WO2021260065A1 - Mva-based vaccine against covid-19 expressing sars-cov-2 antigens - Google Patents

Mva-based vaccine against covid-19 expressing sars-cov-2 antigens Download PDF

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WO2021260065A1
WO2021260065A1 PCT/EP2021/067245 EP2021067245W WO2021260065A1 WO 2021260065 A1 WO2021260065 A1 WO 2021260065A1 EP 2021067245 W EP2021067245 W EP 2021067245W WO 2021260065 A1 WO2021260065 A1 WO 2021260065A1
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mva
cov
sars
composition
vaccine
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PCT/EP2021/067245
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Juan Francisco GARCÍA ARRIAZA
Mariano ESTEBAN RODRÍGUEZ
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Consejo Superior De Investigaciones Científicas (Csic)
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
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    • C12N2710/24011—Poxviridae
    • C12N2710/24041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
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    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention is directed to a recombinant modified vaccinia virus Ankara (MVA), which carries a nucleic acid sequence coding for an antigenic protein useful for vaccinating against SARS-CoV-2.
  • MVA modified vaccinia virus Ankara
  • the present invention is further directed to a vaccine composition containing said recombinant MVA as well as to a method for generating a protective immune response in a mammal against SARS-CoV-2.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • MERS Middle East respiratory syndrome
  • FIG. 1 Design and generation and in vitro characterization of vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S).
  • A Genome map of vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S), expressing the coronavirus SARS-CoV-2 S- protein (this vector is also referred indistinctively as MVA-CoV2-S or MVA-S). The different regions of the MVA genome are indicated with capital letters and the central conserved region and the left and right terminal regions are shown. Below the map, the deleted or fragmented MVA genes are depicted as black boxes.
  • the MVA genes C6L, K7R, and A46R which have been deleted in one of the parental viruses (MVA-GFP), used for generating the vaccine candidate MVA-A-COVID19(S), are indicated.
  • the coronavirus SARS-CoV-2 S genes inserted within the MVA TK viral locus (J2R) of MVA-WT or MVA- GFP parental viruses (to generated MVA-COVID19(S) and MVA-A-COVID19(S), respectively) and driven by the sE/L virus promoter are indicated.
  • TK-L TK left; TK-R: TK right.
  • B PCR analysis of MVA TK locus.
  • Viral DNA was extracted from DF-1 cells that were not infected (mock) or infected with 5 PFU/cell of vaccine candidates MVA- COVID19(S) (referred to as MVA-S) and MVA-A-COVID19(S) (referred to as MVA-A-S), MVA-GFP, or MVA-WT.
  • Oligonucleotides which hybridize in the flanking regions of the TK locus were used for the PCR analysis of the coronavirus SARS-CoV-2 S gene inserted in the TK locus.
  • a molecular weight marker (1 kb) with the corresponding bp sizes is indicated on the left side of the images, and the amplified DNA products are indicated with arrows on the right side.
  • DF-1 cells were left uninfected (mock) or infected for 4 or 24 hours (in the absence or presence of tunicamycin) at 5 PFU/cell with vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) and MVA-WT.
  • DF-1 cells were left uninfected (mock) or infected with vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S), and MVA-WT, at 5 PFU/cell.
  • the cell extracts and the supernatants were collected 4 and 24 hours post- infection (the supernatants were first precipitated with 10% TCA), lysed in Laemmli 1X + b-mercaptoethanol buffer, fractioned with 7% SDS-PAGE, and analyzed by Western blotting using a mouse monoclonal antibody against SARS-CoV (against SARS-CoV S2 region) or a rabbit polyclonal antibody against SARS-CoV-2 (against SARS-CoV-2 S1 region).
  • the arrows on the right side indicate the S protein.
  • the sizes of standards in kilodaltons [kDa]) (Precision Plus protein standards; Bio-Rad Laboratories) are indicated on the left.
  • FIG. 1 In vitro characterization of vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S).
  • A Expression of SARS-CoV-2 S protein under reducing and nonreducing conditions. DF-1 cells were infected with MVA-S and MVA-A-S vaccine candidates and at 24 hpi, cells were lysed under reducing (Laemmli 1X + b- mercaptoethanol (BME) buffer) or nonreducing conditions (Laemmli 1X - b- mercaptoethanol buffer), fractionated by 7% SDS-PAGE, and analyzed by Western blotting using a mouse monoclonal antibody against SARS-CoV (against SARS-CoV S2 region) or a rabbit polyclonal antibody against SARS-CoV-2 (against SARS-CoV-2 S1 region).
  • BME b- mercaptoethanol
  • DF-1 cells were the left uninfected ⁇ mock) or infected with vaccine candidates MVA- COVID19(S) and MVA-A-COVID19(S) from the different passages, or with MVA-WT, and the cells were lysed at 24 hours post-infection in Laemmli 1X + b-mercaptoethanol buffer, fractionated by 7% SDS-PAGE, and analyzed by Western Blotting using a rabbit polyclonal antibody against SARS-CoV-2 (against SARS-CoV-2 S1 region).
  • the arrows on the right side indicate the position of the S protein.
  • the sizes of standards in kilodaltons [kDa]) (Precision Plus protein standards; Bio-Rad Laboratories) are indicated on the left.
  • FIG. Immunization schedule of the heterologous (DNA/MVA) and homologous (MVA/MVA) prime/boost immunization protocols performed in C57BL/6 mice immunized with vaccine candidates MVA-COVID19(S) or MVA-A-COVID19(S).
  • mice per group were inoculated at days 0 and 15 (prime/boost) with the different indicated vaccine candidates (either DNA-S or MVAs in the prime or the different MVAs in the boost).
  • mice were sacrificed 11 days after the boost (day 26) and the following was obtained: i) spleens of the mice for studying adaptive T cell-mediated immune responses against SARS-CoV-2 by means of ELISpot and ICS, and ii) serum samples originating from blood for studying adaptive humoral immune responses against SARS-CoV-2, by means of ELISA and a neutralization assay.
  • PFU Plaque forming units; i.m.: intramuscular.
  • FIG. 4 ELISpot analysis of the adaptive SARS-CoV-2 S-specific cell-mediated immune responses induced by vaccine candidates MVA-COVID19(S) or MVA-D- COVID19(S) in C57BL/6 mice immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost immunization protocol.
  • Six mice per group were sacrificed at 11 days post-boost, and the splenic SARS-CoV-2-specific cells secreting IFN-y were evaluated by an ELISpot assay, as described in Materials and Methods. Samples from each group of mice were analysed in triplicate, and the bars indicate the mean values and the standard deviation.
  • the responses obtained have been obtained by subtracting the RPMI values for each condition and are represented as IFN- Y positive cells per million of splenocytes.
  • A Magnitude of SARS-CoV-2-specific cells secreting IFN-g and directed against SARS-CoV-2 S1 and S2 peptide pools.
  • B Magnitude of VACV E3-specific cells secreting IFN-g.
  • SARS-CoV-2 S-specific adaptive CD4 + (left) and CD8 + (right) T cell- mediated immune responses induced by vaccine candidates MVA-COVID19(S) or MVA-A-COVID19(S) in C57BL/6 mice immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost immunization protocol.
  • the data is depicted with its corresponding confidence intervals.
  • A The total magnitude of SARS-CoV-2 S (S1 + S2)- specific adaptive CD4 + T cell-mediated (left) and CD8 + T cell-mediated (right) immune responses.
  • the y-axis shows the percentage measured as the sum of SARS-CoV-2 S (mixture of S1 + S2 peptide pools)-specific T cells expressing CD107a and/or producing IFN-Y and/or TNF-a and/or IL-2.
  • B Breadth of SARS-CoV-2-specific CD4 + and CD8 + T cell adaptive immune responses. S1 or S2 peptide pool-specific adaptive CD4 + T cell- mediated (left) and CD8 + T cell-mediated (right) immune responses.
  • the y-axis shows the percentage measured as the sum of SARS-CoV-2 S1 or S2 peptide pool-specific T cells expressing CD107a and/or producing IFN-g and/or TNF-a and/or IL-2.
  • C Adaptive CD4 + T cell-mediated (left) and CD8 + T cell-mediated (right) immune responses shown as the percentage of SARS-CoV-2 S (S1 + S2)-specific cells expressing CD107a or producing IFN-g or TNF-a or IL-2.
  • FIG. 7 Phenotype profile of SARS-CoV-2 S-specific adaptive CD4 + and CD8 + T cells induced by vaccine candidates MVA-COVID19(S) or MVA-A-COVID19(S), in C57BL/6 mice immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost immunization protocol.
  • the y-axis indicates the percentage of SARS-CoV-2 S (S1 +S2)-specific CD4 + and CD8 + central memory T cells (TCM; CD127 + , CD62L + ), effector memory T cells (TEM; CD127 + , CD62L ), and effector T cells (TE; CD127 , CD62L ) expressing CD107a and/or IFN-g and/or TNF-a and/or IL-2 (total response).
  • TCM central memory T cells
  • TEM effector memory T cells
  • TE effector T cells
  • the data is depicted with its corresponding confidence intervals.
  • B Percentages of total SARS-CoV-2-specific CD4+ Tfh cells expressing CD40L or producing IFN-g or IL-21 against a mixture of SARS-CoV-2 S protein plus S1 , and S2 SARS-CoV-2 S peptide pools.
  • C Polyfunctionality of total SARS-CoV-2-specific adaptive CD4+ Tfh cells directed against a mixture of SARS-CoV-2 S protein plus S1 and
  • SARS-CoV-2 S protein plus S1 and S2 peptide pools are shown on the y axis.
  • FIG. 9 SARS-CoV-2 S-specific and RBD-specific humoral immune responses induced by vaccine candidates MVA-COVID19(S) or MVA-A-COVID19(S) in C57BL/6 mice immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost immunization protocol.
  • Groups of C57BL/6 mice (n 6) were immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost protocol.
  • mice were sacrificed, serum samples were collected, and the levels of total coronavirus SARS-CoV-2 S-specific and RBD- specific IgG antibodies (A) or antibodies of subclasses lgG1, lgG2c, and lgG3 (B) in pooled sera from mice derived from each immunization group were analyzed by ELISA. Mean absorbance values (measured at 450 nm) and standard deviations of duplicate pooled serum dilutions are represented.
  • mice were sacrificed, serum samples were collected, and their capacity to inhibit the entry in VeroE6 cells of lentivirus-based pseudoparticles expressing the coronavirus SARS-CoV-2 S protein was analyzed.
  • RPMI was used as a negative control.
  • Each dot represents the mean luciferase units of triplicates obtained for each individual mouse serum sample from each immunization group, and analyzed at a 1/50 dilution. Mean values and SEM obtained for each immunization group is represented.
  • MVA-COVID19(S) protects humanized K18-hACE2 mice from SARS-CoV-
  • mice were immunized with one or two doses of 1 x 10 7 PFU of MVA-S or MVA-WT by intramuscular route at weeks 0 and 4. At 10 days after the last immunization (day 38) mice were bleed and serum samples were obtained. At week 9 (day 63) mice were intranasally (i.n.) challenged with 1 x 10 5 PFU of SARS-CoV-2 (MAD6 isolate). MVA-WT-inoculated (group 3) and unvaccinated (group 4) mice were also i.n.
  • mice were challenged with SARS-CoV-2; unvaccinated and unchallenged mice were used as a negative control (group 5). At days 2 and 4 post-challenge 3 mice per group were sacrificed and lungs and serum samples were collected as indicated. In surviving mice, serum was collected at day 15 post- challenge. All mice were monitored daily for change in the percentage of initial body weight (B) and survival (C) during 14 days after virus challenge.
  • MVA-S vaccine candidate is highly immunogenic in prechallenged K18- hACE2 humanized mice.
  • A Titers of IgG antibodies specific for the S (left) and RBD (right) proteins. Titers were determined by ELISA in individual mouse serum samples collected from immunized K18-hACE2 mice at 10 days after the last immunization (prechallenge) and were calculated as the serum dilution at which the absorbance at 450 nm was at least three times higher than a naive serum value in each of the indicated immunization regimens. Mean values and SEM is represented for each immunization group. The dashed line represents the detection limit.
  • the ID50 titers were evaluated in individual mouse serum samples collected from immunized K18-hACE2 mice at 10 days after the last immunization (prechallenge) using retrovirus- based pseudoparticles expressing the SARS-CoV-2 S protein (left) or live-virus microneutralization (right) assays.
  • the ID50 titers in the retrovirus-based pseudoparticle assay were determined as the highest serum dilution which resulted in a 50% reduction of luciferase units compared with pseudotyped viruses not incubated with serum.
  • the ID50 titers in the live-virus microneutralization assay were calculated as the reciprocal dilution resulting in 50% inhibition of cell death. Mean ID50 values and SEM for each immunization group is represented. Dotted line represented the detection limit.
  • the NIBSC 20/136 international standard containing pooled plasma obtained from eleven individuals recovered from SARS-CoV-2 infection is included.
  • (C) Lung inflammation scores and percentage of lung area with lesions. Analyzed in lung samples (n 3) obtained at 4 days postchallenge from mice vaccinated and infected as in Fig. 12A. Mean values and SEM is represented for each immunization group.
  • MVA-S vaccine candidate is highly immunogenic in challenged K18-hACE2 humanized mice.
  • A Titers of IgG antibodies specific for the S (left) and RBD (right) proteins. Titers were determined by ELISA in individual mouse serum samples collected at days 2, 4 and 15 post-challenge from immunized K18-hACE2 mice and were calculated as the serum dilution at which the absorbance at 450 nm was at least three times higher than a naive serum value in each of the indicated immunization regimens. Mean values and SEM is represented for each immunization group. The dashed line represents the detection limit.
  • B Ratio of lgG2c/lgG1 specific for the S and RBD proteins.
  • the ratio was calculated by ELISA in pooled mice serum samples collected at days 4 and 15 post challenge, and were calculated from the mean optical density at 450 nm obtained for lgG2c and lgG1 at a dilution 1/9765. A ratio >1 indicates a Th1 response.
  • C Neutralizing antibody titers. The ID50 titers were evaluated in individual mouse serum samples collected from immunized K18-hACE2 mice at 4 and 15 days post-challenge using retrovirus-based pseudoparticles expressing the SARS-CoV-2 S protein, and were determined as the highest serum dilution which resulted in a 50% reduction of luciferase units compared with pseudotyped viruses not incubated with serum.
  • mice were monitored for change of body weights (B) and mortality (C) for 6 days. At day 6 post-challenge mice were sacrificed and lungs and serum samples were collected as indicated.
  • D SARS-CoV-2 RNA. Detected by RT-qPCR targeting genomic viral RdRp gene with normalization to 28S in lungs obtained at day 6 days post-rechallenge from mice vaccinated and infected as in panel A. Mean RNA levels (in A.U.) and SEM from duplicates of each lung sample is represented for each immunization group; relative values are referred to uninfected mice.
  • E SARS-CoV-2 infectious virus.
  • MVA-S vaccine candidate is highly immunogenic in re-challenged K18- hACE2 humanized mice.
  • A mRNA levels of proinflam matory cytokines. Detected by RT-qPCR with normalization to 28S in lungs obtained at day 6 days post-rechallenge from mice vaccinated and infected as in Fig. 16A. Mean RNA levels (in A.U.) and SEM from duplicates of each lung sample is represented for each immunization group; relative values are referred to uninfected mice.
  • B Titers of IgG antibodies specific for the S (left) and RBD (right) proteins.
  • Titers were determined by ELISA in individual mouse serum samples collected at day 6 post-rechallenge from mice vaccinated and infected as in Fig. 16A and were calculated as the serum dilution at which the absorbance at 450 nm was at least three times higher than a naive serum value in each of the indicated immunization regimens. Mean values and SEM is represented for each immunization group. The dashed line represents the detection limit. (C) Ratio of lgG2c/lgG1 specific for the S and RBD proteins.
  • the ratio was calculated by ELISA in pooled mice serum samples collected at day 6 post-rechallenge, and were calculated from the mean optical density at 450 nm obtained for lgG2c and lgG1 at a dilution 1/9765. A ratio >1 indicates a Th1 response.
  • D Neutralizing antibody titers. The ID50 titers were evaluated in individual mouse serum samples collected from immunized K18-hACE2 mice at day 6 post-rechallenge using retrovirus-based pseudoparticles expressing the SARS-CoV-2 S protein, and were determined as the highest serum dilution which resulted in a 50% reduction of luciferase units compared with pseudotyped viruses not incubated with serum.
  • Mean ID50 values and SEM for each immunization group is represented. Dotted line represented the detection limit.
  • the ratio was calculated by ELISA in pooled mice serum samples collected at 6 months post-boost, and were calculated from the mean optical density at 450 nm obtained for lgG2c and lgG1 at a dilution 1/9765. A ratio >1 indicates a Th1 response.
  • C Neutralizing antibody titers. The ID50 titers were evaluated in individual mouse serum samples at 6 months post-boost using retrovirus-based pseudoparticles expressing the SARS-CoV-2 S protein, and were determined as the highest serum dilution which resulted in a 50% reduction of luciferase units compared with pseudotyped viruses not incubated with serum. Mean ID50 values and SEM for each immunization group is represented. Dotted line represented the detection limit.
  • Fig. 20 Immunogenicity and efficacy study schedule with MVA-S vaccine candidate in Syrian-gold hamsters.
  • mice were intranasally challenged with 2 x 10 5 TCID50 of SARS-CoV-2 (BetaCoV/Belgium/GHB-03021/2020 isolate).
  • SARS-CoV-2 BetaCoV/Belgium/GHB-03021/2020 isolate.
  • serum samples were obtained, previously to the challenge.
  • the challenged hamsters were monitored for change of body weights for 14 days, and at days 2, 4 and 14 post-challenge 4 hamsters per group were sacrificed; lungs were obtained for analysis of SARS-CoV-2 RNA and infectious virus and serum samples were also obtained.
  • Fig. 21 Change in body weight in hamsters immunized with MVA-S and challenge with SARS-CoV-2. Hamsters were monitored for change of body weights previously to the challenge (data not shown), and after challenge with SARS-CoV-2.
  • Fig. 22 Binding IgG antibody titers against S protein induced in hamsters immunized with MVA-S. Titers of IgG antibodies specific for the S protein were determined by UFA in individual hamster serum samples collected at day 39, and were calculated as the serum dilution (Y axis) at which the absorbance was three times higher than the naive serum value in each of the indicated immunization regimens, shown in the X axis. Dotted line represented the detection limit.
  • Fig. 23 Neutralizing antibody titers against SARS-CoV-2 induced in hamsters immunized with MVA-S. Neutralizing antibody titers were determined by neutralization of VSV pseudotype virus using Vero-adaptive spike mutations in individual hamster serum samples collected at day 39, and were calculated as the serum dilution (Y axis) that neutralize 50%. Dotted line represented the detection limit.
  • Fig. 24 SARS-CoV-2 RNA and infectious virus in lungs of hamsters vaccinated with MVA-S.
  • Fig. 26 Lung histopathology lesions. Cumulative lung score of lesions observed in lung samples obtained from hamsters at 2, 4- and 14-days post-challenge.
  • Fig. 27 Immunogenicity and efficacy study schedule with MVA-S vaccine candidate in rhesus macaques.
  • MVA-S or MVA-WT control
  • c 10 8 PFU in the prime and boost
  • a technical grade lot from Biofabri, Spain
  • week 8 day 56
  • macaques were intranasally + intratracheally challenged with 1 c 10 6 PFU of SARS-CoV-2.
  • serum samples were obtained, previously to the challenge.
  • the challenged macaques were monitored for 14 days, and at different days post-challenge several samples were taken. Animals were sacrificed at day 14 post challenge.
  • Fig. 28 MVA-S elicit SARS-CoV-2 S-specific cell immune responses in immunized rhesus macaques.
  • IFN-g was evaluated by an ELISpot assay from PBMCs derived from the 6 immunized macaques at 14 days post-prime. Samples were analyzed in triplicate; bars show the triplicate mean values and the standard deviation.
  • Fig. 29 MVA-S prevented SARS-CoV-2 virus replication in throat and bronchoalveolar samples.
  • SARS-CoV-2 RNA was analyzed by RT-qPCR targeting genomic viral RdRp gene and subgenomic N gene. Mean RNA levels (in A.U.) and SEM from duplicates of each sample is represented for each immunization group.
  • Fig. 30 Intranasal immunization with MVA-S induce robust SARS-CoV-2 S-specific T-cell and humoral immune responses in immunized mice.
  • A Magnitude of SARS- CoV-2 S-specific CD4+ and CD8+ T-cell immune responses evaluated in splenocytes and lung cells at 10 days post-immunization. Percentages of CD4+ or CD8+ T cells expressing CD107a and/or producing IFN-g and/or TNF-a and/or IL-2 against a mixture of S1 and S2 peptide pools in mice immunized by the intranasal route with the indicative regimen. Cell percentages were determined by ICS.
  • C Titers of IgA antibodies specific for the S protein.
  • Titers were determined by ELISA in bronchoalveolar mouse samples collected at 10 days post-immunization from intranasally immunized C57BL/6 mice and were calculated as the serum dilution at which the absorbance at 450 nm was at least three times higher than a naive serum value in each of the indicated immunization regimens. Mean values and SEM is represented for each immunization group.
  • the ID50 titers were evaluated in individual mouse serum samples at 10 days post-immunization using retrovirus-based pseudoparticles expressing the SARS-CoV-2 S protein, and were determined as the highest serum dilution which resulted in a 50% reduction of luciferase units compared with pseudotyped viruses not incubated with serum. Mean ID50 values and SEM for each from intranasally immunization group is represented. Dotted line represented the detection limit.
  • the conjunctive term "and/or" between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by "and/or", a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term "and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term "and/or.”
  • subtype herein can be replaced with “species”. It includes strains, isolates, clades or variants of any SARS-CoV-2.
  • strain "clade” or “isolate” are technical terms, well known to the practitioner, referring to the taxonomy of microorganisms. The taxonomic system classifies all so far characterized microorganisms into the hierarchic order of Families, Genera, Species, Strains (Fields Virology, ed. by Fields B. N., Lippincott-Raven Publishers, 4th edition 2001).
  • a Genera comprises all members which share common characteristics
  • a Species is defined as a polythetic class that constitutes a replicating lineage and occupies a particular ecological niche.
  • the term "strain” or “clade” describes a microorganism, i.e. , virus, which shares common characteristics, like basic morphology or genome structure and organization, but varies in biological properties, like host range, tissue tropism, geographic distribution, attenuation or pathogenicity.
  • subject as used herein is a living multi-cellular vertebrate organism, including, for example, humans, non-human mammals and (non-human) primates.
  • subject may be used interchangeably with the term “animal” herein.
  • the term "enhanced" when used with respect to an immune response against SARS-CoV-2 refers to an increase in the immune response in an animal administered with a homologous prime-boost combination vaccine of recombinant MVA relative to the corresponding immune response observed from the animal administered with a homologous prime-boost combination vaccine of MVA vectors, wherein the MVA vectors do not express any SARS-CoV-2 protein or refers to an increase in the immune response in an animal administered with a heterologous prime-boost combination vaccine of recombinant DNA and MVA vectors according to the invention, relative to the corresponding immune response observed from the animal administered with a heterologous prime-boost combination vaccine of DNA and MVA vectors according to the invention, wherein the MVA vector does not express any SARS
  • Modified vaccinia virus Ankara (MVA) vector as used herein is an attenuated replication- deficient vaccinia virus that has been genetically modified to include in its genome foreign DNA (that is, DNA that does not naturally belong to poxviruses).
  • the modified vaccinia virus Ankara vector of the present invention has been genetically modified to express SARS-CoV-2 protein(s).
  • the MVA vector of the present invention refers to viral particles of MVA that comprise inside the viral particle the MVA genome, wherein at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype has been introduced in the MVA genome.
  • MVA-COVID19(S) “MVA-S” and “MVA-CoV2-S” are synonymous names to refer to a recombinant MVA vector comprising at least a nucleic acid encoding the structural protein S of the SARS-CoV-2 virus, or an antigenic fragment thereof.
  • M VA-A-C O V I D 19( S ) “MVA-A-S” and “MVA-A-CoV2-S” are synonymous names used to refer to a recombinant MVA vector further modified by deletion or inactivation of the MVA immunomodulatory genes C6L, K7R, and A46R, comprising at least a nucleic acid encoding the structural protein S of the SARS-CoV-2 virus, or an antigenic fragment thereof.
  • Recombinant MVA virus vaccine compositions are synonymous names used to refer to a recombinant MVA vector further modified by deletion or inactivation of the MVA immunomodulatory genes C6L, K7R, and A46R, comprising at least a nucleic acid encoding the structural protein S of the SARS-CoV-2 virus, or an antigenic fragment thereof.
  • Vaccinia virus is amongst the most extensively evaluated live vectors and has particular features in support of its use as recombinant vaccine: It is highly stable, cheap to manufacture, easy to administer, and it can accommodate large amounts of foreign DNA. It has the advantage of inducing both antibody and cytotoxic T cell responses and allows presentation of antigens to the immune system in a more natural way, and it was successfully used as vector vaccine protecting against several infectious diseases in a broad variety of animal models. Additionally, vaccinia vectors are extremely valuable research tools to analyse structure-function relationships of recombinant proteins, determine targets of humoral and cell- mediated immune responses, and investigate the type of immune parameters needed to protect against a specific disease.
  • VACV is infectious for humans and its use as expression vector in the laboratory has been affected by safety concerns and regulations. Furthermore, possible future applications of recombinant VACV e.g. to generate recombinant proteins or recombinant viral particles for novel therapeutic or prophylactic approaches in humans, are hindered by the productive replication of the recombinant VACV vector. Most of the recombinant VACVs described in the literature are based on the Western Reserve (WR) strain of W. On the other hand, it is known that this strain is highly neurovirulent and is thus poorly suited for use in humans and animals (Morita et al., Vaccine 5, 65-70 [1987]).
  • WR Western Reserve
  • VACV vectors from highly attenuated virus strains which are characterized by their restricted replicative capacity in vitro and their avirulence in vivo. Strains of viruses specially cultured to avoid undesired side effects have been known for a long time. Thus, it has been possible, by long-term serial passages of the chorioallantoic VACV Ankara (CVA) strain on chicken embryo fibroblasts, to culture MVA (for review see Mayr, A., Hochstein-Mintzel, V. and Stickl, H. (1975) Infection 3, 6-14; Swiss Patent No. 568 392).
  • CVA chorioallantoic VACV Ankara
  • the MVA virus was deposited in compliance with the requirements of the Budapest Treaty at CNCM (Institut Pasteur, Collectione Nationale de Cultures de Microorganisms, 25, rue de Dondel Roux, 75724 Paris Cedex 15) on Dec. 15, 1987 under Depositary No. 1-721.
  • the MVA virus has been analysed to determine alterations in the genome relative to the wild type CVA strain.
  • Six major deletions (deletion I, II, III, IV, V, and VI) have been identified (Meyer, H., Sutter, G. and Mayr A. (1991) J. Gen. Virol. 72, 1031-1038).
  • This MVA has only low virulence, that is to say it is followed by no side effects when used for vaccination. Hence it is particularly suitable for the initial vaccination of immunocompromised subjects.
  • the excellent properties of the MVA strain have been demonstrated in a number of clinical trials (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org.
  • MVA is a valuable tool as safe viral vector for expression of recombinant genes and can be used for such different purposes as the in vitro study of protein functions or the in vivo induction of antigen-specific cellular or humoral immune responses.
  • a major advantage of MVA is to allow for high level gene expression despite being replication defective in human and most mammalian cells.
  • MVA as a vaccine has an excellent safety track-record, can be handled under biosafety level 1 conditions and has proven to be immunogenic and protective when delivering heterologous antigens in animals, and first human candidate vaccines have proceeded into clinical trials.
  • MVA Although unable to multiply in most mammalian cell lines, MVA retains its genome plasticity that allows the insertion of large amounts of foreign DNA (heterologous genes) (Sutter and Moss, 1992). Absence of pathogenicity for humans, inherent avirulence even in immunocompromised hosts, high-level expression of foreign antigens and adjuvant effect for immune responses make recombinant MVA (rMVA), expressing heterologous genes, an ideal vector for both prophylactic and therapeutic vaccination, as demonstrated by the wide use in prime-boost immunization strategies
  • recombinant MVA (rMVA)-based vaccines comprising heterologous genes and therefore expressing heterologous proteins are able to elicit both humoral and cell- mediated adaptive immune responses (Ramirez et al., 2000) and have been proved to be protective in animal models of several infectious diseases (Garcia-Arriaza et al., 2014, Journal of Virology (Chikungunya) Perez et al., 2018, Scientific Reports (Zika) Lazaro- Frias et al., 2018 (Ebola) Gomez et al., 2013; Marin et al., 2019, Journal of Virology (Hepatitis C)).
  • VACV recombinant VACV
  • rMVA-based vaccines would or not be capable of generating a strong immunogenic and protective immune response against the SARS- CoV-2 virus in a subject in need thereof.
  • the authors of the present invention have found and demonstrated that a vaccine comprising:
  • a recombinant MVA vector comprising at least a nucleic acid encoding an antigenic protein, or an antigenic fragment thereof, of at least one SARS-CoV-2 virus subtype, preferably the structural protein S of the SARS-CoV-2 virus, optionally together with a pharmaceutically acceptable carrier, diluent and/or additive (from hereinafter such vaccine candidate shall be referred to as MVA-COVID19); and/or
  • antigenic protein or "antigenic determinant” refers to any molecule that stimulates a host's immune system to make an antigen-specific immune response, whether a cellular response or a humoral antibody response.
  • Antigenic determinants may include proteins, polypeptides, antigenic protein fragments, antigens, and epitopes which still elicit an immune response in a host and form part of an antigen, homologues or variants of proteins, polypeptides, and antigenic protein fragments, antigens and epitopes including, for example, glycosylated proteins, polypeptides, antigenic protein fragments, antigens and epitopes, and nucleotide sequences encoding such molecules.
  • proteins, polypeptides, antigenic protein fragments, antigens and epitopes are not limited to particular native nucleotide or amino acid sequences but encompass sequences identical to the native sequence as well as modifications to the native sequence, such as deletions, additions, insertions and substitutions.
  • epitope refers to a site on an antigen to which B- and/or T-cells respond, either alone or in conjunction with another protein such as, for example, a major histocompatibility complex ("MHC") protein or a T-cell receptor.
  • MHC major histocompatibility complex
  • Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by secondary and/or tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 5, 6, 7, 8, 9, 10 or more amino acids - but generally less than 20 amino acids - in a unique spatial conformation.
  • Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., "Epitope Mapping Protocols" in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).
  • a homologue or variant has at least about 50%, at least about 60% or 65%, at least about 70% or 75%, at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically, at least about 90%, 91 %, 92%, 93%, or 94% and even more typically at least about 95%, 96%, 97%, 98% or 99%, most typically, at least about 99% identity with the referenced protein, polypeptide, antigenic protein fragment, antigen and epitope at the level of nucleotide or amino acid sequence.
  • sequence identity between nucleic acids and amino acids are known in the art. Two or more sequences can be compared by determining their "percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100.
  • Percent (%) amino acid sequence identity with respect to proteins, polypeptides, antigenic protein fragments, antigens and epitopes described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence (i.e. , the protein, polypeptide, antigenic protein fragment, antigen or epitope from which it is derived), 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 for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publically available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full-length of the sequences being compared.
  • nucleic acid sequences For example, an appropriate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, (1981 ), Advances in Applied Mathematics 2:482-489. This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov (1986), Nucl. Acids Res. 14(6):6745-6763. An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application.
  • a preferred method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, California). From this suite of packages, the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six).
  • BLAST BLAST
  • Another alignment program is BLAST, used with default parameters.
  • the heterologous nucleic acid of any of the vaccine compositions described herein may also optionally encode antigenic domains or antigenic protein fragments rather than the entire antigenic protein. These fragments can be of any length sufficient to be antigenic or immunogenic. Fragments can be at least 8 amino acids long, preferably 10-20 amino acids, but can be longer, such as, e.g., at least 50, 100, 200, 500, 600, 800, 1000, 1200, 1600, 2000 amino acids long, or any length in between. In some embodiments, at least one nucleic acid fragment encoding an antigenic protein fragment or immunogenic polypeptide thereof is inserted into the genome of the recombinant viral vector of the invention.
  • nucleic acids encoding different antigenic proteins are inserted into the genome of one or more of the recombinant viral vectors.
  • multiple immunogenic fragments or subunits of various proteins can be used. For example, several different epitopes from different sites of a single protein or from different proteins of the same SARS-CoV-2 strain, or from a protein orthologue from different strains can be expressed from the vectors.
  • vaccine candidates MVA-COVID19 and MVA-A-COVID19 may comprise any nucleic acid encoding an antigenic protein, or an antigenic fragment thereof, of at least one SARS-CoV-2 virus subtype; wherein such antigenic proteins are preferably selected from the list consisting of structural proteins S (spike), E (envelope), M (membrane), RBD (receptor binding domain) and/or N (nucleocapsid) of SARS-CoV-2, preferably such antigenic proteins are those selected from any of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8 and SEQ ID NO 10, preferably, respectively, encoded by SEQ ID No 1 , SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7 and SEQ ID NO 9.
  • a vaccine comprising:
  • MVA-COVID19(S) an immunologically effective amount of a recombinant MVA vector comprising at least a nucleic acid encoding the structural protein S of the SARS-CoV-2 virus, or an antigenic fragment thereof, preferably, optionally together with a pharmaceutically acceptable carrier, diluent and/or additive (from hereinafter such vaccine candidate shall be referred to as MVA-COVID19(S)); and/or
  • MVA-A-COVID19(S) provides a SARS-CoV-2 vaccine capable of generating a remarkably strong immunogenic immune response against the SARS-CoV-2 virus in a subject in need thereof.
  • vaccine candidates comprising priming and/or boosting compositions which in turn comprise MVA-COVID19(S) or MVA-A-COVID19(S), do not only induced high levels of total IgG, lgG1 , lgG2c, and lgG3 antibodies against SARS-CoV-2 S- and RBD proteins, with a Th1 response (Figure 9), but the results showed that individual serum samples obtained from all mice vaccinated with vaccine compositions MVA- COVID19(S) or MVA-A-COVID19(S) neutralized SARS-CoV-2 pseudoparticles, significantly reducing the levels of luciferase units, compared to serum from the control group MVA-WT/MVA-WT, where no neutralization was observed (Figure 10).
  • vaccine candidates comprising priming and/or boosting vaccine compositions MVA- COVID19(S) or MVA-A-COVID19(S), are capable of inducing broad and polyfunctional coronavirus SARS-CoV-2 S-protein-specific adaptive CD4+ and CD8+ T cell-mediated immune responses of great magnitude ( Figures 5, 6, 7 and 8).
  • a recombinant MVA vector comprising in its genome a nucleotide sequence encoding an antigenic determinant of SARS-CoV-2, preferably encoding the structural protein S of the SARS-CoV-2 virus, reveals very good and specific immune responses against SARS-CoV-2 strains.
  • the MVA- based vaccine would also induce a protective efficacy in animal models and will be able to generate immunogenic and effective immune responses in humans.
  • Figure 12 show that a single dose of MVA-COVID19(S) and a posterior challenge with the SARS-CoV-2 virus induces firstly a small body weight decrease in mice (first 4 days post-challenge), but then the mice fully recover their normal weight showing 100% of survival and demonstrating that a single dose is enough to protect mice from a lethal dose of SARS-CoV-2.
  • Figure 12 also shows that two doses of MVA- COVID19(S) also induced protection upon a lethal dose of SARS-CoV-2, where the vaccinated and challenged mice do not suffer from any weight loss and where the survival is also 100%.
  • control mice vaccinated with the MVA-WT parental vector and subsequently challenged with SARS-CoV-2 virus suffer from an accentuated body weight loss that eventually leads to 0% survival rate.
  • MVA-based vaccines disclose herein confer, both in a single and in two doses, a full protection in challenged mice.
  • a vaccine combination comprising: a. a first composition comprising an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably MVA- COVID19(S) or MVA-A-COVID19(S); and b. a second composition comprising an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably MVA- COVID19(S) or MVA-A-COVID19(S); as a homologous prime and boost generates a strong protective immune response against a SARS-CoV-2 immunogen.
  • compositions comprises an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 composition comprising at least a nucleic acid encoding the S antigenic protein of at least one SARS-CoV-2 virus subtype, preferably encoding for the S antigenic protein of SEQ ID NO 2 (please refer to the examples and figures).
  • a first composition comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype (from hereinafter DNA vaccine); and b. a second composition comprising an immunologically effective amount of a MVA-COVID1 9 or MVA-A-COVID19, preferably MVA-COVID19(S) or MVA- A-COVID19(S); as a heterologous prime and boost generates a protective immune response against a SARS-CoV-2 immunogen.
  • Such response is particularly strong when both compositions comprise at least a nucleic acid encoding the S antigenic protein of at least one SARS- CoV-2 virus subtype, preferably encoding for the S antigenic protein of SEQ ID NO 2.
  • the recombinant MVA and/or DNA heterologous or homologous vaccines may be either monovalent, i.e. , the nucleic acid comprised in the recombinant MVA and/or DNA vectors may comprise only one heterologous sequence encoding an antigenic determinant of SARS-CoV-2, or multivalent, i.e., the nucleic acid comprised in the recombinant MVA and/or DNA vectors may comprise at least two heterologous sequences encoding antigenic determinants of SARS-CoV-2, such as those selected from the list consisting of structural proteins S, E or M of SARS-CoV-2, preferably such antigenic proteins are those selected from any of SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6, respectively encoded by nucleic acid sequences SEQ ID NO 1 , SEQ ID NO 3 and SEQ ID NO 5.
  • nucleic acid comprised in the recombinant MVA/or DNA vectors may encode for structural proteins such as RBD and/or N (nucleocapsid) of SARS-CoV-2 that can also be useful as heterologous sequences to work the present invention, preferably such antigenic proteins are those selected from any of SEQ ID NO 8 and SEQ ID NO 10, respectively encoded by nucleic acid sequences SEQ ID NO 7 and SEQ ID NO 9.
  • the invention thus provides, for the first time, effective vaccines or vaccine combinations for use in generating a protective immune response against infections by at least SARS- CoV-2 and vaccines or vaccine combinations which can be used for manufacturing of a vaccine against SARS-CoV-2.
  • a first aspect of the present invention provides a vaccine composition
  • a vaccine composition comprising a. an immunologically effective amount of a MVA vector comprising at least one nucleic acid encoding an antigenic protein, or an antigenic fragment thereof, of at least one SARS-CoV-2 virus subtype, optionally together with a pharmaceutically acceptable carrier, diluent and/or additive (from hereinafter such vaccine candidate shall be referred to as MVA-COVID19); and/or b.
  • MVA-A-COVID19 an immunologically effective amount of the MVA vector of a), wherein said MVA vector is further modified by deletion of the MVA immunomodulatory genes C6L, K7R, and A46R, optionally together with a pharmaceutically acceptable carrier, diluent and/or additive (from hereinafter such vaccine candidate shall be referred to as MVA-A-COVID19).
  • the nucleic acid encoding the antigenic protein is incorporated into a non-essential region of the genome of the MVA. In an embodiment of the present invention, the nucleic acid encoding the antigenic protein is incorporated into the MVA by substituting a non-essential region of the genome of the MVA. In a preferred embodiment of the present invention, the nucleic acid encoding the antigenic protein is incorporated into the MVA thymidine kinase (TK) locus or hemagglutinin (HA) locus.
  • TK thymidine kinase
  • HA hemagglutinin
  • the transfer vector is the pCyA vector.
  • the pCyA vector has been previously described in WO 2014/162031 A1.
  • the intermediate MVA recombinant viruses Upon homologous recombination by means of an infection with parental virus followed by a transfection with the plasmid transfer vector, selection and purification of recombinant viruses encoding the antigenic protein is performed.
  • the intermediate MVA recombinant viruses also expresses marker genes that facilitate the tracking and selection of the desired final recombinant viruses.
  • the intermediate recombinant MVA viruses might express b-galactosidase gene, a fluorescent protein such as the green fluorescent protein (GFP), or an antibiotic resistance gene.
  • VACV vectors early expression of antigens by VACV vectors is crucial for efficient antigen-specific CD8 T cell responses.
  • the specific properties of the early elements poxviral promoters might thus be crucial for induction of an antigen-specific T cell response.
  • higher amounts of antigen are beneficial for induction of stronger antigen-specific immune responses.
  • the recombinant MVA vector comprises at least one nucleic acid encoding an antigenic protein, or an antigenic fragment thereof, of at least one SARS-CoV-2 virus subtype that is operably linked and under the control of a VACV- specific promoter, an orthopox virus-specific promoter, a poxvirus-specific promoter or a synthetic promoter.
  • a VACV- specific promoter an orthopox virus-specific promoter
  • a poxvirus-specific promoter or a synthetic promoter.
  • promoters may be used for the present invention.
  • promoters such as the 30K and 40K promoters (US 5,747,324, A Strong Synthetic Early-Late Promoter (Sutter, et al.
  • the recombinant MVA vector comprises at least one nucleic acid encoding an antigenic protein, or an antigenic fragment thereof, of at least one SARS-CoV-2 virus subtype that is operably linked and under the control of a synthetic promoter.
  • Synthetic promoters are DNA sequences that do not exist in nature and which are designed to regulate the activity of genes to which they are operably linked, controlling a gene's ability to produce its encoded protein.
  • the synthetic promoter drives the expression of the antigenic protein early during infection. For instance, in an embodiment, the expression of the antigenic protein can be detected as early as 3-hour post-infection.
  • the synthetic promoter may also drive the expression of the antigenic protein late during infection.
  • the expression of the antigenic protein is operably linked and under the control of a synthetic promoter that drives the expression of the antigenic protein both, early and late, during viral infection.
  • the synthetic promoter may be comprised by one or more elements driving early expression of said antigen.
  • the synthetic promoter may be comprised by one or more elements driving late expression of said antigen.
  • Different types of synthetic early/late promoters are further described in WO 2010/102822 A1.
  • the synthetic promoter is formed by both early and late elements, referred as synthetic early/late (sE/L) promoter (Chakrabarti, S.; Sisler, J.R.; Moss, B. Compact, synthetic, vaccinia virus early/late promoter for protein expression. Biotechniques 1997, 23, 1094- 1097) or the LEO160 promoter (Di Pilato, M.; Sanchez-Sampedro, L; Mejias-Perez, E.;
  • the nucleic acid encoding the antigenic protein inserted in MVA genome according to the invention can be selected from any group of nucleic acids encoding proteins from SARS-CoV-2 virus subtype.
