Temperature-controllable self-replicating RNA vaccines for viral diseases

Abstract

The present disclosure relates to mRNAs, self-replicating RNAs and temperature-sensitive self-replicating RNAs encoding coronavirus nucleocapsid proteins or influenza virus nucleocapsid proteins in operative combination with a mammalian signal peptide. The present disclosure relates to mRNAs, self-replicating RNAs and temperature-sensitive self-replicating RNAs encoding other viral nucleocapsid proteins in operative combination with a mammalian signal peptide. The RNA constructs are suitable for active immunization against viruses in mammalian subjects, such as human subjects.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 275,398, filed November 3, 2021, U.S. Provisional Application No. 63 / 240,278, filed September 2, 2021, and U.S. Provisional Application No. 63 / 211,974, filed June 17, 2021, each of which is incorporated by reference in its entirety herein.

[0002] Submission of sequence listing as an ASCII text file The contents of the following submission regarding an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing Computer Readable Form (CRF) (Filename: 699442001440SEQLIST.TXT, Recorded: June 16, 2022, Size: 126,113 bytes).

[0003] Field The present disclosure relates to mRNAs, self-replicating RNAs and temperature-sensitive self-replicating RNAs encoding coronavirus nucleocapsid proteins or influenza virus nucleocapsid proteins in operative combination with a mammalian signal peptide. The present disclosure relates to mRNAs, self-replicating RNAs and temperature-sensitive self-replicating RNAs encoding other viral nucleocapsid proteins in operative combination with a mammalian signal peptide. The RNA constructs are suitable for active immunization against viruses in mammalian subjects, such as human subjects. [Background technology]

[0004] The βcoronavirus genus includes Severe Acute Respiratory Syndrome (SARS)-CoV-2, which caused the COVID-19 pandemic, SARS-CoV-1, which caused the SARS pandemic in 2002–2004, and Middle East Respiratory Syndrome (MERS)-CoV. The COVID-19 pandemic has made the design and production of a vaccine urgently necessary for immunization of much of the world's population.

[0005] Currently, SARS-CoV-2 vaccines approved by the U.S. Food and Drug Administration are designed to induce neutralizing antibodies (nAbs) against the spike (S) protein or the receptor-binding domain (RBD) of the S protein prior to infection. However, this approach faces significant challenges in that the S protein is not well conserved between SARS-CoV-1 and SARS-CoV-2 strains. In particular, small amino acid changes that occur between mutant strains often result in conformational changes in the S protein, which can significantly reduce the efficacy of nAbs elicited by the specific S protein of COVID-19 vaccines.

[0006] Therefore, progress on a vaccine targeting only the S protein of βcoronaviruses is expected, similar to seasonal influenza vaccines. This means that new vaccines will likely need to be developed and produced periodically as variants continue to emerge. Although annual production of a βcoronavirus vaccine may be technically feasible, a global vaccination effort to administer a new vaccine every year is economically and logistically impractical. The challenges posed by administering a new vaccine every year place a particularly disproportionate burden on low- and middle-income countries.

[0007] Consequently, there is a need in the art for a betacoronavirus vaccine that safely induces a broadly reactive, long-lasting immune response against variants of SARS-CoV-2. Preferably, the long-lasting immune response is broadly reactive with other betacoronaviruses that cause human disease. There is also a need in the art for a safe and effective influenza virus vaccine that induces a broadly reactive immune response against influenza A and / or influenza B viruses. Summary of the Invention

[0008] overview The present disclosure relates to the use of nucleoproteins (also referred to herein as nucleocapsid proteins) from beta coronaviruses as vaccine antigens to induce cellular immune responses that are broadly reactive with mutant strains of beta coronaviruses. In some embodiments, a temperature-controllable self-replicating RNA (herein referred to as srRNAts and c-srRNA) vaccine platform is utilized. The c-srRNA vaccine platform is advantageous for the induction of a strong cellular immune response after intradermal administration. In some embodiments, a nucleoprotein from SARS-CoV-2 is expressed in host cells to combat infection with both SARS-CoV-2 and SARS-CoV-1 and their mutant strains. In some embodiments, a nucleoprotein from a coronavirus is expressed in host cells fused with a signal peptide of the human CD5 antigen to enhance the cellular immune response elicited against coronaviruses. In some embodiments, a nucleoprotein from a first coronavirus is fused to a nucleoprotein from a second coronavirus that is different from the first coronavirus. In some embodiments, the fusion protein comprises a tandem array of nucleoproteins of two or three coronaviruses. In a subset of these embodiments, the fusion protein comprises a SARS-CoV-2 nucleoprotein and a MERS-CoV nucleoprotein. In some embodiments, the fusion protein further comprises a coronavirus spike protein or a fragment thereof. In this manner, a more broadly reactive coronavirus-specific immune response is stimulated.

[0009] The present disclosure also relates to the use of nucleoprotein (also referred to herein as nucleocapsid protein) from influenza virus as a vaccine antigen to induce a cellular immune response that is broadly reactive to influenza A and / or influenza B viruses that change rapidly over time as a result of antigenic drift and antigenic shift. In some embodiments, a temperature-controllable self-replicating RNA vaccine platform is utilized. The c-srRNA vaccine platform is advantageous for the induction of a strong cellular immune response after intradermal administration. In some embodiments, a nucleoprotein from one subtype of influenza A (FluA) virus is expressed in host cells to combat infection with the same and different subtypes of FluA. In some embodiments, a nucleoprotein from one lineage of influenza B (FluB) virus is expressed in host cells to combat infection with the same and different lineages of FluB. In some embodiments, a nucleoprotein from influenza virus is fused with a signal peptide of human CD5 antigen to express in host cells to enhance the cellular immune response elicited against influenza virus. In some embodiments, a nucleoprotein from a FluA virus is fused to a nucleoprotein from a FluB virus. In some embodiments, the fusion protein comprises a tandem array of two or three nucleoproteins from one or more FluA lineages and / or one or more FluB lineages. 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.

[0010] The present disclosure also relates to the use of nucleoprotein (also referred to herein as nucleocapsid protein) from Ebola virus as a vaccine antigen to induce a cellular immune response broadly reactive with two, three or four species of Ebola virus that infect humans. In some embodiments, a temperature-controllable self-replicating RNA vaccine platform is utilized. The c-srRNA vaccine platform is advantageous for the induction of a strong cellular immune response after intradermal administration. In some embodiments, the nucleoprotein from Ebola virus is fused to a signal peptide of human CD5 antigen and expressed in host cells to enhance the cellular immune response elicited against Ebola virus. In some embodiments, the nucleoprotein from a first Ebola virus species is fused to a nucleoprotein from a second Ebola virus species, optionally fused to a nucleoprotein from a third Ebola virus species, and optionally fused to a nucleoprotein from 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 species of Ebola virus. In some embodiments, the fusion protein further comprises an Ebola virus envelope glycoprotein or a fragment thereof, thus stimulating a more broadly reactive Ebola virus-specific immune response.

[0011] Among other embodiments, the present disclosure provides a composition 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 lactate. In some embodiments, the composition does not comprise a liposome or lipid nanoparticle. [Brief description of the drawings]

[0012] [Figure 1]FIG. 1 shows a schematic of the mechanism of induction of cellular (CD4+ and CD8+ T cell) immune responses following intradermal injection of temperature-controllable self-replicating RNA (herein referred to as srRNAts and c-srRNA) vaccines.

[0013] [Diagram 2] FIG. 2 shows a schematic diagram of the SARS-CoV-2 nucleocapsid (N) protein expressed from mRNA, self-replicating RNA or temperature-sensitive self-replicating RNA (srRNAts) delivered to a mammalian host cell. In an exemplary embodiment, the coding region of the N protein is a gene of interest (GOI) inserted within the srRNAts. The amino acid sequence of the G5004 antigen is set forth as SEQ ID NO:5. The G5004 antigen is the N protein of SARS-CoV-2 without a signal peptide. The amino acid sequence of the G5005 antigen is set forth as SEQ ID NO:6. The G5005 antigen is a fusion protein comprising a signal peptide sequence from the human CD5 antigen (CD5-SP) set forth as SEQ ID NO:8 and the N protein of SARS-CoV-2 in which CD5-SP replaces the initiating methionine at position 1 of the N protein. The amino acid sequence of the G5006 antigen is set forth as SEQ ID NO:7. The G5006 antigen is a fusion protein containing a signal peptide sequence from CD5-SP, the N protein of SARS-CoV-2 and the N protein of MERS-CoV. The nucleotide sequence encoding the G5004 antigen is set forth as SEQ ID NO: 1. The nucleotide sequence encoding the G5005 antigen is set forth as SEQ ID NO: 2. The nucleotide sequence encoding the G5006 antigen is set forth as SEQ ID NO: 3 and as a codon-optimized version in SEQ ID NO: 4.

[0014] [Diagram 3]3 shows a schematic of an exemplary method for stimulating an immune response to coronavirus in a human subject. Temperature-sensitive agents (ts-agents) such as srRNAts function at permissive temperatures but not at non-permissive temperatures. The temperature at or just below the surface of the human subject (body surface temperature) is a permissive temperature, while the core body temperature of the human subject is a higher, non-permissive temperature. Thus, a ts-agent administered intradermally to a human subject functions while located just below the body surface of the human subject at the permissive temperature.

[0015] [Figure 4] Figures 4A and 4B show the frequency of cytokine-secreting cells in samples of splenocytes obtained from CD-1 outbred mice immunized by a single intradermal injection of a 100 μL solution containing 5 μg or 25 μg of temperature-controllable self-replicating RNA (srRNA1ts2 [PCT / US20 / 67506]) encoding the G5004 antigen or a placebo (PBO: buffer only). Figure 4A shows the frequency of interferon-γ (INF-γ) spot-forming cells (SFCs) and Figure 4B shows the frequency of interleukin-4 (IL-4) SFCs in 1 x 10^6 splenocytes after restimulation by culturing splenocytes in the presence or absence of a pool of SARS-CoV-2 nucleoprotein peptides. The frequencies obtained in the presence of peptides are displayed on the graphs after subtracting the frequencies obtained in the absence of peptides (background). For each group, the mean and standard deviation (error bars) of 5 mice (n=5) are shown. Splenocytes were isolated 14 days after intradermal injection.

[0016] [Diagram 5]Figures 5A and 5B show the frequency of cytokine-secreting cells in samples of splenocytes obtained from CD-1 outbred mice immunized by a single intradermal injection of a 100 μL solution containing 5 μg or 25 μg of temperature-controllable self-replicating RNA (srRNA1ts2 [PCT / US20 / 67506]) encoding the G5005 antigen or a placebo (PBO: buffer only). Figure 5A shows the frequency of interferon-γ (INF-γ) spot-forming cells (SFCs) and Figure 5B shows the frequency of interleukin-4 (IL-4) SFCs in 1 x 10^6 splenocytes after restimulation by culturing splenocytes in the presence or absence of a pool of SARS-CoV-2 nucleoprotein peptides. The frequencies obtained in the presence of peptides are displayed on the graphs after subtracting the frequencies obtained in the absence of peptides (background). For each group, the mean and standard deviation (error bars) of 5 mice (n=5) are shown. Splenocytes were isolated 14 days after intradermal injection.

[0017] [Figure 6] Figures 6A and 6B show the frequency of cytokine-secreting cells in samples of splenocytes obtained from BALB / c mice immunized by a single intradermal injection of a 100 μL solution containing 5 μg or 25 μg of temperature-controllable self-replicating RNA encoding the G5005 antigen (srRNA1ts2 [PCT / US20 / 67506]) or a placebo (PBO: buffer only). ) Figure 6A shows the frequency of interferon-γ (INF-γ) spot-forming cells (SFCs) and Figure 6B shows the frequency of interleukin-4 (IL-4) SFCs in 1x10^6 splenocytes after restimulation by culturing splenocytes in the presence or absence of a pool of SARS-CoV-2 nucleoprotein peptides. The frequencies obtained in the presence of peptides are displayed on the graphs after subtracting the frequencies obtained in the absence of peptides (background). For each group, the mean and standard deviation (error bars) of 5 mice (n=5) are shown. Splenocytes were isolated 30 days after vaccination.

[0018] [Figure 7]Figure 7 shows the levels of SARS-CoV-2 antigen-reactive immunoglobulin G (IgG) in the serum of BALB / c mice immunized by a single intradermal injection of a 100 μL solution containing 5 μg or 25 μg of temperature-controllable self-replicating RNA encoding the G5005 antigen (srRNA1ts2 [PCT / US20 / 67506]) or a placebo (PBO: buffer only). IgG levels are expressed by OD450 in ELISA. IgG levels are shown before (day -1) and after (day 30) vaccination (day 0). For each group, the mean and standard deviation (error bars) of 5 mice (n=5) are shown.

[0019] [Figure 8] Figure 8 shows the frequency of interferon-gamma (INF-γ)-secreting cells in samples of splenocytes obtained from BALB / c mice immunized by a single intradermal injection of a 100 μL solution containing 5 μg or 25 μg of temperature-controllable self-replicating RNA encoding the G5006 antigen (srRNA1ts2 [PCT / US20 / 67506]) or a placebo (PBO: buffer only). Specifically, Figure 8 shows the frequency of INF-γ spot-forming cells (SFC) in 1 × 10^6 splenocytes after restimulation by culturing the splenocytes in the presence or absence of a pool of SARS-CoV-2 nucleoprotein peptides. The frequency obtained in the presence of peptides is displayed on the graph after subtracting the frequency obtained in the absence of peptides (background). For each group, the mean and standard deviation (error bars) of 5 mice (n = 5) are shown. Splenocytes were isolated 14 days after vaccination.

[0020] [Figure 9]9 shows a schematic diagram of an exemplary pan-influenza vaccine: a fusion protein comprising a nucleoprotein from influenza A virus (FluA) and a nucleoprotein from influenza B virus (FluB) is expressed from mRNA, self-replicating RNA or temperature-sensitive self-replicating RNA (srRNAts) that is delivered to a mammalian host cell. In an exemplary embodiment, the coding region of the fusion protein is a gene of interest (GOI) inserted within the srRNAts. Specifically, G5010 is a fusion protein comprising the human CD5 antigen (CD5-SP) set forth as SEQ ID NO: 8, a signal peptide sequence derived from FluA nucleoprotein (influenza A, H5N8 subtype [A / breeder duck / Korea / Gochang1 / 2014], GenBank No. KJ413835.1, ProteinID No. AHL21420.1) and FluB nucleoprotein (influenza B [B / Florida / 4 / 2006], GenBank No. CY033879.1, ProteinID No. ACF54251.1). In G5010, CD5-SP replaces the start methionine of the FluA nucleoprotein, and the FluA nucleoprotein is fused to the start codon methionine of the FluB nucleoprotein.

[0021] [Figure 10] FIG. 10 shows an alignment of the nucleoprotein (SEQ ID NO: 13) of influenza A (H5N8 strain; ProteinID AHL21420.1) used as the vaccine antigen for G5010 and the nucleoprotein (SEQ ID NO: 17) of influenza A (NP / AnnArbor H2N2; ProteinID P21433) used as the source of peptide pools for the ELISpot assay.

[0022] [Figure 11]Figures 11A and 11B show the frequency of cytokine-secreting cells in samples of splenocytes obtained from BALB / c mice immunized by a single intradermal injection of 100 μL of a solution containing 5 μg or 25 μg of temperature-controllable self-replicating RNA (srRNA1ts2 [PCT / US20 / 67506]) encoding the G5010 antigen or a placebo (PBO: buffer only). Figure 11A shows the frequency of interferon-γ (INF-γ) spot-forming cells (SFCs) and Figure 11B shows the frequency of interleukin-4 (IL-4) SFCs in 1 x 10^6 splenocytes after restimulation by culturing splenocytes in the presence or absence of a pool of influenza A (H2N2) nucleoprotein peptides. The frequencies obtained in the presence of peptides are displayed on the graphs after subtracting the frequencies obtained in the absence of peptides (background). For each group, the mean and standard deviation (error bars) of 5 mice (n=5) are shown. Splenocytes were isolated 14 days after vaccination.

