WO2022269003A1 - Vaccin à base de mva exprimant une protéine s du sars-cov-2 stabilisée par préfusion - Google Patents

Vaccin à base de mva exprimant une protéine s du sars-cov-2 stabilisée par préfusion Download PDF

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WO2022269003A1
WO2022269003A1 PCT/EP2022/067271 EP2022067271W WO2022269003A1 WO 2022269003 A1 WO2022269003 A1 WO 2022269003A1 EP 2022067271 W EP2022067271 W EP 2022067271W WO 2022269003 A1 WO2022269003 A1 WO 2022269003A1
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mva
cov
protein
sars
vaccine
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Juan Francisco GARCÍA ARRIAZA
Mariano ESTEBAN RODRÍGUEZ
Patricia PÉREZ RAMÍREZ
Adrian LÁZARO FRÍAS
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Consejo Superior De Investigaciones Cientificas
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Priority to EP22737464.2A priority Critical patent/EP4358999A1/fr
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    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • A61K39/12—Viral antigens
    • A61K39/275—Poxviridae, e.g. avipoxvirus
    • A61K39/285—Vaccinia virus or variola virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention is directed to a recombinant modified vaccinia virus Ankara (MVA) vector comprising a nucleic acid sequence coding for a prefusion-stabilized full- length spike (S) protein of at least one SARS-CoV-2 variant that is 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 at least one SARS-CoV-2 variant.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • SARS-CoV-2 Middle East respiratory syndrome coronaviruses
  • Coronavirus virions are decorated with a spike (S) glycoprotein that binds to host cell receptors and mediates cell entry via fusion of the host and viral membranes.
  • S spike
  • This protein is also the main target of neutralizing antibodies.
  • SARS-CoV-2 S ectodomain is unstable and difficult to produce reliably in mammalian cells, hampering the development of S-based vaccines.
  • Prefusion stabilization techniques are aimed at increasing the recombinant expression of viral fusion glycoproteins, possibly by preventing triggering or misfolding that results from a tendency to adopt the more stable postfusion structure.
  • prefusion-stabilized viral glycoproteins are also superior immunogens to their wild-type counterparts, since they represent the primary target of neutralizing antibodies.
  • FIG. 1 Design, generation, and in vitro characterization of MVA-S(3P) vaccine candidate in permissive DF-1 cells.
  • A Genome map of the vaccine candidate MVA- S(3P) [also termed MVA-CoV2-S(3P)]) expressing a prefusion-stabilized coronavirus SARS-CoV-2 S protein. 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 coronavirus SARS-CoV-2 S(3P) gene inserted within the MVA TK viral locus (J2R) of MVA-WT parental virus and driven by the sE/L virus promoter are indicated.
  • TK-L TK left
  • TK-R TK right.
  • An scheme of the prefusion-stabilized full-length S protein inserted in the MVA genome is included.
  • S1 and S2 regions are indicated, together with the amino acid mutations in the furin cleavage site and changes to prolines in the S2 region.
  • NTD N-terminal domain
  • RBD receptor binding domain
  • TM transmembrane
  • CT cytoplasmic tail.
  • 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-S and MVA-S(3P), 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 infected for 24 hours (in the absence or presence of tunicamycin) at 5 PFU/cell with vaccine candidates MVA-S and MVA-S(3P) and MVA-WT.
  • vaccine candidates MVA-S and MVA-S(3P) and MVA-WT.
  • 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 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.
  • DF-1 cells were infected with MVA-S and MVA-S(3P) vaccine candidates and at 24 hpi, cells were lysed under nonreducing conditions (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). 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.
  • E 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-S and MVA-S(3P).
  • DF-1 cells were infected with vaccine candidates MVA-S and MVA-S(3P), and MVA-WT, at 5 PFU/cell.
  • the cell extracts and the supernatants were collected 4, 7 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 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 candidate MVA-S(3P) in nonpermissive human HeLa cells.
  • A Expression of the coronavirus SARS-CoV-2 S- protein. HeLa cells were infected for 24 hours (in the absence or presence of tunicamycin) at 5 PFU/cell with vaccine candidates MVA-S and MVA-S(3P) and MVA- WT. At 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 rabbit polyclonal antibody against SARS-CoV-2 (against SARS-CoV-2 S1 region).
  • the cell extracts and the supernatants were collected 4, 7 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 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 Pius protein standards; Bio-Rad Laboratories) are indicated on the left.
  • C Expression of SARS-CoV-2 S protein under nonreducing conditions.
  • HeLa cells were infected with MVA-S and MVA-S(3P) vaccine candidates and at 4, 7 and 24 hpi, cells were lysed under nonreducing conditions (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). 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. Figure 3.
  • MVA-S(3P) is stable, express S in the membrane of infected cells and replicates in permissive DF-1 cells.
  • A Genetic stability of vaccine candidate MVA- S(3P). DF-1 cell monolayers were infected with the P2 stock of MVA-S(3P) at a low MOI for 8 serial passages.
  • chick DF-1 cells were the left uninfected (mock) or infected with vaccine candidate MVA-S(3P) 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). 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 indicate the position of the S protein or the VACV E3 protein.
  • DF-1 cell monolayers were infected with MVA-WT, MVA-S, and MVA-S(3P) 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.
  • Figure 4 Expression of the S protein in the cell membrane of HeLa cells infected with MVA-CoV2-S(3P), and analyzed by immunofluorescence. Subcellular distribution of the SARS-CoV-2 S protein in cells infected with MVA-S and MVA-S(3P).
  • mice were sacrificed 10 days after the boost (day 25) 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.
  • B and C Titers of binding IgG antibodies specific for the S and RBD proteins at 10 days post-prime (B) or 10 days post-boost (C).
  • Titers were determined by ELISA in individual mice serum samples collected 10 days post-prime or 10 days postboost immunization, and were calculated as the serum dilution (Y axis) at which the absorbance at 450 nm 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.
  • Figure 6 SARS-CoV-2 S-specific and RBD-specific lgG1, lgG2b, lgG2c, and lgG3 antibodies induced by vaccine candidate MVA-S(3P) in C57BL/6 mice immunized.
  • Groups of C57BL/6 mice were immunized as represented in Figure 5A.
  • serum samples were collected, and the levels of total coronavirus SARS-CoV-2 S-specific and RBD-specific lgG1, lgG2b, IgG2c, and igG3 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.
  • A retrovirus-based pseudoparticles expressing the SARS-CoV-2 S protein
  • B live-virus microneutralization
  • 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. 8 ELISpot analysis of the adaptive SARS-CoV-2 S-specific cell-mediated immune responses induced by vaccine candidate MVA-S(3P) in C57BL/6 mice.
  • Six mice per group were sacrificed at 10 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-g positive ceils per million of splenocytes. The magnitude of SARS- CoV-2-specific cells secreting IFN-g and directed against SARS-CoV-2 S1 and S2 peptide pools is represented.
  • FIG. 9 SARS-CoV-2 S-specific adaptive CD4+ (left) and CD8+ (right) T cell- mediated immune responses induced by vaccine candidate MVA-S(3P) in C57BL/6 mice. CD4+ and CD8+ T cell-mediated responses in the splenocytes of mice
  • FIG. 10 Polyfunctionality of SARS-CoV-2 S-specific adaptive CD4+ and CD8+ T cell-mediated immune responses induced by vaccine candidate MVA-S(3P) in C57BL/6 mice. Six mice per group were sacrificed at 10 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.
  • MVA-S(3P) homologous prime-boost 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.
  • Figure 12 SARS-CoV-2 S-specific adaptive CD4+ T follicular helper (Tfh) cell- mediated immune responses induced by vaccine candidate MVA-S(3P) in C57BL/6 mice.
  • 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-g 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-y 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 S2 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- Y and/or IL-21 against SARS-CoV-2 S protein plus S1 and S2 peptide pools are shown on the y axis.
  • FIG. 13 One dose of MVA-S(3P) protects transgenic K18-hACE2 mice from SARS-CoV-2 infection.
  • mice per group were sacrificed and lungs and serum samples collected as indicated. Serum was also collected at 14 days after immunization and in mice alive at 15 days postchallenge (groups 1 and 2).
  • B, C The challenged mice were monitored for change of body weights (B) and mortality (C) for 15 days.
  • ⁇ mice were euthanized due to loss of more than 25% of initial body weight.
  • D Virus replication in lung samples.
  • Subgenomic (E) and genomic (RdRp) SARS-CoV-2 RNA detected by RT-qPCR at 4 days after virus infection (n 6).
  • RNA levels in arbitrary units [A.U.]) and SEM from duplicates of each lung sample; relative values are referred to uninfected mice.
  • E SARS-CoV-2 infectious virus in lung samples. Mean (PFU/g of lung tissue) and SEM from triplicates of each lung sample. Student’s t-test: ** P ⁇ 0.005.
  • Fig. 14 One dose of MVA-S(3P) reduced SARS-CoV-2 lung pathology and diminished levels of pro-inflammatory cytokines in K18-hACE2 transgenic mice.
  • B Representative lung histopathological sections (H&E staining) from K18-hACE2 transgenic mice euthanized at day 4 postchallenge. A general view of the lung area (magnification: 4x) along with histopathological details from selected lung areas (black boxes) have been displayed (magnification: 10x).
  • mice vaccinated with one dose of MVA-S and MVA-WT displayed more extensive and severe lung inflammatory lesions compared to mice immunized with one dose of MVA- S(3P) (c).
  • lung inflammatory lesions were characterized by the presence of moderate diffuse thickening of the alveolar septae, perivascular oedema (white arrowheads), moderate presence of mononuclear cell infiltrates within alveolar spaces (black arrows), hyaline membranes and cell debris within alveoli (black arrowheads) as well as the presence of large multifocal perivascular and peribronchiolar mononuclear infiltrates (red arrows).
  • MVA-S(3P) vaccine candidate induced high levels of humoral responses in vaccinated and challenged K18-hACE2 transgenic mice.
  • B SARS-CoV-2 neutralizing antibody titers.
  • NT50 titers were evaluated in individual mouse serum samples collected at day 14 pi and at days 4 and 15 pc, using a live virus microneutralization assay (MAD6 strain, having D614G mutation). Mean NT50 values and SEM are represented. Upper dotted line represents the levels obtained with the NIBSC 20/136 international standard plasma (containing pooled plasma obtained from 11 individuals recovered from SARS- CoV-2 infection). Bottom dotted line represented the limit of detection. (C) SARS-CoV-2 neutralizing antibody titers against SARS-CoV-2 VoC.
  • NT50 titers were evaluated in pooled mouse serum samples collected at days 14 pi (left) and 15 pc (right), using VSV- based pseudoparticles expressing the SARS-CoV-2 S protein of different VoC. Mean NT50 values and 95% confidence intervals are represented. Dashed line represents the limit of detection. Student’s t-test: ** , P ⁇ 0.005; *** P ⁇ 0.001.
  • FIGURE 16 SARS-CoV-2 S-specific T-cell immune responses elicited in C57BL/6 mice immunized with one intranasal dose of MVA-S(3P).
  • (A) Magnitude of S-specific CD4 + (left) and CD8 + (right) T-cell immune responses evaluated at 14 days post-immunization in spleen, lungs and bronchial lymph nodes (BLN). Percentages of CD4 + or CD8 + T cells expressing CD107a and/or producing IFN-y and/or TNF-a and/or IL-2 against a mixture of S1 and S2 peptide pools in immunized mice. Cell percentages were determined by ICS from pools of cells obtained from the different tissues.
  • FIGURE 18 One intranasal dose of MVA-S(3P) protects transgenic K18-hACE2 mice from SARS-CoV-2 infection.
  • A Efficacy schedule.
  • mice were challenged i.n. with 1 x 10 5 PFUs of SARS-CoV-2 (MAD6 isolate).
  • MVA- WT-inoculated mice were also challenged with SARS-CoV-2 and used as a control.
  • mice per group were sacrificed and lungs, nasal washes and serum samples collected as indicated. Serum was also collected at 14 days after immunization and at 14 days postchallenge (in groups 1 , 2 and 3).
  • B, C The challenged mice were monitored for change of body weights (B) and mortality (C) for 14 days. ⁇ : mice were euthanized due to loss of more than 20% of initial body weight.
  • RNA levels in arbitrary units [A.U.]) and SEM from duplicates of each lung and nasal washes samples; relative values are referred to uninfected mice.
  • F, G SARS-CoV-2 infectious virus in lung samples (F) and nasal washes (G).
  • Mean PFUs/g of lung tissue or PFUs per ml of nasal wash) and SEM from triplicates of each sample. Student’s t-test: * , p ⁇ 0.05; ** , p ⁇ 0.005; *** , p ⁇ 0.001.
  • FIGURE 19 One intranasal dose of MVA-S(3P) reduced SARS-CoV-2 lung pathology in K18-hACE2 transgenic mice.
  • FIG. 1 Representative lung histopathological sections (H&E staining) from K18-hACE2 mice euthanized at day 5 postchallenge (magnification: 10x).
  • mice immunized with MVA-S(3P) (c) only displayed small lung areas with mild inflammatory lesions such as focal thickening of alveolar septae, occasional presence of mononuclear cell infiltrates within alveolar spaces (black arrowheads) and mild perivascular or peribronchiolar mononuclear infiltrates (black arrows).
  • FIGURE 20 One intranasal dose of MVA-S(3P) diminished levels of pro- inflammatory cytokines in lung samples and nasal washes from K18-hACE2 transgenic mice.
  • mRNA levels detected by RT-qPCR in lungs (A) and nasal washes (B) (n 4/group) obtained at 5 days postchallenge.
  • FIGURE 21 One intranasal dose of MVA-S(3P) induced high levels of humoral responses in vaccinated and challenged K18-hACE2 transgenic mice.
  • B SARS-CoV-2 neutralizing antibody titers.
  • NTso titers were evaluated in individual mouse serum samples collected at day 14 pi and at days 5 and 14 pc, using a live virus microneutralization assay (MAD6 strain, having D614G mutation). Mean NTso values and SEM are represented. Dotted line represented the limit of detection.
  • C SARS-CoV-2 neutralizing antibody titers against SARS-CoV-2 VoC. NTso titers were evaluated in pooled mouse serum samples collected at days 14 pi (left) and 14 pc (right), using VSV-based pseudoparticles expressing the SARS-CoV-2 S protein of different VoC. Mean NT50 values and 95% confidence intervals are represented. Dashed line represents the limit of detection. Student’s t-test: * , p ⁇ 0.05;
  • FIGURE 22 MVA-S(3P) protects transgenic K18-hACE2 mice from infection with SARS-CoV-2 B.1.351 variant.
