Nucleic acids for vaccination encoding antigen-displaying protein nanoparticles

Abstract

The present invention relates to an RNA or DNA molecule, such as a messenger RNA (mRNA), a self-amplifying RNA (saRNA) or an expression plasmid, encoding a fusion protein comprising a disease-associated antigen that is joined to a subunit of a self-assembling protein nanoparticle, and to medical uses thereof.

Description

[0001] NUCLEIC ACIDS FOR VACCINATION ENCODING ANTIGEN-DISPLAYING PROTEIN NANOPARTICLES

[0002] Technical Field

[0003] The present invention relates to the field of vaccines. More specifically, the invention relates to a pharmaceutical composition comprising an RNA or DNA molecule, particularly a messenger RNA (mRNA), a self-amplifying RNA (saRNA), or an expression plasmid, encoding fusion proteins in which a vaccine antigen is fused to a subunit of a self-assembling protein nanoparticle. The invention further relates to pharmaceutical compositions comprising such an RNA or DNA molecule, as well as to their medical uses in the prevention of diseases.

[0004] Background

[0005] Recombinant viral vectors encoding foreign disease-associated antigens have long been described as efficient inducers of potent and specific immune responses, and they have successfully been used as vaccines against infectious diseases such as Ebola virus disease (e.g., Mvabea®) and coronavirus disease 2019 (COVID-19; e.g., Vaxzevria®). Such viral vectors deliver genetic material of an antigen foreign to the vector backbone ( / .e., a transgene), which is transcribed and translated into the respective antigenic protein by the vaccine recipient’s host cells.

[0006] A well-characterized viral vector is MVA-BN®, which was developed from a Modified Vaccinia Virus Ankara (MVA) virus stock. MVA which was derived from the prototype species vaccinia virus (VACV) of the Orthopoxvirus genus within the family Poxviridae. The dermal VACV Ankara strain (Chorioallantois vaccinia virus Ankara, CVA) is a replicating vaccinia virus [1]. By serial propagation of CVA over more than 570 passages on primary chicken embryo fibroblasts (CEFs), the attenuated CVA-derived virus MVA was obtained. This MVA was further passaged by Bavarian Nordic resulting in a further attenuated MVA strain, namely MVA-BN® [2], MVA-BN® lacks approximately 15% of the genome compared to ancestral CVA virus (loss of 31 kb resulting in six major deletion sites). These deletions affect a number of virulence and host range genes, as well as the gene for Type A inclusion bodies. MVA-BN® can attach to and enter human cells and can express very efficiently virally encoded genes in the infected human cells. However, assembly and release of progeny virus does not occur in human cells. Therefore, MVA-BN® is a safe and versatile vaccine vector able to efficiently express antigenencoding transgenes for use in vaccination approaches that target diseases with hitherto unmet medical need (e.g., Mvabea® against Ebola virus disease [3]). Preparations of MVA- BN® and derivatives have been administered to many types of animals and to more than 10.500 human subjects in clinical studies, including immunodeficient individuals, without any serious adverse events.

[0007] Virus replicon particles (VRPs) are alphavirus derived viral vectors that contain a selfamplifying positive sense single strand RNA genome that is packaged in a shell consisting of a capsid protein as well as an envelope with a heterodimeric envelope protein, which is embedded in lipid membrane derived from the producing cell. VRPs used for vaccination encode a foreign antigen in place of the viral capsid and envelope genes. As they lack the genes for these structural proteins, VRPs cannot replicate in transduced cells but they induce expression of large quantities of the transgene, resulting in the induction of robust immune responses.

[0008] Upon administration to a recipient, viral vector vaccines exhibit multiple characteristics of a true virus infection and thereby induce strong immune responses against the delivered vaccine antigen as well as against antigens of the vector itself, without the need of support by an adjuvant. Viral vector vaccines are excellent inducers of T-helper (Th) 1 skewed T-cell responses. In contrast to this, antibody responses induced by viral vector vaccines against the transgene-encoded antigens tend to be lower than those induced by antigens delivered as protein in adjuvant or as a virus like particle (VLP) at least in short interval vaccination schedules for rapid induction of protective antibody levels.

[0009] However, induction of a robust and long-lived antibody response generally is a highly desirable characteristic of vaccines against infectious diseases. There is a broad scientific consensus that strongest antibody responses are induced upon immunization with protein antigens that are multimerized in a regular array, either as a VLP or displayed on any other kind of nanoparticle [4, 5]. However, T-cell responses induced by these antigens tend to be weak and Th2 skewed, a disadvantage that can be overcome to some extent by formulation with a suitable and strong adjuvant.

[0010] Thus, there is a need for advanced vaccines inducing improved immune responses. Summary of Invention

[0011] It is an objective of the present invention to provide means and methods for improving the immune response of a vaccine recipient to a vaccine antigen.

[0012] The objective of the present invention is solved by the provision of RNA and DNA vaccines comprising RNA and DNA molecules, respectively, expressing at least one foreign vaccine antigen and, additionally, subunits of a self-assembling protein nanoparticle such that the antigen is displayed on the protein nanoparticle’s surface.

[0013] In particular, the invention is defined by the appended claims and by the following aspects and embodiments.

[0014] In a first aspect, the invention provides a pharmaceutical composition comprising an RNA or DNA molecule, the RNA or DNA molecule comprising a nucleic acid section encoding a fusion protein comprising a disease-associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle.

[0015] In a second aspect, the invention provides a process for preparing a pharmaceutical composition as described herein, comprising the steps of:

[0016] (1 ) providing a nucleic acid encoding a fusion protein comprising a disease- associated antigen, or an antigenic part thereof, joined to a subunit of a selfassembling protein nanoparticle;

[0017] (2) generating an RNA or DNA molecule comprising the nucleic acid provided in step (1 );

[0018] (3) obtaining the RNA or DNA molecule generated in step (2);

[0019] (4) combining the RNA or DNA molecule obtained in step (3) with a pharmaceutically acceptable carrier or excipient.

[0020] In another aspect, the invention provides a pharmaceutical composition comprising an RNA or DNA molecule as described herein for use in the prevention or treatment of a disease, preferably an infectious disease or cancer.

[0021] In a further aspect, the invention provides a method of prevention or treatment of an infectious disease or cancer comprising the step of administering to a subject a composition as described herein, such as a composition comprising an RNA or DNA molecules as described herein.

[0022] In a further aspect, the invention provides a method for inducing an immune response to an infectious disease or cancer comprising the step of administering to a subject a composition as described herein, such as a composition comprising an RNA or DNA molecule as described herein.

[0023] Brief Description of Drawings / Figures

[0024] Figure 1 schematically illustrates SARS-CoV-2 Spike RBD antigen constructs.

[0025] Construct- 1 : Monomeric SARS-CoV-2 Spike RBD (“RBD monomer”). The receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein is fused at its N-terminus to the secretion signal peptide of human tissue plasminogen activator (htPA SP).

[0026] Construct-2: SARS-CoV-2 Spike RBD-Bullfrog / / - / . pylori hybrid ferritin fusion protein (“RBD-BFF”). The RBD of SARS-CoV-2 S protein is fused at its C-terminus to Bullfrog / / - / , pylori hybrid ferritin (BFF) via a 9 amino acid linker and at its N- terminus to the htPA SP.

[0027] Figure 2 shows an analysis of protein expression of SARS-CoV-2 Spike RBD monomer and SARS-CoV-2 Spike RBD-BFF fusion protein encoded by recombinant MVA.

[0028] HeLa cells were infected with recombinant MVA encoding either construct-1 or construct-2. Cell lysates and supernatants were analyzed by Western blot using an anti-RBD antibody. Lysates were subjected to poly-acrylamide gel electrophoresis (PAGE) under denaturing conditions (A); supernatants were subjected to applied to poly-acrylamide gel electrophoresis (PAGE) under denaturing (B) or non-denaturing (native) conditions (C). Lanes are labelled by construct-# (1 : RBD monomer; 2: RBD-BFF).

[0029] Figure 3 shows an analysis of protein expression of SARS-CoV-2 Spike RBD monomer and SARS-CoV-2 Spike RBD-BFF fusion protein encoded by VRPs.

[0030] HEK293T cells were transfected with a plasmid encoding a VEEV replicon under control of a CMV promotor, encoding the VEEV TrD83 nonstructural proteins as well as either construct- 1 or construct-2 under the VEEV subgenomic promotor. Cell lysates and supernatants were analyzed by Western blot using an anti-RBD antibody. Lysates were subjected to polyacrylamide gel electrophoresis (PAGE) under denaturing conditions (A); supernatants were subjected to poly-acrylamide gel electrophoresis (PAGE) under denaturing (B) or nondenaturing (native) conditions (C). Lanes are labelled by construct-# (1 : RBD monomer; 2: RBD-BFF). Figure 4 shows in vivo immunogenicity of SARS-CoV-2 Spike RBD monomer and BFF fusion protein encoded by recombinant MVA or VRPs.

[0031] A-C Balb / c mice were immunized intramuscularly on day 0 (prime) and day 21 with 1 x108Infll of recombinant MVA encoding either RBD monomer (MVA-RBD monomer) or RBD-BFF (MVA-RBD-BFF), or buffer (control). D-F Balb / c mice were immunized intramuscularly on day 0 (prime) and day 21 with 1x108Til of VRPs encoding either RBD monomer (VRP-RBD monomer) or RBD-BFF (VRP-RBD-BFF), or buffer (control). A-B, D-E Blood for serum isolation was drawn on day 34 after prime immunization. A, D Anti-RBD Ig titers in the serum were analyzed by ELISA on RBD-coated plates with a starting dilution of 1 :300 to determine total anti-RBD Ig titers using anti-mouse IgG (H+L) antibodies. Arbitrary titers (AU = arbitrary units) were calculated via a 4PL-fit curve with an intercept at OD = 0.3. B, E ePass SARS- CoV-2 neutralization antibody detection kit (Genscript) was used to assess RBD-binding antibody titers and the titer at 50% inhibition compared to negative control is plotted. C, F Mice were sacrificed on day 35 after prime immunization and single-cell suspensions from splenocytes were prepared. 0.5 x 106cells were seeded in coated IFN-y ELISpot plates and restimulated with Spike Peptide Pool A (containing the RBD region) or control medium. ELISpot plates were incubated overnight at 37°C before development. Data are shown as Mean ± SEM.

[0032] Figure 5 shows improved germinal center formation and RBD-specific memory B cell induction by MVA-RBD-BFF.

[0033] Balb / c mice were immunized intramuscularly on day 0 with 1 x108InfU of MVA-RBD monomer or MVA-RBD-BFF, or buffer (control). Inguinal and popliteal lymph nodes were isolated and pooled on day 7 and day 14 after immunization. Single cells were stained with fluorochrome- labelled antibodies and fluorochrome-labelled RBD protein and analyzed by flow cytometry to identify (A) germinal center (GO) B cells and (B) memory B cells. Data are shown as Mean ± SEM.

[0034] Figure 6 shows an analysis of protein expression of SARS-CoV-2 Spike RBD monomer and BFF fusion protein encoded by mRNA, saRNA or plasmid DNA.

[0035] HEK293T cells were transfected with mRNA, self-amplifying RNA (saRNA) or plasmid DNA, each encoding either construct- 1 or construct-2. Cell lysates and supernatants were analyzed by Western blot using an anti-RBD antibody. Lysates were r subjected to poly-acrylamide gel electrophoresis (PAGE) under denaturing conditions (A); supernatants were subjected to polyacrylamide gel electrophoresis (PAGE) under denaturing (B) or non-denaturing (native) conditions (C). Lanes are labelled by construct-# (1 : RBD monomer; 2: RBD-BFF). Figure 7-1 shows in vivo immunogenicity of SARS-CoV-2 Spike RBD antigen encoded by saRNA.

[0036] A-B Balb / c mice were immunized intramuscularly on day 0 (prime) and day 21 with 2.5 pg saRNA-RBD monomer or saRNA-RBD-BFF mixed with in vivo-jetPEI®, or buffer (control). A Serum was isolated on day 41 after prime immunization. Anti-RBD Ig titers in the serum were analyzed by ELISA. An anti-RBD antibody was used to establish a standard for serum antibody quantification. B Mice were sacrificed on day 42 after prime immunization and single-cell suspensions from splenocytes were prepared. 0.5 x 106cells were seeded in coated IFN-y ELISpot plates and restimulated with spike Peptide Pool A (containing the RBD region) or control medium. IFNy-producing cells were revealed with biotin-conjugated anti-IFNy antibody in combination with streptavidin-HRP and AEC substrate. Spots were counted using an ELISpot reader. Data are shown as Mean ± SEM.

[0037] Figure 7-2 shows in vivo immunogenicity of SARS-CoV-2 Spike RBD antigen encoded by saRNA and mRNA.

[0038] A-B Balb / c mice were immunized intramuscularly on day 0 (prime) and day 22 with 1 pg or 0.1 pg mRNA or saRNA packaged in LNP (Genscript), 1 x 108TU VRP or buffer (control). A Serum was isolated on day 34 after prime immunization. Anti-RBD IgG titers in the serum were analyzed by ELISA. An anti-RBD antibody was used to establish a standard for serum antibody quantification. B Mice were sacrificed on day 34 after prime immunization and single-cell suspensions from splenocytes were prepared. 0.5 x 106cells were seeded in coated IFN-y ELISpot plates and restimulated with Spike Peptide Pool A (containing the RBD region), Ferritin Peptide Pool (Genscript) or control medium. Data are shown as Mean ± SEM.

[0039] Figure 8 schematically illustrates EBV gp350 antigen constructs.