  • the nucleic acid encoding the antigenic protein according to the present invention may preferably be the protein S, M, E.
  • the nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype encodes for the protein S.
  • the at least one nucleotide sequence encoding an antigenic protein of at least one SARS-CoV-2 virus subtype is preferably the structural protein S of SARS-CoV-2, and said nucleic acid encoding the structural protein S of SARS-CoV-2, if present, is inserted into the TK locus of the MVA genome; and wherein the MVA vector regulates the expression of the nucleic acid encoding the antigenic protein and is operably linked to said nucleotide sequence.
  • the antigenic protein is selected from the any of the group of structural proteins S, E or M of SARS-CoV-2, or any combination thereof. It is noted that if at least one nucleotide sequence encodes for any of antigenic proteins E or M of SARS-CoV-2, such nucleic acids, if present, should be preferably inserted into the HA locus of the MVA genome; wherein the MVA vector regulates the expression of the nucleic acids encoding the antigenic proteins and should be operably linked to said nucleotide sequences.
  • the antigenic protein is the structural protein S of SARS-CoV-2 resulting in vaccine compositions MVA- COVID19(S) and/or MVA-A-COVID19(S), preferably the antigenic protein is the protein of SEQ ID NO 2, more preferably the antigenic protein of is the protein of SEQ ID NO 2 encoded by the nucleic acid consisting of SEQ ID NO 1.
  • any of vaccine compositions MVA-COVID19(S) and/or MVA-A-COVID19(S) are further characterized by comprising at least one nucleotide sequence encoding for any of antigenic proteins E or M of SARS-CoV-2; wherein such nucleic acids, if present, should be preferably inserted into the HA locus of the MVA genome.
  • the recombinant MVA viruses described herein are highly replication restricted and, thus, highly attenuated, and thus they are ideal candidates for the treatment of a wide range of mammals including humans and even immune-compromised humans.
  • pharmaceutical compositions and vaccines as described above in the first aspect of the invention, suitable for inducing an immune response in a living animal body, including a human against the SARS-CoV-2 virus.
  • the vaccines of the first aspect preferably comprise any of the recombinant MVA viruses described herein formulated in solution in a concentration range of from 1 x 10 7 to 1 x 10 8 PFUs per dosis. This is illustrated in Figures 4-11 which show that immunogenicity was induced in vaccinated mice with a dose of 1 x 10 7 PFUs per dose (see immunization schedule in Figure 3). Further, as shown in Figure 12, Groups 1 and 2 were vaccinated with 1x10 7 PFUs of virus per dose, which indicate that this dose is sufficient to induce protective immunity that leads to 100% survival in both groups upon a lethal challenge with SARS-Cov-2.
  • the vaccine compositions provided herein in the first aspect may generally include one or more pharmaceutically acceptable and/or approved carriers, additives, antibiotics, preservatives, adjuvants, diluents and/or stabilizers.
  • auxiliary substances can be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, or the like.
  • Suitable carriers are typically large, slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, or the like.
  • the recombinant MVA viruses provided herein can be converted into a physiologically acceptable form. This can be done based on experience in the preparation of poxvirus vaccines used for vaccination against smallpox as described by H. Stickl et al. , Dtsch. med. Wschr. 99:2386-2392 (1974).
  • purified viruses can be stored at -80°C with a titer of 5x10 8 PFU/ml formulated in about 10 mM Tris, 140 mM NaCI pH 7.4.
  • a titer of 5x10 8 PFU/ml formulated in about 10 mM Tris, 140 mM NaCI pH 7.4.
  • particles of the virus can be lyophilized in 100 ml of phosphate-buffered saline (PBS) in the presence of 2% peptone and 1% human albumin in an ampoule, preferably a glass ampoule.
  • the vaccine shots can be produced by stepwise freeze-drying of the virus in a formulation.
  • This formulation can contain additional additives such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone or other aids such as antioxidants or inert gas, stabilizers or recombinant proteins (e.g., human serum albumin) suitable for in vivo administration.
  • additional additives such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone or other aids such as antioxidants or inert gas, stabilizers or recombinant proteins (e.g., human serum albumin) suitable for in vivo administration.
  • the glass ampoule is then sealed and can be stored between 45°C and room temperature for several months. Flowever, as long as no need exists, the ampoule is stored preferably at temperatures below -20°C.
  • the lyophilisate can be dissolved in an aqueous solution, preferably physiological saline or Tris buffer, and administered either systemically or locally, i.e. , parenteral, subcutaneous, intravenous, intramuscular, intranasal, or any other path of administration known to the skilled practitioner.
  • aqueous solution preferably physiological saline or Tris buffer
  • parenteral i.e. , parenteral, subcutaneous, intravenous, intramuscular, intranasal, or any other path of administration known to the skilled practitioner.
  • the mode of administration, the dose and the number of administrations can be optimized by those skilled in the art in a known manner. Flowever, most commonly a patient is vaccinated with a second shot about one month to six weeks after the first vaccination shot, although booster doses can also be administered years after the first dose.
  • any avirulent MVA vector suitable for human vaccination could be used.
  • “Derivatives” or “variants” of MVA are also suitable for working the present invention.
  • the MVA vector is further modified by deletion of one or more vaccinia immunomodulatory genes; preferably one, two, or three of the genes C6L, K7R, and A46R. Most preferably, all three genes are deleted. Deletion of these genes has been shown to enhance the innate immune response triggered by the viral vector, and also adaptive antigen-specific immune responses.
  • “Derivatives” or “variants” of MVA refer to viruses exhibiting essentially the same replication characteristics as MVA but exhibiting differences in one or more parts of their genomes.
  • the term "fails to reproductively replicate” refers to a virus that has a virus amplification ratio at 4 days after infection of less than 1 .
  • Assays described in WO 02/42480 or in U.S. Patent No. 6,761 ,893 are applicable for the determination of the virus amplification ratio.
  • the amplification or replication of a virus is normally expressed as the ratio of virus produced from an infected cell (output) to the amount originally used to infect the cell in the first place (input) referred to as the "amplification ratio".
  • An amplification ratio of "1" defines an amplification status where the amount of virus produced from the infected cells is the same as the amount initially used to infect the cells, meaning that the infected cells are permissive for virus infection and reproduction.
  • an amplification ratio of less than 1 i.e. , a decrease in output compared to the input level, indicates a lack of reproductive replication and therefore attenuation of the virus.
  • the recombinant MVA vector of any of the embodiments used for generating the recombinant virus is a MVA virus or a derivative having the capability of reproductive replication in vitro in chicken embryo fibroblasts (CEF) cells and in established chick cells DF-1 , but no capability of reproductive replication in human cervix adenocarcinoma cell line FleLa.
  • CEF chicken embryo fibroblasts
  • the recombinant MVA vector of any of the embodiments used for generating the recombinant virus is a MVA vector further modified by deletion of one or more vaccinia immunomodulatory genes; preferably one, two, or three of the genes C6L, K7R, and A46R. Most preferably, all three genes are deleted.
  • MVA vectors useful for the present invention can be prepared using methods known in the art, such as those described in WO 02/042480 and WO 02/24224, both of which are incorporated by reference herein.
  • a MVA viral strain suitable for generating the recombinant viruses of the present invention may be strain MVA-572, MVA-575 or any similarly attenuated MVA strain. Also suitable may be a mutant MVA, such as the deleted chorioallantoic vaccinia virus Ankara (dCVA).
  • dCVA comprises del I, del II, del III, del IV, del V, and del VI deletion sites of the MVA genome. The sites are particularly useful for the insertion of multiple heterologous sequences.
  • the dCVA can reproductively replicate (with an amplification ratio of greater than 10) in a human cell line (such as human 293, 143B, and MRC-5 cell lines), which then enable the optimization by further mutation useful for a virus-based vaccination strategy (see WO 201 1/092029).
  • a human cell line such as human 293, 143B, and MRC-5 cell lines
  • the Combination Vaccines and methods described herein may be used as part of a homologous prime-boost regimen.
  • a first priming vaccination is given followed by one or more subsequent boosting vaccinations.
  • the boosting vaccinations are configured to boost the immune response generated in the first vaccination by administration of the same recombinant MVA that was used in the first vaccination.
  • a homologous prime-boost regimen may be employed wherein a MVA viral vector as defined herein in the first aspect of the invention is administered in a first dosage.
  • One or more subsequent administrations of a MVA viral vector as defined herein in the first aspect of the invention can be given to boost the immune response provided in the first administration.
  • the one or more antigenic determinants are the same or similar to those of the first administration.
  • the MVA recombinant viral vectors according to the present invention may also be used in prime-boost regimens in which one or more of the initial prime vaccinations are done with either the MVA or the DNA vector (see definition of DNA vaccine below) as defined herein and one or more subsequent boosting vaccinations are done with the MVA vector not used in the prime vaccination, e.g., if a MVA vector defined herein is given in a prime boost, then subsequent boosting vaccinations would be DNA vectors and vice versa.
  • a second aspect of the invention refers to a vaccine combination (from hereinafter referred to as homologous vaccine combination) comprising: a. a first composition comprising an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably a MVA-COVID1 9(S) or MVA-A-COVID19(S) vaccine composition; and b.
  • a second composition comprising an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably a MVA-COVID1 9(S) or MVA-A-COVID19(S) composition; wherein the first compositions is a priming composition and the second composition is a boosting composition, preferably wherein the boosting composition comprises two or more doses of the vector of the boosting composition.
  • an alternative second aspect of the invention refers to a vaccine combination (from hereinafter referred to as heterologous vaccine combination) comprising: a. a first composition comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype (from hereinafter DNA vaccine); preferably DNA-S and b.
  • a second composition comprising an immunologically effective amount of a MVA-COVID1 9 or MVA-A-COVID19, preferably MVA-COVID19(S) or MVA- A-COVID19(S); wherein the first compositions is a priming composition and the second composition is a boosting composition or vice versa, and preferably wherein the boosting composition comprises two or more doses of the vector of the boosting composition.
  • the recombinant heterologous or homologous vaccine combinations may be either monovalent, i.e. , comprising only one heterologous sequence encoding an antigenic determinant of SARS-CoV-2, or multivalent, i.e., comprising at least two heterologous sequences encoding antigenic determinants of SARS-CoV-2, such as those selected from the list consisting of structural proteins S, E or M of SARS-CoV-2, preferably such antigenic proteins are those selected from any of SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6, respectively encoded by nucleic acid sequences SEQ ID NO 1 , SEQ ID NO 3 and SEQ ID NO 5.
  • the antigenic protein of any of the homologous or heterologous vaccine combinations is selected from any of the group of structural proteins S, E or M of SARS-CoV-2, or any combination thereof.
  • the at least one nucleotide sequence encoding an antigenic protein of at least one SARS-CoV-2 virus subtype is preferably the structural protein S of SARS-CoV- 2, and said nucleic acid encoding the structural protein S of SARS-CoV-2, if present, should be preferably inserted into the TK locus of the MVA genome.
  • nucleotide sequence encodes for any of antigenic proteins E or M of SARS-CoV-2
  • such nucleic acids should be preferably inserted into the HA locus of the MVA genome.
  • cells preferably muscle cells
  • the plasmid may be efficiently transfected and produce a relatively large amount of antigen that may be secreted or otherwise released.
  • the plasmid inserts itself into the transfected cell nucleus, it codes for the antigen of interest.
  • the transfected cell displays on its surface the foreign antigen in both histocompatibility complex (MHC) classes I and class II molecules.
  • MHC histocompatibility complex
  • the antigen-presenting cell primed with the antigen travels to the lymph nodes and presents the antigen peptide and costimulatory molecules, initiating the immune response.
  • DNA-based vectors suitable for used as DNA vaccines are known in the art.
  • Hobernik and Bros Int J Mol Sci. 2018 Nov 15; 19(11 ):3605 summarize the knowledge on the course of action of DNA vaccines and the methods for DNA vaccine immunogenicity optimization.
  • Lee et al. Acta Biomater. 2018 Oct 15;80:31 -47 relates to engineering DNA vaccines against infectious diseases.
  • a DNA-based vaccine which comprises antigen expressing plasmid DNA vectors
  • DNA vaccines may be introduced into animal tissues by multiple methods. For instance, naked DNA can be administrated intramuscularly. Other examples are DNA delivered by electroporation, by gene gun or using a liposoluble carrier. Methods of DNA-based vaccines administration are not limited and are known in the art.
  • the DNA-based vaccine vector encodes for one or more antigens against which the immune response is to be elicited. Further, the DNA vector can also encode other co stimulatory molecules, such as interleukins, cytokines, etc.
  • the promoter operably linked to the antigen of interest would need to be recognized by mammal cellular transcription machinery.
  • Promoters for expressing proteins in mammalian cells are also known in the art. For instance, some viral promoters might be used, such as cytomegalovirus promoter or T7 phage promoter.
  • the DNA backbone to construct the DNA-based vaccine of the present invention is not limited. Any DNA, linear or plasmid, able to express the antigen in a mammalian cell may be used. Mammalian expression DNA vectors are widely known in the art.
  • the mammalian expression vector is a plasmid vector and produces high levels of the antigen.
  • suitable mammalian expression plasmid vectors that can be used in the present inventions are, but not limited to, pCDNA, pCAGGS, etc.
  • the mammalian expression vector can be grown in bacterial cells for high- yield production.
  • the DNA-based plasmid vaccine has been produced by endo-free means, for instance, by using EndoFree Plasmid Kits by Qiagen.
  • the mammalian expression plasmid vector is a pCDNA vector encoding an antigenic protein of at least one SARS-CoV-2 virus subtype.
  • the mammalian expression plasmid vector is a pCDNA vector encoding at least one or a combination of the proteins S, M, E of SARS-CoV-2 virus subtype.
  • the plasmid vector encodes for the protein S of SARS-CoV-2 virus subtype.
  • the plasmid vector encoding an antigenic protein of at least one SARS-CoV-2 virus subtype has a SEQ ID 1 encoding for SEQ ID NO 2.
  • kits comprising any one or more of the recombinant MVAs described herein.
  • the kit can comprise one or multiple containers or vials of the recombinant MVA, together with instructions for the administration of the recombinant MVA to a subject at risk of SARS-CoV-2 infection.
  • the subject is a human.
  • the instructions indicate that the recombinant MVA is administered to the subject in a single dose, or in multiple (i.e. , 2, 3, 4, etc.) doses.
  • the instructions indicate that the recombinant MVA virus is administered in a single dose to naive or non-naive subjects.
  • the vaccines and kits comprising recombinant MVA viruses as described in the present invention comprise a single dose to be administrated to a subject in need thereof.
  • the instructions indicate that the recombinant MVA virus is administered in a first (priming) and second (boosting) administration to naive or non- naive subjects.
  • a kit comprises at least two vials for prime/boost immunization comprising the recombinant MVAs as described herein for a first inoculation ("priming inoculation”) in a first vial/container and for an at least second and/or third and/or further inoculation ("boosting inoculation”) in a second and/or further vial/container.
  • the vaccines and kits described herein comprise a first (priming) and second (boosting) dose of an immunologically effective amount of the recombinant MVAs as described herein, preferably of MVA-COVID19 and MVA-A-COVID19, even more preferably MVA-COVID19(S) or MVA-A-COVID19(S), for use in homologous or heterologous prime boost inoculations.
  • kits comprising one, preferably two, or more doses of any of the vaccine compositions as defined in the first aspect of the invention, or of any of the homologous or heterologous vaccine combinations of the second aspect of the invention.
  • the kit comprises an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably MVA-COVID19(S) or MVA-A-COVID19(S), according to the first aspect of the invention in a first vial or container for a first administration (priming) and in a second vial or container for a second administration (boosting).
  • kits comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype, preferably the structural protein S of SARS-CoV-2, in a first vial or container for a first administration (priming), and an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably MVA-COVID19(S) or MVA-D- COVID19(S), according to the first aspect of the invention in a second vial or container for a second administration (boosting).
  • a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype, preferably the structural protein S of SARS-CoV-2
  • a MVA-COVID19 or MVA-A-COVID19 vaccine composition preferably MVA-COVID19(
  • kits comprising an immunologically effective amount of a a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably MVA-COVID19(S) or MVA-A-COVID19(S), according to the first aspect of the invention, in a first vial or container for a first administration (priming), and an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype, preferably the structural protein S of SARS-CoV-2, in a second vial or container for a second administration (boosting).
  • a MVA-COVID19 or MVA-A-COVID19 vaccine composition preferably MVA-COVID19(S) or MVA-A-COVID19(S)
  • a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV
  • any of the kits referred to herein comprise a third, fourth or further vial or container comprising any of the vaccine compositions indicated throughout the present invention for a third, fourth or further administration.
  • the vaccines compositions and kits provided in any of the previous aspects are for use in generating a protective immune response against at least one SARS-CoV-2 subtype, wherein the kits or vaccine composition comprise one dose of an immunologically effective amount of the vaccines provided herein.
  • the vaccines compositions and kits provided in any of the previous aspects are for use in generating a protective immune response against at least one SARS-CoV-2 subtype, wherein the kits or vaccine composition comprise one dose of an immunologically effective amount of the vector MVA-COVID19(S) or MVA-A-COVID19(S) vaccines.
  • the vaccines, vaccine combinations and kits provided in any of the previous aspects are for use in generating a protective immune response against at least one SARS-CoV-2 subtype, wherein if the kits or vaccine combinations are used, the first composition is used for priming said immune response and the second composition is used for boosting said immune response or for use in generating a protective immune response against at least one SARS-CoV-2 subtype, wherein the second composition is used for priming said immune response and the first composition is used for boosting said immune response.
  • the boosting composition can comprise two or more doses of the vector of the boosting composition.
  • the present invention also relates to heterologous vaccination regimes using two different non-replicating viral vectors.
  • the present invention provides a combination vaccine and/or vaccination kit which comprises: a. a first composition comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype (from hereinafter DNA vaccine); preferably DNA-S and b.
  • a second composition comprising an immunologically effective amount of a MVA-COVID1 9 or MVA-A-COVID19, preferably MVA-COVID19(S) or MVA- A-COVID19(S); wherein the first compositions is a priming composition and the second composition is a boosting composition or vice versa, and preferably wherein the boosting composition comprises two or more doses of the vector of the boosting composition.
  • the present invention also provides homologous vaccination regimes using two equal non-replicating viral vectors.
  • the present invention thus provides a combination vaccine and/or vaccination kit which comprises: a. a first composition comprising an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably a MVA-COVID1 9(S) or MVA-A-COVID19(S) vaccine composition; and b.
  • a second composition comprising an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably a MVA-COVID1 9(S) or MVA-A-COVID19(S) composition; wherein the first compositions is a priming composition and the second composition is a boosting composition, preferably wherein the boosting composition comprises two or more doses of the vector of the boosting composition.
  • the combination vaccines and/or kit comprises at least two vials for prime/boost immunization comprising the recombinant MVAs/DNAs as described herein for a first inoculation ("priming inoculation") in a first vial/container and for an at least second and/or third and/or further inoculation ("boosting inoculation”) in a second and/or further vial/container.
  • the combination vaccine and/or kit can comprise multiple containers or vials of the recombinant MVA/DNA, together with instructions for the administration of the recombinant MVA/DNA to a subject at risk of SARS-CoV-2 infection.
  • the subject is a human.
  • the instructions indicate that the recombinant MVA/DNA is administered to the subject in a single dose, or in multiple (i.e. , 2, 3, 4, etc.) doses.
  • the instructions indicate that the recombinant MVA/DNA virus is administered in a first (priming) and second (boosting) administration to naive or non-naive subjects.
  • the first and/or second composition or MVA and/or DNA of any combination vaccine, vaccination kit and/or any homologous or heterologous vaccine program of the invention can comprise any of the MVA and/or DNA vector described herein and any combination thereof.
  • Methods and Uses of Recombinant MVA Viruses Also provided herein are methods of use of any of the recombinant MVA-COVID19 or MVA-A-COVID19 vaccine compositions, preferably the MVA-COVID19(S) or MVA-D- COVID19(S) vaccine compositions, as described in the first aspect of the invention, for immunizing a subject animal or for affecting an immune response in a subject against SARS-CoV-2 infection.
  • recombinant vaccine compositions described herein for the preparation of a medicament or pharmaceutical for the immunization of a subject animal, in particular for the preparation of a medicament or vaccine for treating and/or preventing a SARS-CoV-2-caused disease in a subject.
  • recombinant MVAs according to any embodiment herein for use in priming or boosting an immune response against a SARS-CoV-2 infection, preferably wherein the recombinant MVA is administered once, twice, three times or four times.
  • vaccine combinations of any of the embodiments for use as a medicament or vaccine for inducing an enhanced immune response against a SARS- CoV-2 infection wherein the combination is capable of producing the antigenic determinant, and/or SARS-CoV-2 -like particles in the subject to be treated, preferably, wherein the combination vaccine is producing the encoded antigenic determinant in the subject to be treated.
  • a fourth aspect of the present invention provides a method of generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject, the method comprising administering to the subject the recombinant MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably the MVA-COVID19(S) or MVA-A-COVID19(S) vaccine composition, as described in the first aspect of the invention; or a kit or any of the vaccine combinations, including the homologous or heterologous vaccination regimes, as described in any of the second or third aspects of the invention.
  • the term “immunogenic and protective immune response”, “protective immunity” or “protective immune response” means that the vaccinated subject is able to control an infection with the pathogenic agent against which the vaccination was done.
  • the subject having developed a “protective immune response” develops only mild to moderate clinical symptoms or no symptoms at all.
  • a subject having a "protective immune response” or “protective immunity” against a certain agent will not die as a result of the infection with said agent.
  • the subject animal is a mammal.
  • the mammal may be an adult cow, a calf, in particular a juvenile calf, a rat, rabbit, pig, mouse, but preferably a human, and the method comprises administering a dose of any one or more of the recombinant MVAs or combinations thereof as described in the fourth aspect of the invention.
  • the subject is a human.
  • the subject is an adult.
  • the adult is immune-compromised.
  • the adult is over the age of 10, 15, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 years.
  • any of the recombinant MVAs provided in the first aspect of the invention, or any of the kits or vaccine combinations, including the homologous or heterologous vaccination regimes, as described in any of the second or third aspects of the invention, may be preferably administered to the subject at a dose of 1x10 7 or 1x10 8 PFU/dose.
  • any of the recombinant MVAs provided in the first aspect of the invention, or any of the kits or vaccine combinations, including the homologous or heterologous vaccination regimes, as described in any of the second or third aspects of the invention are administered to the subject at any of the doses provided herein prior to SARS-CoV-2 virus exposure as, e.g., 1 , 2, 3, or 4 weeks or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months before SARS-CoV-2 virus exposure.
  • any of the said vaccine compositions or kits provided herein is administered to the subject at any of the doses provided herein after SARS-CoV-2 virus exposure as, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9. 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 hours or 1 , 2, 3, 4, 5, 6, or 7 days after SARS-CoV-2 exposure.
  • the recombinant MVAs provided in the first aspect of the invention, or any of the kits or vaccine combinations, including the homologous or heterologous vaccination regimes, as described in any of the second or third aspects of the invention are administered to the subject in a single dose, or in multiple (i.e., 2, 3, 4, etc.) doses.
  • the said kits or vaccine compositions provided herein are administered in a first (priming) and second (boosting) administration.
  • a single dose of MVA viral vector as defined herein in the first aspect of the present invention may be given to induce protective immune response, as shown in Figure 12 (vaccinated mice Group 1 ).
  • more than one dose of an immunologically effective amount of an MVA viral vector as defined herein in the first aspect of the present invention may be given to induce protective immune response, as also shown in Figure 12 (vaccinated mice Group 2).
  • a single dose or more than one dose of an immunologically effective amount of MVA-COVID19(S) or MVA-D- COVID19(S) is preferred.
  • Boosting compositions are generally administered once or multiple times weeks or months after administration of the priming composition, for example, about 1 or 2 weeks or 3 weeks, or 4 weeks, or 6 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks or one to two years.
  • the initial boosting inoculation is administered 1-12 weeks or 2-12 weeks after priming, more preferably 1, 2, 4 or 8 weeks after priming.
  • the initial boosting inoculation is administered 4 or 8 weeks after priming.
  • the initial boosting is conducted at least 2 weeks or at least 4 weeks after priming.
  • the initial boosting is conducted 4-12 weeks or 4-8 weeks after priming.
  • the recombinant MVAs provided in the first aspect of the invention, or any of the kits or vaccine combinations, including the homologous or heterologous vaccination regimes, as described in any of the second or third aspects of the invention, can be administered systemically or locally.
  • the said recombinant vaccine compositions are administered parenterally, subcutaneously, intravenously, intramuscularly, or intranasally, in particular subcutaneously or intramuscularly.
  • the recombinant MVAs are administered intranasally.
  • the said recombinant vaccine compositions are administered by any other path of administration known to the skilled practitioner.
  • the said recombinant vaccine composition is administered intramuscularly, preferably the recombinant vaccine composition is administered intramuscularly in a volume ranging between about 0.10 and 1.0 ml, preferably containing concentrations of e.g., about 10 7 to 10 9 virus particles/ml.
  • the said recombinant vaccine composition is administered in a volume ranging between 0.25 and 1.0 ml. More preferably, the said recombinant vaccine composition is administered in a volume of about 0.5 ml.
  • DMEM Dulbecco’s modified Eagle’s medium
  • FCS fetal calf serum
  • DMEM Dulbecco’s modified Eagle’s medium
  • Opti-MEM medium was used as a transient culture medium during plasmid transfection.
  • DMEM was used as a medium for absorbing the virus and was removed after 1 hour, and DMEM-2 % FCS was used as a medium for incubating infections.
  • the solid infection medium was used to select the recombinant virus plaques and consisted of a 1:1 ratio of DMEM 2X-4 % FCS and 1.9 % previously melted agarose (Conda). All the cells were kept in a humidified incubator at 37°C and 5 % CO2.
  • the bacterial strains used for plasmid growth and transformations during cloning were chemically competent Escherichia coli DH5a cells (CNB) and electrocompetent Escherichia coli DH10B cells (New England Biolabs).
  • the bacteria were cultured in Luria- Bertani (LB) medium supplemented with 1 % bacto-tryptone (BD Biosciences), 1 % NaCI (Sigma-Aldrich), and 0.5 % yeast extract (BD Biosciences) at pH 7 in the presence of ampicillin (100 pg/ml, Merck) or kanamycin (100 pg/ml, Merck) (Green MR, Sambrook J. 2012. Molecular Cloning: a laboratory manual, 4 ed. Cold Spring Harbor Laboratory Press, New York, USA)
  • Coronavirus SARS-CoV-2 S antigen SEQ ID NO 2
  • the antigen used to generate recombinant MVA viruses is the coronavirus SARS-CoV-2 S gene (Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1 ; the most contemporary isolate available at the time we initiated this work. GenBank accession number: MN908947.3). The nucleotide sequence of this protein was edited with codons optimized for humans by GeneArt (Thermo Fisher Scientific). 1.4. Virus
  • the parental modified vaccinia virus Ankara (MVA) used for generating MVA-COVID- 19(S) vaccine candidates was a wild-type MVA (MVA-WT) donated by Dr. G. Sutter of the Institute of Molecular Virology of Kunststoff (Germany), or an MVA-GFP previously generated from MVA-WT, by inserting the gene encoding green fluorescent protein (GFP) in the thymidine kinase (TK) locus and by eliminating immunomodulatory genes C6L, K7R, and A46R of MVA.
  • the GFP present in the MVA-GFP TK locus was then replaced by the coronavirus SARS-CoV-2 S gene to generate the different vaccine candidates MVA-COVID1 9(S) or MVA-A-COVID19(S).
  • the S gene was inserted in the viral TK locus (gene J2R), and they are under the control of the synthetic early/late viral promoter (pE/L).
  • SARS-CoV-2 virus SARS-CoV-2 strain MAD6 (kindly provided by Jose M. Honrubia and Dr. Luis Enjuanes, CNB-CSIC, Madrid, Spain).
  • SARS-CoV-2 MAD6 is a virus isolated from a patient from Hospital 12 de Octubre in Madrid. The virus was isolated, plaque cloned three times and amplified at CNB-CSIC.
  • Plasmid transfer vector used to generate recombinant MV As
  • a plasmid transfer vector pCyA was used for inserting the coronavirus SARS-CoV-2 S gene into the MVA genome.
  • the cells were first infected with the parental MVA virus (MVA-WT or MVA-GFP) and then transfected with these plasmids.
  • the recombinant MVA viruses were subsequently selected after several consecutive plaque purification steps.
  • the process whereby a heterologous gene is inserted into the MVA genome is called homologous recombination, a type of genetic recombination in which nucleotides sequences are exchanged between two similar or identical DNA molecules. In this case, recombination occurs between the genetic sequences of the transfer plasmid and the viral genome.
  • the transfer plasmids In order to insert the heterologous genes into the MVA genome, the transfer plasmids must contain the following elements: • Flanking regions of the desired insertion area: These are sequences including the left (L) and right (R) regions of the MVA TK gene such that they allow recombination processes whereby the antigens of interest are inserted in the TK locus.
  • Antigens of interest These must be located between the L and R flanking regions of the TK gene, such that as a consequence of recombination, the antigens of interest are inserted in the viral genome. These antigens were inserted into the plasmid multiple cloning site (MCS) under the control of the VACV synthetic early/late (sE/L) promoter cloned into that same site.
  • MCS plasmid multiple cloning site
  • Selectable marker gene in cell culture This is located in the area adjacent to the genes of the antigens of interest and within the flanking regions, such that it is inserted into the viral genome next to the genes of interest. Another area of recombination (L flanking region) necessary for the later removal of the marker gene by additional recombination processes is found to be repeated between the antigens of interest and the marker. Recombinant MVAs containing only the antigen of interest are therefore generated, which is necessary for use as vaccine candidates in humans.
  • the LacZ (b-galactosidase) marker gene was used to select in cell culture the intermediary recombinant viruses forming blue lysis plaques, after insertion in the TK locus. Furthermore, in the case of insertion of the antigens of interest in the TK locus, the genetic sequence of GFP will be removed, and loss of green fluorescence in recombinant virus plaques may therefore be used as an additional selection criterion.
  • the plasmid transfer vector used to introduce the coronavirus SARS-CoV-2 S protein gene into the MVA TK locus is briefly described below:
  • Plasmid transfer vector pCyA-S was used to generate MVA-COVID19(S) and MVA-A-COVID19(S) recombinant viruses and was developed by the CNB. It directs the insertion of the coronavirus SARS-CoV-2 S gene sequence, under the control of the VACV sE/L promoter, in the TK locus of the parental MVA-WT or MVA-A-GFP virus.
  • the S gene was introduced between the VACV TK-L and TK-R flanking regions, under the control of the VACV sE/L promoter, in plasmid pCyA-20 MCS (Gomez et al., 2013, J Virol 87:7282- 7300). Furthermore, this plasmid also contains the selectable marker genes for ampicillin and b-galactosidase (LacZ gene). Polymerase chain reaction (PCR) and sequencing techniques confirmed that plasmid pCyA-S (11,322 bp) had been generated correctly.
  • a DNA plasmid expressing SARS-CoV-2 S protein was generated by inserting human codon optimized SARS-CoV-2 S gene (see section 1.3) into mammalian expression vector pcDNA 3.1.
  • the plasmid generated was termed DNA-S, and was used in in vivo animal studies. Plasmid DNA-S was generated by Dr. Jose Maria Casasnovas (CNB, Madrid).
  • mice of strain C57BL/6JOIaFlsd (Envigo Laboratories) 6-8 weeks of age at the start of the experiment were used for the immunogenicity studies. These tests were approved by the Ethics Committee for Animal Testing (CEEA, Comite Etico de Experimentacion Animal) of the CNB (Spanish National Center for Biotechnology), and by the Division of Animal Protection of the Considad de Madrid (PROEX 49/20), in accordance with national and international guidelines and Spanish Royal Decree (RD 53/2013), and were carried out in the animal facility of the National Center for Biotechnology (CNB; Madrid, Spain) in a pathogen-free area.
  • mice Female transgenic humanized K18-hACE2 mice, expressing human ACE2, were obtained from the Jackson Laboratory (034860-B6.Cg-Tg(K18-ACE2)2Prlman/J) and efficacy experiments were performed at the BSL-3 laboratory of CISA-INIA (Madrid, Spain).
  • the experiments performed in K18-hACE2 humanized transgenic mice at the CNB-CSIC and CISA-INIA were approved by the Ethical Committee of Animal Experimentation (CEEA) of the CNB-CSIC and CISA-INIA (Madrid, Spain) and by the Division of Animal Protection of the Considad de Madrid (PROEX 169.4/20). Animal procedures were conformed to international guidelines (European Union (EU) Directive 2010/63EU and Recommendation 2007/526/EC) and to the Spanish law under the Royal
  • Table 1 shows the different oligonucleotides used for recombinant virus cloning, sequencing, and generation processes and those used in the PCR for detecting mycoplasma contamination.
  • SARS-CoV-2 S peptide pools These peptides are grouped into two mixtures, S1 (158 peptides) and S2 (157 peptides) (JPT). They are overlapping peptides of the coronavirus SARS-CoV-2 S-protein formed by 15 amino acids (15-mers), 11 of which are overlapping. Each peptide pool (S1 or S2) is at a concentration of 500 pg/ml.
  • coronavirus SARS-CoV-2 S and RBD proteins were used in enzyme-linked immunosorbent assays (ELISA) at a concentration of 2 pg/ml. Those proteins were provided by Dr. Jose Maria Casasnovas (CNB, Madrid) 2.3. Antibodies
  • plasmid DNA from bacterial cultures To extract plasmid DNA from a bacterial culture, the bacteria were previously grown in LB and then DNA was then extracted by means of different commercial kits. The purification of plasmid DNA during the molecular cloning process was performed using the Qiagen Plasmid Mini kit (Qiagen), from 2 ml of positive bacterial clone cultures, following the manufacturer’s instructions. Larger amount of purified plasmid DNA were obtained (from 200-ml bacterial cultures) for use in transfections and infections-transfections in cell cultures using the Qiagen Plasmid Maxi kit (Qiagen), following the manufacturer’s protocol. The plasmid DNAs used for in vivo experiments were purified by means of the Qiagen Plasmid endo-free Mega kit (Qiagen) from 500-ml bacterial cultures, following the manufacturer’s recommendations.
  • Qiagen Plasmid Mini kit Qiagen Plasmid Mini kit
  • RNAse A 40 pg/ml, PanReac AppliChem
  • RNAse A 40 pg/ml, PanReac AppliChem
  • saturated NaCI was added. It was mixed by inverting the tube and centrifuged at 13000 rpm for 10 min at room temperature. The supernatant was collected, mixed with isopropanol at a ratio of 1:0.7 to precipitate the DNA, and centrifuged at 13,000 rpm for 10 min at room temperature. The precipitate was washed with 75 % ethanol and centrifuged again at 13,000 rpm for 10 min at room temperature. Lastly the supernatant was carefully removed and the precipitated DNA was left to dry at room temperature, after which it was resuspended in sterile distilled water.
  • DNA concentrations were measured in a NanoDrop® ND-1000 full-spectrum spectrophotometer (Thermo Fisher Scientific). Isolated DNA was later used for different experiments, including PCR, sequencing, transfections, infections-transfections, and in vivo assays in mice.
  • the PCR technique was used to amplify DNA fragments for later cloning or to check for the gene insertion in transfer plasmids or in the recombinant MVA genome.
  • the polymerase used was Phusion® High Fidelity (New England Biolabs) with its corresponding buffers and reagents and following the manufacturer’s recommendations. About 10-100 ng of template DNA together with 0.4 mM of the corresponding oligonucleotides, 1-2.5 units of Phusion® High Fidelity polymerase with the corresponding buffer, and 0.2 mM of each of the four deoxynucleotide triphosphates (dNTPs) (Roche Diagnostics GmbH) were used for each reaction.
  • dNTPs deoxynucleotide triphosphates
  • the temperature used for annealing was selected according to the oligonucleotides used, and the extension time used depended on the length of the fragment to be amplified.
  • the reactions were carried out in a 96-well VeritiTM thermocycler (Applied Biosystems).
  • Coronavirus SARS-CoV-2 S gene was cloned into transfer plasmid pCyA to generate plasmid transfer vector pCyA-S by GeneArt (Thermo Fisher Scientific).
  • the infected or transfected cell samples were collected, centrifuged at 2,000 rpm for 5 min at room temperature, and the precipitate was resuspended in Laemmli IC-b-mercaptoethanol loading buffer.
  • the cell supernatants were also centrifuged at 2,000 rpm for 5 min at 4°C to separate them from T cells that might be present.
  • the proteins were precipitated from the supernatant with 10 % trichloroacetic acid (TCA) (Sigma-Aldrich), centrifuged at 13,000 rpm for 15 min at room temperature, and the precipitate was resuspended in Laemmli 1C-b- mercaptoethanol.
  • TCA % trichloroacetic acid
  • both the cell extract and supernatant samples were denatured at 95°C for 10 min and were loaded in 7 % polyacrylamide gels in the presence of SDS.
  • the proteins were separated by means of one-dimensional electrophoresis at 100 V and at room temperature for about 90 min.
  • the SDS-PAGE gels were transferred to nitrocellulose membranes (GE Healthcare), following the wet system protocol recommended by the manufacturer (Mini Trans-Blot® Cell, Bio-Rad).
  • the gel and previously hydrated nitrocellulose membrane were arranged between two Whattman-3MM® filter papers (GE Healthcare), which were also hydrated. They were then mounted in the transfer system in the presence of the transfer buffer, and the transfer was performed at a current intensity of 100 V for 50 min. Once this ended, checking for the presence of proteins was performed by staining the membrane with the reversible Ponceau S stain (0.2 % Ponceau in 3 % TCA; Sigma-Aldrich).
  • nitrocellulose membrane was then blocked with a 5 % skim milk powder solution prepared in PBS 1X-0.05 % Tween20 (Sigma-Aldrich) (PBS-T) at room temperature for 1 hour with gentle stirring.
  • PBS-T PBS 1X-0.05 % Tween20
  • the corresponding primary antibodies prepared at the desired dilution in the blocking buffer, were added and incubated under stirring at 4°C overnight.
  • the membrane was incubated with the appropriate secondary antibody (suitably diluted in the same blocking buffer) for 1 hour at room temperature and was then washed again four times with PBS-T.
  • the membranes were developed with the ECL® luminol system (Amersham), exposing Carestream Kodak BioMax XAR autoradiography film (Kodak) or directly in the ChemiDocTM Imaging System (Bio-Rad). The proteins bands were analyzed and quantified by means of Image Lab software (Bio-Rad). 3.3. Virus manipulation techniques
  • viruses All the viruses were kept at -70°C and thawed at 37°C in a bath prior to use. Once thawed, they were stirred with a vortex and sonicated with a 3-cycle 10-second sonication and 10- second pause program (Misonix Incorporated S-3000 Sonicator, Cole-Parmer). The viruses were then added to the cell monolayer with the minimum volume of (serum-free) DMEM needed to cover the cell monolayer at the desired MOI. After 1 hour of adsorption at 37°C, the inoculum was removed and fresh DMEM-2 % FCS medium was added. The time the different infections were maintained varied with the objective of the infection, and the manner in which the cells were collected also varied.
  • MVA viruses expressing coronavirus SARS-CoV-2 S- protein were generated in DF-1 cells according to the previously described standard infection/transfection protocol (Earl et al. , 2001, Curr Protoc Mol Biol, 16:16 17), with certain modifications.
  • MVA-WT was used as the parental virus
  • MVA-A-COVID19(S) was used as a parental virus, as previously described (Garcia-Arriaza et al., 2014, J Virol 88:3527-3547).
  • Figure 11 shows a diagram summarizing the process followed for generating the recombinant MVA viruses.
  • the DF-1 cell monolayer cultured in p60 dishes (Nunc) at 80-90 % confluence was infected at a MOI of 0.05 PFU/cell with the corresponding parental virus.
  • a mixture of DNA-lipofectamine 2000 was prepared by mixing 10 pg of the corresponding transfer plasmid with the suitable volume of lipofectamine 2000 (Invitrogen) in Opti-MEM (Gibco) (1.5 pi lipofectamine 2000/pg of DNA) for 20 min at room temperature to form DNA-liposome complexes.
  • the inoculum was removed, the cells were washed two times with Opti-MEM and incubated with 1 ml of the DNA-lipofectamine mixture for 4-6 hours at 37°C and 5 % CO2.
  • the transfection medium was removed, the cells were washed two times with Opti-MEM and incubated with DMEM-2 % FCS medium at 37°C and 5 % CO2.
  • DMEM-2 % FCS medium 37°C and 5 % CO2.
  • the cells were then frozen (at -70°C) and thawed (at 37°C) three times, stirring with a vortex between each freeze-thaw cycle to lyse them and release the virus into the supernatant, which was obtained after centrifuging at 3,000 rpm for 5 min at room temperature.
  • the virus was then used to infect confluent monolayers of DF-1 cells grown in 6-well plates (M6) (Nunc) at different serial dilutions (from 10 1 to 10 6 ) in serum-free DMEM.
  • the inoculum was removed and replaced with 3 ml of solid infection medium consisting of a mixture of 1.9 % (previously melted) agarose (Conda) with DMEM 2X-4 % FCS medium at a 1 :1 ratio.
  • 3 ml of solid infection medium consisting of a mixture of 1.9 % (previously melted) agarose (Conda) with DMEM 2X-4 % FCS medium at a 1 :1 ratio.
  • the selected lysis plaques were obtained with a 150-mm glass Pasteur pipette (Deltalab) and added to 500 pi of serum-free DMEM. After three freeze-thaw cycles and subsequent stirring with a vortex and sonication, these plaques were used as an inoculum to infect monolayers of confluent cells in 24-well plates (M24) (Nunc) as explained in section 3.3.1. These infections were performed to re-grow the virus, and they were collected when a cytopathic effect was observed.
  • the initial viral stock (stock P1 ) consists of the generated recombinant virus originating from cell monolayers infected with the final plaque and collected after observing a cytopathic effect.
  • stock P2 5 p150 plates (Nunc) containing confluent DF-1 cells were infected at an MOI of 0.05 PFU/cell and cells were collected when a cytopathic effect was observed.
  • Stock P2 was used in all the experiments for characterizing the virus in vitro.
  • the purified viral stock (stock P3) was generated from stock P2 (described in section 3.3.4.).
  • Viral titration Titration of the MVA viruses was carried out according to the previously described standard protocols (Ramirez et al. , 2000, J. Virol, 74:923-933).