  • A Efficacy schedule.
  • At week 8 (day 56) mice were challenged i.n. with 1 x 10 5 PFUs of SARS-CoV-2 (B.1.351 variant of concern).
  • MVA-S(3P) protects transgenic K18-hACE2 mice from infection with SARS-CoV-2 B.1.351 variant.
  • mice WT-inoculated mice were also challenged with SARS-CoV-2 and used as a control. At day 4 postchallenge 3-6 mice per group were sacrificed and lungs, BAL and serum samples collected as indicated. Serum was also collected at 14 days after prime immunization, 21 days after boost, and at days 4 and 10 days postchallenge.
  • B, C The challenged mice were monitored for change of body weights (B) and mortality (C) for 10 days.
  • ⁇ mice were euthanized due to loss of more than 20% of initial body weight.
  • D, E Virus replication in lung samples (D) and BAL (E).
  • RNA levels in arbitrary units [A.U.]
  • SEM SARS-CoV-2 infectious virus in lung samples
  • BAL BAL
  • Mean PFUs/g of lung tissue or PFUs per ml of BAL
  • FIGURE 23 MVA-S(3P) diminished levels of pro-inflammatory cytokines in lung samples from K18-hACE2 transgenic mice challenged with SARS-CoV-2 B.1.351 variant.
  • mRNA levels detected by RT-qPCR in lungs (n 3-6/group) obtained at 4 days postchallenge.
  • FIGURE 24 MVA-S(3P) induced high levels of humoral responses in vaccinated K18-hACE2 transgenic mice challenged with SARS-CoV-2 B.1.351 variant.
  • B Titers of IgG antibodies against RBD from different SARS-CoV-2 VoC. Determined by ELISA in pooled sera samples collected at days 14 postprime (left) and 21 postboost (right). FIGURE 25.
  • Monkeypox-specific CD8+ T cell-mediated immune responses induced by MVA-S(3P) vaccine candidate in C57BL/6 mice Monkeypox-specific CD8+ T cell-mediated immune responses induced by MVA-S(3P) vaccine candidate in C57BL/6 mice.
  • the y-axis shows the percentage of monkeypox-specific T cells expressing CD107a and/or producing IFN-y and/or TNF-a and/or IL-2.
  • B Adaptive CD8+ T cell-mediated immune responses shown as the percentage of Monkeypox-specific cells expressing CD107a or producing IFN-g or TNF-a or IL-2.
  • C Phenotype profile of monkeypox-specific adaptive CD8+ T ceils.
  • the y-axis indicates the percentage of monkeypox-specific 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
  • D Polyfunctional profiles of total monkeypox-specific CD8+ T cell adaptive immune responses.
  • SARS-CoV-2 S(3P) protein is expressed at higher levels than a S(2P) protein.
  • Concentration of S(3P) and S(2P) proteins obtained from transfected HEK-293 cells. Measured by ELISA. Optical density at 490 nm in the ELISA assays were corrected by the background signals determined in wells with untransfected cell supernatants. Average of two independent transfections are shown.
  • 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.
  • the expression “at least” or “at least one of as used herein includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use.
  • 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.
  • 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 recombinant MVA according to the present invention relative to the corresponding immune response observed from the animal administered with a vector that does not express any SARS-CoV-2 proteins, e.g., a MVA-WT vector.
  • 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 MVA 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.
  • 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.
  • the reference genome in the context of the present invention is GenBank accession number MN908947.3.
  • the reference protein or polypeptide in the context of the present invention is obtained from the translation of the reference genome (GenBank accession number MN908947.3). More preferably, for the S protein, the reference protein is the amino acid sequence of PDB ID 6VSB.
  • 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.
  • 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.
  • subtype herein includes strains, isolates, clades, lineages, linages, and/or variants of any severe acute respiratory syndrome coronavirus, namely SARS-CoV-2.
  • strain “clade”, “lineage or linage”, “isolate” and/or “variant” are technical terms, well known to the skilled person, referring to the taxonomy of microorganisms, that is, referring to all characterized microorganisms into the hierarchic order of Families, Genera, Species, Strains.
  • 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.
  • strain or "clade” describes a microorganism, for instance, a virus, which shares common characteristics with other microorganisms, like basic morphology or genome structure and organization, but varies in biological properties, like host range, tissue tropism, geographic distribution, attenuation or pathogenicity.
  • variant describes a microorganism, in the present invention, a virus, which replicates and introduces one or more new mutations into its genome which results in differences from the original virus.
  • the terms “variant” and “subtype” are synonymous and are used interchangeably.
  • the term “lineage” or “linage” describes a cluster of viral sequences derived from a common ancestor, which are associated with an epidemiological event, for instance, an introduction of the virus into a distinct geographic area with evidence of onward spread. Lineages are designed to capture the emerging edge of a pandemic and are at a fine-grain resolution suitable to genomic epidemiological surveillance and outbreak investigation.
  • the SARS-CoV-2 lineage nomenclature is described for example, in Rambaut A. et al. A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology. Nat Microbiol. 2020; 5(11): 1403-1407.
  • At least one variant of severe acute respiratory syndrome coronavirus 2 is meant at least one variant, strain, isolate, lineage or linage, and/or clade of the SARS-CoV-2 virus.
  • SARS-CoV-2 can be found in databases such as Emma B. Hodcroft. 2021. “CoVariants: SARS-CoV-2 Mutations and Variations of Interest” (covariants.org/variants) or O’Toole A. et al., 2020 “A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology”, PANGO lineages (cov-lineages.org/).
  • WHO World Health Organization
  • the term “variant” or “linage” includes all SARS-CoV-2 viral sequences that encode for a S protein with a percentage of amino acid sequence identity of at least 90%, 91%, 92%, 93%, 94%, preferably of at least 95%, 96%, 97%, 98%, or 99% from the reference S protein of the reference strain SARS-CoV-2 Wuhan-Hu-1 (GenBank accession No QHD43416.1 or Uniprot ID: P0DTC2), when both S proteins are locally aligned, for example, by using Basic Local Alignment Search Tool (BLAST).
  • BLAST Basic Local Alignment Search Tool
  • an “homologous prime-boost combination” is referred herein as the administration of at least two doses of a vaccine, wherein the vaccine is the same in every dose.
  • heterologous prime-boost combination is referred herein as the administration of at least two doses of a vaccine, wherein the first dose comprises a vaccine that is different from the vaccine comprised in the second or subsequent doses.
  • different is meant herein that either the immunogen is different (different antigen) or that, even if the antigen is the same, it is carried by a different vector, or both (different vector and different antigen).
  • a heterologous prime-boost combination may be a first dose with a DNA-based vaccine, and a second dose with a MVA-based vaccine.
  • Immunogen is referred herein to a protein or a portion thereof that is capable of inducing an immune response in a mammal, such as a mammal infected or at risk of infection with a pathogen.
  • synthetic early/late promoter is referred herein as a vaccinia virus promoter comprising both early and late regulatory elements, wherein said elements overlap in a region of 5 base pairs.
  • Said promoter is widely known in the field, see e.g., Chakrabarti, S.; Sisler, J.R.; Moss, B. Compact, synthetic, vaccinia virus early/!ate promoter for protein expression. Biotechniques 1997, 23, 1094-1097.
  • the synthetic early/late promoter as referred herein comprises or consists of SEQ ID NO 21 or SEQ ID NO 26.
  • Vaccinia virus is amongst the most extensively evaluated live vectors and has particular features in support of its use as a 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 VACV. 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. B 167, 375-390 [1987], Stickl et al., Dtsch. med. Wschr. 99, 2386-2392 [1974]).
  • 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 aviru!ence 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 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) Garcia.-Arriaza et al., 2021 (SARS-CoV-2)).
  • VACV recombinant VACV
  • Highly immunogenic viral proteins such as the S protein of SARS-CoV-2 virus
  • S protein of SARS-CoV-2 virus are usually elusive and present several mechanisms to mislead the immune system and avoid the straightforward generation of neutralizing antibodies.
  • Some of the mechanisms used to evade the immune system of the host imply conformational changes that may also be important for viral cycle.
  • it is particularly relevant the transition between the prefusion state of the S protein, that is naturally present in the viral particles when they are free in the media, to the postfusion state, that occurs after the binding of the S protein to the host cell receptor.
  • the S protein is first produced as a precursor that trimerizes and is thought to be cleaved by a furin-like protease into two fragments: the receptor-binding fragment S1 and the fusion fragment S2.
  • Binding through the receptor-binding domain (RBD) comprised in the S1 to a host cell and further proteolytic cleavage at a second site in S2 are believed to trigger dissociation of S1 and irreversible refolding of S2 into a postfusion conformation, a trimeric hairpin structure formed by heptad repeat 1 (HR1 ) and heptad repeat 2 (HR2). These large structural rearrangements bring together the viral and cellular membranes, ultimately leading to fusion of the two bilayers.
  • rMVA-based vaccines would or not be capable of generating a strong immunogenic and protective immune response against the prefusion-stabilized S protein from SARS-CoV-2 virus in a subject in need thereof.
  • the authors of the present invention have redesigned the S protein of SARS-CoV-2 virus by introducing modifications into the wildtype S amino acid sequence and they have found and demonstrated that a vaccine comprising an immunologically effective amount of a MVA vector expressing a prefusion-stabilized SARS-CoV-2 S protein (termed MVA-CoV2-S(3P), which will also be referred to as MVA-S(3P) throughout the text and figures) is capable of generating a strong immunogenic and protective immune response against the SARS-CoV-2 virus in a subject in need thereof, remarkably only after one dose of the vaccine.
  • the prefusion- stabilized S protein disclosed herein comprises at least the substitutions R682G, R683S, R685S, A942P, K986P and V98
  • mice vaccinated with one intramuscular dose of MVA-S(3P) generated higher neutralizing antibodies and higher anti-S and anti-RBD IgG antibodies than the parental MVA-CoV2-S vector (also termed MVA-S) (Fig. 5, 7, and 15), in which the S protein does not comprise the substitutions R682G, R683S, R685S, A942P, K986P and V987P and therefore the vector MVA- CoV2-S does not produce a prefusion-stabilized S protein.
  • MVA-S(3P) enhances the titers of neutralizing antibodies against parental Wuhan strain and variants of concern (Fig. 15C).
  • mice vaccinated with MVA-S(3P) did not present a reduction in body weight due to the infection, while mice vaccinated with MVA-CoV2-S suffered loss of body weight, indicating that the virus caused an infection in these animals (see Fig. 13B).
  • vaccination with one dose of MVA-S(3P) was highly effective to prevent SARS-CoV-2 replication and virus yields at 4 days postchallenge in lung samples (Fig. 13D and 13E), to reduce lung pathology (Fig.
  • the vector MVA-S(3P) is able to generate higher neutralizing antibody titers than parental MVA-S after one dose (10 days post prime) (Fig. 7A). All these higher humoral immune responses elicited by MVA-S(3P) are in accordance with the findings described above that the vector MVA- S(3P) protects more efficiently and without the course of symptoms than MVA-S after a single dose of the vaccine prior a SARS-CoV-2 challenge.
  • the present invention thus provides a homologous prime/boost vaccine combination comprising:
  • composition comprising an immuno!ogical!y effective amount of the rMVA vector provided herein;
  • compositions comprising an immunologically effective amount of the rMVA vector provided herein; wherein one of the compositions is a priming composition and the other composition is a boosting composition.
  • Such response is particularly strong when the composition comprises the MVA-S(3P) provided herein.
  • Intranasal administration of one dose of MVA-S(3P) of the present invention also prevented morbidity (see Fig. 18B) and mortality (see Fig. 18C) in SARS-CoV-2 challenged K18-hACE2 mice, reducing SARS-CoV-2 virus replication in lungs and nasal washes (see Fig. 18D to 18G), thereby reducing lung pathology (see Fig. 19) and the levels of pro-inflammatory cytokines in lungs and nasal washes (see Fig. 20).
  • MVA-S(3P) of the present invention also prevented morbidity (see Fig. 22B) and mortality (see Fig. 22C) of K18-hACE2 mice challenged with SARS-CoV-2 B.1.351 (beta) variant, reducing SARS-CoV-2 virus beta variant replication in lungs and BAL (see Fig. 22D to 22G), thereby reducing the levels of pro-inflammatory cytokines in lungs (see Fig. 23).
  • high titers of binding IgG antibodies that were directed not only against Wuhan variant, but also against other variants of concerns were induced (see Fig. 24).
  • the present invention also provides a heterologous prime/boost vaccine combination comprising:
  • composition comprising an immunologically effective amount of the rMVA vector provided herein;
  • composition comprising an immunologically effective amount of at least one immunogen of at least one SARS-CoV-2 variant; wherein one of the compositions is a priming composition and the other composition is a boosting composition, in any order.
  • a composition comprising an immunologically effective amount of at least one immunogen of at least one SARS-CoV-2 variant; wherein one of the compositions is a priming composition and the other composition is a boosting composition, in any order.
  • Such response may be particularly strong when the composition comprising the MVA-S(3P) provided herein is given as a boost.
  • the invention provides, for the first time, enhanced and effective vaccines or vaccine combinations for use in generating a protective immune response against infections by at least one SARS-CoV-2 variant or linage and vaccines or vaccine combinations which can be used for manufacturing of a vaccine against at least one SARS-CoV-2 variant or linage.
  • a first aspect of the present invention provides a vaccine composition
  • a vaccine composition comprising an immunologically effective amount of a modified vaccinia virus Ankara (MVA) vector comprising at least one nucleic acid encoding a SARS-CoV-2 spike (S) protein, or an immunogenic fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant or linage, wherein said S protein or fragment comprise amino acid substitutions aimed at enhancing the stability of the prefusion state of the S protein or fragment therefrom, and wherein the MVA vector regulates the expression of the nucleic acid encoding said stabilized prefusion S protein or fragment thereof.
  • MVA modified vaccinia virus Ankara
  • the redesigned S protein encoded by the genome of MVA contains amino acid substitutions that enhance stability of the prefusion S structure. These include mutations that inactivate the S1/S2 cleavage site, and mutations in HR1 that remove any strain in the turn region between HR1 and CH, i.e., to prevent the formation of a straight helix during fusion.
  • the resigned S protein can additionally contain a truncation of the HR2 motif. Truncation of the HR2 domain leads to disruption of the HR1/HR2 fusion core and also stabilizes the prefusion S structure.
  • the S protein or an immunogenic fragment of said S protein comprising at least one epitope comprises at least one, two, preferably three, amino acid substitutions to inactivate the S1/S2 furin cleavage site. Inactivation of this cleavage site can be achieved by a number of sequence alterations (e.g., deletions or substitutions) within or around the site.