[0040] Construct-3A: Tetrameric EBV gp350-GCN4 (“gp350-GCN4”) in MVA-mBN443. A fragment (aa 1 -434) of glycoprotein 350 (gp350) of Epstein-Barr virus (EBV) is fused to a GCN4 derived tetramerization domain at the C-terminus via a 13 amino acid linker.

[0041] Construct-3B: Tetrameric EBV gp350-GCN4(“gp350-GCN4”) in VRP. A fragment (aa 4-434) of glycoprotein 350 (gp350) of Epstein-Barr virus (EBV) is fused to a GCN4 derived tetramerization domain at the C-terminus via a 13 amino acid linker and to signal peptide IgK LC at its N-terminus.

[0042] Construct-4: EBV gp350-Bullfrog / H pylori hybrid ferritin fusion protein (“gp350-BFF”). A fragment (aa 2-434) of EBV gp350 is fused at its C-terminus to Bullfrog / / - / , pylori hybrid ferritin (BFF) via a 10 amino acid linker and to signal peptide IgK LC at its N-terminus. Figure 9 shows an analysis of protein expression of tetrameric EBV gp350 and BFF fusion protein encoded by recombinant MVA.

[0043] HeLa cells were infected with recombinant MVA encoding either construct-3A, or construct-4. Cell lysates and supernatants were analyzed by Western blot using an anti-gp350 antibody. Lysates were subjected to poly-acrylamide gel electrophoresis (PAGE) under denaturing conditions (A); supernatants were subjected to poly-acrylamide gel electrophoresis (PAGE) under denaturing (B) or non-denaturing (native) conditions (C). Lanes are labelled by construct-# (3A: gp350 tetramer; 4: gp350-BFF).

[0044] Figure 10 shows in vivo immunogenicity of EBV gp350-GCN4 and BFF fusion protein encoded by recombinant MVA or VRPs.

[0045] A, B Balb / c mice were immunized intramuscularly on day 0 (prime) and day 21 with 1 x107InfU with MVA-gp350-GCN4 (mBN443B) or MVA-gp350-BFF (mBN510), or buffer (control). C, D Balb / c mice were immunized intramuscularly on day 0 (prime) and day 21 with 1 x108TU VRP- gp350-GCN4 [+gHgL] or VRP-gp350-BFF [+gHgL], or buffer (control). A, C Blood for serum isolation was drawn on day 35 (MVA) or day 42 (VRPs) after prime immunization. Serum was analyzed the same day for anti-gp350 IgG titers by ELISA. An anti-gp350 antibody was used to establish a standard for serum antibody quantification. B, D Splenocytes were isolated on day 35 (MVA) or day 42 (VRPs) after prime immunization and restimulated with gp350 peptide pool on anti-IFNy-coated ELISpot plates. IFNy-producing cells were revealed with biotin- conjugated anti-IFNy antibody in combination with streptavidin-HRP and AEC substrate. Spots were counted using an ELISPOT reader. Data are shown as Mean ± SEM.

[0046] Figure 11 shows in vivo immunogenicity of EBV gp350 tetramer and BFF fusion protein encoded by recombinant MVA in outbred mice.

[0047] A, B Outbred CD1 mice were immunized intramuscularly on day 0 (prime) and day 21 with 1 x108InfU of MVA-gp350-GCN4, MVA-gp350-BFF (mBN510), or TBS buffer (control). A Blood for serum isolation was drawn on day 40 after prime immunization for analysis of anti-gp350 IgG titers by ELISA. An anti-gp350 antibody was used to establish a standard for serum antibody quantification. B Splenocytes were isolated on day 41 after prime immunization and restimulated with a gp350 peptide pool on anti-IFNy-coated ELISpot plates. IFNy-producing cells were revealed with biotin-conjugated anti-IFNy antibody in combination with streptavidin- HRP and AEC substrate. Spots were counted using an ELISpot reader. Data are shown as Mean ± SEM. Figure 12 schematically illustrates Lyme disease antigen constructs.

[0048] Construct-5: Monomeric OspA serotypes 1 , 5 and 6 C-terminal fragment string (“OspA ST 1 - 5-6”). The signal peptide of human tissue plasminogen activator (htPA SP) is fused to a string of the C-terminal fragments (CF) of OspA serotype 1 , serotype 5 and serotype 6. The OspA CFs are separated from each other by (GGGGS)4 linkers.

[0049] Construct-6: OspA serotypes 1 , 5 and 6 C-terminal fragment string-Bu Ilfrog / H pylori hybrid ferritin fusion protein (“OspA ST1 -5-6-BFF”). The OspA C-terminal fragment string described above is fused at its C-terminus to Bullfrog / / - / , pylori hybrid ferritin (BFF) via a 9 amino acid linker.

[0050] Figure 13 shows in vivo immunogenicity of OspA-ST1 -5-6 and BFF fusion protein encoded by recombinant VRPs.

[0051] Balb / c mice were immunized intramuscularly on day 0 (prime) and day 21 with 1 x108Til of VRPs encoding either VRP-OspA-ST1 -5-6 or VRP-OspA-ST1 -5-6-BFF, or buffer (control). Blood for serum isolation was drawn on day 35 after prime immunization. Anti-OspA-ST1 Ig titers in the serum were analyzed by ELISA on OspA-ST 1 -coated plates with a starting dilution of 1 :300 to determine total anti-OspA-ST1 Ig titers using anti-mouse IgG (Fc) antibody conjugated to HRP. Arbitrary titers (AU = arbitrary units) were calculated via a 4PL-fit curve with an intercept at OD = 0.3. Data are shown as Mean ± SEM.

[0052] Figure 14 describes further nanoparticle candidates for fusion to antigens.

[0053] Nanoparticle candidates HisB, PdhC, DPS, SOR, and MrsD were identified using Protein Data Base (PDB). Gaussian surface representation is shown to scale. White circles illustrate N- termini, black circles illustrate C-termini. The table gives details about the particles’ donor organism, protein characteristics and their natural function. Images were generated using PyMol using PDB biological assemblies 5XDS, 1 B5S, 1 JTS, 2CB2 and 1 P3Y.

[0054] Figure 15 schematically illustrates SARS-CoV-2 Spike RBD antigen constructs in which the antigen is fused to the N-terminus of a nanoparticle candidate.

[0055] Construct-7: SARS-CoV-2 Spike RBD-HisB fusion protein (“RBD-HisB”).

[0056] Construct-8: SARS-CoV-2 Spike RBD-PdhC fusion protein (“RBD-PdhC”).

[0057] Construct-9: SARS-CoV-2 Spike RBD-DPS fusion protein (“RBD-DPS”).

[0058] Construct-10: SARS-CoV-2 Spike RBD-SOR fusion protein (“RBD-SOR”).

[0059] Construct-11 : SARS-CoV-2 spike RBD-MrsD fusion protein (“RBD-MrsD”).

[0060] In construct-7 to construct-11 , the RBD of SARS-CoV-2 spike protein is fused to the N- terminus of one of the nanoparticle candidates shown in Figure 14 via a 9 amino acid linker and at the RBD N-terminus to htPA SP. Figure 16 shows an analysis of protein expression of SARS-CoV-2 Spike RBD- nanoparticle candidate fusion proteins.

[0061] HEK293T cells were transfected with a plasmid encoding a VEEV replicon under control of a CMV promotor encoding the VEEV TrD83 nonstructural proteins as well as one of construct- 5 to construct-9 under the VEEV subgenomic promotor. Cell lysates and supernatants were analyzed by Western blot using an anti-RBD antibody. Lysates were subjected to polyacrylamide gel electrophoresis (PAGE) under denaturing conditions (A); supernatants were subjected to poly-acrylamide gel electrophoresis (PAGE) under denaturing (B) or nondenaturing (native) conditions (C). Lanes are labelled by construct-# (7: RBD-HisB; 8: RBD- PdhC; 9: RBD-DPS; 10: RBD-SOR; 11 : RBD-MrsD).

[0062] Figure 17 schematically illustrates SARS-CoV-2 Spike RBD antigen constructs in which the antigen is fused to the C-terminus of a nanoparticle candidate.

[0063] Construct- 12 HisB-SARS-CoV-2 Spike RBD fusion protein (“HisB-RBD”).

[0064] Construct-13 DPS-SARS-CoV-2 Spike RBD fusion protein (“DPS-RBD”).

[0065] Construct- 14: SOR-SARS-CoV-2 Spike RBD fusion protein (“SOR-RBD”).

[0066] Construct- 15: MrsD-SARS-CoV-2 spike RBD fusion protein (“MrsD-RBD”).

[0067] In construct-12 to construct-15, the RBD of SARS-CoV-2 Spike protein is fused via a 9 amino acid linker to the C-terminus of a nanoparticle candidate, which in turn is fused at its N- terminus to htPA SP.

[0068] Figure 18 shows an analysis of protein expression of SARS-CoV-2 Spike RBD- nanoparticle candidate fusion proteins with the antigen fused to the C-terminus of a nanoparticle candidate.

[0069] HEK293T cells were transfected with a plasmid encoding a VEEV replicon under control of a CMV promotor, encoding the VEEV TrD83 nonstructural proteins as well as one of construct- 10 to construct-13 under the VEEV subgenomic promotor. Cell lysates and supernatants were analyzed by Western blot using an anti-RBD antibody. Lysates were subjected to polyacrylamide gel electrophoresis (PAGE) under denaturing conditions (A); supernatants were subjected to poly-acrylamide gel electrophoresis (PAGE) under denaturing (B) or nondenaturing (native) conditions (C). Lane 12: HisB-RBD; Lanes are labelled by construct-# (13: DPS-RBD; 14: SOR-RBD; 15: MrsD-RBD).

[0070] Figure 19 shows in vivo immunogenicity of SARS-CoV-2 Spike RBD antigen fused to a nanoparticle candidate encoded by VRP.

[0071] A-C Balb / c mice were immunized intramuscularly on day 0 (prime) and day 21 with 1 x108TU with VRP-RBD monomer, VRP encoding the RBD C-terminally (VRP-RBD-DPS) or N- terminally (VRP-DPS-RBD) fused to DPS, or VRP encoding the RBD N-terminally fused to HisB (VRP-HisB-RBD). A-B Blood for serum isolation was drawn on day 35 after prime immunization. A Anti-RBD Ig titers in the serum were analyzed by ELISA on RBD-coated plates with a starting dilution of 1 :300 to determine total anti-RBD Ig titers using anti-mouse IgG (H+L) antibodies. Arbitrary titers (AU = arbitrary units) were calculated via a 4P L-f it curve with an intercept at OD = 0.3. B ePass SARS-CoV-2 neutralization antibody detection kit (Genscript) was used to assess RBD-binding antibody titers and the titer at 50% inhibition compared to negative control is plotted. C Mice were sacrificed on day 35 after prime immunization and single-cell suspensions from splenocytes were prepared. 0.5 x 106cells were seeded in coated IFNy ELISpot plates and restimulated with the spike peptide QPYRVVVLSFELLHAPA or control medium. ELISpot plates were incubated overnight at 37°C before development. Data are shown as Mean ± SEM.

[0072] Figure 20 shows in vivo immunogenicity of SARS-CoV-2 Spike RBD antigen encoded by VRPs in outbred mice.

[0073] A, B Outbred CD1 mice were immunized intramuscularly on day 0 (prime) and day 21 with 1 x108TU of VRP-RBD monomer, VRP-HisB-RBD, VRP-DPS-RBD, and VRP-RBD-BFF, or buffer (control). A Blood for serum isolation was drawn on day 42 after prime immunization for analysis of anti-RBD IgG titers by ELISA. An anti-RBD antibody was used to establish a standard for serum antibody quantification. B Splenocytes were isolated on day 42 after prime immunization and restimulated with RBD peptide pool on anti-IFNy-coated ELISpot plates. IFNy-producing cells were revealed with biotin-conjugated anti-IFNy antibody in combination with streptavidin-HRP and AEC substrate. Spots were counted using an ELISpot reader. Data are shown as Mean ± SEM.

[0074] Figure 21 schematically illustrates EBV gp350 antigen constructs in which the antigen is fused to the N-terminus of a nanoparticle candidate.

[0075] Construct- 16: EBV gp350-PdhC fusion protein (“gp350-PdhC”).

[0076] Construct- 17: EBV gp 350-DPS fusion protein (“gp350-DPS”).

[0077] In construct-16 and construct-17, a fragment (aa 2-434) of EBV gp350 is fused at its C- terminus to PdhC and DPS, respectively via a 10 amino acid linker and to signal peptide IgK LC at its N-terminus.

[0078] Figure 22 shows in vivo immunogenicity of gp350 antigen fused to a nanoparticle candidate encoded by VRP.

[0079] Balb / c mice were prime-boost immunized intramuscularly with 5 x 107TU of VRP-gp350- GCN4, VRP-gp350-BFF, VRP- DPS-gp350 or VRP-PdhC-gp350. Buffer was injected as control. Serum was analyzed three weeks after boost immunization (day 40) for anti-gp350 IgG titers by ELISA. For this, ELISA plates were coated with gp350 protein and serum was added in different dilutions. Anti-gp350 antibody (clone 2L10; Merck) was used to establish a standard for serum antibody quantification by standard curve calculation in GraphPad Prism. Data are shown as Mean ± SEM.

[0080] Figure 23 schematically illustrates a construct expressing a multi-antigen nanoparticle containing SARS-CoV-2 Spike RBD and EBV gp350 each fused to BFF.

[0081] Construct- 18: EBV gp350-BFF fusion protein and SARS-CoV-2 Spike RBD-BFF fusion protein separated by a T2A site (“RBD-BFF-gp350-BFF”). EBV gp350 is fused to BFF via a 10 amino acid linker and to IgK LC SP, the sequence for the fusion protein is followed by a T2A site and the RBD of SARS-CoV-2 Spike protein is fused via a 9 amino acid linker to BFF and to the htPA SP.

