains (Yamagata, victoria) are very similar. In contrast, the amino acid sequence of the nucleoprotein of influenza a is significantly different from that of influenza b.
T cell epitopes are present in short linear peptides (typically in the size range of 8-11 residues for MHC class I and 10-30 residues for MHC class II). Unlike many B cell epitopes, the conformation or 3D structure of T cell epitopes is not important for the recognition of immune cell receptors. Thus, one representative nucleoprotein from influenza a is expected to include many T-cell epitopes common to many influenza a strains. Also, one representative nucleoprotein from influenza b is expected to include a number of T-cell epitopes common to a number of influenza b strains. Thus, the inventors speculate that nucleoproteins from different influenza strains may be fused together in the absence of long linkers (greater than 10 amino acids in length) for use as vaccine antigens to elicit immune responses against different influenza viruses (e.g., different strains of influenza a and different strains of influenza b).
The amino acid sequences of the nucleoproteins of representative influenza a strains (H1N 1, H3N2, H5N8, H7N7, H7N9, H9N2 and H10N 8) were found to be similar to each other. The nucleoprotein of influenza strain H5N8 was chosen because it has minimal differences from the nucleoproteins of the other strains (H1N 1, H3N2, H7N7, H7N9, H9N2 and H10N 8). The nucleoprotein of influenza B strain (B/Florida/4/2006;GenBank CY033879.1) was selected as a representative influenza B nucleoprotein. A fusion protein comprising a human CD5 signal peptide, one FluA nucleoprotein and one FluB nucleoprotein (see G5010 in fig. 9) was designed and the coding region of the fusion protein was cloned downstream of the subgenomic promoter of srRNA1ts 2. mRNA is then produced by in vitro transcription. The amino acid sequence of FluA nucleoprotein is shown as SEQ ID NO. 13 (influenza A, subtype H5N8 [ A/b.sub.d./Korea/Gochang 1/2014], genBank number KJ413835.1, proteinID number AHL 21420.1), and the amino acid sequence of FluB nucleoprotein is shown as SEQ ID NO. 14 (influenza B [ B/Florida/4/2006], genBank number CY033879.1, proteinID number ACF 54251.1).
Mice were vaccinated with srRNA1ts2-G5010 by intradermal injection and antigen-specific cellular immune responses were measured by ELISPot assay. To recall nucleoprotein-reactive T-cell immunity, splenocytes harvested from mice 14 days post vaccination were restimulated using 122 overlapping peptide pools from peptide scans of the influenza a nucleoprotein sequence as shown in SEQ ID NO: 17. Despite the differences between the influenza a nucleoprotein sequence of G5010 and that of the peptide pool (fig. 10), the srRNA1ts2-G5010 vaccine induced a strong INF- γ secreting T cell response against the FluA nucleoprotein (fig. 11). Importantly, there was little induction of IL-4 secreting T cells against FluA nucleoprotein. These results indicate that the srRNA1ts2-G5010 vaccine induces a Th1 (INF-gamma) dominant response (Th 1> Th2 balance), which is an advantageous feature of vaccines against viral diseases.
In summary, when fusion proteins comprising nucleoproteins from representative influenza a and influenza b strains are expressed by intradermally injected temperature-controllable self-replicating RNAs, the fusion proteins induce a strong antigen-specific cellular immune response.
EXAMPLE 7 cellular immunity induced by srRNA1ts 2-ubiquitin (ubiquitin vaccine)
This example describes the following findings: when fusion proteins comprising nucleoprotein fragments from four ebola virus species (zaire-type ebola virus, sudan-type ebola virus, bundi Jiao Xingai ebola virus, tayi forest-type ebola virus) are used as vaccine antigens, the fusion proteins can induce strong cellular immunity against ebola virus. This example uses temperature-controlled self-replicating RNA as an expression vector.
Materials and methods
BALB/c female mice.
In vitro transcription of temperature controlled self-replicating RNA vectors (srRNA 1ts2[ PCT/US20/67506 ]) encoding the pan-type Ebola antigens produced srRNA1ts 2-pan-type Ebola mRNA (FIG. 12).
From the nuclear protein of Ebola virus by Ebola virus-Talin type (Swiss-Prot ID: B8XCN 6) [ JPT peptide; pepnix takie forest type ebola virus (NP); JPT product code: PM-TEBOV-NP ] was scanned for a pool of 182 peptides (15 mer with 11 amino acid overlap).
ELISPot assay plates and reagents for interferon gamma (INF-gamma) and interleukin-4 (IL-4) (Cellular Technology Limited, ohio).
Immunospot S6 Entry Analyzer (Cellular Technology Limited, ohio, U.S.A.).
Results
Ebola virus causes a highly fatal hemorrhagic fever. Four ebola virus species are known to cause disease in humans: ebola virus (zaire ebola virus), sudan virus (sudan ebola virus), bund Jiao Bingdu (bund Jiao Xingai ebola virus), and taeda forest virus (taeda forest type ebola virus, formerly known as coltadalaw ebola virus).
Currently, only one licensed vaccine (rVSV-ZEBOV) is available for ebola virus. This vaccine is an attenuated recombinant Vesicular Stomatitis Virus (VSV) that expresses a major Glycoprotein (GP) from zaire ebola virus. Although vaccines can induce neutralizing antibodies against ebola virus, the protein sequence of GP is highly different among four ebola virus species that infect humans. Thus, the rVSV-ZEBOV vaccine is effective against only zaire-type ebola virus. It is desirable to have a universal ebola virus vaccine that can provide protection against all four ebola virus species.
The Nucleoprotein (NP) sequence is more conserved among the four ebola virus species than GP. However, unlike GP, NP is not a surface protein, and thus antibodies raised against NP are not neutralizing antibodies. Importantly, it has been shown that mice vaccinated with zaire-type ebola virus NP can be protected from zaire-type ebola virus challenge, which is mediated by cellular rather than humoral immunity (Wilson and Hart, J Virol,75:2660-2664,2001). Protection was also shown to be mediated by MHC class I restricted cd8+ killer T cells (cytotoxic T lymphocytes) rather than MHC class II restricted cd4+ helper T cells (Wilson and Hart, supra, 2001).
The use of the fusion proteins of NPs of all four ebola virus species as vaccine antigens provides protection against all four ebola virus species. However, each NP is about 740 amino acids in length. Thus, fusing four intact NPs together will result in a relatively large protein of about 3,000 amino acids. Smaller sized antigens are desirable for many vaccine platforms.
The amino acid sequences of the nucleoprotein of the four ebola viruses were compared using NCBI BlastP (zaire ebola virus NP (GenBank ID: AF 272001), sudan ebola virus NP (GenBank ID: AF 173836), bunsen Jiao Xingai ebola virus NP (GenBank ID: FJ 217161), and tay forest ebola virus NP (GenBank ID: FJ 217162)). The sequences of the N-terminal half of NPs (designated region A) were found to be similar to each other (88% -92% identical), while the sequences of the C-terminal half of NPs (designated region B) were found to be diverse (42% -54%) (Table 7-1). Thus, zaire (a) was chosen as representative of zaire type (a), sudan type (a), bunsen type (a) and tazilin type (a). For region B, the present dibuca type (B) and the tazicar type (B) were found to be similar to each other (80% and 86% identity) except for the middle portion (40% identity) (referred to as region C). Thus, zaire (B), sudan (B), bendiburley (B) and taisen (C) were selected for inclusion in the pan-type ebola vaccine. Before the four nucleoproteins are assembled into a single fusion protein, an additional 8 amino acid sequences are added to both sides so that the possible T cell epitopes at the ends of the nucleoprotein fragment are not destroyed. A schematic representation of fusion proteins of the pan ebola antigen is shown in fig. 12 and comprises NP fragments of zaire type (a), zaire type (B), sudan type (B), bendio type (B) and taisen type (C), and a human CD5 signal peptide. A graph showing the percent identity of ebola virus NP sequences is shown in fig. 13. The amino acid sequence of the ubiquitin Ebola antigen is shown as SEQ ID NO. 22, and the nucleic acid sequence of the coding ubiquitin Ebola antigen is shown as SEQ ID NO. 23.
TABLE 7-1 percent identity between Ebola virus NP domain sequences
Virus (virus) Zaire type (A) Zaire type (B)
Sudan type pill 88％ 42％
Bendi cloth coke type 92％ 53％
Forest type tower 92％
The srRNA1ts 2-ubiquitin-type ebola vaccine was generated by cloning the ubiquitin fusion protein downstream of the subgenomic promoter of srRNA1ts 2. mRNA was produced by in vitro transcription and used for intradermal vaccination of BALB/c mice in vivo. Antigen-specific cellular immune responses were measured by ELISpot assay. To recall the nucleoprotein-reactive T cell immunity, splenocytes harvested from mice 14 days post vaccination were restimulated using a pool of 182 peptides scanned from the peptide of nucleoprotein (ebola virus-talin type ebola virus nucleoprotein (Swiss-Prot ID: B8XCN 6)). The srRNA1ts 2-ubiquitin ebola vaccine induced a strong INF-gamma secreting T cell response against Taoism nucleoprotein (FIG. 14A). Surprisingly, only a small fraction (169 aa) of the taverine-type nucleoprotein was contained in the mRNA vaccine, while the peptide pool for restimulation covered the entire taverine-type nucleoprotein sequence. Importantly, there was little induction of IL-4 secreting T cells against the taisen-type nucleoprotein (FIG. 14B). These results indicate that srRNA1ts 2-pan-ebola vaccine induces a Th1 (INF-gamma) dominant response (Th 1> Th2 balance), which is an advantageous feature of vaccines against viral diseases.
In summary, when fusion proteins comprising nucleoprotein fragments from four ebola virus species are expressed from intradermally injected temperature-controllable self-replicating RNAs, the fusion proteins induce a strong antigen-specific cellular immune response. The examples show that the size of fusion proteins used as a ubiquitin ebola vaccine can be reduced by removing more well-conserved portions of one or more of the nucleoproteins that make up the vaccine. The pan-ebola antigen is also suitable for use in other vaccine platforms (e.g., adenovirus, adeno-associated virus, recombinant proteins, etc.).
Example 8 cellular immunity induced by srRNA1ts2-G5003o (Omikovia vaccine)
This example describes the following findings: intradermal delivery of c-srRNA encoding RBD of SARS-CoV-2 (amikates strain B.1.1.529) can induce strong cellular immunity in mice.
Materials and methods
C57BL/6 female mice.
srRNA1ts2-G5003o (mRNA) was produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA 1ts2[ WO 2021/138447A1 ]) encoding the G5003o antigen (FIG. 15).
From RBD (S-RBD B.1.1.529) by the SARS-CoV-2 omnikon variety [ JPT Peptides: PM-SARS2-RBDMUT08-1] peptide was scanned to obtain a pool of peptides (15 mer with 11 amino acid overlap).
ELISPot assay plates and reagents for interferon gamma (INF-gamma) and interleukin-4 (IL-4) (Cellular Technology Limited, ohio).
Immunospot S6 Entry Analyzer (Cellular Technology Limited, ohio, U.S.A.).
Results
In this example, c-srRNA encoding the RBD of SARS-CoV-2 Oryctolagkis Rong Bianchong (G5003 o) was generated (FIG. 15). RNA was administered intradermally to C57BL/6 mice and spleen cells were collected after 14 days to examine cellular immunity against SARS-CoV-2RBD (Omik Rong Bianchong). Induction of INF- γ secreting T cells was clearly observed in the c-srRNA-G5003o receptor (fig. 16A), whereas induction of IL-4 secreting T cells was not observed in the c-srRNA-G5003o receptor (fig. 16B).
Conclusion(s)
By using the obreck Rong Bianchong specific RBD as an antigen, we have demonstrated that an obreck Rong Bianchong specific cellular immune response can be induced when proteins are expressed from intradermally injected temperature controlled self-replicating RNAs. A favorable Th1 (INF-. Gamma.) Th2 (IL-4) response was also observed.
EXAMPLE 9.c efficacy of srRNA priming protein boosting protocol
This example describes the following findings: administration of a c-srRNA vaccine encoding a protein antigen of a primary virus is capable of eliciting a humoral immune response against the protein antigen of a variant virus.
Materials and methods
BALB/c female mice.
srRNA1ts2-G5003 (mRNA) was produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA 1ts2[ WO 2021/138447A1 ]) encoding the G5003 antigen (FIG. 15).
Recombinant SARS-CoV-2B.1.617.2 spike GCN4-IZ protein (R & D Systems, catalog number 10878-CV).
Based on AddaVax TM An oil-in-water adjuvant of squalene was obtained from InvivoGen.
From RBD [ JPT Peptides ] by SARS-CoV-2 (original strain): pepmix SARS-CoV-2 (S-RBD) PM-WCPV-S-RBD-2] peptide pool of peptides (15 mer with 11 amino acid overlap)
ELISPot assay plates and reagents for interferon gamma (INF-gamma) and interleukin-4 (IL-4) (Cellular Technology Limited, ohio).
Immunospot S6 Entry Analyzer (Cellular Technology Limited, ohio, U.S.A.).
Vero76 cells for plaque reduction neutralization assay (PRNT).
SARS-CoV-2 delta variant live virus for PRNT assay
For the PRNT assay, vero76 cells were first treated with serial dilutions of mouse serum and then infected with the live virus of SARS-CoV-2 (delta variant strain). In this assay, infected cells die and form plaques after fixation and staining with crystal violet. If the serum contains neutralizing antibodies, the viral infection is inhibited, resulting in a reduced number of plaques. The results are shown as serum dilution titer (PRNT) showing a 50% reduction in plaque number 50 )。
Results
A composition comprising c-srRNA encoding the G5003 antigen (RBD of SARS-CoV-2 original strain) was administered intradermally as naked mRNA into the skin of BALB/c mice (FIG. 17A). That is, the srRNA1ts2-G5003 composition does not contain any nanoparticles or transfection reagents. Subsequently, a composition comprising spike protein of SARS-CoV-2 (delta variant B.1.617.2) in admixture with an adjuvant was administered intradermally (FIG. 17A).
Cellular immunity against the SARS-CoV-2RBD protein was detected in mouse spleen cells 14 days after a single intradermal injection of the c-srRNA-G5003 composition (FIG. 17B). As early as day 7 after protein antigen exposure, subsequent exposure of immunized mice to spike protein of a different strain of SARS-CoV-2 (delta variant b.1.617.2) induced neutralizing antibodies to delta variant of SARS-CoV-2 (detected by PRNT assay) (fig. 17C). In contrast, mice that did not receive the c-srRNA-G5003 encoding the RBD of SARS-CoV-2 (original strain) did not mount a neutralizing antibody response against the delta variant of SARS-CoV-2. Early induction of neutralizing antibodies is a characteristic of secondary immune responses, suggesting that c-srRNA elicits humoral immune responses prior to exposure to adjuvanted RBD proteins.
Conclusion(s)
The results indicate that the c-srRNA immunogen can induce an effective immune response that is broadly reactive to both the antigen encoded by the c-srRNA and to different variant antigens. Thus, the c-srRNA SARS-CoV-2RBD immunogen is suitable for use in immunization protocols against a broad spectrum of SARS-CoV-2 strains.
EXAMPLE 10 efficacy of the protein priming c-srRNA boost regimen
This example describes the following findings: the c-srRNA vaccine can enhance antibody titers when used as a booster vaccine for other vaccines.
Materials and methods
C57BL/6 female mice.
RBD protein (Sino Biological SARS-CoV-2[2019-nCoV ] spike RBD-His recombinant protein, catalog number 40592-V08B)
Based on AddaVax TM An oil-in-water adjuvant of squalene was obtained from InvivoGen.
srRNA1ts2-G5003 (mRNA) was produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA 1ts2[ WO 2021/138447A1 ]) encoding the G5003 antigen (FIG. 15).
srRNA1ts2-G5003o (mRNA) was produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA 1ts2[ WO 2021/138447A1 ]) encoding the G5003o antigen (FIG. 15).
From RBD [ JPT Peptides ] by SARS-CoV-2 (original strain): pepmix SARS-CoV-2 (S-RBD) PM-WCPV-S-RBD-2] peptide pool of peptides (15 mer with 11 amino acid overlap)
ELISPot assay plates and reagents for interferon gamma (INF-gamma) and interleukin-4 (IL-4) (Cellular Technology Limited, ohio).
Immunospot S6 Entry Analyzer (Cellular Technology Limited, ohio, U.S.A.).
ELISA assay plates (ENZO SARS-CoV-2IgG ELISA KIT [ catalog number ENZ-KIT 170-0001; plates were coated with SARS-CoV-2 (original strain) S1 antigen RBD protein ]).
Results
To test the possibility that the c-srRNA vaccine can be used as a booster vaccine, mice were first vaccinated with adjuvanted protein (in this case, RBD of SARS-CoV-2[ original strain "). Fourteen days later (day 14), mice were further treated with intradermal injections of placebo (PBO: buffer only), c-srRNA encoding the G5003 antigen, c-srRNA encoding the G5003o antigen, or adjuvanted RBD protein (FIG. 18A).
On day 28, cellular immunity was assessed by ELISpot assay. As expected, RBD (1 st) +pbo (2 nd) groups were unable to induce cellular immunity, whereas RBD (1 st) +rbd (2 nd) groups induced cellular immunity (fig. 18B, fig. 18C). Interestingly, RBD (1st) +c-srRNA-G5003 and C-srRNA-G5003o groups also induced cellular immunity (FIG. 18B, FIG. 18C). This is expected because the c-srRNA vaccine alone can induce cellular immunity.
On day 28, serum antibody levels against RBD of SARS-CoV-2 virus (original strain) were assessed by ELISA assay (fig. 19). The first vaccination with adjuvanted RBD protein alone may weakly induce antibodies. In another aspect, the c-srRNA vaccine is capable of inducing antibodies at a level similar to that of the second vaccination adjuvanted protein.
Conclusion(s)
The results indicate that the c-srRNA vaccine can function as a booster vaccine for both cellular and humoral immunity.
Example 11 potent cellular immune response induced by srRNA1ts2-G5006
This example describes the following findings: when a fusion protein comprising SARS-CoV-2 nucleoprotein and MERS-CoV nucleoprotein is expressed by intradermally injected temperature-controllable self-replicating RNA, the fusion protein can induce strong cellular immunity against SARS-CoV-2 and MERS-CoV. Vaccinated mice can eliminate implanted tumor cells that express fusion proteins comprising SARS-CoV-2 nucleoprotein and MERS-CoV nucleoprotein.
Materials and methods
BALB/c female mice.
srRNA1ts2-G5006 mRNA was produced by in vitro transcription of a temperature controlled self-replicating RNA vector encoding the G5006 antigen (srRNA 1ts2 as described in WO 2021/138447 A1) (FIG. 2).
From nucleoprotein (UniProt: P0DTC 9) [ JPT peptide product code) by SARS-CoV-2: PM-WCPV-NCAP ] peptides the resulting pool of peptides (15 mer with 11 amino acid overlap) was scanned.
Pools of peptides (15 MERS with 11 amino acid overlap) were scanned from peptides of nucleoprotein by MERS-CoV (YP 009047211.1). Peptides were tailored by JPT Peptides.
ELISPot assay plates and reagents for interferon gamma (INF-gamma) and interleukin-4 (IL-4) (Cellular Technology Limited, ohio).
Immunospot S6 Entry Analyzer (Cellular Technology Limited, ohio, U.S.A.).
A4T 1 breast cancer cell line (catalog number CRL-2539) derived from BALB/c mice and known as a model for triple negative stage IV human breast cancer was purchased from ATCC.
Plasmid DNA encoding the fusion protein of the nucleoprotein of SARS-CoV-2 and MERS-CoV under the CMV promoter (non-secreted form of G5006, i.e. without CD5 signal peptide) and the hygromycin resistance gene under the SV40 early promoter was transfected into 4T1 cells. Cells expressing the fusion protein of SARS-CoV-2 and MERS-CoV (referred to as 4T 1-SMN) were isolated by culturing the cells in the presence of 200 μg/mL hygromycin B.
Results
To model virus-infected cells, we used a 4T1 breast cancer cell line derived from BALB/c mice and known as a model for triple negative stage IV human breast cancer. When injected into BALB/c mice, 4T1 cells grew rapidly and formed tumors. This syngeneic mouse model was used to model the rapid increase in infected cells. To this end, we first prepared a plasmid vector encoding a fusion protein of the nucleoprotein of SARS-CoV-2 and MERS-CoV under the CMV promoter (designated SMN protein), thus allowing constitutive expression of the protein. This fusion protein is identical to G5006, but the CD5 signal peptide is removed from the N-terminus of the protein. Naturally, nucleoproteins do not have a signal peptide and reside within the cytoplasm of the cell. 4T1 cells expressing SMN protein (designated 4T 1-SMN) were established after hygromycin selection, as the plasmid vector also carries the hygromycin resistance gene.
BALB/c mice were vaccinated with c-srRNA-G5006 and induction of cellular immunity was demonstrated by the presence of T cells that responded to both SARS-CoV-2 nucleoprotein (FIG. 20A) and MERS-CoV nucleoprotein (FIG. 20B).
On day 24 (24 days after vaccination), 4T1-SMN cells were injected into BALB/c mice vaccinated with c-srRNA-G5006 (FIG. 21). As expected, 4T1-SMN cells grew rapidly in mice receiving placebo (no vaccinated group) 4T1-SMN cells. In another aspect, the growth of the 4T1-SMN tumor is inhibited in c-srRNA-G5006 vaccinated mice. Two mice receiving the 25 μ G c-srRNA-G5006 vaccine became tumor-free and survived, although the tumor initially grew. Furthermore, even after the second round of injection of 4T1-SMN tumor at day 143 post-vaccination, no tumor grew and mice were tumor-free and continued to survive (fig. 21).