  • One mutation that inactivates the cleavage site without otherwise impacting the structure of the protein is substitution of residues 682 RRAR 685.
  • the amino acid numbering used as reference is the one based on cryo-EM model PDB ID 6VSB or GenBank accession number MN908947.3.
  • said inactivation of the S1/S2 cleavage site is achieved by introducing at least one, two, or three of the amino acid substitutions selected from the list consisting of R682G, R683S or R685S. More preferably, the S protein or the immunogenic fragment thereof comprises at least the three amino acid substitutions R682G, R683S and R685S.
  • the soluble SARS-CoV-2 immunogen polypeptides can additionally contain a double mutation in the HR1 region that remove strain in the turn region (between HR1 and CH motifs) during fusion by preventing the formation of a straight helix.
  • the S protein, or an immunogenic fragment thereof, encoded by the nucleic acid comprised in the MVA vector of the present invention further comprises other amino acid substitutions aimed at maintaining the quaternary structure of the S protein in a prefusion state or form.
  • a “prefusion protein” or a “protein in a prefusion state or form” is meant a S protein or a fragment of said protein that are designed to be stabilized in a prefusion conformation, preventing structural rearrangements that usually take place when the protein interacts with its receptor or other protein.
  • a strategy to stabilize the S protein in a prefusion conformation is eliminating the causes of metastability by amino acid substitutions in various regions of S, particularly HR1 and in HR2.
  • amino acid substitutions to proline are performed in the S protein to provide a prefusion S protein.
  • said amino acid substitutions aimed at maintaining the prefusion quaternary structure of the S protein are selected from the group including, but not limited to, T961D, G769E, L938F, F817P, A942P, S884C, A892P, A899P, A893C, T859K, Q957E, Q895P, T912P, K921P, G946P, or S975P, or any combination thereof.
  • additional mutations of the wildtype soluble S sequence can be introduced to destabilize the postfusion S structure.
  • one or more proline and/or glycine substitution can be engineered in the region of HR1 that interacts with HR2 to form the fusion core. These substitutions function to disrupt the six-helix- bundle fusion core.
  • the mutations can include A942P, S943P, A944P, A942G, S943G and A944G.
  • the substitutions function to disrupt the six-helix-bundle fusion core.
  • the mutations can include S924P, T925P, A926P, S924G, T925G, and A926G.
  • one or more extra amino acid residues can be inserted into the region of HR1 that interacts with HR2 to form the fusion core. Similarly, these insertions also function to disrupt helical pattern of the fusion core.
  • the insertions can include insertion of G or GS between any residues in A942-A944.
  • the substitutions may be double amino acid substitutions such as K986G/V987G, K986P/V987P, K986G/V987P or K986PA/987G.
  • the S protein or the immunogenic fragment thereof comprise at least one, two or three of the substitutions A942P, K986P and V987P.
  • the S protein or the immunogenic fragment thereof encoded by the nucleic acid comprised in the MVA vector provided herein comprise at least the three substitutions A942P, K986P and V987P.
  • the S protein or the immunogenic fragment thereof encoded by the nucleic acid comprised in the MVA vector provided herein comprises the above mentioned substitutions to avoid the furin cleavage in combination with the above mentioned substitutions to maintain the S protein in a prefusion state.
  • the vaccine composition according to the first aspect or any of its embodiments comprises an immunologically effective amount of a MVA vector comprising at least one nucleic acid encoding the S protein, or a fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant or linage, wherein said S protein or fragment thereof, comprises at least the substitutions R682G, R683S, R685S, A942P, K986P and V987P, and wherein the MVA vector regulates the expression of the nucleic acid encoding the S protein, or the fragment thereof.
  • the S protein encoded by the nucleic acid comprised in the MVA vector comprises, consists or consists essentially of the full-length S protein of at least a SARS-CoV-2 variant or linage.
  • the full-length S protein comprises or consists of amino acids 1 to 1273.
  • the S protein of at least a SARS-CoV-2 virus encoded by the nucleic acid comprised in the MVA-S(3P) vector according to the first aspect of the present invention comprises, consists, or consists essentially of SEQ ID NO: 1 or a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over the full length with SEQ ID NO: 1.
  • nucleic acid of the vaccine composition described herein may also optionally encode antigenic domains or antigenic protein fragments rather than the entire antigenic S protein.
  • These fragments can be of any length sufficient to be antigenic or immunogenic as long as they comprise at least the amino acid substitutions R682G, R683S, R685S, A942P, K986P and V987P. Fragments can be at least 300 amino acids long, but can be longer, such as, e.g., at least 350, 400, 500, 600, 700, 800, 900, 1000, 1200 amino acids long, or any length in between.
  • At least one nucleic acid fragment encoding an antigenic S protein fragment or immunogenic S polypeptide thereof is inserted into the genome of the recombinant viral vector of the invention.
  • about 2-6 different nucleic acids encoding different antigenic proteins are inserted into the genome of the recombinant viral vector.
  • multiple immunogenic fragments or subunits of various proteins can be inserted. 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 S protein orthologue from different strains can be expressed from the vectors.
  • the S protein consists of a truncated protein lacking at least 5, 10, 15, 20, 15, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, or more than 400 amino acid of the full-length S protein.
  • the S protein consists of a truncated protein where the N-terminal domain has been removed.
  • the S protein consists of a truncated protein where the C-terminal domain has been removed.
  • the S protein consists of a truncated protein consisting of only the S1 and S2 subunits.
  • the S protein consists of a truncated protein where the cytosolic domain has been removed.
  • the S protein consists of a truncated protein where the transmembrane domain has been removed. In some embodiments, the S protein consists of a soluble S protein wherein the transmembrane and the cytosolic domains has been removed.
  • a "fragment" of the S protein of at least a SARS-CoV-2 virus according to the present invention is a partial amino acid sequence of the SARS-CoV-2 S protein or a functional equivalent of such a fragment.
  • a fragment is shorter than the full-length complete SARS-CoV-2 S protein and preferably comprises or consists of the amino acids 682-987 of the full-length S protein.
  • a fragment of the SARS-CoV-2 S protein also includes peptides having at least 15, 20 or 25 contiguous amino acid residues having at least about 70%, at least about 80%, at least about 90%, preferably at least about 95%, more preferably at least 98% sequence identity with at least about 15, 20 or 25 contiguous amino acid residues of SEQ ID NO: 2 having about the same length as said peptides.
  • a fragment that "corresponds substantially to" a fragment of the SARS-CoV-2 S protein is a fragment that has substantially the same amino acid sequence and has substantially the same functionality as the specified fragment of the SARS-CoV-2 S protein.
  • a fragment that has "substantially the same amino acid sequence" as a fragment of the SARS-CoV-2 S protein typically has more than 90% amino acid identity with this fragment. Included in this definition are conservative amino acid substitutions.
  • the antigenic protein is the structural protein S of SARS-CoV-2 resulting in vaccine compositions comprising the MVA-S(3P) vector, preferably the antigenic protein is the protein of SEQ ID NO 2, more preferably the antigenic protein is the protein of SEQ ID NO 2 encoded by the nucleic acid consisting of SEQ ID NO 1, or a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over the full length with SEQ ID NO: 1.
  • the S protein of at least a SARS-CoV-2 virus encoded by the nucleic acid comprised in the MVA-S(3P) vector according to the first aspect of the present invention comprises, consists, or consists essentially of SEQ ID NO: 2 or a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over the full length with SEQ ID NO: 2 (amino acids 1 to 1273 encoded by the reference genome of GenBank accession number MN908947.3, or amino acids of the reference S protein PDB ID 6VSB).
  • the nucleic acid encoding the antigenic protein is incorporated or inserted into a non-essential region of the genome of the MVA.
  • the nucleic acid encoding the antigenic protein is incorporated into the MVA by substituting a non-essential region of the genome of the MVA.
  • the nucleic acid encoding the S protein is incorporated or inserted into the MVA thymidine kinase (TK) locus or hemagglutinin (HA) locus.
  • the transfer vector is the pCyA vector.
  • the pCyA vector has been previously described in WO 2014/162031 A1.
  • the vaccine composition comprises an immunologically effective amount of a MVA vector comprising at least one nucleic acid encoding the S protein, or a fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant or linage, wherein said S protein or fragment thereof, comprises at least the substitutions R682G, R683S, R685S, A942P, K986P and V987P, wherein the S protein or the fragment thereof are inserted into the TK locus of the MVA genome, and wherein the MVA vector regulates the expression of the nucleic acid encoding the S protein, or the fragment thereof.
  • 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 and/or CD4 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.
  • the nucleic acid encoding for the S protein, or an immunogenic fragment thereof, comprising at least the substitutions R682G, R683S, R685S, A942P, K986P and V987P and of at least one SARS-CoV-2 variant or linage 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.
  • promoters such as 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), a promoter derived from the cow pox virus type A (inclusion ATI gene) (Lee et al., J. Gen. Virol. 1998), 79, 613), and the new synthetic late-early optimized (LEO) promoter (Di Pilato et al. J Gen Virol.
  • the promoter is the synthetic early-late (sE/L) promoter.
  • the nucleic acid encoding for the prefusion-stabilized S protein, or a fragment thereof, comprising at least the substitutions R682G, R683S, R685S, A942P, K986P and V987P of at least one SARS-CoV-2 variant or linage 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/!ate promoters are further described in WO 2010/102822 A1.
  • the synthetic promoter is formed by both early and late elements, referred as sE/L promoter (Chakrabarti, S.; Sisler, J.R.; Moss, B. Compact, synthetic, vaccinia virus early/late promoter for protein expression.
  • the vaccine composition comprises an immunologically effective amount of a MVA vector comprising at least one nucleic acid encoding the S protein, or a fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant or linage, wherein said S protein or fragment thereof, comprises at least the substitutions R682G, R683S, R685S, A942P, K986P and V987P; wherein the nucleic acid encoding for the S protein or the fragment is operab!y linked to an early/late promoter from VACV, preferably the synthetic early/late promoter; and wherein the MVA vector regulates the expression of the nucleic acid encoding the S protein, or the fragment thereof.
  • a MVA vector comprising at least one nucleic acid encoding the S protein, or a fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant or linage, wherein said S protein or fragment thereof, comprises at least the substitutions
  • the nucleic acid encoding for the prefusion-stabilized S protein, or a fragment thereof, comprising at least the substitutions R682G, R683S, R685S, A942P, K986P and V987P of at least one SARS- CoV-2 variant or linage is operably linked and under the control of the synthetic early/late promoter, wherein the synthetic early/late promoter comprises or consists of SEQ ID NO: 21 or 26, or a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over the full length with SEQ ID NO: 21 or 26.
  • the MVA vector provided in the first aspect or the present invention or in any of its embodiments comprises at least one nucleic acid encoding the S protein, or a fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant or linage, wherein said S protein or fragment comprises at least the substitutions R682G, R683S, R685S, A942P, K986P and V987P; wherein the nucleic acid encoding for the S protein or the fragment is operably linked to an early/!ate promoter from VACV, preferably the synthetic early/late promoter; wherein said nucleic acid is inserted into the TK locus of the MVA genome; and wherein the MVA vector regulates the expression of the nucleic acid encoding the S protein, or the fragment thereof.
  • a Kozak sequence can be added downstream the synthetic early/late promoter.
  • downstream refers to a nucleotide sequence that is located 3’ to a reference nucleotide sequence, in this case to the synthetic early/late promoter.
  • the nucleic acid encoding the S protein, or a fragment of said S protein comprising at least one epitope, and that is operably linked to the synthetic early/late promoter further comprises a Kozak sequence located downstream the synthetic early/late promoter.
  • said Kozak sequence comprises or consists of SEQ ID NO: 22, or a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over the full length with SEQ ID NO: 22.
  • the nucleic acid inserted in MVA genome according to the invention can further encode for one or more antigenic proteins that can be any protein(s) from any SARS-CoV-2 virus variant or linage.
  • the nucleic acid comprised in the MVA vector further encodes for one or more antigenic proteins selected from the group consisting of structural proteins E (envelope), receptor-binding domain (RBD), N (nucleocapsid) or M (membrane), or any combination thereof, from at least one SARS-CoV-2 variant.
  • said further antigenic proteins are inserted in a different locus of the MVA genome, preferably in the HA locus.
  • said further antigenic proteins are placed under the control of a synthetic VACV promoter, preferably the synthetic early/late promoter.
  • the MVA vector comprising at least one nucleic acid encoding the S protein, or a fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant or linage according to the first aspect or any of its embodiments is derived from a variant of concern (VOC) as defined by the Centers for Disease Control and Prevention (CDC) “SARS-CoV-2 Variant Classifications and Definitions”.
  • VOC variant of concern
  • CDC Centers for Disease Control and Prevention
  • VOC variant of concern
  • the S protein, or a fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant or linage is derived from the Wuhan-Hu-1 seafood market pneumonia virus isolate (GenBank accession number: MN908947).
  • the S protein, or a fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant or linage is derived from the United Kingdom SARS-CoV-2 variant VOC 202012/01 (Lineage B.1.1.7, also known as VOC alpha).
  • VOC-202012/01 variant has been reported in 31 other countries/territories/areas in five of the six WHO regions.
  • the S protein, or a fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant or linage according to the first aspect or any of its embodiments is derived from the South African SARS-CoV-2 variant (Lineage B.1.351, also known as VOC beta).
  • South Africa On 18 December 2020, national authorities in South Africa announced the detection of a new variant of SARS-CoV-2 that is rapidly spreading in three provinces of South Africa. South Africa has named this variant as Lineage B.1.351 , also known as 501Y.V2. This variant is described in the scientific literature, see, e.g., Tegally et al.
  • SARS-CoV-2 severe acute respiratory syndrome-related coronavirus 2
  • SARS-CoV-2 South African variant is characterised by three mutations K417N, E484K and N501Y in the RBD. While SARS-CoV-2 VOC 202012/01 from the UK also has the N501Y mutation, phylogenetic analysis has shown that the virus from South Africa are different virus variants.
  • the S protein, or a fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant or linage according to the first aspect or any of its embodiments is derived from the Brazilian SARS-CoV-2 variant VOC-202101/02 (Linage B.1.1.28, also known as VOC gamma).
  • Brazilian variant is also known as Lineage P.1, also known as 20J/501Y.V3, Variant of Concern 202101/02 (VOC-202101/02). This variant is described in the scientific literature, see, e.g., Faria, et al. Genomic Characterisation of an Emergent SARS-CoV-2 Lineage in Manaus: Preliminary Findings.
  • SARS-CoV-2 has 17 unique amino acid changes, ten of which are in its S protein, including these three designated to be of particular concern: N501Y, E484K and K417T.