  • DF-1 cells seeded in M6 plates were infected in duplicate with serial dilutions of the virus in serum-free DMEM, as described in section 3.3.1.
  • the medium was removed 30-40 hours post-infection and the cells were fixed for 3 min using a 1 : 1 mixture of methanol and acetone and the titer was determined by means of an immunostaining assay which allows detecting and counting lysis plaques formed by the virus.
  • the monolayers were thereby incubated with a rabbit anti-VACV WR polyclonal primary antibody (diluted 1 : 1.000 in PBS-3 % FCS; CNB) for 1 hour at room temperature; it was then washed three times with PBS 1X and incubated for 1 hour at room temperature with the HRP-conjugated goat anti-rabbit secondary antibody (diluted 1 : 1.000 in PBS-3 % FCS; Sigma-Aldrich).
  • the plates were developed using 1 mg/ml of 3,3’-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich), as the HRP substrate, with 30 % hydrogen peroxide (Sigma- Aldrich) and 3 % nickel sulfate (NiSC ) (Sigma-Aldrich). After counting the lysis plaques, the titer was referenced as PFU/ml. ⁇ Replication kinetics
  • the genetic stability of vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) was analyzed.
  • Monolayers of DF-1 cells were infected with the different recombinant viruses at an MOI of 0.05 PFU/cell.
  • the cells were collected 72 hours post-infection, frozen-thawed three times, and briefly sonicated.
  • the cell extracts were then centrifuged at 1 ,500 rpm for 5 minutes, and the supernatant was used for a new round of infection at a low MOI. The same method was repeated nine times.
  • Expression of the coronavirus SARS-CoV-2 S-protein was analyzed by means of Western blot (see section 3.2.3.) after infecting DF-1 cells with the viral stocks from each passage.
  • Purified virus stocks (stock P3) were used for the in vivo assays according to the method first described by Joklik (Joklik, 1962, Virology, 18:9-18) and modified by Esteban (Esteban, 1984, Virology, 133:220-227).
  • 30 p150 plates containing confluent CEF cells were infected with stock P2 at an MOI of 0.01 PFU/cell. The cells were collected when a cytopathic effect was observed (usually after 3 days), centrifuged at 1 ,500 rpm for 5 min at 4°C, and the precipitate was washed with PBS and resuspended in 10 mM Tris-HCI buffer pH 9.
  • the infected cells were then lysed by means of three sonication cycles, stirring with a vortex, and centrifugation to release the viral particles into the supernatant.
  • this supernatant was centrifuged for 1 hour at 20,000 rpm at 4°C in the SW28 rotor (Beckman) on a 36 % sucrose pad in 10 mM Tris-HCI pH 9.
  • the obtained precipitate was resuspended in 10 mM Tris-HCI pH 9 and centrifuged again on another 36 % sucrose pad under the same conditions.
  • the precipitate obtained in this second centrifugation was resuspended in 10 mM Tris-HCI pH 9, aliquoted, and frozen at -70°C until use.
  • the purified viruses were tittered in duplicate and checked for the absence of mycoplasma contamination (through PCR with mycoplasma-specific oligonucleotides; see Table 1), bacteria (by means of growth in LB agar plates), and fungi (by means of growth in blood agar plates, Oxoid). Furthermore, checking for the correct expression of the antigens of stocks P3 was performed by means of PCR and Western blot before use in the in vivo assays. 3.4. In vivo immunological techniques 3.4.1. Immunization protocols in mice
  • a heterologous prime-boost immunization regimen was used, which consisted of a first inoculation with 100 pg (50 pg/leg) of a plasmid vector expressing the coronavirus SARS-CoV-2 S-protein (DNA-S), followed by a 1 x 10 7 PFU booster dose of vaccine candidates MVA- COVID19(S) and MVA-A-COVID19(S) 2 weeks later. The mice were sacrificed 11 days after the booster dose as well. Moreover, to evaluate the long-term memory immunogenicity of the MVA-S vaccine candidate, a MVA-S prime/MVA-S boost immunization protocol was performed in female C57BL/6 mice (6 to 8 weeks old) as described above.
  • mice received two doses of 1 x 10 7 PFU of MVA-S or MVA-WT (used as negative control mice) by the intramuscular route in 100 pi of PBS (50 mI/leg) at 0 and 2 weeks. At 6 months after the last immunization, mice were sacrificed using CO2.
  • MVA prime/MVA boost immunization protocol was performed in female C57BL/6 mice (6 to 8 weeks old).
  • Blood, bronchoalveolar lavage (BAL) samples, spleen and lungs were collected and processed.
  • Blood from each individual mouse was collected directly from heart and was allowed to clot 60 min at 37°C; after leaving samples at 4°C overnight, they were centrifuge 20 minutes at 3600 rpm, and sera were removed and stored at -20°C until used in ELISA and neutralization experiments.
  • lung and bronchoalveolar lavage (BAL) samples were collected and processed.
  • BAL from each individual mouse were taken by flushing 1ml of RPMI 1640 (Gibco-Life Technologies) medium supplemented with HEPES pH 7.4 (10 mM, Merck), b-mercaptoethanol (10 mM, Sigma-Aldrich), L-glutamine (2 mM, Merck), and 10% FCS into the trachea; then samples were spun down to remove cellular debris and supernatants kept at -20°C until used for ELISA and neutralization experiments. Spleens and lungs were extracted, processed mechanically or enzyme-digested, respectively, blood-cell depleted and filtered through 40pm cell strainers until obtain single-cell samples.
  • the ELISpot assay was used to detect S antigen-specific IFNy-secreting T cells (300).
  • the spleens of each group of immunized animals were collected in RPMI-10 % FCS medium and processed together to obtain the splenocytes as previously described (Garcia- Arriaza et al., 2013, PLoS One, 8:e66894; Najera et al. , 2010, PLoS One, 5:e11406).
  • the spleens were homogenized by means of mechanical disintegration through a 40-pm cell strainer (Falcon). The disintegrated cells were centrifuged for 5 min at 1,500 rpm at 4°C and washed with RPMI-10 % FCS.
  • the erythrocytes were lysed by adding 0.1 M NH4CI (2 ml/spleen) for 5 min in ice. After this time, the cells were washed three times with RPMI-10 % FCS to eliminate fat and resuspended in 12 ml of RPMI- 10 % FCS, and the number of living splenocytes was counted by means of staining with trypan blue (4 % in water; Sigma-Aldrich).
  • 10 6 splenocytes per condition were re-stimulated with 1 pg/ml of the S1 or S2 peptide pool.
  • 10 6 splenocytes were stimulated with 2.5 pg/ml of VACV E3 peptide.
  • the plates were incubated with the peptides for 48 hours at 37°C in 5 % CO2 atmosphere, washed five times with PBS-T, and incubated with 2 pg/ml of biotinylated rat anti-mouse IFNy monoclonal antibody XMG1.2 (Pharmingen) diluted in PBS-T, for 2 hours at room temperature.
  • the plates were then washed five times with PBS-T and a 1 :800 dilution of HRP-conjugated streptavidin (0.5 mg/ml; Sigma-Aldrich) was added. After 1 hour at room temperature, it was washed three times with PBS-T and two times with PBS, and finally 1 pg/ml of the DAB substrate (Sigma-Aldrich), resuspended in 50 mM Tris-CI pH 7.5 and 0.015 % H2O2, was added to develop the plates.
  • HRP-conjugated streptavidin 0.5 mg/ml; Sigma-Aldrich
  • the reaction was stopped by washing the plate with abundant water and once it was dry, the spots were counted using the ELISpot Reader System -ELR02- plate reader (AID Autoimmun Diagnostika GmbH) with the aid of AID ELISpot reader system software (Vitro).
  • brefeldin A Protein Transport Inhibitor BD GolgiPlug, BD Biosciences
  • BFA Protein Transport Inhibitor BD GolgiPlug, BD Biosciences
  • a 1 :300 dilution of anti-CD107a-FITC antibody 4x 10 6 cells were added in M96 conical bottom wells and were incubated for 6 hours in the presence of 1 pg/ml of the S1 or S2 peptide pool, or with 5 pg/ml of VACV E3 peptide to determine the response to the MVA vector.
  • the splenocytes or lung cells were stimulated with 2 pl/ml of LAC (Leukocyte Activation Cocktail, BD Biosciences) and Monensin 1X.
  • LAC Leukocyte Activation Cocktail
  • the splenocytes or lung cells were washed with IB buffer and incubated with Violet Dye (0.5 pl/ml, Invitrogen) for 20 min in the dark at 4°C to evaluate cell viability. Next, two washes were performed with IB buffer and the splenocytes or lung cells were incubated for 20 min in the dark at 4°C with 50 pi of the primary surface antibodies
  • the analysis strategy was performed by selecting the living cells expressing CD3 on their surface; then those expressing CD4, CD8, CD127, or CD62L; and finally those secreting the different cytokines (IFN-y, TNF-a, and IL-2) or expressing degranulation marker CD107a.
  • spleens were extracted from sacrificed animals and processed as explained above. The splenocytes were then resuspended in RPMI-10% FCS medium with 1 mI/ml of BFA (BD Biosciences), 1 mI/ml of Monensin 1X (eBioscience), and a 1:100 dilution of the anti- CD154 (CD40L)-PE antibody.
  • splenocytes were stimulated with 5 pg/ml of S-protein and 1 pg/ml of S1 and S2 peptide pools for 6 hours at 37°C in a conical-bottom 96-well plate.
  • the cells were then stained with Fixable Viability Stain (FVS) 520 (BD Biosciences) for 20 min at 4°C in the dark to analyze cell viability.
  • FVS Fixable Viability Stain
  • the splenocytes were then washed two times with IB buffer and stained for 20 min at 4°C in the dark with 50 pi of the antibodies specific against the surface markers (described in Table 2), using the dilutions recommended by the manufacturer.
  • the splenocytes were again washed two times with IB buffer, fixed, and permeabilized with the BD Cytofix/CytopermTM kit (BD Biosciences) for 20 min at 4°C in the dark, and they were left in IB buffer overnight at 4°C in the dark.
  • the cells were washed the next day with Permwash 1X (BD Biosciences) and the Fc receptors were blocked with 25 pi of Fc Block (diluted to 1 : 100 in Permwash 1X) for 5 min at 4°C in the dark.
  • the analysis strategy was performed by selecting living cells expressing CD3, CD4, and CD44 on their surface, followed by cells expressing CXCR5, PD1, and CD154 (CD40L), and finally cells secreting the different cytokines (IFN-y, IL-4, and IL-21).
  • the humoral immune response was determined by analyzing the levels of IgG or IgA antibodies in the sera or BAL samples, respectively, of immunized mice and evaluating the capacity of these antibodies to neutralize the SARS-CoV-2 virus.
  • blood was collected from the animals after sacrificing them by means of intracardiac puncture. Next, the blood samples were maintained at 37°C for 1 hour and kept at 4°C overnight. The tubes of blood were centrifuged the next day at 3600 rpm for 20 min at 4°C and the serum which was kept at -20°C until use was obtained.
  • Serial dilutions of each serum or BAL sample (or group of sera) were then performed to measure the levels of coronavirus SARS-CoV-2 S-protein-specific and RBD-specific antibodies by means of ELISA, or to measure their SARS-CoV-2 neutralizing capacity by means of a luciferase assay or a live-virus SARS-CoV-2 neutralization assay.
  • ELISA assays were performed to analyze the levels of antibodies in the serum or BAL samples of immunized animals.
  • NUNC MaxiSorpTM 96-well plates (Thermo Fisher Scientific) were coated with 2 pg/ml of the coronavirus SARS-CoV-2 S-protein and RBD in PBS and incubated at 4°C overnight. The plates were washed three times the next day with PBS-T and blocked for 2 hours at room temperature with 5% skim milk prepared in PBS-T.
  • the plates were washed three times with PBS-T, 50 pi of the serial dilutions of the serum or BAL samples diluted in PBS with 1% skim milk and 0.01% Tween20 were added, and the plates were incubated for 1.5 hours at room temperature.
  • the plates were again washed three times with PBS-T and incubated with 50 mI of HRP-conjugated mouse anti-lgG, -lgG1, -lgG2c, or-lgG3 antibodies (diluted to 1:1000 in PBS with 1% skim milk and 0.01% Tween20) for 1 hour at room temperature.
  • the plates were developed by adding 100 mI of TMB substrate (Sigma-Aldrich) and the reaction was stopped by adding 50 mI of 1 M H2SO4. Absorbance was read at 450 nm by means of an EZ Read 400 microplate reader (Biochrom).
  • live-virus SARS-CoV-2 neutralization antibody titers were assessed by a microneutralization test (MNT), using SARS-CoV-2 MAD6 strain in a BSL-3 laboratory at the CNB-CSIC. Serum samples were inactivated at 56°C for 30 min and then serially diluted two-fold in duplicate in serum-free DMEM and incubated at a 1:1 ratio with 100 TCID50 (50% Tissue Culture Infectious Dose) of SARS-CoV-2 MAD6 isolate at 37° C for 1 hour.
  • MNT microneutralization test
  • the virus/serum mixture were added to VeroE6 cell monolayers, seeded in 96- well plates (40.000-50.000 cells per well), and incubated at 37° C, in a 5% CO2 incubator for 3 days. Then, cells were fixed with 4% formaldehyde and stained with crystal violet. ID50 titers were calculated as the reciprocal dilution resulting in 50% inhibition of cell death.
  • mice Female K18-hACE2 mice (10 weeks old at the beginning of the study) immunized with one or two doses of MVA-S were used to evaluate the efficacy of the MVA-S vaccine candidate.
  • Mice primed and boosted with nonrecombinant MVA-WT or not vaccinated were used as control groups.
  • mice were challenged with a lethal dose (1 x 10 5 PFU) of SARS-CoV- 2 (MAD6 strain) by the intranasal route in 50 mI of PBS, and one group of unvaccinated mice were left uninfected. Mice were monitored for body weight change and mortality for 15 days postchallenge. Animals with more than a 25% of weight loss were euthanized. At days 2 and 4 postchallenge, three mice per group were euthanized, and lungs and serum samples were collected. One lung was inactivated in zinc formalin (Sigma-Aldrich) for 48 hours and then included in paraffin and processed for histopathological analysis.
  • the other lung was divided longitudinally in two, with one part placed in RNALater stabilization reagent (Sigma-Aldrich) and stored at -80°C until RNA extraction, and the other part stored also at -80°C until analysis of virus yields.
  • Blood was collected from the animals by submandibular bleeding, maintained at 37°C for 1 hour, kept at 4°C overnight, and centrifuged at 3600 rpm for 20 min at 4°C to obtain the serum samples, which was then inactivated at 56°C for 30 min and kept at -20°C until use.
  • Mice were monitored for body weight change and mortality for 6 days postchallenge, moment at what control infected mice lost more than a 25% of the initial body weight and have to be sacrificed. At day 6 postchallenge, all mice were euthanized, and lungs and serum samples were collected and processed similarly as above.
  • RNALater Sigma-Aldrich
  • b-mercaptoethanol Sigma-Aldrich
  • First-strand cDNA synthesis and subsequent real-time PCR were performed in one step using NZYSpeedy One-step RT-qPCR Master Mix (NZYTech) according to the manufacturer’s specifications using ROX as reference dye.
  • SARS-CoV- 2 viral RNA content was determined using previously validated set of primers and probes specific for the SARS-CoV-2 subgenomic RNA for the protein E, the genomic virus RNA dependent RNA polymerase (RdRp) gene and the cellular 28S rRNA for normalization. Data were acquired with a 7500 real-time PCR system (Applied Biosystems) and analyzed with 7500 software v2.0.6. Relative RNA arbitrary units (A.U.) were quantified relative to the negative group (uninfected mice) and were performed using the 2-AACt method. All samples were tested in triplicate.
  • RNA expression levels of the genes for IL-6, TNF-a, IL-10, IP-10 and IFN-y were analyzed by real-time PCR with specific oligonucleotides (sequences are available upon request). Specific gene expression was expressed relative to the expression of the cellular 28S ribosomal RNA gene in fold change units using the 2-AACt method. All samples were tested in triplicate.
  • Lung histopathology For histological examination, lung tissues were fixed in zinc formalin embedded in paraffin. Tissue sections (5 pm) were stained with hematoxylin and eosin and analyzed for lung damage.
  • hamsters were anaesthetized by intraperitoneal injection of a xylazine (16 mg kg— 1 , XYL-M, V.M.D.), ketamine (40 mg kg— 1 , Nimatek, EuroVet) and atropine (0.2 mg kg— 1 , Sterop) solution.
  • the animals were monitored daily for signs of disease (lethargy, heavy breathing or ruffled fur).
  • the iTaq Universal Probes One-Step RT-qPCR kit (BioRad) was used to perform reactions.
  • the 2-AACq method was used to calculate the relative RNA fold-change, using housekeeping gene b-actin for normalization. 3.6.4. End-point virus titrations.
  • End-point titrations to quantify infectious SARS-CoV-2 particles were performed on confluent Vero E6 cells in 96-well plates. Lung tissues were homogenized using bead disruption (Precellys) in 350 mI minimal essential medium and centrifuged (10,000 rpm, 5 min, 4 °C) to pellet the cell debris. Supernatant was serially diluted into the 96-well plates and scoring for cytopathic effect performed after 3 days. Viral titers were calculated by the Spearman and Karber method.
  • Lung histology For histological examination, lung tissues were fixed in formalin and embedded in paraffin. Tissue sections (5 pm) were stained with hematoxylin and eosin and analyzed for lung damage.
  • ELISPOT assay Quantification of antigen-specific cytokine-secreting cells in macaques was performed by IFN-y and IL-4 enzyme-linked immunospot (ELISpot) assays on freshly isolated peripheral blood mononuclear cells (PBMC), according to the manufacturer's instructions (U-Cytech, Utrecht, The Netherlands). Antigen-specific responses were measured against 5-pg/ml of S1 and S2 peptide pools. Results are expressed as the mean number of spot-forming cells (SFC) per 10 6 PBMC from triplicate assays minus background values (mean number of SFC plus two standard deviations [SD] of triplicate assays with medium alone). Mean numbers of spots for each animal or individual per antigen are reported.
  • SFC spot-forming cells
  • RNA assay RT-PCR assays were utilized to monitor viral loads. Briefly, RNA was extracted using a QIAcube FIT (Qiagen, Germany) and the Cador pathogen FIT kit from bronchoalveolar lavage (BAL) supernatant and throat swabs. RNA was reverse transcribed using superscript VILO (Invitrogen) and ran in duplicate using the QuantStudio 6 and 7 Flex Real-Time PCR System (Applied Biosystems) according to manufacturer’s specifications. Viral loads were calculated of viral RNA copies per ml_ or per swab and the assay sensitivity was 50 copies. The target for amplification was the SARS-CoV2 N (nucleocapsid) subgenomic gene or genomic RdRp gene. RESULTS
  • new MVA viral vector-based vaccines have been designed and developed against the coronavirus SARS-CoV-2 causing the COVID-19 pandemic. Once generated, these recombinant MVA viruses have been characterized in vitro and their capacity to stimulate adaptive SARS-CoV-2-specific T cell-mediated immune responses and humoral immune responses in vivo in immunized mice has been analysed.
  • MVA-based vaccine candidates expressing the SARS-CoV-2 full- length S structural gene (Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1 , Genbank number: MN908947.3; the most contemporary isolate available at the time we initiated this work) [termed MVA-COVID19(S) and MVA-A-COVID19(S)] (see Materials and Methods).
  • Vaccine candidate MVA-COVID19(S) was generated following the insertion of the complete sequence of the coronavirus SARS-CoV-2 S gene in the genome of the parental virus MVA-WT, whereas the other vaccine candidate, MVA-D- COVID19(S), was generated following the insertion of the complete sequence of the coronavirus S gene in the genome of an MVA, referred to as MVA-GFP, expressing the GFP protein and containing complete deletions in the vaccinia virus immunomodulatory genes C6L, K7R, and A46R.
  • the expression of the SARS-CoV-2 gene is under the transcriptional control of a synthetic early/late (sE/L) promoter.
  • an MVA viral vector lacking said immunomodulatory genes C6L, K7R, and A46R and expressing the structural genes of the Chikungunya virus is capable of providing full protection to mice and non-human primates after a Chikungunya virus challenge (Garcia-Arriaza et al. , 2014, J. Virol, 88:3527-3547; Roques et al., 2017, JCI Insight, 2(6):e83527). Additionally,
  • Zika and Ebola vaccine candidates based on this MVA viral vector lacking said immunomodulatory genes are capable of providing protection to mice after a Zika virus challenge (Perez et al., 2018, Scientific Reports 8:17385) or an Ebola virus challenge (Lazaro-Frias et al., 2018, J. Virol, 92:e00363-18).
  • FIG. 1A shows a diagram of the generated vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S).
  • MVA-COVID19(S) will be referred to as MVA-S or MVA-CoV2-S and MVA-A-COVID19(S) as MVA-A-S or MVA-A-CoV2-S throughout the text and figures.
  • the methodology used for generating these vaccine candidates has been described above in Materials and Methods.
  • the correct insertion of the coronavirus SARS-CoV-2 S gene and the purity of the generated vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) were confirmed by means of PCR and sequencing, after amplifying the DNA obtained from DF-1 cells that were not infected (mock) or infected with 5 PFU/cell of MVA-WT, MVA-GFP, MVA-COVID19(S), and MVA- A-COVID19(S).
  • Oligonucleotides (described in Table 3 of Materials and Methods) which hybridize in the flanking regions of the MVA TK gene ( J2R gene) were used in the PCR, and the obtained results confirmed the presence of the S gene in MVA-COVID19(S) and MVA-A-COVID19(S), without contamination with the parental MVA-WT or MVA-GFP, respectively ( Figure 1 B).
  • the TK locus of parental viruses MVA-WT or MVA-GFP was amplified as PCR control, with the TK gene and the GFP gene being amplified, respectively.
  • the correct presence of the S gene inserted in the TK locus was also confirmed by means of DNA sequencing.
  • MVA-S and MVA-A-S constitutively express the SARS-CoV-2 S protein
  • DF-1 infected cells were leave in the presence or absence of tunicamycin, an inhibitor of protein N-glycosylation, to analyze the glycosylation pattern of SARS-CoV-2 S protein expressed from MVA.
  • vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) efficiently express the coronavirus SARS-CoV-2 S-protein.
  • DF-1 cells were infected with vaccine candidates MVA- COVID19(S) and MVA-A-COVID19(S), and parental viruses MVA-WT and MVA-GFP, and cell extracts were collected at different times post-infection (0, 24, 48, and 72 hours), and the amount of virus present was determined.
  • the results showed that the viral growth kinetics of vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S), was similar ( Figure 2B).
  • Vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) are stable in cell culture
  • the immunogenicity of vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) in C57BL/6 mice was then evaluated using heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost immunization regimens, and the coronavirus SARS-CoV-2 S- protein-specific adaptive cell-mediated immune responses and humoral immune responses induced by those vaccine candidates were analysed in detail.
  • the mice were immunized with two doses of MVA-WT.
  • Splenocytes originating from the spleens of each group were stimulated ex vivo with a panel of peptide pools covering the complete sequence of the coronavirus SARS-CoV-2 S-protein (S1 and S2 peptide pools).
  • the samples were stimulated with RPMI as a negative control, and with a VACV E3 protein peptide as a positive control.
  • the cells were labeled with antibodies to identify IFN-y-secreting cells specific for these peptides ( Figure 4).
  • heterologous (DNA/MVA) immunizations induced better response than homologous (MVA/MVA) immunizations, and vaccine candidate MVA-A-S, increased the responses in comparison with MVA-S, in both heterologous immunizations and homologous immunizations ( Figure 4A).
  • the response against MVA vector (E3 peptide) showed that homologous (MVA/MVA) immunizations induced a better response than heterologous (DNA/MVA) immunizations, with the MVA-A-S/MVA-A-S group inducing the highest levels of IFN-y-secreting cells ( Figure 4B).
  • splenocytes originating from the spleens of each immunization group were stimulated ex vivo with a panel of peptide pools covering the complete sequence of the coronavirus SARS-CoV-2 S-protein (S1 and S2 peptide pools).
  • the samples were stimulated with RPMI as a negative control.
  • the cells were labelled with specific antibodies to identify (CD4 + and CD8 + ) T cell populations and respondent cells (expressing CD107a on the surface of the T cells as an indirect cytotoxicity marker and/or producing cytokines IFN-y, TNF-a, and IL-2) by ICS.
  • the polyfunctionality of the CD4 + and CD8 + T cell-mediated immune response generated by the different immunization groups was analyzed by measuring the simultaneous production pattern of cytokines (IFN-g, TNF-a, and/or IL-2) plus the cytotoxic potential thereof (CD107a as a degranulation marker).
  • the coronavirus SARS- CoV-2 S-protein (S1+S2)-specific cell-mediated responses generated by the different immunization groups were characterized by a high polyfunctionality ( Figure 6).
  • the CD4 + T cell-mediated response was mainly characterized by cells which are capable of carrying out all 4 functions (secreting all 3 cytokines and expressing the marker, CD107a), followed by cells capable of carrying out 3 functions (IFN-g, IL-2, and TNF-a), and cells capable of carrying out 2 functions (IFN-g and TNF-a) ( Figure 6A).
  • the CD8 + T cell-mediated response was mainly characterized by cells which are capable of carrying out 3 functions (CD107a, IFN-g and TNF-a), followed by cells capable of carrying out 4 functions (CD107a, IFN-y, IL-2, and TNF-a) ( Figure 6B).
  • vaccine candidates MVA-COVID19(S) or MVA-A-COVID19(S) are capable of inducing broad and polyfunctional coronavirus SARS-CoV-2 S-protein-specific adaptive CD4 + and CD8 + T cell-mediated immune responses of great magnitude.
  • Tfollicular helper (Tfh) cells was analyzed by means of ICS 11 days after the boost in C57BL/6 mice immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost protocol, as described above.
  • Splenocytes isolated from the spleen were stimulated ex vivo with a combination of the coronavirus SARS-CoV-2 S-protein, together with the S1 and S2 peptide pools covering the complete sequence of said protein.
  • the samples were stimulated with RPMI as a negative control.
  • the cells were labelled with specific antibodies to identify the population of S-specific Tfh cells (CD4 + , CXCR5 + , and PD1 + ) expressing the marker CD40L (marker related to T cell effector functions) and/or producing the cytokines IFN-g and/or IL-21.
  • the polyfunctionality of the Tfh cell-mediated immune response generated by the different immunization groups was analyzed by measuring the simultaneous production pattern of cytokines (IFN-y and/or IL-21 ) plus the marker, CD40L.
  • the coronavirus SARS- CoV-2 S-protein-specific Tfh cell-mediated responses generated by the different immunization groups were characterized by a high polyfunctionality ( Figure 9B).
  • the Tfh cell-mediated response was mainly characterized by cells which are capable of carrying out all 3 functions (secreting the 2 cytokines and expressing the marker, CD40L), followed by cells capable of carrying out 2 functions (IFN-g, IL-21) ( Figure 9B).
  • vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) induce Tfh cell-mediated responses.
  • vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) induced high levels of total IgG, lgG1, lgG2c, and lgG3 antibodies against SARS-CoV-2 S- and RBD proteins, with a Th1 response.
  • the efficacy of the MVA-S vaccine candidate was analysed in K18-hACE2 mice, a mouse model for SARS-CoV-2 infection and lethality.
  • Mice primed and boosted with MVA-WT or unvaccinated, and unchallenged mice were used as a control groups.
  • the challenged K18-hACE2 mice were monitored for change in body weight (Fig.
  • MVA-S vaccine candidate is highly immunogenic in K18-hACE2 humanized mice.
  • Fig 13A K18-hACE2 mice vaccinated with MVA-S induced high titers of S- and RBD-specific binding IgG antibodies, with the two-dose regimen triggering significantly higher levels than the one-dose treatment.
  • the analysis of different IgG isotypes against S and RBD proteins showed higher lgG2c levels than lgG1 , leading to a lgG2c/lgG1 ratio higher than 1 (Fig. 13B), suggestive of a Th1-like protective humoral response (Visciano et al., 2012).
  • the two-dose regimen induced significant higher neutralizing antibody ID50 titers than the one-dose treatment, either using a retrovirus-based pseudoparticle assay (Fig. 13C, left) or a live SARS-CoV-2 microneutralization assay (Fig. 13C, right), with titers superior to the titers of a WFIO International Standard for anti-SARS-CoV-2 human immunoglobulin (NIBSC 20/136) (Fig. 13C).
  • MVA-CoV2-S vaccine candidate is highly efficacious in K18-hACE2 humanized mice, preventing SARS-CoV-2 virus replication, reducing lung pathology and the levels of pro-inflammatory cytokines.
  • MVA-CoV-2 a lethal dose of SARS-CoV-2 [MAD6 isolate, 10 5 PFU/mouse)]
  • MVA-S vaccine candidate in virus replication, three mice per group were sacrificed at days 2 and 4 after SARS-CoV-2 virus challenge, lung tissue samples were collected and processed, and we analysed the presence of SARS-CoV-2 genomic RNA (Fig. 14A) and of live infectious virus (Fig. 14B).
  • Two doses of MVA-S were highly effective to completely prevent SARS-CoV-2 replication and virus yields at 2 and 4 days postchallenge, in comparison with the high levels of SARS-CoV-2 RNA and infectious virus detected in control infected mice (Fig. 14A and 14B), with a significantly remarkable reduction in infectious virus (Fig. 14B).
  • a single dose of MVA-S has also an inhibitory effect reducing the levels of SARS-CoV-2 RNA and of virus loads, mainly at 4 days postchallenge (Fig. 14A and 14B).
  • mice vaccinated with two doses of MVA-S have significantly less lung histopathological inflammatory lesions (Fig. 14C, left panel) and less percentage of lung area with lesions (Fig. 14C, right panel) at 4 days postchallenge, compared to control MVA-WT inoculated mice or mice immunized with one dose of MVA-S.
  • Fig. 14C left panel
  • Fig. 14C right panel
  • MVA-CoV2-S vaccine candidate induced high levels of binding IgG antibodies and neutralizing antibodies in SARS-CoV-2 challenged-K18-hACE2 humanized mice.
  • SARS-CoV-2-specific humoral immunogenicity triggered by the MVA-S vaccine candidate after SARS-CoV-2 challenge.
  • mice vaccinated with two doses of MVA- S lacked an anamnestic response, supporting severe inhibition of virus replication, with similar anti-S and anti-RBD IgG titers at 2, 4 and 15 days postchallenge, contrary to mice immunized with one dose of MVA-S, where an increase in the IgG titers against S and RBD was observed between days 4 and 15 postchallenge (Fig. 15A).
  • the analysis of the levels of different IgG isotypes against S and RBD proteins at days 4 and 15 postchallenge showed higher lgG2c titers than lgG1 , leading to a lgG2c/lgG1 ratio higher than 1 (Fig.
  • mice immunized with one or two doses of MVA-S were protected against a second SARS-CoV-2 infection
  • surviving mice were challenged at week 16 with SARS-CoV-2 (MAD6 isolate, 10 5 PFU/mouse), as indicated (Fig. 16A) and changes in body weight and mortality were evaluated for 6 days postchallenge, time at what all animals were sacrificed.
  • All mice vaccinated with one or two doses of MVA-S did not lose body weight (Fig. 16B) and survived (Fig. 16C), while unvaccinated and challenged mice lost body weight and died at 6 days postchallenge (Fig. 16B and 16C).
  • SARS-CoV-2-specific humoral immunogenicity triggered by one and two doses of MVA-S at day 6 after SARS-CoV-2 reinfection was evaluated in serum samples.
  • Mice immunized with one or two doses of MVA-S elicited similar high titers of S- and RBD-specific binding IgG antibodies (Fig. 17B), with a S- and RBD-specific lgG2c/lgG1 ratio higher than 1 (Fig. 17C), indicative of a Th1 response.
  • similar high titers of SARS-CoV-2 neutralizing antibodies were elicited at day 6 after SARS-CoV-2 reinfection in mice vaccinated with one or two doses of MVA-S (Fig. 17D).
  • mice with MVA-CoV2-S vaccine candidate induces long-term strong, broad, and polyfunctional SARS-CoV-2 S-specific CD4+ and CD8+ T-cell memory immune responses.
  • MVA-S induce long-term S- specific T-cell immune responses.
  • groups of C57BL/6 mice were immunized twice with MVA-S (intramuscularly, 1 x 10 7 PFU/mouse) at weeks 0 and 2 following a homologous MVA-S/MVA-S prime/boost immunization regimen, as shown in materials and methods, and 6 months later animals were sacrificed.
  • SARS-CoV-2 S1 and S2 peptide pools spanning the entire S protein, to evaluate by an enzyme-linked immunosorbent spot (ELISpot) assay the induction of SARS-CoV-2 S-specific gamma interferon (IFNy)-secreting cells; or by an intracellular cytokine staining (ICS) assay the induction of SARS-CoV-2 S-specific CD4+ and CD8+ T cells expressing CD107a, IFNy, tumor necrosis factor alpha (TNFa), and/or interleukin 2 (IL-2).
  • ELISpot enzyme-linked immunosorbent spot
  • IFNy gamma interferon-secreting cells
  • ICS intracellular cytokine staining
  • mice vaccinated with MVA-S elicited high levels of long-term IFNy-secreting cells against a mixture of SARS-CoV-2 S1+S2 peptide pools (Fig. 18A).
  • long-term S-specific CD4+ and CD8+ T-cell responses were broad and directed mainly against the S1 peptide pool (Fig. 18C).
  • mice were intranasally challenged with 2 c 10 5 TCID50 of SARS-CoV-2 (Fig. 20). Flamsters primed and boosted with MVA-WT were used as a control group. The challenged hamsters were monitored for change in body weight previously to the challenge and postchallenge (Fig. 21). All animals gain weight similarly, previous to the challenge (data not shown). After challenge, all hamsters at 2 days post-infection lost some body weight, but vaccinated animals recovered faster than control at day 4 (Fig. 21 ).
  • SARS-CoV-2 virus replication in lungs is prevented by vaccination of hamsters with MVA-S.
  • MVA-S MVA-S.
  • mice were intranasally and intratracheally challenged with 1 c 10 6 PFU of SARS-CoV-2 (Fig. 27).
  • serum samples were obtained, previously to the challenge.
  • the challenged macaques were monitored for 14 days, and at different days post-challenge several samples were taken. Animals were sacrificed at day 14 post challenge.
  • PBMCs were obtained at day 14 post-boost and were stimulated with SARS-CoV-2 S1 and S2 peptide pools, spanning the entire S protein, to evaluate by an enzyme-linked immunosorbent spot (ELISpot) assay the induction of SARS-CoV-2 S-specific IFNy- and IL-4-secreting cells.
  • ELISpot enzyme-linked immunosorbent spot
  • SARS-CoV-2 virus replication in throat swabs and BAL is controlled by vaccination of rhesus macaques with MVA-S.
  • MVA-S vaccine in virus replication To analyze the effect of the MVA-S vaccine in virus replication, at different days after SARS-CoV-2 virus challenge, throat swabs and BAL samples from rhesus macaques were collected, RNA was extracted and analyzed by RT-qPCR for SARS-CoV-2 replication (Fig. 29). The peak of RNA was obtained at day 2 post-challenge, and the detailed analysis at this timepoint showed that vaccination with MVA-S significantly reduced SARS-CoV-2 replication in throat swabs and BAL samples (Fig. 29).
  • mice were immunized twice with MVA-S (intranasally, 1 x 10 7 PFU/mouse) at weeks 0 and 2 following a homologous MVA-S/MVA-S prime/boost immunization regimen, as shown in materials and methods, and 11 days post-boost animals were sacrificed.
  • mice vaccinated with MVA-S and MVA-A-S elicited potent levels of S-specific CD4+ and CD8+ T-cell responses, which were higher in lung cells than in splenocytes (Fig. 30A and 30B), and with a higher overall response mediated by the CD8+ T-cell repertoire in both cells (Fig. 30A and 30B).
  • mice vaccinated with two doses of MVA-S and MVA-A-S by intranasal route induced high titers of S- and RBD-specific binding IgG antibodies in serum samples (Fig. 30C), as well as IgA antibodies in BAL samples (Fig. 30D).
  • high titers of SARS-CoV-2 neutralizing antibodies were induced (Fig. 30E).
  • the SARS-CoV-2 S sequence inserted in vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) described in the present examples was synthetized by GeneArt and was human codon optimized. Previous to the beginning of the S sequence there is a Kozak sequence (GCCACC).
  • GCCACC Kozak sequence
  • GenBank: MN908947.3 The sequence derived from Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1 , GenBank: MN908947.3, https://www.ncbi.nlm.nih.qov/nuccore/MN908947
  • SEQ ID NO 1 The S nucleotide sequence reads as follows (SEQ ID NO 1 ):
  • VNNATNVVIKVCEFQFCNDPFL GVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFL
  • the M protein sequence reads as follows (SEQ ID NO 6): MADSNGTITVEELKKLLEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPVT
  • RBD nucleotide sequence reads as follows (SEQ ID NO 7):
  • the RBD protein sequence reads as follows (SEQ ID NO 8):
  • N nucleotide sequence reads as follows (SEQ ID NO 9):
  • N protein sequence reads as follows (SEQ ID NO 10):
  • a vaccine composition comprising a. an immunologically effective amount of a modified vaccinia virus Ankara (MVA) vector comprising at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype, optionally together with a pharmaceutically acceptable carrier, diluent and/or additive; and/or b.
  • MVA modified vaccinia virus Ankara
  • MVA modified vaccinia virus Ankara
  • said MVA vector is further modified by deletion of the MVA immunomodulatory genes C6L, K7R, and A46R, optionally together with a pharmaceutically acceptable carrier, diluent and/or additive; wherein the nucleotide sequence encoding an antigenic protein of at least one SARS-CoV-2 virus subtype is inserted into the thymidine kinase (TK) locus of the MVA genome; and wherein the MVA vector regulates the expression of the nucleic acid encoding the antigenic protein and is operably linked to said nucleotide sequence.
  • TK thymidine kinase
  • MVA vector further comprises an antigenic protein selected from the any of the group of structural proteins E (envelope), receptor-binding domain (RBD), N (nucleocapsid) or M (membrane) of SARS-CoV-2.
  • E envelope
  • RBD receptor-binding domain
  • N nucleocapsid
  • M membrane membrane
  • the MVA used for generating the recombinant virus is a MVA virus or a derivative having the capability of reproductive replication in vitro in chicken embryo fibroblasts (CEF) cells and in established chick cells DF-1 , but no capability of reproductive replication in human cervix adenocarcinoma cell line FleLa.
  • the modified (MVA) vector is the MVA as defined in claim 1a).
  • the vaccine further comprises one or more pharmaceutically acceptable carriers, additives, antibiotics, preservatives, adjuvants, diluents and/or stabilizers.
  • a vaccine combination comprising: c. a first composition comprising an immunologically effective amount of a modified vaccinia virus Ankara (MVA) vector as defined in any of the precedent clauses; and d. a second composition comprising an immunologically effective amount of a modified vaccinia virus Ankara (MVA) vector as defined in any of the precedent clauses; wherein one of the compositions is a priming composition and the other composition is a boosting composition.
  • a first composition comprising an immunologically effective amount of a modified vaccinia virus Ankara (MVA) vector as defined in any of the precedent clauses
  • a second composition comprising an immunologically effective amount of a modified vaccinia virus Ankara (MVA) vector as defined in any of the precedent clauses
  • one of the compositions is a priming composition and the other composition is a boosting composition.
  • a vaccine combination comprising: a. a first composition comprising an immunologically effective amount of a modified vaccinia virus Ankara (MVA) vector as defined in any of clauses 1 to 8; and b. a second composition comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype; wherein one of the compositions is a priming composition and the other composition is a boosting composition.
  • MVA modified vaccinia virus Ankara
  • the vaccine combination comprising: a. a first composition comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype; and b. a second composition comprising an immunologically effective amount of a modified vaccinia virus Ankara (MVA) vector as defined in any of clauses 1 to 8; wherein the first compositions is a priming composition and the second composition is a boosting composition.
  • a first composition comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype
  • a second composition comprising an immunologically effective amount of a modified vaccinia virus Ankara (MVA) vector as defined in any of clauses 1 to 8; wherein the first compositions is a priming composition and the second composition is a boosting composition.
  • the vaccine composition according to clauses 1-8 for use in generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject, wherein said composition is used for priming said immune response and/or boosting said immune response and wherein the MVA is capable of producing at least the structural protein S of SARS-CoV-2, preferably of SEQ ID NO 2, in the subject to be treated.
  • the vaccine combination according to clauses 9 to 11 for use in generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject, wherein said combination is capable of producing at least the structural protein S of SARS-CoV-2, preferably of SEQ ID NO 2, in the subject to be treated.
  • the vaccine combination according to clause 11 for use in generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject, wherein said combination is capable of producing at least the structural protein S of SARS-CoV-2, in the subject to be treated.
  • the MVA is capable of producing any of the group of structural proteins S, E, receptor-binding domain (RBD), N and/or M of SARS- CoV-2, or any combination thereof, in the subject to be treated.
  • composition or combination is administered via the intramuscular route.
  • kits comprising one, preferably two, or more doses of any of the vaccine compositions as defined in any of clauses 1 to 8, or the vaccine combination of any of clauses 9 to 13.
  • a kit comprising an immunologically effective amount of a MVA vector according to any of clauses 1 to 8 in a first vial or container for a first administration (priming) and in a second vial or container for a second administration (boosting).
  • a kit comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype, preferably the structural protein S of SARS-CoV- 2, in a first vial or container for a first administration (priming), and an immunologically effective amount of a MVA vector according to any of clauses 1 to 8 in a second vial or container for a second administration (boosting).
  • a kit comprising an immunologically effective amount of a MVA vector according to any of clauses 1 to 8, in a first vial or container for a first administration (priming), and an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype, , preferably the structural protein S of SARS-CoV-2, in a second vial or container for a second administration (boosting).
  • kit according to any of clauses 23 to 26, comprising in a third, fourth or further vial or container a MVA vector according to any of clauses 1 to 8 for a third, fourth or further administration.
  • kit according to clause 28 wherein the combination of compositions contained in the vials is capable of producing at least the structural protein S of SARS-CoV- 2, in the subject to be treated, and generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof.
  • said method comprises administering any of the vaccine compositions according to any of clauses 1 to 8 for priming said immune response and/or boosting said immune response.
  • compositions or combinations of compositions contained in the vials are administered via the intramuscular route.
  • said compositions or combinations of compositions contained in the vials are administered via the intramuscular route.