[0082] Figure 24 shows an analysis of protein expression of a multi-antigen nanoparticle containing SARS-CoV-2 Spike RBD and EBV gp350 each fused to BFF.

[0083] HEK293T cells were transfected with a plasmid encoding a VEEV replicon under control of a CMV promotor, encoding the VEEV TrD83 nonstructural proteins as well as either construct- 18, construct-2 or construct-4 under the VEEV subgenomic promotor. Cell supernatants were subjected to poly-acrylamide gel electrophoresis (PAGE) under non-denaturing (native) conditions and analyzed by Western blot using an anti-RBD antibody, an anti-gp350 antibody or an anti-ferritin antibody. Lanes are labelled by construct-# (18: RBD-BFF-gp350-BFF; 2: RBD-BFF, 4: gp350-BFF).

[0084] Figure 25 shows in vivo immunogenicity of a multi-antigen nanoparticle containing SARS- CoV-2 Spike RBD and EBV gp350 each fused to BFF.

[0085] A-D Balb / c mice were immunized intramuscularly with 1 x108Til on day 0 (prime) and day 21 with VRP-RBD-BFF, VRP-gp350-BFF or VRP-RBD-BFF-gp350-BFF, or buffer (control). A, B Blood for serum isolation was drawn at day 35 after prime immunization. Serum was analyzed for anti-gp350 and anti-RBD IgG titers by ELISA. An anti-RBD antibody (A) or anti-gp350 antibody (B) was used to establish a standard for serum antibody quantification. C, D Mice were sacrificed on day 35 after prime immunization and single-cell suspensions from splenocytes were prepared. 0.5 x 106cells were seeded in coated IFN-y ELISpot plates and restimulated with Spike Peptide Pool A (C) or gp350 pool (D). ELISpot plates were incubated overnight at 37°C before development. Data are shown as Mean ± SEM. Figure 26 schematically illustrates Lyme disease antigen constructs, including a construct for the display of two distinct Lyme disease antigens on BFF encoded by recombinant VRPs.

[0086] Construct-19: OspA serotypes 2, 4 and 3 C-terminal fragment String-Bullfrog / / - / , pylori hybrid ferritin fusion protein (“OspA ST2-4-3-BFF”). The signal peptide of human tissue plasminogen activator (htPA SP) is fused to a string of the C-terminal fragments (CF) of OspA serotype 2, serotype 4 and serotype 3. The OspA CFs are separated from each other by (GGGGS)4 linkers, and the string is fused at its C-terminus to Bullfrog / / - / , pylori hybrid ferritin (BFF) via a 9 amino acid SSGGASVLA linker.

[0087] Construct-20: OspA ST2-4-3-BFF fusion protein and OspA ST2-4-3-BFF fusion protein separated by a T2A site.

[0088] Figure 27 shows in vivo immunogenicity of OspA-ST5-6-1 and OspA-ST5-6-1 fused to BFF encoded by VRPs.

[0089] A-B Balb / c mice were immunized intramuscularly on day 0 (prime) and day 21 with 1 x108TU VRP-OspA-ST1 -5-6-BFF plus 1x108TU VRP-OspA-ST2-4-3-BFF or 1 x108TU VRP-OspA- ST1 -5-6-BFF-ST2-4-3-BFF. Blood for serum isolation was drawn on day 35 after prime immunization. A Anti-OspA-ST 1 Ig titers in the serum were analyzed by ELISA on OspA-ST 1 - coated plates with a starting dilution of 1 :300 to determine total anti-OspA-ST1 Ig titers using anti-mouse IgG (Fc) antibody conjugated to HRP. B Anti-OspA-ST2 Ig titers in the serum were analyzed by ELISA on OspA-ST2-coated plates with a starting dilution of 1 :300 to determine total anti-OspA-ST2 Ig titers using anti-mouse IgG (Fc) antibody conjugated to HRP. Arbitrary titers (AU = arbitrary units) were calculated via a 4PL-fit curve with an intercept at OD = 0.3. Data are shown as Mean ± SEM.

[0090] Figure 28 illustrates the design of recombinant MVAs.

[0091] Schematic representation of the transgenes contained in recombinant MVA-resO65 and MVA- res067 coding for a secreted RBD from the SARS-CoV-2 S protein (isolate Wuhan-Hu-1 ) and the same RBD fused to the nanoparticle forming BFF protein, respectively. Also shown are recombinant MVA mBN443B containing the indicated transgenes derived from Epstein-Barr virus (EBV). The gp350 in mBN443 is fused to a GCN4 domain leading to tetramerization of the expressed gp350 domain. MVA-mBN510 contains the same transgenes as mBN443B arranged in a different design. In addition, in MVA-mBN510, amino acids 2-434 of EBV gp350 are fused to the nanoparticle-forming BFF protein instead of the GCN4 tetramerization domain. Brief Description of Sequences

[0092] SEQ ID NO: 1 is a nucleic acid sequence encoding SARS-CoV-2 spike RBD.

[0093] SEQ ID NO: 2 is the amino acid sequence of SARS-CoV-2 spike RBD.

[0094] SEQ ID NO: 3 is a nucleic acid sequence encoding EBV gp350.

[0095] SEQ ID NO: 4 is the amino acid sequence of EBV gp350.

[0096] SEQ ID NO: 5 is a nucleic acid sequence of Bullfrog / / - / . pylori hybrid ferritin.

[0097] SEQ ID NO: 6 is the amino acid sequence of Bullfrog / / - / , pylori hybrid ferritin.

[0098] SEQ ID NO: 7 is a nucleic acid sequence encoding HisB.

[0099] SEQ ID NO: 8 is the amino acid sequence of HisB.

[0100] SEQ ID NO: 9 is a nucleic acid sequence encoding PdhC.

[0101] SEQ ID NO: 10 is the amino acid sequence of PdhC.

[0102] SEQ ID NO: 11 is a nucleic acid sequence encoding DPS.

[0103] SEQ ID NO: 12 is the amino acid sequence of DPS.

[0104] SEQ ID NO: 13 is a nucleic acid sequence encoding SOR.

[0105] SEQ ID NO: 14 is the amino acid sequence of SOR.

[0106] SEQ ID NO: 15 is a nucleic acid sequence encoding MrsD.

[0107] SEQ ID NO: 16 is the amino acid sequence of MrsD.

[0108] SEQ ID NO: 17 is the nucleic acid sequence encoding SARS-CoV-2 spike RBD with htPA signal peptide (“construct-1 ”) in MVA.

[0109] SEQ ID NO: 18 is the nucleic acid sequence encoding SARS-CoV-2 spike RBD with htPA signal peptide (“construct-1 ”) in VRPs.

[0110] SEQ ID NO: 19 is the nucleic acid sequence encoding SARS-CoV-2 spike RBD with htPA signal peptide (“construct-1 ”) in mRNA, saRNA, and plasmid DNA.

[0111] SEQ ID NO: 20 is the amino acid sequence of SARS-CoV-2 spike RBD with htPA signal peptide (“construct-1 ”).

[0112] SEQ ID NO: 21 is the nucleic acid sequence encoding SARS-CoV-2 spike RBD- Bullfrog / / - / . pylori hybrid ferritin fusion protein with htPA signal peptide (“construct-2”) in MVA.

[0113] SEQ ID NO: 22 is the nucleic acid sequence encoding SARS-CoV-2 spike RBD- Bullfrog / / - / . pylori hybrid ferritin fusion protein with htPA signal peptide (“construct-2”) in VRPs.

[0114] SEQ ID NO: 23 is the nucleic acid sequence encoding SARS-CoV-2 spike RBD- Bullfrog / / - / . pylori hybrid ferritin fusion protein with htPA signal peptide (“construct-2”) in mRNA, saRNA, and plasmid DNA. SEQ ID NO: 24 is the amino acid sequence of SARS-CoV-2 spike RBD-Bullfrog / / - / . pylori hybrid ferritin fusion protein with htPA signal peptide (“construct-2”).

[0115] SEQ ID NO: 25 is the nucleic acid sequence encoding EBV gp350-GCN4 fusion protein (“construct-3A”) in MVA.

[0116] SEQ ID NO: 26 is the nucleic acid sequence encoding EBV gp350-GCN4 fusion protein with IgK LC signal peptide (“construct-3B”) in VRPs.

[0117] SEQ ID NO: 27 is the amino acid amino acid sequence of EBV gp350-GCN4 fusion protein (“construct-3A, 3B”).

[0118] SEQ ID NO: 28 is the nucleic acid sequence encoding EBV gp350- Bu Ilf rog / H. pylori hybrid ferritin fusion protein with IgK LC signal peptide (“construct-4”) in MVA.

[0119] SEQ ID NO: 29 is the nucleic acid sequence encoding EBV gp350- Bu Ilf rog / H. pylori hybrid ferritin fusion protein with IgK LC signal peptide (“construct-4”) in VRPs.

[0120] SEQ ID NO: 30 is the amino acid sequence of.EBV gp350-Bullfrog / / - / . pylori hybrid ferritin fusion protein with IgK LC signal peptide (“construct-4”).

[0121] SEQ ID NO: 31 is the nucleic acid sequence encoding an OspA serotypes 1 , 5 and 6 C-terminal fragment string (OspA ST1-5-6) with htPA signal peptide (“construct-5”) in VRPs.

[0122] SEQ ID NO: 32 is the amino acid sequence of OspA serotypes 1 , 5 and 6 C- terminal fragment string (OspA ST1-5-6) with htPA signal peptide (“construct-5”) in VRPs.

[0123] SEQ ID NO: 33 is the nucleic acid sequence encoding an OspA serotypes 1 , 5 and 6 C-terminal fragment string (OspA ST1 -5-6)-Bullfrog / / - / . pylori hybrid ferritin fusion protein with htPA signal peptide (“construct- 6”) in VRPs.

[0124] SEQ ID NO: 34 is the amino acid sequence of an OspA serotypes 1 , 5 and 6 C- terminal fragment string (OspA ST1 -5-6)-Bullfrog / / - / . pylori hybrid ferritin fusion protein with htPA signal peptide (“construct-6”) in VRPs.

[0125] SEQ ID NO: 35 is the nucleic acid sequence encoding SARS-CoV-2 spike RBD- HisB fusion protein with htPA signal peptide (“construct-7”) in VRPs.

[0126] SEQ ID NO: 36 is the amino acid sequence of SARS-CoV-2 spike RBD-HisB fusion protein with htPA signal peptide (“construct-7”). SEQ ID NO: 37 is the nucleic acid sequence encoding SARS-CoV-2 spike RBD- PdhC fusion protein with htPA signal peptide (“construct-8”) in VRPs.

[0127] SEQ ID NO: 38 is the amino acid sequence of SARS-CoV-2 spike RBD-PdhC fusion protein with htPA signal peptide (“construct-8”).

[0128] SEQ ID NO: 39 is the nucleic acid sequence encoding SARS-CoV-2 spike RBD- DPS fusion protein with htPA signal peptide (“construct-9”) in VRPs.

[0129] SEQ ID NO: 40 is the amino acid sequence of SARS-CoV-2 spike RBD-DPS fusion protein with htPA signal peptide (“construct-9”).

[0130] SEQ ID NO: 41 is the nucleic acid sequence encoding SARS-CoV-2 spike RBD- SOR fusion protein with htPA signal peptide (“construct-10”) in VRPs.

[0131] SEQ ID NO: 42 is the amino acid sequence of SARS-CoV-2 spike RBD-SOR fusion protein with htPA signal peptide (“construct-10”).

[0132] SEQ ID NO: 43 is the nucleic acid sequence encoding SARS-CoV-2 spike RBD- MrsD fusion protein with htPA signal peptide (“construct-11”) in VRPs.

[0133] SEQ ID NO: 44 is the amino acid sequence of SARS-CoV-2 spike RBD-MrsD fusion protein with htPA signal peptide (“construct-11 ”).

[0134] SEQ ID NO: 45 is the nucleic acid sequence encoding HisB-SARS-CoV-2 spike RBD fusion protein with htPA signal peptide (“construct- 12”) in VRPs.

[0135] SEQ ID NO: 46 is the amino acid sequence of HisB-SARS-CoV-2 spike RBD fusion protein with htPA signal peptide (“construct-12”).

[0136] SEQ ID NO: 47 is the nucleic acid sequence encoding DPS-SARS-CoV-2 spike RBD fusion protein with htPA signal peptide (“construct- 13”) in VRPs.

[0137] SEQ ID NO: 48 is the amino acid sequence of DPS-SARS-CoV-2 spike RBD fusion protein with htPA signal peptide (“construct-13”).

[0138] SEQ ID NO: 49 is the nucleic acid sequence encoding SOR-SARS-CoV-2 spike RBD fusion protein with htPA signal peptide (“construct- 14”) in VRPs.

[0139] SEQ ID NO: 50 is the amino acid sequence of SOR-SARS-CoV-2 spike RBD fusion protein with htPA signal peptide (“construct-14”). SEQ ID NO: 51 is the nucleic acid sequence encoding MrsD-SARS-CoV-2 spike RBD fusion protein with htPA signal peptide (“construct- 15”) in VRPs.

[0140] SEQ ID NO: 52 is the amino acid sequence of MrsD-SARS-CoV-2 spike RBD fusion protein with htPA signal peptide (“construct-15”).

[0141] SEQ ID NO: 53 is the nucleic acid sequence encoding EBVgp350-PdhC fusion protein with IgK LC signal peptide (“construct-16”) in VRPs.

[0142] SEQ ID NO: 54 is the amino acid sequence of EBVgp350-PdhC fusion protein with IgK LC signal peptide (“construct-16”) in VRPs.