Conclusion(s)
The c-srRNA vaccine can induce stronger cellular immunity, and the cellular immunity can kill and eliminate cells expressing the antigen. This result suggests that c-srRNA acts as a vaccine by eliminating infected cells.
EXAMPLE 12 cellular immunity induced by srRNA1ts 2-ubiquitin coronavirus vaccine
This example describes the following findings: when a fusion protein comprising the CD5 signal peptide, the spike-RBD of SARS-CoV-2, the nucleoprotein of MERS-CoV and the spike-RBD of MERS-CoV is expressed by intradermally injected temperature-controllable self-replicating RNA, the fusion protein can induce strong cellular immunity against all of these antigens.
Materials and methods
C57BL/6 female mice.
srRNA1ts2-G5006 mRNA was produced by in vitro transcription of a temperature controlled self-replicating RNA vector encoding the G5006 antigen (srRNA 1ts2 as described in WO 2021/138447 A1) (FIG. 2).
srRNA1ts2-G5006d mRNA was produced by in vitro transcription of a temperature controlled self-replicating RNA vector encoding G5006d antigen (srRNA 1ts2 as described in WO 2021/138447 A1) (FIG. 22).
From RBD [ JPT Peptides ] by SARS-CoV-2 (original strain): pepmix SARS-CoV-2 (S-RBD) PM-WCPV-S-RBD-2] peptide pool of peptides (15 mer with 11 amino acid overlap)
From nucleoprotein (UniProt: P0DTC 9) [ JPT peptide product code) by SARS-CoV-2: PM-WCPV-NCAP ] peptides the resulting pool of peptides (15 mer with 11 amino acid overlap) was scanned.
Pools of peptides (15 MERS with 11 amino acid overlap) were scanned from peptides of nucleoprotein by MERS-CoV (YP 009047211.1). Peptides were tailored by JPT Peptides.
From the spike glycoprotein (Swiss-Prot ID: K9N5Q 8) of MERS-CoV (middle east respiratory syndrome associated coronavirus) [ JPT peptide product code: PM-MERS-CoV-S-1] peptide scan resulted in a pool of 336 (168+168) peptides (15 mer with 11 amino acid overlap).
ELISPot assay plates and reagents for interferon gamma (INF-gamma) and interleukin-4 (IL-4) (Cellular Technology Limited, ohio).
Immunospot S6 Entry Analyzer (Cellular Technology Limited, ohio, U.S.A.).
Results
Here we designed a new booster vaccine, a c-srRNA vaccine (called c-srRNA-G5006 d) encoding a fusion protein comprising the CD5 signal peptide, the spike-RBD of SARS-CoV-2, the nucleoprotein of MERS-CoV, and the spike-RBD of MERS-CoV (FIG. 22).
Mice were vaccinated intradermally with placebo (PBO: buffer only), c-srRNA encoding the G5006 antigen, and c-srRNA encoding the G5006d antigen. On day 14 post-vaccination, cellular immunity was assessed by ELISpot assay.
As shown in fig. 23A, c-srRNA-G5006d can stimulate cellular immunity against all of the following proteins encoded on this vaccine: spike-RBD of SARS-CoV-2, nucleoprotein of MERS-CoV and spike-RBD of MERS-CoV.
Conclusion(s)
The results indicate that the c-srRNA vaccine can function as a booster vaccine for both cellular and humoral immunity.
EXAMPLE 13 srRNA1 ts2-ubiquitin-type influenza Virus vaccine
This example describes the design of a pan-type influenza booster vaccine based on the unique features of the c-srRNA vaccine platform. The antigen encoded on c-srRNA (G5012) is a fusion protein of the CD5 signal peptide (residues 1-24), a portion of the hemagglutinin of influenza A (HA), the nucleoprotein of influenza A, the nucleoprotein of influenza B, and a portion of the hemagglutinin of influenza B (HA).
Materials and methods
C57BL/6 female mice.
The c-srRNA-G5012 mRNA was produced by in vitro transcription of a temperature controllable self-replicating RNA vector encoding the G5012 antigen (srRNA 1ts2 as described in WO 2021/138447 A1) (FIG. 24).
A pool of peptides (15 mers with 11 amino acid overlaps) obtained from peptide scans through a portion of Hemagglutinin (HA) of influenza a, nucleoprotein of influenza b, and a portion of Hemagglutinin (HA) of influenza b.
ELISPot assay plates and reagents for interferon gamma (INF-gamma) and interleukin-4 (IL-4) (Cellular Technology Limited, ohio).
Immunospot S6 Entry Analyzer (Cellular Technology Limited, ohio, U.S.A.).
Results
Mice were vaccinated with intradermal injections of placebo (PBO: buffer only) and c-srRNA encoding the G5012 antigen. On day 14 post-vaccination, cellular immunity was assessed by ELISpot assay.
c-srRNA-G5012 stimulated cellular immunity against all of the following antigens encoded on this vaccine: hemagglutinin of influenza a (HA), nucleoprotein of influenza a, nucleoprotein of influenza b, and hemagglutinin of influenza b (HA).
Conclusion(s)
The results indicate that the c-srRNA vaccine can function as a booster vaccine for both cellular and humoral immunity.
EXAMPLE 14 Chitosan enhances luciferase expression by srRNA1ts2-LUC2
This example describes the following findings: the chitosan oligomer is capable of enhancing in vivo expression of a gene of interest (GOI) encoded by the c-srRNA construct.
Materials and methods
C57BL/6 female mice.
srRNA1ts2-LUC2 (mRNA) is produced by in vitro transcription of a temperature-controllable self-replicating RNA vector encoding a luciferase gene(s) (e.g., srRNA1ts2 as described in WO 2021/138447 A1).
Chitosan oligomer (molecular weight: 5kDa, deacetylation: heppe Medical Chitosan GmbH: product number 44009).
Chitosan oligosaccharide lactic acid (molecular weight about 5kDa, >90% deacetylated: sigma-Aldrich: product number 523682).
Bioluminescence imaging systems, AMI HTX (Spectral Instruments Imaging, atlasen, arizona).
Results
To test whether chitosan oligomer could enhance expression of the GOI encoded on C-srRNA in vivo, 5. Mu.g of C-srRNA (also referred to as srRNA1ts 2) encoding a luciferase gene as GOI was mixed with chitosan and intradermally administered to each C57BL/6 mouse (FIG. 25). In lactate ringer's solution, c-srRNA is formulated as naked RNA in the absence of lipid nanoparticles or any other transfection reagent. Luciferase activity was visualized and quantified by using the bioluminescence imaging system AMI HTX (Spectral Instruments Imaging, atlasen, arizona).
Five mice each were tested in the following groups: 1, control-c-srRNA only; 2, c-srRNA mixed with chitosan oligosaccharide (0.001. Mu.g/mL); 3, c-srRNA mixed with chitosan oligosaccharide (0.01. Mu.g/mL); 4, c-srRNA mixed with chitosan oligosaccharide (0.5. Mu.g/mL); 5, c-srRNA mixed with chitosan oligosaccharide lactic acid (0.1. Mu.g/mL).
As shown in FIG. 27, all conditions with chitosan oligomers at concentrations of 0.001. Mu.g/mL, 0.01. Mu.g/mL and 0.5. Mu.g/mL and conditions with chitosan oligosaccharide lactic acid at a concentration of 0.1. Mu.g/mL showed about 10-fold higher levels of luciferase activity compared to the control condition (i.e., c-srRNA: no chitosan).
Conclusion(s)
When mixed with c-srRNA prior to intradermal injection into mouse skin, low molecular weight chitosans (e.g., chitosan oligomers and chitosan oligosaccharide lactic acid) can enhance expression of GOI encoded on the c-srRNA. Chitosan oligomers provide about 10-fold enhancement of gene expression even at very low concentrations (0.001. Mu.g/mL or about 0.2 nM). This surprising finding provides an effective means of enhancing the in vivo therapeutic expression of GOI encoded on c-srRNA.
Sequence(s)
SEQ ID NO:1
>G5004(SARS-COV-2_N，mRNA)
SEQ ID NO:2
G5005 (CD 5-SP_SARS-COV-2_N, mRNA Synthesis)
SEQ ID NO:3
G5006A (CD 5-SP_SARS2-N_MERS-N, mRNA synthesized)
SEQ ID NO:4
G5006B (CD 5-SP_SARS2-N_MERS-N: CODON-OPTIMIZED, synthesis of mRNA)
SEQ ID NO:5
G5004 (SARS-COV-2_N, protein)
SEQ ID NO:6
G5005 (CD 5-SP_SARS-CoV-2_N, synthetic protein)
SEQ ID NO:7
G5006a (CD 5-SP_SARS2-N_MERS-N, synthetic protein)
SEQ ID NO:8
Human CD5 Signal peptide (Homo sapiens, protein)
MPMGSLQPLATLYLLGMLVASCLG
SEQ ID NO:9
MERS-N protein
SEQ ID NO:10
SARS-COV-1-N protein
SEQ ID NO:11
Cloning site (synthetic DNA)
GGCGCGCCattggccaccGCGGCCGC
The gene of interest (e.g., the beta-coronavirus nucleoprotein open reading frame) is inserted between nucleotides 18 and 19 of SEQ ID NO: 11. GGCGCGCC is an AscI restriction site and GCGGCCGC is a NotI restriction site.
SEQ ID NO:12
Artificial work
Attggccacc (synthetic DNA)
This nucleotide sequence was added to the 5' end of the β coronavirus nucleoprotein encoding region to provide a Kozak consensus sequence for initiating mRNA translation.
SEQ ID NO:13
Influenza A virus (H5N 8) nucleocapsid protein
>
SEQ ID NO:14
Influenza b virus nucleocapsid protein
SEQ ID NO:15
G5010 (synthetic mRNA) [ codon optimized sequence for pan-influenza antigen ]
SEQ ID NO:16
G5010 (synthetic protein) [ pandemic influenza antigen ]
SEQ ID NO:17
Nucleoprotein of influenza a virus (H2N 2) (Swiss-Protein ID P21433) [ JPT peptide product code: PM-INFA-NPH2N2]
ASQGTKRSYEQMETDGERQNANEIRASVGKMIGGIGRFYIQMCTELKLSDYEGRLIQNSLTIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYKRVDGKWMRELVLYDKEEIRRIWRQANNGDDATAGLTHMMIWHSNLNDTTYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELIRMIKRGINDRNFWRGENGRKTRNAYERMCNILKGKFQTAAQRAMMDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACVYGPAVASGYDFEKEGYSLVGIDPFKLLQNSQVYSLIRPNENPAHKSQLVWMACNSAAFEDLRVSSFIRGTKVIPRGKLSTRGVQIASNENMDTMGSSTLELRSRYWAIRTRSGGNTNQQRASAGQISVQPTFSVQRNLPFDKPTIMAAFTGNAEGRTSDMRAEIIRMMEGAKPEEVSFQGRGVFELSDEKATNPIVPSFDMSNEGSYFFGDNAEEYDN
SEQ ID NO:18
Nucleoprotein fragments of zaire ebola virus
SEQ ID NO:19
Nuclear protein fragment of Sudan type ebola virus
SEQ ID NO:20
Nuclear protein fragment of Bendi cloth Jiao Xingai Bola virus
SEQ ID NO:21
Nuclear protein fragment of Tayi forest type ebola virus
SEQ ID NO:22
General Ebola antigen (synthetic protein)
SEQ ID NO:23
General Ebola antigen (synthetic mRNA)
SEQ ID NO:24
G5003o (synthetic mRNA) [ CD5sp+RBD2 (Omikovin) ]
SEQ ID NO:25
G5003o (synthetic protein) [ CD5sp+RBD2 (Omikovin) ]
SEQ ID NO:26
G5006d (Synthesis of mRNA)
SEQ ID NO:27
G5006d (synthetic protein)
SEQ ID NO:28
G50012 (Synthesis of mRNA)
SEQ ID NO:29
G50012 (synthetic protein)
Sequence listing
<110> illix root treatment Co
<120> temperature-controllable self-replicating RNA for viral diseases
Vaccine
<130> 69944-20014.40
<140> not yet allocated
<141> along with the submission
<150> US 63/275,398
<151> 2021-11-03
<150> US 63/240,278
<151> 2021-09-02
<150> US 63/211,974
<151> 2021-06-17
<160> 29
<170> FastSEQ version 4.0 for Windows
<210> 1
<211> 1260
<212> DNA
<213> SARS-COV-2
<400> 1
atgtctgata atggacccca aaatcagcga aatgcacccc gcattacgtt tggtggaccc 60
tcagattcaa ctggcagtaa ccagaatgga gaacgcagtg gggcgcgatc aaaacaacgt 120
cggccccaag gtttacccaa taatactgcg tcttggttca ccgctctcac tcaacatggc 180
aaggaagacc ttaaattccc tcgaggacaa ggcgttccaa ttaacaccaa tagcagtcca 240
gatgaccaaa ttggctacta ccgaagagct accagacgaa ttcgtggtgg tgacggtaaa 300
atgaaagatc tcagtccaag atggtatttc tactacctag gaactgggcc agaagctgga 360
cttccctatg gtgctaacaa agacggcatc atatgggttg caactgaggg agccttgaat 420
acaccaaaag atcacattgg cacccgcaat cctgctaaca atgctgcaat cgtgctacaa 480
cttcctcaag gaacaacatt gccaaaaggc ttctacgcag aagggagcag aggcggcagt 540
caagcctctt ctcgttcctc atcacgtagt cgcaacagtt caagaaattc aactccaggc 600
agcagtaggg gaacttctcc tgctagaatg gctggcaatg gcggtgatgc tgctcttgct 660
ttgctgctgc ttgacagatt gaaccagctt gagagcaaaa tgtctggtaa aggccaacaa 720
caacaaggcc aaactgtcac taagaaatct gctgctgagg cttctaagaa gcctcggcaa 780
aaacgtactg ccactaaagc atacaatgta acacaagctt tcggcagacg tggtccagaa 840
caaacccaag gaaattttgg ggaccaggaa ctaatcagac aaggaactga ttacaaacat 900
tggccgcaaa ttgcacaatt tgcccccagc gcttcagcgt tcttcggaat gtcgcgcatt 960
ggcatggaag tcacaccttc gggaacgtgg ttgacctaca caggtgccat caaattggat 1020
gacaaagatc caaatttcaa agatcaagtc attttgctga ataagcatat tgacgcatac 1080
aaaacattcc caccaacaga gcctaaaaag gacaaaaaga agaaggctga tgaaactcaa 1140
gccttaccgc agagacagaa gaaacagcaa actgtgactc ttcttcctgc tgcagatttg 1200
gatgatttct ccaaacaatt gcaacaatcc atgagcagtg ctgactcaac tcaggcctaa 1260
<210> 2
<211> 1329
<212> DNA
<213> artificial sequence
<220>
<223> synthetic construct
<400> 2
atgcccatgg ggtctctgca accgctggcc accttgtacc tgctggggat gctggtcgct 60
tcctgcctcg gatctgataa tggaccccaa aatcagcgaa atgcaccccg cattacgttt 120
ggtggaccct cagattcaac tggcagtaac cagaatggag aacgcagtgg ggcgcgatca 180
aaacaacgtc ggccccaagg tttacccaat aatactgcgt cttggttcac cgctctcact 240
caacatggca aggaagacct taaattccct cgaggacaag gcgttccaat taacaccaat 300
agcagtccag atgaccaaat tggctactac cgaagagcta ccagacgaat tcgtggtggt 360
gacggtaaaa tgaaagatct cagtccaaga tggtatttct actacctagg aactgggcca 420
gaagctggac ttccctatgg tgctaacaaa gacggcatca tatgggttgc aactgaggga 480
gccttgaata caccaaaaga tcacattggc acccgcaatc ctgctaacaa tgctgcaatc 540
gtgctacaac ttcctcaagg aacaacattg ccaaaaggct tctacgcaga agggagcaga 600
ggcggcagtc aagcctcttc tcgttcctca tcacgtagtc gcaacagttc aagaaattca 660
actccaggca gcagtagggg aacttctcct gctagaatgg ctggcaatgg cggtgatgct 720
gctcttgctt tgctgctgct tgacagattg aaccagcttg agagcaaaat gtctggtaaa 780
ggccaacaac aacaaggcca aactgtcact aagaaatctg ctgctgaggc ttctaagaag 840
cctcggcaaa aacgtactgc cactaaagca tacaatgtaa cacaagcttt cggcagacgt 900
ggtccagaac aaacccaagg aaattttggg gaccaggaac taatcagaca aggaactgat 960
tacaaacatt ggccgcaaat tgcacaattt gcccccagcg cttcagcgtt cttcggaatg 1020
tcgcgcattg gcatggaagt cacaccttcg ggaacgtggt tgacctacac aggtgccatc 1080
aaattggatg acaaagatcc aaatttcaaa gatcaagtca ttttgctgaa taagcatatt 1140
gacgcataca aaacattccc accaacagag cctaaaaagg acaaaaagaa gaaggctgat 1200
gaaactcaag ccttaccgca gagacagaag aaacagcaaa ctgtgactct tcttcctgct 1260
gcagatttgg atgatttctc caaacaattg caacaatcca tgagcagtgc tgactcaact 1320
caggcctaa 1329
<210> 3
<211> 2568
<212> DNA
<213> artificial sequence
<220>
<223> synthetic construct
<400> 3
atgcccatgg ggtctctgca accgctggcc accttgtacc tgctggggat gctggtcgct 60
tcctgcctcg gatctgataa tggaccccaa aatcagcgaa atgcaccccg cattacgttt 120
ggtggaccct cagattcaac tggcagtaac cagaatggag aacgcagtgg ggcgcgatca 180
aaacaacgtc ggccccaagg tttacccaat aatactgcgt cttggttcac cgctctcact 240
caacatggca aggaagacct taaattccct cgaggacaag gcgttccaat taacaccaat 300
agcagtccag atgaccaaat tggctactac cgaagagcta ccagacgaat tcgtggtggt 360
gacggtaaaa tgaaagatct cagtccaaga tggtatttct actacctagg aactgggcca 420
gaagctggac ttccctatgg tgctaacaaa gacggcatca tatgggttgc aactgaggga 480
gccttgaata caccaaaaga tcacattggc acccgcaatc ctgctaacaa tgctgcaatc 540
gtgctacaac ttcctcaagg aacaacattg ccaaaaggct tctacgcaga agggagcaga 600
ggcggcagtc aagcctcttc tcgttcctca tcacgtagtc gcaacagttc aagaaattca 660
actccaggca gcagtagggg aacttctcct gctagaatgg ctggcaatgg cggtgatgct 720
gctcttgctt tgctgctgct tgacagattg aaccagcttg agagcaaaat gtctggtaaa 780
ggccaacaac aacaaggcca aactgtcact aagaaatctg ctgctgaggc ttctaagaag 840
cctcggcaaa aacgtactgc cactaaagca tacaatgtaa cacaagcttt cggcagacgt 900
ggtccagaac aaacccaagg aaattttggg gaccaggaac taatcagaca aggaactgat 960
tacaaacatt ggccgcaaat tgcacaattt gcccccagcg cttcagcgtt cttcggaatg 1020
tcgcgcattg gcatggaagt cacaccttcg ggaacgtggt tgacctacac aggtgccatc 1080
aaattggatg acaaagatcc aaatttcaaa gatcaagtca ttttgctgaa taagcatatt 1140
gacgcataca aaacattccc accaacagag cctaaaaagg acaaaaagaa gaaggctgat 1200
gaaactcaag ccttaccgca gagacagaag aaacagcaaa ctgtgactct tcttcctgct 1260
gcagatttgg atgatttctc caaacaattg caacaatcca tgagcagtgc tgactcaact 1320
caggccatgg catcccctgc tgcacctcgt gctgtttcct ttgccgataa caatgatata 1380
acaaatacaa acctatctcg aggtagagga cgtaatccaa aaccacgagc tgcaccaaat 1440
aacactgtct cttggtacac tgggcttacc caacacggga aagtccctct tacctttcca 1500
cctgggcagg gtgtacctct taatgccaat tctacccctg cgcaaaatgc tgggtattgg 1560
cggagacagg acagaaaaat taataccggg aatggaatta agcaactggc tcccaggtgg 1620
tacttctact acactggaac tggacccgaa gcagcactcc cattccgggc tgttaaggat 1680
ggcatcgttt gggtccatga agatggcgcc actgatgctc cttcaacttt tgggacgcgg 1740
aaccctaaca atgattcagc tattgttaca caattcgcgc ccggtactaa gcttcctaaa 1800
aacttccaca ttgaggggac tggaggcaat agtcaatcat cttcaagagc ctctagctta 1860
agcagaaact cttccagatc tagttcacaa ggttcaagat caggaaactc tacccgcggc 1920
acttctccag gtccatctgg aatcggagca gtaggaggtg atctacttta ccttgatctt 1980
ctgaacagac tacaagccct tgagtctggc aaagtaaagc aatcgcagcc aaaagtaatc 2040
actaagaaag atgctgctgc tgctaaaaat aagatgcgcc acaagcgcac ttccaccaaa 2100
agtttcaaca tggtgcaagc ttttggtctt cgcggaccag gagacctcca gggaaacttt 2160