  • the method according to any of clause 39 to 41 , wherein the boosting composition is administered at least 2 weeks after administering the priming composition.
  • the method according to any of clauses 39 to 41 wherein the boosting composition is administered 2-12 weeks, preferably 2, 3 or 4 weeks, after administering the priming composition.
  • a method of inducing an enhanced immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject comprising administering to the subject any of the vaccine compositions according to any of clauses 1 to 8 or any of the vaccine combinations according to any of clauses 9 to 13.
  • the method according to claim 45 wherein said compositions or combinations of compositions contained in the vials are administered via the intramuscular route.

Abstract

The present invention is directed to a recombinant modified vaccinia virus Ankara (MVA), which carries one or more nucleic acid sequence/s coding for an antigenic protein of SARS-CoV-2 useful for vaccinating against COVID19. The present invention is further directed to a vaccine composition containing said recombinant MVA as well as to a method for enhancing T cell and humoral immune responses in a mammal against COVID19.

Description

MVA-BASED VACCINE AGAINST COVID-19 EXPRESSING SARS-CoV-2
ANTIGENS
TECHNICAL FIELD OF THE INVENTION The present invention is directed to a recombinant modified vaccinia virus Ankara (MVA), which carries a nucleic acid sequence coding for an antigenic protein useful for vaccinating against SARS-CoV-2. The present invention is further directed to a vaccine composition containing said recombinant MVA as well as to a method for generating a protective immune response in a mammal against SARS-CoV-2.
BACKGROUND OF THE INVENTION
The emergence of the strain of coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and its high impact on global health have made imperative the development of safe and effective vaccines for this lethal strain, the causal agent of COVID-19 (coronavirus disease 2019). SARS-CoV-2 now adds to the list of coronavirus diseases that have threatened global health, along with the SARS (severe acute respiratory syndrome) and MERS (Middle East respiratory syndrome) coronaviruses that emerged in 2002/2003 and 2012, respectively. As of June 2020, no vaccine is commercially available for these coronavirus strains. Nevertheless, the knowledge obtained from the vaccine development efforts for SARS and MERS can be of high value for COVID-19.
Here, we provide an effective vaccine, as well as vaccine regimens, against SARS-CoV- 2. BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Design and generation and in vitro characterization of vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S). (A) Genome map of vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S), expressing the coronavirus SARS-CoV-2 S- protein (this vector is also referred indistinctively as MVA-CoV2-S or MVA-S). The different regions of the MVA genome are indicated with capital letters and the central conserved region and the left and right terminal regions are shown. Below the map, the deleted or fragmented MVA genes are depicted as black boxes. The MVA genes C6L, K7R, and A46R which have been deleted in one of the parental viruses (MVA-GFP), used for generating the vaccine candidate MVA-A-COVID19(S), are indicated. The coronavirus SARS-CoV-2 S genes inserted within the MVA TK viral locus (J2R) of MVA-WT or MVA- GFP parental viruses (to generated MVA-COVID19(S) and MVA-A-COVID19(S), respectively) and driven by the sE/L virus promoter are indicated. TK-L: TK left; TK-R: TK right. (B) PCR analysis of MVA TK locus. Viral DNA was extracted from DF-1 cells that were not infected (mock) or infected with 5 PFU/cell of vaccine candidates MVA- COVID19(S) (referred to as MVA-S) and MVA-A-COVID19(S) (referred to as MVA-A-S), MVA-GFP, or MVA-WT. Oligonucleotides which hybridize in the flanking regions of the TK locus were used for the PCR analysis of the coronavirus SARS-CoV-2 S gene inserted in the TK locus. A molecular weight marker (1 kb) with the corresponding bp sizes is indicated on the left side of the images, and the amplified DNA products are indicated with arrows on the right side. (C) Expression of the coronavirus SARS-CoV-2 S-protein. DF-1 cells were left uninfected (mock) or infected for 4 or 24 hours (in the absence or presence of tunicamycin) at 5 PFU/cell with vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) and MVA-WT. At 4 and 24 hours post-infection cells were lysed in Laemmli 1X + b-mercaptoethanol buffer, fractionated by 7% SDS-PAGE, and the presence of the S protein was analyzed by Western blotting using a mouse monoclonal antibody against SARS-CoV (against SARS-CoV S2 region) or a rabbit polyclonal antibody against SARS-CoV-2 (against SARS-CoV-2 S1 region). A rabbit anti-VACV E3 protein antibody was used as a loading control for the amount of viral proteins. The arrows on the right side of the images indicate the S protein (glycosylated or unglycosylated), together with the VACV E3 protein, whereas the sizes of standards (in kilodaltons [kDa]) (Precision Plus protein standards; Bio-Rad Laboratories) are indicated on the left side. (D) Kinetics of expression of the coronavirus SARS-CoV-2 S protein in cell extracts
(pellet) and supernatants (SN) from cells infected with vaccine candidates MVA- COVID19(S) and MVA-A-COVID19(S). DF-1 cells were left uninfected (mock) or infected with vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S), and MVA-WT, at 5 PFU/cell. The cell extracts and the supernatants were collected 4 and 24 hours post- infection (the supernatants were first precipitated with 10% TCA), lysed in Laemmli 1X + b-mercaptoethanol buffer, fractioned with 7% SDS-PAGE, and analyzed by Western blotting using a mouse monoclonal antibody against SARS-CoV (against SARS-CoV S2 region) or a rabbit polyclonal antibody against SARS-CoV-2 (against SARS-CoV-2 S1 region). The arrows on the right side indicate the S protein. The sizes of standards (in kilodaltons [kDa]) (Precision Plus protein standards; Bio-Rad Laboratories) are indicated on the left.
Figure 2. In vitro characterization of vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S). (A) (A) Expression of SARS-CoV-2 S protein under reducing and nonreducing conditions. DF-1 cells were infected with MVA-S and MVA-A-S vaccine candidates and at 24 hpi, cells were lysed under reducing (Laemmli 1X + b- mercaptoethanol (BME) buffer) or nonreducing conditions (Laemmli 1X - b- mercaptoethanol buffer), fractionated by 7% SDS-PAGE, and analyzed by Western blotting using a mouse monoclonal antibody against SARS-CoV (against SARS-CoV S2 region) or a rabbit polyclonal antibody against SARS-CoV-2 (against SARS-CoV-2 S1 region). Arrows on the right indicates the SARS-CoV-2 S protein in the form of monomers or oligomers. The sizes of standards (in kilodaltons [kDa]) (Precision Plus protein standards; Bio-Rad Laboratories) are indicated on the left. (B) Viral growth kinetics of vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S). DF-1 cell monolayers were infected with MVA-WT, MVA-GFP, and vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) at 0.01 PFU/cell. Cells were collected at different times post infection (0, 24, 48, and 72 hours) and the viral titers in the cell lysates were quantified by a plaque immunostaining assay with anti-VACV antibodies. The means and standard deviations of the results obtained from two independent experiments are shown. (C) Genetic stability of vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S). DF- 1 cell monolayers were infected with the P2 stocks of vaccine candidates MVA- COVID19(S) and MVA-A-COVID19(S) at a low MOI for 9 serial passages. Then, DF-1 cells were the left uninfected {mock) or infected with vaccine candidates MVA- COVID19(S) and MVA-A-COVID19(S) from the different passages, or with MVA-WT, and the cells were lysed at 24 hours post-infection in Laemmli 1X + b-mercaptoethanol buffer, fractionated by 7% SDS-PAGE, and analyzed by Western Blotting using a rabbit polyclonal antibody against SARS-CoV-2 (against SARS-CoV-2 S1 region). The arrows on the right side indicate the position of the S protein. The sizes of standards (in kilodaltons [kDa]) (Precision Plus protein standards; Bio-Rad Laboratories) are indicated on the left.
Figure 3. Immunization schedule of the heterologous (DNA/MVA) and homologous (MVA/MVA) prime/boost immunization protocols performed in C57BL/6 mice immunized with vaccine candidates MVA-COVID19(S) or MVA-A-COVID19(S). Six
C57BL/6 mice per group were inoculated at days 0 and 15 (prime/boost) with the different indicated vaccine candidates (either DNA-S or MVAs in the prime or the different MVAs in the boost). Immunization groups are indicated (n=6 mice per group), together with the time points at which animals were immunized (dose and route of administration are shown) and sacrificed to analyze the adaptive SARS-CoV-2-specific T cell and humoral immune responses. The mice were sacrificed 11 days after the boost (day 26) and the following was obtained: i) spleens of the mice for studying adaptive T cell-mediated immune responses against SARS-CoV-2 by means of ELISpot and ICS, and ii) serum samples originating from blood for studying adaptive humoral immune responses against SARS-CoV-2, by means of ELISA and a neutralization assay. PFU: Plaque forming units; i.m.: intramuscular.
Figure 4. ELISpot analysis of the adaptive SARS-CoV-2 S-specific cell-mediated immune responses induced by vaccine candidates MVA-COVID19(S) or MVA-D- COVID19(S) in C57BL/6 mice immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost immunization protocol. Six mice per group were sacrificed at 11 days post-boost, and the splenic SARS-CoV-2-specific cells secreting IFN-y were evaluated by an ELISpot assay, as described in Materials and Methods. Samples from each group of mice were analysed in triplicate, and the bars indicate the mean values and the standard deviation. The responses obtained have been obtained by subtracting the RPMI values for each condition and are represented as IFN- Y positive cells per million of splenocytes. (A) Magnitude of SARS-CoV-2-specific cells secreting IFN-g and directed against SARS-CoV-2 S1 and S2 peptide pools. (B) Magnitude of VACV E3-specific cells secreting IFN-g.
Figure 5. SARS-CoV-2 S-specific adaptive CD4+ (left) and CD8+ (right) T cell- mediated immune responses induced by vaccine candidates MVA-COVID19(S) or MVA-A-COVID19(S) in C57BL/6 mice immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost immunization protocol. CD4+ and CD8+ T cell- mediated responses in the splenocytes of mice (n=6) immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost protocol were analysed by ICS, as described in Materials and Methods 11 days after the boost. The data is depicted with its corresponding confidence intervals. (A) The total magnitude of SARS-CoV-2 S (S1 + S2)- specific adaptive CD4+ T cell-mediated (left) and CD8+ T cell-mediated (right) immune responses. The y-axis shows the percentage measured as the sum of SARS-CoV-2 S (mixture of S1 + S2 peptide pools)-specific T cells expressing CD107a and/or producing IFN-Y and/or TNF-a and/or IL-2. (B) Breadth of SARS-CoV-2-specific CD4+ and CD8+ T cell adaptive immune responses. S1 or S2 peptide pool-specific adaptive CD4+ T cell- mediated (left) and CD8+ T cell-mediated (right) immune responses. The y-axis shows the percentage measured as the sum of SARS-CoV-2 S1 or S2 peptide pool-specific T cells expressing CD107a and/or producing IFN-g and/or TNF-a and/or IL-2. (C) Adaptive CD4+ T cell-mediated (left) and CD8+ T cell-mediated (right) immune responses shown as the percentage of SARS-CoV-2 S (S1 + S2)-specific cells expressing CD107a or producing IFN-g or TNF-a or IL-2.
Figure 6. Polyfunctionality of SARS-CoV-2 S-specific adaptive CD4+ and CD8+ T cell-mediated immune responses induced by vaccine candidates MVA-COVID19(S) or MVA-A-COVID19(S) in C57BL/6 mice immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost immunization protocol. Six mice per group were sacrificed at 11 days post-boost, and the splenic SARS-CoV-2-specific CD4+ and CD8+ T cell immune responses were analysed by ICS, as described in Materials and Methods. Polyfunctional profiles of total SARS-CoV-2-specific CD4+ (A) and CD8+ (B) T cell adaptive immune responses directed against a mixture of S1 , and S2 SARS-CoV-2 S peptide pools. All of the possible combinations of responses are shown on the x axis, while the percentages of T cells expressing CD107a and/or IFN-y and/or TNF-a and/or IL-2 against S1 plus S2 peptide pools are shown on the y axis.
Figure 7. Phenotype profile of SARS-CoV-2 S-specific adaptive CD4+ and CD8+ T cells induced by vaccine candidates MVA-COVID19(S) or MVA-A-COVID19(S), in C57BL/6 mice immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost immunization protocol. Splenocytes of mice (n=6) immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost protocol were obtained 11 days after the boost, and SARS-CoV-2 S (S1 +S2 peptide pools)-specific CD4+ T cell-mediated (A) and CD8+ T cell-mediated (B) immune responses were analysed by ICS. The y-axis indicates the percentage of SARS-CoV-2 S (S1 +S2)-specific CD4+ and CD8+ central memory T cells (TCM; CD127+, CD62L+), effector memory T cells (TEM; CD127+, CD62L ), and effector T cells (TE; CD127 , CD62L ) expressing CD107a and/or IFN-g and/or TNF-a and/or IL-2 (total response). The data is depicted with its corresponding confidence intervals.
Figure 8. SARS-CoV-2 S-specific adaptive CD4+ T follicular helper (Tfh) cell- mediated immune responses induced by vaccine candidates MVA-COVID19(S) or M VA-A-COVI D 19(S), in C57BL/6 mice immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost immunization protocol. Splenocytes of mice (n=6) immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime- boost protocol were obtained 11 days after the boost, and SARS-CoV-2 S (S protein plus
51 +S2 peptide pools)-specific CD4+ Tfh cell-mediated immune responses were analysed by ICS. The data is depicted with its corresponding confidence intervals. (A) Magnitude of total SARS-CoV-2-specific adaptive CD4+ Tfh cell immune responses directed against SARS-CoV-2 S protein plus S1 and S2 peptide pools. Percentages of CD4+ Tfh cells (CXCR5+, PD1+) expressing CD40L and/or producing IFN-y and/or IL-21 against a mixture of SARS-CoV-2 S protein plus S1, and S2 SARS-CoV-2 S peptide pools are represented. (B) Percentages of total SARS-CoV-2-specific CD4+ Tfh cells expressing CD40L or producing IFN-g or IL-21 against a mixture of SARS-CoV-2 S protein plus S1 , and S2 SARS-CoV-2 S peptide pools. (C) Polyfunctionality of total SARS-CoV-2-specific adaptive CD4+ Tfh cells directed against a mixture of SARS-CoV-2 S protein plus S1 and
52 peptide pools. All of the possible combinations of responses are shown on the x axis, while the percentages of T cells expressing CD40L and/or IFN-g and/or IL-21 against
SARS-CoV-2 S protein plus S1 and S2 peptide pools are shown on the y axis.
Figure 9. SARS-CoV-2 S-specific and RBD-specific humoral immune responses induced by vaccine candidates MVA-COVID19(S) or MVA-A-COVID19(S) in C57BL/6 mice immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost immunization protocol. Groups of C57BL/6 mice (n=6) were immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost protocol. Eleven (11) days after the last immunization, the mice were sacrificed, serum samples were collected, and the levels of total coronavirus SARS-CoV-2 S-specific and RBD- specific IgG antibodies (A) or antibodies of subclasses lgG1, lgG2c, and lgG3 (B) in pooled sera from mice derived from each immunization group were analyzed by ELISA. Mean absorbance values (measured at 450 nm) and standard deviations of duplicate pooled serum dilutions are represented.
Figure 10. Levels of SARS-CoV-2-specific neutralizing antibodies induced by vaccine candidates MVA-COVID19(S) or MVA-A-COVID19(S) in C57BL/6 mice immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime- boost immunization protocol. Groups of C57BL/6 mice (n=6) were immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost protocol. Eleven (11) days after the last immunization, the mice were sacrificed, serum samples were collected, and their capacity to inhibit the entry in VeroE6 cells of lentivirus-based pseudoparticles expressing the coronavirus SARS-CoV-2 S protein was analyzed. RPMI was used as a negative control. Each dot represents the mean luciferase units of triplicates obtained for each individual mouse serum sample from each immunization group, and analyzed at a 1/50 dilution. Mean values and SEM obtained for each immunization group is represented.
Figure 11. Generation of recombinant MVA viruses. After infection-transfection in DF-
1 cells, three rounds of purification were performed selecting blue plaques (LacZ+), followed by another three rounds selecting colorless plaques (without a marker) to finally generate the recombinant MVA virus. Figure 12. MVA-COVID19(S) protects humanized K18-hACE2 mice from SARS-CoV-
2 infection. (A) Efficacy study schedule. K18-hACE2 mice (n=11 per group) were immunized with one or two doses of 1 x 107 PFU of MVA-S or MVA-WT by intramuscular route at weeks 0 and 4. At 10 days after the last immunization (day 38) mice were bleed and serum samples were obtained. At week 9 (day 63) mice were intranasally (i.n.) challenged with 1 x 105 PFU of SARS-CoV-2 (MAD6 isolate). MVA-WT-inoculated (group 3) and unvaccinated (group 4) mice were also i.n. challenged with SARS-CoV-2; unvaccinated and unchallenged mice were used as a negative control (group 5). At days 2 and 4 post-challenge 3 mice per group were sacrificed and lungs and serum samples were collected as indicated. In surviving mice, serum was collected at day 15 post- challenge. All mice were monitored daily for change in the percentage of initial body weight (B) and survival (C) during 14 days after virus challenge.
Figure 13. MVA-S vaccine candidate is highly immunogenic in prechallenged K18- hACE2 humanized mice. (A) Titers of IgG antibodies specific for the S (left) and RBD (right) proteins. Titers were determined by ELISA in individual mouse serum samples collected from immunized K18-hACE2 mice at 10 days after the last immunization (prechallenge) and were calculated as the serum dilution at which the absorbance at 450 nm was at least three times higher than a naive serum value in each of the indicated immunization regimens. Mean values and SEM is represented for each immunization group. The dashed line represents the detection limit. (B) Ratio of lgG2c/lgG1 specific for the S and RBD proteins. The ratio was calculated by ELISA in pooled mice serum samples collected 10 days after the last immunization (prechallenge), and were calculated from the mean optical density at 450 nm obtained for lgG2c and lgG1 at a dilution 1/9765. A ratio >1 indicates a Th1 response. (C) Neutralizing antibody titers. The ID50 titers were evaluated in individual mouse serum samples collected from immunized K18-hACE2 mice at 10 days after the last immunization (prechallenge) using retrovirus- based pseudoparticles expressing the SARS-CoV-2 S protein (left) or live-virus microneutralization (right) assays. The ID50 titers in the retrovirus-based pseudoparticle assay were determined as the highest serum dilution which resulted in a 50% reduction of luciferase units compared with pseudotyped viruses not incubated with serum. The ID50 titers in the live-virus microneutralization assay were calculated as the reciprocal dilution resulting in 50% inhibition of cell death. Mean ID50 values and SEM for each immunization group is represented. Dotted line represented the detection limit. The NIBSC 20/136 international standard containing pooled plasma obtained from eleven individuals recovered from SARS-CoV-2 infection is included.
Fig. 14. MVA-S vaccine candidate is highly efficacious in K18-hACE2 humanized mice, preventing SARS-CoV-2 virus replication, reducing lung pathology and the levels of pro-inflammatory cytokines. (A) SARS-CoV-2 RNA. Detected by RT-qPCR targeting genomic viral RdRp gene with normalization to 28S in lungs obtained at 2 (n = 3) and 4 (n = 3) days post-challenge from mice vaccinated and infected as in Fig. 12A. Mean RNA levels (in arbitrary units [A.U.]) and SEM from duplicates of each lung sample is represented for each immunization group; relative values are referred to uninfected mice.
(B) SARS-CoV-2 infectious virus. Detected by a plaque assay in Vero E6 cells in homogenate lung samples obtained at 2 (n = 3) and 4 (n = 3) days postchallenge from mice vaccinated and infected as in Fig. 12A. Mean levels (in PFU/g of lung tissue) and SEM from triplicates of each lung sample is represented for each immunization group.
(C) Lung inflammation scores and percentage of lung area with lesions. Analyzed in lung samples (n = 3) obtained at 4 days postchallenge from mice vaccinated and infected as in Fig. 12A. Mean values and SEM is represented for each immunization group. (D) mRNA levels of proinflammatory cytokines. Detected by RT-qPCR with normalization to 28S in lungs obtained at 2 (n = 3) and 4 (n = 3) days postchallenge from mice vaccinated and infected as in Fig. 12A. Mean RNA levels (in A.U.) and SEM from duplicates of each lung sample is represented for each immunization group; relative values are referred to uninfected mice.
Fig. 15. MVA-S vaccine candidate is highly immunogenic in challenged K18-hACE2 humanized mice. (A) Titers of IgG antibodies specific for the S (left) and RBD (right) proteins. Titers were determined by ELISA in individual mouse serum samples collected at days 2, 4 and 15 post-challenge from immunized K18-hACE2 mice and were calculated as the serum dilution at which the absorbance at 450 nm was at least three times higher than a naive serum value in each of the indicated immunization regimens. Mean values and SEM is represented for each immunization group. The dashed line represents the detection limit. (B) Ratio of lgG2c/lgG1 specific for the S and RBD proteins. The ratio was calculated by ELISA in pooled mice serum samples collected at days 4 and 15 post challenge, and were calculated from the mean optical density at 450 nm obtained for lgG2c and lgG1 at a dilution 1/9765. A ratio >1 indicates a Th1 response. (C) Neutralizing antibody titers. The ID50 titers were evaluated in individual mouse serum samples collected from immunized K18-hACE2 mice at 4 and 15 days post-challenge using retrovirus-based pseudoparticles expressing the SARS-CoV-2 S protein, and were determined as the highest serum dilution which resulted in a 50% reduction of luciferase units compared with pseudotyped viruses not incubated with serum. Mean ID50 values and SEM for each immunization group is represented. Dotted line represented the detection limit. Fig. 16. Re-challenge study in humanized K18-hACE2 mice. (A) Female K18-hACE2 mice (n = 5 per group) were immunized with one or two doses of 1 c 107 PFU of MVA-S by the intramuscular (i.m.) route at weeks 0 and 4. At week 9 and 16, mice were intranasally challenged with 1 c 105 PFU of SARS-CoV-2 (MAD6 isolate). Unvaccinated mice (n = 5) were also intranasally challenged with 1 c 105 PFU of SARS-CoV-2 at week 16, and were used as a control. The challenged mice were monitored for change of body weights (B) and mortality (C) for 6 days. At day 6 post-challenge mice were sacrificed and lungs and serum samples were collected as indicated. (D) SARS-CoV-2 RNA. Detected by RT-qPCR targeting genomic viral RdRp gene with normalization to 28S in lungs obtained at day 6 days post-rechallenge from mice vaccinated and infected as in panel A. Mean RNA levels (in A.U.) and SEM from duplicates of each lung sample is represented for each immunization group; relative values are referred to uninfected mice. (E) SARS-CoV-2 infectious virus. Detected by a plaque assay in Vero E6 cells in homogenate lung samples obtained at day 6 days post-rechallenge from mice vaccinated and infected as in panel A. Mean levels (in PFU/g of lung tissue) and SEM from triplicates of each lung sample is represented for each immunization group. Mean values and SEM is represented for each immunization group.
Fig. 17. MVA-S vaccine candidate is highly immunogenic in re-challenged K18- hACE2 humanized mice. (A) mRNA levels of proinflam matory cytokines. Detected by RT-qPCR with normalization to 28S in lungs obtained at day 6 days post-rechallenge from mice vaccinated and infected as in Fig. 16A. Mean RNA levels (in A.U.) and SEM from duplicates of each lung sample is represented for each immunization group; relative values are referred to uninfected mice. (B) Titers of IgG antibodies specific for the S (left) and RBD (right) proteins. Titers were determined by ELISA in individual mouse serum samples collected at day 6 post-rechallenge from mice vaccinated and infected as in Fig. 16A and were calculated as the serum dilution at which the absorbance at 450 nm was at least three times higher than a naive serum value in each of the indicated immunization regimens. Mean values and SEM is represented for each immunization group. The dashed line represents the detection limit. (C) Ratio of lgG2c/lgG1 specific for the S and RBD proteins. The ratio was calculated by ELISA in pooled mice serum samples collected at day 6 post-rechallenge, and were calculated from the mean optical density at 450 nm obtained for lgG2c and lgG1 at a dilution 1/9765. A ratio >1 indicates a Th1 response. (D) Neutralizing antibody titers. The ID50 titers were evaluated in individual mouse serum samples collected from immunized K18-hACE2 mice at day 6 post-rechallenge using retrovirus-based pseudoparticles expressing the SARS-CoV-2 S protein, and were determined as the highest serum dilution which resulted in a 50% reduction of luciferase units compared with pseudotyped viruses not incubated with serum. Mean ID50 values and SEM for each immunization group is represented. Dotted line represented the detection limit. Fig. 18. MVA-S elicit long-lasting SARS-CoV-2 S-specific T-cell immune responses in immunized mice. (A) Magnitude of SARS-CoV-2 S-specific cell responses. Cells secreting IFN-y per million of splenocytes and directed against S1 + S2 peptide pools in mice immunized with the indicate prime/boost protocols. IFN-g was evaluated by an ELISpot assay from a pool of splenocytes derived from 5 immunized mice per group at 6 months post-boost, as described in Materials and Methods. Samples were analyzed in triplicate; bars show the triplicate mean values and the standard deviation. (B) Magnitude of SARS-CoV-2 S-specific CD4+ and CD8+ T-cell immune responses evaluated at 6 months post-immunization. Percentages of CD4+ or CD8+ T cells expressing CD107a and/or producing IFN-g and/or TNF-a and/or IL-2 against a mixture of S1 and S2 peptide pools in mice immunized with the indicative regimen. Cell percentages were determined by ICS from splenocyte pools. (C) SARS-CoV-2 S-specific T-cell immune responses against S1 and S2 regions. Percentages of S1- or S2-specific CD4+ and CD8+ T cells determined as in panel B. (D) Percentages of Naive (CD127-/CD62L-), T central memory (Tern, CD127+/CD62L+), T effector memory (Tern, CD127+/CD62L ), and T effector (Te, CD127 CD62L ) CD8+ T cells specific for S1 and S2 peptide pools, and expressing any of the markers CD107a, IFN-g, TNF-a and IL-2. T-cell responses were analyzed by ICS in splenocyte pools. (E) Polyfunctional profiles (based on expression of selected markers CD107a, IFN-Y, TNF-a and IL-2) of total SARS-CoV-2-specific CD4+ or CD8+ T-cell immune responses directed against a mixture of S1 and S2 peptide pools. T-cell responses were analyzed by ICS in splenocyte pools. The response profiles are shown in the X axis, and the percentages of T cells for each of the immunization regimens in the Y axis. The pie charts summarize the percentage of S-specific T cells exhibiting 1, 2, 3, or 4 markers, which are shown color-coded. Fig. 19. MVA-S elicit long-lasting SARS-CoV-2 S-specific humoral immune responses in immunized mice. (A) Titers of IgG antibodies specific for the S (left) and RBD (right) proteins. Titers were determined by ELISA in individual mouse serum samples collected at 6 months post-boost from immunized C57BL/6 mice and were calculated as the serum dilution at which the absorbance at 450 nm was at least three times higher than a naive serum value in each of the indicated immunization regimens. Mean values and SEM is represented for each immunization group. The dashed line represents the detection limit. (B) Ratio of lgG2c/lgG1 specific for the S and RBD proteins. The ratio was calculated by ELISA in pooled mice serum samples collected at 6 months post-boost, and were calculated from the mean optical density at 450 nm obtained for lgG2c and lgG1 at a dilution 1/9765. A ratio >1 indicates a Th1 response. (C) Neutralizing antibody titers. The ID50 titers were evaluated in individual mouse serum samples at 6 months post-boost using retrovirus-based pseudoparticles expressing the SARS-CoV-2 S protein, and were determined as the highest serum dilution which resulted in a 50% reduction of luciferase units compared with pseudotyped viruses not incubated with serum. Mean ID50 values and SEM for each immunization group is represented. Dotted line represented the detection limit.
Fig. 20. Immunogenicity and efficacy study schedule with MVA-S vaccine candidate in Syrian-gold hamsters. Syrian-gold female hamsters (6-8 weeks-old, n = 12 per group) were immunized with one or two doses of MVA-S (2 c 107 PFU in the prime and 4 c 107 PFU in the boost, with a technical grade lot from Biofabri, Spain) or two doses of MVA-WT (2 c 107 PFU in the prime and 4 c 107 PFU in the boost) by the intraperitoneal route at weeks 0 and 3. At week 6 (day 42), mice were intranasally challenged with 2 x 105 TCID50 of SARS-CoV-2 (BetaCoV/Belgium/GHB-03021/2020 isolate). At 21 and 39 days, serum samples were obtained, previously to the challenge. The challenged hamsters were monitored for change of body weights for 14 days, and at days 2, 4 and 14 post-challenge 4 hamsters per group were sacrificed; lungs were obtained for analysis of SARS-CoV-2 RNA and infectious virus and serum samples were also obtained. Fig. 21. Change in body weight in hamsters immunized with MVA-S and challenge with SARS-CoV-2. Hamsters were monitored for change of body weights previously to the challenge (data not shown), and after challenge with SARS-CoV-2.
Fig. 22. Binding IgG antibody titers against S protein induced in hamsters immunized with MVA-S. Titers of IgG antibodies specific for the S protein were determined by UFA in individual hamster serum samples collected at day 39, and were calculated as the serum dilution (Y axis) at which the absorbance was three times higher than the naive serum value in each of the indicated immunization regimens, shown in the X axis. Dotted line represented the detection limit.
Fig. 23. Neutralizing antibody titers against SARS-CoV-2 induced in hamsters immunized with MVA-S. Neutralizing antibody titers were determined by neutralization of VSV pseudotype virus using Vero-adaptive spike mutations in individual hamster serum samples collected at day 39, and were calculated as the serum dilution (Y axis) that neutralize 50%. Dotted line represented the detection limit.
Fig. 24. SARS-CoV-2 RNA and infectious virus in lungs of hamsters vaccinated with MVA-S. SARS-CoV-2 RNA and infectious virus were determined by RT-qPCR and a plaque assay, respectively, at days 2 and 4 post-challenge (n=4 samples per day).
Fig. 25. Lung histopathology. Histopathological score of lung samples obtained from hamsters at 2, 4- and 14-days post-challenge. (n=4 samples per day).
Fig. 26. Lung histopathology lesions. Cumulative lung score of lesions observed in lung samples obtained from hamsters at 2, 4- and 14-days post-challenge.
Fig. 27. Immunogenicity and efficacy study schedule with MVA-S vaccine candidate in rhesus macaques. Rhesus macaques (n = 6 per group) were immunized with two doses of MVA-S or MVA-WT (control) (2 c 108 PFU in the prime and boost, with a technical grade lot from Biofabri, Spain) by the intramuscular route at weeks 0 and 4. At week 8 (day 56), macaques were intranasally + intratracheally challenged with 1 c 106 PFU of SARS-CoV-2. At week 0, 4 and 6, serum samples were obtained, previously to the challenge. The challenged macaques were monitored for 14 days, and at different days post-challenge several samples were taken. Animals were sacrificed at day 14 post challenge.
Fig. 28. MVA-S elicit SARS-CoV-2 S-specific cell immune responses in immunized rhesus macaques. Magnitude of SARS-CoV-2 S-specific cell responses from PBMCs. Cells secreting IFN-y per million of PBMCs and directed against S1 and S2 peptide pools in rhesus macaques immunized is indicated. IFN-g was evaluated by an ELISpot assay from PBMCs derived from the 6 immunized macaques at 14 days post-prime. Samples were analyzed in triplicate; bars show the triplicate mean values and the standard deviation.
Fig. 29. MVA-S prevented SARS-CoV-2 virus replication in throat and bronchoalveolar samples. SARS-CoV-2 genomic and subgenomic RNA in throat and bronchoalveolar samples at day 2 post-challenge. SARS-CoV-2 RNA was analyzed by RT-qPCR targeting genomic viral RdRp gene and subgenomic N gene. Mean RNA levels (in A.U.) and SEM from duplicates of each sample is represented for each immunization group.
Fig. 30. Intranasal immunization with MVA-S induce robust SARS-CoV-2 S-specific T-cell and humoral immune responses in immunized mice. (A) Magnitude of SARS- CoV-2 S-specific CD4+ and CD8+ T-cell immune responses evaluated in splenocytes and lung cells at 10 days post-immunization. Percentages of CD4+ or CD8+ T cells expressing CD107a and/or producing IFN-g and/or TNF-a and/or IL-2 against a mixture of S1 and S2 peptide pools in mice immunized by the intranasal route with the indicative regimen. Cell percentages were determined by ICS. (B) Titers of IgG antibodies specific for the S (left) and RBD (right) proteins. Titers were determined by ELISA in individual mouse serum samples collected at 10 days post-immunization from intranasally immunized C57BL/6 mice and were calculated as the serum dilution at which the absorbance at 450 nm was at least three times higher than a naive serum value in each of the indicated immunization regimens. Mean values and SEM is represented for each immunization group. The dashed line represents the detection limit. (C) Titers of IgA antibodies specific for the S protein. Titers were determined by ELISA in bronchoalveolar mouse samples collected at 10 days post-immunization from intranasally immunized C57BL/6 mice and were calculated as the serum dilution at which the absorbance at 450 nm was at least three times higher than a naive serum value in each of the indicated immunization regimens. Mean values and SEM is represented for each immunization group. (D) The ID50 titers were evaluated in individual mouse serum samples at 10 days post-immunization using retrovirus-based pseudoparticles expressing the SARS-CoV-2 S protein, and were determined as the highest serum dilution which resulted in a 50% reduction of luciferase units compared with pseudotyped viruses not incubated with serum. Mean ID50 values and SEM for each from intranasally immunization group is represented. Dotted line represented the detection limit.
DESCRIPTION OF THE INVENTION General definitions
It must be noted that, as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "an antigenic determinant" includes one or more antigenic determinants and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.
Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having". Any of the aforementioned terms (comprising, containing, including, having), whenever used herein in the context of an aspect or embodiment of the present invention may be substituted with the term "consisting of", though less preferred.
When used herein "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.
As used herein, the conjunctive term "and/or" between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by "and/or", a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term "and/or" as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term "and/or."
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
The term "subtype" herein can be replaced with "species". It includes strains, isolates, clades or variants of any SARS-CoV-2. The terms "strain" "clade" or "isolate" are technical terms, well known to the practitioner, referring to the taxonomy of microorganisms. The taxonomic system classifies all so far characterized microorganisms into the hierarchic order of Families, Genera, Species, Strains (Fields Virology, ed. by Fields B. N., Lippincott-Raven Publishers, 4th edition 2001). While the criteria for the members of a Family is their phylogenetic relationship, a Genera comprises all members which share common characteristics, and a Species is defined as a polythetic class that constitutes a replicating lineage and occupies a particular ecological niche. The term "strain" or "clade" describes a microorganism, i.e. , virus, which shares common characteristics, like basic morphology or genome structure and organization, but varies in biological properties, like host range, tissue tropism, geographic distribution, attenuation or pathogenicity.
The term "subject" as used herein is a living multi-cellular vertebrate organism, including, for example, humans, non-human mammals and (non-human) primates. The term "subject" may be used interchangeably with the term "animal" herein. As used herein, the term "enhanced" when used with respect to an immune response against SARS-CoV-2, such as an antibody response (e.g., neutralizing antigen specific antibody response), a cytokine response or a CD8 T cell response (e.g., immunodominant CD8 T cell response), refers to an increase in the immune response in an animal administered with a homologous prime-boost combination vaccine of recombinant MVA relative to the corresponding immune response observed from the animal administered with a homologous prime-boost combination vaccine of MVA vectors, wherein the MVA vectors do not express any SARS-CoV-2 protein or refers to an increase in the immune response in an animal administered with a heterologous prime-boost combination vaccine of recombinant DNA and MVA vectors according to the invention, relative to the corresponding immune response observed from the animal administered with a heterologous prime-boost combination vaccine of DNA and MVA vectors according to the invention, wherein the MVA vector does not express any SARS-CoV-2 protein.
“Modified vaccinia virus Ankara (MVA) vector” as used herein is an attenuated replication- deficient vaccinia virus that has been genetically modified to include in its genome foreign DNA (that is, DNA that does not naturally belong to poxviruses). In particular, the modified vaccinia virus Ankara vector of the present invention has been genetically modified to express SARS-CoV-2 protein(s). Thus, the MVA vector of the present invention refers to viral particles of MVA that comprise inside the viral particle the MVA genome, wherein at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype has been introduced in the MVA genome.
“MVA-COVID19(S)”, “MVA-S” and “MVA-CoV2-S” are synonymous names to refer to a recombinant MVA vector comprising at least a nucleic acid encoding the structural protein S of the SARS-CoV-2 virus, or an antigenic fragment thereof. “ M VA-A-C O V I D 19( S )” , “MVA-A-S” and “MVA-A-CoV2-S” are synonymous names used to refer to a recombinant MVA vector further modified by deletion or inactivation of the MVA immunomodulatory genes C6L, K7R, and A46R, comprising at least a nucleic acid encoding the structural protein S of the SARS-CoV-2 virus, or an antigenic fragment thereof. Recombinant MVA virus vaccine compositions
Vaccinia virus (VACV) is amongst the most extensively evaluated live vectors and has particular features in support of its use as recombinant vaccine: It is highly stable, cheap to manufacture, easy to administer, and it can accommodate large amounts of foreign DNA. It has the advantage of inducing both antibody and cytotoxic T cell responses and allows presentation of antigens to the immune system in a more natural way, and it was successfully used as vector vaccine protecting against several infectious diseases in a broad variety of animal models. Additionally, vaccinia vectors are extremely valuable research tools to analyse structure-function relationships of recombinant proteins, determine targets of humoral and cell- mediated immune responses, and investigate the type of immune parameters needed to protect against a specific disease.
However, VACV is infectious for humans and its use as expression vector in the laboratory has been affected by safety concerns and regulations. Furthermore, possible future applications of recombinant VACV e.g. to generate recombinant proteins or recombinant viral particles for novel therapeutic or prophylactic approaches in humans, are hindered by the productive replication of the recombinant VACV vector. Most of the recombinant VACVs described in the literature are based on the Western Reserve (WR) strain of W. On the other hand, it is known that this strain is highly neurovirulent and is thus poorly suited for use in humans and animals (Morita et al., Vaccine 5, 65-70 [1987]).
Concerns with the safety of standard strains of VACV have been addressed by the development of VACV vectors from highly attenuated virus strains which are characterized by their restricted replicative capacity in vitro and their avirulence in vivo. Strains of viruses specially cultured to avoid undesired side effects have been known for a long time. Thus, it has been possible, by long-term serial passages of the chorioallantoic VACV Ankara (CVA) strain on chicken embryo fibroblasts, to culture MVA (for review see Mayr, A., Hochstein-Mintzel, V. and Stickl, H. (1975) Infection 3, 6-14; Swiss Patent No. 568 392). The MVA virus was deposited in compliance with the requirements of the Budapest Treaty at CNCM (Institut Pasteur, Collectione Nationale de Cultures de Microorganisms, 25, rue de Docteur Roux, 75724 Paris Cedex 15) on Dec. 15, 1987 under Depositary No. 1-721.
The MVA virus has been analysed to determine alterations in the genome relative to the wild type CVA strain. Six major deletions (deletion I, II, III, IV, V, and VI) have been identified (Meyer, H., Sutter, G. and Mayr A. (1991) J. Gen. Virol. 72, 1031-1038). This MVA has only low virulence, that is to say it is followed by no side effects when used for vaccination. Hence it is particularly suitable for the initial vaccination of immunocompromised subjects. The excellent properties of the MVA strain have been demonstrated in a number of clinical trials (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167, 375-390 [1987], Stickl et al., Dtsch. med. Wschr. 99, 2386-2392 [1974]). Thus, MVA is a valuable tool as safe viral vector for expression of recombinant genes and can be used for such different purposes as the in vitro study of protein functions or the in vivo induction of antigen-specific cellular or humoral immune responses. A major advantage of MVA is to allow for high level gene expression despite being replication defective in human and most mammalian cells. MVA as a vaccine has an excellent safety track-record, can be handled under biosafety level 1 conditions and has proven to be immunogenic and protective when delivering heterologous antigens in animals, and first human candidate vaccines have proceeded into clinical trials.
Although unable to multiply in most mammalian cell lines, MVA retains its genome plasticity that allows the insertion of large amounts of foreign DNA (heterologous genes) (Sutter and Moss, 1992). Absence of pathogenicity for humans, inherent avirulence even in immunocompromised hosts, high-level expression of foreign antigens and adjuvant effect for immune responses make recombinant MVA (rMVA), expressing heterologous genes, an ideal vector for both prophylactic and therapeutic vaccination, as demonstrated by the wide use in prime-boost immunization strategies
Indeed, recombinant MVA (rMVA)-based vaccines comprising heterologous genes and therefore expressing heterologous proteins are able to elicit both humoral and cell- mediated adaptive immune responses (Ramirez et al., 2000) and have been proved to be protective in animal models of several infectious diseases (Garcia-Arriaza et al., 2014, Journal of Virology (Chikungunya) Perez et al., 2018, Scientific Reports (Zika) Lazaro- Frias et al., 2018 (Ebola) Gomez et al., 2013; Marin et al., 2019, Journal of Virology (Hepatitis C)).
Standard procedures to generate recombinant VACV (rVACV) have been described in detail (Earl et al., 1998) and rely on in vivo homologous recombination between acceptor VACV DNA and the transfected transfer plasmid, in which the foreign gene is flanked by VACV sequences. Additional approaches for the generation of recombinant MVA are, for example, disclosed in WO 04074493, WO 03023040 and WO 9702355.
An important question is thus whether rMVA-based vaccines would or not be capable of generating a strong immunogenic and protective immune response against the SARS- CoV-2 virus in a subject in need thereof. In this regard, the authors of the present invention have found and demonstrated that a vaccine comprising:
- an immunologically effective amount of a recombinant MVA vector comprising at least a nucleic acid encoding an antigenic protein, or an antigenic fragment thereof, of at least one SARS-CoV-2 virus subtype, preferably the structural protein S of the SARS-CoV-2 virus, optionally together with a pharmaceutically acceptable carrier, diluent and/or additive (from hereinafter such vaccine candidate shall be referred to as MVA-COVID19); and/or
- an immunologically effective amount of such a MVA vector further modified by deletion or inactivation of the MVA immunomodulatory genes C6L, K7R, and A46R, comprising at least a nucleic acid encoding an antigenic protein, or an antigenic fragment thereof, of at least one SARS-CoV-2 virus subtype, preferably the structural protein S of the SARS-CoV-2 virus, optionally together with a pharmaceutically acceptable carrier, diluent and/or additive (from hereinafter such vaccine candidate shall be referred to as MVA-A-COVID19), provides a SARS-CoV-2 vaccine capable of generating a strong immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof.