[0023] [Figure 12]Figure 12 shows a schematic of an exemplary pan-ebolavirus vaccine: a fusion protein containing the nucleoproteins of four different Ebola virus strains is expressed from mRNA, self-replicating RNA or temperature-sensitive self-replicating RNA (srRNAts) delivered to a mammalian host cell. In an exemplary embodiment, the coding region of the fusion protein is a gene of interest (GOI) inserted within the srRNAts. Specifically, exemplary PanEbola antigens include a signal peptide sequence from human CD5 antigen (CD5-SP) set forth as SEQ ID NO:8, a portion of the nucleoprotein of Zaire Ebola virus set forth as SEQ ID NO:18 (residues 2-739; 738 aa in total; GenBank ID: AF272001), a portion of the nucleoprotein of Sudan Ebola virus set forth as SEQ ID NO:19 (residues 403-738; 336 aa in total; GenBank ID: AF173836), a portion of the nucleoprotein of Bundibugyo Ebola virus set forth as SEQ ID NO:20 (residues 403-739; 337 aa in total; GenBank ID: FJ217161), and a portion of the nucleoprotein of Tai Forest Ebola virus set forth as SEQ ID NO:21 (residues 483-651; 169 aa in total; GenBank ID: FJ217162). The amino acid sequence of the PanEbola antigen is set forth as SEQ ID NO:22, and the nucleic acid sequence encoding the PanEbola antigen is set forth as SEQ ID NO:23.

[0024] [Figure 13]Figure 13 shows the amino acid sequence similarity as percent identities among the four Ebolaviruses. The amino acid sequences of Zaire Ebolavirus NP (GenBank ID: AF272001), Sudan Ebolavirus NP (GenBank ID: AF173836), Bundibugyo Ebolavirus NP (GenBank ID: FJ217161), and Tai Forest Ebolavirus NP (GenBank ID: FJ217162) were compared with each other by using the NCBI BlastP algorithm. Based on the sequence alignment, the proteins were divided into well-conserved regions (A) and less-conserved regions (B). The amino acid sequence identity between Zaire Ebolavirus NP and Sudan Ebolavirus NP was 88% in region A, whereas it was 42% in region B. The amino acid sequence identity of Zaire Ebolavirus NP and Tai Forest Ebolavirus NP was 92% in region A, whereas it was 54% in region B. With respect to region B, the Bundibugyo (B) and Tai Forest (B) sequences shared a relatively high level of sequence similarity. Based on sequence alignment of region B, the proteins were divided into well-conserved regions (80% and 86% similarity; not labeled) and less conserved regions (40% identity; referred to herein as region C).

[0025] [Figure 14]Figures 14A and 14B show the frequency of cytokine-secreting cells in samples of splenocytes obtained from BALB / c mice immunized by a single intradermal injection of a 100 μL solution containing 25 μg of temperature-controllable self-replicating RNA encoding the PanEbola antigen (srRNA1ts2-PanEbola, also known as G5011) (srRNA1ts2, also known as c-srRNA as described in WO 2021 / 138447 A1) or placebo (PBO: buffer only). Figure 14A shows the frequency of interferon-gamma (INF-gamma) spot forming cells (SFCs) and Figure 14B shows the frequency of interleukin-4 (IL-4) SFCs in 1x10^6 splenocytes after restimulation by culturing splenocytes in the presence or absence of a pool of 182 peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the nucleoprotein (Swiss-Prot ID: B8XCN6) of Tai Forest Ebola Virus [JPT peptides; PepMix Tai Forest Ebola Virus (NP); JPT product code: PM-TEBOV-NP]. The frequency obtained in the presence of peptide is displayed on the graph after subtracting the frequency obtained in the absence of peptide (background). For each group, the mean and standard deviation (error bars) of 5 mice (n=5) are shown. Splenocytes were isolated 14 days after vaccination.

[0026] [Figure 15]FIG. 15 displays 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 Wuhan 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 (Omicron strain B.1.1.529: Science Brief: Omicron (B.1.1.529) Variant | CDC). The nucleotide sequence of the G5003o open reading frame is set forth as SEQ ID NO:24.

[0027] [Figure 16]Figures 16A and 16B show the frequency of cytokine-secreting cells in samples of splenocytes obtained from C57BL / 6 mice immunized by a single intradermal injection of a 100 μL solution containing 25 μg of temperature-controllable self-replicating RNA encoding the G5003o antigen (srRNA1ts2 as described in WO 2021 / 138447 A1) or placebo (PBO: buffer only). Figure 16A shows the frequency of interferon-gamma (INF-gamma) spot-forming cells (SFCs) and Figure 16B shows the frequency of interleukin-4 (IL-4) SFCs in 1x10^6 splenocytes from immunized mice restimulated by culturing in the presence or absence of a pool of 53 peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the RBD of the Omicron variant (B.1.1.529) of SARS-CoV-2 [JPT peptide product code: PM-SARS2-RBDMUT08-1]. The assay was performed by ELISpot assay. The frequency obtained in the presence of peptide is displayed on the graph after subtracting the frequency obtained in the absence of peptide (background). For each group, the mean and standard deviation (error bars) of 5 mice (n=5) are shown. Splenocytes were isolated 14 days after vaccination.

[0028] [Figure 17]Figures 17A-17C show the induction of both cellular and humoral immunity in mice as a result of administering a composition containing c-srRNA encoding an antigen followed by a composition containing a protein antigen. Figure 17A is a schematic diagram of the experimental procedure. On day -40, blood was collected from female BALB / c mice for plaque reduction neutralization test (PRNT). On day -36, the mice were treated with c-srRNA encoding the G5003 antigen. The c-srRNA was injected intradermally into the skin of the mice as naked RNA without any nanoparticles or transfection reagents. On day 22 (14 days after c-srRNA-G5003 vaccination), half of the mice were sacrificed to obtain splenocytes for ELISpot assay. On day 0, the remaining mice were intradermally injected with spike protein of the delta variant of SARS-CoV-2 (B.1.617.2) mixed with adjuvant (AddaVax™ adjuvant sold by Invivogen). On day 7 (7 days after spike protein injection), blood was collected for PRNT assay. Figure 17B shows the induction of cellular immunity against RBD protein by a single intradermal vaccination with c-srRNA-G5003 vaccine. The figure shows the frequency of interferon-gamma (INF-gamma) spot-forming cells (SFCs) in 1x10^6 splenocytes from immunized mice restimulated by culturing in the presence or absence of a pool of 53 peptides (11 amino acid overlapping 15mers) covering the SARS-CoV-2 RBD (original Wuhan strain). The assay was performed by ELISpot assay. The frequencies obtained in the presence of peptides are displayed in the graphs after subtracting the frequencies obtained in the absence of peptides (background). For each group, the mean and standard deviation (error bars) of 5 mice (n=5) are shown. Splenocytes were isolated on day -22 (14 days after vaccination). Figure 17C shows the titers of serum antibodies capable of neutralizing (50%) the SARS-CoV-2 virus (delta variant B.1.617.2) as measured by 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 specifically against the delta variant of the SARS-CoV-2 virus only in mice vaccinated with the vaccine c-srRNA-G5003, which encodes the RBD of SARS-CoV-2 (original Wuhan strain).

[0029] [Figure 18] Figures 18A-18C show the induction of cellular immunity in mice as a result of administering a composition comprising a protein antigen followed by administration of a composition comprising c-srRNA encoding the antigen. Figure 18A displays a schematic of the experimental procedure. On day 0 (first treatment), female C57BL / 6 mice were treated with an intradermal injection of 10 μg of RBD protein (Sino Biological SARS-CoV-2 [2019-nCoV]) + adjuvant (AddaVax™ adjuvant sold by Invivogen). On day 14 (second treatment), mice were treated with an intradermal injection of placebo (PBO: buffer only), 25 μg c-srRNA encoding the G5003 antigen, 25 μg c-srRNA encoding the G5003o antigen, or 10 μg RBD protein (Sino Biological SARS-CoV-2 [2019-nCoV]) + adjuvant (AddaVax™ adjuvant). On day 28, mice were sacrificed and splenocytes and serum were collected. Figure 18B shows the frequency of interferon-γ (INF-γ), and Figure 18C shows the frequency of interleukin-4 (IL-4) spot-forming cells (SFCs) in 1×10^6 splenocytes restimulated by culturing in the presence or absence of a pool of 53 peptides (15mers containing 11 amino acid overlaps) covering the RBD of SARS-CoV-2 (original Wuhan strain). The assay was carried out by ELISpot assay. The frequencies obtained in the presence of peptides are displayed in the graphs after subtraction of the frequencies obtained in the absence of peptides (background).

[0030] [Figure 19] Figure 19 shows the levels of serum antibodies against the RBD of the SARS-CoV-2 virus (original Wuhan strain) as determined by ELISA assay (represented by OD450 measurements). For each group, the mean and standard deviation (error bars) of five mice (n=5) are shown. For each group, data are shown for day -1 (before the first treatment) and day 28 (after the second treatment).

[0031] [Figure 20] Figure 20A-D show the frequency of interferon-gamma (INF-gamma) or interleukin 4 (IL-4) secreting cells in samples of splenocytes obtained from BALB / c mice immunized by a single intradermal injection of a 100 μL solution containing 5 μg (n=1) or 25 μg (n=4) of temperature-controllable self-replicating RNA (srRNA1ts2 as described in WO 2021 / 138447 A1) encoding the G5006 antigen (Figure 2) or a placebo (PBO: buffer only: n=5). The frequency obtained in the presence of peptide is displayed on the graph after subtracting the frequency obtained in the absence of peptide (background). For each group, the mean and standard deviation (error bars) of one mouse (n=1) or four mice (n=4) are shown. Splenocytes were isolated 14 days after vaccination. Figures 20A and 20B show the results after restimulation of splenocytes by culturing them in the presence or absence of a pool of SARS-CoV-2 nucleoprotein peptides, and Figures 20C and 20D show the results after restimulation of splenocytes by culturing them in the presence or absence of a pool of MERS-CoV-2 nucleoprotein peptides.

[0032] [Figure 21] FIG. 21 shows the survival rate (%) of female BALB / c mice vaccinated with c-srRNA-G5006 and subsequently injected with tumor cells expressing the G5006 antigen.

[0033] [Figure 22]22 displays a schematic diagram showing an exemplary srRNA1ts2 construct encoding a fusion protein of the 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 RBD of MERS-CoV (designated herein as G5006d). The amino acid sequence of the pan-coronavirus antigen (G5006d) is set forth as SEQ ID NO:27, and the nucleotide sequence of its open reading frame is set forth as SEQ ID NO:26.

[0034] [Diagram 23]Figures 23A-B show the frequency of cytokine-secreting cells in samples of splenocytes obtained from female C57BL / 6 mice immunized by a single intradermal injection of a 100 μL solution containing placebo (PBO: buffer only), 25 μg of a temperature-controllable self-replicating RNA encoding the G5006 antigen (srRNA1ts2 as described in WO 2021 / 138447 A1), or 25 μg of a temperature-controllable self-replicating RNA encoding the G5006d antigen (srRNA1ts2 as described in WO 2021 / 138447 A1). Figure 23A shows the frequency of interferon-gamma (INF-gamma) spot forming cells (SFCs) and Figure 23B shows the frequency of interleukin-4 (IL-4) SFCs in 1x10^6 splenocytes from immunized mice restimulated by culturing in the presence or absence of a pool of peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through (A) the RBD of the spike protein of SARS-CoV-2 [JPT peptide product code: PM-WCPV-S-RBD-2]; (B) the nucleoprotein of SARS-CoV-2 [JPT peptide product code: PM-WCPV-NCAP]; (C) the nucleoprotein of MERS-CoV [JPT peptide, custom made]; and (D) the spike protein of MERS-CoV [JPT peptide product code: PM-MERS-CoV-S-1). Assays were performed by ELISpot assay. The frequencies obtained in the presence of peptides are displayed on the graphs after subtracting the frequencies obtained in the absence of peptides (background). For each group, the mean and standard deviation (error bars) of 5 mice (n=5) for PBO, 4 mice (n=4) for G5006, and 5 mice (n=5) for G5006d are shown. Splenocytes were isolated 14 days after vaccination.

[0035] [Figure 24]24 displays a schematic diagram showing an exemplary srRNA1ts2 construct encoding a fusion protein (G5012) of the signal peptide of CD5 (residues 1-24), a portion of the hemagglutinin (HA) (residues 25-165) of influenza A (A / New Caledonia / 20 / 1999 (H1N1)), a nucleoprotein (residues 166-662) of influenza A (A / Breeder Duck / Korea / Gochang1 / 2014 (H5N8)), a nucleoprotein (residues 663-1222) of influenza B (B / Florida / 4 / 2006), and a portion of the hemagglutinin (HA) (residues 1223-1365) of influenza B (B / Florida / 4 / 2006). The amino acid sequence of the pan-influenza virus antigen (G5012) is set forth as SEQ ID NO:29, and the nucleotide sequence of its open reading frame is set forth as SEQ ID NO:28.

[0036] [Diagram 25] Figure 25 shows the effect of chitosan oligomer on gene (luciferase) expression from srRNA1ts2 (an exemplary c-srRNA) in mice. The c-srRNA encoding luciferase was intradermally injected into mice under the following conditions: 1, control-c-srRNA only; 2, c-srRNA mixed with chitosan oligosaccharide (0.001 μg / mL); 3, c-srRNA mixed with chitosan oligosaccharide (0.01 μg / mL); 4, c-srRNA mixed with chitosan oligosaccharide (0.5 μg / mL); and 5, c-srRNA mixed with chitosan oligosaccharide lactate (0.1 μg / mL). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description The best way to achieve broader and longer-lasting protection against SARS-CoV-1, SARS-CoV-2, MERS-CoV and their variants is a vaccine that induces cell-mediated immunity (i.e., a T cell-inducing vaccine that includes CD8+ killer T cells and CD4+ helper T cells). This is a departure from the current neutralizing antibody-focused COVID-19 vaccine paradigm, as described in the background section. The crucial importance of cell-mediated immunity in the fight against coronaviruses has been experimentally proven and widely discussed [Sette and Crotty 2021]. Cell-mediated immunity alone can provide protection via CD8+ killer T cells [Matchett et al. 2021]. Also, cell-mediated immunity depends on linear T cell epitopes, whereas humoral immunity depends on conformational (as well as linear) B cell epitopes. Therefore, cell-mediated immunity is much more robust against variants than humoral immunity. Moreover, memory T cells last longer than memory B cells and may confer lifelong immunity. This requires both appropriate antigen and cell-mediated immunity-based vaccine platforms.

[0038] Cellular immunity-based mRNA vaccine platform The vaccine platform is described in Elixirgen's previous patent application [PCT / US20 / 67506, now published as WO 2021 / 138447 A1]. This vaccine platform is optimized to induce cell-mediated immunity, made possible by combining existing knowledge of vaccine biology with temperature-controllable self-replicating mRNA (srRNAts) based on alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV). The terms c-srRNA and srRNAts are used interchangeably throughout this disclosure, with srRNA1ts2 (described in WO 2021 / 138447 A1) being an exemplary embodiment. srRNAts are based on srRNA, also known as self-amplifying mRNA (saRNA or SAM), by the incorporation of small amino acid changes in the alphavirus replicase that provide temperature sensitivity. Elixirgen Therapeutic Inc.'s srRNAts functions at 30-35°C, but not at or above 37°C±0.5°C. It has all the advantages of the mRNA platform: no genomic integration, rapid development and deployment and simple good manufacturing process (GMP), and the additional advantages of the srRNA platform compared to the mRNA platform, especially longer expression [Johanning et al., 1995], higher immunogenicity at low doses [Brito et al., 2014]. However, this simple temperature-controllable feature allows for the compilation of many desirable features of a T cell-inducing vaccine as described herein.