[0143] SEQ ID NO: 55 is the nucleic acid sequence encoding EBVgp350-DPS fusion protein with IgK LC signal peptide (“construct-17”) in VRPs

[0144] SEQ ID NO: 56 is the amino acid sequence of EBVgp350-DPS fusion protein with IgK LC signal peptide (“construct-17”) in VRPs.

[0145] SEQ ID NO: 57 is the nucleic acid sequence encoding EBV gp350- Bu Ilf rog / H. pylori hybrid ferritin fusion protein with IgK LC signal peptide and SARS- CoV-2 spike RBD-Bullfrog / / - / . pylori hybrid ferritin fusion protein with htPA signal peptide (“construct-18”) in VRPs.

[0146] SEQ ID NO: 58 is the amino acid sequence of EBV gp350-Bullfrog / / - / . pylori hybrid ferritin fusion protein with IgK LC signal peptide and SARS-CoV-2 spike RBD-Bullfrog / / - / . pylori hybrid ferritin fusion protein with htPA signal peptide separated by a T2A site (“construct-18”).

[0147] SEQ ID NO: 59 is the nucleic acid sequence encoding an OspA serotypes 2, 4 and 3 C-terminal fragment string (OspA ST2-4-3) -Bullfrog / / - / , pylori hybrid ferritin fusion protein with htPA signal peptide (“construct- 19”) in VRPs.

[0148] SEQ ID NO: 60 is the amino acid sequence of an OspA serotypes 2, 4 and 3 C- terminal fragment string (OspA ST2-4-3) -Bullfrog / / - / , pylori hybrid ferritin fusion protein with htPA signal peptide (“construct-19”) in VRPs.

[0149] SEQ ID NO: 61 is the nucleic acid sequence encoding an OspA serotypes 1 , 5 and 6 C-terminal fragment string (OspA ST1 -5-6) -Bullfrog / / - / , pylori hybrid ferritin fusion protein with htPA signal peptide and an OspA serotypes 2, 4 and 3 C-terminal fragment string (OspA ST2-4-3)- Bullfrog / / - / . pylori hybrid ferritin fusion protein with htPA signal peptide separated by a T2A site (“construct-20”) in VRPs.

[0150] SEQ ID NO: 62 is the amino acid sequence of an OspA serotypes 1 , 5 and 6 C- terminal fragment string (OspA ST1-5-6) -Bullfrog / / - / , pylori hybrid ferritin fusion protein with htPA signal peptide and an OspA serotypes 2, 4 and 3 C-terminal fragment string (OspA ST2-4-3) - Bullfrog / / - / , pylori hybrid ferritin fusion protein with htPA signal peptide separated by a T2A site (“construct-20”) in VRPs (T2A site indicated in bold).

[0151] SEQ ID NO: 63 is a nucleic acid sequence encoding SARS-CoV-2 spike RBD- Bullfrog / / - / . pylori hybrid ferritin fusion protein.

[0152] SEQ ID NO: 64 is the amino acid sequence of SARS-CoV-2 spike RBD- Bullfrog / / - / . pylori hybrid ferritin fusion protein.

[0153] SEQ ID NO: 65 is a nucleic acid sequence encoding EBV gp350-Bullfrog / / - / . pylori hybrid ferritin fusion protein.

[0154] SEQ ID NO: 66 is the amino acid sequence of EBV gp350- Bullfrog / / - / , pylori hybrid ferritin fusion protein.

[0155] SEQ ID NO: 67 is the nucleic acid sequence encoding an OspA serotypes 1 , 5 and 6 C-terminal fragment string (OspA ST1 -5-6)-Bullfrog / / - / . pylori hybrid ferritin fusion protein.

[0156] SEQ ID NO: 68 is the amino acid sequence of an OspA serotypes 1 , 5 and 6 C- terminal fragment string (OspA ST1 -5-6)-Bullfrog / / - / . pylori hybrid ferritin fusion protein.

[0157] SEQ ID NO: 69 is the nucleic acid sequence encoding SARS-CoV-2 Spike RBD- PdhC fusion protein.

[0158] SEQ ID NO: 70 is the amino acid sequence of SARS-CoV-2 Spike RBD-PdhC fusion protein.

[0159] SEQ ID NO: 71 is the nucleic acid sequence encoding SARS-CoV-2 Spike RBD- DPS fusion protein.

[0160] SEQ ID NO: 72 is the amino acid sequence of SARS-CoV-2 Spike RBD-DPS fusion protein.

[0161] SEQ ID NO: 73 is the nucleic acid sequence encoding HisB-SARS-CoV-2 Spike RBD fusion protein.

[0162] SEQ ID NO: 74 is the amino acid sequence of HisB-SARS-CoV-2 Spike RBD fusion protein.

[0163] SEQ ID NO: 75 is the nucleic acid sequence encoding DPS-SARS-CoV-2 Spike RBD fusion protein.

[0164] SEQ ID NO: 76 is the amino acid sequence of DPS-SARS-CoV-2 Spike RBD fusion protein.

[0165] SEQ ID NO: 77 is the nucleic acid sequence encoding EBVgp350-PdhC fusion protein. SEQ ID NO: 78 is the amino acid sequence of EBVgp350-PdhC fusion protein.

[0166] SEQ ID NO: 79 is the nucleic acid sequence encoding EBV gp350-DPS fusion protein.

[0167] SEQ ID NO: 80 is the amino acid sequence of EBVgp350-DPS fusion protein.

[0168] SEQ ID NO: 81 is the nucleic acid encoding an OspA serotypes 2, 4 and 3 C- terminal fragment string (OspA ST2-4-3)-Bullfrog / / - / . pylori hybrid ferritin fusion protein.

[0169] SEQ ID NO: 82 is the amino acid sequence of an OspA serotypes 2, 4 and 3 C- terminal fragment string (OspA ST2-4-3) -Bullfrog / / - / , pylori hybrid ferritin fusion protein.

[0170] SEQ ID NO: 83 is a nucleic acid sequence encoding htPA signal peptide.

[0171] SEQ ID NO: 84 is the amino acid sequence of htPA signal peptide.

[0172] SEQ ID NO: 85 is a nucleic acid sequence encoding IgK LC signal peptide.

[0173] SEQ ID NO: 86 is the amino acid sequence of IgK LC signal peptide.

[0174] SEQ ID NO: 87 is a nucleic acid sequence encoding 9aa linker sequence.

[0175] SEQ ID NO: 88 is the amino acid sequence of 9aa linker sequence.

[0176] SEQ ID NO: 89 is a nucleic acid sequence encoding 10aa linker sequence.

[0177] SEQ ID NO: 90 is the amino acid sequence of 10aa linker sequence.

[0178] SEQ ID NO: 91 is nucleic acid sequence encoding 13aa linker sequence.

[0179] SEQ ID NO: 92 is the amino acid sequence of 13aa linker sequence.

[0180] SEQ ID NO: 93 is a nucleic acid sequence encoding (GGGGS)4 linker sequence.

[0181] SEQ ID NO: 94 is the amino acid sequence of 1 (GGGGS)4 linker sequence.

[0182] SEQ ID NO: 95 is a nucleic acid sequence encoding T2A site.

[0183] SEQ ID NO: 96 is the amino acid sequence of T2A site.

[0184] SEQ ID NO: 97 Nucleic acid sequence of Pr13.5 promoter.

[0185] SEQ ID NO: 98 Nucleic acid sequence of GCN4 multimerization domain.

[0186] SEQ ID NO: 99 Amino acid sequence of GCN4 multimerization domain.

[0187] SEQ ID NO: 100 is a nucleic acid sequence encoding a fusion protein of OspA C- terminal fragments ST1-5-6.

[0188] SEQ ID NO: 101 is the amino acid sequence of a fusion protein of OspA C-terminal fragments ST1-5-6.

[0189] SEQ ID NO: 102 is a nucleic acid sequence encoding a fusion protein of OspA C- terminal fragments ST2-4-3.

[0190] SEQ ID NO: 103 is the amino acid sequence of a fusion protein of OspA C-terminal fragments ST2-4-3. Detailed Description of Invention

[0191] The objective underlying the invention was to provide improved vaccines, particularly inducing improved antibody responses. Strong antibody responses were known to be elicited by protein antigens multimerized in a regular array, for example displayed on protein nanoparticles [4, 5]. One example of a self-assembling nanoparticle used for antigen multimerization is the Bullfrog / H pylori hybrid ferritin (BFF), a hybrid protein containing a portion of Bullfrog ferritin and a portion of ferritin from H. pylori, as described by Kanekiyo et al. 2015 [6]; WO 2015 / 054639.

[0192] Using three different model antigens, i.e., the receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein, a fragment of the N-terminal domain of Epstein-Barr virus (EBV) surface glycoprotein 350 (gp350), and a fusion protein of outer surface protein A (OspA) fragments from three different Borrelia strains, here we report enhanced antibody responses in mice observable when the antigen expressed by a viral vector such as Modified Vaccina Virus Ankara (MVA) or Venezuelan Equine Encephalitis Virus (VEEV) derived virus replicon particles (VRPs) was conjugated to BFF and delivered in the form of an antigen-nanoparticle fusion protein. Likewise, the robust T cell responses inherent to these viral vector vaccines were maintained. Importantly, the increased antibody responses as well as the T cell responses induced by MVA or VRPs were obtained without the need for an adjuvant.

[0193] Thus, viral vectors delivering the genes of nanoparticle conjugated antigens combine two highly desirable properties of a vaccine, namely potent induction of humoral and cellular immunity.

[0194] Additionally, we also showed that similar immune responses were achieved when SARS-CoV-2 spike RBD presenting BFF was encoded by RNA or DNA in the absence of a viral vector.

[0195] Furthermore, antibody responses against both SARS-CoV-2 RBD and EBV gp350 could be elicited when the antigens were displayed on the same BFF nanoparticle. Similarly, antibody responses against two different OspA antigens displayed on the same BFF nanoparticle were observed.

[0196] However, the antigen-nanoparticle conjugate approach is not universal to all protein nanoparticles. Here we show that nanoparticles formed of imidazoleglycerol-phosphate dehydratase (HisB), acetyltransferase of pyruvate dehydrogenase complex (PdhC) or DNA binding protein from starved cells (DPS) proved to be useful alternatives to BFF. Both enhanced antibody responses and maintained T cell responses were observed in mice when the SARS-CoV-2 spike RBD antigen was displayed on HisB or DPS nanoparticles, and enhanced antibody responses were also observed when the EBV gp350 antigen was displayed on PdhC or DPS nanoparticles. In contrast, antigen-nanoparticle fusion proteins with sulphur oxidase / reductase (SOR) or mersacidin decarboxylase (MrsD), also known to form self-assembling nanoparticles, failed to be secreted successfully in cell culture.

[0197] BFF and PdhC can be fused to an antigen only via the antigen’s C-terminus. HisB and DPS, however, have both N- and C-termini exposed, and antigens can thus be fused via their C- or N-termini to the nanoparticle core protein. Here we show that conjugation of DPS to SARS- CoV-2 Spike RBD enhanced antibody responses in mice while maintaining T cell responses irrespective of the orientation in which nanoparticle and antigen were fused together. In contrast, HisB fused to SARS-CoV-2 Spike RBD was shown in cell culture to be successfully secreted and assembled only with the antigen’s N-terminus being joined to the nanoparticle’s C-terminus. As some antigens are naturally anchored at their N-terminus, nanoparticles like HisB and DPS thus provide the advantage that nanoparticulate antigen presentation can be realized also with such antigens maintaining the orientation of the protein on the surface of either bacterial or eucaryotic cells, viruses or, here, nanoparticles.

[0198] Definitions

[0199] It must be noted that, as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a nucleic acid sequence” includes one or more nucleic acid sequences.

[0200] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or”, a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0201] Throughout this specification and the appended claims, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated feature but not the exclusion of any other feature. When used in the context of an aspect or embodiment in the description of the present invention the term “comprising” can be amended and thus replaced with the term “containing” or “including” or when used herein with the term “having.” Similarly, any of the afore-mentioned terms (comprising, containing, including, having), whenever used in the context of an aspect or embodiment in the description of the present invention include, by virtue, the terms “consisting of” or “consisting essentially of,” which each denotes specific legal meaning depending on jurisdiction.

[0202] When used herein “consisting of” excludes any feature, element, step, or ingredient not specified in the claim. When used herein, “consisting essentially of” does not exclude features, materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0203] An “aspect” refers to a conception of the invention in its broadest sense; it may map to an independent claim. An “embodiment” is a specific version or implementation or a concrete example of the invention; it may map to a dependent claim.

[0204] The term “recombinant” as used herein refers to nucleic acids not occurring naturally but being the result of genetic engineering. For example, “recombinant MVA” refers to an MVA comprising a nucleic acid sequence inserted in its genome, which is not naturally present in the wildtype virus ( / .e., is foreign or heterologous to MVA). A recombinant MVA thus refers to MVA made by an artificial combination of two or more segments of nucleic acid sequence of synthetic or semisynthetic origin which does not occur in nature or is linked to another nucleic acid in an arrangement not found in nature. A recombinant MVA is a genetically engineered or a genetically modified MVA. The term “recombinant MVA” as used herein includes MVA which has integrated at least one recombinant nucleic acid, preferably in the form of a transcriptional unit, in its genome. Recombinant MVA may express heterologous peptides, polypeptides, or proteins (antigens) upon induction of the regulatory elements, e.g., the promoter.

[0205] The term “heterologous nucleic acid” as used herein in the context of viral vectors means that the nucleic acid is foreign to, i.e., not naturally present in the viral vector.

[0206] The term “construct” as used herein refers to an artificial nucleic acid, peptide or protein being the result of genetic engineering. A nucleic acid construct may be comprised by a viral vector, an RNA or a plasmid DNA as described herein.