ggtgatcttc aattgaataa actcggcact gaggacccac gttggcccca aattgctgag 2220
cttgctccta cagccagtgc ttttatgggt atgtcgcaat ttaaacttac ccatcagaac 2280
aatgatgatc atggcaaccc tgtgtacttc cttcggtaca gtggagccat taaacttgac 2340
ccaaagaatc ccaactacaa taagtggttg gagcttcttg agcaaaatat tgatgcctac 2400
aaaaccttcc ctaagaagga aaagaaacaa aaggcaccaa aagaagaatc aacagaccaa 2460
atgtctgaac ctccaaagga gcagcgtgtg caaggtagca tcactcagcg cactcgcacc 2520
cgtccaagtg ttcagcctgg tccaatgatt gatgttaaca ctgattag 2568
<210> 4
<211> 2568
<212> DNA
<213> artificial sequence
<220>
<223> synthetic construct
<400> 4
atgcctatgg gctctctgca gcccctggcc accctgtacc tgctgggcat gctggtggcc 60
agctgcctgg gaagcgacaa cggcccccag aaccagagaa acgcccctag aatcacattt 120
ggcggcccta gtgatagcac cggatctaat caaaacggcg agagaagcgg cgctcggtct 180
aaacagagac ggccacaggg actgcctaac aacaccgcca gctggttcac cgccctgacc 240
cagcacggca aggaggacct taagttccct cggggacagg gcgtgccaat caacaccaac 300
tctagtcccg acgaccagat cggctattat agaagagcca caagacgcat cagaggtggc 360
gacggcaaga tgaaggacct gagccctcgc tggtactttt actacctggg gaccggccct 420
gaagccggcc tgccttacgg cgccaacaag gacggaatca tctgggtcgc caccgagggc 480
gccctgaata cccctaagga ccacatcggc accagaaacc ctgctaataa tgccgctatc 540
gtgctgcagc tgcctcaggg caccaccctg cctaagggct tctacgccga gggctcccgg 600
ggaggttccc aggctagcag cagatcttcc agccggagca gaaacagctc caggaacagc 660
acacctggca gcagcagagg tacgagccct gcccggatgg ccggaaacgg cggcgatgcc 720
gccctggccc tgctgctgct ggacagactg aaccagctcg agagcaagat gtctggcaag 780
ggccagcagc agcagggcca gacagtgacc aagaaatccg ccgctgaggc cagcaaaaaa 840
cccagacaga aaagaaccgc tacaaaggcc tacaacgtta cccaggcctt tggcagacgg 900
ggcccagagc agacccaggg aaacttcggc gaccaggagc tgatccggca gggcacggac 960
tacaagcact ggcctcaaat cgcccagttt gccccttccg ccagcgcttt cttcggaatg 1020
agcagaatcg gcatggaagt gacacctagt ggcacctggc tgacctacac cggtgccatt 1080
aagctggatg acaaggaccc caacttcaag gatcaggtga tcctgctgaa caagcacatt 1140
gatgcttaca agaccttccc acctaccgag ccaaaaaaag ataagaagaa aaaagccgat 1200
gagacacaag ccctgcccca gaggcagaag aagcaacaaa ccgtcaccct gctgcctgct 1260
gccgacctgg acgacttcag caaacagctg cagcagagca tgagctctgc tgatagcacc 1320
caggccatgg cctctccagc cgctcccaga gctgtgtcct tcgccgataa taacgacatc 1380
acaaacacca acctgagccg gggcagaggc agaaacccta aacctagagc cgcccccaac 1440
aacaccgtga gctggtatac aggcctcacc cagcatggca aggtgcctct gacattcccc 1500
cctgggcagg gcgtgcccct gaacgccaac agcacccctg cccagaatgc cggctactgg 1560
cggaggcagg acagaaagat caacactggt aacggcatca agcagctggc cccacggtgg 1620
tatttctact acaccggcac cggcccggaa gccgccctcc ccttcagagc cgtgaaggac 1680
ggcatcgtgt gggtgcacga ggacggcgcc acagatgccc cgtctacatt tggcactcgg 1740
aatcccaata acgacagcgc catcgtgacc cagttcgccc ctggcaccaa gctgcctaag 1800
aactttcaca tcgagggcac aggaggcaac agccagagca gcagccgggc ttcgagcctg 1860
tctcggaata gctcccggtc cagctctcag ggcagccgca gtggaaattc cacccggggc 1920
acatctcctg gccccagcgg catcggcgct gtgggcggag acctgctcta cctggacctg 1980
ctgaacagac tgcaggcact tgaaagcggc aaagttaagc aatctcaacc taaggtgatc 2040
accaaaaagg acgccgccgc cgctaagaac aagatgagac acaagagaac aagcacaaag 2100
agcttcaaca tggtgcaagc cttcggcctg cggggacctg gcgacctgca gggcaacttc 2160
ggcgacctgc agctgaacaa gctgggcaca gaggatcctc gatggcccca gatcgccgaa 2220
ctagctccaa ccgccagcgc cttcatgggc atgagccagt tcaagctgac acaccagaac 2280
aatgacgatc acggaaatcc tgtgtacttc ctgagataca gcggcgccat caagctggat 2340
cctaagaacc ccaactacaa caagtggctg gaactgctgg aacagaacat cgacgcctac 2400
aagaccttcc ccaagaagga aaagaagcag aaggccccta aagaggaaag cacagatcag 2460
atgagcgagc ctcccaagga acagagagtg cagggatcta tcacccagag aacaagaaca 2520
agacccagcg tgcagcctgg ccctatgatt gacgtgaaca ccgactag 2568
<210> 5
<211> 419
<212> PRT
<213> SARS-COV-2
<400> 5
Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr
1 5 10 15
Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg
20 25 30
Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn
35 40 45
Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu
50 55 60
Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro
65 70 75 80
Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly
85 90 95
Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr
100 105 110
Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp
115 120 125
Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp
130 135 140
His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln
145 150 155 160
Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser
165 170 175
Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn
180 185 190
Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala
195 200 205
Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu
210 215 220
Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln
225 230 235 240
Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys
245 250 255
Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln
260 265 270
Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp
275 280 285
Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile
290 295 300
Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile
305 310 315 320
Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala
325 330 335
Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu
340 345 350
Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro
355 360 365
Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln
370 375 380
Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu
385 390 395 400
Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser
405 410 415
Thr Gln Ala
<210> 6
<211> 442
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 6
Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly
1 5 10 15
Met Leu Val Ala Ser Cys Leu Gly Ser Asp Asn Gly Pro Gln Asn Gln
20 25 30
Arg Asn Ala Pro Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly
35 40 45
Ser Asn Gln Asn Gly Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg
50 55 60
Pro Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr
65 70 75 80
Gln His Gly Lys Glu Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro
85 90 95
Ile Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg
100 105 110
Ala Thr Arg Arg Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser
115 120 125
Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu
130 135 140
Pro Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly
145 150 155 160
Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn
165 170 175
Asn Ala Ala Ile Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys
180 185 190
Gly Phe Tyr Ala Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg
195 200 205
Ser Ser Ser Arg Ser Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser
210 215 220
Ser Arg Gly Thr Ser Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala
225 230 235 240
Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys
245 250 255
Met Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys
260 265 270
Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr
275 280 285
Lys Ala Tyr Asn Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln
290 295 300
Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp
305 310 315 320
Tyr Lys His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala
325 330 335
Phe Phe Gly Met Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr
340 345 350
Trp Leu Thr Tyr Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn
355 360 365
Phe Lys Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys
370 375 380
Thr Phe Pro Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp
385 390 395 400
Glu Thr Gln Ala Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr
405 410 415
Leu Leu Pro Ala Ala Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln
420 425 430
Ser Met Ser Ser Ala Asp Ser Thr Gln Ala
435 440
<210> 7
<211> 855
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 7
Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly
1 5 10 15
Met Leu Val Ala Ser Cys Leu Gly Ser Asp Asn Gly Pro Gln Asn Gln
20 25 30
Arg Asn Ala Pro Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly
35 40 45
Ser Asn Gln Asn Gly Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg
50 55 60
Pro Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr
65 70 75 80
Gln His Gly Lys Glu Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro
85 90 95
Ile Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg
100 105 110
Ala Thr Arg Arg Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser
115 120 125
Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu
130 135 140
Pro Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly
145 150 155 160
Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn
165 170 175
Asn Ala Ala Ile Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys
180 185 190
Gly Phe Tyr Ala Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg
195 200 205
Ser Ser Ser Arg Ser Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser
210 215 220
Ser Arg Gly Thr Ser Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala
225 230 235 240
Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys
245 250 255
Met Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys
260 265 270
Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr
275 280 285
Lys Ala Tyr Asn Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln
290 295 300
Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp
305 310 315 320
Tyr Lys His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala
325 330 335
Phe Phe Gly Met Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr
340 345 350
Trp Leu Thr Tyr Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn
355 360 365
Phe Lys Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys
370 375 380
Thr Phe Pro Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp
385 390 395 400
Glu Thr Gln Ala Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr
405 410 415
Leu Leu Pro Ala Ala Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln
420 425 430
Ser Met Ser Ser Ala Asp Ser Thr Gln Ala Met Ala Ser Pro Ala Ala
435 440 445
Pro Arg Ala Val Ser Phe Ala Asp Asn Asn Asp Ile Thr Asn Thr Asn
450 455 460
Leu Ser Arg Gly Arg Gly Arg Asn Pro Lys Pro Arg Ala Ala Pro Asn
465 470 475 480
Asn Thr Val Ser Trp Tyr Thr Gly Leu Thr Gln His Gly Lys Val Pro
485 490 495
Leu Thr Phe Pro Pro Gly Gln Gly Val Pro Leu Asn Ala Asn Ser Thr
500 505 510
Pro Ala Gln Asn Ala Gly Tyr Trp Arg Arg Gln Asp Arg Lys Ile Asn
515 520 525
Thr Gly Asn Gly Ile Lys Gln Leu Ala Pro Arg Trp Tyr Phe Tyr Tyr
530 535 540
Thr Gly Thr Gly Pro Glu Ala Ala Leu Pro Phe Arg Ala Val Lys Asp
545 550 555 560
Gly Ile Val Trp Val His Glu Asp Gly Ala Thr Asp Ala Pro Ser Thr
565 570 575
Phe Gly Thr Arg Asn Pro Asn Asn Asp Ser Ala Ile Val Thr Gln Phe
580 585 590
Ala Pro Gly Thr Lys Leu Pro Lys Asn Phe His Ile Glu Gly Thr Gly
595 600 605
Gly Asn Ser Gln Ser Ser Ser Arg Ala Ser Ser Leu Ser Arg Asn Ser
610 615 620
Ser Arg Ser Ser Ser Gln Gly Ser Arg Ser Gly Asn Ser Thr Arg Gly
625 630 635 640
Thr Ser Pro Gly Pro Ser Gly Ile Gly Ala Val Gly Gly Asp Leu Leu
645 650 655
Tyr Leu Asp Leu Leu Asn Arg Leu Gln Ala Leu Glu Ser Gly Lys Val
660 665 670
Lys Gln Ser Gln Pro Lys Val Ile Thr Lys Lys Asp Ala Ala Ala Ala
675 680 685
Lys Asn Lys Met Arg His Lys Arg Thr Ser Thr Lys Ser Phe Asn Met
690 695 700
Val Gln Ala Phe Gly Leu Arg Gly Pro Gly Asp Leu Gln Gly Asn Phe
705 710 715 720
Gly Asp Leu Gln Leu Asn Lys Leu Gly Thr Glu Asp Pro Arg Trp Pro
725 730 735
Gln Ile Ala Glu Leu Ala Pro Thr Ala Ser Ala Phe Met Gly Met Ser
740 745 750
Gln Phe Lys Leu Thr His Gln Asn Asn Asp Asp His Gly Asn Pro Val
755 760 765
Tyr Phe Leu Arg Tyr Ser Gly Ala Ile Lys Leu Asp Pro Lys Asn Pro
770 775 780
Asn Tyr Asn Lys Trp Leu Glu Leu Leu Glu Gln Asn Ile Asp Ala Tyr
785 790 795 800
Lys Thr Phe Pro Lys Lys Glu Lys Lys Gln Lys Ala Pro Lys Glu Glu
805 810 815
Ser Thr Asp Gln Met Ser Glu Pro Pro Lys Glu Gln Arg Val Gln Gly
820 825 830
Ser Ile Thr Gln Arg Thr Arg Thr Arg Pro Ser Val Gln Pro Gly Pro
835 840 845
Met Ile Asp Val Asn Thr Asp
850 855
<210> 8
<211> 24
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 8
Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly
1 5 10 15
Met Leu Val Ala Ser Cys Leu Gly
20
<210> 9
<211> 413
<212> PRT
<213> MERS
<400> 9
Met Ala Ser Pro Ala Ala Pro Arg Ala Val Ser Phe Ala Asp Asn Asn
1 5 10 15
Asp Ile Thr Asn Thr Asn Leu Ser Arg Gly Arg Gly Arg Asn Pro Lys
20 25 30
Pro Arg Ala Ala Pro Asn Asn Thr Val Ser Trp Tyr Thr Gly Leu Thr
35 40 45
Gln His Gly Lys Val Pro Leu Thr Phe Pro Pro Gly Gln Gly Val Pro
50 55 60
Leu Asn Ala Asn Ser Thr Pro Ala Gln Asn Ala Gly Tyr Trp Arg Arg
65 70 75 80
Gln Asp Arg Lys Ile Asn Thr Gly Asn Gly Ile Lys Gln Leu Ala Pro
85 90 95
Arg Trp Tyr Phe Tyr Tyr Thr Gly Thr Gly Pro Glu Ala Ala Leu Pro
100 105 110
Phe Arg Ala Val Lys Asp Gly Ile Val Trp Val His Glu Asp Gly Ala
115 120 125
Thr Asp Ala Pro Ser Thr Phe Gly Thr Arg Asn Pro Asn Asn Asp Ser
130 135 140
Ala Ile Val Thr Gln Phe Ala Pro Gly Thr Lys Leu Pro Lys Asn Phe
145 150 155 160
His Ile Glu Gly Thr Gly Gly Asn Ser Gln Ser Ser Ser Arg Ala Ser
165 170 175
Ser Leu Ser Arg Asn Ser Ser Arg Ser Ser Ser Gln Gly Ser Arg Ser
180 185 190
Gly Asn Ser Thr Arg Gly Thr Ser Pro Gly Pro Ser Gly Ile Gly Ala
195 200 205
Val Gly Gly Asp Leu Leu Tyr Leu Asp Leu Leu Asn Arg Leu Gln Ala
210 215 220
Leu Glu Ser Gly Lys Val Lys Gln Ser Gln Pro Lys Val Ile Thr Lys
225 230 235 240
Lys Asp Ala Ala Ala Ala Lys Asn Lys Met Arg His Lys Arg Thr Ser
245 250 255
Thr Lys Ser Phe Asn Met Val Gln Ala Phe Gly Leu Arg Gly Pro Gly
260 265 270
Asp Leu Gln Gly Asn Phe Gly Asp Leu Gln Leu Asn Lys Leu Gly Thr
275 280 285
Glu Asp Pro Arg Trp Pro Gln Ile Ala Glu Leu Ala Pro Thr Ala Ser
290 295 300
Ala Phe Met Gly Met Ser Gln Phe Lys Leu Thr His Gln Asn Asn Asp
305 310 315 320
Asp His Gly Asn Pro Val Tyr Phe Leu Arg Tyr Ser Gly Ala Ile Lys
325 330 335
Leu Asp Pro Lys Asn Pro Asn Tyr Asn Lys Trp Leu Glu Leu Leu Glu
340 345 350
Gln Asn Ile Asp Ala Tyr Lys Thr Phe Pro Lys Lys Glu Lys Lys Gln
355 360 365
Lys Ala Pro Lys Glu Glu Ser Thr Asp Gln Met Ser Glu Pro Pro Lys
370 375 380
Glu Gln Arg Val Gln Gly Ser Ile Thr Gln Arg Thr Arg Thr Arg Pro
385 390 395 400
Ser Val Gln Pro Gly Pro Met Ile Asp Val Asn Thr Asp
405 410
<210> 10
<211> 422
<212> PRT
<213> SARS-COV-1
<400> 10
Met Ser Asp Asn Gly Pro Gln Ser Asn Gln Arg Ser Ala Pro Arg Ile
1 5 10 15
Thr Phe Gly Gly Pro Thr Asp Ser Thr Asp Asn Asn Gln Asn Gly Gly
20 25 30
Arg Asn Gly Ala Arg Pro Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn
35 40 45
Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Glu
50 55 60
Leu Arg Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Gly
65 70 75 80
Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Val Arg
85 90 95
Gly Gly Asp Gly Lys Met Lys Glu Leu Ser Pro Arg Trp Tyr Phe Tyr
100 105 110
Tyr Leu Gly Thr Gly Pro Glu Ala Ser Leu Pro Tyr Gly Ala Asn Lys
115 120 125
Glu Gly Ile Val Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys
130 135 140
Asp His Ile Gly Thr Arg Asn Pro Asn Asn Asn Ala Ala Thr Val Leu
145 150 155 160
Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly
165 170 175
Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg
180 185 190
Gly Asn Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Asn Ser Pro
195 200 205
Ala Arg Met Ala Ser Gly Gly Gly Glu Thr Ala Leu Ala Leu Leu Leu
210 215 220
Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Val Ser Gly Lys Gly Gln
225 230 235 240
Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser
245 250 255
Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Gln Tyr Asn Val Thr
260 265 270
Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly
275 280 285
Asp Gln Asp Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln
290 295 300
Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg
305 310 315 320
Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr His Gly
325 330 335
Ala Ile Lys Leu Asp Asp Lys Asp Pro Gln Phe Lys Asp Asn Val Ile
340 345 350
Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu
355 360 365
Pro Lys Lys Asp Lys Lys Lys Lys Thr Asp Glu Ala Gln Pro Leu Pro
370 375 380
Gln Arg Gln Lys Lys Gln Pro Thr Val Thr Leu Leu Pro Ala Ala Asp
385 390 395 400
Met Asp Asp Phe Ser Arg Gln Leu Gln Asn Ser Met Ser Gly Ala Ser
405 410 415