[0039] Briefly, srRNA1ts2 is a temperature-sensitive self-replicating VEEV-based RNA replicon developed for the transient expression of heterologous proteins. Temperature sensitivity is conferred by inserting five amino acid residues into the nonstructural protein 2 (nsP2) of VEEV. The nsP2 protein is a helicase / proteinase and, together with nsP1, nsP3 and nsP4, constitutes the VEEV replicase. The disclosure of WO 2021 / 138447 A1 of Elixirgen Therapeutics, Inc. is incorporated herein by reference. In particular, Example 3, FIG. 12 and SEQ ID NOs: 29-49 are incorporated herein by reference.

[0040] Overall, the compelling potential of the srRNAts platform for immunogenicity (dose-sparing) and safety advantages (temperature control and naked delivery), provision of long-lasting baseline cellular immunity and the ability to provide a rapid humoral response across variants make it a strong candidate for large-scale deployment to meet the global need for an inexpensive and safe variant-addressing vaccine providing long-term immunity.

[0041] General Techniques and Definitions The 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.

[0042] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless otherwise indicated. For example, "an" excipient includes one or more excipients.

[0043] As used herein, the phrase "comprising" is open ended and indicates that such embodiments may include additional elements. In contrast, the phrase "consisting of" is closed and indicates that such embodiments do not include additional elements (except for trace impurities). The phrase "consisting essentially of" is partially closed and indicates that such embodiments may include further elements that do not materially alter the basic characteristics of such embodiment.

[0044] As used herein with respect to a value, the term "about" encompasses 90% to 110% of that value (e.g., when used with respect to chitosan oligosaccharide, a molecular weight of about 5,000 daltons refers to 4,500 daltons to 5,500 daltons).

[0045] The term "antigen" refers to a substance that is specifically recognized and bound by an antibody or a T-cell antigen receptor. Antigens may include peptides, polypeptides, proteins, glycoproteins, polysaccharides, glycoconjugates, sugars, gangliosides, lipids and phospholipids; portions thereof and combinations thereof. In the context of the present disclosure, the term "antigen" refers to a polypeptide or protein antigen, typically at least 8 amino acid residues in length, and may include one or more post-translational modifications.

[0046] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a particular length unless otherwise specified. A polypeptide can contain natural amino acid residues or a combination of natural and non-natural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In some embodiments, a polypeptide can contain modifications with respect to a native or natural sequence, so long as the protein maintains a desired activity (e.g., antigenicity).

[0047] As used herein, the terms "isolated" and "purified" refer to a material that is removed from at least one component with which it is naturally associated (e.g., removed from its original environment). The term "isolated," when used in reference to a recombinant protein, refers to a protein that has been removed from the culture medium of a host cell that produced the protein. In some embodiments, an isolated protein (e.g., spike protein of SARS-CoV-2) is at least 75%, 90%, 95%, 96%, 97%, 98% or 99% pure as determined by HPLC.

[0048] An "effective amount" or "sufficient amount" of a substance is an amount sufficient to effect beneficial or desired results, including clinical results, and as such, "effective amount" will depend on the context in which it is applied. In the context of administering a composition of the present disclosure that includes an mRNA encoding an antigen, an effective amount includes sufficient mRNA to stimulate an immune response, preferably a cellular immune response to the antigen.

[0049] In this disclosure, the terms "individual" and "subject" refer to a mammal. "Mammals" include, but are not limited to, humans, non-human primates (e.g., monkeys), farm animals, sport animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats). In some preferred embodiments, the subject is a human subject.

[0050] As used herein with respect to a composition comprising an mRNA encoding an antigen, the term "dose" refers to the measured portion taken (administered or received) by a subject 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 an mRNA encoding an antigen to stimulate an immune response to the antigen in the subject.

[0051] "Stimulation" of a response or parameter includes eliciting and / or enhancing that response or parameter when compared to otherwise the same conditions except for the parameter of interest, or alternatively, when compared to another condition (e.g., increased antigen-specific cytokine secretion following administration of a composition containing or encoding an antigen compared to administration of a control composition not containing or encoding the antigen). For example, "stimulation" of an immune response (e.g., a Th1 response) refers to an increase in the response. Depending on the parameter being measured, 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, 1000, 5000, or 10,000-fold.

[0052] Conversely, "inhibition" of a response or parameter includes reducing and / or suppressing that response or parameter when compared to otherwise the same conditions except for the parameter of interest, or alternatively, when compared to another condition. For example, "inhibition" of an immune response (e.g., a Th2 response) refers to a decrease in the response. Depending on the parameter being measured, the decrease may be 2-200 fold, 5-500 fold or more, 10-1000 fold or more, or 2, 5, 10, 50, or 100 fold to 200, 500, 1000, 2000, 5000, or 10000 fold.

[0053] The relative terms "higher" and "lower" refer to a measurable increase or decrease, respectively, in a response or parameter when compared to the same conditions except for the parameter of interest, or alternatively, when compared to another condition. For example, a "higher antibody titer" refers to an antigen-reactive antibody titer resulting from administration of a composition of the present disclosure comprising an mRNA encoding an antigen that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than the antigen-reactive antibody titer resulting from a control condition (e.g., administration of a comparator composition that does not comprise an mRNA or that comprises a control mRNA that does not encode the antigen). Similarly, a "lower antibody titer" refers to an antigen-reactive antibody titer resulting from administration of a composition of the present disclosure comprising an mRNA encoding an antigen that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times lower than the antigen-reactive antibody titer resulting from administration of a composition of the present disclosure comprising an mRNA encoding the antigen.

[0054] As used herein, the term "immunization" refers to the process of mounting a mammalian subject's response to an antigen, thus improving its ability to resist or overcome infection and / or resist disease.

[0055] As used herein, the term "vaccination" refers to the introduction of a vaccine into a mammalian subject.

[0056] As used herein, "percent (%) amino acid sequence identity" and "percent identity" and "sequence identity" when used in reference to an amino acid sequence (reference polypeptide sequence) are defined as the percentage of amino acid residues in a candidate sequence (e.g., an antigen of interest) that are identical to amino acid residues in a 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. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0057] Amino acid substitutions can involve replacing one amino acid in a polypeptide with another amino acid. Amino acid substitutions can be introduced into an antigen of interest and the products screened for a desired activity, e.g., increased stability and / or immunogenicity.

[0058] Amino acids can generally be grouped according to the following common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that influence chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0059] Conservative amino acid substitutions involve exchanging a member of one of these classes for another member of the same class. Non-conservative amino acid substitutions involve exchanging a member of one of these classes for a member of another class.

[0060] As used herein, the term "excipient" refers to a compound present in a composition that includes an active ingredient (e.g., an mRNA encoding an antigen). Pharmaceutically acceptable excipients are inert pharmaceutical agents and may include, for example, a solvent, a bulking agent, a buffering agent, a tonicity adjusting agent, and a preservative (Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the compositions of the present disclosure include an excipient that functions as one or more of a solvent, a bulking agent, a buffering agent, and a tonicity adjusting agent (e.g., sodium chloride in saline can function as both an aqueous vehicle and a tonicity adjusting agent).

[0061] Optimization for intradermal delivery Intradermal vaccination results in long-lasting cellular immunity and improved immunogenicity [Hickling 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 5-10 times more effective than subcutaneous or intramuscular vaccination because it targets APCs present in the skin [Hickling and Jones, 2009], thereby activating the T cell immune pathway for long-lasting immunity. Upon intradermal injection, srRNAts is primarily taken up by skin APCs, where it replicates, produces antigens, digests antigens into peptides, and presents peptides to T cells (Figure 1). Peptides presented via this pathway stimulate MHC-I-restricted CD8+ killer T cells. In another pathway, APCs also take up antigens produced by surrounding skin cells. Peptides presented via this pathway stimulate MHC-II-restricted CD4+ helper T cells and B cells to produce neutralizing antibodies (nAbs) and help fight viral infection.

[0062] Problems and solutions for intradermal injection The potential problems we identified and the solutions the srRNAts platform offers are as follows:

[0063] (1) A significant unrecognized hurdle for the application of srRNA as an intradermal vaccine platform is that neither mRNA nor srRNA express antigens well at skin temperature [PCT / US20 / 67506]. Counterintuitively, the temperature of human skin is lower (approximately 30-35°C) than the human core body temperature (approximately 37°C); this means that vectors and platforms developed at 37°C are not optimal for intradermal injection. The innovation of the srRNAts platform is that it robustly expresses antigens at skin temperature [PCT / US20 / 67506]. Moreover, this temperature control also minimizes safety risks caused by unintended systemic distribution of srRNAts, since srRNAts is inactivated when its temperature rises above an acceptable threshold (approaching the body core). In other words, the srRNAts platform expresses antigens that are optimal for intradermal injection compared to mRNA and srRNA, and it is safer: the ability of the vector to spread and be produced in other parts of the subject's body is limited or inactivated.

[0064] (2) Another challenge of intradermal vaccination is the lack of suitable additives. Adjuvants such as aluminum salts and oil-in-water types are too reactive locally when delivered by the intradermal route, so no clinically approved intradermal vaccines incorporate adjuvants, resulting in lower immunogenicity [Hickling and Jones, 2009]. Lipid nanoparticles (LNPs) used for intramuscularly administered mRNA and srRNA vaccines are also oil-in-water types, which may cause skin reactogenicity and increase the risk of allergic reactions to LNP components such as PEG. The c-srRNA platform is a solution to this problem, since it is injected as naked c-srRNA (no LNPs, no adjuvant). First, the self-replication of RNA in cells, especially in APCs, induces strong innate immunity, replacing the primary function of adjuvants. Second, data in the literature and data obtained during the development of this disclosure demonstrate that naked mRNA / srRNA is equally efficient in producing antigens compared to electroporation of mRNA / srRNA [Johansson et al., 2012] and mRNA / srRNA combined with LNPs [Golombek et al., 2018], particularly with respect to intradermal injection.

[0065] (3) The third challenge is the limited number of precedents for intradermal vaccines. Only the BCG vaccine is routinely administered intradermally, and all currently available COVID-19 vaccines are administered intramuscularly. One way to lower the hurdle to adopting intradermal injections is to use specialized equipment such as MicronJet600 (Nanopath) and Immucise (Terumo), which are now available for simple, stable intradermal injections. These equipment are also well adapted for large-scale production and deployment. However, these specialized equipment are relatively expensive, so intradermal injections using the Mantoux technique with standard needles and syringes are also an option.

[0066] Designing the right antigen Because humoral immunity, i.e. the induction of neutralizing antibodies, is not the primary consideration, an approach focused on cellular immunity has made it possible to reconsider all proteins encoded on the viral genome as potential antigens.

[0067] When selecting an antigen that would provide broader protection against SARS-CoV-1, SARS-CoV-2, MERS-CoV and their variants, the nucleoprotein (N) was determined to be the most appropriate because (1) in the virus particle, N is the most abundant protein, followed by the membrane (M) and spike (S) [Finkel et al., 2021], (2) N is the most conserved protein overall among the above-mentioned β-coronaviruses [Grifoni et al., 2020], and (3) B and T cell epitopes are most abundant in S and N [Grifoni et al., 2020]. This is consistent with previous suggestions that N is the optimal antigen for a vaccine [Dutta et al., 2020]. Of note, recent reports have clearly demonstrated that a vaccine using only N as an antigen can provide S-independent protective immunity in both hamsters and mice. [Matchett et al., 2021] Enhanced disease has previously been observed with N vaccines as well as S vaccines [Lambert et al., 2020], but these data were obtained by using a different vector with an unfavorable Th2>Th1 profile.

[0068] An exemplary vaccine candidate, srRNA1ts2-G5005, was designed to express the N protein of SARS-CoV-2 (SARS2-N). However, MERS-N forms a separate group and shows only 48% identity [Tilocca et al., 2020]. With this in mind, a further exemplary vaccine candidate, srRNA1ts2-G5006, was designed to express a fusion protein of SARS2-N and MERS-N. The G5005 and G5006 antigens are shown diagrammatically in Figure 2. srRNA1ts2-G5005 is suitable for inducing immune responses against SARS-CoV-1, SARS-CoV-2 and their variants. In contrast, srRNA1ts2-G5006 is suitable for inducing a pan-coronavirus immune response (e.g., against SARS-CoV-1, SARS-CoV-2, MERS-CoV and their variants).

[0069] To address the emergence of variants (mutations) of the SARS-CoV-2 virus, c-srRNA (G5003o) encoding the RBD of the SARS-CoV-2 Omicron variant was generated and administered intradermally to C57BL / 6 mice (Example 8 and Figure 15). Cellular immunity was evaluated 14 days after vaccination. The results clearly show that c-srRNA can induce Omicron variant-specific cellular immunity when the open reading frame of the receptor binding domain (RBD) of the Omicron variant is included in the c-srRNA. Importantly, c-srRNA encoding the G5003o antigen was found to induce a Th1-biased response [Th1 (INF-γ)>Th2 (IL-4)] as shown in Figures 16A-16B, which favors the vaccine.

[0070] Prime-boost immunization regimen included One of the unique features of intradermally administered c-srRNA vaccines is their ability to induce cellular immunity without overtly inducing humoral immunity (i.e., antibodies). As determined during the development of the present disclosure, c-srRNA vaccines can prime humoral immune responses to subsequently encountered protein antigens. That is, as described in Example 9 and shown in FIG. 17A, mice were first treated with c-srRNA encoding an antigen (i.e., the RBD of the SARS-CoV-2 Wuhan strain) and then with an adjuvanted mutant RBD protein (i.e., the RBD of the SARS-CoV-2 delta mutant strain).

[0071] Cellular immunity, assessed by measuring the presence of antigen-specific IFN-γ-secreting T cells, was already induced by day 14 (prime) after the first vaccination, as shown in FIG. 17B. Antigen-specific antibodies were not detected at this time point. After treatment with adjuvanted protein antigen, antibodies were induced as early as day 7 (boost) after the second vaccination, as shown in FIG. 17C. This early induction of antibodies is consistent with a secondary immune response, indicating that c-srRNA has already primed humoral immunity. Importantly, antibodies induced by protein antigen boost were able to neutralize virus mutants with RBD sequences different from the RBD antigen encoded by the c-srRNA vaccine. This surprising finding indicates that c-srRNA vaccines can induce protective immune responses against pathogens with antigen sequences different from those encoded by the c-srRNA vaccine. Thus, c-srRNA vaccines are expected to induce broadly reactive immune responses, which are important for providing protection against mutant pathogens.

[0072] Subunit vaccines against pathogens generally do not provide long-lasting humoral immunity (i.e., pathogen-specific antibodies), and therefore require one or more booster vaccines. As determined during the development of the present disclosure, c-srRNA vaccines are suitable for use as booster vaccines when adjuvanted proteins are administered as a prime vaccine. That is, mice were first treated with adjuvanted proteins (i.e., RBD of SARS-CoV-2 Wuhan strain) and then treated with placebo (PBO: buffer only), c-srRNA encoding G5003 antigen (Wuhan RBD), c-srRNA encoding G5003o antigen (Omicron RBD) or adjuvanted protein antigen (Wuhan RBD), as described in Example 10 and shown in Figure 18A.

[0073] As shown in Figure 17C, the c-srRNA vaccine alone does not induce humoral immunity in the form of neutralizing antibody responses (see PBO on day 7). However, when humoral immunity is primed by adjuvanted protein (as a model for initial vaccination), the c-srRNA booster vaccine can induce both antigen-specific cytokine responses (Figures 18B-18C) and antigen-specific antibody responses (Figure 19). It is noteworthy that in the present experimental conditions, a single dose of adjuvanted protein did not induce RBD-specific antibodies. Apparently, the cellular immunity induced by c-srRNA is capable of stimulating antibody production against previously encountered protein antigens. This observation indicates that an important interplay is occurring between the cellular and humoral immune responses.