[0207] The term “virus replicon particle” or “VRP” refers to genetically engineered infectious virions incapable of generating progeny virus due to partial or complete deletion of at least one structural gene. VRPs fulfil the criteria of a safe vaccine and gene delivery system. The term “replicon” as used herein refers to a nucleic acid comprising the 5’ UTR of an alphavirus, such as Venezuelan Equine Encephalitis Virus (VEEV), a gene encoding the non- structural proteins (nsp) 1 , 2, 3 and 4 of an alphavirus, the alphavirus subgenomic promotor and the subgenomic 5’ UTR, the gene(s) for the expression of heterologous peptides, polypeptides, or proteins (antigens), the alphavirus 3’UTR, and a poly(A) sequence.

[0208] The term “fusion protein” refers to a recombinant protein comprising at least two separate stretches of amino acids, or proteins, e.g., protein domains, that have been joined artificially so that they are transcribed and translated as a single protein. Mostly, the two separate stretches of amino acids are joined using a linker sequence, but they could also be joined directly to each other. As used herein, the term “fusion protein” generally describes a protein resulting from the fusion of an antigen and a protein nanoparticle subunit.

[0209] The term “signal peptide” as used herein refers to a peptide added N-terminally to a protein newly synthesized in a cell that allows the protein’s translocation to the cellular membrane with subsequent secretion.

[0210] The term “self-assembling protein nanoparticle”, sometimes briefly “self-assembling nanoparticle”, “protein nanoparticle” or “nanoparticle”, as used herein refers to a polymeric assembly of monomeric polypeptides referred to as “subunits” that are capable of directing their self-assembly into the nanoparticle.

[0211] The term “multi-antigen protein nanoparticle” as used herein refers to a self-assembling protein nanoparticle, assembled from two or more distinct fusion proteins, displaying two or more different antigens but sharing the same nanoparticle forming subunit, resulting in a nanoparticle displaying multiple antigens on its surface, in contrast to a homotypic selfassembling protein nanoparticle displaying one single type of antigen.

[0212] A “2A site”, a“T2A site” or a “P2A site” is a peptide sequence that co-translationally leads to a stop-and-restart of translation, leaving away one peptide bond allowing for expression of separate proteins in one open reading frame (ORF).

[0213] The term “adjuvant” as used herein refers to a compound capable of enhancing an immune response to an antigen. For example, a pharmaceutical composition containing a vaccine antigen and additionally an adjuvant would elicit an immune response in a subject that is enhanced as compared to pharmaceutical composition containing the vaccine antigen but not the adjuvant. Abbreviations

[0214] AU arbitrary units

[0215] BFF Bullfrog / / - / , pylori hybrid ferritin

[0216] CEF chicken embryo fibroblast

[0217] CMV cytomegalovirus

[0218] EBV Epstein-Barr virus

[0219] GMFI geometric mean fluorescence intensity

[0220] HEK293T human embryo kidney cell line containing the sequence of the large T antigen of Simian Virus 40 SV40 htPA SP signal peptide of human tissue plasminogen activator

[0221] IgK LC SP signal peptide of murine immunoglobulin kappa light chain

[0222] MOI multiplicity of infection mRNA messenger RNA

[0223] MVA Modified Vaccinia Virus Ankara

[0224] MVA-BN MVA-BN® of Bavarian Nordic

[0225] P2A peptide of the 2A protein of porcine teschovirus-1

[0226] T2A peptide of the 2A protein of thosea asigna virus

[0227] RBD receptor binding domain of SARS-CoV-2 S protein S1 domain saRNA self-amplifying RNA

[0228] SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2

[0229] S protein spike protein of SARS-CoV-2

[0230] SFV Shope fibroma virus

[0231] SP signal peptide

[0232] VACV vaccinia virus

[0233] VRP virus replicon particle or virus-derived replicon particle

[0234] VEEV Venezuelan Equine Encephalitis Virus

[0235] Embodiments and further aspects

[0236] Aspects and embodiments relating to a recombinant virus-based vector

[0237] In one aspect, provided is a recombinant virus-based vector comprising a heterologous nucleic acid operably linked to a promoter, wherein the heterologous nucleic acid encodes a fusion protein comprising a disease-associated protein, or a part thereof, joined to a subunit of a selfassembling protein nanoparticle. In one embodiment, the recombinant virus-based vector is derived from a DNA virus or an RNA virus.

[0238] In one embodiment, the recombinant virus-based vector is a recombinant DNA virus, preferably is a recombinant poxvirus.

[0239] In one embodiment, the recombinant virus-based vector is a recombinant RNA virus, preferably is a recombinant virus replicon particle (VRP).

[0240] In one embodiment, the recombinant virus-based vector further comprises another heterologous nucleic acid operably linked to a promoter, wherein the other heterologous nucleic acid encodes a fusion protein comprising another disease-associated protein, or a part thereof, joined to subunit of a self-assembling protein nanoparticle, preferably wherein the disease-associated protein and the other disease-associated protein are different. More preferably, the subunit of a self-assembling protein nanoparticle, which the disease-associated protein is joined to and the subunit of a self-assembling protein nanoparticle, which the other disease-associated protein is joined to are of the same type of self-assembling protein nanoparticle.

[0241] In another aspect, provided is a recombinant virus-based vector comprising:

[0242] (a) a first heterologous nucleic acid operably linked to a promoter, wherein the first heterologous nucleic acid encodes a first fusion protein comprising a first disease-associated protein, or a part thereof, joined to a subunit of a selfassembling protein nanoparticle; and

[0243] (b) a second heterologous nucleic acid operably linked to a promoter, wherein the second heterologous nucleic acid encodes a second fusion protein comprising a second disease-associated protein, or apart thereof, joined to a subunit of a self-assembling protein nanoparticle. preferably wherein the first and second disease-associated proteins are different, more preferably wherein the subunit of a self-assembling protein nanoparticle, which the first disease-associate protein is joined to and the subunit of a self-assembling protein nanoparticle, which the second disease-associate protein is joined to, are of the same type of self-assembling protein nanoparticle.

[0244] In one embodiment of the recombinant virus-based vector, the first heterologous nucleic acid and the second heterologous nucleic acid are joined by a heterologous nucleic acid encoding a 2A peptide, preferably a T2A peptide as depicted in SEQ ID NO: 96. Alternatively, the first heterologous nucleic acid and the second heterologous nucleic acid may be joined by an internal ribosomal entry site (IRES). Aspects and embodiments relating to a recombinant poxyirus

[0245] In one aspect, provided is a recombinant poxvirus comprising a heterologous nucleic acid operably linked to a promoter, wherein the heterologous nucleic acid encodes a fusion protein comprising a disease-associated protein, or a part thereof, joined to a subunit of a selfassembling protein nanoparticle.

[0246] In one embodiment, the poxvirus is an Orthopoxvirus, preferably is a vaccinia virus, more preferably is Modified Vaccinia Virus Ankara (MVA), most preferably is MVA-BN®.

[0247] In one embodiment, the recombinant poxvirus is derived from a member of the Avipoxvirus, Orthopoxvirus or Parapoxvirus genus.

[0248] In one embodiment of the recombinant poxvirus, the member of the Avipoxvirus genus is selected from the group consisting of canarypox virus, fowlpox virus, mynahpox virus, pigeonpox virus, and quailpox virus.

[0249] In one embodiment, the member of the Parapoxvirus genus is selected from the group consisting of bovine papular stomatitis virus, ORF virus, parapoxvirus of New Zealand red deer, and pseudocowpox virus.

[0250] In one embodiment, the member of the Orthopoxvirus genus is selected from the group consisting of buffalopox virus, camelpox virus, cowpox virus, ectromelia virus, monkeypox virus, raccoonpox virus, smallpox virus (variola virus), or vaccinia virus (VACV).

[0251] In one embodiment, the recombinant poxvirus is a recombinant vaccinia virus.

[0252] In one embodiment, the recombinant vaccinia virus is derived from a wild-type vaccinia virus or an attenuated vaccinia virus strain, preferably selected from the group consisting of vaccinia virus-Western Reserve, vaccinia virus-Copenhagen, Dryvax (vaccinia virus-Wyeth), ACAM2000, chorioallantois vaccinia virus Ankara (CVA), or Modified Vaccinia Virus Ankara (MVA).

[0253] In one embodiment, the recombinant poxvirus is derived from an attenuated poxvirus vector selected from the group consisting of ALVAC (a canarypox virus-based vector), NYVAC (a vaccinia virus vector) and MVA.

[0254] In one embodiment, the recombinant poxvirus is recombinant MVA.

[0255] In one embodiment, the recombinant MVA is derived from wild-type MVA or an MVA derivative having the capability of reproductive replication in vitro in chicken embryo fibroblast (CEF) cells, but no capability of reproductive replication in the human keratinocyte cell line HaCaT, the human bone osteosarcoma cell line 143B, the human embryo kidney cell line 293, and the human cervix adenocarcinoma cell line HeLa.

[0256] In one embodiment, the recombinant MVA is derived from MVA-BN® as deposited at the European Collection of Animal Cell cultures (ECACC) under accession number V00083008 on 30 August 2000.

[0257] In one embodiment of the recombinant poxvirus, the promoter is a poxviral promoter.

[0258] In one embodiment, the poxviral promoter is selected from the group consisting of promoters Pr11 , Pr7.5, PrSSL, PrATI, and PrS; Pr13.5long / Pr13.5, PrHyb, Pr1328, PrH2R and PrH5m, preferably is Pr13.5long.

[0259] In one embodiment, the poxviral promoter comprises or consists of a nucleic acid as depicted in SEQ ID NO: 97.

[0260] In one embodiment, the recombinant poxvirus is recombinant MVA, and the heterologous nucleic acid is inserted into the MVA genome at an intergenic region (IGR) selected from the group consisting of IGR 44 / 45, 51 / 52, 64 / 65, 88 / 89, and 148 / 149, preferably at IGR 64 / 65.

[0261] In one embodiment, the recombinant poxvirus is a recombinant MVA comprising a heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes a disease-associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle, wherein the poxviral promoter is Pr13.5long and the heterologous nucleic acid is inserted into the MVA genome at IGR 64 / 65. Preferably, the disease-associated antigen is from SARS-CoV-2, preferably SARS-CoV-2 S RBD, or from EBV, preferably EBV gp350. Preferably, the self-assembling protein nanoparticle is BFF.

[0262] In one embodiment, the recombinant poxvirus is a recombinant MVA comprising a heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes an EBV gp350 antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle. Preferably, the poxviral promoter is Pr13.5long. Preferably, the heterologous nucleic acid is inserted into the MVA genome at IGR 64 / 65. Preferably, the self-assembling protein nanoparticle is BFF.

[0263] In one embodiment, the recombinant poxvirus is a recombinant MVA comprising:

[0264] (i) a first heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes an EBV gp350 antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle; preferably wherein the poxviral promoter is Pr13.5long; preferably, wherein the self-assembling protein nanoparticle is BFF; and (ii) a second heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes EBV gH and / or EBV gL, preferably gH and gL separated from each other by a P2A site, or an antigenic part thereof; preferably wherein the poxviral promoter is PrS; preferably wherein the first and second heterologous nucleic acids are inserted into the MVA genome at IGR 64 / 65.

[0265] In one embodiment, the recombinant poxvirus is a recombinant MVA comprising (i) a first heterologous nucleic acid as described above and (ii) a second heterologous nucleic acid as described above, further comprising:

[0266] (iii) a third heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes a BZLF1 -BRLF1 fusion protein, or an antigenic part thereof, preferably wherein the poxviral promoter is Pr13.5long; and

[0267] (iv) a fourth heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes EBNA3A, or an antigenic part thereof, preferably wherein the poxviral promoter is Pr1328; preferably wherein the third and fourth heterologous nucleic acids are inserted into the MVA genome at IGR 88 / 89.

[0268] In one embodiment, the recombinant poxvirus further comprises another heterologous nucleic acid operably linked to a promoter, wherein the other heterologous nucleic acid encodes a fusion protein comprising another disease-associated protein, or a part thereof, joined to subunit of a self-assembling protein nanoparticle, preferably wherein the disease-associated protein and the other disease-associated protein are different. More preferably, the subunit of a self-assembling protein nanoparticle, which the disease-associated protein is joined to and the subunit of a self-assembling protein nanoparticle, which the other disease-associated protein is joined to are of the same type of self-assembling protein nanoparticle.

[0269] In another aspect, provided is a recombinant poxvirus comprising:

[0270] (a) a first heterologous nucleic acid operably linked to a promoter, wherein the first heterologous nucleic acid encodes a first fusion protein comprising a first disease-associated protein, or a part thereof, joined to a subunit of a selfassembling protein nanoparticle; and

[0271] (b) a second heterologous nucleic acid operably linked to a promoter, wherein the second heterologous nucleic acid encodes a second fusion protein comprising a second disease-associated protein, or a part thereof, joined to a subunit of a self-assembling protein nanoparticle; preferably wherein the first and second disease-associated proteins are different; more preferably wherein the subunit of a self-assembling protein nanoparticle, which the first disease-associate protein is joined to and the subunit of a self-assembling protein nanoparticle, which the second disease-associate protein is joined to, are of the same type of self-assembling protein nanoparticle.

[0272] In one embodiment of the recombinant poxvirus, the first heterologous nucleic acid and the second heterologous nucleic acid are joined by a heterologous nucleic acid encoding a 2A peptide, preferably a T2A peptide as depicted in SEQ ID NO: 96. Alternatively, the first heterologous nucleic acid and the second heterologous nucleic acid may be joined by an internal ribosomal entry site (IRES).

[0273] In yet another aspect, provided is a process for preparing a recombinant poxvirus as described herein, comprising the steps of:

[0274] (1 ) providing a transcription unit as described herein.

[0275] (2) inserting the transcription unit prepared in step (1 ) into a poxviral genome;

[0276] (3) obtaining the recombinant poxvirus.