Ala Asp Ser Thr Gln Ala
420
<210> 11
<211> 26
<212> DNA
<213> artificial sequence
<220>
<223> synthetic construct
<400> 11
ggcgcgccat tggccaccgc ggccgc 26
<210> 12
<211> 10
<212> DNA
<213> artificial sequence
<220>
<223> synthetic construct
<400> 12
attggccacc 10
<210> 13
<211> 497
<212> PRT
<213> influenza A Virus
<400> 13
Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Gly Gly
1 5 10 15
Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Arg Met Val
20 25 30
Gly Gly Ile Gly Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys Leu
35 40 45
Ser Asp Tyr Glu Gly Arg Leu Ile Gln Asn Ser Ile Thr Ile Glu Arg
50 55 60
Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu Glu
65 70 75 80
His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile Tyr
85 90 95
Arg Arg Arg Asp Gly Lys Trp Val Arg Glu Leu Ile Leu Tyr Asp Lys
100 105 110
Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly Glu Asp Ala
115 120 125
Thr Ala Gly Leu Thr His Leu Met Ile Trp His Ser Asn Leu Asn Asp
130 135 140
Ala Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp Pro
145 150 155 160
Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser Gly
165 170 175
Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu Leu
180 185 190
Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg Gly
195 200 205
Glu Asn Gly Arg Arg Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn Ile
210 215 220
Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp Gln
225 230 235 240
Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu Ile
245 250 255
Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His Lys
260 265 270
Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Ala Val Ala Ser Gly Tyr
275 280 285
Asp Phe Glu Arg Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe Arg
290 295 300
Leu Leu Gln Asn Ser Gln Val Phe Ser Leu Ile Arg Pro Asn Glu Asn
305 310 315 320
Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala Ala
325 330 335
Phe Glu Asp Leu Arg Val Ser Ser Phe Ile Arg Gly Thr Arg Val Val
340 345 350
Pro Arg Gly Gln Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn Glu
355 360 365
Asn Met Glu Thr Met Asp Ser Ser Thr Leu Glu Leu Arg Ser Arg Tyr
370 375 380
Trp Ala Ile Arg Thr Arg Ser Gly Gly Thr Thr Asn Gln Gln Arg Ala
385 390 395 400
Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg Asn
405 410 415
Leu Pro Phe Glu Arg Ala Thr Ile Met Ala Ala Phe Thr Gly Asn Thr
420 425 430
Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Ile Ile Arg Met Met Glu
435 440 445
Ser Ala Lys Pro Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe Glu
450 455 460
Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp Met
465 470 475 480
Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr Asp
485 490 495
Asn
<210> 14
<211> 560
<212> PRT
<213> influenza b virus
<400> 14
Met Ser Asn Met Asp Ile Asp Gly Ile Asn Thr Gly Thr Ile Asp Lys
1 5 10 15
Thr Pro Glu Glu Ile Thr Pro Gly Thr Ser Gly Thr Thr Arg Pro Ile
20 25 30
Ile Arg Pro Ala Thr Leu Ala Pro Pro Ser Asn Lys Arg Thr Arg Asn
35 40 45
Pro Ser Pro Glu Arg Ala Thr Thr Ser Ser Glu Asp Asp Val Gly Arg
50 55 60
Lys Thr Gln Lys Lys Gln Thr Pro Thr Glu Ile Lys Lys Ser Val Tyr
65 70 75 80
Asn Met Val Val Lys Leu Gly Glu Phe Tyr Asn Gln Met Met Val Lys
85 90 95
Ala Gly Leu Asn Asp Asp Met Glu Arg Asn Leu Ile Gln Asn Ala His
100 105 110
Ala Val Glu Arg Ile Leu Leu Ala Ala Thr Asp Asp Lys Lys Thr Glu
115 120 125
Phe Gln Lys Lys Lys Asn Ala Arg Asp Val Lys Glu Gly Lys Glu Glu
130 135 140
Ile Asp His Asn Lys Thr Gly Gly Thr Phe Tyr Lys Met Val Arg Asp
145 150 155 160
Asp Lys Thr Ile Tyr Phe Ser Pro Ile Arg Ile Thr Phe Leu Lys Glu
165 170 175
Glu Val Lys Thr Met Tyr Lys Thr Thr Met Gly Ser Asp Gly Phe Ser
180 185 190
Gly Leu Asn His Ile Met Ile Gly His Ser Gln Met Asn Asp Val Cys
195 200 205
Phe Gln Arg Ser Lys Ala Leu Lys Arg Val Gly Leu Asp Pro Ser Leu
210 215 220
Ile Ser Thr Phe Ala Gly Ser Thr Ile Pro Arg Arg Ser Gly Ala Thr
225 230 235 240
Gly Val Ala Ile Lys Gly Gly Gly Thr Leu Val Ala Glu Ala Ile Arg
245 250 255
Phe Ile Gly Arg Ala Met Ala Asp Arg Gly Leu Leu Arg Asp Ile Lys
260 265 270
Ala Lys Thr Ala Tyr Glu Lys Ile Leu Leu Asn Leu Lys Asn Lys Cys
275 280 285
Ser Ala Pro Gln Gln Lys Ala Leu Val Asp Gln Val Ile Gly Ser Arg
290 295 300
Asn Pro Gly Ile Ala Asp Ile Glu Asp Leu Thr Leu Leu Ala Arg Ser
305 310 315 320
Met Val Val Val Arg Pro Ser Val Ala Ser Lys Val Val Leu Pro Ile
325 330 335
Ser Ile Tyr Ala Lys Ile Pro Gln Leu Gly Phe Asn Val Glu Glu Tyr
340 345 350
Ser Met Val Gly Tyr Glu Ala Met Ala Leu Tyr Asn Met Ala Thr Pro
355 360 365
Val Ser Ile Leu Arg Met Gly Asp Asp Ala Lys Asp Lys Ser Gln Leu
370 375 380
Phe Phe Met Ser Cys Phe Gly Ala Ala Tyr Glu Asp Leu Arg Val Leu
385 390 395 400
Ser Ala Leu Thr Gly Thr Glu Phe Lys Pro Arg Ser Ala Leu Lys Cys
405 410 415
Lys Gly Phe His Val Pro Ala Lys Glu Gln Val Glu Gly Met Gly Ala
420 425 430
Ala Leu Met Ser Ile Lys Leu Gln Phe Trp Ala Pro Met Thr Arg Ser
435 440 445
Gly Gly Asn Glu Val Gly Gly Asp Gly Gly Ser Gly Gln Ile Ser Cys
450 455 460
Ser Pro Val Phe Ala Val Glu Arg Pro Ile Ala Leu Ser Lys Gln Ala
465 470 475 480
Val Arg Arg Met Leu Ser Met Asn Ile Glu Gly Arg Asp Ala Asp Val
485 490 495
Lys Gly Asn Leu Leu Lys Met Met Asn Asp Ser Met Ala Lys Lys Thr
500 505 510
Ser Gly Asn Ala Phe Ile Gly Lys Lys Met Phe Gln Ile Ser Asp Lys
515 520 525
Asn Lys Thr Asn Pro Val Glu Ile Pro Ile Lys Gln Thr Ile Pro Asn
530 535 540
Phe Phe Phe Gly Arg Asp Thr Ala Glu Asp Tyr Asp Asp Leu Asp Tyr
545 550 555 560
<210> 15
<211> 3246
<212> DNA
<213> artificial sequence
<220>
<223> synthetic construct
<400> 15
atgcctatgg gcagcctgca gccactggct acactgtacc tgctgggcat gctggtggcc 60
tcttgtctgg gcgccagcca aggcactaag agaagctacg agcagatgga aaccggaggc 120
gaacggcaga acgccacaga gatcagagcc tctgtgggcc gtatggtcgg cggcatcggc 180
agattctaca tccagatgtg caccgaactg aagctgagcg actacgaggg ccgcctgatc 240
cagaacagca tcacaatcga gagaatggtg ctgtccgcct ttgacgagcg gagaaacaaa 300
tacctggaag agcaccctag cgccggaaaa gatcctaaga aaaccggcgg acctatctac 360
agaagaagag atggtaagtg ggtgagagag ctgattctgt acgataagga agagattcga 420
agaatctgga gacaggccaa caacggcgag gatgccaccg caggcctgac acacctgatg 480
atctggcaca gcaacctgaa cgatgcgacc taccagcgca cgcgggccct ggtcagaacc 540
ggcatggatc ctcggatgtg tagcctgatg cagggcagca cactgccaag acggagtggg 600
gccgccggcg ctgcagtgaa gggcgtcgga accatggtga tggagctgat ccggatgata 660
aagcggggca tcaacgacag aaacttctgg cgaggcgaga acggccgaag aacccggatc 720
gcctacgaga gaatgtgcaa catcctgaaa ggaaaattcc agaccgccgc ccagcgggcc 780
atgatggacc aggtgcgcga gagcagaaac cccggcaatg ccgagatcga ggacctgatc 840
ttcctggcca gaagcgccct cattcttaga ggctctgtgg cccacaagag ctgtctgcct 900
gcctgtgtgt acggcctggc agtggcctca ggctacgact tcgagcggga aggatacagt 960
ctggtgggca tcgacccttt cagactcctg cagaatagcc aggtgtttag cctgatcaga 1020
ccaaacgaaa accccgccca taagagccag ctggtgtgga tggcctgcca cagcgccgcc 1080
tttgaggatc tgagagtgag ctcttttatc agaggcaccc gggtggttcc acgaggtcaa 1140
ctgtctacaa gaggtgtgca gatcgccagc aacgagaaca tggagaccat ggatagcagc 1200
accctggaac tgagatccag atactgggcc atcaggacac ggagcggcgg caccaccaat 1260
cagcagcgcg ccagcgccgg ccagatctct gtccagccta cgtttagcgt gcagcggaat 1320
ttgcccttcg aacgcgccac aatcatggct gctttcaccg gcaatacaga gggcagaacc 1380
agcgatatga gaacagaaat tatccgtatg atggagtccg caaaacctga ggacgtgtcc 1440
ttccaaggca gaggcgtgtt cgagctgagc gacgagaagg ccaccaaccc tatcgtgcct 1500
agcttcgata tgtctaatga gggcagctac tttttcggag ataacgccga agagtacgac 1560
aacatgtcta atatggatat cgacggcatt aacaccggca ccatcgacaa aacccctgag 1620
gagatcaccc ctggcaccag cggcacaacc cggcccatca tccgccccgc tacactggct 1680
ccacctagca acaagcggac cagaaatccc tcgccagaaa gagccacaac ctccagcgag 1740
gacgacgtgg gacggaagac acaaaagaag cagaccccta cagagatcaa gaagtctgtt 1800
tacaacatgg tggtgaaact gggcgagttc tacaaccaga tgatggtgaa ggccggcctg 1860
aacgacgata tggaaagaaa tctgatccag aacgcccacg ccgtggagcg gattctgctg 1920
gccgccaccg atgataagaa gaccgaattc cagaaaaaga aaaacgccag agacgtgaag 1980
gaaggcaagg aagagatcga ccacaacaag acaggcggca cattctacaa gatggtccgg 2040
gacgacaaga ccatctactt cagccctatc cggataacat tcctgaaaga agaagtgaag 2100
accatgtaca aaaccacaat gggctctgac ggcttcagcg gcctgaatca catcatgatc 2160
ggccactctc aaatgaacga tgtgtgcttc cagagaagca aggctctgaa gcgcgtgggc 2220
ctggatccta gcctgatctc taccttcgcc ggcagcacca tccccagaag atcgggcgct 2280
accggcgtgg ctatcaaggg aggaggcaca ctggtggctg aagccatcag attcatcgga 2340
agagccatgg ccgacagagg actcctgaga gatatcaaag ccaaaaccgc ctacgaaaaa 2400
atcctgctga acctgaagaa caagtgcagc gcgcctcaac agaaggccct ggtggaccag 2460
gttatcggct ctagaaaccc tggaatcgcc gatatcgagg acctgacact gctggccaga 2520
tctatggtgg tggtgagacc ctccgtggcc agcaaggtgg tgctgcctat cagcatctac 2580
gccaagatcc ctcagctggg atttaacgtg gaagaataca gcatggttgg ttatgaggcc 2640
atggccctgt acaacatggc cacacctgtg tccatcctga gaatgggcga cgatgccaaa 2700
gacaagagcc agctgttctt catgagctgc ttcggcgctg cctatgagga cctgagagtg 2760
ctgtccgctc ttacaggaac agagttcaag cctaggagcg cactgaagtg caagggcttc 2820
cacgtgcccg ccaaggaaca ggtggaaggc atgggagctg ctctgatgtc catcaagctg 2880
caattttggg ctcctatgac ccggagcggc ggaaatgagg tgggtggcga cggaggcagc 2940
ggacagattt cttgcagccc cgtatttgcc gtggagagac caatcgccct gtccaagcag 3000
gccgtgagaa gaatgctgag catgaacatc gagggccggg acgccgacgt gaagggcaac 3060
ctgttgaaga tgatgaacga cagcatggcc aagaagacca gtggcaatgc cttcatcggc 3120
aagaagatgt tccagatctc cgacaagaac aagaccaacc ccgtggaaat ccccatcaag 3180
cagacaatcc ctaacttctt cttcggcaga gacaccgccg aagactatga cgacctggac 3240
tactga 3246
<210> 16
<211> 1081
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 16
Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly
1 5 10 15
Met Leu Val Ala Ser Cys Leu Gly Ala Ser Gln Gly Thr Lys Arg Ser
20 25 30
Tyr Glu Gln Met Glu Thr Gly Gly Glu Arg Gln Asn Ala Thr Glu Ile
35 40 45
Arg Ala Ser Val Gly Arg Met Val Gly Gly Ile Gly Arg Phe Tyr Ile
50 55 60
Gln Met Cys Thr Glu Leu Lys Leu Ser Asp Tyr Glu Gly Arg Leu Ile
65 70 75 80
Gln Asn Ser Ile Thr Ile Glu Arg Met Val Leu Ser Ala Phe Asp Glu
85 90 95
Arg Arg Asn Lys Tyr Leu Glu Glu His Pro Ser Ala Gly Lys Asp Pro
100 105 110
Lys Lys Thr Gly Gly Pro Ile Tyr Arg Arg Arg Asp Gly Lys Trp Val
115 120 125
Arg Glu Leu Ile Leu Tyr Asp Lys Glu Glu Ile Arg Arg Ile Trp Arg
130 135 140
Gln Ala Asn Asn Gly Glu Asp Ala Thr Ala Gly Leu Thr His Leu Met
145 150 155 160
Ile Trp His Ser Asn Leu Asn Asp Ala Thr Tyr Gln Arg Thr Arg Ala
165 170 175
Leu Val Arg Thr Gly Met Asp Pro Arg Met Cys Ser Leu Met Gln Gly
180 185 190
Ser Thr Leu Pro Arg Arg Ser Gly Ala Ala Gly Ala Ala Val Lys Gly
195 200 205
Val Gly Thr Met Val Met Glu Leu Ile Arg Met Ile Lys Arg Gly Ile
210 215 220
Asn Asp Arg Asn Phe Trp Arg Gly Glu Asn Gly Arg Arg Thr Arg Ile
225 230 235 240
Ala Tyr Glu Arg Met Cys Asn Ile Leu Lys Gly Lys Phe Gln Thr Ala
245 250 255
Ala Gln Arg Ala Met Met Asp Gln Val Arg Glu Ser Arg Asn Pro Gly
260 265 270
Asn Ala Glu Ile Glu Asp Leu Ile Phe Leu Ala Arg Ser Ala Leu Ile
275 280 285
Leu Arg Gly Ser Val Ala His Lys Ser Cys Leu Pro Ala Cys Val Tyr
290 295 300
Gly Leu Ala Val Ala Ser Gly Tyr Asp Phe Glu Arg Glu Gly Tyr Ser
305 310 315 320
Leu Val Gly Ile Asp Pro Phe Arg Leu Leu Gln Asn Ser Gln Val Phe
325 330 335
Ser Leu Ile Arg Pro Asn Glu Asn Pro Ala His Lys Ser Gln Leu Val
340 345 350
Trp Met Ala Cys His Ser Ala Ala Phe Glu Asp Leu Arg Val Ser Ser
355 360 365
Phe Ile Arg Gly Thr Arg Val Val Pro Arg Gly Gln Leu Ser Thr Arg
370 375 380
Gly Val Gln Ile Ala Ser Asn Glu Asn Met Glu Thr Met Asp Ser Ser
385 390 395 400
Thr Leu Glu Leu Arg Ser Arg Tyr Trp Ala Ile Arg Thr Arg Ser Gly
405 410 415
Gly Thr Thr Asn Gln Gln Arg Ala Ser Ala Gly Gln Ile Ser Val Gln
420 425 430
Pro Thr Phe Ser Val Gln Arg Asn Leu Pro Phe Glu Arg Ala Thr Ile
435 440 445
Met Ala Ala Phe Thr Gly Asn Thr Glu Gly Arg Thr Ser Asp Met Arg
450 455 460
Thr Glu Ile Ile Arg Met Met Glu Ser Ala Lys Pro Glu Asp Val Ser
465 470 475 480
Phe Gln Gly Arg Gly Val Phe Glu Leu Ser Asp Glu Lys Ala Thr Asn
485 490 495
Pro Ile Val Pro Ser Phe Asp Met Ser Asn Glu Gly Ser Tyr Phe Phe
500 505 510
Gly Asp Asn Ala Glu Glu Tyr Asp Asn Met Ser Asn Met Asp Ile Asp
515 520 525
Gly Ile Asn Thr Gly Thr Ile Asp Lys Thr Pro Glu Glu Ile Thr Pro
530 535 540
Gly Thr Ser Gly Thr Thr Arg Pro Ile Ile Arg Pro Ala Thr Leu Ala
545 550 555 560
Pro Pro Ser Asn Lys Arg Thr Arg Asn Pro Ser Pro Glu Arg Ala Thr
565 570 575
Thr Ser Ser Glu Asp Asp Val Gly Arg Lys Thr Gln Lys Lys Gln Thr
580 585 590
Pro Thr Glu Ile Lys Lys Ser Val Tyr Asn Met Val Val Lys Leu Gly
595 600 605
Glu Phe Tyr Asn Gln Met Met Val Lys Ala Gly Leu Asn Asp Asp Met
610 615 620
Glu Arg Asn Leu Ile Gln Asn Ala His Ala Val Glu Arg Ile Leu Leu
625 630 635 640
Ala Ala Thr Asp Asp Lys Lys Thr Glu Phe Gln Lys Lys Lys Asn Ala
645 650 655
Arg Asp Val Lys Glu Gly Lys Glu Glu Ile Asp His Asn Lys Thr Gly
660 665 670
Gly Thr Phe Tyr Lys Met Val Arg Asp Asp Lys Thr Ile Tyr Phe Ser
675 680 685
Pro Ile Arg Ile Thr Phe Leu Lys Glu Glu Val Lys Thr Met Tyr Lys
690 695 700
Thr Thr Met Gly Ser Asp Gly Phe Ser Gly Leu Asn His Ile Met Ile
705 710 715 720
Gly His Ser Gln Met Asn Asp Val Cys Phe Gln Arg Ser Lys Ala Leu
725 730 735
Lys Arg Val Gly Leu Asp Pro Ser Leu Ile Ser Thr Phe Ala Gly Ser
740 745 750
Thr Ile Pro Arg Arg Ser Gly Ala Thr Gly Val Ala Ile Lys Gly Gly
755 760 765
Gly Thr Leu Val Ala Glu Ala Ile Arg Phe Ile Gly Arg Ala Met Ala
770 775 780
Asp Arg Gly Leu Leu Arg Asp Ile Lys Ala Lys Thr Ala Tyr Glu Lys
785 790 795 800
Ile Leu Leu Asn Leu Lys Asn Lys Cys Ser Ala Pro Gln Gln Lys Ala
805 810 815
Leu Val Asp Gln Val Ile Gly Ser Arg Asn Pro Gly Ile Ala Asp Ile
820 825 830
Glu Asp Leu Thr Leu Leu Ala Arg Ser Met Val Val Val Arg Pro Ser
835 840 845
Val Ala Ser Lys Val Val Leu Pro Ile Ser Ile Tyr Ala Lys Ile Pro
850 855 860
Gln Leu Gly Phe Asn Val Glu Glu Tyr Ser Met Val Gly Tyr Glu Ala
865 870 875 880
Met Ala Leu Tyr Asn Met Ala Thr Pro Val Ser Ile Leu Arg Met Gly
885 890 895
Asp Asp Ala Lys Asp Lys Ser Gln Leu Phe Phe Met Ser Cys Phe Gly
900 905 910
Ala Ala Tyr Glu Asp Leu Arg Val Leu Ser Ala Leu Thr Gly Thr Glu
915 920 925
Phe Lys Pro Arg Ser Ala Leu Lys Cys Lys Gly Phe His Val Pro Ala
930 935 940
Lys Glu Gln Val Glu Gly Met Gly Ala Ala Leu Met Ser Ile Lys Leu
945 950 955 960
Gln Phe Trp Ala Pro Met Thr Arg Ser Gly Gly Asn Glu Val Gly Gly
965 970 975
Asp Gly Gly Ser Gly Gln Ile Ser Cys Ser Pro Val Phe Ala Val Glu
980 985 990
Arg Pro Ile Ala Leu Ser Lys Gln Ala Val Arg Arg Met Leu Ser Met
995 1000 1005
Asn Ile Glu Gly Arg Asp Ala Asp Val Lys Gly Asn Leu Leu Lys Met
1010 1015 1020
Met Asn Asp Ser Met Ala Lys Lys Thr Ser Gly Asn Ala Phe Ile Gly
1025 1030 1035 1040
Lys Lys Met Phe Gln Ile Ser Asp Lys Asn Lys Thr Asn Pro Val Glu
1045 1050 1055
Ile Pro Ile Lys Gln Thr Ile Pro Asn Phe Phe Phe Gly Arg Asp Thr
1060 1065 1070
Ala Glu Asp Tyr Asp Asp Leu Asp Tyr
1075 1080
<210> 17
<211> 497
<212> PRT
<213> influenza A Virus
<400> 17
Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp Gly
1 5 10 15
Glu Arg Gln Asn Ala Asn Glu Ile Arg Ala Ser Val Gly Lys Met Ile
20 25 30
Gly Gly Ile Gly Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys Leu
35 40 45
Ser Asp Tyr Glu Gly Arg Leu Ile Gln Asn Ser Leu Thr Ile Glu Arg
50 55 60
Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu Glu
65 70 75 80
His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile Tyr
85 90 95
Lys Arg Val Asp Gly Lys Trp Met Arg Glu Leu Val Leu Tyr Asp Lys
100 105 110
Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly Asp Asp Ala
115 120 125
Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn Asp
130 135 140
Thr Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp Pro
145 150 155 160
Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser Gly
165 170 175
Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu Leu
180 185 190
Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg Gly
195 200 205
Glu Asn Gly Arg Lys Thr Arg Asn Ala Tyr Glu Arg Met Cys Asn Ile
210 215 220
Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp Gln
225 230 235 240
Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu Ile
245 250 255
Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His Lys
260 265 270
Ser Cys Leu Pro Ala Cys Val Tyr Gly Pro Ala Val Ala Ser Gly Tyr
275 280 285
Asp Phe Glu Lys Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe Lys