  • This variant of SARS-CoV-2 was first detected by the National Institute of Infectious Diseases (NIID), Japan, on 6 January 2021 in four people who had arrived in Tokyo having visited Amazonas, Brazil four days earlier. It was subsequently declared to be in circulation in Brazil and spreading around the world.
  • NIID National Institute of Infectious Diseases
  • the S protein, or a fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant or linage according to the first aspect or any of its embodiments is derived from the Californian SARS-CoV-2 (Linage B.1.427 or B.1.429, also known as variant of interest epsilon).
  • This variant is characterized by the mutations S131 , W152C in the N-terminal domain (NTD) of the S protein and by the L452R mutation in the RBD of the S protein. This variant was originally detected in
  • the S protein, or a fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant or linage according to the first aspect or any of its embodiments is derived from a SARS-CoV-2 variant or linage selected from the group including, but not limited to, Wuhan-Hu-1 seafood market pneumonia virus isolate (GenBank accession number: MN908947), Linage B.1.1.28 (Brazilian variant), Linage B.1.351 (South African variant), Linage B.1.427 or Linage B.1.429 (California variant), Linage B.1.617 (Indian variant), or Linage B.1.1.7 (United Kingdom variant), or Linage B.1.1.529 (Omicron variant) or any combination thereof.
  • Wuhan-Hu-1 seafood market pneumonia virus isolate GenBank accession number: MN908947
  • Linage B.1.1.28 Brazilian variant
  • Linage B.1.351 South African variant
  • Linage B.1.427 or Linage B.1.429 Californ
  • the S protein, or a fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant or linage is derived from a SARS-CoV-2 variant selected from the group consisting of Wuhan-Hu-1 seafood market pneumonia virus isolate (GenBank accession number: MN908947), Linage B.1.1.28 (Brazilian variant), Linage B.1.351 (South African variant), Linage B.1.427 or Linage B.1.429 (California variant), Linage B.1.617 (Indian variant), or Linage B.1.1.7 (United Kingdom variant), or Linage B.1.1.529 (Omicron variant), or any combination thereof.
  • Wuhan-Hu-1 seafood market pneumonia virus isolate GenBank accession number: MN908947
  • Linage B.1.1.28 Brazilian variant
  • Linage B.1.351 South African variant
  • Linage B.1.427 or Linage B.1.429 California variant
  • Linage B.1.617 Indian variant
  • the Indian variant or B.1.617 linage includes the Delta variant or Linage B.1.617.2.
  • the Omicron variant or linage B.1.1.529 includes BA.1 , BA.2, BA.3, BA.4, BA.5 and descendent lineages.
  • the S protein, or a fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant or linage is derived from a SARS-CoV-2 variant or linage selected from the group including or consisting of Wuhan-Hu-1 seafood market pneumonia virus isolate (GenBank accession number: MN908947), linage B.1.1.7 (alpha VOC), linage B.1.351 (beta VOC), linage B.1.1.28 or P1 (gamma VOC), linage B.1.617.2 (delta VOC), linage B.1.1.529 (omicron VOC), or other lineages such as linage B.1.427 or Linage B.1.429 (epsilon variant of interest), or any combination thereof.
  • Wuhan-Hu-1 seafood market pneumonia virus isolate GenBank accession number: MN908947
  • linage B.1.1.7 alpha VOC
  • linage B.1.351 beta VOC
  • the MVA vector provided in the first aspect of the present invention or in any of its embodiments comprises at least one nucleic acid encoding the S protein of Wuhan-Hu-1 seafood market pneumonia virus isolate (GenBank accession number: MN908947), or a fragment of said S protein comprising at least one epitope, wherein said S protein or fragment comprises at least the substitutions R682G, R683S, R685S, A942P, K986P and V987P; wherein the nucleic acid encoding for the S protein or the fragment is operably linked to an early/late promoter from VACV, preferably the synthetic early/late promoter; wherein said nucleic acid is inserted into the TK locus of the MVA genome; and wherein the MVA vector regulates the expression of the nucleic acid encoding the S protein, or the fragment thereof.
  • the MVA vector provided in the first aspect or the present invention or in any of its embodiments comprises at least one nucleic acid encoding the S protein, or a fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant or linage, preferably Wuhan variant, wherein said S protein or fragment comprises at least the substitutions R682G, R683S, R685S, A942P, K986P and V987P; wherein the nucleic acid encoding for the S protein or the fragment is operably linked to an early/late promoter from VACV, preferably the synthetic early/late promoter; wherein said nucleic acid is inserted into the TK locus of the MVA genome; wherein the MVA vector regulates the expression of the nucleic acid encoding the S protein, or the fragment thereof, and wherein said nucleic acid is codon optimized for
  • the MVA vector provided in the first aspect or the present invention or in any of its embodiments comprises, from 5 ' to 3 ' , at least the following sequences: i) the synthetic early/late promoter comprising or consisting of SEQ ID NO: 21 or 26, or a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over the full length with SEQ ID NO: 21 or 26, ii) a Kozak sequence comprising or consisting of SEQ ID NO: 22, or a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over the full length with SEQ ID NO: 22, iii) at least one nucleic acid encoding the S protein, or a fragment of said S protein comprising
  • the MVA vector provided in the first aspect or the present invention or in any of its embodiments comprises, from 5 ' to 3 ' , at least the following sequences: i) the synthetic early/late promoter comprising or consisting of SEQ ID NO: 21 or 26, or a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over the full length with SEQ ID NO: 21 or 26, ii) a Kozak sequence comprising or consisting of SEQ ID NO: 22, or a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over the full length with SEQ ID NO: 22, and iii) at least one nucleic acid encoding the S protein of at least one SARS-CoV-2 variant or linage, preferably Wu
  • the MVA vector provided in the first aspect or the present invention or in any of its embodiments comprises, preferably from 5 ' to 3 ' , at least the following sequences: i) the synthetic early/late promoter comprising or consisting of SEQ ID NO: 21 or 26, or a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over the full length with SEQ ID NO: 21 or 26, ii) a Kozak sequence comprising or consisting of SEQ ID NO: 22, or a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over the full length with SEQ ID NO: 22, and iii) at least one nucleic acid encoding the S protein, wherein the amino acid protein of said S protein comprises or consists of S
  • nucleic acid encoding for the S protein or the fragment as defined in iii) is operably linked to the synthetic early/late promoter as defined in i), wherein said nucleic acid is inserted into the TK locus of the MVA genome; and wherein the MVA vector regulates the expression of the nucleic acid encoding the S protein.
  • the nucleic acid comprised in the MVA vector comprises, consists, or consists essentially of SEQ ID NO: 23, or a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over the full length with SEQ ID NO: 23.
  • Rational design of S-based vaccines also provides the generation of chimeric S proteins comprising the most dominant antigenic epitopes from more than one SARS-CoV-2 variants or linages.
  • the S protein, or a fragment of said S protein comprising at least one epitope comprises or consists of the sequences of two or more SARS-CoV-2 variants or linages.
  • the S protein, or a fragment of said S protein comprising at least one epitope comprises or consists of a consensus sequence derived from two or more different SARS-CoV-2 variants or linages.
  • compositions and vaccines 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 the MVA vector described herein formulated in solution in a concentration range of from 1 x 10 7 to 1 x 10 8 PFUs per dose.
  • FIGs 5-15 show that immunogenicity was induced in vaccinated mice with an intramuscular dose of 1 x 10 7 PFUs per dose (see immunization schedule in Figures 5 and 13). Further, as shown in Figure 13, mice were vaccinated with a single dose of 1x10 7 PFUs of MVA-S(3P), which indicates that this dose is sufficient to induce protective immunity that leads to 100% survival upon a lethal challenge with SARS-CoV-2.
  • mice vaccinated with MVA-S(3P) did not show any weight loss nor disease symptoms, indicating that the vaccine is not only able to protect mice from a lethal challenge, but it is able to stop and/or reduce viral infection and the symptoms associated to it at early times upon challenge, as it is described in Figure 13D and 13E.
  • one single intramuscular dose of MVA-S(3P) reduced lung pathology and levels of proinflammatory cytokines, as shown in Figure 14.
  • one single intranasal dose of 1 x 10 7 PFUs of MVA-S(3P) induced in mice local and systemic SARS-CoV-2-S-specific CD4+ and CD8+ T-cell immune responses see Fig.
  • Intranasal administration of one dose of 1 x 10 7 PFUs of MVA-S(3P) of the present invention also prevented morbidity (see Fig. 18B) and mortality (see Fig. 18C) in SARS-CoV-2 challenged K18-hACE2 mice, reducing SARS-CoV-2 virus replication in lungs and nasal washes (see Fig. 18D to 18G), thereby reducing lung pathology (see Fig. 19) and the levels of pro-inflammatory cytokines in lungs and nasal washes (see Fig. 20).
  • 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 a!., Dtsch. med. Wschr. 99:2386-2392 (1974).
  • purified viruses can be stored at -80°C with a titer of 5 x 10 8 PFUs/ml formulated in about 10 mM Tris, 140 mM NaCI pH 7.4.
  • a titer of 5 x 10 8 PFUs/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. However, as long as no need exists, the ampoule is stored preferably at temperatures below -20°C.
  • the lyophilizate 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. However, 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.
  • 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.
  • the present invention provides a vaccine composition comprising an immunologically effective amount of a MVA vector comprising at least one nucleic acid encoding the S protein, or a fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant or linage, wherein said S protein or fragment thereof, comprises at least the substitutions R682G, R683S, R685S, A942P, K986P and V987P, wherein the MVA vector regulates the expression of the nucleic acid encoding the S protein, or the fragment thereof, and wherein 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.
  • “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.
  • 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 of the present invention is characterized for being replication deficient in mammalian cells, particularly human cells.
  • 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 HeLa.
  • the recombinant MVA vector of any of the embodiments used for generating the recombinant virus is a MVA vector may be 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.
  • 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).
  • 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 or in any of its embodiments is administered in a first dosage.
  • One or more subsequent administrations of a MVA viral vector as defined herein in the first aspect or any of its embodiments 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 vector provided herein is administered as a boost (i.e., as a second, third, fourth, or successive vaccinations).
  • the MVA recombinant viral vectors according to the present invention may also be used in prime-boost regimens in combination with other immunogens (heterologous prime/boost combination), such as in combination with a DNA or RNA vector or another viral vector, such as an adenoviruses.
  • other immunogens heterologous prime/boost combination
  • one or more subsequent boosting vaccinations may be done with the MVA vector provided herein and not used in the prime vaccination, e.g., if another immunogenic vector is given in a prime, then subsequent boosting vaccinations may be performed with the MVA provided herein, and vice versa.
  • a vaccine combination (from hereinafter referred to as homologous vaccine combination) comprising:
  • compositions comprising an immunologically effective amount of a MVA vector as defined in the first aspect or any of its embodiments; and 2. a composition comprising an immunologically effective amount of a MVA vector as defined in the first aspect or any of its embodiments; wherein one of the compositions is a priming composition and the other composition is a boosting composition, preferably wherein the boosting composition comprises or consists of two or more doses of the vector of the boosting composition.
  • a vaccine combination from hereinafter referred to as heterologous vaccine combination
  • heterologous vaccine combination comprising:
  • composition comprising an immunologically effective amount of a MVA vector as defined in the first aspect or any of its embodiments;
  • compositions comprising an immunologically effective amount of at least one immunogen of at least one SARS-CoV-2 variant or linage, preferably selected from the group consisting of viral vectors, recombinant proteins or polypeptides, nucleic acids, virus-like particles, nanoparticles, or any combination thereof, wherein one of the compositions is a priming composition and the other composition is a boosting composition, in any order.
  • the composition comprising the MVA vector as defined in the first aspect or any of its embodiments is administered as a boost, after a prime with a composition comprising an immunologically effective amount of an immunogen of at least one SARS-CoV-2 variant or linage.
  • the boost composition comprises or consists of two or more doses of the vector or immunogen 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, RBD, E, N or M 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, respectively encoded by nucleic acid sequences SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7 and SEQ ID NO 9.
  • the immunologically effective amount of an immunogen of at least one SARS-CoV-2 variant or linage comprises an antigenic protein or nucleic acid encoding for an antigenic protein, wherein the antigenic protein is selected from any of the group of structural proteins S, RBD, E, N or M of SARS-CoV-2, or any combination or fragments thereof.
  • the at least one immunogen of at least one SARS-CoV-2 variant or linage is a nucleotide sequence of a DNA or RNA vector that encodes for an antigenic protein of at least one SARS-CoV-2 variant or linage.
  • said nucleotide sequence encoding for an antigenic protein is preferably the structural protein S of SARS-CoV-2, or a fragment thereof such as the RBD.
  • heterologous vaccine compositions it is important to note that upon injection, preferably intramuscularly, of the DNA/RNA 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 or the RNA reaches the cell, the antigen of interest is produced. Next, the transfected cell displays on its surface the foreign antigen in both major 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.
  • MHC major histocompatibility complex
  • DNA/RNA-based vectors suitable for used as 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.
  • the DNA/RNA-based vaccine vector encode for one or more antigens against which the immune response is to be elicited. Further, the DNA/RNA 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.
  • Any DNA, linear or plasmid, able to express the antigen in a mammalian cell may be used to construct a DNA/RNA vaccine.
  • Mammalian expression DNA and RNA vectors are widely known in the art.
  • the other immunogen that is combined with the MVA vector provided herein is a mammalian expression vector (a DNA plasmid vector) and produces high levels of the antigen.
  • a mammalian expression vector a DNA plasmid vector
  • 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 other immunogen that is combined with the MVA vector provided herein is a RNA molecule.
  • the other immunogen that is combined with the MVA vector provided herein is an adenovirus.
  • the other immunogen that is combined with the MVA vector provided herein is a recombinant protein or polypeptide.
  • DNA/RNA-based vaccine has been produced by endo-free means, for instance, by using EndoFree Plasmid Kits by Qiagen.
  • Vaccines and kits comprising recombinant MVA viruses
  • 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
  • the vaccines and kits comprising recombinant MVA vectors or vaccine combinations as described in the first or second aspects or any of their embodiments 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.
  • the instructions indicate that the recombinant MVA virus is administered as a boost to naive or non-naive subjects.
  • a kit comprises at least two vials or container 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, for use in homologous or heterologous prime boost inoculations.
  • kits comprising one, two, or more doses of any of the vaccine compositions as defined in the first aspect of the invention or any of its embodiments, or of any of the homologous or heterologous vaccine combinations of the second aspect of the invention or any of its embodiments.