As used herein, term “antigenic protein” or "antigenic determinant" refers to any molecule that stimulates a host's immune system to make an antigen-specific immune response, whether a cellular response or a humoral antibody response. Antigenic determinants may include proteins, polypeptides, antigenic protein fragments, antigens, and epitopes which still elicit an immune response in a host and form part of an antigen, homologues or variants of proteins, polypeptides, and antigenic protein fragments, antigens and epitopes including, for example, glycosylated proteins, polypeptides, antigenic protein fragments, antigens and epitopes, and nucleotide sequences encoding such molecules. Thus, proteins, polypeptides, antigenic protein fragments, antigens and epitopes are not limited to particular native nucleotide or amino acid sequences but encompass sequences identical to the native sequence as well as modifications to the native sequence, such as deletions, additions, insertions and substitutions.
The term "epitope" refers to a site on an antigen to which B- and/or T-cells respond, either alone or in conjunction with another protein such as, for example, a major histocompatibility complex ("MHC") protein or a T-cell receptor. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by secondary and/or tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 5, 6, 7, 8, 9, 10 or more amino acids - but generally less than 20 amino acids - in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., "Epitope Mapping Protocols" in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).
Preferably, a homologue or variant has at least about 50%, at least about 60% or 65%, at least about 70% or 75%, at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically, at least about 90%, 91 %, 92%, 93%, or 94% and even more typically at least about 95%, 96%, 97%, 98% or 99%, most typically, at least about 99% identity with the referenced protein, polypeptide, antigenic protein fragment, antigen and epitope at the level of nucleotide or amino acid sequence.
Techniques for determining sequence identity between nucleic acids and amino acids are known in the art. Two or more sequences can be compared by determining their "percent identity." The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100.
"Percent (%) amino acid sequence identity" with respect to proteins, polypeptides, antigenic protein fragments, antigens and epitopes described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence (i.e. , the protein, polypeptide, antigenic protein fragment, antigen or epitope from which it is derived), 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 for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publically available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full-length of the sequences being compared.
The same applies to "percent (%) nucleotide sequence identity", mutatis mutandis.
For example, an appropriate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, (1981 ), Advances in Applied Mathematics 2:482-489. This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov (1986), Nucl. Acids Res. 14(6):6745-6763. An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.). A preferred method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, California). From this suite of packages, the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+ GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found at the following internet address: http://wvw.ncbi.nlm.gov/cqi-bin/BLAST.
In the context of the present invention, the heterologous nucleic acid of any of the vaccine compositions described herein may also optionally encode antigenic domains or antigenic protein fragments rather than the entire antigenic protein. These fragments can be of any length sufficient to be antigenic or immunogenic. Fragments can be at least 8 amino acids long, preferably 10-20 amino acids, but can be longer, such as, e.g., at least 50, 100, 200, 500, 600, 800, 1000, 1200, 1600, 2000 amino acids long, or any length in between. In some embodiments, at least one nucleic acid fragment encoding an antigenic protein fragment or immunogenic polypeptide thereof is inserted into the genome of the recombinant viral vector of the invention. In another embodiment, about 2-6 different nucleic acids encoding different antigenic proteins are inserted into the genome of one or more of the recombinant viral vectors. In some embodiments, multiple immunogenic fragments or subunits of various proteins can be used. For example, several different epitopes from different sites of a single protein or from different proteins of the same SARS-CoV-2 strain, or from a protein orthologue from different strains can be expressed from the vectors.
It is herein thus noted that, as described above, vaccine candidates MVA-COVID19 and MVA-A-COVID19, may comprise any nucleic acid encoding an antigenic protein, or an antigenic fragment thereof, of at least one SARS-CoV-2 virus subtype; wherein such antigenic proteins are preferably selected from the list consisting of structural proteins S (spike), E (envelope), M (membrane), RBD (receptor binding domain) and/or N (nucleocapsid) of SARS-CoV-2, preferably such antigenic proteins are those selected from any of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8 and SEQ ID NO 10, preferably, respectively, encoded by SEQ ID No 1 , SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7 and SEQ ID NO 9.
More particularly, the present inventors have found that a vaccine comprising:
- an immunologically effective amount of a recombinant MVA vector comprising at least a nucleic acid encoding the structural protein S of the SARS-CoV-2 virus, or an antigenic fragment thereof, preferably, optionally together with a pharmaceutically acceptable carrier, diluent and/or additive (from hereinafter such vaccine candidate shall be referred to as MVA-COVID19(S)); and/or
- an immunologically effective amount of such a MVA vector further modified by deletion or inactivation of the MVA immunomodulatory genes C6L, K7R, and A46R, comprising at least a nucleic acid encoding the structural protein S of the SARS-CoV-2 virus, or an antigenic fragment thereof, preferably, optionally together with a pharmaceutically acceptable carrier, diluent and/or additive (from hereinafter such vaccine candidate shall be referred to as MVA-A-COVID19(S)), provides a SARS-CoV-2 vaccine capable of generating a remarkably strong immunogenic immune response against the SARS-CoV-2 virus in a subject in need thereof. In particular, and as illustrated in the figures of the present invention (i.e. Figures 5, 6, 7, 8, 9 and 10), vaccine candidates comprising priming and/or boosting compositions which in turn comprise MVA-COVID19(S) or MVA-A-COVID19(S), do not only induced high levels of total IgG, lgG1 , lgG2c, and lgG3 antibodies against SARS-CoV-2 S- and RBD proteins, with a Th1 response (Figure 9), but the results showed that individual serum samples obtained from all mice vaccinated with vaccine compositions MVA- COVID19(S) or MVA-A-COVID19(S) neutralized SARS-CoV-2 pseudoparticles, significantly reducing the levels of luciferase units, compared to serum from the control group MVA-WT/MVA-WT, where no neutralization was observed (Figure 10). Moreover, vaccine candidates comprising priming and/or boosting vaccine compositions MVA- COVID19(S) or MVA-A-COVID19(S), are capable of inducing broad and polyfunctional coronavirus SARS-CoV-2 S-protein-specific adaptive CD4+ and CD8+ T cell-mediated immune responses of great magnitude (Figures 5, 6, 7 and 8).
Thus, a recombinant MVA vector comprising in its genome a nucleotide sequence encoding an antigenic determinant of SARS-CoV-2, preferably encoding the structural protein S of the SARS-CoV-2 virus, reveals very good and specific immune responses against SARS-CoV-2 strains. In fact, from the data generated by the present inventors and their observations, it is more than reasonable and plausible to conclude that the MVA- based vaccine would also induce a protective efficacy in animal models and will be able to generate immunogenic and effective immune responses in humans. In this sense, the results in Figure 12 show that a single dose of MVA-COVID19(S) and a posterior challenge with the SARS-CoV-2 virus induces firstly a small body weight decrease in mice (first 4 days post-challenge), but then the mice fully recover their normal weight showing 100% of survival and demonstrating that a single dose is enough to protect mice from a lethal dose of SARS-CoV-2. Furthermore, Figure 12 also shows that two doses of MVA- COVID19(S) also induced protection upon a lethal dose of SARS-CoV-2, where the vaccinated and challenged mice do not suffer from any weight loss and where the survival is also 100%. On the other hand, as shown in the same figure, control mice vaccinated with the MVA-WT parental vector and subsequently challenged with SARS-CoV-2 virus suffer from an accentuated body weight loss that eventually leads to 0% survival rate. This indicates that the MVA-based vaccines disclose herein confer, both in a single and in two doses, a full protection in challenged mice.
In line with the above, in the study underlying the present invention it has been thus found that the use of a vaccine combination comprising: a. a first composition comprising an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably MVA- COVID19(S) or MVA-A-COVID19(S); and b. a second composition comprising an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably MVA- COVID19(S) or MVA-A-COVID19(S); as a homologous prime and boost generates a strong protective immune response against a SARS-CoV-2 immunogen. Such response is particularly strong when both compositions comprises an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 composition comprising at least a nucleic acid encoding the S antigenic protein of at least one SARS-CoV-2 virus subtype, preferably encoding for the S antigenic protein of SEQ ID NO 2 (please refer to the examples and figures).
Furthermore, in view of the results obtained in Figure 12, it is also plausible that a single dose of an immunologically effective amount of MVA-COVID19 or MVA-A-COVID19 combined in a prime or boost with an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype also elicits protective immune responses against a lethal dose of SARS-CoV-2 virus subtype. In line with this, in the study underlying the present invention it has been further found that the use of vaccine combination comprising: a. a first composition comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype (from hereinafter DNA vaccine); and b. a second composition comprising an immunologically effective amount of a MVA-COVID1 9 or MVA-A-COVID19, preferably MVA-COVID19(S) or MVA- A-COVID19(S); as a heterologous prime and boost generates a protective immune response against a SARS-CoV-2 immunogen. Such response is particularly strong when both compositions comprise at least a nucleic acid encoding the S antigenic protein of at least one SARS- CoV-2 virus subtype, preferably encoding for the S antigenic protein of SEQ ID NO 2.
It is noted that the recombinant MVA and/or DNA heterologous or homologous vaccines may be either monovalent, i.e. , the nucleic acid comprised in the recombinant MVA and/or DNA vectors may comprise only one heterologous sequence encoding an antigenic determinant of SARS-CoV-2, or multivalent, i.e., the nucleic acid comprised in the recombinant MVA and/or DNA vectors may comprise at least two heterologous sequences encoding antigenic determinants of SARS-CoV-2, such as those selected from the list consisting of structural proteins S, E or M of SARS-CoV-2, preferably such antigenic proteins are those selected from any of SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6, respectively encoded by nucleic acid sequences SEQ ID NO 1 , SEQ ID NO 3 and SEQ ID NO 5. Further, the nucleic acid comprised in the recombinant MVA/or DNA vectors may encode for structural proteins such as RBD and/or N (nucleocapsid) of SARS-CoV-2 that can also be useful as heterologous sequences to work the present invention, preferably such antigenic proteins are those selected from any of SEQ ID NO 8 and SEQ ID NO 10, respectively encoded by nucleic acid sequences SEQ ID NO 7 and SEQ ID NO 9.
The invention thus provides, for the first time, effective vaccines or vaccine combinations for use in generating a protective immune response against infections by at least SARS- CoV-2 and vaccines or vaccine combinations which can be used for manufacturing of a vaccine against SARS-CoV-2.
Thus, a first aspect of the present invention provides a vaccine composition comprising a. an immunologically effective amount of a MVA vector comprising at least one nucleic acid encoding an antigenic protein, or an antigenic fragment thereof, of at least one SARS-CoV-2 virus subtype, optionally together with a pharmaceutically acceptable carrier, diluent and/or additive (from hereinafter such vaccine candidate shall be referred to as MVA-COVID19); and/or b. an immunologically effective amount of the MVA vector of a), wherein said MVA vector is further modified by deletion of the MVA immunomodulatory genes C6L, K7R, and A46R, optionally together with a pharmaceutically acceptable carrier, diluent and/or additive (from hereinafter such vaccine candidate shall be referred to as MVA-A-COVID19).
In a preferred embodiment, the nucleic acid encoding the antigenic protein is incorporated into a non-essential region of the genome of the MVA. In an embodiment of the present invention, the nucleic acid encoding the antigenic protein is incorporated into the MVA by substituting a non-essential region of the genome of the MVA. In a preferred embodiment of the present invention, the nucleic acid encoding the antigenic protein is incorporated into the MVA thymidine kinase (TK) locus or hemagglutinin (HA) locus. Integration of the nucleic acid encoding the antigenic protein is performed preferentially by homologous recombination of the flanking regions of a so-called transfer vector, which initially comprises the nucleic acid encoding the antigenic protein according to the invention prior to its integration into the parental MVA genome. In a preferred embodiment, the transfer vector is the pCyA vector. The pCyA vector has been previously described in WO 2014/162031 A1.
Upon homologous recombination by means of an infection with parental virus followed by a transfection with the plasmid transfer vector, selection and purification of recombinant viruses encoding the antigenic protein is performed. In an embodiment, the intermediate MVA recombinant viruses also expresses marker genes that facilitate the tracking and selection of the desired final recombinant viruses. For instance, the intermediate recombinant MVA viruses might express b-galactosidase gene, a fluorescent protein such as the green fluorescent protein (GFP), or an antibiotic resistance gene. Methods for generating recombinant MVAs by homologous recombination by means of intermediate MVAs encoding for selectable genes are described in WO 2014/162031 A1 and WO 2016/086980 A1.
On the other hand, early expression of antigens by VACV vectors is crucial for efficient antigen-specific CD8 T cell responses. The specific properties of the early elements poxviral promoters might thus be crucial for induction of an antigen-specific T cell response. Moreover, it is commonly known that higher amounts of antigen are beneficial for induction of stronger antigen-specific immune responses.
Therefore, in a further embodiment, the recombinant MVA vector comprises at least one nucleic acid encoding an antigenic protein, or an antigenic fragment thereof, of at least one SARS-CoV-2 virus subtype that is operably linked and under the control of a VACV- specific promoter, an orthopox virus-specific promoter, a poxvirus-specific promoter or a synthetic promoter. A number of promoters may be used for the present invention. For the expression of the nucleic acid encoding an antigenic protein according to the invention several promoters, such the 30K and 40K promoters (US 5,747,324, A Strong Synthetic Early-Late Promoter (Sutter, et al. , Vaccine (1994), 12, 1032-1040)), the P7.5 promoter (Endo et al., J. Gen. Virol. (1991) 72, 699-703) and a promoter derived from the cow pox virus type A (inclusion ATI gene) (Lee et al., J. Gen. Virol. 1998), 79, 613) can be used.
In another preferred embodiment, the recombinant MVA vector comprises at least one nucleic acid encoding an antigenic protein, or an antigenic fragment thereof, of at least one SARS-CoV-2 virus subtype that is operably linked and under the control of a synthetic promoter. Synthetic promoters are DNA sequences that do not exist in nature and which are designed to regulate the activity of genes to which they are operably linked, controlling a gene's ability to produce its encoded protein. In a preferred embodiment, the synthetic promoter drives the expression of the antigenic protein early during infection. For instance, in an embodiment, the expression of the antigenic protein can be detected as early as 3-hour post-infection. In another embodiment, the synthetic promoter may also drive the expression of the antigenic protein late during infection. In a preferred embodiment, the expression of the antigenic protein is operably linked and under the control of a synthetic promoter that drives the expression of the antigenic protein both, early and late, during viral infection.
The synthetic promoter may be comprised by one or more elements driving early expression of said antigen. The synthetic promoter may be comprised by one or more elements driving late expression of said antigen. Different types of synthetic early/late promoters are further described in WO 2010/102822 A1. In another embodiment, the synthetic promoter is formed by both early and late elements, referred as synthetic early/late (sE/L) promoter (Chakrabarti, S.; Sisler, J.R.; Moss, B. Compact, synthetic, vaccinia virus early/late promoter for protein expression. Biotechniques 1997, 23, 1094- 1097) or the LEO160 promoter (Di Pilato, M.; Sanchez-Sampedro, L; Mejias-Perez, E.;
Sorzano, C.O.S.; Esteban, M. Modification of promoter spacer length in vaccinia virus as a strategy to control the antigen expression. J. Gen. Virol. 2015, 96, 2360-2371. Perez et al. , 2019, Vaccines 2019, 7, 208). In another embodiment, the early and late elements are overlapping. On the other hand, the nucleic acid encoding the antigenic protein inserted in MVA genome according to the invention can be selected from any group of nucleic acids encoding proteins from SARS-CoV-2 virus subtype. The nucleic acid encoding the antigenic protein according to the present invention may preferably be the protein S, M, E. In a preferred embodiment, the nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype encodes for the protein S.
In another preferred embodiment, the at least one nucleotide sequence encoding an antigenic protein of at least one SARS-CoV-2 virus subtype is preferably the structural protein S of SARS-CoV-2, and said nucleic acid encoding the structural protein S of SARS-CoV-2, if present, is inserted into the TK locus of the MVA genome; and wherein the MVA vector regulates the expression of the nucleic acid encoding the antigenic protein and is operably linked to said nucleotide sequence.
In another preferred embodiment of the first aspect of the invention, the antigenic protein is selected from the any of the group of structural proteins S, E or M of SARS-CoV-2, or any combination thereof. It is noted that if at least one nucleotide sequence encodes for any of antigenic proteins E or M of SARS-CoV-2, such nucleic acids, if present, should be preferably inserted into the HA locus of the MVA genome; wherein the MVA vector regulates the expression of the nucleic acids encoding the antigenic proteins and should be operably linked to said nucleotide sequences.
In another preferred embodiment of the first aspect of the invention, the antigenic protein is the structural protein S of SARS-CoV-2 resulting in vaccine compositions MVA- COVID19(S) and/or MVA-A-COVID19(S), preferably the antigenic protein is the protein of SEQ ID NO 2, more preferably the antigenic protein of is the protein of SEQ ID NO 2 encoded by the nucleic acid consisting of SEQ ID NO 1. Still, more preferably, any of vaccine compositions MVA-COVID19(S) and/or MVA-A-COVID19(S) are further characterized by comprising at least one nucleotide sequence encoding for any of antigenic proteins E or M of SARS-CoV-2; wherein such nucleic acids, if present, should be preferably inserted into the HA locus of the MVA genome. It is noted that the recombinant MVA viruses described herein are highly replication restricted and, thus, highly attenuated, and thus they are ideal candidates for the treatment of a wide range of mammals including humans and even immune-compromised humans. Hence, provided herein are pharmaceutical compositions and vaccines, as described above in the first aspect of the invention, suitable for inducing an immune response in a living animal body, including a human against the SARS-CoV-2 virus.
The vaccines of the first aspect preferably comprise any of the recombinant MVA viruses described herein formulated in solution in a concentration range of from 1 x 107 to 1 x 108 PFUs per dosis. This is illustrated in Figures 4-11 which show that immunogenicity was induced in vaccinated mice with a dose of 1 x 107 PFUs per dose (see immunization schedule in Figure 3). Further, as shown in Figure 12, Groups 1 and 2 were vaccinated with 1x107 PFUs of virus per dose, which indicate that this dose is sufficient to induce protective immunity that leads to 100% survival in both groups upon a lethal challenge with SARS-Cov-2.
The vaccine compositions provided herein in the first aspect may generally include one or more pharmaceutically acceptable and/or approved carriers, additives, antibiotics, preservatives, adjuvants, diluents and/or stabilizers. Such auxiliary substances can be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, or the like. Suitable carriers are typically large, slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, or the like.
For the preparation of vaccines, the recombinant MVA viruses provided herein can be converted into a physiologically acceptable form. This can be done based on experience in the preparation of poxvirus vaccines used for vaccination against smallpox as described by H. Stickl et al. , Dtsch. med. Wschr. 99:2386-2392 (1974).
For example, purified viruses can be stored at -80°C with a titer of 5x108 PFU/ml formulated in about 10 mM Tris, 140 mM NaCI pH 7.4. For the preparation of vaccine shots, e.g., 102-108 or 102-109 particles of the virus can be lyophilized in 100 ml of phosphate-buffered saline (PBS) in the presence of 2% peptone and 1% human albumin in an ampoule, preferably a glass ampoule. Alternatively, the vaccine shots can be produced by stepwise freeze-drying of the virus in a formulation. This formulation can contain additional additives such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone or other aids such as antioxidants or inert gas, stabilizers or recombinant proteins (e.g., human serum albumin) suitable for in vivo administration. The glass ampoule is then sealed and can be stored between 45°C and room temperature for several months. Flowever, as long as no need exists, the ampoule is stored preferably at temperatures below -20°C.
For vaccination or therapy, the lyophilisate can be dissolved in an aqueous solution, preferably physiological saline or Tris buffer, and administered either systemically or locally, i.e. , parenteral, subcutaneous, intravenous, intramuscular, intranasal, or any other path of administration known to the skilled practitioner. The mode of administration, the dose and the number of administrations can be optimized by those skilled in the art in a known manner. Flowever, most commonly a patient is vaccinated with a second shot about one month to six weeks after the first vaccination shot, although booster doses can also be administered years after the first dose.
In addition, for working the present invention any avirulent MVA vector suitable for human vaccination could be used. "Derivatives" or "variants" of MVA are also suitable for working the present invention. In preferred embodiments, the MVA vector is further modified by deletion of one or more vaccinia immunomodulatory genes; preferably one, two, or three of the genes C6L, K7R, and A46R. Most preferably, all three genes are deleted. Deletion of these genes has been shown to enhance the innate immune response triggered by the viral vector, and also adaptive antigen-specific immune responses. In the context of the present invention, "Derivatives" or "variants" of MVA refer to viruses exhibiting essentially the same replication characteristics as MVA but exhibiting differences in one or more parts of their genomes. These "Derivatives" or "variants" of MVA must fail to reproductively replicate in vivo in humans and mice, even in severely immune suppressed mice. Tests and assays for the properties of MVA variants are described in WO 02/42480 (US 2003/0206926) and WO 03/048184 (US 2006/0159699).
The term "fails to reproductively replicate" refers to a virus that has a virus amplification ratio at 4 days after infection of less than 1 . Assays described in WO 02/42480 or in U.S. Patent No. 6,761 ,893 are applicable for the determination of the virus amplification ratio.
The amplification or replication of a virus is normally expressed as the ratio of virus produced from an infected cell (output) to the amount originally used to infect the cell in the first place (input) referred to as the "amplification ratio". An amplification ratio of "1" defines an amplification status where the amount of virus produced from the infected cells is the same as the amount initially used to infect the cells, meaning that the infected cells are permissive for virus infection and reproduction. In contrast, an amplification ratio of less than 1 , i.e. , a decrease in output compared to the input level, indicates a lack of reproductive replication and therefore attenuation of the virus.
The advantages of MVA-based vaccine include their safety profile as well as availability for large scale vaccine production. In a preferred embodiment, the recombinant MVA vector of any of the embodiments used for generating the recombinant virus is a MVA virus or a derivative having the capability of reproductive replication in vitro in chicken embryo fibroblasts (CEF) cells and in established chick cells DF-1 , but no capability of reproductive replication in human cervix adenocarcinoma cell line FleLa. Also preferably, the recombinant MVA vector of any of the embodiments used for generating the recombinant virus is a MVA vector further modified by deletion of one or more vaccinia immunomodulatory genes; preferably one, two, or three of the genes C6L, K7R, and A46R. Most preferably, all three genes are deleted.
MVA vectors useful for the present invention can be prepared using methods known in the art, such as those described in WO 02/042480 and WO 02/24224, both of which are incorporated by reference herein.
In another aspect, a MVA viral strain suitable for generating the recombinant viruses of the present invention may be strain MVA-572, MVA-575 or any similarly attenuated MVA strain. Also suitable may be a mutant MVA, such as the deleted chorioallantoic vaccinia virus Ankara (dCVA). A dCVA comprises del I, del II, del III, del IV, del V, and del VI deletion sites of the MVA genome. The sites are particularly useful for the insertion of multiple heterologous sequences. The dCVA can reproductively replicate (with an amplification ratio of greater than 10) in a human cell line (such as human 293, 143B, and MRC-5 cell lines), which then enable the optimization by further mutation useful for a virus-based vaccination strategy (see WO 201 1/092029).
Combination Vaccines Using Homoloqous/Heteroloqous Prime-Boost Regimens
The Combination Vaccines and methods described herein may be used as part of a homologous prime-boost regimen. In the homologous prime-boost, a first priming vaccination is given followed by one or more subsequent boosting vaccinations. The boosting vaccinations are configured to boost the immune response generated in the first vaccination by administration of the same recombinant MVA that was used in the first vaccination.
In one exemplary embodiment a homologous prime-boost regimen may be employed wherein a MVA viral vector as defined herein in the first aspect of the invention is administered in a first dosage. One or more subsequent administrations of a MVA viral vector as defined herein in the first aspect of the invention can be given to boost the immune response provided in the first administration. Preferably, the one or more antigenic determinants are the same or similar to those of the first administration.
The MVA recombinant viral vectors according to the present invention may also be used in prime-boost regimens in which one or more of the initial prime vaccinations are done with either the MVA or the DNA vector (see definition of DNA vaccine below) as defined herein and one or more subsequent boosting vaccinations are done with the MVA vector not used in the prime vaccination, e.g., if a MVA vector defined herein is given in a prime boost, then subsequent boosting vaccinations would be DNA vectors and vice versa.
Thus, a second aspect of the invention refers to a vaccine combination (from hereinafter referred to as homologous vaccine combination) comprising: a. a first composition comprising an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably a MVA-COVID1 9(S) or MVA-A-COVID19(S) vaccine composition; and b. a second composition comprising an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably a MVA-COVID1 9(S) or MVA-A-COVID19(S) composition; wherein the first compositions is a priming composition and the second composition is a boosting composition, preferably wherein the boosting composition comprises two or more doses of the vector of the boosting composition.
Furthermore, an alternative second aspect of the invention refers to a vaccine combination (from hereinafter referred to as heterologous vaccine combination) comprising: a. a first composition comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype (from hereinafter DNA vaccine); preferably DNA-S and b. a second composition comprising an immunologically effective amount of a MVA-COVID1 9 or MVA-A-COVID19, preferably MVA-COVID19(S) or MVA- A-COVID19(S); wherein the first compositions is a priming composition and the second composition is a boosting composition or vice versa, and preferably wherein the boosting composition comprises two or more doses of the vector of the boosting composition.
It is noted that the recombinant heterologous or homologous vaccine combinations may be either monovalent, i.e. , comprising only one heterologous sequence encoding an antigenic determinant of SARS-CoV-2, or multivalent, i.e., comprising at least two heterologous sequences encoding antigenic determinants of SARS-CoV-2, such as those selected from the list consisting of structural proteins S, E or M of SARS-CoV-2, preferably such antigenic proteins are those selected from any of SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6, respectively encoded by nucleic acid sequences SEQ ID NO 1 , SEQ ID NO 3 and SEQ ID NO 5.
In one preferred embodiment of the second aspect of the invention, the antigenic protein of any of the homologous or heterologous vaccine combinations, is selected from any of the group of structural proteins S, E or M of SARS-CoV-2, or any combination thereof. It is noted that the at least one nucleotide sequence encoding an antigenic protein of at least one SARS-CoV-2 virus subtype is preferably the structural protein S of SARS-CoV- 2, and said nucleic acid encoding the structural protein S of SARS-CoV-2, if present, should be preferably inserted into the TK locus of the MVA genome. It is further noted that if at least one nucleotide sequence encodes for any of antigenic proteins E or M of SARS-CoV-2, such nucleic acids, if present, should be preferably inserted into the HA locus of the MVA genome. As regards the heterologous vaccine compositions it is important to note that upon injection, preferably intramuscularly, of the DNA vaccine, cells, preferably muscle cells, may be efficiently transfected and produce a relatively large amount of antigen that may be secreted or otherwise released. Once the plasmid inserts itself into the transfected cell nucleus, it codes for the antigen of interest. Next, the transfected cell displays on its surface the foreign antigen in both histocompatibility complex (MHC) classes I and class II molecules. The antigen-presenting cell primed with the antigen travels to the lymph nodes and presents the antigen peptide and costimulatory molecules, initiating the immune response.
DNA-based vectors suitable for used as DNA vaccines, as disclosed herein for the heterologous regimes or heterologous vaccine combinations, are known in the art. For instance, Hobernik and Bros Int J Mol Sci. 2018 Nov 15; 19(11 ):3605 summarize the knowledge on the course of action of DNA vaccines and the methods for DNA vaccine immunogenicity optimization. Lee et al. Acta Biomater. 2018 Oct 15;80:31 -47 relates to engineering DNA vaccines against infectious diseases. In one aspect of the present invention, a DNA-based vaccine, which comprises antigen expressing plasmid DNA vectors, is provided. DNA vaccines may be introduced into animal tissues by multiple methods. For instance, naked DNA can be administrated intramuscularly. Other examples are DNA delivered by electroporation, by gene gun or using a liposoluble carrier. Methods of DNA-based vaccines administration are not limited and are known in the art.
The DNA-based vaccine vector encodes for one or more antigens against which the immune response is to be elicited. Further, the DNA vector can also encode other co stimulatory molecules, such as interleukins, cytokines, etc.
If the DNA-based vaccine is to be administrated to a mammal, the promoter operably linked to the antigen of interest would need to be recognized by mammal cellular transcription machinery. Promoters for expressing proteins in mammalian cells are also known in the art. For instance, some viral promoters might be used, such as cytomegalovirus promoter or T7 phage promoter. The DNA backbone to construct the DNA-based vaccine of the present invention is not limited. Any DNA, linear or plasmid, able to express the antigen in a mammalian cell may be used. Mammalian expression DNA vectors are widely known in the art.
In a preferred embodiment, the mammalian expression vector is a plasmid vector and produces high levels of the antigen. For instance, suitable mammalian expression plasmid vectors that can be used in the present inventions are, but not limited to, pCDNA, pCAGGS, etc. The mammalian expression vector can be grown in bacterial cells for high- yield production.
Methods for generating, producing and isolating plasmid DNA vectors are known in the art. In a preferred embodiment of the present invention, the DNA-based plasmid vaccine has been produced by endo-free means, for instance, by using EndoFree Plasmid Kits by Qiagen.
In an embodiment of the present invention, the mammalian expression plasmid vector is a pCDNA vector encoding an antigenic protein of at least one SARS-CoV-2 virus subtype. In another embodiment of the present invention, the mammalian expression plasmid vector is a pCDNA vector encoding at least one or a combination of the proteins S, M, E of SARS-CoV-2 virus subtype. In a more preferred embodiment, the plasmid vector encodes for the protein S of SARS-CoV-2 virus subtype. In a more preferred embodiment, the plasmid vector encoding an antigenic protein of at least one SARS-CoV-2 virus subtype has a SEQ ID 1 encoding for SEQ ID NO 2.
Vaccines and Kits Comprising Recombinant MVA Viruses
Also provided herein are vaccines and kits comprising any one or more of the recombinant MVAs described herein. The kit can comprise one or multiple containers or vials of the recombinant MVA, together with instructions for the administration of the recombinant MVA to a subject at risk of SARS-CoV-2 infection. In certain embodiments, the subject is a human. In certain embodiments, the instructions indicate that the recombinant MVA is administered to the subject in a single dose, or in multiple (i.e. , 2, 3, 4, etc.) doses. In certain embodiments, the instructions indicate that the recombinant MVA virus is administered in a single dose to naive or non-naive subjects. Thus, in a preferred embodiment, the vaccines and kits comprising recombinant MVA viruses as described in the present invention comprise a single dose to be administrated to a subject in need thereof. In certain embodiments, the instructions indicate that the recombinant MVA virus is administered in a first (priming) and second (boosting) administration to naive or non- naive subjects. Preferably, a kit comprises at least two vials for prime/boost immunization comprising the recombinant MVAs as described herein for a first inoculation ("priming inoculation") in a first vial/container and for an at least second and/or third and/or further inoculation ("boosting inoculation") in a second and/or further vial/container. More preferably, the vaccines and kits described herein comprise a first (priming) and second (boosting) dose of an immunologically effective amount of the recombinant MVAs as described herein, preferably of MVA-COVID19 and MVA-A-COVID19, even more preferably MVA-COVID19(S) or MVA-A-COVID19(S), for use in homologous or heterologous prime boost inoculations.
Thus, a third aspect of the invention refers to kits comprising one, preferably two, or more doses of any of the vaccine compositions as defined in the first aspect of the invention, or of any of the homologous or heterologous vaccine combinations of the second aspect of the invention. Preferably, the kit comprises an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably MVA-COVID19(S) or MVA-A-COVID19(S), according to the first aspect of the invention in a first vial or container for a first administration (priming) and in a second vial or container for a second administration (boosting).
A preferred embodiment of the third aspect of the invention refers to kits comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype, preferably the structural protein S of SARS-CoV-2, in a first vial or container for a first administration (priming), and an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably MVA-COVID19(S) or MVA-D- COVID19(S), according to the first aspect of the invention in a second vial or container for a second administration (boosting).
Another preferred embodiment of the third aspect of the invention refers to kits comprising an immunologically effective amount of a a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably MVA-COVID19(S) or MVA-A-COVID19(S), according to the first aspect of the invention, in a first vial or container for a first administration (priming), and an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype, preferably the structural protein S of SARS-CoV-2, in a second vial or container for a second administration (boosting). In another preferred embodiment of the third aspect of the invention, any of the kits referred to herein, comprise a third, fourth or further vial or container comprising any of the vaccine compositions indicated throughout the present invention for a third, fourth or further administration. In an embodiment, the vaccines compositions and kits provided in any of the previous aspects are for use in generating a protective immune response against at least one SARS-CoV-2 subtype, wherein the kits or vaccine composition comprise one dose of an immunologically effective amount of the vaccines provided herein. Preferably, the vaccines compositions and kits provided in any of the previous aspects are for use in generating a protective immune response against at least one SARS-CoV-2 subtype, wherein the kits or vaccine composition comprise one dose of an immunologically effective amount of the vector MVA-COVID19(S) or MVA-A-COVID19(S) vaccines.
In a further preferred embodiment the vaccines, vaccine combinations and kits provided in any of the previous aspects are for use in generating a protective immune response against at least one SARS-CoV-2 subtype, wherein if the kits or vaccine combinations are used, the first composition is used for priming said immune response and the second composition is used for boosting said immune response or for use in generating a protective immune response against at least one SARS-CoV-2 subtype, wherein the second composition is used for priming said immune response and the first composition is used for boosting said immune response. In any of the vaccines, vaccine combinations and kits provided herein the boosting composition can comprise two or more doses of the vector of the boosting composition.
As discussed previously above, the present invention also relates to heterologous vaccination regimes using two different non-replicating viral vectors. For heterologous vaccine programs, the present invention provides a combination vaccine and/or vaccination kit which comprises: a. a first composition comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype (from hereinafter DNA vaccine); preferably DNA-S and b. a second composition comprising an immunologically effective amount of a MVA-COVID1 9 or MVA-A-COVID19, preferably MVA-COVID19(S) or MVA- A-COVID19(S); wherein the first compositions is a priming composition and the second composition is a boosting composition or vice versa, and preferably wherein the boosting composition comprises two or more doses of the vector of the boosting composition.
The present invention also provides homologous vaccination regimes using two equal non-replicating viral vectors. For homologous vaccine programs, the present invention thus provides a combination vaccine and/or vaccination kit which comprises: a. a first composition comprising an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably a MVA-COVID1 9(S) or MVA-A-COVID19(S) vaccine composition; and b. a second composition comprising an immunologically effective amount of a MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably a MVA-COVID1 9(S) or MVA-A-COVID19(S) composition; wherein the first compositions is a priming composition and the second composition is a boosting composition, preferably wherein the boosting composition comprises two or more doses of the vector of the boosting composition.
In this embodiment, the combination vaccines and/or kit comprises at least two vials for prime/boost immunization comprising the recombinant MVAs/DNAs as described herein for a first inoculation ("priming inoculation") in a first vial/container and for an at least second and/or third and/or further inoculation ("boosting inoculation") in a second and/or further vial/container.
The combination vaccine and/or kit can comprise multiple containers or vials of the recombinant MVA/DNA, together with instructions for the administration of the recombinant MVA/DNA to a subject at risk of SARS-CoV-2 infection. In certain embodiments, the subject is a human. In certain embodiments, the instructions indicate that the recombinant MVA/DNA is administered to the subject in a single dose, or in multiple (i.e. , 2, 3, 4, etc.) doses. In certain embodiments, the instructions indicate that the recombinant MVA/DNA virus is administered in a first (priming) and second (boosting) administration to naive or non-naive subjects.
The first and/or second composition or MVA and/or DNA of any combination vaccine, vaccination kit and/or any homologous or heterologous vaccine program of the invention can comprise any of the MVA and/or DNA vector described herein and any combination thereof.
Methods and Uses of Recombinant MVA Viruses Also provided herein are methods of use of any of the recombinant MVA-COVID19 or MVA-A-COVID19 vaccine compositions, preferably the MVA-COVID19(S) or MVA-D- COVID19(S) vaccine compositions, as described in the first aspect of the invention, for immunizing a subject animal or for affecting an immune response in a subject against SARS-CoV-2 infection. Also covered are uses of the said recombinant vaccine compositions described herein for the preparation of a medicament or pharmaceutical for the immunization of a subject animal, in particular for the preparation of a medicament or vaccine for treating and/or preventing a SARS-CoV-2-caused disease in a subject. Provided are also said recombinant MVAs according to any embodiment herein for use in priming or boosting an immune response against a SARS-CoV-2 infection, preferably wherein the recombinant MVA is administered once, twice, three times or four times.
Further covered herein are vaccine combinations of any of the embodiments for use as a medicament or vaccine for inducing an enhanced immune response against a SARS- CoV-2 infection wherein the combination is capable of producing the antigenic determinant, and/or SARS-CoV-2 -like particles in the subject to be treated, preferably, wherein the combination vaccine is producing the encoded antigenic determinant in the subject to be treated.
Accordingly, a fourth aspect of the present invention provides a method of generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject, the method comprising administering to the subject the recombinant MVA-COVID19 or MVA-A-COVID19 vaccine composition, preferably the MVA-COVID19(S) or MVA-A-COVID19(S) vaccine composition, as described in the first aspect of the invention; or a kit or any of the vaccine combinations, including the homologous or heterologous vaccination regimes, as described in any of the second or third aspects of the invention.
As used herein, the term “immunogenic and protective immune response", "protective immunity" or "protective immune response" means that the vaccinated subject is able to control an infection with the pathogenic agent against which the vaccination was done. Usually, the subject having developed a "protective immune response" develops only mild to moderate clinical symptoms or no symptoms at all. Usually, a subject having a "protective immune response" or "protective immunity" against a certain agent will not die as a result of the infection with said agent. In certain embodiments, the subject animal is a mammal. The mammal may be an adult cow, a calf, in particular a juvenile calf, a rat, rabbit, pig, mouse, but preferably a human, and the method comprises administering a dose of any one or more of the recombinant MVAs or combinations thereof as described in the fourth aspect of the invention.
In certain embodiments of the fourth aspect of the invention, the subject is a human. In certain embodiments, the subject is an adult. In certain embodiments, the adult is immune-compromised. In certain embodiments, the adult is over the age of 10, 15, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 years.
Any of the recombinant MVAs provided in the first aspect of the invention, or any of the kits or vaccine combinations, including the homologous or heterologous vaccination regimes, as described in any of the second or third aspects of the invention, may be preferably administered to the subject at a dose of 1x107 or 1x108 PFU/dose.
In certain embodiments, any of the recombinant MVAs provided in the first aspect of the invention, or any of the kits or vaccine combinations, including the homologous or heterologous vaccination regimes, as described in any of the second or third aspects of the invention, are administered to the subject at any of the doses provided herein prior to SARS-CoV-2 virus exposure as, e.g., 1 , 2, 3, or 4 weeks or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months before SARS-CoV-2 virus exposure. In certain embodiments, any of the said vaccine compositions or kits provided herein is administered to the subject at any of the doses provided herein after SARS-CoV-2 virus exposure as, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9. 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 hours or 1 , 2, 3, 4, 5, 6, or 7 days after SARS-CoV-2 exposure.
In certain embodiments, the recombinant MVAs provided in the first aspect of the invention, or any of the kits or vaccine combinations, including the homologous or heterologous vaccination regimes, as described in any of the second or third aspects of the invention, are administered to the subject in a single dose, or in multiple (i.e., 2, 3, 4, etc.) doses. In certain embodiments, the said kits or vaccine compositions provided herein are administered in a first (priming) and second (boosting) administration.
In an embodiment, a single dose of MVA viral vector as defined herein in the first aspect of the present invention may be given to induce protective immune response, as shown in Figure 12 (vaccinated mice Group 1 ). In a further embodiment, more than one dose of an immunologically effective amount of an MVA viral vector as defined herein in the first aspect of the present invention may be given to induce protective immune response, as also shown in Figure 12 (vaccinated mice Group 2). In particular, a single dose or more than one dose of an immunologically effective amount of MVA-COVID19(S) or MVA-D- COVID19(S) is preferred.
Boosting compositions are generally administered once or multiple times weeks or months after administration of the priming composition, for example, about 1 or 2 weeks or 3 weeks, or 4 weeks, or 6 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks or one to two years. Preferably, the initial boosting inoculation is administered 1-12 weeks or 2-12 weeks after priming, more preferably 1, 2, 4 or 8 weeks after priming. In a preferred embodiment, the initial boosting inoculation is administered 4 or 8 weeks after priming. In additional preferred embodiments, the initial boosting is conducted at least 2 weeks or at least 4 weeks after priming. In still another preferred embodiment, the initial boosting is conducted 4-12 weeks or 4-8 weeks after priming.
The recombinant MVAs provided in the first aspect of the invention, or any of the kits or vaccine combinations, including the homologous or heterologous vaccination regimes, as described in any of the second or third aspects of the invention, can be administered systemically or locally. In certain embodiments, the said recombinant vaccine compositions are administered parenterally, subcutaneously, intravenously, intramuscularly, or intranasally, in particular subcutaneously or intramuscularly. Preferably, the recombinant MVAs are administered intranasally. In other embodiments, the said recombinant vaccine compositions are administered by any other path of administration known to the skilled practitioner. In a further preferred embodiment, the said recombinant vaccine composition is administered intramuscularly, preferably the recombinant vaccine composition is administered intramuscularly in a volume ranging between about 0.10 and 1.0 ml, preferably containing concentrations of e.g., about 107to 109 virus particles/ml. Preferably, the said recombinant vaccine composition is administered in a volume ranging between 0.25 and 1.0 ml. More preferably, the said recombinant vaccine composition is administered in a volume of about 0.5 ml.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the appended claims. Examples
MATERIALS AND METHODS 1. Materials
1.1. Culture media DF-1 and HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with non-essential amino acids (1X, Sigma-Aldrich) and L-glutamine (2 mM, Merck) and with 10 % fetal calf serum (FCS) (Gibco). Opti-MEM medium (Gibco) was used as a transient culture medium during plasmid transfection. DMEM was used as a medium for absorbing the virus and was removed after 1 hour, and DMEM-2 % FCS was used as a medium for incubating infections. The solid infection medium was used to select the recombinant virus plaques and consisted of a 1:1 ratio of DMEM 2X-4 % FCS and 1.9 % previously melted agarose (Conda). All the cells were kept in a humidified incubator at 37°C and 5 % CO2.