[0074] Elimination of antigen-expressing cells in vivo c-srRNA vaccines can induce strong cellular immune responses (i.e., antigen-specific CD8+ cytotoxic T lymphocytes and CD4+ helper T lymphocytes). Antigen-specific CD8+ CTLs lyse cells on which the antigen is expressed. Antigen recognition by CD8+ CTLs is based on the presentation of short peptide fragments (T cell epitopes) by MHC class I molecules, and therefore does not require that the antigen be expressed on the surface of the target cell. In the case of a vaccine against a pathogen, the vaccine is expected to lyse cells infected by the pathogen. In the case of a vaccine against cancer, the vaccine is expected to lyse cancer cells.

[0075] A c-srRNA vaccine was created that encodes a fusion protein of the SARS-CoV-2 nucleoprotein and the MERS-CoV nucleoprotein (called the SMN protein or G5006) as an antigen. To model cells infected with the virus, the 4T1 breast cancer cell line, which arises from BALB / c mice and is known to be a model of triple-negative stage 4 human breast cancer, was selected. When injected into BALB / c mice, 4T1 cells rapidly proliferate and form tumors. This syngeneic mouse model was used to mimic the rapid increase in infected cells. 4T1 cells expressing the SMN protein (named 4T1-SMN) were established by transfecting a plasmid vector encoding the SMN protein under the CMV promoter. The fusion protein is the same as G5006, except that the CD5 signal peptide was removed from the N-terminus of the SMN protein expressed in 4T1 cells.

[0076] BALB / c mice were vaccinated with c-srRNA-G5006, and induction of cellular immunity was manifested by the presence of T cells responding to both the SARS-CoV-2 nucleoprotein (Figures 20A-20B) and the MERS-CoV nucleoprotein (Figures 20C-20D). 4T1-SMN cells were then injected into the c-srRNA-G5006-vaccinated BALB / c mice on day 24 (24 days after vaccination). As expected, 4T1-SMN cells proliferated rapidly in placebo-treated mice (non-vaccinated group). In contrast, 4T1-SMN tumor growth was suppressed in the c-srRNA-G5006-vaccinated mice. In two mice that received 25 μg of the c-srRNA-G5006 vaccine, tumors initially grew but eventually disappeared and survived long after the placebo-vaccinated mice had died. Moreover, even after a second injection of 4T1-SMN tumors on day 143 post-vaccination, tumors did not grow and mice remained alive and tumor-free for the duration of the study (Figure 21). This result suggests that c-srRNA vaccines encoding the G5006 antigen (i.e., SMN protein) can induce protective immune responses by eliminating cells infected with SARS-CoV-2 or MERS-CoV.

[0077] Pancoronavirus Booster Vaccine For infectious diseases such as COVID-19, World Health Organization guidelines require that licensed vaccines have the ability to induce neutralizing antibodies (nAbs). This requirement is reasonable because nAbs can prevent cells from becoming infected, and therefore, nAbs can efficiently control the spread of infection. However, nAb levels generally decline rapidly, and therefore booster vaccines are required periodically (e.g., once or twice a year) after the completion of the initial vaccination series (first and second vaccinations) to maintain adequate nAb levels. The high mutation rate of SARS-CoV-2, especially within the RBD of the spike protein, which is the target of nAbs, is a major concern that is usually associated with the use of first generation COVID-19 vaccines that target the spike protein of SARS-CoV-2.

[0078] To address these issues, a new booster vaccine, c-srRNA-G5006d, was developed that encodes a fusion protein containing the CD5 signal peptide, the Spike-RBD of SARS-CoV-2, the nucleoprotein of SARS-CoV-2, the nucleoprotein of MERS-CoV, and the Spike-RBD of MERS-CoV (Example 12 and Figure 22). The amino acid sequence of the pan-coronavirus antigen (G5006d) is set forth as SEQ ID NO: 27, and the nucleotide sequence of its open reading frame is set forth as SEQ ID NO: 26. The order of each sequence segment (SARS-CoV-2 RBD; SARS-CoV-2 nucleoprotein; MERS-CoV nucleoprotein; MERS-CoV RBD) of the fusion protein can be varied, and the amino acid sequence of each segment does not need to be 100% identical to the exemplary sequences provided herein.

[0079] The c-srRNA-G5006d vaccine is intended to be used as a booster vaccine after a first series of vaccines targeting the spike antigen or a fragment thereof (RBD) (first vaccination or first and second vaccinations). However, the c-srRNA-G5006d vaccine can also be used as part of the first series of vaccines.

[0080] The c-srRNA-G5006d vaccine increases nAb levels and provides cell-mediated immunity against human-infecting β-coronaviruses, which are important for providing long-lasting protection from severe disease, hospitalization, and death.

[0081] As described in Example 10, a c-srRNA vaccine encoding Spike-RBD can increase the levels of antibodies or nAbs against Spike-RBD when used as a booster vaccine following administration of a vaccine that can prime or induce humoral immunity.

[0082] c-srRNA-G5006d encodes both the Spike-RBD protein of SARS-CoV-2 and the Spike-RBD protein of MERS-CoV. Therefore, c-srRNA-G5006d can be used as a booster vaccine for both SARS-CoV-2 and MERS-CoV.

[0083] The spike proteins of SARS-CoV-2 and SARS-CoV are similar (about 76% identity) (Grifoni et al., 2020). Therefore, c-srRNA-G5006d is effective as a booster against SARS-CoV-2, SARS-CoV, and their mutant strains. On the other hand, 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 the Spike-RBD of MERS-CoV. Therefore, c-srRNA-G5006d is effective as a booster against MERS-CoV and its mutant strains. Taken together, c-srRNA-G5006d is effective as a booster against SARS-CoV-2, SARS-CoV, MERS-CoV, and their mutant strains.

[0084] c-srRNA-G5006d also encodes the nucleoproteins of SARS-CoV-2 and MERS-CoV. Therefore, c-srRNA-G5006d can induce strong cellular immunity against SARS-CoV-2 and MERS-CoV. The nucleoproteins of SARS-CoV-2 and SARS-CoV are highly similar to each other (about 90% identity) (Grifoni et al., 2020). Therefore, c-srRNA-G5006d provides strong cellular immunity against SARS-CoV-2, SARS-CoV and their mutant strains. In contrast, the nucleoproteins of SARS-CoV-2 and MERS-CoV are divergent (about 48% identity) (Grifoni et al., 2020). However, c-srRNA-G5006d also encodes the nucleoprotein of MERS-CoV. Therefore, c-srRNA-G5006d appears to provide strong cellular immunity against MERS-CoV and its mutants. Taken together, c-srRNA-G5006d induces strong immune responses against SARS-CoV-2, SARS-CoV, MERS-CoV and their mutants.

[0085] As described in Examples 9 and 10, c-srRNA vaccines have a surprising mode of action: the encoded antigens do not appear to directly stimulate B cells, and therefore there is no need to consider the three-dimensional structure of the encoded antigen. This is different from conventional vaccines designed to directly stimulate B cells to produce antibodies against conformational epitopes (three-dimensional structures of antigens). While this is why the use of fusion proteins in c-srRNA vaccines is appropriate, the use of fusion proteins in conventional subunit vaccines is complicated by the fact that the natural three-dimensional structure of each antigen may be destroyed when expressed as a fusion protein. c-srRNA booster vaccines stimulate antibody production through the activation of CD4+ helper T cells, and therefore rely on short peptide epitopes (~15mers). Therefore, for antigens encoded by c-srRNA vaccines, it is possible to simply combine two or more different antigens into one fusion protein, but this mechanism may be problematic for the design of subunit vaccines.

[0086] The fact that c-srRNA relies on short peptide epitopes for the induction of cellular and humoral immune responses also provides an advantage for more broadly reactive vaccines that induce protection against mutant pathogens. Many T cell epitopes are present in one protein, and therefore any one mutation is unlikely to cause loss of immunogenicity. On the other hand, traditional subunit vaccines rely on the three-dimensional structure of protein antigens, and therefore even a single mutation can change the conformation of the protein, leading to loss of immunogenicity.

[0087] As shown in Figures 23A-23B, c-srRNA-G5006d can stimulate cellular immunity against all proteins encoded by the following vaccines: Spike-RBD of SARS-CoV-2, nucleoprotein of SARS-CoV-2, nucleoprotein of MERS-CoV and Spike-RBD of MERS-CoV.

[0088] Pan-influenza booster vaccine As determined during the development of this disclosure (see, e.g., Example 6), fusion proteins containing nucleoproteins from representative influenza A and B strains were able to induce strong antigen-specific cellular immune responses when the fusion proteins were expressed from temperature-controllable self-replicating RNA injected intradermally. It is generally believed that protection is primarily mediated by neutralizing antibodies against hemagglutinin (HA), one of the surface proteins of influenza viruses. Therefore, FDA-approved influenza vaccines contain HA as an antigen, either alone or in combination with other influenza antigens. c-srRNA-based booster vaccines require only CD4+ T cell epitopes on the HA protein to boost Ab production, so there is no need to consider the three-dimensional structure of the HA protein. It is known that only certain portions of the HA protein of the H1N1 influenza virus can function as CD4+ T cell epitopes (Knowlden et al., Pathogens. 8(4):220, 2019). B cell epitopes and CD4+ T cell epitopes in both influenza A and B have been identified (Terajima et al., Virol J, 10:244, 2013). The sequences of the HA proteins of representative H1N1 influenza viruses were aligned (Darricarrere et al., J Virol, 92(22):e01349-18, 2018), and regions containing well-conserved sequences were identified. Based on these studies, the HA protein fragment (residues 316-456) of influenza A virus (A / New Caledonia / 20 / 1999(H1N1)) [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. Nucleoproteins from influenza A and B, previously described in Example 6 and designated as G5010 antigens, were also included.

[0089] FIG. 24 shows the design of a pan-influenza booster vaccine. c-srRNA-G5012 encodes a fusion protein (G5012) containing the signal peptide of CD5 (residues 1-24), a portion of influenza A hemagglutinin (HA), influenza A nucleoprotein, influenza B nucleoprotein, and a portion of influenza B hemagglutinin (HA). The amino acid sequence of the pan-influenza virus antigen (G5012) is set forth as SEQ ID NO: 29, and the nucleotide sequence of its open reading frame is set forth as SEQ ID NO: 28. The order of each sequence segment of the fusion protein (part of influenza A HA; influenza A nucleoprotein; influenza B nucleoprotein; part of influenza B HA) can be changed, and the amino acid sequence of each segment does not have to be 100% identical to the exemplary sequence provided herein.

[0090] This c-srRNA-G5012 influenza vaccine increases nAb levels through the enhancement of HA-specific CD4+ helper T cells. It also provides cell-mediated immunity to essentially all influenza viruses through the evolutionarily conserved nucleoprotein. Cell-mediated immunity is known to provide long-lasting protection from severe disease, hospitalization, and death.

[0091] Chitosan-enhancement of gene expression in vivo RNase inhibitors (proteins purified from human placenta) slightly enhance immunogenicity against antigens encoded on c-srRNA by enhancing the expression of c-srRNA-derived antigens in vivo when injected intradermally in mice (see, for example, Figure 25C of WO 2021 / 138447 A1). RNase inhibitors may protect c-srRNA from RNase-mediated degradation in vivo. However, it is difficult to use protein-based RNase inhibitors as excipients in injectable formulations, so it is desirable to find alternative drugs that can enhance the expression of genes of interest (GOI) in vivo for therapeutic purposes.

[0092] Low molecular weight chitosan (molecular weight ~6 kDa) was shown to inhibit the activity of RNase with an inhibition constant in the range of 30-220 nM (Yakovlev et al., Biochem Biophys Res Commun, 357(3):584-8, 2007). This was only shown in vitro and also for artificially made polynucleotides such as poly(A) / poly(U), but whether chitosan oligosaccharides could enhance the expression of GOIs derived from c-srRNA needed to be tested in vivo by intradermal injection of c-srRNA in mice. As shown in Example 14, two different chitosan oligomers were tested: chitosan oligomer (molecular weight ≦5 kDa, ≧75% deacetylated: Heppe Medical Chitosan GmbH: product no. 44009) and chitosan oligosaccharide lactate (molecular weight about 5 kDa, >90% deacetylated: Sigma-Aldrich: product no. 523682). Surprisingly, it was found that even very low levels of chitosan oligomers, such as 0.001 μg / mL (approximately 0.2 nM, approximately 1 / 100 of the inhibition constant found by Yakovlev et al., supra, 2007), could enhance the expression of luciferase encoded on c-srRNA by ∼10-fold (Figure 25). Similar enhancement of GOI expression was achieved with chitosan oligomers up to 0.5 μg / mL and chitosan oligosaccharide lactate at 0.1 μg / mL.

[0093] Chitosan has been used as a nucleotide (DNA and RNA) delivery vector because it can form complexes or nanoparticles (reviewed in Buschmann et al., Adv Drug Deliv Rev, 65(9):1234-70, 2013; and Cao et al., Drugs, 17:381, 2019). However, it is worth noting that the enhancement of GOI expression by chitosan oligomers is unlikely to be mediated by nanoparticles or complex formation between c-srRNA and chitosan oligomers. First, the low concentration of chitosan oligomers does not allow complex formation with RNA. Second, chitosan oligomers are added to c-srRNA just before intradermal injection, and therefore there is not enough time for complex formation.