  • the kit comprises an immunologically effective amount of a MVA-based vaccine composition, preferably MVA-S(3P) vector 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 a kit comprising an immunologically effective amount of a MVA vector according to the first aspect or any of its embodiment in a container for a first or a second or subsequent administrations, wherein the kit further comprises at least another container comprising an immunologically effective amount of another immunogen of at least one SARS-CoV-2 variant or linage for a first or a second or subsequent administrations.
  • kits comprising an immunologically effective amount of at least one immunogen of at least one SARS-CoV-2 variant or linage in a first vial or container for a first administration (priming), and an immunologically effective amount of a MVA vector according to the first aspect or any of its embodiments, in a second vial or container for a second administration (boosting).
  • 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 MVA vector provided herein is used in a second, third, fourth, or further administrations.
  • the combination of the combination of compositions contained in the containers is capable of producing at least the S protein, or a fragment thereof, comprising the substitutions R682G, R683S, R685S, A942P, K986P and V987P derived from at least one SARS-CoV-2 variant or linage, in the subject to be treated, and generating an immunogenic and/or protective immune response against at least one SARS-CoV-2 variant or linage in a subject in need thereof.
  • the vaccines compositions and combinations and kits provided in any of the previous aspects are used in therapy.
  • the vaccines compositions and combinations and kits provided in any of the previous aspects are also for use as a medicament or as a vaccine for the treatment and/or prevention of a coronavirus-caused disease, preferably a SARS-CoV-2-caused disease.
  • 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 or linage, wherein the kits or vaccine composition comprise one dose of an immunologically effective amount of the vaccine compositions 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 variant or linage, wherein the kits or vaccine composition comprise at least one dose of an immunologically effective amount of the vector MVA-S(3P).
  • the vaccine composition according to the first aspect or any of its embodiment or the kits of the third aspect or any of its embodiments is for use in generating an immunogenic and/or protective immune response in a subject in need thereof, preferably in a human subject, wherein said immune response is against at least the S protein, or a fragment of said S protein, comprising the substitutions R682G, R683S, R685S, A942P, K986P and V987P and derived from at least one SARS-CoV-2 variant or linage, wherein said composition is used for priming and/or for boosting said immune response, and wherein the MVA is capable of producing said S protein, or fragment thereof, in the subject to be treated.
  • the vaccine composition, combination, or kits are for use in generating or inducing an immunogenic and/or protective immune response in a subject in need thereof, preferably in a human subject, preferably wherein said immunogenic and/or protective immune response is generated in the subject after at least a single administration (be it as a prime or as a boost) of the vaccine composition, combinations, or kits described herein.
  • the vaccine composition according to the first aspect, the vaccine combinations according to the second aspect or the kits according to the third aspect, or any of their related embodiments are used to induce an immunogenic and/or protective immune response against at least one SARS-CoV-2 variant or linage with only a single dose of an immunologically effective amount of the MVA-S(3P) of the invention, wherein preferably the single dose contains concentrations of MVA of about 1 x 10 7 to 2 x 10 8 PFUs.
  • said single dose of the vaccine composition or combination or the kits of the first or second or third aspects, or any of their related embodiments is administered as a boost, preferably after a vaccination with a different immunogen (such as a RNA vaccine, DNA vaccine, protein-based vaccine, etc.).
  • a different immunogen such as a RNA vaccine, DNA vaccine, protein-based vaccine, etc.
  • 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 variant or linage, 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 variant or linage, 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 vaccine combination according to the second aspect or any of its embodiments is for use in generating an immunogenic and/or protective immune response in a subject in need thereof, preferably in a human subject, wherein said immune response is against at least the S protein, or a fragment of said S protein, comprising the substitutions R682G, R683S, R685S, A942P, K986P and V987P and derived from at least one SARS-CoV-2 variant or linage, and wherein said combination is capable of producing said S protein, or fragment thereof, in the subject to be treated.
  • the vaccine composition according to the first aspect or the vaccine combinations according to the second aspect or any of their embodiments are used to induce an enhanced immune response against at least one SARS-CoV-2 variant or linage in a subject in need thereof, preferably in a human subject, wherein the MVA is capable of producing one or more proteins, or fragments thereof, selected from the group consisting of proteins S, E, RBD, N and/or M, or any combination thereof, derived from at least one SARS-CoV-2 variant or linage, in the subject to be treated.
  • the present invention provides heterologous vaccine programs that consists of a combination vaccine and/or vaccination kit which comprises:
  • compositions comprising an immunologically effective amount of a MVA vector as defined in the first aspect or any of its embodiments, and 2. a composition comprising an immunologically effective amount of at least one immunogen of at least one SARS-CoV-2 variant or linage, wherein one of the compositions is a priming composition and the other composition is a boosting composition, in any order, and preferably wherein the boosting composition comprises two or more doses.
  • the MVA vector as defined in the first aspect or any of its embodiments is 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:
  • composition comprising an immunologically effective amount of a MVA vector as defined in the first aspect or any of its embodiments, and
  • compositions comprising an immunologically effective amount of a MVA vector as defined in the first aspect or any of its embodiments, wherein one of the compositions is a priming composition and the other composition is a boosting composition, preferably wherein the boosting composition comprises two or more doses.
  • the combination vaccines and/or kit comprises at least two vials or containers for prime/boost immunization comprising the recombinant MVA according to the first aspect or any of its embodiments for a first inoculation ("priming inoculation") in a first vial or container and for an at least second and/or third and/or further inoculation ("boosting inoculation”) in a second and/or further vial or container.
  • prime/boost immunization comprising the recombinant MVA according to the first aspect or any of its embodiments for a first inoculation ("priming inoculation") in a first vial or container and for an at least second and/or third and/or further inoculation ("boosting inoculation”) in a second and/or further vial or container.
  • the heterologous combination vaccine and/or kit can comprise multiple containers or vials of the recombinant MVA/other immunogen, together with instructions for the administration of the recombinant MVA/other immunogen to a subject at risk of SARS- CoV-2 infection.
  • the subject is a human.
  • the instructions indicate that the recombinant MVA/other immunogen 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/other immunogen virus is administered in a first (priming) and second (boosting) administration to naive or non-naive subjects.
  • the other immunogen of at least one SARS-CoV-2 variant or linage is selected from the group including, but not limited to, viral vectors, recombinant proteins or polypeptides, nucleic acids, virus like particles, nanoparticles, or any combination thereof.
  • said immunogen is selected from the group consisting of viral vectors, recombinant proteins or polypeptides, nucleic acids, virus-like particles, nanoparticles, or any combination thereof.
  • the first and/or second composition or MVA and/or other immunogen of any combination vaccine, vaccination kit and/or any homologous or heterologous vaccine program of the invention can comprise any of the MVA vector described herein and any combination thereof.
  • 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/or protective immune response against at least one SARS-CoV-2 variant or linage in a subject in need thereof, preferably in a human subject, the method comprising administering to the subject the recombinant MVA vaccine composition as described in the first aspect of the invention or any of its embodiments; 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 or any of their embodiments.
  • said method is for priming the immune response and/or for boosting said immune response.
  • Figs. 15B, 15C, 21 B, and 21 C neutralizing antibody titers against SARS- CoV-2 parental Wuhan strain, D614G mutant, and VoCs alpha (B.1.1.7), beta (B.1.351), gamma (P.1), and delta (B.1.167.2) were detected in serum from mice immunized with MVA-S(3P), markedly higher than the titers in MVA-S- or MVA-A-S vaccinated mice.
  • the vaccine composition according to the first aspect or the vaccine combination according to the second aspect or the kits of the third aspect, or any of their related embodiments are for use in generating an immunogenic and/or protective immune response that confers protection (including cross protection) against infections caused by different SARS-CoV-2 variants or linages, preferably against infections caused at least by SARS-CoV-2 Wuhan strain, D614G mutant, VoCs alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.167.2) and omicron (B.1.1.529) linages, and/or any combinations thereof.
  • the vaccine composition according to the first aspect or the vaccine combination according to the second aspect or the kits of the third aspect or any of their related embodiments are for use in generating an immunogenic and/or protective immune response against the S protein of at least one, two, three, four, five, six, or more SARS-CoV-2 different variants or linages, preferably selected from Wuhan strain GenBank accession number MN908947, Wuhan strain D614G mutant, linage B.1.1.7, linage B.1.351, linage P.1, linage B.1.167.2, linage B.1.1.529, and/or any combination thereof.
  • the vaccine composition according to the first aspect or the vaccine combinations according to the second aspect or the kit of the third aspect or any of their related embodiments are used to induce an immunogenic and/or protective immune response against the S protein of at least one SARS-CoV-2 variant or linage, wherein preferably said immunogenic and/or protective immune response is directed against at least a SARS-CoV-2 variant or linage selected from the group consisting of Wuhan strain GenBank accession number MN908947, Wuhan strain D614G mutant, linage B.1.1.7, linage B.1.351 , linage P.1, linage B.1.167.2, linage B.1.1.529, and/or any combination thereof.
  • the protective immune response elicited by the vaccine composition according to the first aspect or the vaccine combination according to the second aspect or the kits of the third aspect or any of their related embodiments protects a subject in need thereof, preferably after a single dose, preferably administered as a prime or as a boost, against at least one or more infections caused by at least a SARS-CoV-2 virus, preferably selected from the list consisting of SARS- CoV-2 Wuhan strain, D614G mutant, VoCs alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.167.2), and/or any combination thereof.
  • said protective immune response protects a subject in need thereof, preferably after a single dose, preferably administered as a prime or as a boost, against at least one, two, three, four, five, six, or more infections caused by at least one SARS-CoV-2 variant or linage, preferably selected from Wuhan strain GenBank accession number MN908947, Wuhan strain D614G mutant, linage B.1.1.7, linage B.1.351, linage P.1 , linage B.1.167.2, linage B.1.1.529, and/or any combination thereof. Further, since immunization with the MVA-
  • S(3P) of the invention elicited SARS-CoV-2 S-specific CD4+ and CD8+ T cell-mediated immune response with memory phenotype (see, e.g, Fig. 11), it is more than plausible that they are protective not only against a first SARS-CoV-2 infection, but also after subsequent SARS-CoV-2 infections.
  • the vaccine composition according to the first aspect or the vaccine combinations according to the second aspect or the kit of the third aspect or any of their related embodiments are used to induce in a subject in need thereof a protective immune response against an infection caused by at least one SARS-CoV-2 variant or linage, preferably selected from the group consisting of Wuhan strain GenBank accession number MN908947, Wuhan strain D614G mutant, linage B.1.1.7, linage B.1.351, linage P.1 , linage B.1.167.2, linage B.1.1.529, and/or any combination thereof; preferably wherein said protective immune response is capable of protecting said subject against said SARS-CoV-2 infections for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months or years.
  • 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 the recombinant MVA or combinations thereof as described in the fourth aspect of the invention.
  • the protective immune response with a single dose (be it as a boost or as a prime) is capable of, or is characterized by being, sufficient to prevent a vaccinated subject from contracting an infection that leads to the morbility and/or mortality of said subject, when infected by at least one SARS-CoV-2 variant or linage.
  • infection is herein understood, but not limited to, as the presence of replication of the virus in the body’s cells, preferably lung cells, of the host. More preferably, the term “infection” is understood as the presence of replication of the virus in the body’s cells, preferably lung cells, of the host, wherein said replication leads to the morbidity and/or mortality of said subject.
  • 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.
  • the recombinant MVA 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 1 x 10 7 , 5 x 10 7 , 1 x 10 8 or 2 x 10 8 PFUs/dose.
  • any of the recombinant MVA 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 MVA 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, either intramuscularly (as shown in Figures 13-15; vaccinated mice Group 2) or intranasally (as shown in Figures 16-21).
  • 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.
  • a single dose of an immunologically effective amount of MVA-S(3P) is preferred, although two doses might be a choice for longer term immune responses.
  • 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, 3, 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 MVA 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, or in any of their embodiments, can be administered systemically or locally.
  • the said recombinant vaccine compositions are administered parenterally, subcutaneously, intravenously, intramuscularly, or intranasally, in particular intranasally or intramuscularly.
  • the recombinant MVA or the containers comprising the recombinant MVA 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 1 x 10 7 to 2 x 10 8 PFUs/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.
  • the MVA vector of the present invention also elicits an immune response against the poxviral E3 protein, which is a host range protein encoded by the E3L gene comprised in VACV genome.
  • the E3 protein is typical from poxviruses, and thus not only the MVA virus encodes for it, but also other poxviruses such as monkeypox or smallpox. This means that an immune response generated against said E3 protein elicited by one or more MVA immunizations would also target E3 protein from monkeypox or smallpox viruses.
  • the MVA vectors described herein provide dual immunogenicity: they elicit an immunogenic and/or protective response against at least one SARS-CoV-2 variant or linage, and at the same time they elicit an immune response against a poxvirus, such as the monkeypox or smallpox viruses.
  • the present invention provides an MVA vector expressing the E3 protein of poxviruses for use in generating an immunogenic and/or protective immune response against at least a poxvirus, preferably against smallpox and/or monkeypox virus.
  • the MVA vector expressing the poxviral E3 protein for use according to the fifth aspect is for use in generating an immunogenic and/or protective immune response in a subject in need thereof, preferably in a human subject, wherein said immune response is against at least the poxviral E3 protein, or a fragment of said protein, wherein preferably said vector is used for priming and/or for boosting said immune response, and wherein the MVA is capable of producing said poxviral E3 protein, or fragment thereof, in the subject to be treated.
  • the MVA vector used according to the fifth aspect comprises at least one nucleic acid that encodes for the E3 protein of poxviruses, preferably operably linked and under the promoter of the wildtype promoter of E3L gene.
  • the nucleotide sequence of the poxviral E3 protein comprises or consists of SEQ ID NO: 24, or a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over the full length with SEQ ID NO: 24.
  • the amino acid sequence of the poxviral E3 protein comprises or consists of SEQ ID NO: 25, or a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over the full length with SEQ ID NO: 25.
  • a further aspect of the present invention refers to the use of said MVA-S(3P) virus in generating an immunogenic and/or protective immune response against at least a poxvirus, preferably against smallpox and/or monkeypox virus.
  • the MVA vector for use not only expresses the poxviral E3 protein, but also expresses the S protein of at least one SARS-CoV-2 variant or linage, wherein said S protein comprises at least the amino acid substitutions R682G, R683S, R685S, A942P, K986P and V987P as defined using the amino acid numbering of the reference Wuhan S protein PDB ID 6VSB.
  • the vaccines compositions and/or combinations and/or kits provided in any of the previous aspects and their related embodiments are for use as a medicament or as a vaccine for the treatment and/or prevention of a poxvirus-caused disease.