290 295 300
Leu Leu Gln Asn Ser Gln Val Tyr Ser Leu Ile Arg Pro Asn Glu Asn
305 310 315 320
Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys Asn Ser Ala Ala
325 330 335
Phe Glu Asp Leu Arg Val Ser Ser Phe Ile Arg Gly Thr Lys Val Ile
340 345 350
Pro Arg Gly Lys Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn Glu
355 360 365
Asn Met Asp Thr Met Gly Ser Ser Thr Leu Glu Leu Arg Ser Arg Tyr
370 375 380
Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln Gln Arg Ala
385 390 395 400
Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg Asn
405 410 415
Leu Pro Phe Asp Lys Pro Thr Ile Met Ala Ala Phe Thr Gly Asn Ala
420 425 430
Glu Gly Arg Thr Ser Asp Met Arg Ala Glu Ile Ile Arg Met Met Glu
435 440 445
Gly Ala Lys Pro Glu Glu Val Ser Phe Gln Gly Arg Gly Val Phe Glu
450 455 460
Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp Met
465 470 475 480
Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr Asp
485 490 495
Asn
<210> 18
<211> 738
<212> PRT
<213> zaire Ebola Virus
<400> 18
Asp Ser Arg Pro Gln Lys Ile Trp Met Ala Pro Ser Leu Thr Glu Ser
1 5 10 15
Asp Met Asp Tyr His Lys Ile Leu Thr Ala Gly Leu Ser Val Gln Gln
20 25 30
Gly Ile Val Arg Gln Arg Val Ile Pro Val Tyr Gln Val Asn Asn Leu
35 40 45
Glu Glu Ile Cys Gln Leu Ile Ile Gln Ala Phe Glu Ala Gly Val Asp
50 55 60
Phe Gln Glu Ser Ala Asp Ser Phe Leu Leu Met Leu Cys Leu His His
65 70 75 80
Ala Tyr Gln Gly Asp Tyr Lys Leu Phe Leu Glu Ser Gly Ala Val Lys
85 90 95
Tyr Leu Glu Gly His Gly Phe Arg Phe Glu Val Lys Lys Arg Asp Gly
100 105 110
Val Lys Arg Leu Glu Glu Leu Leu Pro Ala Val Ser Ser Gly Lys Asn
115 120 125
Ile Lys Arg Thr Leu Ala Ala Met Pro Glu Glu Glu Thr Thr Glu Ala
130 135 140
Asn Ala Gly Gln Phe Leu Ser Phe Ala Ser Leu Phe Leu Pro Lys Leu
145 150 155 160
Val Val Gly Glu Lys Ala Cys Leu Glu Lys Val Gln Arg Gln Ile Gln
165 170 175
Val His Ala Glu Gln Gly Leu Ile Gln Tyr Pro Thr Ala Trp Gln Ser
180 185 190
Val Gly His Met Met Val Ile Phe Arg Leu Met Arg Thr Asn Phe Leu
195 200 205
Ile Lys Phe Leu Leu Ile His Gln Gly Met His Met Val Ala Gly His
210 215 220
Asp Ala Asn Asp Ala Val Ile Ser Asn Ser Val Ala Gln Ala Arg Phe
225 230 235 240
Ser Gly Leu Leu Ile Val Lys Thr Val Leu Asp His Ile Leu Gln Lys
245 250 255
Thr Glu Arg Gly Val Arg Leu His Pro Leu Ala Arg Thr Ala Lys Val
260 265 270
Lys Asn Glu Val Asn Ser Phe Lys Ala Ala Leu Ser Ser Leu Ala Lys
275 280 285
His Gly Glu Tyr Ala Pro Phe Ala Arg Leu Leu Asn Leu Ser Gly Val
290 295 300
Asn Asn Leu Glu His Gly Leu Phe Pro Gln Leu Ser Ala Ile Ala Leu
305 310 315 320
Gly Val Ala Thr Ala His Gly Ser Thr Leu Ala Gly Val Asn Val Gly
325 330 335
Glu Gln Tyr Gln Gln Leu Arg Glu Ala Ala Thr Glu Ala Glu Lys Gln
340 345 350
Leu Gln Gln Tyr Ala Glu Ser Arg Glu Leu Asp His Leu Gly Leu Asp
355 360 365
Asp Gln Glu Lys Lys Ile Leu Met Asn Phe His Gln Lys Lys Asn Glu
370 375 380
Ile Ser Phe Gln Gln Thr Asn Ala Met Val Thr Leu Arg Lys Glu Arg
385 390 395 400
Leu Ala Lys Leu Thr Glu Ala Ile Thr Ala Ala Ser Leu Pro Lys Thr
405 410 415
Ser Gly His Tyr Asp Asp Asp Asp Asp Ile Pro Phe Pro Gly Pro Ile
420 425 430
Asn Asp Asp Asp Asn Pro Gly His Gln Asp Asp Asp Pro Thr Asp Ser
435 440 445
Gln Asp Thr Thr Ile Pro Asp Val Val Val Asp Pro Asp Asp Gly Ser
450 455 460
Tyr Gly Glu Tyr Gln Ser Tyr Ser Glu Asn Gly Met Asn Ala Pro Asp
465 470 475 480
Asp Leu Val Leu Phe Asp Leu Asp Glu Asp Asp Glu Asp Thr Lys Pro
485 490 495
Val Pro Asn Arg Ser Thr Lys Gly Gly Gln Gln Lys Asn Ser Gln Lys
500 505 510
Gly Gln His Ile Glu Gly Arg Gln Thr Gln Phe Arg Pro Ile Gln Asn
515 520 525
Val Pro Gly Pro His Arg Thr Ile His His Ala Ser Ala Pro Leu Thr
530 535 540
Asp Asn Asp Arg Arg Asn Glu Pro Ser Gly Ser Thr Ser Pro Arg Met
545 550 555 560
Leu Thr Pro Ile Asn Glu Glu Ala Asp Pro Leu Asp Asp Ala Asp Asp
565 570 575
Glu Thr Ser Ser Leu Pro Pro Leu Glu Ser Asp Asp Glu Glu Gln Asp
580 585 590
Arg Asp Gly Thr Ser Asn Arg Thr Pro Thr Val Ala Pro Pro Ala Pro
595 600 605
Val Tyr Arg Asp His Ser Glu Lys Lys Glu Leu Pro Gln Asp Glu Gln
610 615 620
Gln Asp Gln Asp His Thr Gln Glu Ala Arg Asn Gln Asp Ser Asp Asn
625 630 635 640
Thr Gln Ser Glu His Ser Leu Glu Glu Met Tyr Arg His Ile Leu Arg
645 650 655
Ser Gln Gly Pro Phe Asp Ala Val Leu Tyr Tyr His Met Met Lys Asp
660 665 670
Glu Pro Val Val Phe Ser Thr Ser Asp Gly Lys Glu Tyr Thr Tyr Pro
675 680 685
Asp Ser Leu Glu Glu Glu Tyr Pro Pro Trp Leu Thr Glu Lys Glu Ala
690 695 700
Met Asn Glu Glu Asn Arg Phe Val Thr Leu Asp Gly Gln Gln Phe Tyr
705 710 715 720
Trp Pro Val Met Asn His Lys Asn Lys Phe Met Ala Ile Leu Gln His
725 730 735
His Gln
<210> 19
<211> 336
<212> PRT
<213> Sudan type ebola virus
<400> 19
Ala Lys Leu Thr Glu Ala Ile Thr Thr Ala Ser Lys Ile Lys Val Gly
1 5 10 15
Asp Arg Tyr Pro Asp Asp Asn Asp Ile Pro Phe Pro Gly Pro Ile Tyr
20 25 30
Asp Asp Thr His Pro Asn Pro Ser Asp Asp Asn Pro Asp Asp Ser Arg
35 40 45
Asp Thr Thr Ile Pro Gly Gly Val Val Asp Pro Tyr Asp Asp Glu Ser
50 55 60
Asn Asn Tyr Pro Asp Tyr Glu Asp Ser Ala Glu Gly Thr Thr Gly Asp
65 70 75 80
Leu Asp Leu Phe Asn Leu Asp Asp Asp Asp Asp Asp Ser Arg Pro Gly
85 90 95
Pro Pro Asp Arg Gly Gln Asn Lys Glu Arg Ala Ala Arg Thr Tyr Gly
100 105 110
Leu Gln Asp Pro Thr Leu Asp Gly Ala Lys Lys Val Pro Glu Leu Thr
115 120 125
Pro Gly Ser His Gln Pro Gly Asn Leu His Ile Thr Lys Ser Gly Ser
130 135 140
Asn Thr Asn Gln Pro Gln Gly Asn Met Ser Ser Thr Leu His Ser Met
145 150 155 160
Thr Pro Ile Gln Glu Glu Ser Glu Pro Asp Asp Gln Lys Asp Asn Asp
165 170 175
Asp Glu Ser Leu Thr Ser Leu Asp Ser Glu Gly Asp Glu Asp Gly Glu
180 185 190
Ser Ile Ser Glu Glu Asn Thr Pro Thr Val Ala Pro Pro Ala Pro Val
195 200 205
Tyr Lys Asp Thr Gly Val Asp Thr Asn Gln Gln Asn Gly Pro Ser Ser
210 215 220
Thr Val Asp Ser Gln Gly Ser Glu Ser Glu Ala Leu Pro Ile Asn Ser
225 230 235 240
Lys Lys Ser Ser Ala Leu Glu Glu Thr Tyr Tyr His Leu Leu Lys Thr
245 250 255
Gln Gly Pro Phe Glu Ala Ile Asn Tyr Tyr His Leu Met Ser Asp Glu
260 265 270
Pro Ile Ala Phe Ser Thr Glu Ser Gly Lys Glu Tyr Ile Phe Pro Asp
275 280 285
Ser Leu Glu Glu Ala Tyr Pro Pro Trp Leu Ser Glu Lys Glu Ala Leu
290 295 300
Glu Lys Glu Asn Arg Tyr Leu Val Ile Asp Gly Gln Gln Phe Leu Trp
305 310 315 320
Pro Val Met Ser Leu Arg Asp Lys Phe Leu Ala Val Leu Gln His Asp
325 330 335
<210> 20
<211> 337
<212> PRT
<213> Bendi cloth Jiao Xingai Bola virus
<400> 20
Ala Lys Leu Thr Glu Ala Ile Thr Ser Thr Ser Ile Leu Lys Thr Gly
1 5 10 15
Arg Arg Tyr Asp Asp Asp Asn Asp Ile Pro Phe Pro Gly Pro Ile Asn
20 25 30
Asp Asn Glu Asn Ser Gly Gln Asn Asp Asp Asp Pro Thr Asp Ser Gln
35 40 45
Asp Thr Thr Ile Pro Asp Val Ile Ile Asp Pro Asn Asp Gly Gly Tyr
50 55 60
Asn Asn Tyr Ser Asp Tyr Ala Asn Asp Ala Ala Ser Ala Pro Asp Asp
65 70 75 80
Leu Val Leu Phe Asp Leu Glu Asp Glu Asp Asp Ala Asp Asn Pro Ala
85 90 95
Gln Asn Thr Pro Glu Lys Asn Asp Arg Pro Ala Thr Thr Lys Leu Arg
100 105 110
Asn Gly Gln Asp Gln Asp Gly Asn Gln Gly Glu Thr Ala Ser Pro Arg
115 120 125
Val Ala Pro Asn Gln Tyr Arg Asp Lys Pro Met Pro Gln Val Gln Asp
130 135 140
Arg Ser Glu Asn His Asp Gln Thr Leu Gln Thr Gln Ser Arg Val Leu
145 150 155 160
Thr Pro Ile Ser Glu Glu Ala Asp Pro Ser Asp His Asn Asp Gly Asp
165 170 175
Asn Glu Ser Ile Pro Pro Leu Glu Ser Asp Asp Glu Gly Ser Thr Asp
180 185 190
Thr Thr Ala Ala Glu Thr Lys Pro Ala Thr Ala Pro Pro Ala Pro Val
195 200 205
Tyr Arg Ser Ile Ser Val Asp Asp Ser Val Pro Ser Glu Asn Ile Pro
210 215 220
Ala Gln Ser Asn Gln Thr Asn Asn Glu Asp Asn Val Arg Asn Asn Ala
225 230 235 240
Gln Ser Glu Gln Ser Ile Ala Glu Met Tyr Gln His Ile Leu Lys Thr
245 250 255
Gln Gly Pro Phe Asp Ala Ile Leu Tyr Tyr His Met Met Lys Glu Glu
260 265 270
Pro Ile Ile Phe Ser Thr Ser Asp Gly Lys Glu Tyr Thr Tyr Pro Asp
275 280 285
Ser Leu Glu Asp Glu Tyr Pro Pro Trp Leu Ser Glu Lys Glu Ala Met
290 295 300
Asn Glu Asp Asn Arg Phe Ile Thr Met Asp Gly Gln Gln Phe Tyr Trp
305 310 315 320
Pro Val Met Asn His Arg Asn Lys Phe Met Ala Ile Leu Gln His His
325 330 335
Arg
<210> 21
<211> 169
<212> PRT
<213> Tayi forest type ebola virus
<400> 21
Leu Val Leu Phe Asp Leu Glu Asp Gly Asp Glu Asp Asp His Arg Pro
1 5 10 15
Ser Ser Ser Ser Glu Asn Asn Asn Lys His Ser Leu Thr Gly Thr Asp
20 25 30
Ser Asn Lys Thr Ser Asn Trp Asn Arg Asn Pro Thr Asn Met Pro Lys
35 40 45
Lys Asp Ser Thr Gln Asn Asn Asp Asn Pro Ala Gln Arg Ala Gln Glu
50 55 60
Tyr Ala Arg Asp Asn Ile Gln Asp Thr Pro Thr Pro His Arg Ala Leu
65 70 75 80
Thr Pro Ile Ser Glu Glu Thr Gly Ser Asn Gly His Asn Glu Asp Asp
85 90 95
Ile Asp Ser Ile Pro Pro Leu Glu Ser Asp Glu Glu Asn Asn Thr Glu
100 105 110
Thr Thr Ile Thr Thr Thr Lys Asn Thr Thr Ala Pro Pro Ala Pro Val
115 120 125
Tyr Arg Ser Asn Ser Glu Lys Glu Pro Leu Pro Gln Glu Lys Ser Gln
130 135 140
Lys Gln Pro Asn Gln Val Ser Gly Ser Glu Asn Thr Asp Asn Lys Pro
145 150 155 160
His Ser Glu Gln Ser Val Glu Glu Met
165
<210> 22
<211> 1604
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 22
Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly
1 5 10 15
Met Leu Val Ala Ser Cys Leu Gly Asp Ser Arg Pro Gln Lys Ile Trp
20 25 30
Met Ala Pro Ser Leu Thr Glu Ser Asp Met Asp Tyr His Lys Ile Leu
35 40 45
Thr Ala Gly Leu Ser Val Gln Gln Gly Ile Val Arg Gln Arg Val Ile
50 55 60
Pro Val Tyr Gln Val Asn Asn Leu Glu Glu Ile Cys Gln Leu Ile Ile
65 70 75 80
Gln Ala Phe Glu Ala Gly Val Asp Phe Gln Glu Ser Ala Asp Ser Phe
85 90 95
Leu Leu Met Leu Cys Leu His His Ala Tyr Gln Gly Asp Tyr Lys Leu
100 105 110
Phe Leu Glu Ser Gly Ala Val Lys Tyr Leu Glu Gly His Gly Phe Arg
115 120 125
Phe Glu Val Lys Lys Arg Asp Gly Val Lys Arg Leu Glu Glu Leu Leu
130 135 140
Pro Ala Val Ser Ser Gly Lys Asn Ile Lys Arg Thr Leu Ala Ala Met
145 150 155 160
Pro Glu Glu Glu Thr Thr Glu Ala Asn Ala Gly Gln Phe Leu Ser Phe
165 170 175
Ala Ser Leu Phe Leu Pro Lys Leu Val Val Gly Glu Lys Ala Cys Leu
180 185 190
Glu Lys Val Gln Arg Gln Ile Gln Val His Ala Glu Gln Gly Leu Ile
195 200 205
Gln Tyr Pro Thr Ala Trp Gln Ser Val Gly His Met Met Val Ile Phe
210 215 220
Arg Leu Met Arg Thr Asn Phe Leu Ile Lys Phe Leu Leu Ile His Gln
225 230 235 240
Gly Met His Met Val Ala Gly His Asp Ala Asn Asp Ala Val Ile Ser
245 250 255
Asn Ser Val Ala Gln Ala Arg Phe Ser Gly Leu Leu Ile Val Lys Thr
260 265 270
Val Leu Asp His Ile Leu Gln Lys Thr Glu Arg Gly Val Arg Leu His
275 280 285
Pro Leu Ala Arg Thr Ala Lys Val Lys Asn Glu Val Asn Ser Phe Lys
290 295 300
Ala Ala Leu Ser Ser Leu Ala Lys His Gly Glu Tyr Ala Pro Phe Ala
305 310 315 320
Arg Leu Leu Asn Leu Ser Gly Val Asn Asn Leu Glu His Gly Leu Phe
325 330 335
Pro Gln Leu Ser Ala Ile Ala Leu Gly Val Ala Thr Ala His Gly Ser
340 345 350
Thr Leu Ala Gly Val Asn Val Gly Glu Gln Tyr Gln Gln Leu Arg Glu
355 360 365
Ala Ala Thr Glu Ala Glu Lys Gln Leu Gln Gln Tyr Ala Glu Ser Arg
370 375 380
Glu Leu Asp His Leu Gly Leu Asp Asp Gln Glu Lys Lys Ile Leu Met
385 390 395 400
Asn Phe His Gln Lys Lys Asn Glu Ile Ser Phe Gln Gln Thr Asn Ala
405 410 415
Met Val Thr Leu Arg Lys Glu Arg Leu Ala Lys Leu Thr Glu Ala Ile
420 425 430
Thr Ala Ala Ser Leu Pro Lys Thr Ser Gly His Tyr Asp Asp Asp Asp
435 440 445
Asp Ile Pro Phe Pro Gly Pro Ile Asn Asp Asp Asp Asn Pro Gly His
450 455 460
Gln Asp Asp Asp Pro Thr Asp Ser Gln Asp Thr Thr Ile Pro Asp Val
465 470 475 480
Val Val Asp Pro Asp Asp Gly Ser Tyr Gly Glu Tyr Gln Ser Tyr Ser
485 490 495
Glu Asn Gly Met Asn Ala Pro Asp Asp Leu Val Leu Phe Asp Leu Asp
500 505 510
Glu Asp Asp Glu Asp Thr Lys Pro Val Pro Asn Arg Ser Thr Lys Gly
515 520 525
Gly Gln Gln Lys Asn Ser Gln Lys Gly Gln His Ile Glu Gly Arg Gln
530 535 540
Thr Gln Phe Arg Pro Ile Gln Asn Val Pro Gly Pro His Arg Thr Ile
545 550 555 560
His His Ala Ser Ala Pro Leu Thr Asp Asn Asp Arg Arg Asn Glu Pro
565 570 575
Ser Gly Ser Thr Ser Pro Arg Met Leu Thr Pro Ile Asn Glu Glu Ala
580 585 590
Asp Pro Leu Asp Asp Ala Asp Asp Glu Thr Ser Ser Leu Pro Pro Leu
595 600 605
Glu Ser Asp Asp Glu Glu Gln Asp Arg Asp Gly Thr Ser Asn Arg Thr
610 615 620
Pro Thr Val Ala Pro Pro Ala Pro Val Tyr Arg Asp His Ser Glu Lys
625 630 635 640
Lys Glu Leu Pro Gln Asp Glu Gln Gln Asp Gln Asp His Thr Gln Glu
645 650 655
Ala Arg Asn Gln Asp Ser Asp Asn Thr Gln Ser Glu His Ser Leu Glu
660 665 670
Glu Met Tyr Arg His Ile Leu Arg Ser Gln Gly Pro Phe Asp Ala Val
675 680 685
Leu Tyr Tyr His Met Met Lys Asp Glu Pro Val Val Phe Ser Thr Ser
690 695 700
Asp Gly Lys Glu Tyr Thr Tyr Pro Asp Ser Leu Glu Glu Glu Tyr Pro
705 710 715 720
Pro Trp Leu Thr Glu Lys Glu Ala Met Asn Glu Glu Asn Arg Phe Val
725 730 735
Thr Leu Asp Gly Gln Gln Phe Tyr Trp Pro Val Met Asn His Lys Asn
740 745 750
Lys Phe Met Ala Ile Leu Gln His His Gln Ala Lys Leu Thr Glu Ala
755 760 765
Ile Thr Thr Ala Ser Lys Ile Lys Val Gly Asp Arg Tyr Pro Asp Asp
770 775 780
Asn Asp Ile Pro Phe Pro Gly Pro Ile Tyr Asp Asp Thr His Pro Asn
785 790 795 800
Pro Ser Asp Asp Asn Pro Asp Asp Ser Arg Asp Thr Thr Ile Pro Gly
805 810 815
Gly Val Val Asp Pro Tyr Asp Asp Glu Ser Asn Asn Tyr Pro Asp Tyr
820 825 830
Glu Asp Ser Ala Glu Gly Thr Thr Gly Asp Leu Asp Leu Phe Asn Leu
835 840 845
Asp Asp Asp Asp Asp Asp Ser Arg Pro Gly Pro Pro Asp Arg Gly Gln
850 855 860
Asn Lys Glu Arg Ala Ala Arg Thr Tyr Gly Leu Gln Asp Pro Thr Leu
865 870 875 880
Asp Gly Ala Lys Lys Val Pro Glu Leu Thr Pro Gly Ser His Gln Pro
885 890 895
Gly Asn Leu His Ile Thr Lys Ser Gly Ser Asn Thr Asn Gln Pro Gln
900 905 910
Gly Asn Met Ser Ser Thr Leu His Ser Met Thr Pro Ile Gln Glu Glu
915 920 925
Ser Glu Pro Asp Asp Gln Lys Asp Asn Asp Asp Glu Ser Leu Thr Ser
930 935 940
Leu Asp Ser Glu Gly Asp Glu Asp Gly Glu Ser Ile Ser Glu Glu Asn
945 950 955 960
Thr Pro Thr Val Ala Pro Pro Ala Pro Val Tyr Lys Asp Thr Gly Val
965 970 975
Asp Thr Asn Gln Gln Asn Gly Pro Ser Ser Thr Val Asp Ser Gln Gly
980 985 990
Ser Glu Ser Glu Ala Leu Pro Ile Asn Ser Lys Lys Ser Ser Ala Leu
995 1000 1005
Glu Glu Thr Tyr Tyr His Leu Leu Lys Thr Gln Gly Pro Phe Glu Ala
1010 1015 1020
Ile Asn Tyr Tyr His Leu Met Ser Asp Glu Pro Ile Ala Phe Ser Thr
1025 1030 1035 1040
Glu Ser Gly Lys Glu Tyr Ile Phe Pro Asp Ser Leu Glu Glu Ala Tyr
1045 1050 1055
Pro Pro Trp Leu Ser Glu Lys Glu Ala Leu Glu Lys Glu Asn Arg Tyr
1060 1065 1070
Leu Val Ile Asp Gly Gln Gln Phe Leu Trp Pro Val Met Ser Leu Arg
1075 1080 1085
Asp Lys Phe Leu Ala Val Leu Gln His Asp Ala Lys Leu Thr Glu Ala
1090 1095 1100
Ile Thr Ser Thr Ser Ile Leu Lys Thr Gly Arg Arg Tyr Asp Asp Asp
1105 1110 1115 1120
Asn Asp Ile Pro Phe Pro Gly Pro Ile Asn Asp Asn Glu Asn Ser Gly
1125 1130 1135
Gln Asn Asp Asp Asp Pro Thr Asp Ser Gln Asp Thr Thr Ile Pro Asp
1140 1145 1150
Val Ile Ile Asp Pro Asn Asp Gly Gly Tyr Asn Asn Tyr Ser Asp Tyr
1155 1160 1165
Ala Asn Asp Ala Ala Ser Ala Pro Asp Asp Leu Val Leu Phe Asp Leu
1170 1175 1180
Glu Asp Glu Asp Asp Ala Asp Asn Pro Ala Gln Asn Thr Pro Glu Lys
1185 1190 1195 1200
Asn Asp Arg Pro Ala Thr Thr Lys Leu Arg Asn Gly Gln Asp Gln Asp
1205 1210 1215
Gly Asn Gln Gly Glu Thr Ala Ser Pro Arg Val Ala Pro Asn Gln Tyr
1220 1225 1230
Arg Asp Lys Pro Met Pro Gln Val Gln Asp Arg Ser Glu Asn His Asp
1235 1240 1245
Gln Thr Leu Gln Thr Gln Ser Arg Val Leu Thr Pro Ile Ser Glu Glu
1250 1255 1260
Ala Asp Pro Ser Asp His Asn Asp Gly Asp Asn Glu Ser Ile Pro Pro
1265 1270 1275 1280
Leu Glu Ser Asp Asp Glu Gly Ser Thr Asp Thr Thr Ala Ala Glu Thr
1285 1290 1295
Lys Pro Ala Thr Ala Pro Pro Ala Pro Val Tyr Arg Ser Ile Ser Val
1300 1305 1310
Asp Asp Ser Val Pro Ser Glu Asn Ile Pro Ala Gln Ser Asn Gln Thr
1315 1320 1325
Asn Asn Glu Asp Asn Val Arg Asn Asn Ala Gln Ser Glu Gln Ser Ile
1330 1335 1340
Ala Glu Met Tyr Gln His Ile Leu Lys Thr Gln Gly Pro Phe Asp Ala
1345 1350 1355 1360
Ile Leu Tyr Tyr His Met Met Lys Glu Glu Pro Ile Ile Phe Ser Thr
1365 1370 1375
Ser Asp Gly Lys Glu Tyr Thr Tyr Pro Asp Ser Leu Glu Asp Glu Tyr
1380 1385 1390
Pro Pro Trp Leu Ser Glu Lys Glu Ala Met Asn Glu Asp Asn Arg Phe
1395 1400 1405
Ile Thr Met Asp Gly Gln Gln Phe Tyr Trp Pro Val Met Asn His Arg
1410 1415 1420
Asn Lys Phe Met Ala Ile Leu Gln His His Arg Leu Val Leu Phe Asp
1425 1430 1435 1440
Leu Glu Asp Gly Asp Glu Asp Asp His Arg Pro Ser Ser Ser Ser Glu
1445 1450 1455
Asn Asn Asn Lys His Ser Leu Thr Gly Thr Asp Ser Asn Lys Thr Ser
1460 1465 1470
Asn Trp Asn Arg Asn Pro Thr Asn Met Pro Lys Lys Asp Ser Thr Gln
1475 1480 1485