[0094] Since chitosan oligomers enhance the expression of GOIs in vivo at much lower concentrations compared to their effective concentrations as RNase inhibitors in vitro (Yakovlev et al., supra, 2007), it is conceivable that this enhancement of GOI expression by chitosan oligomers may not be mediated by their RNase inhibitory mechanism. For example, chitosan oligomers may promote the uptake of c-srRNA into cells, thereby enhancing the expression of GOIs derived from c-srRNA. Nevertheless, this surprising finding should provide an effective means of enhancing the therapeutic expression of GOIs encoded on c-srRNA in vivo. Enumerated Embodiments [Embodiment 1] 1. A composition for stimulating an immune response to a coronavirus in a mammalian subject comprising an excipient and a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a fusion protein, the ORF comprising, from 5' to 3', the following: (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a coronavirus nucleocapsid protein; A composition comprising: [Embodiment 2] The composition of embodiment 1, wherein the coronavirus is a β-coronavirus, and optionally, the β-coronavirus is a human β-coronavirus. [Embodiment 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. [Embodiment 4] The composition of embodiment 3, wherein the beta coronavirus comprises severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). [Embodiment 5] 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 first mutant SARS-CoV-2 nucleocapsid protein from a first clade and the second nucleocapsid protein is a second mutant SARS-CoV-2 nucleocapsid protein from a second clade, 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. [Embodiment 6] 1. A composition for stimulating an immune response to a coronavirus in a mammalian subject comprising an excipient and a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a fusion protein, the ORF comprising, 5' to 3', the following: (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding two or more coronavirus nucleocapsid proteins. [Embodiment 7] The composition of embodiment 6, wherein the coronavirus is a β-coronavirus, and optionally, the β-coronavirus is a human β-coronavirus. [Embodiment 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. [Embodiment 9] The composition of embodiment 8, wherein the beta coronavirus comprises severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). [Embodiment 10] 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. [Embodiment 11] 10. The composition of embodiment 9, wherein the two or more coronavirus nucleocapsit proteins comprise a SARS-CoV-2 nucleocapsit protein, a SARS-CoV-1 nucleocapsit protein, and a MERS nucleocapsit protein. [Embodiment 12] The composition of any one of embodiments 6 to 11, wherein the two or more coronavirus nucleocapsit proteins are separated by a linker having a length of 1 to 10 residues. [Embodiment 13] The composition according to any one of embodiments 1 to 12, wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in a mammalian antigen-presenting cell. [Embodiment 14] 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. [Embodiment 15] 15. The composition of any one of embodiments 1 to 14, wherein the amino acid sequence of the nucleocapsid protein comprises residues 2 to 419 of SEQ ID NO:5 or an amino acid sequence at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to residues 2 to 419 of SEQ ID NO:5. [Embodiment 16] 15. The composition of any one of embodiments 1 to 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. [Embodiment 17] The composition according to any one of embodiments 6 to 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. [Embodiment 18] 17. The composition of embodiment 16, wherein the open reading frame comprises the nucleotide sequence of SEQ ID NO:2. [Embodiment 19] 18. The composition of embodiment 17, wherein the open reading frame comprises the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4. [Embodiment 20] 15. The composition of any one of embodiments 1 to 14, wherein the amino acid sequence of the fusion protein comprises residues 2 to 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 to 413 of SEQ ID NO:9. [Embodiment 21] 15. The composition of any one of embodiments 1 to 14, wherein the amino acid sequence of the fusion protein comprises residues 2 to 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 to 422 of SEQ ID NO: 10. [Embodiment 22] The composition of any one of the preceding embodiments, wherein the composition does not comprise liposomes or lipid nanoparticles. [Embodiment 23] The composition of any one of embodiments 1 to 22, wherein the mRNA is a self-replicating mRNA. [Embodiment 24] 24. The composition of embodiment 23, wherein the self-replicating RNA comprises an alphavirus replicon lacking viral structural protein coding regions. [Embodiment 25] 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. [Embodiment 26] The composition of embodiment 25, wherein the alphavirus is Venezuelan Equine Encephalitis virus. [Embodiment 27] The composition of any one of embodiments 23 to 26, wherein the alphavirus replicon comprises a nonstructural protein coding region having an insertion of 12 to 18 nucleotides that results in expression of nonstructural protein 2 (nsP2) comprising 4 to 6 additional amino acids between beta sheet 4 and beta sheet 6 of nsP2. [Embodiment 28] The composition of any one of embodiments 1 to 27, wherein the self-replicating mRNA is a temperature-sensitive agent (ts-agent) capable of expressing a fusion at a permissive temperature but not at a non-permissive temperature. [Embodiment 29] The composition of embodiment 28, wherein the permissive temperature is between 31°C and 35°C and the non-permissive temperature is at least 37°C±0.5°C. [Embodiment 30] A method for stimulating an immune response against coronavirus in a mammalian subject, comprising administering to the mammalian subject a composition described in any one of embodiments 1 to 29, so as to stimulate an immune response against coronavirus nucleocapsid protein in the mammalian subject. [Embodiment 31] The method of embodiment 30, wherein the composition is administered intradermally. [Embodiment 32] The method of embodiment 30 or embodiment 31, wherein the immune response comprises a coronavirus-reactive cellular immune response. [Embodiment 33] 33. The method of embodiment 32, wherein the immune response further comprises a coronavirus-reactive humoral immune response. [Embodiment 34] The method of any one of embodiments 30-33, wherein the mammalian subject is a human subject. [Embodiment 35] below: The composition according to any one of embodiments 1 to 29 or any one of embodiments 37 to 62; and A device for intradermal delivery of a composition to a mammalian subject; Including the kit. [Embodiment 36] The kit of embodiment 35, wherein the device comprises a syringe and a needle. [Embodiment 37] 1. A composition for stimulating an immune response to two or more viruses in a mammalian subject comprising an excipient and a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a fusion protein, said ORF comprising, from 5' to 3', the following: (i) a nucleotide sequence encoding a mammalian signal peptide; and (ii) a nucleotide sequence encoding a first nucleocapsid protein of a first virus and a second nucleocapsid protein of a second virus; A composition comprising: [Embodiment 38] 38. The composition of embodiment 37, wherein the first and second viruses are capable of causing disease upon infection of a human subject. [Embodiment 39] The composition of embodiment 38, wherein the first and second viruses are different variants, subtypes or lineages of the same species. [Embodiment 40] The composition of embodiment 38, wherein the first and second viruses are different species of the same genus. [Embodiment 41] The composition of embodiment 40, wherein the first and second viruses are both members of the betacoronavirus genus. [Embodiment 42] The composition of embodiment 41, wherein the first and second viruses comprise Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2) and Middle East Respiratory Syndrome-related coronavirus (MERS-CoV). [Embodiment 43] The composition of embodiment 38, wherein the first and second viruses are members of different families, orders, classes or phyla of the same kingdom. [Embodiment 44] The composition of embodiment 43, wherein the first and second viruses are both members of the Orthomyxoviridae family. [Embodiment 45] The composition of embodiment 44, wherein the first and second viruses comprise an influenza A virus and an influenza B virus. [Embodiment 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. [Embodiment 47] The composition of embodiment 38, wherein the first and second viruses are both members of the Ortornavirus kingdom, and optionally the first and second viruses 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. [Embodiment 48] The composition of embodiment 40, wherein the first and second viruses are both members of the Ebolavirus genus, and optionally, the first and second viruses are selected from the group consisting of Zaire Ebolavirus, Sudan Ebolavirus, Bundibugyo Ebolavirus, and Tai Forest Ebolavirus. [Embodiment 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, wherein the first, second, third and fourth viruses are Zaire Ebolavirus, Sudan Ebolavirus, Bundibugyo Ebolavirus and Tai Forest Ebolavirus. [Embodiment 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. [Embodiment 51] The composition of embodiment 49, wherein the nucleotide sequence (ii) encodes a shared portion of a first nucleocapsid protein of a first virus for stimulating an immune response against all of the first, second, third and fourth viruses. [Embodiment 52] 52. The composition of embodiment 51, wherein the nucleotide sequence (ii) encodes an individual portion of each of the first, second, third and fourth nucleocapsid proteins for stimulating an immune response against all of the first, second, third and fourth viruses. [Embodiment 53] The composition of embodiment 52, wherein the nucleotide sequence (ii) encodes fragments of individual portions of a second nucleocapsid protein of a second virus for stimulating an immune response against the second and third viruses. [Embodiment 54] The composition of embodiment 37, wherein the nucleotide sequence (ii) encodes a shared portion of a first nucleocapsid protein of a first virus for stimulating an immune response against both the first and second viruses. [Embodiment 55] The composition of embodiment 54, wherein the nucleotide sequence (ii) encodes individual portions of each of the first and second nucleocapsid proteins for stimulating an immune response against both the first and second viruses. [Embodiment 56] The composition of any one of embodiments 37 to 48, wherein the nucleotide sequence of (ii) further encodes at least one additional nucleocapsid protein of at least one additional virus, wherein the at least one additional virus is different from the first and second viruses. [Embodiment 57] 57. The composition according to any one of embodiments 37 to 56, wherein the first and second or first, second and further nucleocapsid proteins are separated by a linker of 1 to 10 residues in length. [Embodiment 58] The composition according to any one of embodiments 37 to 57, wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in a mammalian antigen-presenting cell. [Embodiment 59] The composition of any one of embodiments 37 to 58, wherein the mRNA is a self-replicating mRNA. [Embodiment 60] The composition of embodiment 59, wherein the self-replicating mRNA is a temperature-sensitive agent (ts-agent) that allows the fusion protein to be expressed at a permissive temperature but not at a non-permissive temperature. [Embodiment 61] The composition of embodiment 60, wherein the permissive temperature is 31°C to 35°C and the non-permissive temperature is at least 37°C ± 0.5°C. [Embodiment 62] The composition of any one of embodiments 1-29 or any one of embodiments 37-61, wherein the composition further comprises chitosan. [Embodiment 63] A method for stimulating an immune response against two or more viruses in a mammalian subject, comprising administering to the mammalian subject a composition described in any one of embodiments 37 to 62, thereby stimulating an immune response against nucleocapsid proteins of the two or more viruses in the mammalian subject. [Embodiment 64] The method of embodiment 63, wherein the composition is administered intradermally. [Embodiment 65] The method of embodiment 63 or embodiment 64, wherein the immune response comprises a cellular immune response highly reactive to two or more viruses. [Embodiment 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. [Embodiment 67] 67. The method of embodiment 66, wherein said nucleocapsid protein-specific cytokine secretion comprises secretion of one or both of interferon-gamma and interleukin-4. [Embodiment 68] The method of embodiment 65, wherein the cellular immune response comprises a nucleocapsid protein-specific cytotoxic T lymphocyte (CTL) response. [Embodiment 69] The method of any one of embodiments 65 to 68, wherein the immune response further comprises a humoral immune response highly reactive to two or more viruses. [Embodiment 70] The method of any one of embodiments 63-69, wherein the mammalian subject is a human subject. [Embodiment 71] 1. A composition for stimulating an immune response to a virus in a mammalian subject, comprising an excipient and a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a fusion protein, the ORF comprising, from 5' to 3', the following: (i) a nucleotide sequence encoding a mammalian signal peptide; (ii) a nucleotide sequence encoding a first viral antigen or a fragment thereof of a first virus; and (iii) a nucleotide sequence encoding a second viral antigen or a fragment thereof of the first virus or the 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. [Embodiment 72] 1. A composition for stimulating an immune response to two or more viruses in a mammalian subject, comprising an excipient and a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a fusion protein, the ORF comprising, from 5' to 3', the following: (i) a nucleotide sequence encoding a mammalian signal peptide; (ii) a nucleotide sequence encoding a first viral antigen or a fragment thereof from a first virus; (iii) a nucleotide sequence encoding a second viral antigen or a fragment thereof of the first virus; (iv) a nucleotide sequence encoding a third viral antigen or a fragment thereof of a second virus; (iii) a nucleotide sequence encoding a fourth viral antigen or a fragment thereof of a second virus; 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 The composition, 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. [Embodiment 73] The composition of embodiment 71 or embodiment 72, wherein the mRNA is a self-replicating mRNA. [Embodiment 74] The composition of embodiment 73, wherein the self-replicating RNA comprises an alphavirus replicon lacking viral structural protein coding regions. [Embodiment 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. [Embodiment 76] The composition of embodiment 74, wherein the alphavirus is Venezuelan Equine Encephalitis virus. [Embodiment 77] The composition of any one of embodiments 73 to 76, wherein the self-replicating mRNA is a temperature-sensitive agent capable of expressing a fusion protein at a permissive temperature but not at a non-permissive temperature. [Embodiment 78] The composition of embodiment 77, wherein the permissive temperature is 31°C to 35°C and the non-permissive temperature is at least 37°C ± 0.5°C. [Embodiment 79] The composition of any one of embodiments 74 to 78, wherein the alphavirus replicon comprises a nonstructural protein coding region having an insertion of 12 to 18 nucleotides that results in expression of nonstructural protein 2 (nsP2) that comprises 4 to 6 additional amino acids between beta sheet 4 and beta sheet 6 of nsP2. [Embodiment 80] The composition of any one of embodiments 71 to 79, wherein the first virus and / or the second virus is a coronavirus, optionally, the coronavirus is a beta coronavirus, and optionally, the beta coronavirus is a human beta coronavirus. [Embodiment 81] The composition of embodiment 80, wherein the first and / or 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). [Embodiment 82] The composition of embodiment 80, wherein the first virus is SARS-CoV-2 and the second virus is MERS-CoV. [Embodiment 83] The composition of any one of embodiments 80 to 82, wherein the surface protein, the first surface protein and / or the second surface protein each comprise a receptor binding domain (RBD) of a coronavirus spike protein. [Embodiment 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. [Embodiment 85] The composition of any one of embodiments 71-79, wherein the first virus and / or the second virus is a member of the Orthomyxoviridae family. [Embodiment 86] The composition of embodiment 85, wherein the first and / or second virus is independently selected from the group consisting of influenza A virus (IAV) and influenza B virus (IBV). [Embodiment 87] The composition of embodiment 86, wherein said first virus is IAV and said second virus is IBV. [Embodiment 88] 88. The composition of any one of embodiments 85 to 87, wherein the surface protein, the first surface protein and / or the second surface protein each comprise a portion of influenza hemagglutinin. [Embodiment 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 at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29. [Embodiment 90] The composition of any one of embodiments 71-89, wherein the composition further comprises chitosan. [Embodiment 91] below: (i) the composition according to any one of embodiments 71 to 90; and (ii) a device for intradermal delivery of the composition to a mammalian subject; Kit including: [Embodiment 92] The kit of embodiment 91, wherein the device comprises a syringe and a needle. [Embodiment 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 to one or more of the first viral antigen, the second viral antigen, the third viral antigen, and the fourth viral antigen. [Embodiment 94] A method for stimulating an immune response in a mammalian subject, comprising administering to the mammalian subject a composition described in any one of embodiments 71 to 90, thereby stimulating an immune response in the mammalian subject against one or more of a first viral antigen, a second viral antigen, a third viral antigen and a fourth viral antigen. [Embodiment 95] The method of embodiment 94, wherein the composition is administered intradermally. [Embodiment 96] 96. The method of embodiment 95, wherein the immune response comprises a cellular immune response highly reactive to one or more of the first viral antigen, the second viral antigen, the third viral antigen, and the fourth viral antigen. [Embodiment 97] 97. The method of embodiment 96, wherein the immune response further comprises a humoral immune response highly reactive to one or more of the first viral antigen, the second viral antigen, the third viral antigen, and the fourth viral antigen. [Embodiment 98] The method of any one of embodiments 94-97, wherein the mammalian subject is a human subject. [Embodiment 99] A method for active booster immunization against at least one virus, comprising administering intradermally to a mammalian subject in need thereof a 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 secondary immune response against the virus, wherein the mammalian subject has already received a primary immunization regimen against the virus. [Embodiment 100] The method of embodiment 99, wherein the primary immunization regimen comprises administration of at least one dose of a different vaccine against a virus. [Embodiment 101] 101. The method of embodiment 100, wherein said different vaccines comprise at least one viral protein antigen, optionally wherein said protein antigen is a recombinant protein or fragment thereof or an inactivated virus. [Embodiment 102] 1. A method for active boosting immunization against at least one virus, comprising: (i) administering intradermally to a mammalian subject in need thereof a composition according to any one of embodiments 1 to 29, any one of embodiments 37 to 62, or any one of embodiments 71 to 90 to stimulate a primary immune response against the virus; and (ii) administering to the mammalian subject at least one dose of a different vaccine against the virus to stimulate a secondary immune response against the virus; A method comprising: [Embodiment 103] 103. The method of embodiment 102, wherein said different vaccines comprise at least one viral protein antigen, optionally wherein said protein antigen is a recombinant protein or fragment thereof or an inactivated virus. [Embodiment 104] 1. A method for active primary immunization against at least one virus, comprising: (i) administering intradermally to a mammalian subject in need thereof the composition of any one of embodiments 1 to 29, any one of embodiments 37 to 62, or any one of embodiments 71 to 90 to stimulate a primary immune response against the virus; wherein the mammalian subject has not received a primary immunization regimen against the virus. A method comprising: [Embodiment 105] below: (ii) administering to the mammalian subject at least one dose of a different vaccine against the virus to stimulate a secondary immune response against the virus; 105. The method of embodiment 104, further comprising: [Embodiment 106] 106. The method of embodiment 105, wherein said different vaccines comprise at least one viral protein antigen, optionally wherein said protein antigen is a recombinant protein or fragment thereof or an inactivated virus. [Embodiment 107] The method of any one of embodiments 94-106, wherein the mammalian subject is a human subject. [Embodiment 108] An expression vector comprising an mRNA according to any preceding claim in operative combination with a promoter. [Embodiment 109] The expression vector of embodiment 108, wherein the promoter is a T7 promoter or an SP6 promoter. [Embodiment 110] The expression vector of embodiment 108, wherein the vector is a plasmid. [Embodiment 111] 111. An expression vector according to any one of embodiments 108 to 110, further comprising a selectable marker. EXAMPLES

[0095] Abbreviations: Ab (antibody); APC (antigen presenting cell); CoV (coronavirus); c-srRNA (temperature-controllable self-replicating RNA); CTL (cytotoxic T lymphocyte); FluA or IAV (influenza A virus); FluB or IBV (influenza B virus); IL-4 (interleukin 4); INF-γ (interferon gamma); GOI (gene of interest); HA (hemagglutinin); MERS (Middle East respiratory syndrome associated); nAb (neutralizing antibody); N or NP (nucleocapsid or nucleoprotein); nsP (nonstructural 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 cell); SFU (spot forming unit); srRNAts (temperature-sensitive self-replicating RNA); Th (helper T lymphocyte); and Tx (treatment). The terms c-srRNA and srRNAts are used interchangeably throughout this disclosure, with srRNA1ts2 (described in WO 2021 / 138447 A1) being an exemplary embodiment.