  • the vaccines compositions, combinations and/or kits provided in any of the previous aspects and their related embodiments are for use in generating a protective immune response against at least a poxvirus, wherein the kits or vaccine composition or combinations comprise at least one dose of an immunologically effective amount of the vector MVA wild type or MVA-S(3P).
  • the vaccines compositions and combinations and/or kits provided in any of the previous aspects or in any of their related embodiments are for use as bivalent vaccines to generate an immunogenic and/or protective immune response against both at least a poxvirus and/or at least one SARS-CoV-2 variant or linage, wherein the kits or vaccine composition comprise at least one dose of an immunologically effective amount of the compositions of the first aspect or any of its embodiments.
  • said immunogenic and/or protective immune response is at least against the poxviral E3 protein and/or against at least the S protein from at least one SARS-CoV-2 variant or linage.
  • the vaccine composition of the first aspect or any of its embodiments is used for priming and/or for boosting an immune response
  • the MVA vector is capable of producing a S protein comprising at least the substitutions R682G, R683S, R685S, A942P, K986P and V987P, or fragment thereof, and the poxviral E3 protein, in the subject to be treated.
  • said immunogenic and/or protective immune is achieved after a single administration of the vaccine compositions or combinations.
  • the vector of the fifth aspect may be used to induce an enhanced immune response against at least one SARS-CoV-2 variant or linage and/or against at least a poxvirus in a subject in need thereof, preferably in a human subject, wherein the MVA is capable of producing one or more proteins of said viruses, preferably the S protein of at least a SARS-CoV-2 variant or linage comprising at least the substitutions R682G, R683S, R685S, A942P, K986P and V987P, and the poxviral E3 protein.
  • vaccine compositions and combinations and kits for use in priming or boosting an immune response against a poxviral infection, preferably smallpox and/or monkeypox infection, and/or against a SARS-CoV-2 infection, preferably wherein the recombinant MVA is administered at least once, twice, three times or four times.
  • the vaccine of the first aspect comprising a MVA comprising a heterologous nucleotide sequence encoding the S protein of at least a SARS-CoV-2 variant or linage, wherein said S protein comprises at least the substitutions R682G, R683S, R685S, A942P, K986P and V987P defined using the amino acid numbering of the reference Wuhan S protein PDB ID 6VSB, provides a coronavirus and a poxvirus vaccine capable of inducing both cellular and humoral responses sufficient to confer protective immunity to one or subsequent SARS-CoV-2 virus infections, as well as to one or subsequent poxvirus infections.
  • a vaccine composition comprising an immunologically effective amount of a modified vaccinia virus Ankara (MVA) vector comprising at least one nucleic acid encoding the Spike (S) protein, or a fragment of said S protein comprising at least one epitope, of at least one SARS-CoV-2 variant, wherein said S protein or fragment thereof, comprises at least the substitutions R682G, R683S, R685S, A942P, K986P and V987P, and wherein the MVA vector regulates the expression of the nucleic acid encoding the S protein, or the fragment thereof.
  • MVA modified vaccinia virus Ankara
  • TK thymidine kinase
  • nucleic acid encoding for the S protein, or a fragment thereof, of at least one SARS-CoV-2 variant is operably linked to an early/late promoter from Vaccinia Virus (VACV), preferably the synthetic early/late promoter.
  • VACV Vaccinia Virus
  • nucleic acid encoding for the S protein, or a fragment thereof, of at least one SARS-CoV- 2 variant is inserted into the TK locus of the MVA genome and is operably linked to an early/late promoter from VACV, preferably the synthetic early/late promoter.
  • nucleic acid comprised in the MVA vector further encodes for one or more antigenic proteins selected from the group consisting of structural proteins E (envelope), receptor-binding domain (RBD), N (nucleocapsid) or M (membrane), or any combination thereof, from at least one SARS-CoV-2 variant.
  • At least one SARS-CoV-2 variant is selected from the group consisting of Wuhan-Hu-1 seafood market pneumonia virus isolate (GenBank accession number: MN908947), Linage B.1.1.28 (Brazilian variant), Linage B.1.351 (South African variant), Linage B.1.427 or Linage B.1.429 (California variant), Linage B.1.617 (Indian variant) or Linage B.1.1.7 (United Kingdom variant), or any combination thereof.
  • composition according to any of clauses 1 to 6, wherein the vaccine further comprises one or more pharmaceutically acceptable carriers, additives, antibiotics, preservatives, adjuvants, diluents and/or stabilizers.
  • a vaccine combination comprising: a. a first composition comprising an immunologically effective amount of a MVA vector as defined in any clauses 1 to 7; and b. a second composition comprising an immunologically effective amount of a MVA vector as defined in any of clauses 1 to 7; wherein 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 MVA vector as defined in any of clauses 1 to 7; and b.
  • a second composition comprising an immunologically effective amount of at least one immunogen derived from at least one SARS-CoV-2 variant; wherein one of the compositions is a priming composition and the other composition is a boosting composition.
  • the vaccine combination for use in generating an immunogenic and/or protective immune response in a subject in need thereof, preferably in a human subject, wherein said immune response is against at least the S protein, or a fragment of said S protein, comprising the substitutions R682G, R683S, R685S, A942P, K986P and V987P and derived from at least one SARS-CoV-2 variant, and wherein said combination is capable of producing said S protein, or fragment thereof, in the subject to be treated.
  • a kit comprising one, two, or more doses of the vaccine composition as defined in any of clauses 1 to 7, or the vaccine combination as defined in any of clauses 8 to 10.
  • a kit comprising an immunologically effective amount of a MVA vector according to any of clauses 1 to 7 in a first container for a first administration (priming) and in a second container for a second administration (boosting).
  • a kit comprising an immunologically effective amount of a MVA vector according to any of clauses 1 to 7 in a container for a first or a second administration, wherein the kit further comprises at least another container comprising an immunologically effective amount of another immunogen derived from at least one SARS-CoV-2 variant for a first or a second administration.
  • a kit comprising an immunologically effective amount of an immunogen derived from at least one SARS-CoV-2 variant in a first container for a first administration (priming), and an immunologically effective amount of a MVA vector according to any of clauses 1 to 7 in a second container for a second administration (boosting).
  • the kit according to any of clauses 18 to 21, comprising in a third, fourth or further containers a MVA vector according to any of clauses 1 to 8 for a third, fourth or further administration.
  • kits according to clause 23, wherein the combination of compositions contained in the containers is capable of producing at least the S protein, or a fragment thereof, comprising the substitutions R682G, R683S, R685S, A942P, K986P and V987P derived from at least one SARS-CoV-2 variant, in the subject to be treated, and generating an immunogenic and/or protective immune response against at least one SARS-CoV-2 variant in a subject in need thereof.
  • the kit for use according to any of clauses 23 or 24, wherein said compositions comprised in the containers are administered via the intramuscular or intranasal route.
  • compositions or combinations of compositions contained in the containers are administered via the intramuscular or intranasal route.
  • said method comprises administering the vaccine compositions according to any of clauses 1 to 8 for priming said immune response and/or boosting said immune response.
  • said compositions or combinations of compositions comprised in the containers are administered via the intramuscular or intranasal route.
  • CoV-2 variant in a subject in need thereof, preferably in a human subject, comprising administering to the subject the vaccine composition according to any of clauses 1 to 7 or any of the vaccine combinations according to any of clauses 8 to 10.
  • compositions or combinations of compositions comprised in the containers are administered via the intramuscular or intranasal route.
  • Culture media DF-1 (a spontaneously immortalized chicken embryo fibroblast [CEF] cell line, ATCC catalog no. CRL-12203) and HeLa cells (a human epithelial cervix adenocarcinoma; ATCC catalog no. CCL-2) 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).
  • DMEM Dulbecco’s modified Eagle’s medium
  • FCS 10 % fetal calf serum
  • 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 DM EM-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). Vero-E6 cells (ATCC catalog no.
  • CRL-1586 were maintained in complete medium (DMEM supplemented with 10 mM HEPES [Gibco-Life Technologies], 1 * nonessential amino acids [Gibco-Life Technologies], penicillin [100 U/ml; Sigma-Aldrich], streptomycin [100 mg/ml; Sigma-Aldrich], and 10% heat inactivated fetal bovine serum [FBS, Gibco-Life Technologies]). 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 coii DFI5a 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)
  • coronvirus SARS-CoV-2 S antigen SEQ ID NO 1 and SEQ ID NO 2
  • the SARS-CoV-2 antigen used to generate the recombinant MVA-CoV2-S(3P), also referred as abbreviated MVA-S(3P) vaccine candidate, is a prefusion-stabilized full- length SARS-CoV-2 S gene (Wuhan seafood market pneumonia virus isolate Wuhan- Hu-1. GenBank accession number: MN908947.3).
  • the nucleotide sequence of this S protein was edited with codons optimized for humans by GeneArt (Thermo Fisher Scientific), and contains three mutations in the furin cleavage site (R682G, R683S, R685S) that avoid the cleavage of the S protein in S1 and S2, and three additional mutations in the S2 region that stabilizes the S protein in a prefusion conformation (A942P, K986P, and V987P).
  • the parental MVA used for generating MVA-CoV2-S(3P) vaccine candidate was a wild- type MVA (MVA-WT).
  • the S gene was inserted in the viral TK locus (gene J2R) to generate the MVA-CoV2-S(3P) vaccine candidate.
  • the S gene is under the control of the synthetic early/late viral promoter (pE/L, also called sE/L).
  • SARS-CoV-2 strain MAD6 (kindly provided by Jose M. Honrubia and Dr. Luis Enjuanes, CNB-CSIC, Madrid, Spain) 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).
  • SARS-CoV-2 B.1.351 VOC was obtained from the European Virus Archive Global (EVA-GLOBAL) (Human 2019-nCoV-hCoV-19/France/PDL-IPP01065/2021, clade 20H/501Y.V2/Beta, V2 (B.1.351)) and was then amplified at the CNB-CSIC in Vero-E6 cells.
  • EVA-GLOBAL European Virus Archive Global
  • a plasmid transfer vector pCyA was used for inserting the coronavirus SARS-CoV-2 prefusion-stabilized S(3P) gene into the MVA genome.
  • the cells were first infected with the parental MVA-WT virus and then transfected with this plasmid.
  • the recombinant MVA virus was 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 plasmid In order to insert the heterologous genes into the MVA genome, the transfer plasmid must contain the following elements:
  • Flanking regions of the desired insertion area 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.
  • Antigen of interest This must be located between the L and R flanking regions of the TK gene, such that as a consequence of recombination, the antigen of interest is inserted in the viral genome. This antigen was 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 antigen 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.
  • 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:
  • Plasmid transfer vector pCyA-S was used to generate MVA-S(3P) recombinant virus and was developed by the CNB. It directs the insertion of the coronavirus SARS-CoV-2 prefusion-stabilized S gene sequence, under the control of the VACV sE/L promoter, in the TK locus of the parental MVA-WT virus.
  • pCyA vector was provided to GeneArt (Thermo Fisher Scientific) for optimization of codons, synthesis of the prefusion-stabilized S gene of SARS- CoV-2 and insertion into the plasmid vector.
  • 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(3P)
  • mice of strain C57BL/6JOIaHsd (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 Male and 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 and 161.5/20).
  • 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).
  • 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. 2.2.2. Proteins
  • the coronavirus SARS-CoV-2 S and RBD soluble 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).
  • the S sequence (residues 1 to 1 ,208; Wuhan-Hu-1 strain, GenBank accession number MN908947.3) contained a T4 fibritin trimerization sequence, a Flag epitope, and an 8xHistag at the C-terminus.
  • the furin-recognition motif (RRAR) was replaced by the GSAS sequence, and it also contained the A942P, K986P, and V987P substitutions in the S2 portion.
  • the S protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography from transfected cell supernatants and it was transferred to HEPES buffered saline (HBS), pH 7.5, during concentration or by size- exclusion chromatography (SEC).
  • Ni-NTA nickel-nitrilotriacetic acid
  • the RBD proteins with the S residues 332-534 were produced with an N-terminal HA- tag (YPYDVPDYA) and fused to either a T4 fibritin trimerization sequence, a Flag epitope and an 8xHis-tag (RBD-TFH), or to the human IgGi Fc region (RBD-Fc) at its C-terminus.
  • YPYDVPDYA N-terminal HA- tag
  • RBD-TFH 8xHis-tag
  • RBD-Fc human IgGi Fc region
  • RBD-Fc proteins with RBD substitutions in VoC were also produced: alpha (B.1.1.7; N501Y), beta (B.1.351; K417N-E484K-N501Y), gamma (P.1.; K417T-E484K- N501Y), delta (B.1.617.2; L452R-T478K), kappa (B.1.617.1; L452R-E484Q) and zeta (P.2; E484K).
  • the RBD-TFH and RBD-Fc proteins were purified by affinity chromatography with Ni-NTA and IgSelect (GE Healthcare) columns, respectively.
  • 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.
  • PCR Polymerase chain reaction
  • 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 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).
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel
  • 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.
  • TCA trichloroacetic acid
  • 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).
  • Monolayers of HeLa cells were mock infected or infected at 5 PFU/cell with MVA-WT, MVA-S, or MVA-S(3P).
  • cells were collected by scraping and centrifuged at 2,000 rpm for 5 min, and the cellular pellets were resuspended in phosphate-buffered saline (PBS) staining solution (1X PBS, 0.5% bovine serum albumin [BSA], 1% FCS, 0.065% sodium azide, 2 mM EDTA) and added to a 96-well plate at a rate of 200,000 cells per well. Then, Fc block (BD Pharmingen) was added for 20 min at 4°C.
  • PBS phosphate-buffered saline
  • mice monoclonal antibodies against S and RBD proteins were added to the cells for 30 min at 4°C, and then anti-mouse IgG fluorescein isothiocyanate (FITC)- conjugated antibodies (diluted 1 :100; eBioscience) were added to the cells as secondary antibodies for 20 min in the dark at 4°C.
  • FITC fluorescein isothiocyanate
  • cells were fixed with 4% paraformaldehyde (PFA) for 20 min in the dark at 4°C and were acquired using a Gallios flow cytometer (Beckman Coulter). Data were analyzed using FlowJo software (version 8.5.3; Tree Star, Ashland, OR).
  • Mammalian expression vectors that contained recombinant DNAs coding for the soluble S protein with a T4 fibritin trimerization sequence, a Flag peptide and a His-tag at the C- terminus were used for S expression by transfection of HEK293T cells.
  • S constructs without the furin motif and with the K986P and V987P substitutions in the S2, and without (S2P) or with the mutation A942P (S3P) were transfected into cells for protein expression.