Asn Asn Asp Asn Pro Ala Gln Arg Ala Gln Glu Tyr Ala Arg Asp Asn
1490 1495 1500
Ile Gln Asp Thr Pro Thr Pro His Arg Ala Leu Thr Pro Ile Ser Glu
1505 1510 1515 1520
Glu Thr Gly Ser Asn Gly His Asn Glu Asp Asp Ile Asp Ser Ile Pro
1525 1530 1535
Pro Leu Glu Ser Asp Glu Glu Asn Asn Thr Glu Thr Thr Ile Thr Thr
1540 1545 1550
Thr Lys Asn Thr Thr Ala Pro Pro Ala Pro Val Tyr Arg Ser Asn Ser
1555 1560 1565
Glu Lys Glu Pro Leu Pro Gln Glu Lys Ser Gln Lys Gln Pro Asn Gln
1570 1575 1580
Val Ser Gly Ser Glu Asn Thr Asp Asn Lys Pro His Ser Glu Gln Ser
1585 1590 1595 1600
Val Glu Glu Met
<210> 23
<211> 4815
<212> DNA
<213> artificial sequence
<220>
<223> synthetic construct
<400> 23
atgcccatgg ggtctctgca accgctggcc accttgtacc tgctggggat gctggtcgct 60
tcctgcctcg gagattctcg tcctcagaaa atctggatgg cgccgagtct cactgaatct 120
gacatggatt accacaagat cttgacagca ggtctgtccg ttcaacaggg gattgttcgg 180
caaagagtca tcccagtgta tcaagtaaac aatcttgaag aaatttgcca acttatcata 240
caggcctttg aagcaggtgt tgattttcaa gagagtgcgg acagtttcct tctcatgctt 300
tgtcttcatc atgcgtacca gggagattac aaacttttct tggaaagtgg cgcagtcaag 360
tatttggaag ggcacgggtt ccgttttgaa gtcaagaagc gtgatggagt gaagcgcctt 420
gaggaattgc tgccagcagt atctagtgga aaaaacatta agagaacact tgctgccatg 480
ccggaagagg agacaactga agctaatgcc ggtcagtttc tctcctttgc aagtctattc 540
cttccgaaat tggtagtagg agaaaaggct tgccttgaga aggttcaaag gcaaattcaa 600
gtacatgcag agcaaggact gatacaatat ccaacagctt ggcaatcagt aggacacatg 660
atggtgattt tccgtttgat gcgaacaaat tttctgatca aatttctcct aatacaccaa 720
gggatgcaca tggttgccgg gcatgatgcc aacgatgctg tgatttcaaa ttcagtggct 780
caagctcgtt tttcaggctt attgattgtc aaaacagtac ttgatcatat cctacaaaag 840
acagaacgag gagttcgtct ccatcctctt gcaaggaccg ccaaggtaaa aaatgaggtg 900
aactccttta aggctgcact cagctccctg gccaagcatg gagagtatgc tcctttcgcc 960
cgacttttga acctttctgg agtaaataat cttgagcatg gtcttttccc tcaactatcg 1020
gcaattgcac tcggagtcgc cacagcacac gggagtaccc tcgcaggagt aaatgttgga 1080
gaacagtatc aacaactcag agaggctgcc actgaggctg agaagcaact ccaacaatat 1140
gcagagtctc gcgaacttga ccatcttgga cttgatgatc aggaaaagaa aattcttatg 1200
aacttccatc agaaaaagaa cgaaatcagc ttccagcaaa caaacgctat ggtaactcta 1260
agaaaagagc gcctggccaa gctgacagaa gctatcactg ctgcgtcact gcccaaaaca 1320
agtggacatt acgatgatga tgacgacatt ccctttccag gacccatcaa tgatgacgac 1380
aatcctggcc atcaagatga tgatccgact gactcacagg atacgaccat tcccgatgtg 1440
gtggttgatc ctgatgatgg aagctacggc gaataccaga gttactcgga aaacggcatg 1500
aatgcaccag atgacttggt cctattcgat ctagacgagg acgacgagga cactaagcca 1560
gtgcctaata gatcgaccaa gggtggacaa cagaagaaca gtcaaaaggg ccagcatata 1620
gagggcagac agacacaatt caggccaatt caaaatgtcc caggccctca cagaacaatc 1680
caccacgcca gtgcgccact cacggacaat gacagaagaa atgaaccctc cggctcaacc 1740
agccctcgca tgctgacacc aattaacgaa gaggcagacc cactggacga tgccgacgac 1800
gagacgtcta gccttccgcc cttggagtca gatgatgaag agcaggacag ggacggaact 1860
tccaaccgca cacccactgt cgccccaccg gctcccgtat acagagatca ctctgaaaag 1920
aaagaactcc cgcaagacga gcaacaagat caggaccaca ctcaagaggc caggaaccag 1980
gacagtgaca acacccagtc agaacactcc cttgaggaga tgtatcgcca cattctaaga 2040
tcacaggggc catttgatgc tgttttgtat tatcatatga tgaaggatga gcctgtagtt 2100
ttcagtacca gtgatggcaa agagtacacg tatccagact cccttgaaga ggaatatcca 2160
ccatggctca ctgaaaaaga ggctatgaat gaagagaata gatttgttac attggatggt 2220
caacaatttt attggccggt gatgaatcac aagaataaat tcatggcaat cctgcaacat 2280
catcaggcta aattgaccga agccatcacg actgcatcga agatcaaggt tggagaccgt 2340
tatcctgatg acaatgatat tccatttccc gggccgatct atgatgacac tcaccccaat 2400
ccctctgatg acaatcctga tgattcacgt gatacaacta ttccaggtgg tgttgttgac 2460
ccgtatgatg atgagagtaa taattatcct gactacgagg attcggctga aggcaccaca 2520
ggagatcttg atctcttcaa tttggacgac gacgatgatg acagccgacc aggaccacca 2580
gacagggggc agaacaagga gagggcggcc cggacatatg gcctccaaga tccgaccttg 2640
gacggagcga aaaaggtgcc ggagttgacc ccaggttccc atcaaccagg caacctccac 2700
atcaccaagt cgggttcaaa caccaaccaa ccacaaggca atatgtcatc tactctccat 2760
agtatgaccc ctatacagga agaatcagag cccgatgatc aaaaagataa tgatgacgag 2820
agtctcacat cccttgactc tgaaggtgac gaagatggtg agagcatctc tgaggagaac 2880
accccaactg tagctccacc agcaccagtc tacaaagaca ctggagtaga cactaatcag 2940
cagaatggac caagcagtac tgtagatagt caaggttctg aaagtgaagc tctcccaatc 3000
aactctaaaa agagttccgc actagaagaa acatattatc atctcctaaa aacacagggt 3060
ccatttgagg caatcaatta ttatcaccta atgagtgatg aacccattgc ttttagcact 3120
gaaagtggca aggaatatat ctttccagac tcccttgaag aagcctaccc gccgtggttg 3180
agtgagaagg aggccttaga gaaggaaaat cgttatctgg tcattgatgg ccagcaattc 3240
ctctggccgg taatgagcct acgggacaag ttccttgccg ttcttcaaca tgacgccaaa 3300
ttgaccgaag ctattacttc cacctctatc ctcaaaacag gaaggcggta tgatgatgac 3360
aatgacatac cctttccagg gccaatcaat gataacgaga actctggtca gaacgatgac 3420
gatccaacag actcccagga taccacaatc ccggatgtaa taatcgatcc aaacgatggt 3480
gggtataata attacagcga ttatgcaaat gatgctgcaa gtgctcctga tgacctagtt 3540
ctttttgacc ttgaggacga ggatgatgct gataacccgg ctcaaaacac gccagaaaaa 3600
aatgatagac cagcaacaac aaagctgaga aatggacagg accaggatgg aaaccaaggc 3660
gaaactgcat ccccacgggt agcccccaac caatacagag acaagccaat gccacaagta 3720
caggacagat ccgaaaatca tgaccaaacc cttcaaacac agtccagggt tttgactcct 3780
atcagcgagg aagcagaccc cagcgaccac aacgatggtg acaatgaaag cattcctccc 3840
ctggaatcag acgacgaggg tagcactgat actactgcag cagaaacaaa gcctgccact 3900
gcacctcccg ctcccgtcta ccgaagtatc tccgtagatg attctgtccc ctcagagaac 3960
attcccgcac agtccaatca aacgaacaat gaggacaatg tcaggaacaa tgctcagtcg 4020
gagcaatcca ttgcagaaat gtatcaacat atcttgaaaa cacaaggacc ttttgatgcc 4080
atcctttact accatatgat gaaagaagag cccatcattt tcagcactag tgatgggaag 4140
gagtatacat atccagactc tcttgaagat gagtatccac cctggctcag cgagaaggaa 4200
gccatgaacg aagacaatag attcataacc atggatggtc agcagtttta ctggcctgtg 4260
atgaatcata gaaataaatt catggcaatc ctccagcatc acaggcttgt tctttttgac 4320
cttgaagatg gtgacgagga tgatcaccga ccgtcaagtt catcagagaa caacaacaaa 4380
cacagtctta caggaactga cagtaacaaa acaagtaact ggaatcgaaa cccgactaat 4440
atgccaaaga aagactccac acaaaacaat gacaatcctg cacagcgggc tcaagaatac 4500
gccagggata acatccagga tacaccaaca ccccatcgag ctctaactcc catcagcgaa 4560
gaaaccggct ccaatggtca caatgaagat gacattgata gcatccctcc tttggaatca 4620
gacgaagaaa acaacactga gacaaccatt accaccacaa aaaataccac tgctccacca 4680
gcacctgttt atcggagtaa ttcagaaaag gagcccctcc cgcaagaaaa atcccagaag 4740
caaccaaacc aagtgagtgg tagtgagaat accgacaata aacctcactc agagcaatca 4800
gtggaagaaa tgtaa 4815
<210> 24
<211> 744
<212> DNA
<213> artificial sequence
<220>
<223> synthetic construct
<400> 24
atgcccatgg ggtctctgca accgctggcc accttgtacc tgctggggat gctggtcgct 60
tcctgcctcg gaagagtcca accaacagaa tctattgtta gatttcctaa tattacaaac 120
ttgtgccctt ttgatgaagt ttttaacgcc accagatttg catctgttta tgcttggaac 180
aggaagagaa tcagcaactg tgttgctgat tattctgtcc tatataatct cgcaccattt 240
ttcactttta agtgttatgg agtgtctcct actaaattaa atgatctctg ctttactaat 300
gtctatgcag attcatttgt aattagaggt gatgaagtca gacaaatcgc tccagggcaa 360
actggaaaca ttgctgatta taattataaa ttaccagatg attttacagg ctgcgttata 420
gcttggaatt ctaacaagct tgattctaag gttagtggta attataatta cctgtataga 480
ttgtttagga agtctaatct caaacctttt gagagagata tttcaactga aatctatcag 540
gccggtaaca aaccttgtaa tggtgttgca ggttttaatt gttactttcc tttacgatca 600
tatagtttcc gacccactta tggtgttggt caccaaccat acagagtagt agtactttct 660
tttgaacttc tacatgcacc agcaactgtt tgtggaccta aaaagtctac taatttggtt 720
aaaaacaaat gtgtcaattt ctaa 744
<210> 25
<211> 247
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 25
Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly
1 5 10 15
Met Leu Val Ala Ser Cys Leu Gly Arg Val Gln Pro Thr Glu Ser Ile
20 25 30
Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp Glu Val Phe
35 40 45
Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile
50 55 60
Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu Ala Pro Phe
65 70 75 80
Phe Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu
85 90 95
Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu
100 105 110
Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn
115 120 125
Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser
130 135 140
Asn Lys Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr Leu Tyr Arg
145 150 155 160
Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr
165 170 175
Glu Ile Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val Ala Gly Phe
180 185 190
Asn Cys Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro Thr Tyr Gly
195 200 205
Val Gly His Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu
210 215 220
His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val
225 230 235 240
Lys Asn Lys Cys Val Asn Phe
245
<210> 26
<211> 4056
<212> DNA
<213> artificial sequence
<220>
<223> synthetic construct
<400> 26
atgcccatgg ggtctctgca accgctggcc accttgtacc tgctggggat gctggtcgct 60
tcctgcctcg gaagagtcca accaacagaa tctattgtta gatttcctaa tattacaaac 120
ttgtgccctt ttggtgaagt ttttaacgcc accagatttg catctgttta tgcttggaac 180
aggaagagaa tcagcaactg tgttgctgat tattctgtcc tatataattc cgcatcattt 240
tccactttta agtgttatgg agtgtctcct actaaattaa atgatctctg ctttactaat 300
gtctatgcag attcatttgt aattagaggt gatgaagtca gacaaatcgc tccagggcaa 360
actggaaaga ttgctgatta taattataaa ttaccagatg attttacagg ctgcgttata 420
gcttggaatt ctaacaatct tgattctaag gttggtggta attataatta ccggtataga 480
ttgtttagga agtctaatct caaacctttt gagagagata tttcaactga aatctatcag 540
gccggtagca aaccttgtaa tggtgttgaa ggttttaatt gttactttcc tttacaatca 600
tatggtttcc aacccactaa tggtgttggt taccaaccat acagagtagt agtactttct 660
tttgaacttc tacatgcacc agcaactgtt tgtggaccta aaaagtctac taatttggtt 720
aaaaacaaat gtgtcaattt ctcaggtggt ggcggttcag gcggaggtgg ctctggcggt 780
ggcggatcga tgtctgataa tggaccccaa aatcagcgaa atgcaccccg cattacgttt 840
ggtggaccct cagattcaac tggcagtaac cagaatggag aacgcagtgg ggcgcgatca 900
aaacaacgtc ggccccaagg tttacccaat aatactgcgt cttggttcac cgctctcact 960
caacatggca aggaaggcct taaattccct cgaggacaag gcgttccaat taacaccaat 1020
agcagtccag atgaccaaat tggctactac cgaagagcta ccagacgaat tcgtggtggt 1080
gacggtaaaa tgaaagatct cagtccaaga tggtatttct actacctagg aactgggcca 1140
gaagctggac ttccctatgg tgctaacaaa gacggcatca tatgggttgc aactgaggga 1200
gccttgaata caccaaaaga tcacattggc acccgcaatc ctgctaacaa tgctgcaatc 1260
gtgctacaac ttcctcaagg aacaacattg ccaaaaggct tctacgcaga agggagcaga 1320
ggcggcagtc aagcctcttc tcgttcctca tcacgtagtc gcaacagttc aagaaattca 1380
actccaggca gcagtatggg aacttctcct gctagaatgg ctggcaatgg ctgtgatgct 1440
gctcttgctt tgctgctgct tgacagattg aaccagcttg agagcaaaat gtctggtaaa 1500
ggccaacaac aacaaggcca aactgtcact aagaaatctg ctgctgaggc ttctaagaag 1560
cctcggcaaa aacgtactgc cactaaagca tacaatgtaa cacaagcttt cggcagacgt 1620
ggtccagaac aaacccaagg aaattttggg gaccaggaac taatcagaca aggaactgat 1680
tacaaacatt ggccgcaaat tgcacaattt gcccccagcg cttcagcgtt cttcggaatg 1740
tcgcgcattg gcatggaagt cacaccttcg ggaacgtggt tgacctacac aggtgccatc 1800
aaattggatg acaaagatcc aaatttcaaa gatcaagtca ttttgctgaa taagcatatt 1860
gacgcataca aaacattccc accaacagag cctaaaaagg acaaaaagaa gaaggcttat 1920
gaaactcaag ccttaccgca gagacagaag aaacagcaaa ctgtgactct tcttcctgct 1980
gcagatttgg atgatttctc caaacaattg caacaatcca tgagcagtgc tgactcaact 2040
caggccatgg catcccctgc tgcacctcgt gctgtttcct ttgccgataa caatgatata 2100
acaaatacaa acctatctcg aggtagagga cgtaatccaa aaccacgagc tgcaccaaat 2160
aacactgtct cttggtacac tgggcttacc caacacggga aagtccctct tacctttcca 2220
cctgggcagg gtgtacctct taatgccaat tctacccctg cgcaaaatgc tgggtattgg 2280
cggagacagg acagaaaaat taataccggg aatggaatta agcaactggc tcccaggtgg 2340
tacttctact acactggaac tggacccgaa gcagcactcc cattccgggc tgttaaggat 2400
ggcatcgttt gggtccatga agatggcgcc actgatgctc cttcaacttt tgggacgcgg 2460
aaccctaaca atgattcagc tattgttaca caattcgcgc ccggtactaa gcttcctaaa 2520
aacttccaca ttgaggggac tggaggcaat agtcaatcat cttcaagagc ctctagctta 2580
agcagaaact cttccagatc tagttcacaa ggttcaagat caggaaactc tacccgcggc 2640
acttctccag gtccatctgg aatcggagca gtaggaggtg atctacttta ccttgatctt 2700
ctgaacagac tacaagccct tgagtctggc aaagtaaagc aatcgcagcc aaaagtaatc 2760
actaagaaag atgctgctgc tgctaaaaat aagatgcgcc acaagcgcac ttccaccaaa 2820
agtttcaaca tggtgcaagc ttttggtctt cgcggaccag gagacctcca gggaaacttt 2880
ggtgatcttc aattgaataa actcggcact gaggacccac gttggcccca aattgctgag 2940
cttgctccta cagccagtgc ttttatgggt atgtcgcaat ttaaacttac ccatcagaac 3000
aatgatgatc atggcaaccc tgtgtacttc cttcggtaca gtggagccat taaacttgac 3060
ccaaagaatc ccaactacaa taagtggttg gagcttcttg agcaaaatat tgatgcctac 3120
aaaaccttcc ctaagaagga aaagaaacaa aaggcaccaa aagaagaatc aacagaccaa 3180
atgtctgaac ctccaaagga gcagcgtgtg caaggtagca tcactcagcg cactcgcacc 3240
cgtccaagtg ttcagcctgg tccaatgatt gatgttaaca ctgattctgg tggcggtggc 3300
tcgggcggag gtgggtcggg tggcggcgga tcagaagcaa aaccttctgg ctcagttgtg 3360
gaacaggctg aaggtgttga atgtgatttt tcacctcttc tgtctggcac acctcctcag 3420
gtttataatt tcaagcgttt ggtttttacc aattgcaatt ataatcttac caaattgctt 3480
tcactttttt ctgtgaatga ttttacttgt agtcaaatat ctccagcagc aattgctagc 3540
aactgttatt cttcactgat tttggattac ttttcatacc cacttagtat gaaatccgat 3600
ctcagtgtta gttctgctgg tccaatatcc cagtttaatt ataaacagtc cttttctaat 3660
cccacatgtt tgattttagc gactgttcct cataacctta ctactattac taagcctctt 3720
aagtacagct atattaacaa gtgctctcgt cttctttctg atgatcgtac tgaagtacct 3780
cagttagtga acgctaatca atactcaccc tgtgtatcca ttgtcccatc cactgtgtgg 3840
gaagacggtg attattatag gaaacaacta tctccacttg aaggtggtgg ctggcttgtt 3900
gctagtggct caactgttgc catgactgag caattacaga tgggctttgg tattacagtt 3960
caatatggta cagacaccaa tagtgtttgc cccaagcttg aatttgctaa tgacacaaaa 4020
attgcctctc aattaggcaa ttgcgtggaa tattag 4056
<210> 27
<211> 1351
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 27
Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly
1 5 10 15
Met Leu Val Ala Ser Cys Leu Gly Arg Val Gln Pro Thr Glu Ser Ile
20 25 30
Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe
35 40 45
Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile
50 55 60
Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe
65 70 75 80
Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu
85 90 95
Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu
100 105 110
Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn
115 120 125
Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser
130 135 140
Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Arg Tyr Arg
145 150 155 160
Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr
165 170 175
Glu Ile Tyr Gln Ala Gly Ser Lys Pro Cys Asn Gly Val Glu Gly Phe
180 185 190
Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly
195 200 205
Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu
210 215 220
His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val
225 230 235 240
Lys Asn Lys Cys Val Asn Phe Ser Gly Gly Gly Gly Ser Gly Gly Gly
245 250 255
Gly Ser Gly Gly Gly Gly Ser Met Ser Asp Asn Gly Pro Gln Asn Gln
260 265 270
Arg Asn Ala Pro Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly
275 280 285
Ser Asn Gln Asn Gly Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg
290 295 300
Pro Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr
305 310 315 320
Gln His Gly Lys Glu Gly Leu Lys Phe Pro Arg Gly Gln Gly Val Pro
325 330 335
Ile Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg
340 345 350
Ala Thr Arg Arg Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser
355 360 365
Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu
370 375 380
Pro Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly
385 390 395 400
Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn
405 410 415
Asn Ala Ala Ile Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys
420 425 430
Gly Phe Tyr Ala Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg
435 440 445
Ser Ser Ser Arg Ser Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser
450 455 460
Ser Met Gly Thr Ser Pro Ala Arg Met Ala Gly Asn Gly Cys Asp Ala
465 470 475 480
Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys
485 490 495
Met Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys
500 505 510
Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr
515 520 525
Lys Ala Tyr Asn Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln
530 535 540
Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp
545 550 555 560