[0277] Aspects and embodiments relating to a recombinant VRP

[0278] In one aspect, provided is a recombinant virus replicon particle (VRP) comprising a heterologous nucleic acid operably linked to a promoter, wherein the heterologous nucleic acid encodes a fusion protein comprising a disease-associated protein, or a part thereof, joined to a subunit of a self-assembling protein nanoparticle.

[0279] In one embodiment, the recombinant VRP is a recombinant alphavirus replicon particle.

[0280] In one embodiment, the recombinant VRP is derived from Venezuelan Equine Encephalitis Virus (VEEV), preferably is derived from VEEV strain TC83 and / or TrD.

[0281] In one embodiment of the recombinant VRP, the promoter is the VEEV TC83 genomic pro motor.

[0282] In one embodiment of the recombinant VRP, the promoter is the VEEV TC83 subgenomic pro motor.

[0283] In one embodiment, the recombinant VRP further comprises another heterologous nucleic acid operably linked to a promoter, wherein the other heterologous nucleic acid encodes a fusion protein comprising another disease-associated protein, or a part thereof, joined to subunit of a self-assembling protein nanoparticle, preferably wherein the disease-associated protein and the other disease-associated protein are different. More preferably, the subunit of a self-assembling protein nanoparticle, which the disease-associated protein is joined to and the subunit of a self-assembling protein nanoparticle, which the other disease-associated protein is joined to are of the same type of self-assembling protein nanoparticle.

[0284] In another aspect, provided is a recombinant VRP comprising:

[0285] (a) a first heterologous nucleic acid operably linked to a promoter, wherein the first heterologous nucleic acid encodes a first fusion protein comprising a first disease-associated protein, or a part thereof, joined to a subunit of a selfassembling protein nanoparticle; and

[0286] (b) a second heterologous nucleic acid operably linked to a promoter, wherein the second heterologous nucleic acid encodes a second fusion protein comprising a second disease-associated protein, or a part thereof, joined to a subunit of a self-assembling protein nanoparticle; preferably wherein the first and second disease-associated proteins are different, more preferably wherein the subunit of a self-assembling protein nanoparticle, which the first disease-associate protein is joined to and the subunit of a self-assembling protein nanoparticle, which the second disease-associate protein is joined to, are of the same type of self-assembling protein nanoparticle.

[0287] In one embodiment of the recombinant VRP, the first heterologous nucleic acid and the second heterologous nucleic acid are joined by a heterologous nucleic acid encoding a 2A peptide, preferably a T2A peptide as depicted in SEQ ID NO: 96. Alternatively, the first heterologous nucleic acid and the second heterologous nucleic acid may be joined by an internal ribosomal entry site (IRES).

[0288] In yet another aspect, provided is a process for preparing a recombinant VRP as described herein, comprising the steps of:

[0289] (1 ) providing a plasmid DNA encoding a self-amplifying replicon RNA, preferably a replicon RNA under the control of a cytomegalovirus (CMV) promoter, encoding a fusion protein comprising a disease-associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle as described herein;

[0290] (2) transfecting a VRP production cell, preferably a HEK293T suspension cell, with the recombinant replicon plasmid provided in step (1 ), further transfecting the VRP production cell with a first CMV promoter driven packaging plasmid, preferably derived from pcDNA3.1 , encoding an alphavirus capsid protein and a second CMV promoter driven packaging plasmid, preferably derived from pcDNA3.1 , encoding an alphavirus envelope protein;

[0291] (3) culturing the transfected VRP production cell of step (2); (4) obtaining the recombinant VRP.

[0292] In yet another aspect, provided is a plasmid DNA encoding self-amplifying replicon RNA, preferably a replicon RNA under the control of a cytomegalovirus (CMV), encoding a fusion protein comprising a disease-associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle.

[0293] Aspects and embodiments relating to a transcription unit

[0294] In one aspect, provided is a transcription unit comprising a nucleic acid, operably linked to a promoter, wherein the nucleic acid encodes a fusion protein comprising a disease-associated protein, or a part thereof, joined to a subunit of a self-assembling protein nanoparticle.

[0295] In one embodiment, the transcription unit is comprised by a poxvirus. In this case, the nucleic acid is heterologous with respect to the poxvirus.

[0296] In one embodiment, the transcription unit is comprised by a VRP. In this case, the nucleic acid is heterologous with respect to the VRP.

[0297] In one embodiment of the transcription unit, the promoter is a poxviral promoter.

[0298] In one embodiment of the transcription unit, the poxviral promoter is selected from the group consisting of promoters Pr11 , Pr7.5, PrSSL, PrATI, and PrS; Pr13.5long / Pr13.5, PrHyb, Pr1328, PrH2R and PrH5m, preferably is Pr13.5long.

[0299] In one embodiment, the poxviral promoter comprises or consists of a nucleic acid is as depicted in SEQ ID NO: 97.

[0300] In one embodiment of the transcription unit, the nucleic acid further encodes a signal peptide joined to the fusion protein.

[0301] In another aspect, provided is a use of a transcription unit as described herein for the preparation of a recombinant poxvirus, preferably a recombinant vaccinia virus, more preferably a recombinant MVA.

[0302] In another aspect, provided is a use of a transcription unit as described herein for the preparation of a VRP.

[0303] Aspects and embodiments relating to an RNA molecule

[0304] In one aspect, provided is an RNA molecule encoding a fusion protein comprising a disease- associated protein, or a part thereof, joined to a subunit of a self-assembling protein nanoparticle. In one embodiment, the RNA molecule is a pharmacologically active agent. For example, the nucleic acid is capable of inducing the translation of an antigenic protein in a subject.

[0305] In one embodiment, the RNA molecule is not comprised by a virus-based vector.

[0306] In one embodiment, the RNA molecule is a messenger RNA (mRNA) or a self-amplifying RNA (saRNA). Preferably, the RNA molecule is saRNA.

[0307] In one embodiment, the mRNA or saRNA further comprises a poly(A) tail of multiple adenines, which is located downstream of the heterologous nucleic acid encoding the fusion protein, preferably a poly(A) tail of more than 100 adenines, most preferably 120 adenines.

[0308] In one embodiment, the mRNA or saRNa further comprises a capping structure or a pseudo capping structure.

[0309] In one embodiment, the RNA molecule further encodes a signal peptide joined to the fusion protein.

[0310] In one embodiment, the RNA molecule further comprises another heterologous nucleic acid, wherein the other nucleic acid encodes a fusion protein comprising another disease- associated protein, or a part thereof, joined to subunit of a self-assembling protein nanoparticle, preferably wherein the disease-associated protein and the other disease- associated protein are different, more preferably wherein the nanoparticles are the same.

[0311] In another aspect, provided is an RNA molecule, preferably an mRNA or a saRNA, comprising:

[0312] (a) a first section of the heterologous nucleic acid, with the first section encoding a first fusion protein comprising a first disease-associated protein, or a part thereof, joined to a subunit of a self-assembling protein nanoparticle; and

[0313] (b) a second section of the heterologous nucleic acid, with the second section encoding a second fusion protein comprising a second disease-associated protein, or a part thereof, joined to a subunit of a self-assembling protein nanoparticle; wherein the first and second disease-associated proteins are different, more preferably wherein the self-assembling protein nanoparticles are the same.

[0314] In one embodiment of the recombinant virus-based vector, the first heterologous nucleic acid and the second heterologous nucleic acid are joined by a heterologous nucleic acid encoding a 2A peptide, preferably a T2A peptide as depicted in SEQ ID NO: 96. Alternatively, the first heterologous nucleic acid and the second heterologous nucleic acid may be joined by an internal ribosomal entry site (IRES). In yet another aspect, provided is a process for preparing an RNA molecule as described herein, comprising the steps of:

[0315] (1 ) providing a nucleic acid encoding a fusion protein as described herein comprising a disease-associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle as described herein;

[0316] (2) generating an RNA molecule comprising the nucleic acid provided in step (1 );

[0317] (3) obtaining the RNA molecule generated in step (2).

[0318] Aspects and embodiments relating to an expression plasmid

[0319] In one aspect, provided is a DNA molecule encoding a fusion protein comprising a disease- associated protein, or a part thereof, joined to a subunit of a self-assembling protein nanoparticle.

[0320] In one embodiment, the DNA molecule is an expression plasmid.

[0321] In another aspect, provided is an expression plasmid comprising a heterologous nucleic operably linked to a promoter, wherein the heterologous nucleic acid encodes a fusion protein comprising a disease-associated protein, or a part thereof, joined to a subunit of a selfassembling protein nanoparticle.

[0322] In one embodiment, the expression plasmid is based on pcDNA 3.1.

[0323] In one embodiment, the promoter is cytomegalovirus (CMV) promoter.

[0324] In one embodiment, the expression plasmid further comprises another heterologous nucleic acid, wherein the other nucleic acid encodes a fusion protein comprising another disease- associated protein, or a part thereof, joined to subunit of a self-assembling protein nanoparticle, preferably wherein the disease-associated protein and the other disease- associated protein are different, more preferably wherein the nanoparticles are the same.

[0325] In another aspect, provided is an expression plasmid comprising:

[0326] (a) a first section of the heterologous nucleic acid, with the first section encoding a first fusion protein comprising a first disease-associated protein, or a part thereof, joined to a subunit of a self-assembling protein nanoparticle; and

[0327] (b) a second section of the heterologous nucleic acid, with the second section encoding a second fusion protein comprising a second disease-associated protein, or a part thereof, joined to a subunit of a self-assembling protein nanoparticle; wherein the first and second disease-associated proteins are different, more preferably wherein the self-assembling protein nanoparticles are the same.

[0328] In one embodiment of the expression plasmid, the first heterologous nucleic acid and the second heterologous nucleic acid are joined by a heterologous nucleic acid encoding a 2A peptide, preferably a T2A peptide as depicted in SEQ ID NO: 96. Alternatively, the first heterologous nucleic acid and the second heterologous nucleic acid may be joined by an internal ribosomal entry site (IRES).

[0329] In yet another aspect, provided is a process for preparing an expression plasmid as described herein, comprising the steps of:

[0330] (1 ) providing a nucleic acid encoding a fusion protein as described herein comprising a disease-associated antigen, or an antigenic part thereof, as described herein joined to a subunit of a self-assembling protein nanoparticle as described herein;

[0331] (2) inserting the nucleic acid provided in step (1 ) into an expression plasmid;

[0332] (3) obtaining the expression plasmid generated in step (2).

[0333] Embodiments relating to a heterologous nucleic acid

[0334] The following embodiments may relate to any aspect described herein.

[0335] In one embodiment of the recombinant poxvirus, the heterologous nucleic acid is DNA.

[0336] In one embodiment of the recombinant VRP, the heterologous nucleic acid is RNA.

[0337] In one embodiment, the heterologous nucleic acid further encodes a signal peptide joined to the fusion protein.

[0338] In one embodiment, the heterologous nucleic acid comprises or consists of a nucleic acid as depicted in one of SEQ ID NO: 17, 18, 19, SEQ ID NO: 21 , 22, 23, SEQ ID NO: 25, 26, SEQ ID NO: 28, 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 . In one embodiment, the fusion protein is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the nucleic acid sequence as depicted in in one of SEQ ID NO: 17, 18, 19, SEQ ID NO: 21 , 22, 23, SEQ ID NO: 25, 26, SEQ ID NO: 28, 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61.

[0339] In one embodiment, the heterologous nucleic acid comprises or consists of a nucleic acid encoding an amino acid sequence as depicted in one of SEQ ID NO: 20, SEQ ID NO: 24,

[0340] SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID

[0341] NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,

[0342] SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID

[0343] NO: 60, SEQ ID NO: 62. In one embodiment, the fusion protein is encoded by a nucleic acid sequence encoding an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence as depicted in in one of SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID

[0344] NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46,

[0345] SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID

[0346] NO: 58, SEQ ID NO: 60, SEQ ID NO: 62.

[0347] In one aspect, provided is a use of the heterologous nucleic acid for the preparation of a virusbased vector, a recombinant poxvirus or a recombinant VRP.

[0348] Embodiments relating to a fusion protein

[0349] The following embodiments relate to a fusion protein encoded by a virus-based vector, a recombinant poxvirus, a recombinant VRP, an RNA molecule, or an expression plasmid, and they may relate to any aspect described herein in this respect.

[0350] In one embodiment, the fusion protein has the capability to take part in a self-assembly of fusion proteins into a self-assembling protein nanoparticle, preferably into an oligomeric or multimeric protein composed of monomeric fusion proteins.

[0351] In one embodiment, the fusion protein has maintained the capability of the subunit of a selfassembling protein nanoparticle comprised by the fusion protein to take part in a self-assembly of fusion proteins into a self-assembling protein nanoparticle.

[0352] In one embodiment, a linker is located between the disease-associated protein, or a part thereof, and the subunit of a self-assembling nanoparticle.

[0353] In one embodiment, the linker is a nine amino acid linker, preferably is SSGGASVLA, preferably as depicted in SEQ ID NO: 88 and / or encoded by a nucleic acid as depicted in SEQ ID NO: 87. In one embodiment, the linker is a nine amino acid linker joining an antigen, preferably SARS- CoV-2 RBD or an antigen derived from Borrelia OspA, to BFF.

[0354] In one embodiment, the linker is a nine amino acid linker joining an antigen, preferably SARS- CoV-2 RBD, to HisB, PdhC or DPS.

[0355] In one embodiment, the linker is a ten amino acid linker, preferably is PKPSTPPGSS, preferably as depicted in SEQ ID NO: 90 and / or encoded by a nucleic acid as depicted in SEQ ID NO: 89.