[0096] Example 1. Cellular immunity induced by srRNA1ts2-G5004 This example describes the finding that the SARS-CoV-2 nucleoprotein alone (G5004 antigen without the signal peptide) does not induce a strong cellular immune response when the protein is expressed from temperature-controllable self-replicating RNA injected intradermally.

[0097] Materials and Methods CD-1 outbred female mice.

[0098] The srRNA1ts2-G5004 mRNA was produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA1ts2 as described in PCT / US2020 / 067506) encoding the G5004 antigen (Figure 2).

[0099] A pool of 102 peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the SARS-CoV-2 nucleoprotein (UniProt: P0DTC9) [JPT Peptide Product Code: PM-WCPV-NCAP].

[0100] Interferon gamma (INF-γ) and interleukin-4 (IL-4) ELISpot assay plates and reagents (Cellular Technology Limited, Ohio, USA).

[0101] Immunospot S6 Entry Analyzer (Cellular Technology Limited, Ohio, USA).

[0102] result Recently, it has been shown that vaccination with the nucleoprotein (N) alone induces cell-mediated and spike-independent protective immunity against SARS-CoV-2 in mice and hamsters (Machett et al., bioRxiv.2021.04.26.441518.2021). Vaccination involved the intravenous administration of a human adenovirus serotype 5 (Ad5) vector expressing the N sequence (Ad5-N) originating from the USA-WA1 / 2021 strain.

[0103] To test whether the nucleoprotein (N) alone (without signal peptide) could induce cellular immunity, ELISpot assays were performed 14 days after vaccination of CD-1 outbred mice by a single intradermal injection of 5 μg or 25 μg of srRNA1ts2-G5004 (Figure 2) or placebo (PBO: buffer only). Only a weak induction of interferon-γ (INF-γ)-secreting T cells (Figure 4A) and IL-4-secreting T cells (Figure 4B) was observed. Interestingly, the INF-γ response was not observed to be dose-dependent (5 μg vs. 25 μg).

[0104] It was concluded that the nuclear protein (N) alone, when expressed from temperature-controllable self-replicating RNA injected intradermally, does not induce a strong cellular immune response.

[0105] Example 2. Cellular immunity induced by srRNA1ts2-G5005 This example describes the finding that the addition of the CD5 signal peptide to the nucleoprotein of SARS-CoV-2 induces a robust cellular immune response in CD-1 mice when expressed from temperature-controllable self-replicating RNA injected intradermally.

[0106] Materials and Methods CD-1 outbred female mice.

[0107] The mRNA for srRNA1ts2-G5005 was produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA1ts2 as disclosed in PCT / US2020 / 067506) encoding the G5005 antigen (Figure 2).

[0108] A pool of 102 peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the SARS-CoV-2 nucleoprotein (UniProt: P0DTC9) [JPT Peptide Product Code: PM-WCPV-NCAP].

[0109] Interferon gamma (INF-γ) and interleukin-4 (IL-4) ELISpot assay plates and reagents (Cellular Technology Limited, Ohio, USA).

[0110] Immunospot S6 Entry Analyzer (Cellular Technology Limited, Ohio, USA).

[0111] result The wild-type nuclear protein does not include a signal peptide or a transmembrane domain and is therefore expected not to be directed into the secretory pathway of mammalian host cells. The inventors reasoned that the lack of a signal peptide may be the reason why the wild-type nuclear protein (expressed from srRNA1ts2-G5004 in Example 1) did not induce a strong cellular immune response. With this in mind, the coding region of the signal peptide sequence from the human CD5 gene was added to the coding region of the nuclear protein in place of the start codon (ATG) of the nuclear protein in srRNA1ts2-G5005 (Figure 2). The amino acid sequence of the CD5 signal peptide is MPMGSLQPLATLYLLGMLVASCLG (set forth as SEQ ID NO: 8).

[0112] Cellular immunity was assessed by ELISpot assay 14 days after vaccination of CD-1 outbred mice with a single intradermal injection of 5 μg or 25 μg of srRNA1ts2-G5005 (Figure 2) or placebo (PBO: buffer only).

[0113] As shown in Figure 5A, antigen-specific INF-γ-secreting T cells were strongly induced in a dose-dependent manner (5 μg vs. 25 μg). In contrast, antigen-specific IL-4-secreting T cells were hardly induced (Figure 5B). Th1 cells secrete INF-γ, whereas Th2 cells secrete IL-4. It is generally accepted that Th1>Th2 immune responses are favorable characteristics of vaccines.

[0114] In conclusion, the addition of a signal peptide from human CD5 to the N-terminus of the nucleoprotein (N) resulted in the induction of strong antigen-specific cellular immune responses when the protein was expressed from temperature-controllable self-replicating RNA injected intradermally. The srRNA1ts2-G5005 vaccine also demonstrated a favorable Th1-skewed (Th1>Th2) immune response.

[0115] Example 3. Cellular immunity induced by srRNA1ts2-G5005 This example describes the finding that the addition of the CD5 signal peptide to the SARS-CoV-2 nucleoprotein induces potent cellular immune responses in BALB / c mice when expressed from temperature-controllable self-replicating RNA injected intradermally.

[0116] Materials and Methods BALB / c female mouse.

[0117] The mRNA for srRNA1ts2-G5005 was produced by in vitro transcription of a temperature-controllable self-replicating RNA vector encoding the G5005 antigen (srRNA1ts2 as described in [PCT / US2020 / 067506]) (Figure 2).

[0118] A pool of 102 peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the SARS-CoV-2 nucleoprotein (UniProt: P0DTC9) [JPT Peptide Product Code: PM-WCPV-NCAP].

[0119] Interferon gamma (INF-γ) and interleukin-4 (IL-4) ELISpot assay plates and reagents (Cellular Technology Limited, Ohio, USA).

[0120] Immunospot S6 Entry Analyzer (Cellular Technology Limited, Ohio, USA).

[0121] result To test whether srRNA1ts2-G5005 could induce a strong cellular immune response in another mouse strain, an immunogenicity study was also performed in BALB / c mice. Cellular immunity was assessed by ELISpot assay 30 days after vaccination of BALB / c mice with a single intradermal injection of 5 μg or 25 μg of srRNA1ts2-G5005 (Figure 2) or placebo (PBO: buffer only).

[0122] As shown in Figure 6A, antigen-specific INF-γ-secreting T cells were strongly induced in a dose-dependent manner (5 μg vs. 25 μg). In contrast, antigen-specific IL-4-secreting T cells were not induced (Figure 6B). Therefore, a favorable Th1>Th2 cell response was also observed in BALB / c mice.

[0123] In conclusion, the addition of a signal peptide from human CD5 to the N-terminus of the nucleoprotein (N) significantly enhanced antigen-specific cellular immune responses when the protein was expressed from temperature-controllable self-replicating RNA injected intradermally. In CD-1 mice, the srRNA1ts2-G5005 vaccine induced favorable Th1-skewed (Th1>Th2) immune responses in BALB / c mice.

[0124] Example 4. Humoral immunity induced by srRNA1ts2-G5005 This example describes the finding that the SARS-CoV-2 nucleoprotein, when combined with the human CD5 signal peptide, induces a strong humoral immune response when the protein is expressed from temperature-controllable self-replicating RNA injected intradermally.

[0125] Materials and Methods BALB / c female mouse

[0126] The mRNA for srRNA1ts2-G5005 was produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA1ts2 as described in PCT / US20 / 67506) encoding the G5005 antigen (Fig. 2 ).

[0127] SARS-CoV-2 Nucleocapsid IgG ELISA Kit (ENZO: ENZ-KIT193-0001).

[0128] result To test whether srRNA1ts2-G5005 can induce humoral immunity, 30 days after vaccination of BALB / c mice by a single intradermal injection of 5 μg or 25 μg of srRNA1ts2-G5005 (FIG. 2) or placebo (PBO: buffer only), nucleoprotein-specific IgG levels in serum were measured by ELISA. IgG levels are expressed by OD450 in ELISA. IgG levels were measured before (day -1) and after (day 30) vaccination (day 0).

[0129] As shown in FIG. 7, nucleoprotein-specific serum IgG was strongly induced in a dose-dependent manner (5 μg vs. 25 μg).

[0130] In conclusion, addition of a signal peptide originating from human CD5 to the N-terminus of the nuclear protein (N) induced antigen-specific humoral immune responses when the protein was expressed from temperature-controllable self-replicating RNA injected intradermally.

[0131] Example 5. Cellular immunity induced by srRNA1ts2-G5006 This example describes the findings that a fusion protein containing the SARS-CoV-2 nucleoprotein and the MERS-CoV nucleoprotein, when expressed from temperature-controllable self-replicating RNA injected intradermally, can induce potent cellular immunity against SARS-CoV-2 and MERS-CoV.

[0132] Materials and Methods BALB / c female mouse.

[0133] The srRNA1ts2-G5006 mRNA was produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA1ts2 as described in PCT / US2020 / 067506) encoding the G5006 antigen (Figure 2C).

[0134] Pool of peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the SARS-CoV-2 nucleoprotein (UniProt: P0DTC9) [JPT Peptide Product Code: PM-WCPV-NCAP].

[0135] Pool of peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the nucleoprotein of MERS-CoV.

[0136] Interferon gamma (INF-γ) and interleukin-4 (IL-4) ELISpot assay plates and reagents (Cellular Technology Limited, Ohio, USA).

[0137] Immunospot S6 Entry Analyzer (Cellular Technology Limited, Ohio, USA).

[0138] result T cell epitopes are typically present in short linear peptides ranging in size from 8 to 11 residues for MHC class I and 10 to 30 residues for MHC class II. Unlike many B cell epitopes, the 3-D conformation of T cell epitopes is not important for recognition by immune cell receptors. Therefore, the inventors reasoned that nucleoproteins from different β-coronavirus strains could be fused in the absence of very long linkers (longer than 10 amino acids) for use as vaccine antigens to elicit immune responses against different β-coronaviruses (e.g., SARS-CoV-1 and their mutants, SARS-CoV-2 and their mutants, and MERS-CoV and their mutants).

[0139] To test this concept, we designed a fusion protein containing the human CD5 signal peptide, SARS-CoV-2 nucleoprotein, and MERS-CoV nucleoprotein (see G5006 in Figure 2C). Mice were vaccinated with srRNA1ts2-G5006 by intradermal injection, and antigen-specific cellular immune responses were measured by ELISpot assay. As expected, the srRNA1ts2-G5006 vaccine induced strong INF-γ-secreting T cell responses against both SARS-CoV-2 nucleoprotein (Figure 8) and MERS-CoV nucleoprotein. Furthermore, the cellular immune response is expected to have a Th1>Th2 balance.

[0140] In conclusion, fusion proteins containing nucleoproteins from different β-coronaviruses induced strong antigen-specific cellular immune responses when expressed from temperature-controllable self-replicating RNA injected intradermally.

[0141] Example 6. Cellular immunity induced by srRNA1ts2-G5010 (pan-influenza vaccine) This example describes the evaluation of the immune response induced by a fusion protein containing the nucleoprotein of influenza A virus (FluA) and the nucleoprotein of influenza B virus (FluB) when the protein was expressed from a temperature-controllable self-replicating RNA injected intradermally.

[0142] Materials and Methods BALB / c female mouse.

[0143] The mRNA of srRNA1ts2-G5010 was produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA1ts2 [PCT / US20 / 67506]) encoding the G5010 antigen ( FIG. 9 ). The amino acid sequence of the G5010 fusion protein is set forth as SEQ ID NO: 16. The nucleic acid sequence encoding the G5010 fusion protein was codon-optimized for expression in human cells and set forth as SEQ ID NO: 15.

[0144] A pool of 122 overlapping peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the nucleoprotein (NP) of influenza A (H2N2) (Swiss-Prot ID P21433) [JPT Peptide Product Code: PM-INFA-NPH2N2]. The amino acid sequence of the H2N2 nucleoprotein is set forth as SEQ ID NO: 17.

[0145] Interferon gamma (INF-γ) and interleukin-4 (IL-4) ELISpot assay plates and reagents (Cellular Technology Limited, Ohio, USA).

[0146] Immunospot S6 Entry Analyzer (Cellular Technology Limited, Ohio, USA).

[0147] result Influenza types A and B can infect humans and cause seasonal epidemics or pandemics (see "Types of Influenza Viruses" at the CDC website at www.cdc.gov / flu / about / viruses / types.htm). Compared with the hemagglutinin (HA) and neuraminidase (NA) antigens that are routinely included in influenza vaccines, the nucleoprotein antigens are more conserved among different influenza virus strains. For example, the amino acid sequences of the nucleoproteins of representative influenza type A strains (H1N1, H3N2, H5N8, H7N7, H7N9, H9N2, H10N8) are very similar. Similarly, the amino acid sequences of the nucleoproteins of representative influenza type B strains (Yamagata, Victoria) are very similar. In contrast, the amino acid sequences of the nucleoproteins of influenza type A are significantly different from those of influenza type B.

[0148] T cell epitopes are present in short linear peptides, usually in the size range of 8-11 residues for MHC class I and 10-30 residues for MHC class II. Unlike B cell epitopes, the conformational or 3D structure of T cell epitopes is not important for recognition by immune cell receptors. Therefore, one representative nucleoprotein from influenza A virus is thought to contain many T cell epitopes shared by many influenza A virus strains. Similarly, a representative nucleoprotein from influenza B virus is thought to contain many T cell epitopes shared by many influenza B virus strains. Therefore, the inventors reasoned that nucleoproteins from different influenza strains could be fused in the absence of very long linkers (over 10 amino acids long) for use as vaccine antigens to induce immune responses against different influenza viruses (e.g., different strains of influenza A and different strains of influenza B).

[0149] The amino acid sequences of the nucleoproteins of representative influenza A strains (H1N1, H3N2, H5N8, H7N7, H7N9, H9N2 and H10N8) were found to be similar to each other. The nucleoprotein of influenza strain H5N8 was selected because it showed the smallest difference from the nucleoproteins of other strains (H1N1, H3N2, H7N7, H7N9, H9N2 and H10N8). As a representative influenza B virus nucleoprotein, the nucleoprotein of influenza B strain (B / Florida / 4 / 2006; GenBank CY033879.1) was selected. A fusion protein containing human CD5 signal peptide, one FluA nucleoprotein and one FluB nucleoprotein was designed (see G5010 in Figure 9), and the coding region of the fusion protein was cloned downstream of the subgenomic promoter of srRNA1ts2. The amino acid sequence of the FluA nucleoprotein is listed as SEQ ID NO: 13 (Influenza A, subtype H5N8 [A / Breeder Duck / Korea / Gochang1 / 2014], GenBank No. KJ413835.1, ProteinID No. AHL21420.1), and the amino acid sequence of the FluB nucleoprotein is listed as SEQ ID NO: 14 (Influenza B [B / Florida / 4 / 2006], GenBank No. CY033879.1, ProteinID No. ACF54251.1).