1.2. Bacteria The bacterial strains used for plasmid growth and transformations during cloning were chemically competent Escherichia coli DH5a cells (CNB) and electrocompetent Escherichia coli DH10B cells (New England Biolabs). The bacteria were cultured in Luria- Bertani (LB) medium supplemented with 1 % bacto-tryptone (BD Biosciences), 1 % NaCI (Sigma-Aldrich), and 0.5 % yeast extract (BD Biosciences) at pH 7 in the presence of ampicillin (100 pg/ml, Merck) or kanamycin (100 pg/ml, Merck) (Green MR, Sambrook J. 2012. Molecular Cloning: a laboratory manual, 4 ed. Cold Spring Harbor Laboratory Press, New York, USA)
1.3. Coronavirus SARS-CoV-2 S antigen (SEQ ID NO 2)
The antigen used to generate recombinant MVA viruses (and the mammalian expression vector DNA-S) is the coronavirus SARS-CoV-2 S gene (Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1 ; the most contemporary isolate available at the time we initiated this work. GenBank accession number: MN908947.3). The nucleotide sequence of this protein was edited with codons optimized for humans by GeneArt (Thermo Fisher Scientific). 1.4. Virus
1.4.1. MVA-based viruses
The parental modified vaccinia virus Ankara (MVA) used for generating MVA-COVID- 19(S) vaccine candidates was a wild-type MVA (MVA-WT) donated by Dr. G. Sutter of the Institute of Molecular Virology of Munich (Germany), or an MVA-GFP previously generated from MVA-WT, by inserting the gene encoding green fluorescent protein (GFP) in the thymidine kinase (TK) locus and by eliminating immunomodulatory genes C6L, K7R, and A46R of MVA. The GFP present in the MVA-GFP TK locus was then replaced by the coronavirus SARS-CoV-2 S gene to generate the different vaccine candidates MVA-COVID1 9(S) or MVA-A-COVID19(S).
The S gene was inserted in the viral TK locus (gene J2R), and they are under the control of the synthetic early/late viral promoter (pE/L).
1.4.2. SARS-CoV-2 virus SARS-CoV-2 strain MAD6 (kindly provided by Jose M. Honrubia and Dr. Luis Enjuanes, CNB-CSIC, Madrid, Spain). SARS-CoV-2 MAD6 is a virus isolated from a patient from Hospital 12 de Octubre in Madrid. The virus was isolated, plaque cloned three times and amplified at CNB-CSIC. Full-length virus genome was sequenced, and it was found to be identical to SARS-CoV-2 reference sequence (Wuhan-Hu-1 isolate, GenBank: MN908947), except the silent mutation C3037>T, and two mutations leading to amino acid changes: C14408>T (in nsp12) and A23403>G (D614G in S protein).
1.5. Plasmids
1.5.1. Plasmid transfer vector used to generate recombinant MV As
A plasmid transfer vector pCyA was used for inserting the coronavirus SARS-CoV-2 S gene into the MVA genome. The cells were first infected with the parental MVA virus (MVA-WT or MVA-GFP) and then transfected with these plasmids. The recombinant MVA viruses were subsequently selected after several consecutive plaque purification steps. The process whereby a heterologous gene is inserted into the MVA genome is called homologous recombination, a type of genetic recombination in which nucleotides sequences are exchanged between two similar or identical DNA molecules. In this case, recombination occurs between the genetic sequences of the transfer plasmid and the viral genome. In order to insert the heterologous genes into the MVA genome, the transfer plasmids must contain the following elements: • Flanking regions of the desired insertion area: These are sequences including the left (L) and right (R) regions of the MVA TK gene such that they allow recombination processes whereby the antigens of interest are inserted in the TK locus.
• Antigens of interest: These must be located between the L and R flanking regions of the TK gene, such that as a consequence of recombination, the antigens of interest are inserted in the viral genome. These antigens were inserted into the plasmid multiple cloning site (MCS) under the control of the VACV synthetic early/late (sE/L) promoter cloned into that same site.
• Selectable marker gene in cell culture: This is located in the area adjacent to the genes of the antigens of interest and within the flanking regions, such that it is inserted into the viral genome next to the genes of interest. Another area of recombination (L flanking region) necessary for the later removal of the marker gene by additional recombination processes is found to be repeated between the antigens of interest and the marker. Recombinant MVAs containing only the antigen of interest are therefore generated, which is necessary for use as vaccine candidates in humans. In this invention, the LacZ (b-galactosidase) marker gene was used to select in cell culture the intermediary recombinant viruses forming blue lysis plaques, after insertion in the TK locus. Furthermore, in the case of insertion of the antigens of interest in the TK locus, the genetic sequence of GFP will be removed, and loss of green fluorescence in recombinant virus plaques may therefore be used as an additional selection criterion.
• Selectable gene in bacterial culture: To clone and grow plasmids, the ampicillin resistance gene (b-Lactamase) is included in such plasmids in order to select the bacteria transformed with that gene.
The plasmid transfer vector used to introduce the coronavirus SARS-CoV-2 S protein gene into the MVA TK locus is briefly described below:
■ pCvA-S: Plasmid transfer vector pCyA-S was used to generate MVA-COVID19(S) and MVA-A-COVID19(S) recombinant viruses and was developed by the CNB. It directs the insertion of the coronavirus SARS-CoV-2 S gene sequence, under the control of the VACV sE/L promoter, in the TK locus of the parental MVA-WT or MVA-A-GFP virus. We provided the pCyA vector to GeneArt (Thermo Fisher Scientific) for optimization of codons, synthesis of the S gene of SARS-CoV-2 and insertion into the plasmid vector. More specifically, the S gene was introduced between the VACV TK-L and TK-R flanking regions, under the control of the VACV sE/L promoter, in plasmid pCyA-20 MCS (Gomez et al., 2013, J Virol 87:7282- 7300). Furthermore, this plasmid also contains the selectable marker genes for ampicillin and b-galactosidase (LacZ gene). Polymerase chain reaction (PCR) and sequencing techniques confirmed that plasmid pCyA-S (11,322 bp) had been generated correctly.
1.5.2. DNA plasmid expressing SARS-CoV-2 S protein
A DNA plasmid expressing SARS-CoV-2 S protein was generated by inserting human codon optimized SARS-CoV-2 S gene (see section 1.3) into mammalian expression vector pcDNA 3.1. The plasmid generated was termed DNA-S, and was used in in vivo animal studies. Plasmid DNA-S was generated by Dr. Jose Maria Casasnovas (CNB, Madrid).
1.6. Mice
Female mice of strain C57BL/6JOIaFlsd (Envigo Laboratories) 6-8 weeks of age at the start of the experiment were used for the immunogenicity studies. These tests were approved by the Ethics Committee for Animal Testing (CEEA, Comite Etico de Experimentacion Animal) of the CNB (Spanish National Center for Biotechnology), and by the Division of Animal Protection of the Comunidad de Madrid (PROEX 49/20), in accordance with national and international guidelines and Spanish Royal Decree (RD 53/2013), and were carried out in the animal facility of the National Center for Biotechnology (CNB; Madrid, Spain) in a pathogen-free area.
Female transgenic humanized K18-hACE2 mice, expressing human ACE2, were obtained from the Jackson Laboratory (034860-B6.Cg-Tg(K18-ACE2)2Prlman/J) and efficacy experiments were performed at the BSL-3 laboratory of CISA-INIA (Madrid, Spain). The experiments performed in K18-hACE2 humanized transgenic mice at the CNB-CSIC and CISA-INIA were approved by the Ethical Committee of Animal Experimentation (CEEA) of the CNB-CSIC and CISA-INIA (Madrid, Spain) and by the Division of Animal Protection of the Comunidad de Madrid (PROEX 169.4/20). Animal procedures were conformed to international guidelines (European Union (EU) Directive 2010/63EU and Recommendation 2007/526/EC) and to the Spanish law under the Royal
Decree (RD 53/2013). 2. Reagents
2.1. Oligonucleotides
The following table (Table 1) shows the different oligonucleotides used for recombinant virus cloning, sequencing, and generation processes and those used in the PCR for detecting mycoplasma contamination.
Figure imgf000046_0002
Figure imgf000046_0001
Table 1. Oligonucleotides. Targets of the corresponding restriction enzymes are shown in bold. F = Forward (sense), R = Reverse (antisense).
2.2. Peptides and proteins 2.2.1. Peptides Various coronavirus SARS-CoV-2 S peptides were used for the stimulation of splenocytes isolated from mice immunized with vaccine candidates: ■ SARS-CoV-2 S peptide pools: These peptides are grouped into two mixtures, S1 (158 peptides) and S2 (157 peptides) (JPT). They are overlapping peptides of the coronavirus SARS-CoV-2 S-protein formed by 15 amino acids (15-mers), 11 of which are overlapping. Each peptide pool (S1 or S2) is at a concentration of 500 pg/ml.
2.2.2. Proteins
The coronavirus SARS-CoV-2 S and RBD proteins were used in enzyme-linked immunosorbent assays (ELISA) at a concentration of 2 pg/ml. Those proteins were provided by Dr. Jose Maria Casasnovas (CNB, Madrid) 2.3. Antibodies
The antibodies used in this invention are summarized in the following table (Table 2):
Figure imgf000047_0001
Figure imgf000048_0001
Table 2. Primary and secondary antibodies, a = anti.
3. Methodology
3.1. DNA manipulation techniques
The DNA manipulation techniques used were carried out according to the methodology described in the book Molecular cloning: a laboratory manual (Green MR, Sambrook J. 2012. Molecular Cloning: a laboratory manual, 4 ed. Cold Spring Harbor Laboratory
Press, New York, USA).
3.1.1. DNA purification
The methodology used for DNA purification differed according to the source of the genetic material:
- Purification of plasmid DNA from bacterial cultures: To extract plasmid DNA from a bacterial culture, the bacteria were previously grown in LB and then DNA was then extracted by means of different commercial kits. The purification of plasmid DNA during the molecular cloning process was performed using the Qiagen Plasmid Mini kit (Qiagen), from 2 ml of positive bacterial clone cultures, following the manufacturer’s instructions. Larger amount of purified plasmid DNA were obtained (from 200-ml bacterial cultures) for use in transfections and infections-transfections in cell cultures using the Qiagen Plasmid Maxi kit (Qiagen), following the manufacturer’s protocol. The plasmid DNAs used for in vivo experiments were purified by means of the Qiagen Plasmid endo-free Mega kit (Qiagen) from 500-ml bacterial cultures, following the manufacturer’s recommendations.
- Purification of DNA fragments: The purification of DNA from agarose gels or PCR reactions was carried out with the Wizard® Genomic DNA purification Kit (Promega), following the manufacturer’s recommendations. The purification of these DNA fragments was performed during the plasmid cloning processes and for sequencing regions of interest in plasmids and recombinant viruses to verify that they are generated correctly.
- Purification of genomic DNA of cells infected in culture: This technique was used to obtain the total DNA of cells infected with the recombinant viruses and to check for purity and that such viruses were generated correctly. For that purpose, the infected cells were collected when a cytopathic effect was observed and centrifuged at 3000 revolutions per minute (rpm) for 5 min at room temperature. The supernatant was removed, the precipitate was resuspended in 50 mM Tris-HCI pH 8, and proteinase K (200 pg/ml, Roche) in its corresponding buffer was added. After 1 hour of incubation at 55°C, RNAse A (40 pg/ml, PanReac AppliChem) was added, and it was incubated for 30 min at 37°C. Then saturated NaCI was added. It was mixed by inverting the tube and centrifuged at 13000 rpm for 10 min at room temperature. The supernatant was collected, mixed with isopropanol at a ratio of 1:0.7 to precipitate the DNA, and centrifuged at 13,000 rpm for 10 min at room temperature. The precipitate was washed with 75 % ethanol and centrifuged again at 13,000 rpm for 10 min at room temperature. Lastly the supernatant was carefully removed and the precipitated DNA was left to dry at room temperature, after which it was resuspended in sterile distilled water.
In all cases, DNA concentrations were measured in a NanoDrop® ND-1000 full-spectrum spectrophotometer (Thermo Fisher Scientific). Isolated DNA was later used for different experiments, including PCR, sequencing, transfections, infections-transfections, and in vivo assays in mice.
3.1.2. Polymerase chain reaction (PCR)
The PCR technique was used to amplify DNA fragments for later cloning or to check for the gene insertion in transfer plasmids or in the recombinant MVA genome. The polymerase used was Phusion® High Fidelity (New England Biolabs) with its corresponding buffers and reagents and following the manufacturer’s recommendations. About 10-100 ng of template DNA together with 0.4 mM of the corresponding oligonucleotides, 1-2.5 units of Phusion® High Fidelity polymerase with the corresponding buffer, and 0.2 mM of each of the four deoxynucleotide triphosphates (dNTPs) (Roche Diagnostics GmbH) were used for each reaction. The temperature used for annealing was selected according to the oligonucleotides used, and the extension time used depended on the length of the fragment to be amplified. The reactions were carried out in a 96-well VeritiTM thermocycler (Applied Biosystems).
3.1.3. DNA detection
To separate the different DNA fragments, the electrophoresis technique in 1 % agarose gel (Conda), with 1 % SYBR Safe (Invitrogen) as an intercalating agent was used. The DNA fragments were then viewed by means of the Gel Doc 2000 UV light transilluminator system (Bio-Rad) and QuantityOne software (Bio-Rad).
3.1.4. Gene cloning into plasmids transfer vectors
Coronavirus SARS-CoV-2 S gene was cloned into transfer plasmid pCyA to generate plasmid transfer vector pCyA-S by GeneArt (Thermo Fisher Scientific).
3.2. Protein manipulation techniques
3.2.1. Protein analysis by means of sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis and membrane immunodetection (Western blot) The protein samples of the cell extracts and supernatants were analyzed by means of one-dimensional sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis according to standard protocols previously described (Green MR, Sambrook J. 2012. Molecular Cloning: a laboratory manual, 4 ed. Cold Spring Harbor Laboratory Press, New York, USA). In detail, the infected or transfected cell samples were collected, centrifuged at 2,000 rpm for 5 min at room temperature, and the precipitate was resuspended in Laemmli IC-b-mercaptoethanol loading buffer. The cell supernatants were also centrifuged at 2,000 rpm for 5 min at 4°C to separate them from T cells that might be present. Then the proteins were precipitated from the supernatant with 10 % trichloroacetic acid (TCA) (Sigma-Aldrich), centrifuged at 13,000 rpm for 15 min at room temperature, and the precipitate was resuspended in Laemmli 1C-b- mercaptoethanol. After that, both the cell extract and supernatant samples were denatured at 95°C for 10 min and were loaded in 7 % polyacrylamide gels in the presence of SDS. The proteins were separated by means of one-dimensional electrophoresis at 100 V and at room temperature for about 90 min.
Once electrophoresis ended, the SDS-PAGE gels were transferred to nitrocellulose membranes (GE Healthcare), following the wet system protocol recommended by the manufacturer (Mini Trans-Blot® Cell, Bio-Rad). The gel and previously hydrated nitrocellulose membrane were arranged between two Whattman-3MM® filter papers (GE Healthcare), which were also hydrated. They were then mounted in the transfer system in the presence of the transfer buffer, and the transfer was performed at a current intensity of 100 V for 50 min. Once this ended, checking for the presence of proteins was performed by staining the membrane with the reversible Ponceau S stain (0.2 % Ponceau in 3 % TCA; Sigma-Aldrich).
The nitrocellulose membrane was then blocked with a 5 % skim milk powder solution prepared in PBS 1X-0.05 % Tween20 (Sigma-Aldrich) (PBS-T) at room temperature for 1 hour with gentle stirring. Next, the corresponding primary antibodies, prepared at the desired dilution in the blocking buffer, were added and incubated under stirring at 4°C overnight. After four washes with PBS-T, the membrane was incubated with the appropriate secondary antibody (suitably diluted in the same blocking buffer) for 1 hour at room temperature and was then washed again four times with PBS-T. The membranes were developed with the ECL® luminol system (Amersham), exposing Carestream Kodak BioMax XAR autoradiography film (Kodak) or directly in the ChemiDoc™ Imaging System (Bio-Rad). The proteins bands were analyzed and quantified by means of Image Lab software (Bio-Rad). 3.3. Virus manipulation techniques
3.3.1. Viral infections
All the viruses were kept at -70°C and thawed at 37°C in a bath prior to use. Once thawed, they were stirred with a vortex and sonicated with a 3-cycle 10-second sonication and 10- second pause program (Misonix Incorporated S-3000 Sonicator, Cole-Parmer). The viruses were then added to the cell monolayer with the minimum volume of (serum-free) DMEM needed to cover the cell monolayer at the desired MOI. After 1 hour of adsorption at 37°C, the inoculum was removed and fresh DMEM-2 % FCS medium was added. The time the different infections were maintained varied with the objective of the infection, and the manner in which the cells were collected also varied.
3.3.2. Generation of recombinant MVA viruses
In this invention, 2 recombinant MVA viruses expressing coronavirus SARS-CoV-2 S- protein were generated in DF-1 cells according to the previously described standard infection/transfection protocol (Earl et al. , 2001, Curr Protoc Mol Biol, 16:16 17), with certain modifications. To generate vaccine candidate MVA-COVID19(S), MVA-WT was used as the parental virus, and to generate vaccine candidate MVA-A-COVID19(S), MVA-A-GFP was used as a parental virus, as previously described (Garcia-Arriaza et al., 2014, J Virol 88:3527-3547).
Figure 11 shows a diagram summarizing the process followed for generating the recombinant MVA viruses. In detail, the DF-1 cell monolayer cultured in p60 dishes (Nunc) at 80-90 % confluence was infected at a MOI of 0.05 PFU/cell with the corresponding parental virus. In the meantime, a mixture of DNA-lipofectamine 2000 was prepared by mixing 10 pg of the corresponding transfer plasmid with the suitable volume of lipofectamine 2000 (Invitrogen) in Opti-MEM (Gibco) (1.5 pi lipofectamine 2000/pg of DNA) for 20 min at room temperature to form DNA-liposome complexes. After 1 hour of adsorption of the virus, the inoculum was removed, the cells were washed two times with Opti-MEM and incubated with 1 ml of the DNA-lipofectamine mixture for 4-6 hours at 37°C and 5 % CO2. Next the transfection medium was removed, the cells were washed two times with Opti-MEM and incubated with DMEM-2 % FCS medium at 37°C and 5 % CO2. When a cytopathic effect was observed (usually after 3 days), the cells were collected, centrifuged at 1,500 rpm for 5 min at room temperature, and resuspended in 500 mI of serum-free DMEM. The cells were then frozen (at -70°C) and thawed (at 37°C) three times, stirring with a vortex between each freeze-thaw cycle to lyse them and release the virus into the supernatant, which was obtained after centrifuging at 3,000 rpm for 5 min at room temperature. The virus was then used to infect confluent monolayers of DF-1 cells grown in 6-well plates (M6) (Nunc) at different serial dilutions (from 101 to 106) in serum-free DMEM. After 1 hour of infection, the inoculum was removed and replaced with 3 ml of solid infection medium consisting of a mixture of 1.9 % (previously melted) agarose (Conda) with DMEM 2X-4 % FCS medium at a 1 :1 ratio. After 48 hours of infection, 1 ml of the 1.9 % agarose-DMEM 2X-4 % FCS 1 :1 mixture and 1.2 mg/ml of X- Gal substrate (Sigma-Aldrich) or 0.8 mg/ml of the substrate X-Gluc (Sigma-Aldrich), whichever is appropriate, was added to view the lysis plaques produced by the virus incorporated in the insert of interest along with the LacZ gene or b-Gus, respectively. After 24 hours, the blue plaques that were more isolated from other lysis plaques expressing GFP (green fluorescence), originating from the parental virus, were selected. The selected lysis plaques were obtained with a 150-mm glass Pasteur pipette (Deltalab) and added to 500 pi of serum-free DMEM. After three freeze-thaw cycles and subsequent stirring with a vortex and sonication, these plaques were used as an inoculum to infect monolayers of confluent cells in 24-well plates (M24) (Nunc) as explained in section 3.3.1. These infections were performed to re-grow the virus, and they were collected when a cytopathic effect was observed. These regrown plaques were next used to re-infect monolayers of confluent cells in M24 plates, and once a cytopathic effect was observed, these cells were collected and lysed for PCR and Western Blot analysis to check that the recombinant MVA virus was generated correctly. Oligonucleotides TK-L and TK-R were used in the PCR to amplify the antigens inserted in the TK locus (oligonucleotides described in Table 1). Anti-S-protein rabbit polyclonal or mouse monoclonal antibodies (described in Table 2) were used to analyze expression of the antigens of interest by Western blot.
Once the presence of the antigen and its correct expression by the different plaques were confirmed, one of them was selected and used as an inoculum to infect monolayers of confluent cells in M6 plates at dilutions from 101 to 106. This purification process was carried out three times selecting blue plaques, and three more times selecting colorless plaques (neither blue nor GFP) (Figure 11). A purified recombinant virus containing the antigen of interest in the TK locus and without the LacZ marker, which was lost through homologous recombination, is therefore ultimately obtained.
Three types of viral stocks are distinguished in this invention. The initial viral stock (stock P1 ) consists of the generated recombinant virus originating from cell monolayers infected with the final plaque and collected after observing a cytopathic effect. Once having verified the expression of the antigens of interest by means of Western Blot and the purity of the recombinant virus by means of PCR and sequencing, it was used to generate the working viral stock (stock P2). For that purpose, 5 p150 plates (Nunc) containing confluent DF-1 cells were infected at an MOI of 0.05 PFU/cell and cells were collected when a cytopathic effect was observed. Stock P2 was used in all the experiments for characterizing the virus in vitro. Lastly, for the in vivo assays, the purified viral stock (stock P3) was generated from stock P2 (described in section 3.3.4.).
3.3.3. Characterization of the recombinant MVA viruses The experiments performed to characterize the different recombinant MVA viruses include titration of the viruses, viral growth kinetics, stability analysis of the inserted antigens, and analysis of their apoptotic capacity. As mentioned above, stock P2 was used to perform these characterization experiments.
• Viral titration Titration of the MVA viruses was carried out according to the previously described standard protocols (Ramirez et al. , 2000, J. Virol, 74:923-933). DF-1 cells seeded in M6 plates were infected in duplicate with serial dilutions of the virus in serum-free DMEM, as described in section 3.3.1. The medium was removed 30-40 hours post-infection and the cells were fixed for 3 min using a 1 : 1 mixture of methanol and acetone and the titer was determined by means of an immunostaining assay which allows detecting and counting lysis plaques formed by the virus. The monolayers were thereby incubated with a rabbit anti-VACV WR polyclonal primary antibody (diluted 1 : 1.000 in PBS-3 % FCS; CNB) for 1 hour at room temperature; it was then washed three times with PBS 1X and incubated for 1 hour at room temperature with the HRP-conjugated goat anti-rabbit secondary antibody (diluted 1 : 1.000 in PBS-3 % FCS; Sigma-Aldrich). After three additional washes with PBS 1X, the plates were developed using 1 mg/ml of 3,3’-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich), as the HRP substrate, with 30 % hydrogen peroxide (Sigma- Aldrich) and 3 % nickel sulfate (NiSC ) (Sigma-Aldrich). After counting the lysis plaques, the titer was referenced as PFU/ml. · Replication kinetics
To evaluate the replication capacity of the different recombinant MVA viruses and analyze if the insertion of the S gene affects their replication capacity, monolayers of DF-1 cells were infected with each virus at an MOI of 0.01 PFU/cell, in duplicate. After 1 hour of adsorption, the inoculum was removed and was added to the DMEM-2 % FCS infection medium monolayer. The cells were collected at different times post-infection (0, 24, 48, and 72 hours), subjected to three freeze-thaw cycles (stirred with a vortex between each cycle), and kept at -70°C until titration. Lastly, the viruses were sonicated and titrated using the method described in the preceding section.
• Genetic stability analysis
The genetic stability of vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) was analyzed. Monolayers of DF-1 cells were infected with the different recombinant viruses at an MOI of 0.05 PFU/cell. The cells were collected 72 hours post-infection, frozen-thawed three times, and briefly sonicated. The cell extracts were then centrifuged at 1 ,500 rpm for 5 minutes, and the supernatant was used for a new round of infection at a low MOI. The same method was repeated nine times. Expression of the coronavirus SARS-CoV-2 S-protein was analyzed by means of Western blot (see section 3.2.3.) after infecting DF-1 cells with the viral stocks from each passage.
3.3.4. Virus purification
Purified virus stocks (stock P3) were used for the in vivo assays according to the method first described by Joklik (Joklik, 1962, Virology, 18:9-18) and modified by Esteban (Esteban, 1984, Virology, 133:220-227). In summary, 30 p150 plates containing confluent CEF cells were infected with stock P2 at an MOI of 0.01 PFU/cell. The cells were collected when a cytopathic effect was observed (usually after 3 days), centrifuged at 1 ,500 rpm for 5 min at 4°C, and the precipitate was washed with PBS and resuspended in 10 mM Tris-HCI buffer pH 9. The infected cells were then lysed by means of three sonication cycles, stirring with a vortex, and centrifugation to release the viral particles into the supernatant. Next, this supernatant was centrifuged for 1 hour at 20,000 rpm at 4°C in the SW28 rotor (Beckman) on a 36 % sucrose pad in 10 mM Tris-HCI pH 9. The obtained precipitate was resuspended in 10 mM Tris-HCI pH 9 and centrifuged again on another 36 % sucrose pad under the same conditions. Lastly, the precipitate obtained in this second centrifugation was resuspended in 10 mM Tris-HCI pH 9, aliquoted, and frozen at -70°C until use.
The purified viruses were tittered in duplicate and checked for the absence of mycoplasma contamination (through PCR with mycoplasma-specific oligonucleotides; see Table 1), bacteria (by means of growth in LB agar plates), and fungi (by means of growth in blood agar plates, Oxoid). Furthermore, checking for the correct expression of the antigens of stocks P3 was performed by means of PCR and Western blot before use in the in vivo assays. 3.4. In vivo immunological techniques 3.4.1. Immunization protocols in mice
In assays for analyzing adaptive immune responses, two different immunization protocols were carried out. In all the assays, 6 female C57BL/6 mice per group were inoculated by intramuscular route. On one hand, a homologous prime-boost immunization was used, which consisted of a first inoculation with 1 x 107 PFU of vaccine candidates MVA- COVID19(S) and MVA-A-COVID19(S), followed by a 1 x 107 PFU booster dose of the same vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) 2 weeks later. The mice were sacrificed 11 days after the booster dose. On the other hand, a heterologous prime-boost immunization regimen was used, which consisted of a first inoculation with 100 pg (50 pg/leg) of a plasmid vector expressing the coronavirus SARS-CoV-2 S-protein (DNA-S), followed by a 1 x 107 PFU booster dose of vaccine candidates MVA- COVID19(S) and MVA-A-COVID19(S) 2 weeks later. The mice were sacrificed 11 days after the booster dose as well. Moreover, to evaluate the long-term memory immunogenicity of the MVA-S vaccine candidate, a MVA-S prime/MVA-S boost immunization protocol was performed in female C57BL/6 mice (6 to 8 weeks old) as described above. Groups of animals (n=5) received two doses of 1 x 107 PFU of MVA-S or MVA-WT (used as negative control mice) by the intramuscular route in 100 pi of PBS (50 mI/leg) at 0 and 2 weeks. At 6 months after the last immunization, mice were sacrificed using CO2.
Furthermore, to evaluate the immunogenicity elicited by the MVA-COVID19(S) and MVA- A-COVID19(S) vaccine candidates after intranasal administration, a MVA prime/MVA boost immunization protocol was performed in female C57BL/6 mice (6 to 8 weeks old). Groups of animals (n=5) were slightly anesthetized with Isoflurane (2-chloro-2- (difluoromethoxy)-l ,1,1 -trifluoro-ethane) and received two doses of 1 x 107 PFU of MVA- S, MVA-A-S or MVA-WT (used as negative control mice) by the intranasal route in 50 mI of PBS at 0 and 2 weeks. At 11 days post-boost mice were sacrificed using CO2.
All the preparations of the inocula were performed in endotoxin-free PBS (Gibco). Intramuscular immunization was performed in the triceps of both hind legs in a total volume of 100 mI of PBS (50 mI/leg), using a BD Micro-Fine (30 G) x 8 mm insulin syringe (BD Biosciences). Intranasal immunization was performed in a total volume of 50 mI of PBS, using a micropipette. At the end of each immunization study the corresponding animals were sacrificed using CO2 (or euthanized by isoflurane overdose in mice immunized intranasally). Next, blood, bronchoalveolar lavage (BAL) samples, spleen and lungs were collected and processed. Blood from each individual mouse was collected directly from heart and was allowed to clot 60 min at 37°C; after leaving samples at 4°C overnight, they were centrifuge 20 minutes at 3600 rpm, and sera were removed and stored at -20°C until used in ELISA and neutralization experiments. In the intranasal experiments, also lung and bronchoalveolar lavage (BAL) samples were collected and processed. BAL from each individual mouse were taken by flushing 1ml of RPMI 1640 (Gibco-Life Technologies) medium supplemented with HEPES pH 7.4 (10 mM, Merck), b-mercaptoethanol (10 mM, Sigma-Aldrich), L-glutamine (2 mM, Merck), and 10% FCS into the trachea; then samples were spun down to remove cellular debris and supernatants kept at -20°C until used for ELISA and neutralization experiments. Spleens and lungs were extracted, processed mechanically or enzyme-digested, respectively, blood-cell depleted and filtered through 40pm cell strainers until obtain single-cell samples.
3.4.2. Analysis of the adaptive T cell-mediated immune response by means of ELISpot
The ELISpot assay was used to detect S antigen-specific IFNy-secreting T cells (300). First, 96-well nitrocellulose-bottom plates (Millipore) were covered with 75 pl/well of a solution of the rat anti-mouse IFNy monoclonal antibody (Pharmingen, San Diego, California) at a concentration of 6 pg/ml in PBS. After incubating overnight at room temperature, the wells were washed three times with RPMI medium and blocked with RPMI-10 % FCS for at least one hour at 37°C in 5 %C02 atmosphere. Moreover, the spleens of each group of immunized animals were collected in RPMI-10 % FCS medium and processed together to obtain the splenocytes as previously described (Garcia- Arriaza et al., 2013, PLoS One, 8:e66894; Najera et al. , 2010, PLoS One, 5:e11406). In summary, the spleens were homogenized by means of mechanical disintegration through a 40-pm cell strainer (Falcon). The disintegrated cells were centrifuged for 5 min at 1,500 rpm at 4°C and washed with RPMI-10 % FCS. Next, the erythrocytes were lysed by adding 0.1 M NH4CI (2 ml/spleen) for 5 min in ice. After this time, the cells were washed three times with RPMI-10 % FCS to eliminate fat and resuspended in 12 ml of RPMI- 10 % FCS, and the number of living splenocytes was counted by means of staining with trypan blue (4 % in water; Sigma-Aldrich).
To evaluate the specific response to the different antigens, 106 splenocytes per condition were re-stimulated with 1 pg/ml of the S1 or S2 peptide pool. As a control of the response to the MVA vector, 106 splenocytes were stimulated with 2.5 pg/ml of VACV E3 peptide. The plates were incubated with the peptides for 48 hours at 37°C in 5 % CO2 atmosphere, washed five times with PBS-T, and incubated with 2 pg/ml of biotinylated rat anti-mouse IFNy monoclonal antibody XMG1.2 (Pharmingen) diluted in PBS-T, for 2 hours at room temperature. The plates were then washed five times with PBS-T and a 1 :800 dilution of HRP-conjugated streptavidin (0.5 mg/ml; Sigma-Aldrich) was added. After 1 hour at room temperature, it was washed three times with PBS-T and two times with PBS, and finally 1 pg/ml of the DAB substrate (Sigma-Aldrich), resuspended in 50 mM Tris-CI pH 7.5 and 0.015 % H2O2, was added to develop the plates. The reaction was stopped by washing the plate with abundant water and once it was dry, the spots were counted using the ELISpot Reader System -ELR02- plate reader (AID Autoimmun Diagnostika GmbH) with the aid of AID ELISpot reader system software (Vitro).
3.4.3. Analysis of the adaptive cell-mediated immune response by means of flow cytometry
3.4.3.1. Analysis of the specific responses of CD4+ and CD8+ T cells To study the response of SARS-CoV-2-specific T cells generated by the different immunization protocols (see section 3.4.1.), 6 mice per group were immunized and sacrificed 11 days after the last immunization. After sacrificing the animals, the spleens (or lungs) were removed from the immunized animals in RPMI-10 % FCS medium and processed as described in the preceding section. The splenocytes or lung cells were then resuspended in RPMI-10 % FCS medium with 2 pl/ml of Monensin 1X (Thermo Fisher
Scientific), 2 pl/ml of brefeldin A (BFA) ( Protein Transport Inhibitor BD GolgiPlug, BD Biosciences), and a 1 :300 dilution of anti-CD107a-FITC antibody. 4x 106 cells were added in M96 conical bottom wells and were incubated for 6 hours in the presence of 1 pg/ml of the S1 or S2 peptide pool, or with 5 pg/ml of VACV E3 peptide to determine the response to the MVA vector. As controls, the splenocytes or lung cells were stimulated with 2 pl/ml of LAC (Leukocyte Activation Cocktail, BD Biosciences) and Monensin 1X. After 6 hours of stimulation, the splenocytes or lung cells were washed with IB buffer and incubated with Violet Dye (0.5 pl/ml, Invitrogen) for 20 min in the dark at 4°C to evaluate cell viability. Next, two washes were performed with IB buffer and the splenocytes or lung cells were incubated for 20 min in the dark at 4°C with 50 pi of the primary surface antibodies
(described in the Table 2) diluted as indicated for each batch. The splenocytes or lung cells were then washed two times and 100 pi of Cytofix/Cytoperm (BD Biosciences) was added per well for 20 min in the dark to 4°C for fixing and permeabilizing the cells, and they were then left in IB buffer overnight at 4°C in the dark. A wash was performed the next day with Permwash 1X (BD Biosciences) and 25 mI of Fc Block diluted 1:100 in Permwash 1X were added for 5 min at 4°C in the dark. Next, 25 mI of the antibodies needed for intracellular cytokine staining (ICS) were added to the 25 mI of Fc Block, at different dilutions in Permwash 1X according to the batch of each antibody (see Table 2), with incubation being performed for 20 min at 4°C in the dark. Lastly, two washes were performed with Permwash 1X and the cells resuspended in 200 mI of IB buffer to later be analyzed by means of flow cytometry in a Gallios cytometer (Beckman Coulter) and by means of the FlowJo program ( Tree Star, Ashland, Oregon). The analysis strategy was performed by selecting the living cells expressing CD3 on their surface; then those expressing CD4, CD8, CD127, or CD62L; and finally those secreting the different cytokines (IFN-y, TNF-a, and IL-2) or expressing degranulation marker CD107a.
3.4.3.2. Analysis of the specific responses of follicular helper T cells
To analyze the response of follicular helper T cells (Tfh) against SARS-CoV-2, spleens were extracted from sacrificed animals and processed as explained above. The splenocytes were then resuspended in RPMI-10% FCS medium with 1 mI/ml of BFA (BD Biosciences), 1 mI/ml of Monensin 1X (eBioscience), and a 1:100 dilution of the anti- CD154 (CD40L)-PE antibody. Next, 4 x 106 splenocytes were stimulated with 5 pg/ml of S-protein and 1 pg/ml of S1 and S2 peptide pools for 6 hours at 37°C in a conical-bottom 96-well plate. The cells were then stained with Fixable Viability Stain (FVS) 520 (BD Biosciences) for 20 min at 4°C in the dark to analyze cell viability. The splenocytes were then washed two times with IB buffer and stained for 20 min at 4°C in the dark with 50 pi of the antibodies specific against the surface markers (described in Table 2), using the dilutions recommended by the manufacturer. Next, the splenocytes were again washed two times with IB buffer, fixed, and permeabilized with the BD Cytofix/Cytoperm™ kit (BD Biosciences) for 20 min at 4°C in the dark, and they were left in IB buffer overnight at 4°C in the dark. The cells were washed the next day with Permwash 1X (BD Biosciences) and the Fc receptors were blocked with 25 pi of Fc Block (diluted to 1 : 100 in Permwash 1X) for 5 min at 4°C in the dark. Next, 25 pi of the antibodies (diluted in Permwash 1X at the dilution indicated by the manufacturer) required for ICS (see Table 2) were added on 25 pi of Fc Block, incubating for 20 min at 4°C in the dark. Finally, the splenocytes were washed two times in Permwash 1X, resuspended in 200 pi of IB buffer, and passed through a Gallios flow cytometer (Beckman Coulter). The analysis was performed with the FlowJo software ( Tree Star, Ashland, Oregon). The analysis strategy was performed by selecting living cells expressing CD3, CD4, and CD44 on their surface, followed by cells expressing CXCR5, PD1, and CD154 (CD40L), and finally cells secreting the different cytokines (IFN-y, IL-4, and IL-21).
3.4.4. Analysis of the humoral immune response
The humoral immune response was determined by analyzing the levels of IgG or IgA antibodies in the sera or BAL samples, respectively, of immunized mice and evaluating the capacity of these antibodies to neutralize the SARS-CoV-2 virus. To obtain the sera, blood was collected from the animals after sacrificing them by means of intracardiac puncture. Next, the blood samples were maintained at 37°C for 1 hour and kept at 4°C overnight. The tubes of blood were centrifuged the next day at 3600 rpm for 20 min at 4°C and the serum which was kept at -20°C until use was obtained. Serial dilutions of each serum or BAL sample (or group of sera) were then performed to measure the levels of coronavirus SARS-CoV-2 S-protein-specific and RBD-specific antibodies by means of ELISA, or to measure their SARS-CoV-2 neutralizing capacity by means of a luciferase assay or a live-virus SARS-CoV-2 neutralization assay. These techniques are explained in the following sections.
3.4.4.1. Enzyme-linked immunosorbent assay (ELISA)
ELISA assays were performed to analyze the levels of antibodies in the serum or BAL samples of immunized animals. NUNC MaxiSorp™ 96-well plates (Thermo Fisher Scientific) were coated with 2 pg/ml of the coronavirus SARS-CoV-2 S-protein and RBD in PBS and incubated at 4°C overnight. The plates were washed three times the next day with PBS-T and blocked for 2 hours at room temperature with 5% skim milk prepared in PBS-T. After this time elapsed, the plates were washed three times with PBS-T, 50 pi of the serial dilutions of the serum or BAL samples diluted in PBS with 1% skim milk and 0.01% Tween20 were added, and the plates were incubated for 1.5 hours at room temperature. Next, the plates were again washed three times with PBS-T and incubated with 50 mI of HRP-conjugated mouse anti-lgG, -lgG1, -lgG2c, or-lgG3 antibodies (diluted to 1:1000 in PBS with 1% skim milk and 0.01% Tween20) for 1 hour at room temperature. Finally, after another washing step, the plates were developed by adding 100 mI of TMB substrate (Sigma-Aldrich) and the reaction was stopped by adding 50 mI of 1 M H2SO4. Absorbance was read at 450 nm by means of an EZ Read 400 microplate reader (Biochrom).
3.4.4.2. Neutralization assay The capacity of the sera or BAL samples obtained from mice immunized with vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S), to neutralize the SARS-CoV-2 S virus was determined using lentivirus-based pseudoparticles expressing the SARS- CoV-2 S-protein (generated by Dr. Pablo Gastaminza and Dr. Urtzi Garaigorta; CNB, Madrid). Vero E6 cells were seeded in 96-well plates at 15,000 cells/well. Twenty-four hours post-seeding, a mix of SARS-CoV-2 pseudoparticles and mice serum samples (at different dilutions) were preincubated for 1 hour at 37°C and then added to the cells in triplicates. The DMEM-2%FCS medium was replaced 24 h after infection. Cells were lysed 48 h later and luciferase assays were performed as described: cells were lysed with reporter lysis buffer (Promega, Madison, Wl) and luciferase activity measured for 2.5 seconds in a luminometer (Orion II Microplate Luminometer, Berthold Detection Systems, Pforzheim, Germany).
Furthermore, live-virus SARS-CoV-2 neutralization antibody titers were assessed by a microneutralization test (MNT), using SARS-CoV-2 MAD6 strain in a BSL-3 laboratory at the CNB-CSIC. Serum samples were inactivated at 56°C for 30 min and then serially diluted two-fold in duplicate in serum-free DMEM and incubated at a 1:1 ratio with 100 TCID50 (50% Tissue Culture Infectious Dose) of SARS-CoV-2 MAD6 isolate at 37° C for 1 hour. The virus/serum mixture were added to VeroE6 cell monolayers, seeded in 96- well plates (40.000-50.000 cells per well), and incubated at 37° C, in a 5% CO2 incubator for 3 days. Then, cells were fixed with 4% formaldehyde and stained with crystal violet. ID50 titers were calculated as the reciprocal dilution resulting in 50% inhibition of cell death.
3.5. Efficacy study in K18-hACE2 transgenic mice
Female K18-hACE2 mice (10 weeks old at the beginning of the study) immunized with one or two doses of MVA-S were used to evaluate the efficacy of the MVA-S vaccine candidate. Groups of animals (n=11) received one or two doses of 1 x 107 PFU of MVA- S by the intramuscular route in 100 pi of PBS (50 mI/leg) at 0 and 4 weeks. Mice primed and boosted with nonrecombinant MVA-WT or not vaccinated were used as control groups. At week 9, mice were challenged with a lethal dose (1 x 105 PFU) of SARS-CoV- 2 (MAD6 strain) by the intranasal route in 50 mI of PBS, and one group of unvaccinated mice were left uninfected. Mice were monitored for body weight change and mortality for 15 days postchallenge. Animals with more than a 25% of weight loss were euthanized. At days 2 and 4 postchallenge, three mice per group were euthanized, and lungs and serum samples were collected. One lung was inactivated in zinc formalin (Sigma-Aldrich) for 48 hours and then included in paraffin and processed for histopathological analysis. The other lung was divided longitudinally in two, with one part placed in RNALater stabilization reagent (Sigma-Aldrich) and stored at -80°C until RNA extraction, and the other part stored also at -80°C until analysis of virus yields. Blood was collected from the animals by submandibular bleeding, maintained at 37°C for 1 hour, kept at 4°C overnight, and centrifuged at 3600 rpm for 20 min at 4°C to obtain the serum samples, which was then inactivated at 56°C for 30 min and kept at -20°C until use.