  • the cell supernatants were collected for analysis of protein expression by sandwich ELISA using an anti-Flag antibody and a biotin-labelled antibody that recognize the RBD.
  • Optical density at 490 nm in the ELISA assays were corrected by the background signals determined in wells with untransfected cell supernatants. Two independent transfections were performed. 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-S(3P)j a recombinant MVA virus expressing coronavirus SARS-CoV-2 prefusion-stabilized S protein [MVA-S(3P)j was 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, as previously described (Garcia- Arriaza et al., 2014, J Virol 88:3527-3547).
  • 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.
  • 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.
  • 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) was added to view the lysis plaques. After 24 hours, the blue plaques that were more isolated from other lysis plaques 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.
  • 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-S(3P) virus was carried out according to the previously described standard protocols (Ramirez et ai., 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 (N1SO4) (Sigma- Aldrich). After counting the lysis plaques, the titer was referenced as PFU/ml. Replication kinetics
  • MVA-S(3P) The genetic stability of vaccine candidates MVA-S(3P) was analyzed. Monolayers of DF-1 cells were infected with MVA-S(3P) recombinant virus 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 eight times. Expression of the coronavirus SARS-CoV-2 S protein was analyzed by means of Western blot (see section 3.2.1.) 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).
  • 30 p150 plates containing confluent CEF cells were infected with a stock P2 of MVA-S(3P) 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 virus was titered 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 S antigen of stock P3 was performed by means of PCR and Western blot before use in the in vivo assays.
  • Intramuscular immunization was performed in the triceps of both hind legs in a total volume of 100 pi of PBS (50 mI/leg), using a BD Micro-Fine (30 G) x 8 mm insulin syringe (BD Biosciences).
  • mice 6 female C57BL/6 mice per group were inoculated with a single dose of 1 x 10 7 PFU of vaccine candidates MVA-S, MVA-A-S and MVA-S(3P) and control MVA-WT virus by intranasal route in 50mI of PBS. The mice were sacrificed 14 days after the immunization.
  • the ELISpot assay was used to detect S antigen-specific IFNy-secreting T cells.
  • 96-well nitrocellulose-bottom plates (Millipore) were covered with 75 mI/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 %C0 2 atmosphere.
  • 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).
  • mice per group were immunized and sacrificed 10 days after the last immunization. Moreover, animals immunized with one single intranasal dose were sacrificed at 14 days postimmunization. After sacrificing the animals, spleens, lungs or bronchial lymph nodes were removed from the immunized animals in RPMI-10 % FCS medium and processed as described in the preceding section.
  • the 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.
  • 1-4 x 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 or to monkeypox virus.
  • the cells were stimulated with 2 mI/ml of LAC (Leukocyte Activation Cocktail, BD Biosciences) and Monensin 1X. After 6 hours of stimulation, the cells were washed with IB buffer and incubated with Violet Dye (0.5 mI/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 cells were incubated for 20 min in the dark at 4°C with 50 mI of the primary surface antibodies (described in the Table 2) diluted as indicated for each batch.
  • LAC Leukocyte Activation Cocktail
  • Monensin 1X Monensin 1X
  • the cells were then washed two times and 100 mI 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 m I of Fc Block diluted 1 :100 in Permwash 1X were added for 5 min at 4°C in the dark.
  • Cytofix/Cytoperm BD Biosciences
  • 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 mI 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/Cytoperm TM 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 antibodies in the sera of immunized mice and the capacity of these antibodies to neutralize the SARS-CoV-2 virus. Moreover, levels of IgA antibodies in bronchoalveolar lavages (BAL) were also analyzed. To obtain the sera, blood was collected from the animals by submandibular bleeding or 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 was obtained, inactivated at 56°C for 30 min and kept at -20°C until use.
  • BAL bronchoalveolar lavages
  • BAL samples were obtained in 700 mI of RPM I, after the sacrifice of the mice, inactivated at 56°C for 30 min and kept at -20°C until use. Serial dilutions of each individual serum sample (or group of sera) or group of BAL samples were then performed to measure the titers 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. These techniques are explained in the following sections.
  • Enzyme-linked immunosorbent assay ELISA assays were performed to analyze the levels of antibodies in the serum of immunized animals.
  • NUNC MaxiSorpTM 96-well plates (Thermo Fisher Scientific) were coated with 2 pg/ml of the coronavirus SARS-CoV-2 S and RBD proteins 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 pi of HRP-conjugated mouse anti-lgG, -lgG1, -lgG2b, -lgG2c, -lgG3 or -IgA 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). Total IgG titers were measured as the last dilution that gives an absorbance at least 3 times higher the absorbance of a naive serum.
  • RBD-Fc proteins without (WT, Wuhan) or with mutations in VoC (alpha, beta, gamma, delta, kappa and zeta) were used in ELISA assays to determine the RBD- specific IgG antibody titers. Similar amounts of plastic-bound RBD-Fc proteins were used, as determined with an anti-HA antibody (2.5-0.0025 pg/ml) that recognized an HA tag at the RBD N-terminus. Differences in the HA antibody signal at 450 nm were used to standardize the anti-mouse IgG binding to RBD variants with respect to the WT before IgG titer determination.
  • a mix of retrovirus-based pseudoparticles expressing SARS-CoV-2 S protein and mice serum samples 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, and then, 24 hours later, cells were lysed in passive lysis buffer (Promega, Madison, Wl) and the luciferase activity was measured in a luminometer (Thermo Appliskan Multimode Microplate Reader, Thermo Fisher Scientific).
  • the ID50 titers were determined as the highest dilution of serum which resulted in a 50% reduction of luciferase units compared with serum samples from control group (pseudotyped viruses not incubated with serum).
  • 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.
  • a WHO International Standard containing pooled plasma obtained from eleven individuals recovered from SARS-CoV-2 infection was used for the calibration and harmonisation of serological assays detecting anti-SARS-CoV-2 neutralizing antibodies (NIBSC code: 20/136).
  • SARS-CoV-2 pseudotyped Vesicular Stomatitis Virus (VSV) expressing S protein SARS-CoV-2 pseudotyped Vesicular Stomatitis Virus (VSV) expressing S protein.
  • SARS-CoV-2 S-protein pseudotyped rVSV-luc recombinant viruses (PSV) were produced as described elsewhere.
  • SARS-CoV-2 S variants used were S_614D, S_614G, alpha (B.1.1.7), beta (B.1.351), gamma (P.1) and delta (B.1.617.2).
  • SARS-CoV-2 S variants used were S_614D, S_614G, alpha (B.1.1.7), beta (B.1.351), gamma (P.1) and delta (B.1.617.2).
  • SARS-CoV-2 S variants used were S_614D, S_614G, alpha (B.1.1.7), beta (B.1.351), gamma (P
  • CoV-2 S mutant D614G was generated by site-directed mutagenesis (Q5 Site Directed Mutagenesis Kit; New England Biolabs) following the manufacturer’s instructions and using as an input DNA a pcDNA3.1 expression vector encoding SARS-CoV-2 S_614D.
  • GISAID EPIJSL 19703357
  • GeneArt Thermo Fisher Scientific, GeneArt GmbH, Regensburg, Germany
  • the neutralization activity of serum samples was tested by triplicates at several two-fold dilutions.
  • viruses-containing transfection supernatants were normalized for infectivity to a MOI of 0.5-1 and incubated with the dilutions of serum samples at 37°C for 1 hour in 96-well plates. After the incubation time, 2 x 10 4 Vero-E6 cells were seeded onto the virus-serum mixture and incubated at 37°C for 24 hours.
  • NT50 titers were calculated using a nonlinear regression model fit with settings for log agonist/inhibitor versus normalized response curves, in GraphPad Prism v8.
  • mice Male and/or female K18-hACE2 mice (9 weeks old at the beginning of the study) immunized with one intramuscular dose of MVA-S and MVA-S(3P) were used to evaluate the efficacy of MVA-S(3P) vaccine candidate.
  • Groups of animals (n 11) received one dose of 1 c 10 7 PFU of MVA-S or MVA-S(3P) by the i.m. route in 100 pi of PBS (50 mI/leg) at week 0.
  • Mice primed with the same dose of non-recombinant MVA- WT were used as a control group.
  • mice were lightly anesthetized with isoflurane and infected intranasally with 1 c 10 5 PFU of SARS-CoV-2 MAD6 in a total volume of 50 mI of PBS.
  • mice Female K18-hACE2 mice (9 weeks old at the beginning of the study) immunized with one intranasal dose of MVA-S, MVA-A-S and MVA-S(3P) were used to evaluate the efficacy of MVA-S(3P) vaccine candidate.
  • Groups of animals (n 9) received one dose of 1 c 10 7 PFU of MVA-S, MVA-A-S or MVA-S(3P) by the i.n. route in 50 mI of PBS at week 0.
  • Mice primed with the same dose of non-recombinant MVA-WT were used as a control group.
  • mice were lightly anesthetized with isoflurane and infected intranasally with 1 c 10 5 PFU of SARS-CoV-2 MAD6 in a total volume of 50 m! of PBS.
  • female K18-hACE2 mice 22 weeks old at the beginning of the study) immunized with two intramuscular doses of MVA-S(3P) were used to evaluate the efficacy of MVA-S(3P) vaccine candidate.
  • Groups of animals (n 11) received two doses of 1 x 10 7 PFU of MVA-S(3P) by the i.m. route in 100 mI of PBS (50 mI/leg) at weeks 0 and 4.
  • mice primed with the same doses of non-recombinant MVA-WT were used as a control group.
  • mice were lightly anesthetized with isoflurane and infected intranasally with 1 c 10 5 PFU of SARS-CoV-2 beta VOC (B.1.351) in a total volume of 50 mI of PBS.
  • mice were monitored daily for body weight change and survival for 10-15 days postchallenge. Animals with more than a 25% of weight loss were euthanized. At days 4-5 postchallenge, three-six mice per group were euthanized, and lungs, BAL, nasal washes and serum samples were collected. One 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.
  • RNALater stabilization reagent 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 and RNA level of several cytokines were determined using previously validated set of primers and/or probes specific for the SARS-CoV-2 subgenomic RNA for the protein E, genomic RNA for the RdRp protein, each cytokine gene tested, 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.
  • RNA arbitrary units were quantified relative to the negative group (uninfected mice) and were performed using the 2-AACt method. All samples were tested in triplicate.
  • RT-qPCR reverse transcription of 4000 ng of RNA isolated as described above from lung homogenates of K18-hACE2 mice was performed with the QuantiNova reverse transcription kit (Qiagen), according to the manufacturer’s recommendations.
  • Qiagen QuantiNova reverse transcription kit
  • 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.
  • mRNA expression levels of cytokines IP-10, IL-12b, CCL11, and IFN-y genes 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. 3.5.2. Analysis of SARS-CoV-2 virus yields by plaque assay
  • Lungs from K18-hACE2 mice used in the efficacy studies were harvested at 4-5 days post-challenge, weighted, and stored directly at -80°C until homogenized with a gentleMACS dissociator (Miltenyi Biotec) in 2 ml of PBS buffer and aliquoted. Moreover, nasal washes and BAL samples were also analyzed.
  • a gentleMACS dissociator Miltenyi Biotec
  • Lung histopathology The entire left lung lobe was removed from each K18-hACE2 mouse and immersion- fixed in zinc formalin (Sigma-Aldrich) for 48 hours. After fixation period, samples were routinely processed and embedded in paraffin blocks that were then sectioned at 4 pm thickness on a microtome, mounted onto glass slides and routinely stained with haematoxylin and eosin (H&E). Lung sections were microscopically evaluated using an Olympus BX43 microscope by a single veterinary pathologist who was blinded to the identity and group of individual mice.
  • the histopathological parameters evaluated were the follows: capillary endothelial cell activation; alveolar haemorrhages; alveolar oedema; perivascular oedema; alveolar septal thickening (interstitial pneumonia); alveolar damage and hyaline membranes in alveoli; inflammatory cell infiltration in alveoli; bronchi/bronchioles with epithelial necrosis, detached epithelium or inflammatory cells in the lumen (bronchitis/bronchiolitis); peribronchial/peribronchiolar and perivascular mononuclear infiltrates; pneumocytes hyperplasia; cytopathic effect or syncytia; squamous metaplasia; uniform interstitial fibrosis; organized fibrotic tissue around the bronchi/bronchioles or intrabronchiolar (bronchiolitis obliterans) and pleural thickening.
  • the histopathological parameters were graded following a semi-quantitative scoring system as follows: (0) no lesion; (1) minimal lesion; (2) mild lesion; (3) moderate lesion; (4) severe lesion.
  • the cumulative scores of histopathological lesions provided the total score per animal.
  • the individual scores were used to calculate the group average.
  • H&E-stained sections were visually scored 0-6 based on the percentage of lung area affected by inflammatory lesions as follows: 0% of the lung injured (score 0); ⁇ 5% (score 1); 6-10% (score 2); 11-20% (score 3); 21-30% (score 4); 31-40% (score 5); > 40% (score 6).
  • the individual scores were used to calculate the group average.
  • a new MVA viral vector-based vaccine have been designed and developed against the coronavirus SARS-CoV-2 causing the COVID-19 pandemic. Once generated, this recombinant MVA virus have been characterized in vitro and their capacity to stimulate adaptive SARS-CoV-2-specific T cell and humoral immune responses in vivo in immunized mice has been analysed, as well as its efficacy in humanized K18-hACE2 mice.
  • MVA-based vaccine candidate expressing the SARS-CoV-2 full-length prefusion-stabilized S structural gene (Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1 , Genbank number: MN908947.3) [termed MVA-S(3P)] (see Materials and Methods).
  • Vaccine candidate MVA-S(3P) was generated following the insertion of the complete sequence of the coronavirus SARS-CoV-2 S gene (prefusion- stabilized) in the genome of the parental virus MVA-WT.
  • MVA-CoV2-S(3P) vaccine candidate express a human codon optimized full- length S protein that contains three mutations in the furin cleavage site (R682G, R683S, R685S) that avoid the cleavage of the S protein in S1 and S2, and three additional mutations in the S2 region that stabilizes the S protein in a prefusion conformation (A942P, K986P, and V987P) ( Figure 1A).
  • Figure 1A shows a diagram of the generated vaccine candidate MVA-S(3P).
  • the methodology used for generating this vaccine candidate 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 candidate MVA-S(3P) was 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-S, and MVA-S(3P).
  • Oligonucleotides (described in Table 1 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-S and MVA-S(3P), without contamination with the parental MVA-WT ( Figure 1B).