[0150] 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, a pool of 122 overlapping peptides resulting from a peptide scan of the influenza A nucleoprotein sequence set forth as SEQ ID NO: 17 was used to restimulate splenocytes harvested from mice 14 days after vaccination. Despite the differences between the influenza A nucleoprotein sequence of G5010 and that of the peptide pool (Figure 10), the srRNA1ts2-G5010 vaccine induced a strong INF-γ-secreting T cell response against FluA nucleoprotein (Figure 11). Importantly, there was little to no induction of IL-4-secreting T cells against FluA nucleoprotein. These results indicate that the srRNA1ts2-G5010 vaccine induces a Th1 (INF-γ)-dominant response (Th1>Th2 balance), a favorable characteristic for a vaccine against viral diseases.

[0151] In conclusion, fusion proteins containing nucleoproteins from representative influenza A and B strains induced strong antigen-specific cellular immune responses when the fusion proteins were expressed from temperature-controllable self-replicating RNA injected intradermally.

[0152] Example 7. Cellular immunity induced by srRNA1ts2-PanEbola (pan-Ebola vaccine) This example describes the results of a study that fusion proteins containing fragments of nucleoproteins from four Ebola viruses (Zaire Ebola virus, Sudan Ebola virus, Bundibugyo Ebola virus, and Tai Forest Ebola virus) can induce strong cellular immunity against Ebola virus when the fusion proteins are used as vaccine antigens. This example uses a temperature-controllable self-replicating RNA as an expression vector.

[0153] Materials and Methods BALB / c female mouse.

[0154] srRNA1ts2-PanEbola mRNA was produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA1ts2 [PCT / US20 / 67506]) encoding the PanEbola antigen (Fig. 12 ).

[0155] Ebolavirus - Pool of 182 peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the nucleoprotein (Swiss-Prot ID: B8XCN6) of Tai Forest Ebolavirus [JPT peptides; PepMix Tai Forest Ebolavirus (NP); JPT product code: PM-TEBOV-NP].

[0156] Interferon gamma (INF-γ) and interleukin-4 (IL-4) ELISpot assay plates and reagents (Cellular Technology Limited, Ohio, USA).

[0157] Immunospot S6 Entry Analyzer (Cellular Technology Limited, Ohio, USA).

[0158] result The Ebola virus causes a highly fatal hemorrhagic fever. Four species of Ebola virus are known to cause disease in humans: Ebolavirus (Zaire Ebolavirus species), Sudanvirus (Sudan Ebolavirus species), Bundibugyovirus (Bundibugyo Ebolavirus species), and Tai Forest virus (Taï Forest Ebolavirus species, formerly Côte d'Ivoire Ebolavirus).

[0159] Currently, only one licensed vaccine (rVSV-ZEBOV) is available for Ebola virus. This vaccine is an attenuated recombinant vesicular stomatitis virus (VSV) expressing the major glycoprotein (GP) from Zaire Ebola virus. The vaccine can induce neutralizing antibodies against Ebola virus, but the protein sequence of GP is highly divergent among the four Ebola virus species that infect humans. Therefore, the rVSV-ZEBOV vaccine is only effective against Zaire Ebola virus. A pan-Ebola virus vaccine that can provide protection against all four Ebola virus species is desirable.

[0160] Compared to GP, the nucleoprotein (NP) sequence is more conserved among the four Ebola viruses. However, unlike GP, NP is not a surface protein, and therefore antibodies induced against NP are not neutralizing. Importantly, it has been shown that mice vaccinated with Zaire Ebola virus NP are protected from Zaire Ebola virus challenge, which is mediated by cellular immunity rather than humoral immunity (Wilson and Hart, J Virol, 75:2660-2664, 2001). It has also been shown that protection is 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).

[0161] It was reasoned that using a fusion protein of all four Ebola virus NPs as a vaccine antigen would provide protection against all four Ebola viruses. However, each NP is approximately 740 amino acids in length. Thus, fusing the entire four NPs would result in a relatively large protein of approximately 3,000 amino acids. A smaller size antigen would be desirable for many vaccine platforms.

[0162] The amino acid sequences of the nucleoproteins of the four Ebola virus species were compared using NCBI BlastP (NP of Zaire Ebola virus (GenBank ID: AF272001), NP of Sudan Ebola virus (GenBank ID: AF173836), NP of Bundibugyo Ebola virus (GenBank ID: FJ217161) and NP of Tai Forest Ebola virus (GenBank ID: FJ217162)). The sequences of the N-terminal half of NP (designated as region A) were found to be similar to each other (88%-92% identity), whereas the sequences of the C-terminal half of NP (designated as region B) were found to be diverse (42%-54%) (Table 7-1). Therefore, Zaire (A) was selected as a representative of Zaire (A), Sudan (A), Bundibugyo (A) and Tai Forest (A). With regard to region B, Bundibugyo (B) and Tai Forest (B) were found to be similar to each other (80% and 86% identity), except for the middle part (40% identity), designated as region C. Therefore, Zaire (B), Sudan (B), Bundibugyo (B) and Tai Forest (C) were selected for inclusion in the pan-Ebola vaccine. Before combining the four nucleoproteins to make one fusion protein, an additional 8 amino acid sequence was added on both sides to ensure that possible T cell epitopes at the ends of the nucleoprotein fragments were not destroyed. A schematic diagram of the fusion protein of the pan-Ebola antigen is shown in FIG. 12, which includes the NP fragments of Zaire (A), Zaire (B), Sudan (B), Bundibugyo (B) and Tai Forest (C) and the human CD5 signal peptide. A diagram showing the percent identity of the NP sequences of Ebola viruses is shown in FIG. 13. The amino acid sequence of the PanEbola antigen is set forth as SEQ ID NO:22, and the nucleic acid sequence encoding the PanEbola antigen is set forth as SEQ ID NO:23. [Table 1]

[0163] The srRNA1ts2-PanEbola vaccine was generated by cloning the PanEbola fusion protein downstream of the subgenomic promoter of srRNA1ts2. The mRNA was generated by in vitro transcription and used to intradermally vaccinate BALB / c mice. Antigen-specific cellular immune responses were measured by ELISpot assay. To recall nucleoprotein-reactive T cell immunity, a pool of 182 peptides resulting from a peptide scan of the nucleoprotein ((Swiss-Prot ID: B8XCN6) Ebolavirus-Taiforestin Ebolavirus) was used to restimulate splenocytes harvested from mice 14 days after vaccination. The srRNA1ts2-PanEbola vaccine induced a strong INF-γ-secreting T cell response against the nucleoprotein of Taiiforestin (Figure 14A). This is notable in that only a small portion (169 aa) of the Tai Forest nuclear protein was included in the mRNA vaccine, whereas the peptide pool used for restimulation covered the entire Tai Forest nuclear protein sequence. Importantly, there was little to no induction of IL-4-secreting T cells against the Tai Forest nuclear protein (Figure 14B). These results indicate that the srRNA1ts2-PanEbola vaccine induces a Th1 (INF-γ) dominated response (Th1>Th2 balance), which is a favorable characteristic of a vaccine against viral diseases.

[0164] In conclusion, fusion proteins containing nucleoprotein fragments from four Ebola viruses induced strong antigen-specific cellular immune responses when the fusion proteins were expressed from temperature-controllable self-replicating RNA injected intradermally. The examples demonstrate that the size of fusion proteins used as PanEbola vaccines can be reduced by removing well-conserved portions of one or more of the nucleoproteins that comprise the vaccine. PanEbola antigens are also suitable for other vaccine platforms (e.g., adenovirus, adeno-associated virus, recombinant proteins, etc.).

[0165] Example 8. Cellular immunity induced by srRNA1ts2-G5003o (Omicron vaccine) This example describes the results of a study showing that intradermal administration of c-srRNA encoding the RBD of SARS-CoV-2 (Omicron strain B.1.1.529) induces potent cellular immunity in mice.

[0166] Materials and Methods C57BL / 6 female mice.

[0167] srRNA1ts2-G5003o (mRNA) (Figure 15), produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA1ts2 [WO 2021 / 138447 A1]) encoding the G5003o antigen.

[0168] Pool of peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the RBD of the Omicron strain of SARS-CoV-2 (S-RBD B.1.1.529) [JPT peptide: PM-SARS2-RBDMUT08-1].

[0169] Interferon gamma (INF-γ) and interleukin-4 (IL-4) ELISpot assay plates and reagents (Cellular Technology Limited, Ohio, USA).

[0170] Immunospot S6 Entry Analyzer (Cellular Technology Limited, Ohio, USA).

[0171] result In this example, c-srRNA(G5003o) encoding the RBD of the omicron variant of SARS-CoV-2 was generated (Figure 15). The RNA was administered intradermally to C57BL / 6 mice, and 14 days later, splenocytes were harvested to test cellular immunity against the RBD (omicron variant) of SARS-CoV-2. Induction of IFN-γ-secreting T cells was specifically observed in recipients of c-srRNA-G5003o (Figure 16A), whereas induction of IL-4-secreting T cells was not observed in recipients of c-srRNA-G5003o (Figure 16B).

[0172] conclusion We demonstrated that by using the omicron variant-specific RBD as an antigen, the protein induced an omicron variant-specific cellular immune response when expressed from temperature-controllable self-replicating RNA injected intradermally. A favorable Th1 (INF-γ)>Th2 (IL-4) response was also observed.

[0173] Example 9. Efficacy of c-srRNA prime, protein boost immunization regimen This example describes the findings that administration of a c-srRNA vaccine encoding a protein antigen of the original virus can prime a humoral immune response against a protein antigen of the mutant virus.

[0174] Materials and Methods BALB / c female mouse.

[0175] srRNA1ts2-G5003 (mRNA) (Figure 15), produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA1ts2 [WO 2021 / 138447 A1]) encoding the G5003 antigen.

[0176] Recombinant SARS-CoV-2 B.1.617.2 spike GCN4-IZ protein (R&D Systems, Cat.#10878-CV)

[0177] AddaVax™ squalene-based oil-in-water adjuvant was obtained from InvivoGen.

[0178] Pool of peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the RBD of SARS-CoV-2 (original Wuhan strain) [JPT Peptides:PepMix SARS-CoV-2(S-RBD)PM-WCPV-S-RBD-2].

[0179] Interferon gamma (INF-γ) and interleukin-4 (IL-4) ELISpot assay plates and reagents (Cellular Technology Limited, Ohio, USA).

[0180] Immunospot S6 Entry Analyzer (Cellular Technology Limited, Ohio, USA).

[0181] Vero76 cells for plaque reduction neutralization assay (PRNT).

[0182] SARS-CoV-2 delta variant live virus for PRNT assay

[0183] In the PRNT assay, Vero76 cells were first treated with serially diluted mouse serum, followed by infection with live SARS-CoV-2 (delta mutant) virus. In this assay, infected cells die and form plaques that are stained with crystal violet after fixation. If the serum contains neutralizing antibodies, viral infection is inhibited, resulting in a reduction in the number of plaques. The result is a 50% reduction in the number of plaques (PRNT). 50 ) is expressed as the serum dilution titer.

[0184] result A composition containing c-srRNA encoding the G5003 antigen (the RBD of the original Wuhan strain of SARS-CoV-2) was administered intradermally to the skin of BALB / c mice as naked mRNA (Figure 17A). That is, the srRNA1ts2-G5003 composition did not contain any nanoparticles or transfection reagents. Then, a composition containing the spike protein of SARS-CoV-2 (delta variant B.1.617.2) mixed with an adjuvant was administered intradermally (Figure 17A).

[0185] Cellular immunity against the RBD protein of SARS-CoV-2 was detected in splenocytes of mice 14 days after a single intradermal injection of the c-srRNA-G5003 composition (Figure 17B). Subsequent exposure of immunized mice to the spike protein of a different SARS-CoV-2 strain (delta variant B.1.617.2) induced neutralizing antibodies (detected by PRNT assay) against the delta variant of SARS-CoV-2 as early as 7 days after protein antigen exposure (Figure 17C). In contrast, mice that did not receive c-srRNA-G5003 encoding the RBD of SARS-CoV-2 (original Wuhan strain) did not mount a neutralizing antibody response against the delta variant of SARS-CoV-2. The early induction of neutralizing antibodies is characteristic of a secondary immune response, indicating that c-srRNA primed the humoral immune response before exposure to the adjuvanted RBD protein.

[0186] conclusion The results indicate that c-srRNA immunogens can induce potent broadly reactive immune responses against both c-srRNA-encoded antigens and distinct variant antigens, and thus c-srRNA SARS-CoV-2 RBD immunogens are suitable for immunization regimens against a broad range of SARS-CoV-2 strains.

[0187] Example 10. Efficacy of a protein prime, c-srRNA boost immunization regimen This example describes the results of a study that shows that a c-srRNA vaccine can increase antibody titers when used as a booster vaccine for other vaccines.

[0188] Materials and Methods C57BL / 6 female mice.

[0189] RBD protein (Sino Biological SARS-CoV-2 [2019-nCoV] Spike RBD-His Recombinant Protein, Cat.#40592-V08B)

[0190] AddaVax™ squalene-based oil-in-water adjuvant was obtained from InvivoGen.

[0191] srRNA1ts2-G5003 (mRNA) (Figure 15), produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA1ts2 [WO 2021 / 138447 A1]) encoding the G5003 antigen.

[0192] srRNA1ts2-G5003o (mRNA) (Figure 15), produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA1ts2 [WO 2021 / 138447 A1]) encoding the G5003o antigen.

[0193] Pool of peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the RBD of SARS-CoV-2 (original Wuhan strain) [JPT Peptides:PepMix SARS-CoV-2(S-RBD)PM-WCPV-S-RBD-2].

[0194] Interferon gamma (INF-γ) and interleukin-4 (IL-4) ELISpot assay plates and reagents (Cellular Technology Limited, Ohio, USA).

[0195] Immunospot S6 Entry Analyzer (Cellular Technology Limited, Ohio, USA).

[0196] ELISA assay plate (ENZO SARS-CoV-2 IgG ELISA Kit [Cat.# ENZ-KIT170-0001, the plate was coated with the RBD protein of SARS-CoV-2 (Wuhan strain) S1 antigen)].

[0197] result To test the possibility of using the c-srRNA vaccine as a booster vaccine, mice were first vaccinated with an adjuvanted protein (in this case, the RBD of SARS-CoV-2 [original Wuhan strain]). Fourteen days later (day 14), mice were further treated with an intradermal injection of placebo (PBO: buffer only), c-srRNA encoding the G5003 antigen, c-srRNA encoding the G5003o antigen, or adjuvanted RBD protein (Figure 18A).

[0198] On day 28, cellular immunity was evaluated by ELISpot assay. As expected, RBD(1st)+PBO(2nd) group failed to induce cellular immunity, whereas RBD(1st)+RBD(2nd) group induced cellular immunity (Fig. 18B, C). Interestingly, RBD(1st)+c-srRNA-G5003 and c-srRNA-G5003o groups also induced cellular immunity (Fig. 18B, C). This was expected because c-srRNA vaccine alone can induce cellular immunity.

[0199] On day 28, the levels of serum antibodies against the RBD of the SARS-CoV-2 virus (original Wuhan strain) were assessed by ELISA assay (Figure 19). The first vaccination with the adjuvanted RBD protein alone was able to weakly induce antibodies. On the other hand, the c-srRNA vaccine was able to induce antibodies at a similar level as the second vaccination with the adjuvanted protein.

[0200] conclusion The results indicate that the c-srRNA vaccine can function as a booster vaccine for both cellular and humoral immunity.

[0201] Example 11. Potent cellular immune response induced by srRNA1ts2-G5006 This example describes the results of a study showing that a fusion protein containing the SARS-CoV-2 nucleoprotein and the MERS-CoV nucleoprotein, when expressed from temperature-controllable self-replicating RNA injected intradermally, can induce potent cellular immunity against SARS-CoV-2 and MERS-CoV. Vaccinated mice can eliminate implanted tumor cells expressing a fusion protein containing the SARS-CoV-2 nucleoprotein and the MERS-CoV nucleoprotein.