Tyr Lys His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala
565 570 575
Phe Phe Gly Met Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr
580 585 590
Trp Leu Thr Tyr Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn
595 600 605
Phe Lys Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys
610 615 620
Thr Phe Pro Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Tyr
625 630 635 640
Glu Thr Gln Ala Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr
645 650 655
Leu Leu Pro Ala Ala Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln
660 665 670
Ser Met Ser Ser Ala Asp Ser Thr Gln Ala Met Ala Ser Pro Ala Ala
675 680 685
Pro Arg Ala Val Ser Phe Ala Asp Asn Asn Asp Ile Thr Asn Thr Asn
690 695 700
Leu Ser Arg Gly Arg Gly Arg Asn Pro Lys Pro Arg Ala Ala Pro Asn
705 710 715 720
Asn Thr Val Ser Trp Tyr Thr Gly Leu Thr Gln His Gly Lys Val Pro
725 730 735
Leu Thr Phe Pro Pro Gly Gln Gly Val Pro Leu Asn Ala Asn Ser Thr
740 745 750
Pro Ala Gln Asn Ala Gly Tyr Trp Arg Arg Gln Asp Arg Lys Ile Asn
755 760 765
Thr Gly Asn Gly Ile Lys Gln Leu Ala Pro Arg Trp Tyr Phe Tyr Tyr
770 775 780
Thr Gly Thr Gly Pro Glu Ala Ala Leu Pro Phe Arg Ala Val Lys Asp
785 790 795 800
Gly Ile Val Trp Val His Glu Asp Gly Ala Thr Asp Ala Pro Ser Thr
805 810 815
Phe Gly Thr Arg Asn Pro Asn Asn Asp Ser Ala Ile Val Thr Gln Phe
820 825 830
Ala Pro Gly Thr Lys Leu Pro Lys Asn Phe His Ile Glu Gly Thr Gly
835 840 845
Gly Asn Ser Gln Ser Ser Ser Arg Ala Ser Ser Leu Ser Arg Asn Ser
850 855 860
Ser Arg Ser Ser Ser Gln Gly Ser Arg Ser Gly Asn Ser Thr Arg Gly
865 870 875 880
Thr Ser Pro Gly Pro Ser Gly Ile Gly Ala Val Gly Gly Asp Leu Leu
885 890 895
Tyr Leu Asp Leu Leu Asn Arg Leu Gln Ala Leu Glu Ser Gly Lys Val
900 905 910
Lys Gln Ser Gln Pro Lys Val Ile Thr Lys Lys Asp Ala Ala Ala Ala
915 920 925
Lys Asn Lys Met Arg His Lys Arg Thr Ser Thr Lys Ser Phe Asn Met
930 935 940
Val Gln Ala Phe Gly Leu Arg Gly Pro Gly Asp Leu Gln Gly Asn Phe
945 950 955 960
Gly Asp Leu Gln Leu Asn Lys Leu Gly Thr Glu Asp Pro Arg Trp Pro
965 970 975
Gln Ile Ala Glu Leu Ala Pro Thr Ala Ser Ala Phe Met Gly Met Ser
980 985 990
Gln Phe Lys Leu Thr His Gln Asn Asn Asp Asp His Gly Asn Pro Val
995 1000 1005
Tyr Phe Leu Arg Tyr Ser Gly Ala Ile Lys Leu Asp Pro Lys Asn Pro
1010 1015 1020
Asn Tyr Asn Lys Trp Leu Glu Leu Leu Glu Gln Asn Ile Asp Ala Tyr
1025 1030 1035 1040
Lys Thr Phe Pro Lys Lys Glu Lys Lys Gln Lys Ala Pro Lys Glu Glu
1045 1050 1055
Ser Thr Asp Gln Met Ser Glu Pro Pro Lys Glu Gln Arg Val Gln Gly
1060 1065 1070
Ser Ile Thr Gln Arg Thr Arg Thr Arg Pro Ser Val Gln Pro Gly Pro
1075 1080 1085
Met Ile Asp Val Asn Thr Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly
1090 1095 1100
Gly Ser Gly Gly Gly Gly Ser Glu Ala Lys Pro Ser Gly Ser Val Val
1105 1110 1115 1120
Glu Gln Ala Glu Gly Val Glu Cys Asp Phe Ser Pro Leu Leu Ser Gly
1125 1130 1135
Thr Pro Pro Gln Val Tyr Asn Phe Lys Arg Leu Val Phe Thr Asn Cys
1140 1145 1150
Asn Tyr Asn Leu Thr Lys Leu Leu Ser Leu Phe Ser Val Asn Asp Phe
1155 1160 1165
Thr Cys Ser Gln Ile Ser Pro Ala Ala Ile Ala Ser Asn Cys Tyr Ser
1170 1175 1180
Ser Leu Ile Leu Asp Tyr Phe Ser Tyr Pro Leu Ser Met Lys Ser Asp
1185 1190 1195 1200
Leu Ser Val Ser Ser Ala Gly Pro Ile Ser Gln Phe Asn Tyr Lys Gln
1205 1210 1215
Ser Phe Ser Asn Pro Thr Cys Leu Ile Leu Ala Thr Val Pro His Asn
1220 1225 1230
Leu Thr Thr Ile Thr Lys Pro Leu Lys Tyr Ser Tyr Ile Asn Lys Cys
1235 1240 1245
Ser Arg Leu Leu Ser Asp Asp Arg Thr Glu Val Pro Gln Leu Val Asn
1250 1255 1260
Ala Asn Gln Tyr Ser Pro Cys Val Ser Ile Val Pro Ser Thr Val Trp
1265 1270 1275 1280
Glu Asp Gly Asp Tyr Tyr Arg Lys Gln Leu Ser Pro Leu Glu Gly Gly
1285 1290 1295
Gly Trp Leu Val Ala Ser Gly Ser Thr Val Ala Met Thr Glu Gln Leu
1300 1305 1310
Gln Met Gly Phe Gly Ile Thr Val Gln Tyr Gly Thr Asp Thr Asn Ser
1315 1320 1325
Val Cys Pro Lys Leu Glu Phe Ala Asn Asp Thr Lys Ile Ala Ser Gln
1330 1335 1340
Leu Gly Asn Cys Val Glu Tyr
1345 1350
<210> 28
<211> 4098
<212> DNA
<213> artificial sequence
<220>
<223> synthetic construct
<400> 28
atgcctatgg gcagcctgca gccactggct acactgtacc tgctgggcat gctggtggcc 60
tcttgtctgg gcataggaga gtgtccaaag tatgtcagga gtgcaaaatt aaggatggtt 120
acaggactaa ggaacatccc atccattcaa tccagaggtt tgtttggagc cattgccggt 180
ttcattgaag gggggtggac tggaatggta gatgggtggt atggttatca tcatcagaat 240
gagcaaggat ctggctatgc tgcagatcaa aaaagtacac aaaatgccat taacgggatt 300
acaaacaagg tgaattctgt aattgagaaa atgaacactc aattcacagc tgtgggcaaa 360
gaattcaaca aattggaaag aaggatggaa aacttaaata aaaaagttga tgatgggttt 420
ctagacattt ggacatataa tgcagaattg ttggttctac tggaaaatga aaggactttg 480
gatttccatg actccgccag ccaaggcact aagagaagct acgagcagat ggaaaccgga 540
ggcgaacggc agaacgccac agagatcaga gcctctgtgg gccgtatggt cggcggcatc 600
ggcagattct acatccagat gtgcaccgaa ctgaagctga gcgactacga gggccgcctg 660
atccagaaca gcatcacaat cgagagaatg gtgctgtccg cctttgacga gcggagaaac 720
aaatacctgg aagagcaccc tagcgccgga aaagatccta agaaaaccgg cggacctatc 780
tacagaagaa gagatggtaa gtgggtgaga gagctgattc tgtacgataa ggaagagatt 840
cgaagaatct ggagacaggc caacaacggc gaggatgcca ccgcaggcct gacacacctg 900
atgatctggc acagcaacct gaacgatgcg acctaccagc gcacgcgggc cctggtcaga 960
accggcatgg atcctcggat gtgtagcctg atgcagggca gcacactgcc aagacggagt 1020
ggggccgccg gcgctgcagt gaagggcgtc ggaaccatgg tgatggagct gatccggatg 1080
ataaagcggg gcatcaacga cagaaacttc tggcgaggcg agaacggccg aagaacccgg 1140
atcgcctacg agagaatgtg caacatcctg aaaggaaaat tccagaccgc cgcccagcgg 1200
gccatgatgg accaggtgcg cgagagcaga aaccccggca atgccgagat cgaggacctg 1260
atcttcctgg ccagaagcgc cctcattctt agaggctctg tggcccacaa gagctgtctg 1320
cctgcctgtg tgtacggcct ggcagtggcc tcaggctacg acttcgagcg ggaaggatac 1380
agtctggtgg gcatcgaccc tttcagactc ctgcagaata gccaggtgtt tagcctgatc 1440
agaccaaacg aaaaccccgc ccataagagc cagctggtgt ggatggcctg ccacagcgcc 1500
gcctttgagg atctgagagt gagctctttt atcagaggca cccgggtggt tccacgaggt 1560
caactgtcta caagaggtgt gcagatcgcc agcaacgaga acatggagac catggatagc 1620
agcaccctgg aactgagatc cagatactgg gccatcagga cacggagcgg cggcaccacc 1680
aatcagcagc gcgccagcgc cggccagatc tctgtccagc ctacgtttag cgtgcagcgg 1740
aatttgccct tcgaacgcgc cacaatcatg gctgctttca ccggcaatac agagggcaga 1800
accagcgata tgagaacaga aattatccgt atgatggagt ccgcaaaacc tgaggacgtg 1860
tccttccaag gcagaggcgt gttcgagctg agcgacgaga aggccaccaa ccctatcgtg 1920
cctagcttcg atatgtctaa tgagggcagc tactttttcg gagataacgc cgaagagtac 1980
gacaacatgt ctaatatgga tatcgacggc attaacaccg gcaccatcga caaaacccct 2040
gaggagatca cccctggcac cagcggcaca acccggccca tcatccgccc cgctacactg 2100
gctccaccta gcaacaagcg gaccagaaat ccctcgccag aaagagccac aacctccagc 2160
gaggacgacg tgggacggaa gacacaaaag aagcagaccc ctacagagat caagaagtct 2220
gtttacaaca tggtggtgaa actgggcgag ttctacaacc agatgatggt gaaggccggc 2280
ctgaacgacg atatggaaag aaatctgatc cagaacgccc acgccgtgga gcggattctg 2340
ctggccgcca ccgatgataa gaagaccgaa ttccagaaaa agaaaaacgc cagagacgtg 2400
aaggaaggca aggaagagat cgaccacaac aagacaggcg gcacattcta caagatggtc 2460
cgggacgaca agaccatcta cttcagccct atccggataa cattcctgaa agaagaagtg 2520
aagaccatgt acaaaaccac aatgggctct gacggcttca gcggcctgaa tcacatcatg 2580
atcggccact ctcaaatgaa cgatgtgtgc ttccagagaa gcaaggctct gaagcgcgtg 2640
ggcctggatc ctagcctgat ctctaccttc gccggcagca ccatccccag aagatcgggc 2700
gctaccggcg tggctatcaa gggaggaggc acactggtgg ctgaagccat cagattcatc 2760
ggaagagcca tggccgacag aggactcctg agagatatca aagccaaaac cgcctacgaa 2820
aaaatcctgc tgaacctgaa gaacaagtgc agcgcgcctc aacagaaggc cctggtggac 2880
caggttatcg gctctagaaa ccctggaatc gccgatatcg aggacctgac actgctggcc 2940
agatctatgg tggtggtgag accctccgtg gccagcaagg tggtgctgcc tatcagcatc 3000
tacgccaaga tccctcagct gggatttaac gtggaagaat acagcatggt tggttatgag 3060
gccatggccc tgtacaacat ggccacacct gtgtccatcc tgagaatggg cgacgatgcc 3120
aaagacaaga gccagctgtt cttcatgagc tgcttcggcg ctgcctatga ggacctgaga 3180
gtgctgtccg ctcttacagg aacagagttc aagcctagga gcgcactgaa gtgcaagggc 3240
ttccacgtgc ccgccaagga acaggtggaa ggcatgggag ctgctctgat gtccatcaag 3300
ctgcaatttt gggctcctat gacccggagc ggcggaaatg aggtgggtgg cgacggaggc 3360
agcggacaga tttcttgcag ccccgtattt gccgtggaga gaccaatcgc cctgtccaag 3420
caggccgtga gaagaatgct gagcatgaac atcgagggcc gggacgccga cgtgaagggc 3480
aacctgttga agatgatgaa cgacagcatg gccaagaaga ccagtggcaa tgccttcatc 3540
ggcaagaaga tgttccagat ctccgacaag aacaagacca accccgtgga aatccccatc 3600
aagcagacaa tccctaactt cttcttcggc agagacaccg ccgaagacta tgacgacctg 3660
gactacatag gaaattgccc aatatgggtg aaaacacctt tgaagcttgc caatggaacc 3720
aaatatagac ctcctgcaaa actattaaag gaaaggggtt tcttcggagc tattgctggt 3780
ttcctagaag gaggatggga aggaatgatt gcaggctggc acggatacac atctcacgga 3840
gcacatggag tggcagtggc ggcggacctt aagagtacgc aagaagctat aaacaagata 3900
acaaaaaatc tcaattcttt gagtgagcta gaagtaaaga atcttcaaag actaagtggt 3960
gccatggatg aactccacaa cgaaatactc gagctggatg agaaagtgga tgatctcaga 4020
gctgacacta taagctcgca aatagaactt gcagtcttgc tttccaacga aggaataata 4080
aacagtgaag atgagtga 4098
<210> 29
<211> 1365
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 29
Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly
1 5 10 15
Met Leu Val Ala Ser Cys Leu Gly Ile Gly Glu Cys Pro Lys Tyr Val
20 25 30
Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn Ile Pro Ser
35 40 45
Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly
50 55 60
Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn
65 70 75 80
Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala
85 90 95
Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn
100 105 110
Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Arg Arg
115 120 125
Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp
130 135 140
Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu
145 150 155 160
Asp Phe His Asp Ser Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln
165 170 175
Met Glu Thr Gly Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser
180 185 190
Val Gly Arg Met Val Gly Gly Ile Gly Arg Phe Tyr Ile Gln Met Cys
195 200 205
Thr Glu Leu Lys Leu Ser Asp Tyr Glu Gly Arg Leu Ile Gln Asn Ser
210 215 220
Ile Thr Ile Glu Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn
225 230 235 240
Lys Tyr Leu Glu Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr
245 250 255
Gly Gly Pro Ile Tyr Arg Arg Arg Asp Gly Lys Trp Val Arg Glu Leu
260 265 270
Ile Leu Tyr Asp Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn
275 280 285
Asn Gly Glu Asp Ala Thr Ala Gly Leu Thr His Leu Met Ile Trp His
290 295 300
Ser Asn Leu Asn Asp Ala Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg
305 310 315 320
Thr Gly Met Asp Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu
325 330 335
Pro Arg Arg Ser Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr
340 345 350
Met Val Met Glu Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg
355 360 365
Asn Phe Trp Arg Gly Glu Asn Gly Arg Arg Thr Arg Ile Ala Tyr Glu
370 375 380
Arg Met Cys Asn Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg
385 390 395 400
Ala Met Met Asp Gln Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu
405 410 415
Ile Glu Asp Leu Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly
420 425 430
Ser Val Ala His Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Ala
435 440 445
Val Ala Ser Gly Tyr Asp Phe Glu Arg Glu Gly Tyr Ser Leu Val Gly
450 455 460
Ile Asp Pro Phe Arg Leu Leu Gln Asn Ser Gln Val Phe Ser Leu Ile
465 470 475 480
Arg Pro Asn Glu Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala
485 490 495
Cys His Ser Ala Ala Phe Glu Asp Leu Arg Val Ser Ser Phe Ile Arg
500 505 510
Gly Thr Arg Val Val Pro Arg Gly Gln Leu Ser Thr Arg Gly Val Gln
515 520 525
Ile Ala Ser Asn Glu Asn Met Glu Thr Met Asp Ser Ser Thr Leu Glu
530 535 540
Leu Arg Ser Arg Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Thr Thr
545 550 555 560
Asn Gln Gln Arg Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe
565 570 575
Ser Val Gln Arg Asn Leu Pro Phe Glu Arg Ala Thr Ile Met Ala Ala
580 585 590
Phe Thr Gly Asn Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Ile
595 600 605
Ile Arg Met Met Glu Ser Ala Lys Pro Glu Asp Val Ser Phe Gln Gly
610 615 620
Arg Gly Val Phe Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val
625 630 635 640
Pro Ser Phe Asp Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn
645 650 655
Ala Glu Glu Tyr Asp Asn Met Ser Asn Met Asp Ile Asp Gly Ile Asn
660 665 670
Thr Gly Thr Ile Asp Lys Thr Pro Glu Glu Ile Thr Pro Gly Thr Ser
675 680 685
Gly Thr Thr Arg Pro Ile Ile Arg Pro Ala Thr Leu Ala Pro Pro Ser
690 695 700
Asn Lys Arg Thr Arg Asn Pro Ser Pro Glu Arg Ala Thr Thr Ser Ser
705 710 715 720
Glu Asp Asp Val Gly Arg Lys Thr Gln Lys Lys Gln Thr Pro Thr Glu
725 730 735
Ile Lys Lys Ser Val Tyr Asn Met Val Val Lys Leu Gly Glu Phe Tyr
740 745 750
Asn Gln Met Met Val Lys Ala Gly Leu Asn Asp Asp Met Glu Arg Asn
755 760 765
Leu Ile Gln Asn Ala His Ala Val Glu Arg Ile Leu Leu Ala Ala Thr
770 775 780
Asp Asp Lys Lys Thr Glu Phe Gln Lys Lys Lys Asn Ala Arg Asp Val
785 790 795 800
Lys Glu Gly Lys Glu Glu Ile Asp His Asn Lys Thr Gly Gly Thr Phe
805 810 815
Tyr Lys Met Val Arg Asp Asp Lys Thr Ile Tyr Phe Ser Pro Ile Arg
820 825 830
Ile Thr Phe Leu Lys Glu Glu Val Lys Thr Met Tyr Lys Thr Thr Met
835 840 845
Gly Ser Asp Gly Phe Ser Gly Leu Asn His Ile Met Ile Gly His Ser
850 855 860
Gln Met Asn Asp Val Cys Phe Gln Arg Ser Lys Ala Leu Lys Arg Val
865 870 875 880
Gly Leu Asp Pro Ser Leu Ile Ser Thr Phe Ala Gly Ser Thr Ile Pro
885 890 895
Arg Arg Ser Gly Ala Thr Gly Val Ala Ile Lys Gly Gly Gly Thr Leu
900 905 910
Val Ala Glu Ala Ile Arg Phe Ile Gly Arg Ala Met Ala Asp Arg Gly
915 920 925
Leu Leu Arg Asp Ile Lys Ala Lys Thr Ala Tyr Glu Lys Ile Leu Leu
930 935 940
Asn Leu Lys Asn Lys Cys Ser Ala Pro Gln Gln Lys Ala Leu Val Asp
945 950 955 960
Gln Val Ile Gly Ser Arg Asn Pro Gly Ile Ala Asp Ile Glu Asp Leu
965 970 975
Thr Leu Leu Ala Arg Ser Met Val Val Val Arg Pro Ser Val Ala Ser
980 985 990
Lys Val Val Leu Pro Ile Ser Ile Tyr Ala Lys Ile Pro Gln Leu Gly
995 1000 1005
Phe Asn Val Glu Glu Tyr Ser Met Val Gly Tyr Glu Ala Met Ala Leu
1010 1015 1020
Tyr Asn Met Ala Thr Pro Val Ser Ile Leu Arg Met Gly Asp Asp Ala
1025 1030 1035 1040
Lys Asp Lys Ser Gln Leu Phe Phe Met Ser Cys Phe Gly Ala Ala Tyr
1045 1050 1055
Glu Asp Leu Arg Val Leu Ser Ala Leu Thr Gly Thr Glu Phe Lys Pro
1060 1065 1070
Arg Ser Ala Leu Lys Cys Lys Gly Phe His Val Pro Ala Lys Glu Gln
1075 1080 1085
Val Glu Gly Met Gly Ala Ala Leu Met Ser Ile Lys Leu Gln Phe Trp
1090 1095 1100
Ala Pro Met Thr Arg Ser Gly Gly Asn Glu Val Gly Gly Asp Gly Gly
1105 1110 1115 1120
Ser Gly Gln Ile Ser Cys Ser Pro Val Phe Ala Val Glu Arg Pro Ile
1125 1130 1135
Ala Leu Ser Lys Gln Ala Val Arg Arg Met Leu Ser Met Asn Ile Glu
1140 1145 1150
Gly Arg Asp Ala Asp Val Lys Gly Asn Leu Leu Lys Met Met Asn Asp
1155 1160 1165
Ser Met Ala Lys Lys Thr Ser Gly Asn Ala Phe Ile Gly Lys Lys Met
1170 1175 1180
Phe Gln Ile Ser Asp Lys Asn Lys Thr Asn Pro Val Glu Ile Pro Ile
1185 1190 1195 1200
Lys Gln Thr Ile Pro Asn Phe Phe Phe Gly Arg Asp Thr Ala Glu Asp
1205 1210 1215
Tyr Asp Asp Leu Asp Tyr Ile Gly Asn Cys Pro Ile Trp Val Lys Thr
1220 1225 1230
Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg Pro Pro Ala Lys Leu
1235 1240 1245
Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu Glu Gly
1250 1255 1260
Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly Tyr Thr Ser His Gly
1265 1270 1275 1280
Ala His Gly Val Ala Val Ala Ala Asp Leu Lys Ser Thr Gln Glu Ala
1285 1290 1295
Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu Ser Glu Leu Glu Val
1300 1305 1310
Lys Asn Leu Gln Arg Leu Ser Gly Ala Met Asp Glu Leu His Asn Glu
1315 1320 1325
Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu Arg Ala Asp Thr Ile
1330 1335 1340
Ser Ser Gln Ile Glu Leu Ala Val Leu Leu Ser Asn Glu Gly Ile Ile
1345 1350 1355 1360
Asn Ser Glu Asp Glu
1365

Claims (107)

1. A composition for stimulating an immune response against a coronavirus in a mammalian subject, the composition comprising an excipient and a messenger RNA (mRNA) comprising an Open Reading Frame (ORF) encoding a fusion protein, wherein the ORF comprises from 5 'to 3':
(i) A nucleotide sequence encoding a mammalian signal peptide; and
(ii) Nucleotide sequence encoding a nucleocapsid protein of a coronavirus.