Moreover, those surviving mice were re-infected with SARS-CoV-2 virus. Thus, K18- hACE2 mice previously vaccinated with one or two doses of MVA-S (or control unvaccinated and unchallenged mice) (n=5/group) were challenged at week 16 with a lethal dose (1 x 105 PFU) of SARS-CoV-2 (MAD6 strain) by the intranasal route in 50 pi of PBS. Mice were monitored for body weight change and mortality for 6 days postchallenge, moment at what control infected mice lost more than a 25% of the initial body weight and have to be sacrificed. At day 6 postchallenge, all mice were euthanized, and lungs and serum samples were collected and processed similarly as above.
3.5.1. Analysis of SARS-CoV-2 RNA by quantitative RT-PCR (RT-qPCR). Lungs from K18-hACE2 mice used in the efficacy study were harvested at the indicated timepoints and stored in RNALater (Sigma-Aldrich) at -80°C until homogenized with a gentleMACS dissociator (Miltenyi Biotec) in 2 ml of RLT buffer (Qiagen) plus b-mercaptoethanol (Sigma-Aldrich) and aliquoted. Then, 600 mI of homogenized lung tissue was used to isolate total RNA using the RNeasy minikit (Qiagen), according to the manufacturer's specifications. First-strand cDNA synthesis and subsequent real-time PCR were performed in one step using NZYSpeedy One-step RT-qPCR Master Mix (NZYTech) according to the manufacturer’s specifications using ROX as reference dye. SARS-CoV- 2 viral RNA content was determined using previously validated set of primers and probes specific for the SARS-CoV-2 subgenomic RNA for the protein E, the genomic virus RNA dependent RNA polymerase (RdRp) gene and the cellular 28S rRNA for normalization. Data were acquired with a 7500 real-time PCR system (Applied Biosystems) and analyzed with 7500 software v2.0.6. Relative RNA arbitrary units (A.U.) were quantified relative to the negative group (uninfected mice) and were performed using the 2-AACt method. All samples were tested in triplicate.
3.5.2. Analysis of SARS-CoV-2 virus yields by plaque assay. Lungs from K18-hACE2 mice used in the efficacy study were harvested at the indicated timepoints, weighted, and stored directly at -80°C until homogenized with a gentleMACS dissociator (Miltenyi Biotec) in 2 ml of PBS buffer and aliquoted. Then, undiluted and serial ten-fold dilutions of homogenized lung tissue were added in triplicate to Vero-E6 cell monolayers seeded in 12-well plates at 5 x 105 cells/well and after 1 hour of adsorption the inoculum was removed and plates were incubated at 37°C, 5% CO2 in 2:1 DMEM 2X-4% fetal bovine serum (FBS):2% Agar. After 4 days, cells were fixed for 1 hour with 10% formaldehyde (Sigma-Aldrich), and then the agarose was removed and plaques were visualized by adding 0.5% crystal violet (Sigma-Aldrich). SARS-CoV-2 titers were determined in PFUs per gram of lung tissue.
3.5.3. Lung cytokine profile analysis by RT-qPCR. Reverse transcription of 1000 ng of RNA isolated as described above from lung homogenates of K18-hACE2 mice was performed with the QuantiTect reverse transcription kit (Qiagen, Hilden, Germany), according to the manufacturer’s recommendations. RT-qPCR was performed with a 7500 real-time PCR system (Applied Biosystems) using Power SYBR green PCR Master Mix (Applied Biosystems), as previously described. The m RNA expression levels of the genes for IL-6, TNF-a, IL-10, IP-10 and IFN-y were analyzed by real-time PCR with specific oligonucleotides (sequences are available upon request). Specific gene expression was expressed relative to the expression of the cellular 28S ribosomal RNA gene in fold change units using the 2-AACt method. All samples were tested in triplicate.
3.5.4. Lung histopathology. For histological examination, lung tissues were fixed in zinc formalin embedded in paraffin. Tissue sections (5 pm) were stained with hematoxylin and eosin and analyzed for lung damage.
3.6. In vivo immunological and efficacy techniques in hamsters
3.6.1. Hamsters. Wild-type Syrian gold hamsters (Mesocricetus auratus), purchased from Janvier Laboratories, were housed per 2 in individually ventilated cages (cage type GR900, Sealsafe Plus, Tecniplast), at 21 °C, 55% humidity and 12:12 lightdark cycles. Food and water was provided ad libitum, as well as extra bedding material and wooden gnawing blocks. This project was approved by the KU Leuven ethical committee (project 015/2020), following institutional guidelines approved by the Federation of European Laboratory Animal Science Associations (FELASA). Hamsters were euthanized with an intraperitoneal (i.p.) overdose of 500 pi Dolethal (200 mg ml-1 sodium pentobarbital, Vetoquinol SA).
3.6.2. Immunization, infection and sampling. Hamsters were intraperitoneally primed with 2 x 107 PFU of MVA-S or MVA-WT on day 0, and boosted with 4 c 107 PFU of MVA- S or MVA-WT on day 21. A third group received no prime dose (phosphate-buffered saline) on day 0, but only a dose of 4 c 107 PFU of MVA-S on day 21. On day 42, each hamster was inoculated intranasally by gently adding 50 pi droplets of virus stock containing 2 c 105 TCID50 of SARS-CoV-2 in the nostrils. During infection, hamsters were anaesthetized by intraperitoneal injection of a xylazine (16 mg kg— 1 , XYL-M, V.M.D.), ketamine (40 mg kg— 1 , Nimatek, EuroVet) and atropine (0.2 mg kg— 1 , Sterop) solution. The animals were monitored daily for signs of disease (lethargy, heavy breathing or ruffled fur). On indicated numbers of days after challenge (2, 4 and 14 days), hamsters were euthanized (n=4/group) to collect end serum and lung tissue in RNAIater, MEM or formalin for gene-expression profiling, virus titration or histopathological analysis, respectively. All hamsters were bled at days 21 (before boost) and 39 (before challenge) to analyse serum for binding and neutralizing antibodies against SARS-CoV-2.
3.6.3. SARS-CoV-2 gene expression by RT-qPCR. The presence of SARS-CoV-2 RNA in hamster lung homogenates was quantified by RT-qPCR. RNA was extracted from homogenized lungs (bead disruption by Precellys homogenizer) using the E.Z.N.A.® Total RNA Kit I (Omega Bio-tek) following the manufacturer’s instructions. The iTaq Universal Probes One-Step RT-qPCR kit (BioRad) was used to perform reactions. The 2-AACq method was used to calculate the relative RNA fold-change, using housekeeping gene b-actin for normalization. 3.6.4. End-point virus titrations. End-point titrations to quantify infectious SARS-CoV-2 particles were performed on confluent Vero E6 cells in 96-well plates. Lung tissues were homogenized using bead disruption (Precellys) in 350 mI minimal essential medium and centrifuged (10,000 rpm, 5 min, 4 °C) to pellet the cell debris. Supernatant was serially diluted into the 96-well plates and scoring for cytopathic effect performed after 3 days. Viral titers were calculated by the Spearman and Karber method.
3.6.5. Lung histology. For histological examination, lung tissues were fixed in formalin and embedded in paraffin. Tissue sections (5 pm) were stained with hematoxylin and eosin and analyzed for lung damage.
3.6.6. Detection of total binding IgG and neutralizing antibodies. To detect SARS- CoV-2-specific antibodies in hamster serum, an in-house-developed UFA was used
(Sanchez-Felipe L, et al. , 2020). To quantify SARS-CoV-2 neutralizing antibodies, serial dilutions of serum samples were incubated for 1 h at 37 °C with an equal volume of SARS- CoV-2 pseudotyped VSV particles and inoculated on Vero E6 cells for 18 h. SARS-CoV- 2 VSV pseudotypes were generated as previously described (Sanchez-Felipe L, et al. , 2020).
3.7. In vivo immunological and efficacy techniques in rhesus macaques
3.7.1. Rhesus macaques and study design. 12 outbred Indian-origin adult male and female Rhesus macaques (Macaca mulatta), 6-12 years old, were randomly allocated to groups. Animals received MVA-S (N=6), and sham controls received MVA-WT (N=6). Animals received 2 X 108 PFUs of MVA-S or MVA-WT by intramuscular route at week 0 and week 4. At week 8, all animals were challenged with 1.2 x 106 PFU SARS-CoV-2. Virus was administered as 1 ml by the intranasal route (0.5 ml in each nare) and 1 ml by the intratracheal route. All immunologic and virologic assays were performed blinded. All animal studies were conducted in compliance with all relevant local, state, and federal regulations and were approved.
3.7.2. ELISPOT assay. Quantification of antigen-specific cytokine-secreting cells in macaques was performed by IFN-y and IL-4 enzyme-linked immunospot (ELISpot) assays on freshly isolated peripheral blood mononuclear cells (PBMC), according to the manufacturer's instructions (U-Cytech, Utrecht, The Netherlands). Antigen-specific responses were measured against 5-pg/ml of S1 and S2 peptide pools. Results are expressed as the mean number of spot-forming cells (SFC) per 106 PBMC from triplicate assays minus background values (mean number of SFC plus two standard deviations [SD] of triplicate assays with medium alone). Mean numbers of spots for each animal or individual per antigen are reported.
3.7.3. Viral RNA assay. RT-PCR assays were utilized to monitor viral loads. Briefly, RNA was extracted using a QIAcube FIT (Qiagen, Germany) and the Cador pathogen FIT kit from bronchoalveolar lavage (BAL) supernatant and throat swabs. RNA was reverse transcribed using superscript VILO (Invitrogen) and ran in duplicate using the QuantStudio 6 and 7 Flex Real-Time PCR System (Applied Biosystems) according to manufacturer’s specifications. Viral loads were calculated of viral RNA copies per ml_ or per swab and the assay sensitivity was 50 copies. The target for amplification was the SARS-CoV2 N (nucleocapsid) subgenomic gene or genomic RdRp gene. RESULTS
In this invention, new MVA viral vector-based vaccines have been designed and developed against the coronavirus SARS-CoV-2 causing the COVID-19 pandemic. Once generated, these recombinant MVA viruses have been characterized in vitro and their capacity to stimulate adaptive SARS-CoV-2-specific T cell-mediated immune responses and humoral immune responses in vivo in immunized mice has been analysed.
1. Design and generation and in vitro characterization of vaccine candidates MVA- COVID19(S) and MVA-A-COVID19(S) expressing the coronavirus SARS-CoV-2 S- protein
To generate novel vaccines against SARS-CoV-2, the causative agent of COVID-19 pandemic, that could stimulate SARS-CoV-2-specifc T- and B-cell immune responses, we have developed two MVA-based vaccine candidates expressing the SARS-CoV-2 full- length S structural gene (Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1 , Genbank number: MN908947.3; the most contemporary isolate available at the time we initiated this work) [termed MVA-COVID19(S) and MVA-A-COVID19(S)] (see Materials and Methods). Vaccine candidate MVA-COVID19(S) was generated following the insertion of the complete sequence of the coronavirus SARS-CoV-2 S gene in the genome of the parental virus MVA-WT, whereas the other vaccine candidate, MVA-D- COVID19(S), was generated following the insertion of the complete sequence of the coronavirus S gene in the genome of an MVA, referred to as MVA-GFP, expressing the GFP protein and containing complete deletions in the vaccinia virus immunomodulatory genes C6L, K7R, and A46R. The expression of the SARS-CoV-2 gene is under the transcriptional control of a synthetic early/late (sE/L) promoter.
The authors of the present invention have previously described that an MVA viral vector lacking said immunomodulatory genes C6L, K7R, and A46R and expressing the structural genes of the Chikungunya virus is capable of providing full protection to mice and non-human primates after a Chikungunya virus challenge (Garcia-Arriaza et al. , 2014, J. Virol, 88:3527-3547; Roques et al., 2017, JCI Insight, 2(6):e83527). Additionally,
Zika and Ebola vaccine candidates based on this MVA viral vector lacking said immunomodulatory genes are capable of providing protection to mice after a Zika virus challenge (Perez et al., 2018, Scientific Reports 8:17385) or an Ebola virus challenge (Lazaro-Frias et al., 2018, J. Virol, 92:e00363-18). Furthermore, the elimination of genes C6L and K7R in the H IV/AIDS vaccine candidate, MVA-B, expressing Env, Gag, Pol, and Nef of H IV-1 subtype B, induced an increased in the expression of IFN-b and IFN type 1- inducible genes in cultured immune cells and promoted an increase in the H IV-1 specific immune response in immunized mice (Garcia-Arriaza et al., 2013, PLoS One, 8(6):e66894). Figure 1A shows a diagram of the generated vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S). For the sake of simplicity, MVA-COVID19(S) will be referred to as MVA-S or MVA-CoV2-S and MVA-A-COVID19(S) as MVA-A-S or MVA-A-CoV2-S throughout the text and figures. The methodology used for generating these vaccine candidates has been described above in Materials and Methods. The correct insertion of the coronavirus SARS-CoV-2 S gene and the purity of the generated vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) were confirmed by means of PCR and sequencing, after amplifying the DNA obtained from DF-1 cells that were not infected (mock) or infected with 5 PFU/cell of MVA-WT, MVA-GFP, MVA-COVID19(S), and MVA- A-COVID19(S). Oligonucleotides (described in Table 3 of Materials and Methods) which hybridize in the flanking regions of the MVA TK gene ( J2R gene) were used in the PCR, and the obtained results confirmed the presence of the S gene in MVA-COVID19(S) and MVA-A-COVID19(S), without contamination with the parental MVA-WT or MVA-GFP, respectively (Figure 1 B). The TK locus of parental viruses MVA-WT or MVA-GFP, was amplified as PCR control, with the TK gene and the GFP gene being amplified, respectively. The correct presence of the S gene inserted in the TK locus was also confirmed by means of DNA sequencing.
To demonstrate that MVA-S and MVA-A-S constitutively express the SARS-CoV-2 S protein, we performed a Western blot analysis of cell extracts from MVA-infective permissive chicken DF-1 cells infected at a MOI of 5 PFU/cell for 4 and 24 h with MVA- S, MVA-A-S, or MVA-WT using specific antibodies that recognize the SARS-CoV-2 S protein. Furthermore, DF-1 infected cells were leave in the presence or absence of tunicamycin, an inhibitor of protein N-glycosylation, to analyze the glycosylation pattern of SARS-CoV-2 S protein expressed from MVA. The results proved that MVA-S and MVA- A-S vaccine candidates correctly expressed the SARS-CoV-2 S protein (of an expected molecular weight of about 180 kDa, corresponding to the full-length glycosylated S protein) at 4, and 24 h postinfection (hpi), being detected either using a mouse monoclonal antibody against SARS-CoV (against SARS-CoV S2 region) or a rabbit polyclonal antibody against SARS-CoV-2 (against SARS-CoV-2 S1 region) (Figure 1 C). Furthermore, tunicamycin treatment inhibited SARS-CoV-2 S glycosylation and a single band of about 130 kDa, corresponding to the full-length unglycosylated S protein was detected (Figure 1 C).
The secretion of the S-protein in the supernatant was then analysed at different times post-infection. Cell extracts, or supernatants were collected from DF-1 cells that were not infected or infected with vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S), and with parental viruses MVA-GFP and MVA-WT, at an MOI of 5 PFU/cell 4 and 24 hours post-infection. Next, the expression of the S-protein in cell extracts and the secretion of said protein in the extracellular medium was analysed by means of Western blot (Figure 1 D). The results showed that the expression of the S-protein was detected 24 hours post-infection (Figure 1 D) in the cell extracts originating from vaccine candidates
MVA-COVID19(S) and MVA-A-COVID19(S). Flowever, the expression of the S-protein in the supernatants (Figure 1 D) was not detected because the S-protein inserted in the MVA genome is a transmembrane protein.
In conclusion, vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S), efficiently express the coronavirus SARS-CoV-2 S-protein.
2. In vitro characterization of vaccine candidates MVA-COVID19(S) and MVA-D- COVID19(S)
2.1. Vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) form oligomers
Next, we analyzed the ability of the SARS-CoV-2 S protein expressed from MVA-S and MVA-A-S recombinants to form oligomers. Thus, at 24 hpi, we collected cell extracts under reducing or nonreducing conditions (with or without b-mercaptoethanol, respectively) from DF-1 cells infected at a MOI of 5 PFU/cell with MVA-S, and MVA-A-S, and then we analyzed by Western blotting the expression of SARS-CoV-2 S protein, using either a rabbit monoclonal antibody against SARS-CoV-2 or a mouse monoclonal antibody against SARS-CoV (Figure 2A). Immunoblots showed that under nonreducing conditions, the SARS-CoV-2 S protein expressed from MVA-S and MVA-A-S recombinants existed mainly as high-molecular-mass oligomers; while under reducing conditions, the protein was present as ~180-kDa monomers (Figure 2A).
2.2. Vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) replicate in permissive cells
To determine if the expression of the coronavirus SARS-CoV-2 S-protein affects MVA replication in cell culture, DF-1 cells were infected with vaccine candidates MVA- COVID19(S) and MVA-A-COVID19(S), and parental viruses MVA-WT and MVA-GFP, and cell extracts were collected at different times post-infection (0, 24, 48, and 72 hours), and the amount of virus present was determined. The results showed that the viral growth kinetics of vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S), was similar (Figure 2B). Furthermore, it was also similar and even greater than the growth kinetics of parental viruses MVA-GFP and MVA-WT, which demonstrates that the expression of the S-protein does not negatively affect MVA vector replication in permissive conditions. Moreover, similarly to parental MVA-WT and MVA-GFP viruses and as expected, MVA- S and MVA-A-S does not replicate in human HeLa cells (data not shown).
2.3. Vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) are stable in cell culture
To confirm that vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) are stable and can be maintained in cultured cells without the loss of the S gene, DF-1 cells were infected with MVA-COVID19(S) and MVA-A-COVID19(S) with a low MOI for 9 consecutive serial passages, and the expression of the S-protein was determined by means of Western Blot. The results showed that MVA-COVID19(S) and MVA-D- COVID19(S) efficiently expressed the S-protein for all the passages, which demonstrates that vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S), are genetically very stable (Figure 2C).
3. Analysis of the SARS-CoV-2-specific adaptive immune response of vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) in C57BL/6 mice after heterologous (DNA/MVA) and homologous (MVA/MVA) immunization protocols.
The immunogenicity of vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) in C57BL/6 mice was then evaluated using heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost immunization regimens, and the coronavirus SARS-CoV-2 S- protein-specific adaptive cell-mediated immune responses and humoral immune responses induced by those vaccine candidates were analysed in detail.
Therefore, in the heterologous (DNA/MVA) prime-boost protocols 6 C57BL/6 mice per group were immunized with the plasmid pcDNA-S (DNA-S) followed by vaccine candidates MVA-COVID19(S) or MVA-A-COVID19(S), whereas in the homologous (MVA/MVA) prime-boost protocols 6 C57BL/6 mice per group were immunized with two doses of each vaccine candidate MVA-COVID19(S) or MVA-A-COVID19(S). The mice were sacrificed on day 11 after the boost for measuring adaptive SARS-CoV-2-specific cell-mediated immune responses and humoral immune responses (Figure 3) (see Materials and Methods). 3.1. Immunization of C57BL/6 mice with vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) induces high levels of SARS-CoV-2 S-specific cells secreting IFN-Y
The coronavirus SARS-CoV-2 S-protein-specific adaptive cell-mediated immune response was first analysed by means of ELISpot (see Materials and Methods) 11 days after the boost in C57BL/6 mice (n = 6/group) immunized with the different immunization protocols. As a control group, the mice were immunized with two doses of MVA-WT. Splenocytes originating from the spleens of each group were stimulated ex vivo with a panel of peptide pools covering the complete sequence of the coronavirus SARS-CoV-2 S-protein (S1 and S2 peptide pools). The samples were stimulated with RPMI as a negative control, and with a VACV E3 protein peptide as a positive control. After 48 hours of stimulation, the cells were labeled with antibodies to identify IFN-y-secreting cells specific for these peptides (Figure 4).
The results showed that all the immunization groups including vaccine candidates MVA- COVID19(S) and MVA-A-COVID19(S), induced high levels of S1- and S2-specific IFN-y- secreting cells, with the response being directed to a greater extent against S1 peptide pools and to a lesser extent against S2 peptide pools (Figure 4A). In particular, the DNA- S/MVA-A-S group induced the highest levels of S1- and S2-specific IFN-y-secreting cells. Moreover, heterologous (DNA/MVA) immunizations induced better response than homologous (MVA/MVA) immunizations, and vaccine candidate MVA-A-S, increased the responses in comparison with MVA-S, in both heterologous immunizations and homologous immunizations (Figure 4A). The response against MVA vector (E3 peptide) showed that homologous (MVA/MVA) immunizations induced a better response than heterologous (DNA/MVA) immunizations, with the MVA-A-S/MVA-A-S group inducing the highest levels of IFN-y-secreting cells (Figure 4B).
3.2. Immunization of C57BL/6 mice with vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) induces a strong, broad, and polyfunctional SARS-CoV-2 S- specific adaptive CD4+ and CD8+ T cell-mediated immune response
Next, splenocytes originating from the spleens of each immunization group were stimulated ex vivo with a panel of peptide pools covering the complete sequence of the coronavirus SARS-CoV-2 S-protein (S1 and S2 peptide pools). The samples were stimulated with RPMI as a negative control. After 6 hours of stimulation, the cells were labelled with specific antibodies to identify (CD4+ and CD8+) T cell populations and respondent cells (expressing CD107a on the surface of the T cells as an indirect cytotoxicity marker and/or producing cytokines IFN-y, TNF-a, and IL-2) by ICS.
The results showed that all the immunization regimens (DNA/MVA or MVA/MVA) are capable of inducing coronavirus SARS-CoV-2 S-protein (S1 + S2)-specific adaptive CD4+ T cell-mediated (Figure 5A, left) and CD8+ T cell-mediated (Figure 5A, right) immune responses, with the levels of specific CD8+ T cells being greater than the levels of CD4+ T cells in all the groups. Moreover, heterologous (DNA/MVA) immunizations induced better CD4+ T cell-mediated response and CD8+ T cell-mediated response than homologous (MVA/MVA) immunizations (Figure 5A). Like between groups MVA-S/MVA-S and MVA-A-S/MVA-A-S, the levels of S-protein- specific CD4+ T cells induced by immunization groups DNA-S/MVA-S and DNA-S/MVA- D-S are similar (Figure 5A). However, the levels of S-protein-specific CD8+ T cells induced by immunization groups including MVA-A-S in the boost are greater than groups including MVA-S in the boost (Figure 5A). The detailed analysis of the response showed that, in all the immunization groups, the coronavirus SARS-CoV-2 S-protein-specific CD4+ T cells and CD8+ T cells are directed to a greater extent against S1 peptide pools and to a lesser extent against S2 peptide pools (Figure 5B), maintaining the aforementioned differences between groups.
These aforementioned differences can also be observed when analyzing the percentages of S-protein (S1+S2)-specific CD4+ T cells (Figure 5C, left) or CD8+ T cells (Figure 5C, right) secreting each of the analyzed cytokines (IFN-g, TNF-a, or IL-2) or expressing the cytotoxicity marker, CD107a. It must be pointed out that specific CD8+ T cell responses are characterized by a low IL-2 secretion.
Finally, the polyfunctionality of the CD4+ and CD8+ T cell-mediated immune response generated by the different immunization groups was analyzed by measuring the simultaneous production pattern of cytokines (IFN-g, TNF-a, and/or IL-2) plus the cytotoxic potential thereof (CD107a as a degranulation marker). The coronavirus SARS- CoV-2 S-protein (S1+S2)-specific cell-mediated responses generated by the different immunization groups were characterized by a high polyfunctionality (Figure 6). The CD4+ T cell-mediated response was mainly characterized by cells which are capable of carrying out all 4 functions (secreting all 3 cytokines and expressing the marker, CD107a), followed by cells capable of carrying out 3 functions (IFN-g, IL-2, and TNF-a), and cells capable of carrying out 2 functions (IFN-g and TNF-a) (Figure 6A). On the other hand, the CD8+ T cell-mediated response was mainly characterized by cells which are capable of carrying out 3 functions (CD107a, IFN-g and TNF-a), followed by cells capable of carrying out 4 functions (CD107a, IFN-y, IL-2, and TNF-a) (Figure 6B).
In conclusion, vaccine candidates MVA-COVID19(S) or MVA-A-COVID19(S), are capable of inducing broad and polyfunctional coronavirus SARS-CoV-2 S-protein-specific adaptive CD4+ and CD8+ T cell-mediated immune responses of great magnitude.
3.3. Immunization of C57BL/6 mice with vaccine candidates MVA-COVID19(S) and M VA-A-COVI D 19(S) induces adaptive SARS-CoV-2 S-specific CD4+ and CD8+ T cell- mediated immune response with effector memory phenotype
Next, we determined the phenotype of the adaptive SARS-CoV-2-specific CD4+ and CD8+ T cells by measuring the expression of CD127 and CD62L surface markers, which defined different memory subpopulations: T central memory (TCM, CD127+/CD62L+), T effector memory (TEM, CD127+/CD62L ), and T effector (TE, CD1277CD62L ) cells, and determined the sums of the individual responses expressing CD107a, IFN-g, TNF-a, and/or IL-2 obtained for the S1 , and S2 peptide pools (Figure 7). The results showed that in all vaccinated groups, adaptive SARS-CoV-2-specific CD4+ and CD8+ T cells were mainly of the TEM phenotype (Figure 7), maintaining the magnitude differences between groups previously described (see Figures 5 and 6).
3.4. Immunization of C57BL/6 mice with vaccine candidates MVA-COVID19(S) and M VA-A-COVI D 19(S) induces adaptive SARS-CoV-2 S-specific CD4+ T follicular helper (Tfh) cell-mediated immune response
Next, the proportion of coronavirus SARS-CoV-2 S-specific CD4+ T follicular helper (Tfh) cells was analyzed by means of ICS 11 days after the boost in C57BL/6 mice immunized with a heterologous (DNA/MVA) or homologous (MVA/MVA) prime-boost protocol, as described above.
Splenocytes isolated from the spleen were stimulated ex vivo with a combination of the coronavirus SARS-CoV-2 S-protein, together with the S1 and S2 peptide pools covering the complete sequence of said protein. The samples were stimulated with RPMI as a negative control. After 6 hours of stimulation, the cells were labelled with specific antibodies to identify the population of S-specific Tfh cells (CD4+, CXCR5+, and PD1+) expressing the marker CD40L (marker related to T cell effector functions) and/or producing the cytokines IFN-g and/or IL-21.
The results showed that all vaccinated groups induced SARS-CoV-2-specific CD4+ Tfh cells expressing CD40L and/or secreting IFN-g and/or IL-21 (Figure 8A). Heterologous DNA/MVA immunizations induced higher magnitude of SARS-CoV-2-specific CD4+ Tfh cells than homologous MVA/MVA immunizations (Figure 8A), with DNA-S/MVA-S triggering higher responses than DNA-S/ MVA-A-S and MVA-A-S/MVA-A-S higher responses than MVA-S/MVA-S (Figure 8A).
The pattern of SARS-CoV-2-specific CD4+ Tfh cells showed that in all vaccinated groups most of the CD4+ Tfh secreted IFN-g and IL-21, followed by the presence of CD40L (Figure 8B).
Finally, the polyfunctionality of the Tfh cell-mediated immune response generated by the different immunization groups was analyzed by measuring the simultaneous production pattern of cytokines (IFN-y and/or IL-21 ) plus the marker, CD40L. The coronavirus SARS- CoV-2 S-protein-specific Tfh cell-mediated responses generated by the different immunization groups were characterized by a high polyfunctionality (Figure 9B). The Tfh cell-mediated response was mainly characterized by cells which are capable of carrying out all 3 functions (secreting the 2 cytokines and expressing the marker, CD40L), followed by cells capable of carrying out 2 functions (IFN-g, IL-21) (Figure 9B). In conclusion, vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) induce Tfh cell-mediated responses.
3.5. Immunization of C57BL/6 mice with vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) induces high levels of binding IgG antibodies against SARS- CoV-2 S and RBD proteins Next, the total levels of coronavirus SARS-CoV-2 S-protein-specific and S-protein receptor binding domain (RBD)-specific IgG antibodies and the levels of subclasses thereof, i.e. , lgG1, lgG2c, and lgG3, present in the sera of C57BL/6 mice immunized with the different immunization protocols, were analyzed by means of ELISA 11 days after the second immunization (boost). The animals immunized with two doses of MVA-WT were used as a control group.
The results showed that all the immunization groups induced high levels of coronavirus SARS-CoV-2 S-protein-specific IgG antibodies (Figure 9A, left), as well as high levels of RBD-specific IgG antibodies (Figure 9A, right). In general, homologous (MVA/MVA) immunizations induced slightly higher levels of S-protein-specific and RBD-specific IgG antibodies than heterologous (DNA/MVA) immunizations (Figure 9A). The presence of antibodies against the RBD region suggest that those antibodies could be neutralizing antibodies, as this domain is the main region recognized by neutralizing antibodies. Regarding the IgG subclasses of antibodies, all vaccinated groups elicited higher levels of lgG2c antibodies than lgG1 and lgG3 antibodies against SARS-CoV-2 S protein (Figure 9B, left), and RBD protein (Figure 9B, right); again with homologous MVA/MVA prime/boost immunizations triggering slightly higher levels than heterologous DNA/MVA immunizations regimens, except in the analysis of lgG3 antibodies against SARS-CoV-2 RBD protein where heterologous DNA/MVA immunizations regimens elicited slightly higher levels than homologous MVA/MVA prime/boost immunizations. Moreover, the determination of the lgG2c/lgG1 ratio showed that in all vaccinated groups it was higher than 1, indicating a Th1 response.
In conclusion, vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) induced high levels of total IgG, lgG1, lgG2c, and lgG3 antibodies against SARS-CoV-2 S- and RBD proteins, with a Th1 response.
3.6. Immunization of C57BL/6 mice with vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) induces high levels of SARS-CoV-2-specific neutralizing antibodies
Next, the capacity of the different immunization groups with vaccine candidates MVA- COVID19(S) and MVA-A-COVID19(S), to induce coronavirus SARS-CoV-2-specific neutralizing antibodies in individual sera originating from C57BL/6 mice immunized with the different immunization groups was analyzed as described previously. Sera originating from mice immunized with MVA-WT/MVA-WT were used as a negative control of the experiment. The results showed that individual serum samples obtained from all mice vaccinated with MVA-S and MVA-A-S vaccine candidates neutralized SARS-CoV-2 pseudoparticles, significantly reducing the levels of luciferase units, compared to serum from the control group MVA-WT/MVA-WT, where no neutralization was observed (Figure 10). In detail, heterologous DNA/MVA prime/boost immunization protocols DNA-S/MVA-S and DNA- S/MVA-A-S triggered a positive neutralization, although being variable and of low levels. On the other hand, homologous MVA/MVA prime/boost immunization protocols MVA- S/MVA-S and MVA-A-S/MVA-A-S elicited a high neutralization profile with a reduction in the luciferase units of at least 3 logs, compared to the control group MVA-WT/MVA-WT (Figure 10). In summary, vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) induce coronavirus SARS-CoV-2-specific neutralizing antibodies. 4. K18-hACE2 mice vaccinated with one or two doses of MVA-S are fully (100%) protected against SARS-CoV-2 lethality.
The efficacy of the MVA-S vaccine candidate was analysed in K18-hACE2 mice, a mouse model for SARS-CoV-2 infection and lethality. Groups of K18-hACE2 mice (n=5) were immunized with one or two doses (1 c 107 PFUs by i.m. route) of MVA-S at weeks 0 and 4, and then challenge at week 9 with 1 c 105 PFUs of SARS-CoV-2 (MAD6 isolate) by intranasal route (Fig. 12A). Mice primed and boosted with MVA-WT or unvaccinated, and unchallenged mice were used as a control groups. The challenged K18-hACE2 mice were monitored for change in body weight (Fig. 12B) and mortality (Fig. 12C), for 14 days post challenge. All the K18-hACE2 mice immunised with two doses of MVA-S and challenged with SARS-CoV-2 did not show loose body weight (Fig. 12B) and survived (Fig. 12C), whereas all the MVA-WT- or non-immunised and challenged K18-hACE2 mice showed loss of body weight (more than 25%) (Fig. 12B) and die (Fig. 12C) within 6 days post challenge. All mice immunized with one single dose of MVA-S and challenged with SARS- CoV-2 loss body weight during the first days post-challenge (Fig. 12 B) but by day 4 post challenge they start recovering and all survived (Fig. 12C). Thus, the vaccine MVA-S (also known as MVA-CoV2-S) administered in one or two doses fully protected (100%) humanized mice against the lethal effects of SARS-CoV-2 infection.
4.1. MVA-S vaccine candidate is highly immunogenic in K18-hACE2 humanized mice. Next, we extended the study in K18-hACE2 mice and further evaluated the immunogenicity and efficacy triggered after vaccination with one or two doses of MVA-S. Thus, K18-hACE2 mice (n=11/group), were intramuscularly immunized at weeks 0 and 4 with one or two doses of MVA-S, as indicated (Fig. 12A), and 10 days after the last immunization (day 38) serum samples were obtained and we analyzed the adaptive SARS-CoV-2-specific humoral immune responses elicited before the challenge, as antibodies against SARS-CoV-2 are decisive to control COVID-19 progression (Addetia et al. , 2020; Liu et al., 2020; Salazar et al., 2020). As shown in Fig 13A, K18-hACE2 mice vaccinated with MVA-S induced high titers of S- and RBD-specific binding IgG antibodies, with the two-dose regimen triggering significantly higher levels than the one-dose treatment. In both regimens, the analysis of different IgG isotypes against S and RBD proteins showed higher lgG2c levels than lgG1 , leading to a lgG2c/lgG1 ratio higher than 1 (Fig. 13B), suggestive of a Th1-like protective humoral response (Visciano et al., 2012). Moreover, the two-dose regimen induced significant higher neutralizing antibody ID50 titers than the one-dose treatment, either using a retrovirus-based pseudoparticle assay (Fig. 13C, left) or a live SARS-CoV-2 microneutralization assay (Fig. 13C, right), with titers superior to the titers of a WFIO International Standard for anti-SARS-CoV-2 human immunoglobulin (NIBSC 20/136) (Fig. 13C).
4.2. MVA-CoV2-S vaccine candidate is highly efficacious in K18-hACE2 humanized mice, preventing SARS-CoV-2 virus replication, reducing lung pathology and the levels of pro-inflammatory cytokines. Next, we evaluated the efficacy and immune parameters triggered by the MVA-S vaccine candidate after SARS-CoV-2 infection, through analysis of virus replication in tissues, lung pathology and cytokine storm. Thus, K18-hACE2 mice immunized with one or two doses of MVA-S (n=11/group) were then challenged 5 weeks after the last immunization with a lethal dose of SARS-CoV-2 [MAD6 isolate, 105 PFU/mouse)], by the intranasal route, as indicated (Fig. 12A). Challenged mice primed and boosted with MVA-WT or unvaccinated, and unchallenged mice were used as control groups. Changes in body weight and mortality were evaluated daily for 15 days postchallenge (n=5 mice/group); all mice vaccinated with two doses of MVA-S did not lose body weight and survived, mice immunized with one dose of MVA-S lost body weight during the first 4 days postchallenge, but all recovered; all vaccinated mice survived, whereas all mice inoculated with MVA-WT or unvaccinated lost body weight and died at 6 days postchallenge, as previously reported (Fig. 12B and C).
To examine the effect of the MVA-S vaccine candidate in virus replication, three mice per group were sacrificed at days 2 and 4 after SARS-CoV-2 virus challenge, lung tissue samples were collected and processed, and we analysed the presence of SARS-CoV-2 genomic RNA (Fig. 14A) and of live infectious virus (Fig. 14B). Two doses of MVA-S were highly effective to completely prevent SARS-CoV-2 replication and virus yields at 2 and 4 days postchallenge, in comparison with the high levels of SARS-CoV-2 RNA and infectious virus detected in control infected mice (Fig. 14A and 14B), with a significantly remarkable reduction in infectious virus (Fig. 14B). Furthermore, a single dose of MVA-S has also an inhibitory effect reducing the levels of SARS-CoV-2 RNA and of virus loads, mainly at 4 days postchallenge (Fig. 14A and 14B).
The analysis of the lung histopathology showed that mice vaccinated with two doses of MVA-S have significantly less lung histopathological inflammatory lesions (Fig. 14C, left panel) and less percentage of lung area with lesions (Fig. 14C, right panel) at 4 days postchallenge, compared to control MVA-WT inoculated mice or mice immunized with one dose of MVA-S. At 2 days postchallenge, no lung histopathology differences were observed between vaccinated and unvaccinated mice (data not shown). Next, we evaluated the effect of the MVA-S vaccine candidate in the cytokine pattern induced in mice at days 2 and 4 after SARS-CoV-2 virus challenge, analysing by RT- qPCR the mRNA levels of key cytokines on lung homogenates (Fig. 14D). Two doses of MVA-S induced a significant down-regulation in the mRNA levels of IL-6, TNF-a, IL-10, IP-10 and IFN-y at 2 and 4 days postchallenge, compared to control infected mice or mice immunized with one dose of MVA-S (Fig. 14D), suggesting prevention of a cytokine storm as a result of the vaccine control of virus infection.
4.3. MVA-CoV2-S vaccine candidate induced high levels of binding IgG antibodies and neutralizing antibodies in SARS-CoV-2 challenged-K18-hACE2 humanized mice. We next evaluated the SARS-CoV-2-specific humoral immunogenicity triggered by the MVA-S vaccine candidate after SARS-CoV-2 challenge. Thus, serum samples were obtained from mice sacrificed at days 2 and 4 postchallenge (n=3/group), or at day 15 postchallenge from surviving mice vaccinated with one or two doses of MVA-S, or unvaccinated and unchallenged mice (n=5/group). Mice immunized with MVA-S elicited high titers of S- and RBD-specific binding IgG antibodies, with the two-dose regimen inducing significant higher values than the one- dose treatment at days 2 and 4 postchallenge (similarly to what happened at the prechallenged state, Fig. 13A), whereas at day 15 postchallenge both vaccinated groups elicited similar titers of IgG antibodies (Fig. 15A). Mice vaccinated with two doses of MVA- S lacked an anamnestic response, supporting severe inhibition of virus replication, with similar anti-S and anti-RBD IgG titers at 2, 4 and 15 days postchallenge, contrary to mice immunized with one dose of MVA-S, where an increase in the IgG titers against S and RBD was observed between days 4 and 15 postchallenge (Fig. 15A). The analysis of the levels of different IgG isotypes against S and RBD proteins at days 4 and 15 postchallenge showed higher lgG2c titers than lgG1 , leading to a lgG2c/lgG1 ratio higher than 1 (Fig. 15B), indicative of a Th1 -like protective humoral response, and similarly to what was observed before the challenge (Fig. 15C). Finally, one or two doses of MVA-S induced high titers of SARS-CoV-2 neutralizing antibodies at days 4 and 15 postchallenge, with the two-dose regimen inducing significantly higher neutralizing antibody ID50 titers than the one-dose treatment at day 4 postchallenge (similarly to what happened at the prechallenged state, Fig. 15D), but similar titers at day 15 postchallenge (Fig. 15C).
4.4. Surviving K18-hACE2 humanized mice vaccinated with one or two doses of the MVA-CoV2-S vaccine candidate are fully protected after a second reinfection with SARS-CoV-2. We have demonstrated that one or two doses of MVA-S fully protected K18-hACE2 humanized mice against SARS-CoV-2 infection. However, mice immunized with one dose of MVA-S lost body weight, although they recovered from day 4 postchallenge and at day 15 postchallenge they reached the 100% of initial body weight (Fig. 12B and 12C). Thus, to evaluate whether K18-hACE2 mice immunized with one or two doses of MVA-S were protected against a second SARS-CoV-2 infection, surviving mice (n=5/group) were challenged at week 16 with SARS-CoV-2 (MAD6 isolate, 105 PFU/mouse), as indicated (Fig. 16A) and changes in body weight and mortality were evaluated for 6 days postchallenge, time at what all animals were sacrificed. All mice vaccinated with one or two doses of MVA-S did not lose body weight (Fig. 16B) and survived (Fig. 16C), while unvaccinated and challenged mice lost body weight and died at 6 days postchallenge (Fig. 16B and 16C). The analysis of SARS-CoV-2 virus replication in lung samples at day 6 post-reinfection showed that one or two doses of MVA-S behave similarly and completely abrogated SARS-CoV-2 replication (Fig. 16D) and virus yields (Fig. 16E), in comparison with the high levels of SARS-CoV-2 RNA and infectious virus detected in control infected mice (Fig. 16D and 16E). Furthermore, the analysis of mRNA levels of key cytokines on lung homogenates at 6 days after reinfection showed that one or two doses of MVA-S, compared to control infected mice, significantly down-regulated mRNA levels of IP-10, while there were no differences in the levels of IL- 6 and TNF-a (Fig. 17A).
Finally, SARS-CoV-2-specific humoral immunogenicity triggered by one and two doses of MVA-S at day 6 after SARS-CoV-2 reinfection was evaluated in serum samples. Mice immunized with one or two doses of MVA-S elicited similar high titers of S- and RBD- specific binding IgG antibodies (Fig. 17B), with a S- and RBD-specific lgG2c/lgG1 ratio higher than 1 (Fig. 17C), indicative of a Th1 response. Moreover, similar high titers of SARS-CoV-2 neutralizing antibodies were elicited at day 6 after SARS-CoV-2 reinfection in mice vaccinated with one or two doses of MVA-S (Fig. 17D).
5. Immunization of mice with MVA-CoV2-S vaccine candidate induces long-term strong, broad, and polyfunctional SARS-CoV-2 S-specific CD4+ and CD8+ T-cell memory immune responses. Next, to evaluate whether MVA-S induce long-term S- specific T-cell immune responses, groups of C57BL/6 mice (n=5/group) were immunized twice with MVA-S (intramuscularly, 1 x 107 PFU/mouse) at weeks 0 and 2 following a homologous MVA-S/MVA-S prime/boost immunization regimen, as shown in materials and methods, and 6 months later animals were sacrificed. Then, we stimulated their splenocytes with SARS-CoV-2 S1 and S2 peptide pools, spanning the entire S protein, to evaluate by an enzyme-linked immunosorbent spot (ELISpot) assay the induction of SARS-CoV-2 S-specific gamma interferon (IFNy)-secreting cells; or by an intracellular cytokine staining (ICS) assay the induction of SARS-CoV-2 S-specific CD4+ and CD8+ T cells expressing CD107a, IFNy, tumor necrosis factor alpha (TNFa), and/or interleukin 2 (IL-2).