[0202] Materials and Methods BALB / c female mouse.

[0203] srRNA1ts2-G5006 mRNA was produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA1ts2 as described in WO 2021 / 138447 A1) encoding the G5006 antigen (Figure 2).

[0204] Pool of peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the SARS-CoV-2 nucleoprotein (UniProt: P0DTC9) [JPT Peptide Product Code: PM-WCPV-NCAP].

[0205] Pool of peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the nucleoprotein of MERS-CoV (YP_009047211.1). Peptides were custom made by JPT Peptides.

[0206] Interferon gamma (INF-γ) and interleukin-4 (IL-4) ELISpot assay plates and reagents (Cellular Technology Limited, Ohio, USA).

[0207] Immunospot S6 Entry Analyzer (Cellular Technology Limited, Ohio, USA).

[0208] The 4T1 breast cancer cell line, which arises from BALB / c mice and is a known model of triple-negative stage 4 human breast cancer, was purchased from ATCC (catalog #CRL-2539).

[0209] Plasmid DNA encoding a fusion protein of SARS-CoV-2 and MERS-CoV nucleoproteins (non-secreted form of G5006, i.e., without CD5 signal peptide) under the CMV promoter and a hygromycin resistance gene under the promoter of the SV40 early promoter was transfected into 4T1 cells. Cells expressing the fusion protein of SARS-CoV-2 and MERS-CoV nucleoproteins (called 4T1-SMN) were isolated by culturing in the presence of 200 μg / mL hygromycin B.

[0210] result To model virus-infected cells, we used the 4T1 breast cancer cell line, which originates from BALB / c mice and is known to be a model of triple-negative stage 4 human breast cancer. When injected into BALB / c mice, 4T1 cells grow rapidly and form tumors. We used this syngenic mouse model to mimic the rapid increase in infected cells. For this purpose, we first created a plasmid vector encoding a fusion protein (named SMN protein) of the nucleoproteins of SARS-CoV-2 and MERS-CoV under the CMV promoter so that the protein could be constitutively expressed. This fusion protein is the same as G5006, but the CD5 signal peptide was removed from the N-terminus of the protein. Naturally, the nucleoprotein does not have a signal peptide and remains in the cytoplasm of the cell. The plasmid vector also carried a hygromycin resistance gene, so 4T1 cells expressing the SMN protein (named 4T1-SMN) were established after hygromycin selection.

[0211] BALB / c mice were vaccinated with c-srRNA-G5006, and induction of cellular immunity was demonstrated by the presence of T cells responding to both the SARS-CoV-2 nucleoprotein (Figure 20A) and the MERS-CoV nucleoprotein (Figure 20B).

[0212] 4T1-SMN cells were injected into BALB / c mice vaccinated with c-srRNA-G5006 on day 24 (24 days after vaccination) (Figure 21). As expected, 4T1-SMN cells rapidly grew in mice vaccinated with placebo (non-vaccinated group). Meanwhile, 4T1-SMN tumor growth was suppressed in mice vaccinated with c-srRNA-G5006. Two mice vaccinated with 25 μg of c-srRNA-G5006 vaccine initially grew tumors, but then became tumor-free and survived. Moreover, even after a second 4T1-SMN tumor injection on day 143 after vaccination, tumors did not grow and the mice continued to survive tumor-free (Figure 21).

[0213] conclusion The c-srRNA vaccine can induce strong cell-mediated immunity that can kill and eliminate cells expressing the antigen, indicating that c-srRNA functions as a vaccine by eliminating infected cells.

[0214] Example 12. Cellular immunity induced by srRNA1ts2-PanCoronavirus vaccine This example describes the finding that a fusion protein containing the CD5 signal peptide, the Spike-RBD of SARS-CoV-2, the nucleoprotein of SARS-CoV-2, the nucleoprotein of MERS-CoV and the Spike-RBD of MERS-CoV can induce potent cellular immunity against all of these antigens when the protein is expressed from a temperature-controllable self-replicating RNA injected intradermally.

[0215] Materials and Methods C57BL / 6 female mice.

[0216] The srRNA1ts2-G5006 mRNA was produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA1ts2 as described in WO 2021 / 138447 A1) encoding the G5006 antigen (Figure 2).

[0217] The srRNA1ts2-G5006d mRNA was produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA1ts2 as described in WO 2021 / 138447 A1) encoding the G5006d antigen (Figure 22).

[0218] Pool of peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the RBD of SARS-CoV-2 (original Wuhan strain) [JPT Peptides:PepMix SARS-CoV-2(S-RBD)PM-WCPV-S-RBD-2].

[0219] Pool of peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the SARS-CoV-2 nucleoprotein (UniProt: P0DTC9) [JPT Peptide Product Code: PM-WCPV-NCAP].

[0220] Pool of peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the nucleoprotein of MERS-CoV (YP_009047211.1). Peptides were custom made by JPT Peptides.

[0221] A pool of 336 (168+168) peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through the spike glycoprotein (Swiss-Prot ID: K9N5Q8) of MERS-CoV (Middle East Respiratory Syndrome Coronavirus) [JPT Peptide Product Code: PM-MERS-CoV-S-1].

[0222] Interferon gamma (INF-γ) and interleukin-4 (IL-4) ELISpot assay plates and reagents (Cellular Technology Limited, Ohio, USA).

[0223] Immunospot S6 Entry Analyzer (Cellular Technology Limited, Ohio, USA).

[0224] result We designed a new booster vaccine, a c-srRNA vaccine (called c-srRNA-G5006d) encoding a fusion protein containing the CD5 signal peptide, the Spike-RBD of SARS-CoV-2, the nucleoprotein of SARS-CoV-2, the nucleoprotein of MERS-CoV, and the Spike-RBD of MERS-CoV (Figure 22).

[0225] Mice were vaccinated intradermally with placebo (PBO: buffer only), c-srRNA encoding the G5006 antigen, and c-srRNA encoding the G5006d antigen. Cellular immunity was assessed by ELISpot assay 14 days after vaccination.

[0226] As shown in Figure 23A, c-srRNA-G5006d can stimulate cellular immunity against all proteins encoded in this vaccine: SARS-CoV-2 spike-RBD, SARS-CoV-2 nucleoprotein, MERS-CoV nucleoprotein and MERS-CoV spike-RBD.

[0227] conclusion The results indicate that the c-srRNA vaccine can function as a booster vaccine for both cellular and humoral immunity.

[0228] Example 13. srRNA1ts2-PanInfluenza virus vaccine This example describes the design of a pan-influenza booster vaccine based on the unique features of the c-srRNA vaccine platform. The antigen (G5012) encoded on the c-srRNA is a fusion protein of the CD5 signal peptide (residues 1-24), a portion of influenza A hemagglutinin (HA), influenza A nucleoprotein, influenza B nucleoprotein, and a portion of influenza B hemagglutinin (HA).

[0229] Materials and Methods C57BL / 6 female mice.

[0230] The mRNA for c-srRNA-G5012 was produced by in vitro transcription of a temperature-controllable self-replicating RNA vector (srRNA1ts2 as described in WO 2021 / 138447 A1) encoding the G5012 antigen (Figure 24).

[0231] A pool of peptides resulting from a peptide scan (15mers containing 11 amino acid overlaps) through a portion of influenza A hemagglutinin (HA), influenza A nucleoprotein, influenza B nucleoprotein and a portion of influenza B hemagglutinin (HA).

[0232] Interferon gamma (INF-γ) and interleukin-4 (IL-4) ELISpot assay plates and reagents (Cellular Technology Limited, Ohio, USA).

[0233] Immunospot S6 Entry Analyzer (Cellular Technology Limited, Ohio, USA).

[0234] result Mice were vaccinated intradermally with placebo (PBO: buffer only) and c-srRNA encoding the G5012 antigen. Cellular immunity was assessed by ELISpot assay 14 days after vaccination.

[0235] c-srRNA-G5012 stimulated cellular immunity against all antigens encoded in this vaccine: influenza A hemagglutinin (HA), influenza A nucleoprotein, influenza B nucleoprotein, and influenza B hemagglutinin (HA).

[0236] conclusion The results indicate that the c-srRNA vaccine functions as a booster vaccine for both cellular and humoral immunity.

[0237] Example 14. Chitosan-enhanced luciferase expression from srRNA1ts2-LUC2 This example describes the findings that chitosan oligomers can enhance the in vivo expression of a gene of interest (GOI) encoded by a c-srRNA construct.

[0238] Materials and Methods C57BL / 6 female mice.

[0239] srRNA1ts2-LUC2 (mRNA), produced by in vitro transcription of a temperature-controllable self-replicating RNA vector encoding the luciferase gene (srRNA1ts2 as described in WO 2021 / 138447 A1).

[0240] Chitosan oligomer (molecular weight ≦5 kDa, ≧75.0% deacetylated: Heppe Medical Chitosan GmbH: product no. 44009)

[0241] Chitosan oligosaccharide lactate (molecular weight ~5 kDa, >90% deacetylated: Sigma-Aldrich: product no. 523682)

[0242] Bioluminescence imaging system, AMI HTX (Spectral Instruments Imaging, Tucson, AZ)

[0243] result To test whether chitosan oligomers can enhance the expression of GOIs encoded on c-srRNA in vivo, 5 μg of c-srRNA (also known as srRNA1ts2), encoding the luciferase gene as the GOI, was mixed with chitosan and administered intradermally to each C57BL / 6 mouse ( FIG. 25 ). c-srRNA was formulated as naked RNA without lipid nanoparticles or any other transfection reagents in lactated Ringer's solution ( FIG. 25 ). Luciferase activity was visualized and quantified by using a bioluminescence imaging system, AMI HTX (Spectral Instruments Imaging, Tucson, AZ).

[0244] Five mice were tested in each of the following groups: 1, control-c-srRNA only; 2, c-srRNA mixed with chitosan oligosaccharide (0.001 μg / mL); 3, c-srRNA mixed with chitosan oligosaccharide (0.01 μg / mL); 4, c-srRNA mixed with chitosan oligosaccharide (0.5 μg / mL); 5, c-srRNA mixed with chitosan oligosaccharide lactate (0.1 μg / mL).

[0245] As shown in FIG. 27, compared to the control condition (i.e., c-srRNA only: no chitosan), all conditions using chitosan oligomer at concentrations of 0.001 μg / mL, 0.01 μg / mL, and 0.5 μg / mL and the condition using chitosan oligosaccharide lactate at a concentration of 0.1 μg / mL showed ∼10-fold higher levels of luciferase activity.

[0246] conclusion Low molecular weight chitosans, such as chitosan oligomers and chitosan oligosaccharide lactate, can enhance the expression of GOIs encoded on c-srRNA when mixed with c-srRNA prior to intradermal injection of the c-srRNA into mouse skin. Chitosan oligomers provide approximately 10-fold enhancement of gene expression even at very low concentrations (0.001 μg / mL or approximately 0.2 nM). This surprising discovery provides an effective means to enhance the in vivo therapeutic expression of GOIs encoded on c-srRNA.

[0247] array [Table 2]

[0248] [Table 3]

[0249] [Table 4-1] [Table 4-2]

[0250]

Table 5-1

Table 5-2

[0251]

Table 6

[0252]

Table 7

[0253]

Table 8

[0254]

Table 9

[0255]

Table 10

[0256]

Table 11

[0257]

Table 12

[0258]

Table 13

[0259]

Table 14

[0260]

Table 15

[0261]

Table 16-1

Table 16-2

Table 16-3

[0262]

Table 17

[0263]

Table 18

[0264]

Table 19

[0265]

Table 20

[0266]

Table 21

[0267]

Table 22

[0268]

Table 23-1

Table 23-2

[0269]

Table 24-1

Table 24-2

Table 24-3

Table 24-4

[0270]

Table 25

[0271]

Table 26

[0272]

Table 27-1

Table 27-2

Table 27-3

[0273]

Table 28

[0274]

Table 29-1

Table 29-2

Table 29-3

[0275]

Table 30

Claims

**Claim 1** A composition for stimulating an immune response against two or more viruses in a mammalian subject, comprising messenger RNA (mRNA) containing an excipient and 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) a nucleotide sequence encoding a first nucleocapsid protein of a first virus and a second nucleocapsid protein of a second virus, the composition. **Claim 2** The composition according to claim 1, wherein both the first and second viruses are members of the family Orthomyxoviridae. **Claim 3** The composition according to claim 2, wherein the first and second viruses include influenza A virus and influenza B virus. **Claim 4** The composition according to claim 3, 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. **Claim 5** The composition according to claim 1, wherein both the first and second viruses are members of the order Orthornavirae. **Claim 6** The composition according to claim 1, wherein both the first and second viruses are members of the genus Ebolavirus. **Claim 7** 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, second, third and fourth viruses are Zaire ebolavirus, Sudan ebolavirus, Bundibugyo ebolavirus and Taï Forest ebolavirus, the composition according to claim 6. **Claim 8** The composition according to claim 7, 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. **Claim 9** The composition according to claim 7, wherein the nucleotide sequence (ii) encodes a shared portion of the first nucleocapsid protein of the first virus for stimulating an immune response against all of the first, second, third and fourth viruses. **Claim 10** The nucleotide sequence (ii) is the composition according to claim 9, which encodes individual portions of each of the first, second, third, and fourth nucleocapsid proteins for stimulating an immune response against all the first, second, third, and fourth viruses.

11. The nucleotide sequence (ii) is the composition according to claim 10, which encodes a fragment of an individual portion of the second nucleocapsid protein of the second virus for stimulating an immune response against the second and third viruses.

12. The nucleotide sequence (ii) is the composition according to claim 1, which encodes a shared portion of the first nucleocapsid protein of the first virus for stimulating an immune response against both the first and second viruses.

13. The nucleotide sequence (ii) is the composition according to claim 12, which encodes individual portions of each of the first and second nucleocapsid proteins for stimulating an immune response against both the first and second viruses.

14. The nucleotide sequence (ii) of (ii) further encodes at least one additional nucleocapsid protein of at least one additional virus, and the at least one additional virus is different from the first and second viruses, which is the composition according to claim 1.

15. The composition according to claim 1, wherein the first and second, or the first, second, and additional nucleocapsid proteins are separated by a linker having a length of 1 to 10 residues.

16. The composition according to claim 1, wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in mammalian antigen-presenting cells.

17. The composition according to claim 1, wherein the composition further comprises chitosan.

18. A medicament for stimulating an immune response against two or more viruses in a mammalian subject, which is administered to the mammalian subject to stimulate an immune response against the nucleocapsid proteins of two or more viruses in the mammalian subject, and comprises the composition according to any one of claims 1 to 17.

19. A composition for stimulating an immune response against a virus in a mammalian subject, comprising an excipient and 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 or a fragment thereof of a first virus; and (iii) a nucleotide sequence encoding a second viral antigen or a fragment thereof of the first virus or a 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, composition.

20. A composition for stimulating an immune response against two or more viruses in a mammalian subject, comprising an excipient and 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 or a fragment thereof of a first virus; (iii) a nucleotide sequence encoding a second viral antigen or a fragment thereof of the first virus; (iv) a nucleotide sequence encoding a third viral antigen or a fragment thereof of a second virus; (iii) a nucleotide sequence encoding a fourth viral antigen or a fragment thereof of the second virus, 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 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, composition.

21. The following: (i) The composition according to claim 19 or 20; and (ii) an apparatus for intradermal delivery of the composition to a mammalian subject, A kit comprising:

22. A medicament for stimulating an immune response in a mammalian subject, which is administered to the mammalian subject to stimulate an immune response against one or more first viral antigens, second viral antigens, third viral antigens and fourth viral antigens in the mammalian subject, the medicament comprising the composition according to claim 19 or 20.

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