  • the TK locus of parental virus MVA-WT was amplified as PCR control, with the TK gene being amplified. The correct presence of the S gene inserted in the TK locus was also confirmed by means of DNA sequencing.
  • MVA-S(3P) constitutively express the prefusion-stabilized SARS- CoV-2 S protein
  • vaccine candidates MVA-S and MVA-S(3P) efficiently express the coronavirus SARS-CoV-2 S protein.
  • Vaccine candidate MVA-S(3P) is stable in cell culture
  • Vaccine candidate MVA-S(3P) expresses high levels of S protein in the cell membrane
  • Vaccine candidate MVA-S(3P) replicates well in permissive cells
  • DF-1 cells were infected with vaccine candidates MVA-S and MVA-S(3P), and parental virus MVA-WT, 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 S protein expressed by MVA-S(3P) is present in the cell membrane, similar to what happen with MVA-S ( Figure 4).
  • Figure 4A In permeabilized cells, large amounts of S protein were detected in the cytoplasm of cells infected with MVA-S or MVA-S(3P) ( Figure 4A).
  • Figure 4B S protein cell surface expression, which colocalized with the wheat germ agglutinin (WGA) cell membrane probe.
  • coronavirus SARS-CoV-2 S-protein-specific and S-protein receptor binding domain (RBD)-specific IgG antibodies were analyzed by means of ELISA 10 days after the prime and 10 days after the second immunization (boost).
  • the animals immunized with MVA-WT were used as a control group.
  • vaccine candidate MVA-S(3P) induced higher levels of total IgG, lgG1 , lgG2b, lgG2c, and lgG3 antibodies against SARS-CoV-2 S- and RBD proteins, and higher neutralizing antibodies than MVA-S, after one dose, but similar after two doses.
  • mice were immunized with two doses of MVA-S or MVA-S(3P).
  • 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. After 48 hours of stimulation, the cells were labeled with antibodies to identify IFN-y-secreting cells specific for these peptides (Figure 8).
  • 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 both immunization groups was analyzed by measuring the simultaneous production pattern of cytokines (IFN-y, 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 both immunization groups were characterized by a similar high polyfunctionality ( Figure 10).
  • 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) ( Figure 10A).
  • 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) ( Figure 10B).
  • TCM T central memory
  • TEM T effector memory
  • TE T effector
  • 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-y and/or IL-21.
  • the polyfunctionality of the Tfh cell-mediated immune response generated by both immunization groups was analyzed by measuring the simultaneous production pattern of cytokines (IFN-g and/or IL-21) plus the marker, CD40L.
  • the coronavirus SARS-CoV-2 S-protein-specific Tfh cell-mediated responses generated by both immunization groups were characterized by a high polyfunctionality ( Figure 12C).
  • the Tfh cell-mediated response was mainly characterized by cells which are capable of carrying out all 2 functions (secreting IFN-g and IL-21), followed by cells capable of carrying out 3 functions (secreting the 2 cytokines and expressing the marker, CD40L) ( Figure 12C).
  • vaccine candidates MVA-S and MVA-S(3P) induce Tfh cell- mediated responses.
  • mice were immunized with one dose (1 c 10 7 PFU by the intramuscular route) of MVA-S or MVA-S(3P) at week 0 and then challenged at week 4 with 1 x 10 5 PFU of SARS-CoV-2 (MAD6 isolate) by the intranasal route (Figure 13A). Mice primed with MVA-WT were used as control group.
  • mice were monitored for changes in body weight and mortality for 15 days after the challenge. All K18-hACE2 mice immunized with one dose of MVA-S(3P) and challenged with SARS-CoV-2 did not lose body weight (Figure 13B) and survived ( Figure 13C), whereas mice immunized with one dose of MVA-S lost body weight during the first 4 days postchallenge ( Figure 13B), but recovered and survived ( Figure 13C). On the other hand, mice inoculated with MVA-WT and challenged with SARS-CoV-2 lost body weight (more than 25%) (Figure 13B) and all died at 6-7 days postchallenge (Figure 13C).
  • mice per group were sacrificed at day 4 after SARS-CoV-2 virus challenge, lungs collected and processed, and the presence of SARS-CoV-2 subgenomic E and genomic RdRp RNA (Figure 13D) as well as of live infectious virus ( Figure 13E) was analyzed.
  • One dose of MVA-S(3P) was more effective than MVA-S to prevent SARS-CoV-2 replication, reducing significantly subgenomic and genomic SARS-CoV-2 RNA levels (Figure 13D) in comparison to MVA-WT control infected mice, as well as the infectious virus yields with respect to the MVA-S vaccinated mice and MVA-WT control infected mice ( Figure 13E).
  • mice vaccinated with one dose of MVA-S(3P) exhibited lower lung inflammation scores ( Figure 14A, left panel) and lower percentages of lung area with lesions ( Figure 14A, right panel) than control MVA-WT mice, and significantly lower lesions and lung affected area than mice immunized with one dose of MVA-S ( Figure 14A).
  • Representative images of lung sections are included in Figure 14B; mice vaccinated with one dose of MVA-S(3P) only displayed focal thickening of the alveolar septae, and occasional presence of inflammatory cells within the alveoli.
  • mice immunized with one dose of MVA-S or with control MVA-WT showed more severe diffuse thickening of the alveolar septae, higher presence of mononuclear cell infiltrates within alveolar spaces, as well as the presence of larger multifocal perivascular and peribronchiolar mononuclear infiltrates (Figure 14B).
  • MVA-S(3P) One dose of MVA-S(3P) triggered in K18-hACE2 transgenic mice higher titers of S- and RBD-specific IgGs and neutralizing antibodies against different SARS- CoV-2 variants of concern (VoC) than MVA-S
  • Cells were stimulated ex vivo with S peptide pools, spanning the entire S protein, and an ICS assay was performed to measure the induction of SARS- CoV-2 S-specific CD4 + and CD8 + T cells expressing CD107a, and secreting IFN-y, TNF-a, and/or IL-2.
  • Challenged mice previously inoculated with one dose of MVA-WT were used as a control group.
  • mice were monitored for changes in body weight and mortality for 14 days after the challenge. All K18-hACE2 mice intranasally immunized with one dose of MVA-A-S or MVA-S(3P) and challenged with SARS-CoV-2 did not lose body weight (Figure 18B) and survived ( Figure 18C), whereas mice inoculated with MVA-WT and challenged with SARS-CoV-2 lost body weight (Figure 18B) and all died at 8 days postcha!lenge (Figure 18C). Mice immunized with one dose of MVA-S lost body weight during the first 6 days postchallenge (Figure 18B), but recovered and survived (Figure 18C).
  • mice per group were sacrificed at day 5 after SARS-CoV-2 virus challenge, lungs and nasal washes collected and processed, and the presence of SARS-CoV-2 subgenomic E and genomic RdRp RNA ( Figure 18D and 18E), as well as of live infectious virus ( Figure 18E and 18G) was analyzed.
  • MVA-S(3P) One intranasal dose of MVA-S(3P) was more effective than MVA-S or MVA-A-S to prevent SARS-CoV-2 replication, reducing significantly subgenomic and genomic SARS-CoV-2 RNA levels in lungs ( Figure 18D) and nasal washes (Figure 18E), in comparison to MVA-WT control infected mice.
  • no infectious virus was detected in lungs ( Figure 18F) or nasal washes ( Figure 18G) from vaccinated mice, in comparison to MVA-WT control mice, indicating that sterilizing immunity was achieved.
  • mice intranasally vaccinated with one dose of MVA-S(3P) displayed significant lower lung inflammation scores ( Figure 19A, left panel) and lesser percentages of lung area with lesions ( Figure 19A, right panel) than control MVA-WT mice.
  • mice vaccinated with MVA-S or MVA-A-S showed slightly lower levels of lung inflammation scores and lesser percentages of lung area with lesions than control MVA-WT mice ( Figure 19A), but differences were not significant.
  • mice vaccinated with one dose of MVA-S(3P) only displayed focal thickening of the alveolar septae, and occasional presence of inflammatory cells within the alveoli ( Figure 19B).
  • mice immunized with one dose of MVA-S or MVA-A-S or with control MVA-WT showed more severe diffuse thickening of the alveolar septae, higher presence of mononuclear cell infiltrates within alveolar spaces, as well as the presence of larger multifocal perivascular and peribronchiolar mononuclear infiltrates (Figure 19B).
  • MVA-S(3P) compared to control MVA- WT infected mice, one intranasal dose of MVA-S(3P) induced a significant down- regulation of several pro-inflammatory cytokines, such as IL-6, CXCL10, TNF-a, IFN-y, IL-12b, CXCL5, CCL2, CCL11 and CCL12, either in lung samples ( Figure 20A) or in nasal washes ( Figure 20B).
  • cytokines such as IL-6, CXCL10, TNF-a, IFN-y, IL-12b, CXCL5, CCL2, CCL11 and CCL12
  • MVA-S(3P) induced significantly higher titers of neutralizing antibodies against live SARS-CoV-2 (MAD6 isolate) than MVA-S or MVA-A-S at 14 days postimmunization and at 5 days postchallenge, although at day 14 postchallenge the neutralization titers were enhanced and similar in all groups (Figure 21 B), reflecting the existence of a breakthrough infection mainly in the MVA-S- and MVA-A-S-vaccinated groups and suggesting that MVA-S(3P) vaccination controlled better the infection.
  • Challenged mice previously inoculated with two doses of MVA-WT were used as a control group.
  • mice were monitored for changes in body weight and mortality for 10 days after the challenge. All K18-hACE2 mice immunized with two doses of MVA-S(3P) and challenged with SARS-CoV-2 B.1.351 VOC did not lose body weight (Figure 22B) and survived ( Figure 22C), whereas mice inoculated with MVA-WT and challenged with SARS-CoV-2 lost body weight (Figure 22B) and all died at 4 days postchallenge (Figure 22C).
  • mice per group were sacrificed at day 4 after SARS-CoV-2 virus challenge, lungs and BAL samples collected and processed, and the presence of SARS-CoV-2 subgenomic E and genomic RdRp RNA ( Figure 22D and 22E), as well as of live infectious virus ( Figure 22 E and 22G) was analyzed.
  • MVA-S(3P) prevented SARS-CoV-2 B.1.351 VOC replication, reducing significantly subgenomic and genomic SARS-CoV-2 RNA levels in lungs ( Figure 22D) and BAL ( Figure 22E), in comparison to MVA-WT control infected mice.
  • 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 CD8 + T cell-mediated immune response showed that both immunization groups were characterized by a similar high polyfunctional profile, analyzed by measuring the simultaneous production pattern of cytokines (IFN-y, TNF-a, and/or IL-2) plus the cytotoxic potential thereof (CD107a as a degranulation marker).
  • 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) ( Figure 25D).
  • SARS-CoV-2 S(3P) protein is express at higher levels than a S(2P) protein
  • mammalian expression vectors that contained recombinant DNAs coding for the soluble S protein with a T4 fibritin trimerization sequence, a Flag peptide and a His-tag at the C-terminus were used for S expression by transfection of HEK293T cells.
  • S constructs without the furin motif and with the K986P and V987P substitutions in the S2, and without (S2P) or with the mutation A942P (S3P) were transfected into cells for a comparative of protein expression.
  • the cell supernatants were collected for analysis of protein expression by sandwich ELISA using an anti-Flag antibody and a biotin-labelled antibody that recognize the RBD.
  • the results showed that the S(3P) protein was expressed about 4-fold times than the S(2P) protein (Fig. 26), indicating that the presence of a third mutation in the S protein (A942P) increases the expression levels of the full length protein in transfected cells.
  • the full-length SARS-CoV-2 S sequence inserted in vaccine candidate MVA-S(3P) described in the present examples was synthetized by GeneArt, was human codon optimized, and contains three mutations in the furin cleavage site (R682G, R683S, R685S) that avoid the cleavage of the S protein in S1 and S2, and three additional mutations in the S2 region that stabilizes the S protein in a prefusion conformation (A942P, K986P, and V987P).
  • the sequence was derived from Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1 (GenBank: MN908947.3, https://www.ncbi.nlm.nih.gov/nuccore/MN908947). The sequence is reproduced herein below as SEQ ID NO 1.
  • SEQ ID NO 1 The S nucleotide sequence reads as follows (SEQ ID NO 1):
  • S protein sequence reads as follows (SEQ ID NO 2): MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFF SNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLI VNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFL MDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITR FQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDP LSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWN RKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVR
  • SEQ ID NO 3 The RBD nucleotide sequence described in the specification as SEQ ID NO 3 reads as follows (SEQ ID NO 3):
  • SEQ ID NO 4 The RBD protein sequence described in the specification as SEQ ID NO 4 reads as follows (SEQ ID NO 4):
  • SEQ ID NO 5 The E nucleotide sequence described in the specification as SEQ ID NO 5 reads as follows (SEQ ID NO 5):
  • SEQ ID NO 7 The N nucleotide sequence described in the specification as SEQ ID NO 7 reads as follows (SEQ ID NO 7):
  • SEQ ID NO 8 The N protein sequence described in the specification as SEQ ID NO 8 reads as follows (SEQ ID NO 8):
  • SEQ ID NO 9 The M nucleotide sequence described in the specification as SEQ ID NO 9 reads as follows (SEQ ID NO 9):
  • SEQ ID NO 10 The M protein sequence described in the specification as SEQ ID NO 10 reads as follows (SEQ ID NO 10): MADSNGTITVEELKKLLEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPVT LACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPETNILLN VPLHGTILTRPLLESELVIGAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGA
  • SEQ ID NO 24 The nucleotide sequence of the E3L gene of MVA (MVA050L) described in the specification as SEQ ID NO 24 reads as follows (SEQ ID NO 24):
  • SEQ ID NO 25 The MVA E3 protein sequence described in the specification as SEQ ID NO 25 reads as follows (SEQ ID NO 25):

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Abstract

La présente invention concerne une composition de vaccin comprenant une quantité immunologiquement efficace d'un vecteur du virus modifié de la vaccine Ankara (MVA) comprenant au moins un acide nucléique codant pour la protéine de spicule (S), ou un fragment de ladite protéine S comprenant au moins un épitope, d'au moins un variant du SARS-CoV-2, ladite protéine S ou un fragment de celle-ci comprenant au moins les substitutions R682G, R683S, R685S, A942P, K986P et V987P, et le vecteur de MVA régulant l'expression de l'acide nucléique codant pour la protéine S, ou son fragment. La présente invention concerne également une combinaison de vaccins et leurs utilisations.
PCT/EP2022/067271 2021-06-23 2022-06-23 Vaccin à base de mva exprimant une protéine s du sars-cov-2 stabilisée par préfusion WO2022269003A1 (fr)

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