Mice vaccinated with MVA-S elicited high levels of long-term IFNy-secreting cells against a mixture of SARS-CoV-2 S1+S2 peptide pools (Fig. 18A). Analysis by ICS showed that MVA-S-vaccinated mice induced long-term S-specific CD4+ and CD8+ T-cell responses, with a higher overall response mediated by the CD8+ T-cell repertoire and a CD4+ Th1 profile elicited (Fig. 18B). Furthermore, long-term S-specific CD4+ and CD8+ T-cell responses were broad and directed mainly against the S1 peptide pool (Fig. 18C). S- specific CD4+ and CD8+ T-cell responses were mostly of the T central memory and T effector memory phenotypes, respectively (Fig. 18D). Moreover, the analysis of the quality of the S-specific T-cell responses showed that MVA-S triggered highly polyfunctional long-term CD4+ and CD8+ T cells (Fig. 18E), with 100% and 90% of S- specific CD4+ and CD8+ T cells, respectively, exhibiting three or four functions (Fig. 18E, pie charts), being CD4+ T cells expressing CD107a-IFNy-TNFa-IL-2, and CD8+ T cells expressing CD107a-IFNy-TNFa the most induced populations (Fig. 18E, bars). 5.1. Immunization of mice with MVA-CoV2-S vaccine candidate induces long-term
SARS-CoV-2-specific humoral immune responses. Next, to evaluate the ability of the MVA-S vaccine candidate to elicit long-term humoral immune responses against SARS- CoV-2, we determined by ELISA the levels of S- and RBD-specific binding IgG antibodies in the sera of prime/boost immunized C57BL/6 mice (n=5/group) at 6 months post-boost (Fig. 19A). Mice vaccinated with two doses of MVA-S induced durable humoral immune responses, with high titers of S- and RBD-specific binding IgG antibodies (Fig. 19A). Furthermore, S- and RBD-specific lgG2c/lgG1 ratio was above 1 (Fig. 19B), likely suggestive of Th1 -like immune response. Remarkably, high titers of SARS-CoV-2 neutralizing antibodies were induced at 6 months post-immunization with MVA-S (Fig. 19C).
6. Immunogenicity and efficacy study of MVA-S in Syrian hamsters. Next, we evaluated the immunogenicity and efficacy of the vaccine candidate MVA-CoV2-S in Syrian gold hamsters challenged with SARS-CoV-2, as a second animal model to evaluate the immunogenicity and efficacy of the vaccine MVA-CoV2-S. 6.1. Morbidity induced by SARS-CoV-2 in hamsters vaccinated with MVA-S. For morbidity evaluation, groups of Syrian-gold hamsters (n = 12) were immunized with one or two doses of MVA-S (2 c 107 PFU in the prime and 4 c 107 PFU in the boost) or two doses of MVA-WT (2 c 107 PFU in the prime and 4 c 107 PFU in the boost) by the intraperitoneal route at weeks 0 and 3. At week 6 (day 42), mice were intranasally challenged with 2 c 105 TCID50 of SARS-CoV-2 (Fig. 20). Flamsters primed and boosted with MVA-WT were used as a control group. The challenged hamsters were monitored for change in body weight previously to the challenge and postchallenge (Fig. 21). All animals gain weight similarly, previous to the challenge (data not shown). After challenge, all hamsters at 2 days post-infection lost some body weight, but vaccinated animals recovered faster than control at day 4 (Fig. 21 ).
6.2. Vaccination of hamsters with MVA-S induced high titers of binding IgG antibodies against SARS-CoV-2 S protein. To determine the ability of the MVA-S vaccine candidate to induce SARS-CoV-2-specific humoral immune responses, serum samples from hamsters vaccinated according to the schedule represented in Figure 20 were obtained at day 39 (18 days after the last immunization, and previous to the SARS- CoV-2 challenge) and binding IgG antibodies against S protein were analyzed by an indirect immunofluorescence assay (UFA). The results showed that after one or two doses with MVA-S high titers of IgG antibodies were induced in all hamsters at day 39, with significant higher titers in hamsters immunized with two doses of MVA-S (Fig. 22).
6.3. Vaccination of hamsters with MVA-S induced high titers of SARS-CoV-2 neutralizing antibodies. Next, serum samples from hamsters vaccinated according to the schedule represented in Figure 20 were obtained at day 39 and we analyzed the ability of serum samples to interfere with the entry of VSV pseudotype virus using Vero- adaptive spike mutations. The results showed that after one or two doses of MVA-S similar high titers of neutralizing antibodies were induced at day 39 (Fig. 23).
6.4. SARS-CoV-2 virus replication in lungs is prevented by vaccination of hamsters with MVA-S. To analyze the effect of the vaccine in virus replication, at 2 and 4 days after SARS-CoV-2 virus challenge, four hamsters per group were sacrificed, lung tissue samples were collected, RNA was extracted and analyzed by RT-qPCR for SARS-CoV- 2 replication, and the presence of SARS-CoV-2 infectious virus in lung samples was determine by using a TCID50 assay (Fig. 24). The results showed that vaccination with one or two doses of MVA-S significantly reduced SARS-CoV-2 replication and infectious virus at 2 and 4 days post-challenge, with a major effect at day 4 days post-challenge when compared to MVA-WT vaccinated hamsters, resulting in a reduction of 3 to 5 logs in SARS-CoV-2 infectious virus (Fig. 24).
6.5. Vaccination of hamsters with MVA-S protects against severe pathological changes in the lungs. Next, we analyzed the histopathology of lung samples obtained from hamsters vaccinated according to the schedule represented in Figure 20 and obtained at 2, 4- and 14-days post-challenge. The results showed that in hamsters vaccinated with one or two doses of MVA-S and challenged with SARS-CoV-2 there was a significant reduction in the histopathological lung score, compared to animals immunized with MVA-WT (Fig. 25); effect mainly observed at 4 and 14 days post- challenge. A summary of the different parameters measured (such as perivascular and peribronchiolar inflammation, vasculitis, bronchopneumonia, perivascular cuffs, perivascular edema and apoptotic bodies in bronchial wall) clearly showed that one or two doses of MVA-S diminished lung tissue damage (Fig. 26).
7. Immunogenicity and efficacy study of MVA-S in rhesus macaques. Next, we evaluated the immunogenicity and efficacy of the vaccine candidate MVA-CoV2-S in rhesus macaques challenged with SARS-CoV-2, as a third animal model to evaluate the immunogenicity and efficacy of the vaccine MVA-CoV2-S. Groups of rhesus macaques (n = 6) were immunized with two doses of MVA-S vaccine candidate or MVA-WT as a control (2 c 108 PFU in the prime and in the boost) by the intramuscular route at weeks 0 and 4. At week 8, mice were intranasally and intratracheally challenged with 1 c 106 PFU of SARS-CoV-2 (Fig. 27). At week 0, 2, 4 and 6, serum samples were obtained, previously to the challenge. The challenged macaques were monitored for 14 days, and at different days post-challenge several samples were taken. Animals were sacrificed at day 14 post challenge. 7.1. Vaccination of rhesus macaques with MVA-S induced SARS-CoV-2 S-specific cell immune responses. To evaluate whether MVA-S induce S-specific T-cell immune responses in immunized rhesus macaques, PBMCs were obtained at day 14 post-boost and were stimulated with SARS-CoV-2 S1 and S2 peptide pools, spanning the entire S protein, to evaluate by an enzyme-linked immunosorbent spot (ELISpot) assay the induction of SARS-CoV-2 S-specific IFNy- and IL-4-secreting cells. The results showed that macaques vaccinated with MVA-S elicited high levels of SARS-CoV-2-S-specific IFNy-secreting cells that were mainly directed against S1 peptide pool (Fig. 28, left), and no SARS-CoV-2-S-specific IL-4-secreting cells were detected (Fig. 28, right), indicative of a Thl response. 7.2. SARS-CoV-2 virus replication in throat swabs and BAL is controlled by vaccination of rhesus macaques with MVA-S. To analyze the effect of the MVA-S vaccine in virus replication, at different days after SARS-CoV-2 virus challenge, throat swabs and BAL samples from rhesus macaques were collected, RNA was extracted and analyzed by RT-qPCR for SARS-CoV-2 replication (Fig. 29). The peak of RNA was obtained at day 2 post-challenge, and the detailed analysis at this timepoint showed that vaccination with MVA-S significantly reduced SARS-CoV-2 replication in throat swabs and BAL samples (Fig. 29).
8. Intranasal immunization of mice with MVA-S vaccine candidate induces robust SARS-CoV-2 S-specific CD4+ and CD8+ T-cell and humoral immune responses.
Next, we evaluated whether MVA-S induced robust SARS-CoV-2-specific T-cell and humoral immune responses after intranasal administration. Thus, groups of C57BL/6 mice (n=5/group) were immunized twice with MVA-S (intranasally, 1 x 107 PFU/mouse) at weeks 0 and 2 following a homologous MVA-S/MVA-S prime/boost immunization regimen, as shown in materials and methods, and 11 days post-boost animals were sacrificed.
Then, we stimulated their splenocytes or lung cells with SARS-CoV-2 S1 and S2 peptide pools, spanning the entire S protein, to evaluate by ICS the induction of SARS-CoV-2 S- specific CD4+ and CD8+ T cells expressing CD107a, IFNy, TNFa, and/or IL-2. The results showed that mice vaccinated with MVA-S and MVA-A-S elicited potent levels of S-specific CD4+ and CD8+ T-cell responses, which were higher in lung cells than in splenocytes (Fig. 30A and 30B), and with a higher overall response mediated by the CD8+ T-cell repertoire in both cells (Fig. 30A and 30B).
Moreover, mice vaccinated with two doses of MVA-S and MVA-A-S by intranasal route induced high titers of S- and RBD-specific binding IgG antibodies in serum samples (Fig. 30C), as well as IgA antibodies in BAL samples (Fig. 30D). Remarkably, high titers of SARS-CoV-2 neutralizing antibodies were induced (Fig. 30E). SEQUENCE LISTING
SARS-CoV-2 S nucleotide sequence inserted in MVA-COVID19(S) and MVA-D- COVID19(S)
The SARS-CoV-2 S sequence inserted in vaccine candidates MVA-COVID19(S) and MVA-A-COVID19(S) described in the present examples was synthetized by GeneArt and was human codon optimized. Previous to the beginning of the S sequence there is a Kozak sequence (GCCACC). The sequence derived from Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1 , GenBank: MN908947.3, https://www.ncbi.nlm.nih.qov/nuccore/MN908947
- The S nucleotide sequence reads as follows (SEQ ID NO 1 ):
ATGTTCGTGTTTCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGAACCTGAC
CACCAGAACACAGCTGCCTCCAGCCTACACCAACAGCTTTACCAGAGGCGTGTAC
TACCCCGACAAGGTGTTCAGATCCAGCGTGCTGCACTCTACCCAGGACCTGTTCC
T G C CTTT CTT C AG C AAC GT G AC CT G GTTC C AC G C CAT C C AC GT GTC C G G C AC C AAT
G G C AC C AAG AG ATT C G AC AAC CCCGTGCTGCCCTT C AAC G AC G G G GTGT ACTTT G
CCAGCACCGAGAAGTCCAACATCATCAGAGGCTGGATCTTCGGCACCACACTGGA
C AG C AAG AC C C AG AG C CTG CT GAT C GT G AAC AAC G C C AC C AAC GTG GT CAT C AAA
GTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGGCGTCTACTACCACAAGA
ACAACAAGAGCTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTG
CACCTTCGAGTACGTGTCCCAGCCTTTCCTGATGGACCTGGAAGGCAAGCAGGGC
AACTTCAAGAACCTGCGCGAGTTCGTGTTCAAGAACATCGACGGCTACTTCAAGAT
CTACAGCAAGCACACCCCTATCAACCTCGTGCGGGATCTGCCTCAGGGCTTCTCT
GCTCTGGAACCCCTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAGA
CACTGCTGGCCCTGCACAGAAGCTACCTGACACCTGGCGATAGCAGCAGCGGAT
GGACAGCTGGTGCCGCCGCTTACTATGTGGGCTACCTGCAGCCTAGAACCTTCCT
GCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGATTGTGCTCTGGAT
CCTCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAAAAGGGCATCT
ACCAGACCAGCAACTTCCGGGTGCAGCCCACCGAATCCATCGTGCGGTTCCCCAA
TATCACCAATCTGTGCCCCTTCGGCGAGGTGTTCAATGCCACCAGATTCGCCTCTG
TGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGACTACTCCGTGCT
GTACAACTCCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGTCCCCTACCAAG
CTGAACGACCTGTGCTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGGGGAG ATGAAGTGCGGCAGATTGCCCCTGGACAGACAGGCAAGATCGCCGACTACAACTA
CAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAACAGCAACAACCTG
G ACT C C AAAGT C G GC GG C AACT AC AATT ACCTGTACCGGCT GTT C C G G AAGT C C A
ATCTGAAGCCCTTCGAGCGGGACATCTCCACCGAGATCTATCAGGCCGGCAGCAC
CCCTTGTAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGTCCTACGGC
TTTCAGCCCACAAATGGCGTGGGCTATCAGCCCTACAGAGTGGTGGTGCTGAGCT
TCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGCCCTAAGAAAAGCACCAATCT
CGTGAAGAACAAATGCGTGAACTTCAACTTCAACGGCCTGACCGGCACCGGCGTG
CT G AC AG AG AG C AAC AAG AAGTT C CTG C C ATT C C AG C AGTTT G G C C G G GAT ATC G
C C GAT AC C AC AG AC G C C GTTAG AG AT C C C C AG AC ACT G GAAAT C CTG GAC AT C AC
CCCTTGCAGCTTCGGCGGAGTGTCTGTGATCACCCCTGGCACCAACACCAGCAAT
CAGGTGGCAGTGCTGTACCAGGACGTGAACTGTACCGAAGTGCCCGTGGCCATTC
ACGCCGATCAGCTGACACCTACATGGCGGGTGTACTCCACCGGCAGCAATGTGTT
TCAGACCAGAGCCGGCTGTCTGATCGGAGCCGAGCACGTGAACAATAGCTACGAG
TGCGACATCCCCATCGGCGCTGGCATCTGTGCCAGCTACCAGACACAGACAAACA
G C C C C AGAC G G G C C AG AT CTGTG G C C AG C C AGAG C AT C ATT G C CT AC AC AAT GTC
TCTGGGCGCCGAGAACAGCGTGGCCTACTCCAACAACTCTATCGCTATCCCCACC
AACTTCACCATCAGCGTGACCACAGAGATCCTGCCTGTGTCCATGACCAAGACCA
GCGTGGACTGCACCATGTACATCTGCGGCGATTCCACCGAGTGCTCCAACCTGCT
GCTGCAGTACGGCAGCTTCTGCACCCAGCTGAATAGAGCCCTGACAGGGATCGCC
GTG G AAC AG GAC AAG AAC AC C C AAG AG GT GTTC G C C C AAGT GAAG C AG AT CT AC A
AG AC C C CTC CTAT C AAG G ACTT CGGCGGCTT C AATTT C AG C C AGATT CTGC C C GAT
CCTAGCAAGCCCAGCAAGCGGAGCTTCATCGAGGACCTGCTGTTCAACAAAGTGA
CACTGGCCGACGCCGGCTTCATCAAGCAGTATGGCGATTGTCTGGGCGACATTGC
CGCCAGGGATCTGATTTGCGCCCAGAAGTTTAACGGACTGACAGTGCTGCCTCCT
CTGCTGACCGATGAGATGATCGCCCAGTACACATCTGCCCTGCTGGCCGGCACAA
TCACAAGCGGCTGGACATTTGGAGCTGGCGCCGCTCTGCAGATCCCCTTTGCTAT
G C AG AT GGCCTACCGGTT C AAC G G CAT C G G AGT G AC C C AG AAT GTG CT GTAC G AG
AACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACA
GCCTGAGCAGCACAGCAAGCGCCCTGGGAAAGCTGCAGGACGTGGTCAACCAGA
ATGCCCAGGCACTGAACACCCTGGTCAAGCAGCTGTCCTCCAACTTCGGCGCCAT
CAGCTCTGTGCTGAACGATATCCTGAGCAGACTGGACAAGGTGGAAGCCGAGGTG
CAGATCGACAGACTGATCACCGGAAGGCTGCAGTCCCTGCAGACCTACGTTACCC
AGCAGCTGATCAGAGCCGCCGAGATTAGAGCCTCTGCCAATCTGGCCGCCACCAA
GATGTCTGAGTGTGTGCTGGGCCAGAGCAAGAGAGTGGACTTTTGCGGCAAGGG CTACCACCTGATGAGCTTCCCTCAGTCTGCCCCTCACGGCGTGGTGTTTCTGCAC
GT G AC AT ACGTGCCCGCT C AAG AG AAG AATTT C AC C AC C GCTC C AGC C AT CTG C C
ACGACGGCAAAGCCCACTTTCCTAGAGAAGGCGTGTTCGTGTCCAACGGCACCCA
TTG GTTC GT G AC C C AG C G G AACTT CTAC G AG C C C C AG AT CAT C AC C AC C G AC AAC
ACCTTCGTGTCTGGCAACTGCGACGTCGTGATCGGCATTGTGAACAATACCGTGTA
CGACCCTCTGCAGCCCGAGCTGGACAGCTTCAAAGAGGAACTGGATAAGTACTTT
AAG AAC C AC AC AAG C C C C G AC GT G G AC CT G G G C GAT AT C AG C G G AAT C AAT G C C A
GCGTCGTGAACATCCAGAAAGAGATCGACCGGCTGAACGAGGTGGCCAAGAATCT
GAACGAGAGCCTGATCGACCTGCAAGAACTGGGGAAGTACGAGCAGTACATCAAG
TGGCCCTGGTACATCTGGCTGGGCTTTATCGCCGGACTGATTGCCATCGTGATGG
TCACAATCATGCTGTGTTGCATGACCAGCTGCTGTAGCTGCCTGAAGGGCTGTTGT
AGCTGTGGCAGCTGCTGCAAGTTCGACGAGGACGATTCTGAGCCCGTGCTGAAG
GGCGTGAAACTGCACTACACCTGA
- The S protein sequence reads as follows (SEQ ID NO 2):
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFF
SNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLI
VNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFL
MDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITR
FQTLLALHRSYLTPGDSSSGWFAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDP
LSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWN
RKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPG
QTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEI
YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRWVLSFELLHAPATVCGPKK
STNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDIT
PCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTR
AGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSV
AYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNR
ALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK
VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSG
WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASA
LGKLQDWNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSL
QTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVF
LHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF
VSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASWNIQK EIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCS
CLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT
- The E nucleotide sequence reads as follows (SEQ ID NO 3):
ATGTACAGCTTCGTGTCCGAGGAAACCGGCACACTGATCGTGAACAGCGTGCTGC
TGTTCCTGGCCTTCGTGGTGTTCCTGCTGGTTACCCTGGCTATCCTGACAGCCCTG
AGACTGTGCGCCTACTGCTGCAACATCGTGAACGTGTCCCTCGTGAAGCCCAGCT
TCTACGTGTACAGCAGAGTGAAGAACCTGAACTCCAGCAGGGTGCCCGACCTGCT
GGTCTAA
- The E protein sequence reads as follows (SEQ ID NO 4):
MYSFVSEETGTLIVNSVLLFLAFWFLLVTLAILTALRLCAYCCNIVNVSLVKPSFYVYSR
VKNLNSSRVPDLLV
- The M nucleotide sequence reads as follows (SEQ ID NO 5):
ATG G C C GAT AG C AAT G G C AC CAT C AC C GTG G AAG AACT G AAGAAACTG CT GG AAC
AGTGGAACCTCGTGATCGGCTTCCTGTTCCTGACCTGGATCTGCCTGCTGCAGTT
CGCCTACGC C AAC C G G AAC AG ATT C CTGTATAT CAT C AAG CT GAT CTTT CTGTG G C
TGCTGTGGCCCGTGACACTGGCCTGTTTTGTGCTGGCCGCCGTGTACAGAATCAA
CTGGATCACCGGCGGAATCGCCATTGCCATGGCTTGTCTTGTGGGCCTGATGTGG
CTGAGCTACTTTATCGCCAGCTTCCGGCTGTTCGCCCGGACCAGATCCATGTGGT
C CTT C AAT C C C G AG AC AAAC AT C CTG CT G AAC GT G C C C CTG C AC G G C AC C ATT CT
GACAAGACCTCTGCTGGAAAGCGAGCTGGTTATCGGCGCCGTGATCCTGAGAGGA
CACCTGAGAATTGCCGGCCACCACCTGGGCAGATGCGATATCAAGGACCTGCCTA
AAGAAATCACAGTGGCCACCAGCAGAACCCTGTCCTACTATAAGCTGGGCGCCTC
TCAGAGAGTGGCCGGCGATAGCGGATTTGCCGCCTACAGCAGATACCGGATCGG
CAACTACAAGCTGAACACCGACCACAGCTCCAGCAGCGACAATATCGCACTGCTG
GTGCAGTGA
- The M protein sequence reads as follows (SEQ ID NO 6): MADSNGTITVEELKKLLEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPVT
LACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPETNILLN
VPLHGTILTRPLLESELVIGAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGA
SQRVAGDSGFAAYSRYRIGNYKLNTDHSSSSDNIALLVQ
- The RBD nucleotide sequence reads as follows (SEQ ID NO 7):
ATGGAAACCGACACACTGCTGCTGTGGGTGCTGCTTTTGTGGGTGCCAGGCAGCA
CAGGCGATTACCCCGGGTCTAGATTCCCCAACATCACCAATCTGTGCCCCTTCGG
CGAGGTGTTCAATGCCACAAGATTTGCCAGCGTGTACGCCTGGAACCGGAAGAGA
ATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAATAGCGCCAGCTTCAGCA
CCTTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACCAAT
GTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTTCGGCAGATCGCTCCTG
GACAGACAGGCAAGATCGCCGATTACAACTACAAGCTGCCCGACGACTTCACCGG
CTGCGTGATCGCCTGGAATAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTAC
AACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCTTTCGAGCGGGACA
TCAGCACCGAGATCTATCAGGCCGGCAGCACCCCTTGTAATGGCGTGGAAGGCTT
CAACTGCTACTTCCCACTGCAGTCCTACGGCTTCCAGCCTACAAACGGCGTGGGC
TACCAGCCTTATAGAGTGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCCTGCTA
CCGTGTGTGGCCCTAAGAAGTCCACCAACTGA
- The RBD protein sequence reads as follows (SEQ ID NO 8):
METDTLLLWVLLLWVPGSTGDYPGSRFPNITNLCPFGEVFNATRFASVYAWNRKRISN
CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIA
DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGS
TPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTN
- The N nucleotide sequence reads as follows (SEQ ID NO 9):
AT GAG C G AC AAC G G C C CT C AGAAT C AG C G G AAC G C C C CT AG AAT C AC ATTT G G C G
GCCCTAGCGATAGCACCGGCAGCAATCAGAATGGCGAGAGAAGCGGCGCCAGAA
GCAAACAGAGAAGGCCTCAGGGCCTGCCTAACAACACCGCCTCTTGGTTTACAGC
CCTGACACAGCACGGCAAAGAGGACCTGAAGTTTCCTAGAGGCCAGGGCGTGCC
CAT C AAC AC C AAT AG C AG C C CT GAT GAC C AG AT C G G CTACT AC AG AC G G G C C AC C
AG AAG AATT AG AG G C G G C G AC G G C AAG AT G AAG GAT CT GAG C C C C AGAT G GTACT
TCTACTACCTCGGAACAGGACCCGAGGCCGGACTTCCTTATGGCGCCAACAAGGA TGGCATCATCTGGGTTGCAACAGAAGGCGCCCTGAACACCCCTAAGGACCACATC
G G AAC C AG AAAT C C C G C C AAC AAT G C C G C C ATT GTG CTG C AGTT G C C AC AG G G C A
CAACCCTGCCTAAGGGCTTTTATGCCGAGGGCTCTAGAGGCGGAAGCCAGGCCTC
TAGCAGAAGCAGCAGCAGAAGCCGGAACTCCAGCAGAAATAGCACCCCTGGAAG
CTCCAGAGGCACATCTCCTGCCAGAATGGCCGGAAATGGCGGAGATGCTGCTCTT
GCTCTGCTGCTCCTGGATCGGCTGAACCAGCTGGAAAGCAAGATGTCTGGAAAGG
GCCAGCAGCAACAGGGCCAGACCGTGACAAAAAAATCTGCCGCCGAGGCCAGCA
AGAAGCCCAGACAGAAGAGAACCGCCACCAAGGCCTACAACGTGACCCAGGCCTT
T GG AAG AAG AG G C C C AG AG C AG AC C C AG G G C AATTT C G G C GAT C AAG AG CT GAT
CAGACAGGGCACCGACTACAAGCACTGGCCTCAGATTGCCCAGTTTGCCCCTAGC
GCCTCCGCCTTCTTTGGCATGTCTAGAATCGGCATGGAAGTGACCCCTAGCGGCA
C CTG GTT G AC AT AC AC AG G C G C CAT C AAG CT G G AC GAC AAG G AC C C C AACTT C AA
G G AC C AAGT GAT C CTG CT G AAC AAG C AC AT C G AC G C CTAC AAG AC ATT C C CTC C A
ACCGAGCCTAAGAAGGACAAGAAGAAGAAGGCCGACGAGACACAGGCCCTGCCA
CAGAGACAGAAAAAACAGCAGACAGTGACCCTGCTGCCTGCCGCTGATCTGGACG
ATTTTTCCAAGCAGCTGCAGCAGTCCATGAGCAGCGCAGATAGCACACAGGCCTG
A
- The N protein sequence reads as follows (SEQ ID NO 10):
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTA
LTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYY
LGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGF
YAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRL
NQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQT
QGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLD
DKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAAD
LDDFSKQLQQSMSSADSTQA CLAUSES
1 . A vaccine composition comprising a. an immunologically effective amount of a modified vaccinia virus Ankara (MVA) vector comprising at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype, optionally together with a pharmaceutically acceptable carrier, diluent and/or additive; and/or b. an immunologically effective amount of the modified vaccinia virus Ankara (MVA) vector of a), wherein said MVA vector is further modified by deletion of the MVA immunomodulatory genes C6L, K7R, and A46R, optionally together with a pharmaceutically acceptable carrier, diluent and/or additive; wherein the nucleotide sequence encoding an antigenic protein of at least one SARS-CoV-2 virus subtype is inserted into the thymidine kinase (TK) locus of the MVA genome; and wherein the MVA vector regulates the expression of the nucleic acid encoding the antigenic protein and is operably linked to said nucleotide sequence.
2. The vaccine composition according to clause 1 , wherein the antigenic protein is the structural protein S of SARS-CoV-2.
3. The vaccine composition according to clause 2, wherein the antigenic protein is the S protein of SARS-CoV-2 encoded by the nucleic acid consisting of SEQ ID NO 1. 4. The vaccine composition according to any of the precedent clauses, wherein the
MVA vector further comprises an antigenic protein selected from the any of the group of structural proteins E (envelope), receptor-binding domain (RBD), N (nucleocapsid) or M (membrane) of SARS-CoV-2. 5. The vaccine composition according to any of clauses 1-4, wherein the MVA used for generating the recombinant virus is a MVA virus or a derivative having the capability of reproductive replication in vitro in chicken embryo fibroblasts (CEF) cells and in established chick cells DF-1 , but no capability of reproductive replication in human cervix adenocarcinoma cell line FleLa. 6. The vaccine composition according to any of the precedent clauses, wherein the modified (MVA) vector is the MVA as defined in claim 1a).
7. The vaccine composition according to any of clauses 1 to 5, wherein the modified (MVA) vector is the MVA as defined in claim 1 b)
8. The vaccine composition according to any of clauses 1 to 7, wherein the vaccine further comprises one or more pharmaceutically acceptable carriers, additives, antibiotics, preservatives, adjuvants, diluents and/or stabilizers.
9. A vaccine combination comprising: c. a first composition comprising an immunologically effective amount of a modified vaccinia virus Ankara (MVA) vector as defined in any of the precedent clauses; and d. a second composition comprising an immunologically effective amount of a modified vaccinia virus Ankara (MVA) vector as defined in any of the precedent clauses; wherein one of the compositions is a priming composition and the other composition is a boosting composition.
10. A vaccine combination comprising: a. a first composition comprising an immunologically effective amount of a modified vaccinia virus Ankara (MVA) vector as defined in any of clauses 1 to 8; and b. a second composition comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype; wherein one of the compositions is a priming composition and the other composition is a boosting composition.
11 . The vaccine combination comprising: a. a first composition comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype; and b. a second composition comprising an immunologically effective amount of a modified vaccinia virus Ankara (MVA) vector as defined in any of clauses 1 to 8; wherein the first compositions is a priming composition and the second composition is a boosting composition.
12. The vaccine combination of any of clauses 9 to 11 , wherein the antigenic protein for both compositions is the structural protein S of SARS-CoV-2.
13. The vaccine combination of any of clauses 9 to 11 , wherein the at least one nucleic acid of both compositions consists of SEQ ID NO 1 .
14. The vaccine composition according to clauses 1-8, for use in generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject, wherein said composition is used for priming said immune response and/or boosting said immune response and wherein the MVA is capable of producing at least the structural protein S of SARS-CoV-2, preferably of SEQ ID NO 2, in the subject to be treated.
15. The vaccine combination according to clauses 9 to 11 , for use in generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject, wherein said combination is capable of producing at least the structural protein S of SARS-CoV-2, preferably of SEQ ID NO 2, in the subject to be treated.
16. The vaccine combination according to clauses 9, for use in generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject, wherein said combination is capable of producing at least the structural protein S of SARS-CoV-2, preferably of SEQ ID NO 2, in the subject to be treated. 17. The vaccine combination according to clause 11 , for use in generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject, wherein said combination is capable of producing at least the structural protein S of SARS-CoV-2, in the subject to be treated.
18. The vaccine composition according to clauses 1-8 or the vaccine combination according to claims 9 to 13, for inducing an enhanced immune response against at least one SARS-CoV-2 virus subtype in a subject in need thereof, preferably in a human subject, wherein the MVA is capable of producing any of the group of structural proteins S, E, receptor-binding domain (RBD), N and/or M of SARS- CoV-2, or any combination thereof, in the subject to be treated.
19. The vaccine composition for use according to any of clauses 14 or 18, wherein the MVA vector/s of the vaccine composition/s is/are to be administered once, twice, three times or four times.
20. The vaccine composition or combination for use according to any of clauses 14 to
19, wherein said composition or combination is administered via the intramuscular route.
21 .The vaccine composition or combination for use according to any of clauses 14 to
20, wherein there is a boosting composition and said composition is administered at least 2 weeks after administering the priming composition
22. The vaccine composition or combination for use according to any of clauses 14 to 20, wherein there is a boosting composition and said composition is administered 2-12 weeks, preferably 2, 3 or 4 weeks, after administering the priming composition
23. A kit comprising one, preferably two, or more doses of any of the vaccine compositions as defined in any of clauses 1 to 8, or the vaccine combination of any of clauses 9 to 13. 24. A kit comprising an immunologically effective amount of a MVA vector according to any of clauses 1 to 8 in a first vial or container for a first administration (priming) and in a second vial or container for a second administration (boosting).
25. A kit comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype, preferably the structural protein S of SARS-CoV- 2, in a first vial or container for a first administration (priming), and an immunologically effective amount of a MVA vector according to any of clauses 1 to 8 in a second vial or container for a second administration (boosting).
26. A kit comprising an immunologically effective amount of a MVA vector according to any of clauses 1 to 8, in a first vial or container for a first administration (priming), and an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype, , preferably the structural protein S of SARS-CoV-2, in a second vial or container for a second administration (boosting).
27. The kit according to any of clauses 23 to 26, comprising in a third, fourth or further vial or container a MVA vector according to any of clauses 1 to 8 for a third, fourth or further administration.
28. The kit according to clauses 23 to 27, for use in generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject.
29. The kit according to clause 28, wherein the combination of compositions contained in the vials is capable of producing at least the structural protein S of SARS-CoV- 2, in the subject to be treated, and generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof.
30. The kit for use according to any of clauses 28 or 29, wherein said compositions contained in the vials are administered via the intramuscular route. 31. The kit for use according to any of clauses 28 to 30, wherein the boosting composition is administered at least 2 weeks after administering the priming composition 32. The kit for use according to any of clauses 28 to 30, wherein the boosting composition is administered 2-12 weeks, preferably 2, 3 or 4 weeks, after administering the priming composition.
33. A method of generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject, the method comprising administering to the subject any of the vaccine compositions according to any of clauses 1 to 8 or any of the vaccine combinations according to any of clauses 9 to 13. 34. The method according to claim 33, wherein said method comprises administering any of the vaccine compositions according to any of clauses 1 to 8 for priming said immune response and/or boosting said immune response.
35. The method according to clause 33, wherein said compositions or combinations of compositions contained in the vials are administered via the intramuscular route.
36. The method according to any of clauses 33 to 35, wherein the boosting composition is administered at least 2 weeks after administering the priming composition.
37. The method according to any of clauses 33 to 35, wherein the boosting composition is administered 2-12 weeks, preferably 2, 3 or 4 weeks, after administering the priming composition. 38. The method according to any of clauses 33 to 37, wherein the boosting composition is administered two or more times to the subject.
39. A method for generating an immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably a human subject, comprising administering to the subject any of the vaccine compositions according to any of clauses 1 to 8 or any of the vaccine combinations according to any of clauses 9 to 13. The method according to clause 39, wherein said method comprises administering any of the vaccine compositions according to any of clauses 1 to 8 for priming said immune response and/or boosting said immune response. The method according to clause 39, wherein said compositions or combinations of compositions contained in the vials are administered via the intramuscular route. The method according to any of clause 39 to 41 , wherein the boosting composition is administered at least 2 weeks after administering the priming composition. The method according to any of clauses 39 to 41 , wherein the boosting composition is administered 2-12 weeks, preferably 2, 3 or 4 weeks, after administering the priming composition. The method according to any of clauses 39 to 43, wherein the boosting composition is administered two or more times to the subject. A method of inducing an enhanced immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject, comprising administering to the subject any of the vaccine compositions according to any of clauses 1 to 8 or any of the vaccine combinations according to any of clauses 9 to 13. The method according to claim 45, wherein said compositions or combinations of compositions contained in the vials are administered via the intramuscular route.

Claims

1. A vaccine composition comprising: an immunologically effective amount of a modified vaccinia virus Ankara (MVA) vector comprising at least one nucleic acid encoding an antigenic structural spike (S) protein of at least one SARS-CoV-2 virus subtype, optionally together with a pharmaceutically acceptable carrier, diluent and/or additive; wherein the nucleotide sequence encoding an antigenic structural S protein of at least one SARS-CoV-2 virus subtype is inserted into the thymidine kinase (TK) locus of the MVA genome and is operably linked and under the control of a vaccinia virus synthetic early-late promoter.
2. The vaccine composition according to claim 1, wherein the antigenic protein is the S protein of SARS-CoV-2 encoded by the nucleic acid consisting of SEQ ID NO 1.
3. The vaccine composition according to any of the precedent claims, wherein the MVA vector further comprises an antigenic protein selected from the any of the group of structural proteins E (envelope), receptor-binding domain (RBD), N (nucleocapsid) or M (membrane) of SARS-CoV-2.
4. The vaccine composition according to any of claims 1 -3, wherein the MVA used for generating the recombinant virus is a MVA virus or a derivative having the capability of reproductive replication in vitro in chicken embryo fibroblasts (CEF) cells and in established chick cells DF-1, but no capability of reproductive replication in human cervix adenocarcinoma cell line FleLa.
5. The vaccine composition according to any of claims 1 to 4, wherein the vaccine further comprises one or more pharmaceutically acceptable carriers, additives, antibiotics, preservatives, adjuvants, diluents and/or stabilizers.
6. A vaccine combination comprising: a. a first composition comprising an immunologically effective amount of a MVA vector as defined in any of the precedent claims; and b. a second composition comprising an immunologically effective amount of a MVA vector as defined in any of the precedent claims; wherein one of the compositions is a priming composition and the other composition is a boosting composition.
7. A vaccine combination comprising: a. a first composition comprising an immunologically effective amount of a MVA vector as defined in any of claims 1 to 5; and b. a second composition comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype; wherein one of the compositions is a priming composition and the other composition is a boosting composition.
8. The vaccine combination comprising: a. a first composition comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype; and b. a second composition comprising an immunologically effective amount of a MVA vector as defined in any of claims 1 to 5; wherein the first compositions is a priming composition and the second composition is a boosting composition.
9. The vaccine combination of any of claims 6 to 8, wherein the antigenic protein for both compositions is the structural protein S of SARS-CoV-2.
10. The vaccine combination of any of claims 6 to 8, wherein the at least one nucleic acid of both compositions consists of SEQ ID NO 1 .
11. The vaccine composition according to claims 1-5, for use in generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject, wherein said composition is used for priming said immune response and/or boosting said immune response and wherein the MVA is capable of producing at least the structural protein S of SARS-CoV-2, preferably of SEQ ID NO 2, in the subject to be treated.
12. The vaccine combination according to claims 6 to 8, for use in generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject, wherein said combination is capable of producing at least the structural protein S of SARS- CoV-2, preferably of SEQ ID NO 2, in the subject to be treated.
13. The vaccine combination according to claim 6, for use in generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject, wherein said combination is capable of producing at least the structural protein S of SARS- CoV-2, preferably of SEQ ID NO 2, in the subject to be treated.
14. The vaccine combination according to claim 8, for use in generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject, wherein said combination is capable of producing at least the structural protein S of SARS- CoV-2, in the subject to be treated.
15. The vaccine composition according to claims 1-5 or the vaccine combination according to claims 6 to 10, for inducing an enhanced immune response against at least one SARS-CoV-2 virus subtype in a subject in need thereof, preferably in a human subject, wherein the MVA is capable of producing any of the group of structural proteins S, E, RBD, N and/or M of SARS-CoV-2, or any combination thereof, in the subject to be treated.
16. The vaccine composition for use according to any of claims 11 or 15, wherein the MVA vector/s of the vaccine composition/s is/are to be administered once, twice, three times or four times.
17. The vaccine composition or combination for use according to any of claims 11 to 16, wherein said composition or combination is administered via the intramuscular and/or intranasal route.
18. The vaccine composition or combination for use according to any of claims 11 to 17, wherein there is a boosting composition and said composition is administered at least 2 weeks after administering the priming composition
19. The vaccine composition or combination for use according to any of claims 11 to 17, wherein there is a boosting composition and said composition is administered 2-12 weeks, preferably 2, 3 or 4 weeks, after administering the priming composition.
20. A kit comprising one, preferably two, or more doses of any of the vaccine compositions as defined in any of claims 1 to 5, or the vaccine combination of any of claims 6 to 10.
21. A kit comprising an immunologically effective amount of a MVA vector according to any of claims 1 to 5 in a first vial or container for a first administration (priming) and in a second vial or container for a second administration (boosting).
22. A kit comprising an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype, preferably the structural protein S of SARS-CoV-2, in a first vial or container for a first administration (priming), and an immunologically effective amount of a MVA vector according to any of claims 1 to 5 in a second vial or container for a second administration (boosting).
23. A kit comprising an immunologically effective amount of a MVA vector according to any of claims 1 to 5, in a first vial or container for a first administration
(priming), and an immunologically effective amount of a polynucleotide plasmid that comprises at least one nucleic acid encoding an antigenic protein of at least one SARS-CoV-2 virus subtype, , preferably the structural protein S of SARS- CoV-2, in a second vial or container for a second administration (boosting).
24. The kit according to any of claims 20 to 23, comprising in a third, fourth or further vial or container a MVA vector according to any of claims 1 to 5 for a third, fourth or further administration.
25. The kit according to claims 20 to 24, for use in generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject.
26. The kit according to claim 2825 wherein the combination of compositions contained in the vials is capable of producing at least the structural protein S of SARS-CoV-2, in the subject to be treated, and generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof.
27. The kit for use according to any of claims 25 or 26, wherein said compositions contained in the vials are administered via the intramuscular and/or intranasal route.
28. The kit for use according to any of claims 25 to 27, wherein the boosting composition is administered at least 2 weeks after administering the priming composition
29. The kit for use according to any of claims 25 to 27, wherein the boosting composition is administered 2-12 weeks, preferably 2, 3 or 4 weeks, after administering the priming composition.
30. A method of generating an immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably in a human subject, the method comprising administering to the subject any of the vaccine compositions according to any of claims 1 to 5 or any of the vaccine combinations according to any of claims 6 to 10.
31. The method according to claim 30, wherein said method comprises administering any of the vaccine compositions according to any of claims 1 to 5 for priming said immune response and/or boosting said immune response.
32. The method according to claim 30, wherein said compositions or combinations of compositions contained in the vials are administered via the intramuscular route.
33. The method according to any of claims 30 to 32, wherein the boosting composition is administered at least 2 weeks after administering the priming composition.
34. The method according to any of claims 30 to 32, wherein the boosting composition is administered 2-12 weeks, preferably 2, 3 or 4 weeks, after administering the priming composition.
35. The method according to any of claims 30 to 34, wherein the boosting composition is administered two or more times to the subject.
36. A method for generating an immune response against the SARS-CoV-2 virus in a subject in need thereof, preferably a human subject, comprising administering to the subject any of the vaccine compositions according to any of claims 1 to 5 or any of the vaccine combinations according to any of claims 6 to 10.
37. The method according to claim 36, wherein said method comprises administering any of the vaccine compositions according to any of claims 1 to 5 for priming said immune response and/or boosting said immune response.
38. The method according to claim 36, wherein said compositions or combinations of compositions contained in the vials are administered via the intramuscular and/or intranasal route.
39. The method according to any of claims 36 to 38, wherein the boosting composition is administered at least 2 weeks after administering the priming composition.
40. The method according to any of claims 36 to 38, wherein the boosting composition is administered 2-12 weeks, preferably 2, 3 or 4 weeks, after administering the priming composition.
41. The method according to any of claims 36 to 40, wherein the boosting composition is administered two or more times to the subject.
42. A method of inducing an enhanced immune response against the SARS-CoV- 2 virus in a subject in need thereof, preferably in a human subject, comprising administering to the subject any of the vaccine compositions according to any of claims 1 to 5 or any of the vaccine combinations according to any of claims 6 to 10.
43. The method according to claim 42, wherein said compositions or combinations of compositions contained in the vials are administered via the intramuscular and/or intranasal route.
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