[0356] In one embodiment, the linker is a ten amino acid linker joining an antigen, preferably EBV 350, to BFF, PdhC or DPS.

[0357] In one embodiment, the fusion protein is encoded by a nucleic acid molecule as described herein, for example a heterologous nucleic acid as described herein.

[0358] In one embodiment, the fusion protein is encoded by a nucleic acid comprising or consisting of a nucleic acid as depicted in one of SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 . In one embodiment, the fusion protein is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the nucleic acid sequence as depicted in in one of SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81.

[0359] In one embodiment, the heterologous nucleic acid comprises or consists of a nucleic acid encoding an amino acid sequence as depicted in one of SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQE ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 82. In one embodiment, the fusion protein is encoded by a nucleic acid sequence encoding an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence as depicted in in one of SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQE ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 82.

[0360] Embodiments relating to a disease-associated protein

[0361] The following embodiments may relate to any aspect described herein.

[0362] In one embodiment, the disease-associated protein is selected from the group consisting of a viral, bacterial, fungal, plant, parasite, non-human animal, and human protein. In one embodiment, the disease-associated protein is a gene therapy protein.

[0363] In one embodiment, the disease-associated protein, or a part thereof, comprises one or more antigenic determinants ( / .e., the part of an antigen that is recognized by the immune system, also referred to as epitope).

[0364] In one embodiment, the disease-associated protein is a disease-associated antigen.

[0365] In one embodiment, the part of a disease-associated protein is an antigenic part thereof.

[0366] In one embodiment, the disease-associated protein is an infectious disease-associated antigen.

[0367] In one embodiment, the disease-associated antigen is a viral antigen, preferably is from a virus selected from the group consisting of alpha-virus, adenovirus, Chikungunyavirus Coxsackievirus, Crimean-Congo hemorrhagic fever virus, cytomegalovirus (CMV), dengue virus, Ebola virus, Epstein-Barr virus (EBV), Eastern, Western or Venezuelan equine encephalitis virus (EEV) , Guanarito virus, herpes simplex virus-type 1 (HSV-1 ), herpes simplex virus-type 2 (HSV-2), human herpesvirus-type 8 (HHV-8), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), human immunodeficiency virus (HIV), influenza virus, Junin virus, Lassa virus, Machupo virus, Marburg virus, measles virus, human metapneumovirus, mumps virus, Norwalk virus, human papillomavirus (HPV), parainfluenza virus, parvovirus, poliovirus, rabies virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, rubella virus, Sabia virus, severe acute respiratory syndrome virus 2 (SARS-CoV-2), middle east respiratory syndrome coronavirus (MERS-CoV), varicella zoster virus, variola virus, West Nile virus, yellow fever virus, and a Zika virus.

[0368] In one embodiment, the disease-associated antigen is from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), preferably comprises or consists of a receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein (SARS-CoV-2 S RBD) or an antigenic part thereof.

[0369] In a preferred embodiment, the disease-associated antigen is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 1 .

[0370] In a preferred embodiment, the disease-associated antigen comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 2.

[0371] In a preferred embodiment, the disease-associated antigen is encoded by a nucleic acid encoding an amino acid sequence as depicted in SEQ ID NO: 2. In a more preferred embodiment, the disease-associated antigen comprises or consists of amino acids 331 -524 of SARS-CoV-2 S protein. Most preferably, the disease-associated antigen comprises or consists of or amino acids 320-537 of the full-length SARS-CoV-2 S1 domain.

[0372] In another embodiment, the disease-associated antigen is from Epstein-Barr virus (EBV), preferably selected from the group consisting of EBV proteins BLLF1 a / b (gp350 / 220), BALF4 (gB, gp110), BXLF2 (gH, gp85), BKRF2 (gL, gp25), BZLF2 (gp42), BILF2 (gp78), BDLF3 (gp150), BBRF3 (gM), BLRF1 (gN), BMRF2, EBNA1 , EBNA2, EBNA3, LMP1 , LMP2, BRLF1 or BZLF1 protein, preferably is EBV surface glycoprotein 350 (EBV gp350) or an antigenic part thereof.

[0373] In a preferred embodiment, the disease-associated antigen is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 3.

[0374] In a preferred embodiment, the disease-associated antigen comprises or consists of an amino acid sequence as depicted in in SEQ ID NO: 4.

[0375] In a preferred embodiment, the disease-associated antigen is encoded by a nucleic acid encoding an amino acid sequence as depicted in SEQ ID NO: 4.

[0376] In a more preferred embodiment, the disease-associated antigen comprises or consists of amino acids 2-434 of EBV gp350.

[0377] In one embodiment, the disease-associated antigen is a bacterial antigen, preferably is from a bacterium selected from the group consisting of Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi sensu stricto, Borrelia burgdorferi sensu lato, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diptheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli, Escherichia coli ) 157:H7, Francisella tularensis, Haemophilus influenza, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitides, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae, and Yersinia pestis. In another embodiment, the disease-associated antigen is a Lyme disease antigen. Preferably, the disease-associated antigen is from Borrelia, more preferably is derived from Borrelia outer surface protein A (OspA), or an antigenic part thereof.

[0378] In a preferred embodiment, the disease-associated antigen comprises or consists of one, two, three or more antigenic fragments of OspA. Even more preferably, the disease-associated antigen comprises or consists of a string of three OspA C-terminal fragments, preferably separated from each other by a (GGGGS)4 linker.

[0379] In a more preferred embodiment, the disease-associated antigen comprises or consists of a fusion protein of amino acids 126-273 of OspA serotype 1 (ST1 ) from Borrelia burgdorferi sensu stricto (strain B31 ), amino acids 126-273 of OspA ST5 from Borrelia garinii (strain PHei) and amino acids 126-274 of OspA ST6 from Borrelia garinii (DK29), separated from each other by a (GGGGS)4 linker.

[0380] In another more preferred embodiment, the disease-associated antigen comprises or consists of a fusion protein of amino acids 126-273 of OspA serotype 2 (ST2) from Borrelia afzelii (strain K78), amino acids 126-273 of OspA ST4 from Borrelia bavariensis (strain PBi) and amino acids 126-274 of OspA ST3 from Borrelia garinii (PBr), separated from each other by a (GGGGS)4 linker.

[0381] In a particularly preferred embodiment, the disease-associated antigen is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 100 or SEQ ID NO: 102.

[0382] In a particularly preferred embodiment, the disease-associated antigen comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 101 or SEQ NO: 103.

[0383] In a particularly preferred embodiment, the disease-associated antigen is encoded by a nucleic acid encoding an amino acid sequence as depicted in SEQ ID NO: 101 or SEQ ID NO: 103.

[0384] In one embodiment, the disease-associated antigen is a fungal antigen, preferably is from a fungus selected from the group consisting of Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Blastomyces dermatitidis, Candida albicans, Candida dubliniensis, Candida glabrata, Candida parapsilosis, Candida rugosa, Candida tropicalis, Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, Histoplasma capsulatum, Microsporum canis, Pneumocystis carinii, Pneumocystis jirovecii, Sporothrix schenckii, Stachbotrys chartarum, Tinea barbae, Tinea captitis, Tinea corporis, Tinea cruris, Tinea faciei, Tinea incognito, Tinea nigra, Tinea versicolor, Trichophyton rubrum and Trichophyton tonsurans. In certain embodiments, the disease-associated antigen is a parasite antigen, preferably is from a parasite selected from the group consisting of Anisakis spp. Babesia spp., Baylisascaris procyonis, Cryptosporidium spp., Cyclospora cayetanensis, Diphyllobothrium spp., Dracunculus medinensis, Entamoeba histolytica, Giardia duodenalis, Giardia intestinalis, Giardia lamblia, Leishmania sp., Plasmodium falciparum, Plasmodium vivax, Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Taenia spp., Toxoplasma gondii, Trichinella spiralis, and Trypanosoma cruzi.

[0385] In one embodiment, the disease-associated protein is a tumor specific antigen (TSA) or a tumor associated antigen (TAA).

[0386] Embodiments relating to a self-assembling protein nanoparticle

[0387] The following embodiments may relate to any aspect described herein.

[0388] In one embodiment, the subunit of a self-assembling protein nanoparticle is a monomeric protein.

[0389] In one embodiment, the self-assembling protein nanoparticle is an oligomeric or multimeric protein composed of monomeric subunits.

[0390] In one embodiment, the subunit of a self-assembling protein nanoparticle has the capability to take part in a self-assembly of subunits into the self-assembling protein nanoparticle.

[0391] In one embodiment, the subunit of a self-assembling protein nanoparticle is the full-length subunit of a self-assembling protein nanoparticle.

[0392] In one embodiment, the subunit of a self-assembling protein nanoparticle is a functional fragment of a full-length subunit, i.e., is a fragment having maintained the capability of the full- length subunit to take part in a self-assembly of subunits into the self-assembling protein nanoparticle.

[0393] In one embodiment, the functional fragment comprises at least 50, 75, 100, or 150 contiguous amino acids of the full-length subunit of a self-assembling protein nanoparticle.

[0394] In one embodiment, the functional fragment comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence of the full-length subunit of a self-assembling protein nanoparticle.

[0395] In one embodiment, the self-assembling protein nanoparticle is composed of 12, 24, or 60 subunits. In one embodiment, the subunits, which the self-assembling protein nanoparticle is composed of, are arranged with tetrahedral, octahedral, or icosahedral symmetry.

[0396] In one embodiment, the 12, 24, or 60 subunits, which the self-assembling protein nanoparticle is composed of, are capable of self-assembling into 12mer, 24mer, or a 60mer protein nanoparticle, respectively.

[0397] In one embodiment, the subunit of a self-assembling protein nanoparticle is capable of being joined to a disease-associated protein via the N-terminus and / or the C-terminus of the subunit.

[0398] In one embodiment, the self-assembling protein nanoparticle is selected from the group consisting of hybrid protein Bullfrog / H pylori hybrid ferritin (BFF), imidazoleglycerol-phosphate dehydratase (HisB), acetyltransferase of pyruvate dehydrogenase (PDH) complex (PdhC), and DNA binding protein from starved cells (DPS).

[0399] In one embodiment, the self-assembling protein nanoparticle is hybrid protein BFF. Preferably, BFF comprises or consists of amino acids 2-9 of Bullfrog Rana catesbeiana) ferritin and amino acids 3-167 of the Helicobacter pylori ferritin.

[0400] In one embodiment, the self-assembling nanoparticle is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 5. In one embodiment, the self-assembling nanoparticle is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the nucleic acid sequence as depicted in SEQ ID NO: 5.

[0401] In one embodiment, the self-assembling nanoparticle comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 6. In one embodiment, the self-assembling nanoparticle comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence as depicted in SEQ ID NO: 6.

[0402] In one embodiment, the self-assembling nanoparticle is encoded by a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 6. In one embodiment, the selfassembling nanoparticle is encoded by a nucleic acid sequence encoding an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence as depicted in SEQ ID NO: 6.

[0403] In one embodiment, the self-assembling protein nanoparticle is HisB, PdhC or DPS, preferably is HisB or DPS.

[0404] In one embodiment, the HisB is from Mycobacterium tuberculosis. In one embodiment, the self-assembling protein nanoparticle comprises or consists of amino acids 9-210 of HisB.

[0405] In one embodiment, the self-assembling nanoparticle is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 7. In one embodiment, the self-assembling nanoparticle is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the nucleic acid sequence as depicted in SEQ ID NO: 7.

[0406] In one embodiment, the self-assembling nanoparticle comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 8. In one embodiment, the self-assembling nanoparticle comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence as depicted in SEQ ID NO: 8.

[0407] In one embodiment, the self-assembling nanoparticle is encoded by a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 8. In one embodiment, the selfassembling nanoparticle is encoded by a nucleic acid sequence encoding an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence as depicted in SEQ ID NO: 8.

[0408] In one embodiment, the PdhC is from Geo-) Bacillus stearothermophilus (PdhC).

[0409] In one embodiment, the self-assembling protein nanoparticle comprises or consists of amino acids 185-428 of PdhC.

[0410] In one embodiment, the self-assembling nanoparticle is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 9. In one embodiment, the self-assembling nanoparticle is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the nucleic acid sequence as depicted in SEQ ID NO: 9.

[0411] In one embodiment, the self-assembling nanoparticle comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 10. In one embodiment, the self-assembling nanoparticle comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence as depicted in SEQ ID NO: 10.

[0412] In one embodiment, the self-assembling nanoparticle is encoded by a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 10. In one embodiment, the self-assembling nanoparticle is encoded by a nucleic acid sequence encoding an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence as depicted in SEQ ID NO: 10.

[0413] In one embodiment, the DPS is from Escherichia coli. In one embodiment, the self-assembling protein nanoparticle comprises or consists of amino acids 11 -167 of DPS.

[0414] In one embodiment, the self-assembling nanoparticle is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 11. In one embodiment, the self-assembling nanoparticle is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the nucleic acid sequence as depicted in SEQ ID NO: 11 .

[0415] In one embodiment, the self-assembling nanoparticle comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 12. In one embodiment, the self-assembling nanoparticle is encoded by a nucleic acid sequence encoding an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence as depicted in SEQ ID NO: 12.

[0416] In one embodiment, the self-assembling nanoparticle is encoded by a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 12. In one embodiment, the self-assembling nanoparticle is encoded by a nucleic acid sequence encoding an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence as depicted in SEQ ID NO: 12.

[0417] In one embodiment, the self-assembling protein nanoparticle is BFF, and the disease- associated antigen is SARS-CoV-2 S RBD, EBV gp350, or is derived from OspA.

[0418] In one embodiment, the self-assembling protein nanoparticle is HisB, PdhC or DPS, and the disease-associated antigen is SARS-CoV-2 S RBD.