2. The composition of claim 1, wherein the coronavirus is a beta coronavirus, optionally wherein the beta coronavirus is a human beta coronavirus.
3. The composition of claim 2, wherein the beta coronavirus comprises severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), middle east respiratory syndrome related coronavirus (MERS-CoV), or a combination thereof.
4. The composition of claim 3, wherein the beta coronavirus comprises severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).
5. The composition of claim 4, wherein the coronavirus nucleocapsid protein comprises a first nucleocapsid protein and a second nucleocapsid protein, wherein the first nucleocapsid protein is a SARS-CoV-2 nucleocapsid protein from a first variant of a first clade and the second nucleocapsid protein is a SARS-CoV-2 nucleocapsid protein from a second variant of a second clade, and wherein the first clade and the second clade are different clades defined by one or more of the world health organization, pango, GISAID, and Nextstrain.
6. A composition for stimulating an immune response against a coronavirus in a mammalian subject, the composition comprising an excipient and a messenger RNA (mRNA) comprising an Open Reading Frame (ORF) encoding a fusion protein, wherein the ORF comprises from 5 'to 3':
(i) A nucleotide sequence encoding a mammalian signal peptide; and
(ii) Nucleotide sequences encoding two or more coronavirus nucleocapsid proteins.
7. The composition of claim 6, wherein the coronavirus is a beta coronavirus, optionally wherein the beta coronavirus is a human beta coronavirus.
8. The composition of claim 7, wherein the beta coronavirus comprises severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), middle east respiratory syndrome related coronavirus (MERS-CoV), or a combination thereof.
9. The composition of claim 8, wherein the beta coronavirus comprises severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).
10. The composition of claim 9, wherein the two or more coronavirus nucleocapsid proteins comprise a SARS-CoV-2 nucleocapsid protein and a MERS nucleocapsid protein.
11. The composition of claim 9, wherein the two or more coronavirus nucleocapsid proteins comprise a SARS-CoV-2 nucleocapsid protein, a SARS-CoV-1 nucleocapsid protein, and MERS nucleocapsid protein.
12. The composition of any one of claims 6-11, wherein the two or more coronavirus nucleocapsid proteins are separated by a linker of one to ten residues in length.
13. The composition of any one of claims 1-12, wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in mammalian antigen presenting cells.
14. The composition of claim 13, wherein the mammalian signal peptide is a CD5 signal peptide and the amino acid sequence of the CD5 signal peptide comprises SEQ ID No. 8 or an amino acid sequence at least 90% or 95% identical to SEQ ID No. 8.
15. The composition of any one of claims 1-14, wherein the amino acid sequence of the nucleocapsid protein comprises residues 2-419 of SEQ ID No. 5 or an amino acid sequence that is at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to residues 2-419 of SEQ ID No. 5.
16. The composition of any one of claims 1-14, wherein the amino acid sequence of the fusion protein comprises SEQ ID No. 6 or an amino acid sequence that is at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 6.
17. The composition of any one of claims 6-14, wherein the amino acid sequence of the fusion protein comprises SEQ ID No. 7 or an amino acid sequence at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 7.
18. The composition of claim 16, wherein the open reading frame comprises the nucleotide sequence of SEQ ID No. 2.
19. The composition of claim 17, wherein the open reading frame comprises the nucleotide sequence of SEQ ID No. 3 or SEQ ID No. 4.
20. The composition of any one of claims 1-14, wherein the amino acid sequence of the fusion protein comprises residues 2-413 of SEQ ID No. 9 or an amino acid sequence that is at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to residues 2-413 of SEQ ID No. 9.
21. The composition of any one of claims 1-14, wherein the amino acid sequence of the fusion protein comprises residues 2-422 of SEQ ID No. 10 or an amino acid sequence that is at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to residues 2-422 of SEQ ID No. 10.
22. The composition of any one of claims 1-21, wherein the composition does not comprise a liposome or a lipid nanoparticle.
23. The composition of any one of claims 1-22, wherein the mRNA is a self-replicating mRNA.
24. The composition of claim 23, wherein the self-replicating RNA comprises an alphavirus replicon lacking viral structural protein coding regions.
25. The composition of claim 24, wherein the alphavirus is selected from the group consisting of venezuelan equine encephalitis virus, sindbis virus, and semliki forest virus.
26. The composition of claim 25, wherein the alphavirus is venezuelan equine encephalitis virus.
27. The composition of any one of claims 23-26, wherein the alphavirus replicon comprises a non-structural protein coding region in which 12-18 nucleotides are inserted resulting in expression of nsP2 comprising 4 to 6 additional amino acids between β -sheet 4 and β -sheet 6 of non-structural protein 2 (nsP 2).
28. The composition of any one of claims 1-27, wherein the self-replicating mRNA is a temperature-sensitive agent (ts agent) that is capable of expressing the fusion at a permissive temperature and not expressing the fusion at an non-permissive temperature.
29. The composition of claim 28, wherein the permissible temperature is 31 ℃ to 35 ℃ and the impermissible temperature is at least 37 ℃ ± 0.5 ℃.
30. A method for stimulating an immune response against a coronavirus in a mammalian subject, the method comprising administering the composition of any one of claims 1-29 to a mammalian subject to stimulate an immune response against the coronavirus nucleocapsid protein in the mammalian subject.
31. The method of claim 30, wherein the composition is administered intradermally.
32. The method of claim 30 or claim 31, wherein the immune response comprises a coronavirus-reactive cellular immune response.
33. The method of claim 32, wherein the immune response further comprises a coronavirus-reactive humoral immune response.
34. The method of any one of claims 30-33, wherein the mammalian subject is a human subject.
35. A kit, the kit comprising:
the composition of any one of claims 1-29 or any one of claims 37-62; and
a device for intradermal delivery of the composition to a mammalian subject.
36. The kit of claim 35, wherein the device comprises a syringe and a needle.
37. A composition for stimulating an immune response against two or more viruses in a mammalian subject, the composition comprising an excipient and a messenger RNA (mRNA) comprising an Open Reading Frame (ORF) encoding a fusion protein, wherein the ORF comprises from 5 'to 3':
(i) A nucleotide sequence encoding a mammalian signal peptide; and
(ii) Nucleotide sequences encoding a first nucleocapsid protein of a first virus and a second nucleocapsid protein of a second virus.
38. The composition of claim 37, wherein the first virus and the second virus are capable of causing a disease upon infection of a human subject.
39. The composition of claim 38, wherein the first virus and the second virus are different varieties, subtypes or lineages of the same species.
40. The composition of claim 38, wherein the first virus and the second virus are different species of the same genus.
41. The composition of claim 40, wherein both the first virus and the second virus are members of the genus beta coronavirus.
42. The composition of claim 41, wherein the first virus and the second virus comprise Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and middle east respiratory syndrome associated coronavirus (MERS-CoV).
43. The composition of claim 38, wherein the first virus and the second virus are members of different families, orders, classes, or gates of the same kingdom.
44. The composition of claim 43, wherein both the first virus and the second virus are members of the orthomyxoviridae family.
45. The composition of claim 44, wherein the first virus and the second virus comprise influenza A virus and influenza B virus.
46. The composition of claim 45, wherein the amino acid sequence of the fusion protein comprises SEQ ID NO 16 or an amino acid sequence at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 16.
47. The composition of claim 38, wherein both the first virus and the second virus are members of the orthoriboviridae kingdom, optionally wherein the first virus and the second virus comprise: (a) Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1) or middle east respiratory syndrome related coronavirus (MERS-CoV); and (b) influenza a virus or influenza b virus.
48. The composition of claim 40, wherein both the first virus and the second virus are members of the genus ebola, optionally wherein the first virus and the second virus are selected from the group consisting of zaire-type ebola virus, sudan-type ebola virus, bund Jiao Xingai ebola virus, and tay forest-type ebola virus.
49. The composition of claim 48, wherein the nucleotide sequence further encodes a third nucleocapsid protein of a third virus and a fourth nucleocapsid protein of a fourth virus, and the first virus, the second virus, the third virus, and the fourth virus are zaire-type ebola virus, sudan-type ebola virus, bundi Jiao Xingai bola virus, and tay forest-type ebola virus.
50. The composition of claim 49, wherein the amino acid sequence of the fusion protein comprises SEQ ID NO. 22 or an amino acid sequence that is at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 22.
51. The composition of claim 49, wherein the nucleotide sequence (ii) encodes a shared portion of a first nucleocapsid protein of the first virus for stimulating an immune response against all of the first virus, the second virus, the third virus, and the fourth virus.
52. The composition of claim 51, wherein the nucleotide sequence (ii) encodes a separate portion of the first nucleocapsid protein, the second nucleocapsid protein, the third nucleocapsid protein, and the fourth nucleocapsid protein each for stimulating an immune response against all of the first virus, the second virus, the third virus, and the fourth virus.
53. The composition of claim 52, wherein the nucleotide sequence (ii) encodes a fragment of a separate portion of a second nucleocapsid protein of the second virus for stimulating an immune response against the second virus and the third virus.
54. The composition of claim 37, wherein the nucleotide sequence (ii) encodes a shared portion of a first nucleocapsid protein of the first virus for stimulating an immune response against both the first virus and the second virus.
55. The composition of claim 54, wherein the nucleotide sequence (ii) encodes separate portions of the first nucleocapsid protein and the second nucleocapsid protein each for stimulating an immune response against both the first virus and the second virus.
56. The composition of any one of claims 37-48, wherein the nucleotide sequence (ii) further encodes at least one other nucleocapsid protein of at least one other virus, and wherein the at least one other virus is different from the first virus and the second virus.
57. The composition of any one of claims 37-56, wherein the first nucleocapsid protein and the second nucleocapsid protein, or the first nucleocapsid protein, the second nucleocapsid protein and the other nucleocapsid proteins are separated by a linker of one to ten residues in length.
58. The composition of any one of claims 37-57, wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in mammalian antigen presenting cells.
59. The composition of any one of claims 37-58, wherein the mRNA is a self-replicating mRNA.
60. The composition of claim 59, wherein the self-replicating mRNA is a temperature-sensitive agent (ts agent) that is capable of expressing the fusion protein at a permissive temperature and not expressing the fusion protein at an non-permissive temperature.
61. The composition of claim 60, wherein the permissible temperature is 31 ℃ to 35 ℃ and the impermissible temperature is at least 37 ℃ ± 0.5 ℃.
62. The composition of any one of claims 1-29 or any one of claims 37-61, wherein the composition further comprises chitosan.
63. A method for stimulating an immune response against two or more viruses in a mammalian subject, the method comprising administering to a mammalian subject the composition of any one of claims 37-62 to stimulate an immune response against nucleocapsid proteins of the two or more viruses in the mammalian subject.
64. The method of claim 63, wherein the composition is administered intradermally.
65. The method of claim 63 or claim 64, wherein the immune response comprises a cellular immune response reactive to the two or more viruses.
66. The method of claim 65, wherein the cellular immune response comprises a nucleocapsid protein specific helper T lymphocyte (Th) response comprising nucleocapsid protein specific cytokine secretion.
67. The method of claim 66, wherein the nucleocapsid protein specific cytokine secretion comprises secretion of one or both of interferon-gamma and interleukin-4.
68. The method of claim 65, wherein the cellular immune response comprises a nucleocapsid protein specific Cytotoxic T Lymphocyte (CTL) response.
69. The method of any one of claims 65-68, wherein the immune response further comprises a humoral immune response reactive against the two or more viruses.
70. The method of any one of claims 63-69, wherein the mammalian subject is a human subject.
71. A composition for stimulating an immune response against a virus in a mammalian subject, the composition comprising an excipient and a messenger RNA (mRNA) comprising an Open Reading Frame (ORF) encoding a fusion protein, wherein the ORF comprises from 5 'to 3':
(i) A nucleotide sequence encoding a mammalian signal peptide;
(ii) A nucleotide sequence encoding a first viral antigen of a first virus or a fragment thereof; and
(iii) A nucleotide sequence encoding a second viral antigen of said first virus or second virus or a fragment thereof,
wherein the first viral antigen is a nucleocapsid protein and the second viral antigen is a surface protein, or the first viral antigen is a surface protein and the second viral antigen is a nucleocapsid protein.
72. A composition for stimulating an immune response against two or more viruses in a mammalian subject, the composition comprising an excipient and a messenger RNA (mRNA) comprising an Open Reading Frame (ORF) encoding a fusion protein, wherein the ORF comprises from 5 'to 3':
(i) A nucleotide sequence encoding a mammalian signal peptide;
(ii) A nucleotide sequence encoding a first viral antigen of a first virus or a fragment thereof;
(iii) A nucleotide sequence encoding a second viral antigen of the first virus or a fragment thereof;
(iv) A nucleotide sequence encoding a third viral antigen of a second virus or a fragment thereof;
(iii) A nucleotide sequence encoding a fourth viral antigen of said second virus or a fragment thereof,
wherein the first viral antigen is a first nucleocapsid protein and the second viral antigen is a first surface protein, or the first viral antigen is a first surface protein and the second viral antigen is a first nucleocapsid protein, and
wherein the third viral antigen is a second nucleocapsid protein and the fourth viral antigen is a second surface protein, or the third viral antigen is a second surface protein and the fourth viral antigen is a second nucleocapsid protein.
73. The composition of claim 71 or claim 72, wherein the mRNA is a self-replicating mRNA.
74. The composition of claim 73, wherein the self-replicating RNA comprises an alphavirus replicon lacking viral structural protein coding regions.
75. The composition of claim 74, wherein said alphavirus is selected from the group consisting of venezuelan equine encephalitis virus, sindbis virus, and semliki forest virus.
76. The composition of claim 74, wherein said alphavirus is venezuelan equine encephalitis virus.
77. The composition of any one of claims 73-76, wherein the self-replicating mRNA is a temperature-sensitive agent (ts agent) that is capable of expressing the fusion protein at a permissive temperature and not expressing the fusion protein at an non-permissive temperature.
78. The composition of claim 77, wherein said permissible temperature is 31 ℃ to 35 ℃ and said impermissible temperature is at least 37 ℃ ± 0.5 ℃.
79. The composition of any one of claims 74-78, wherein said alphavirus replicon comprises a non-structural protein coding region in which 12-18 nucleotides are inserted resulting in expression of nsP2 comprising 4 to 6 additional amino acids between β -sheet 4 and β -sheet 6 of non-structural protein 2 (nsP 2).
80. The composition of any one of claims 71-79, wherein the first virus and/or the second virus is a coronavirus, optionally wherein the coronavirus is a beta coronavirus, optionally wherein the beta coronavirus is a human beta coronavirus.
81. The composition of claim 80, wherein said first virus and/or said second virus is a beta coronavirus independently selected from the group consisting of: severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), and middle east respiratory syndrome related coronavirus (MERS-CoV).
82. The composition of claim 80, wherein the first virus is SARS-CoV-2 and the second virus is MERS-CoV.
83. The composition of any one of claims 80-82, wherein each of the surface protein, the first surface protein, and/or the second surface protein comprises a Receptor Binding Domain (RBD) of a coronavirus spike protein.
84. The composition of claim 83, wherein the amino acid sequence of the fusion protein comprises SEQ ID No. 27 or an amino acid sequence at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 27.
85. The composition of any one of claims 71-79, wherein said first virus and/or said second virus is a member of the orthomyxoviridae family.
86. The composition of claim 85, wherein said first virus and/or said second virus are independently selected from the group consisting of Influenza A Virus (IAV) and Influenza B Virus (IBV).
87. The composition of claim 86, wherein the first virus is an IAV and the second virus is an IBV.
88. The composition of any one of claims 85-87, wherein the surface protein, the first surface protein, and/or the second surface protein each comprise a portion of influenza hemagglutinin.
89. The composition of claim 88, wherein the amino acid sequence of the fusion protein comprises SEQ ID No. 29 or an amino acid sequence at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 29.
90. The composition of any one of claims 71-89, wherein the composition further comprises chitosan.
91. A kit, the kit comprising:
(i) The composition of any one of claims 71-90; and
(ii) A device for intradermal delivery of the composition to a mammalian subject.
92. The kit of claim 91, wherein the device comprises a syringe and a needle.
93. The kit of claim 91 or claim 92, further comprising instructions for using the device to administer the composition to a mammalian subject to stimulate an immune response against one or more of the first viral antigen, the second viral antigen, the third viral antigen, and the fourth viral antigen.
94. A method of stimulating an immune response in a mammalian subject, the method comprising administering to a mammalian subject the composition of any one of claims 71-90 to stimulate an immune response in the mammalian subject against one or more of the first viral antigen, the second viral antigen, the third viral antigen, and the fourth viral antigen.
95. The method of claim 94, wherein the composition is administered intradermally.
96. The method of claim 95, wherein the immune response comprises a cellular immune response reactive against one or more of the first viral antigen, the second viral antigen, the third viral antigen, and the fourth viral antigen.
97. The method of claim 96, wherein the immune response further comprises a reactive humoral immune response against one or more of the first, second, third, and fourth viral antigens.
98. The method of any one of claims 94-97, wherein the mammalian subject is a human subject.
99. A method for active boosting against at least one virus, the method comprising intradermally administering to a mammalian subject in need thereof a composition according to any one of claims 1-29, any one of claims 37-62, or any one of claims 71-90 to stimulate a secondary immune response against the virus, wherein the mammalian subject has undergone a primary immunization regimen against the virus.
100. The method of claim 99, wherein the primary immunization regimen comprises administration of at least one dose of a different vaccine against the virus.
101. The method of claim 100, wherein the different vaccine comprises a protein antigen of the at least one virus or an inactivated virus, optionally wherein the protein antigen is a recombinant protein or fragment thereof.
102. A method for active boosting immunity against at least one virus, the method comprising:
(i) Administering intradermally the composition of any one of claims 1-29, any one of claims 37-62, or any one of claims 71-90 to a mammalian subject in need thereof to stimulate a primary immune response against the virus; and
(ii) At least one dose of a different vaccine against the virus is administered to the mammalian subject to stimulate a secondary immune response against the virus.
103. The method of claim 102, wherein the different vaccine comprises a protein antigen of the at least one virus or an inactivated virus, optionally wherein the protein antigen is a recombinant protein or fragment thereof.
104. A method for active primary immunization against at least one virus, the method comprising:
(i) Administering intradermally the composition of any one of claims 1-29, any one of claims 37-62, or any one of claims 71-90 to a mammalian subject in need thereof to stimulate a primary immune response against the virus; wherein the mammalian subject has not undergone a primary immunization regimen against the virus.
105. The method of claim 104, the method further comprising:
(ii) At least one dose of a different vaccine against the virus is administered to the mammalian subject to stimulate a secondary immune response against the virus.
106. The method of claim 105, wherein the different vaccine comprises a protein antigen of the at least one virus or an inactivated virus, optionally wherein the protein antigen is a recombinant protein or fragment thereof.
107. The method of any one of claims 94-106, wherein the mammalian subject is a human subject.
CN202280043353.2A 2021-06-17 2022-06-17 Temperature controllable self-replicating RNA vaccine for viral diseases Pending CN117716036A (en)

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US63/240,278 2021-09-02
US202163275398P 2021-11-03 2021-11-03
US63/275,398 2021-11-03
PCT/US2022/034104 WO2022266511A1 (en) 2021-06-17 2022-06-17 Temperature-controllable, self-replicating rna vaccines for viral diseases

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