[0419] In one embodiment, the self-assembling protein nanoparticle is BFF, wherein the N-terminus of BFF is joined to the C-terminus of the disease-associated antigen.

[0420] In one embodiment, the self-assembling protein nanoparticle is PdhC, wherein the N-terminus of PdhC is joined to the C-terminus of the disease-associated antigen.

[0421] In one embodiment, the self-assembling protein nanoparticle is DPS, wherein the N-terminus of DPS is joined to the C-terminus of the disease-associated antigen, or wherein the C- terminus of DPS is joined to the N-terminus of the disease-associated antigen.

[0422] In one embodiment, the self-assembling protein nanoparticle is HisB, wherein the C-terminus of HisB is joined to the N-terminus of the disease-associated antigen.

[0423] In one embodiment, the N-terminus of BFF is joined to the C-terminus of SARS-CoV-2 S RBD, EBV gp350, or an antigen derived from OspA. In one embodiment, the N-terminus of PdhC is joined to the C-terminus of SARS-CoV-2 S RBD or EBV gp350.

[0424] In one embodiment, the N-terminus of DPS is joined to the C-terminus of SARS-CoV-2 S RBD or EBV gp350.

[0425] In one embodiment, the C-terminus of DPS is joined to the N-terminus of SARS-CoV-2 S RBD.

[0426] In one embodiment, the C-terminus of HisB is joined to the N-terminus of SARS-CoV-2 S RBD.

[0427] Embodiments relating to a signal peptide

[0428] The following embodiments may relate to any aspect as described herein.

[0429] In one embodiment, the signal peptide joined to the fusion protein is capable of allowing or facilitating cellular secretion of the fusion protein.

[0430] In one embodiment, the signal peptide is human tissue plasminogen activator (htPA) signal peptide or murine immunoglobulin kappa light chain (IgK LC) signal peptide.

[0431] In one embodiment, the signal peptide is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 83 or SEQ ID NO: 85.

[0432] In one embodiment, the signal peptide comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 84 or SEQ ID NO: 86.

[0433] In one embodiment, the disease-associated antigen is SARS-CoV-2 S RBD, and the signal peptide is htPA signal peptide.

[0434] In one embodiment, the disease-associated antigen is EBV gp350 and the signal peptide is IgK LC signal peptide.

[0435] In one embodiment, the disease-associated antigen is SARS-CoV-2 S RBD, the selfassembling protein nanoparticle is selected from the group consisting of BFF, HisB, PdhC, and DPS, and the signal peptide is htPA signal peptide.

[0436] In one embodiment, the disease-associated antigen is EBV gp350, the self-assembling protein nanoparticle is BFF, and the signal peptide is IgK LC signal peptide.

[0437] In one embodiment, the signal peptide is joined to the N-terminus of the fusion protein. In one embodiment, the N-terminus of the subunit of a self-assembling protein nanoparticle is joined to the C-terminus of the disease-associated antigen, and the signal peptide is joined to the N-terminus of the disease-associated antigen.

[0438] In one embodiment, the C-terminus of the subunit of a self-assembling protein nanoparticle is joined to the N-terminus of the disease-associated antigen, and the signal peptide is joined to the N-terminus of the subunit of a self-assembling protein nanoparticle.

[0439] Aspects and embodiments relating to a pharmaceutical composition

[0440] In one aspect, provided is a pharmaceutical composition comprising the recombinant virusbased vector, the recombinant poxvirus, the recombinant VRP, the RNA molecule or the expression plasmid as described herein.

[0441] In one embodiment, the pharmaceutical composition is a vaccine.

[0442] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0443] In one embodiment, the pharmaceutical composition comprising the recombinant virus-based vector, the recombinant poxvirus or the recombinant VRP does not comprise an adjuvant.

[0444] In one embodiment, the pharmaceutical composition comprising the RNA molecule or the expression plasmid comprises an adjuvant.

[0445] In one embodiment, the pharmaceutical composition comprises an RNA molecule or an expression plasmid, and further comprises a pharmaceutically acceptable nucleic acid transfection reagent, i.e., a component allowing or supporting the uptake of the nucleic acid into a cell.

[0446] In one embodiment, the pharmaceutical composition comprises an mRNA molecule, or alternatively a saRNA molecule, and further comprises a lipid-based carrier, i.e., a component having the capacity to encapsulate and deliver the nucleic acid to a target tissue, such as lipid nanoparticles (LNPs).

[0447] In another aspect, provided is a process for preparing a pharmaceutical composition as described herein, comprising the steps of:

[0448] (1 ) providing a nucleic acid encoding a fusion protein as described herein comprising a disease-associated protein, or a part thereof, joined to a subunit of a self-assembling protein nanoparticle; (2) generating a recombinant virus-based vector, a recombinant poxvirus, a recombinant VRP, an RNA molecule or an expression plasmid as described herein comprising the nucleic acid provided in step (1);

[0449] (3) obtaining the recombinant virus-based vector, the recombinant poxvirus, the recombinant VRP, the RNA molecule or the expression plasmid generated in step (2);

[0450] (4) combining the recombinant virus-based vector, the recombinant poxvirus, the recombinant VRP, the RNA molecule or the expression plasmid obtained in step (3) with a pharmaceutically acceptable carrier or excipient.

[0451] Aspects and embodiments relating to a kit

[0452] In one aspect, provided is a kit or kit of parts, comprising a first and a second container, optionally at least one further container, each container comprising a pharmaceutical composition as described herein.

[0453] In one embodiment, the kit or kit of parts is designed for use in a prime-boost regimen.

[0454] Aspects and embodiments relating to a medical use

[0455] In one aspect, provided is a recombinant virus-based vector, a recombinant poxvirus, a recombinant VRP, an RNA molecule, or an expression plasmid as described herein for use in the prevention or treatment of a disease, preferably for use in the prevention of a disease.

[0456] In another aspect, provided is a pharmaceutical composition as described herein for use in the prevention or treatment of a disease, preferably for use in the prevention of a disease.

[0457] In yet another aspect, provided is a use of a recombinant virus-based vector, a recombinant poxvirus, a recombinant VRP, an RNA molecule, an expression plasmid or a pharmaceutical composition as described herein for the manufacture of a medicament for the prevention or treatment of a disease, preferably for the prevention of a disease.

[0458] In one embodiment, the disease is an infectious disease or cancer.

[0459] In one embodiment, the disease is cancer related to an infectious disease.

[0460] In one embodiment, the disease is a viral or bacterial infectious disease.

[0461] In one embodiment, the disease is a malignancy associated with or resulting from a viral or bacterial infection.

[0462] In one embodiment, the disease is COVID-19 or long COVID. In one embodiment, the disease is EBV infection, e.g., infectious mononucleosis.

[0463] In one embodiment, the disease is an EBV associated cancer, e.g., Hodgkin lymphoma, Burkitt lymphoma or nasopharyngeal cancer.

[0464] In one embodiment, the disease is Lyme disease or Borrelia infection.

[0465] In one embodiment, the pharmaceutical composition is administered to a subject intramuscularly, subcutaneously or intranasally, preferably intramuscularly.

[0466] In one embodiment, the pharmaceutical composition is administered to a subject once, twice, three times or more often.

[0467] In one embodiment, the pharmaceutical composition is administered to a subject as part of a prime-boost regimen.

[0468] In one embodiment, the subject is a mammal, for example a farm animal, a pet, or a human, preferably is a human.

[0469] Aspects and embodiments relating to medical treatment

[0470] In one aspect, provided is a medical treatment of a disease, or a disease prevention measure, or a vaccination or vaccination regimen, comprising administering to a subject a pharmaceutical composition or a vaccine as described herein.

[0471] In one aspect, the invention provides a method of prevention or treatment of an infectious disease or cancer comprising the step of administering to a subject a composition as described herein, such as a composition comprising a recombinant virus-based vector, a recombinant poxvirus, a recombinant VRP, an RNA molecule or an expression plasmid as described herein. The invention provides a method of prevention or treatment of an infectious disease or cancer comprising the step of administering to a subject a pharmaceutical composition or vaccine as described herein. In some embodiments, the step of administering a composition comprises intramuscular injection of the composition.

[0472] In one aspect, the invention provides a method for inducing an immune response to an infectious disease or cancer comprising the step of administering to a subject a composition as described herein, such as a composition comprising a recombinant virus-based vector, a recombinant poxvirus, a recombinant VRP, an RNA molecule or an expression plasmid as described herein. The invention provides a method for inducing an immune response to an infectious disease or cancer comprising the step of administering to a subject a pharmaceutical composition or vaccine as described herein. In some embodiments, the step of administering a composition comprises intramuscular injection of the composition.

[0473] In some embodiments, the step of administering the composition as described herein to a subject results in an immune response in the subject such as, for example, the production of antibodies (e.g., neutralizing antibodies). In this manner, the invention provides methods of stimulating an immune response in a subject comprising the step of administering a pharmaceutical composition or vaccine to a subject, whereby an immune response is produced. An immune response is said to be produced in a subject, for example, if antibodies specific for the disease-associated protein are present in the subject following administration of the composition. For example, an immune response is said to be produced in a subject following administration of the recombinant virus-based vector, recombinant VRP, fusion protein, and / or self-assembling protein nanoparticle if antibodies are produced in the subject that recognize the full-length disease-associated protein or a part or antigenic determinant thereof. Measurement of antibodies in a subject can be any of a variety of methods well-known in the art.

[0474] In some embodiments, the step of administering the recombinant MVA and / or pharmaceutical composition results in the production of antigen-binding antibodies, the induction of an antigen specific T cell response, preferably a CD8 T cell response, and / or the induction of an antigen specific B cell response. Preferably, the antigen-binding antibodies, the T cell response and / or the B cell response are directed against the full-length disease-associated protein, or a part or an antigenic determinant thereof. In some embodiments, the composition as described herein is administered to a subject more than once, for example, as a priming dose and one or more subsequent “booster” doses.

[0475] Aspects and embodiments relating to a recombinant virus-based vector encoding a multi-antigen protein nanoparticle

[0476] In one aspect, provided is a recombinant virus-based vector comprising:

[0477] (a) a first heterologous nucleic acid operably linked to a promoter, wherein the first heterologous nucleic acid encodes a first fusion protein comprising a first disease-associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle; and

[0478] (b) a second heterologous nucleic acid operably linked to a promoter, wherein the second heterologous nucleic acid encodes a second fusion protein comprising a second disease-associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle; wherein the first and second disease-associated antigens are different, and wherein the selfassembling protein nanoparticle in each case is the same.

[0479] In one embodiment, the recombinant virus-based vector encoding a multi-antigen protein nanoparticle is a recombinant poxvirus, preferably a recombinant vaccinia virus, more preferably a recombinant Modified Vaccinia Virus Ankara (MVA).

[0480] In one embodiment, the recombinant virus-based vector encoding a multi-antigen protein nanoparticle is a recombinant virus replicon particle (VRP), preferably derived from Venezuelan Equine Encephalitis Virus (VEEV), more preferably derived from VEEV strain TC83 and / or TrD.

[0481] In one embodiment of the recombinant virus-based vector encoding a multi-antigen protein nanoparticle, the self-assembling protein nanoparticle is selected from the group consisting of hybrid protein Bullfrog / H py / or / hybrid ferritin (BFF); imidazoleglycerol-phosphate dehydratase (HisB), preferably from Mycobacterium tuberculosis; acetyltransferase of pyruvate dehydrogenase (PDH) complex (PdhC), preferably from Geo-) Bacillus stearothermophilus; and DNA binding protein from starved cells (DPS), preferably from Escherichia coli.

[0482] In one embodiment of the recombinant virus-based vector encoding a multi-antigen protein nanoparticle, the self-assembling protein nanoparticle is HisB, PdhC or DPS.

[0483] In one embodiment of the recombinant virus-based vector encoding a multi-antigen protein nanoparticle, the self-assembling protein nanoparticle is selected from the group consisting of BFF, PdhC and DPS, and wherein in each case the N-terminus of the subunit of the selfassembling nanoparticle is joined to the C-terminus of the disease-associated antigen.

[0484] In one embodiment of the recombinant virus-based vector encoding a multi-antigen protein nanoparticle, the self-assembling protein nanoparticle is HisB or DPS, and wherein in each case the C-terminus of the subunit of the self-assembling nanoparticle is joined to the N- terminus of the disease-associated antigen.

[0485] In one embodiment of the recombinant virus-based vector encoding a multi-antigen protein nanoparticle, the first and / or the second disease-associated antigen is an infectious disease- associated antigen, preferably a viral or bacterial antigen.

[0486] In one embodiment of the recombinant virus-based vector encoding a multi-antigen protein nanoparticle, the first or second infectious disease-associated antigen is from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), preferably comprises or consists of a receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein (SARS-CoV-2 S RBD). In one embodiment of the recombinant virus-based vector encoding a multi-antigen protein nanoparticle, the first or second infectious disease-associated antigen is from Epstein-Barr Virus (EBV), preferably comprises or consists of EBV surface glycoprotein 350 (EBV gp350).

[0487] In one embodiment of the recombinant virus-based vector encoding a multi-antigen protein nanoparticle, the first or second disease-associated antigen is from Borrelia, preferably is derived from Borrelia outer surface protein A (OspA).ln one embodiment of the recombinant virus-based vector encoding a multi-antigen protein nanoparticle, the first or second disease- associated antigen comprises or consists of one, two, three or more antigenic fragments of OspA, preferably as described in more detail above in “Embodiments relating to a disease- associated protein”.

[0488] In one embodiment of the recombinant virus-based vector encoding a multi-antigen protein nanoparticle, the first infectious disease-associated antigen is from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), preferably comprises or consists of a receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein (SARS-CoV-2 S RBD), and the second infectious disease-associated antigen is from Epstein-Barr Virus (EBV), preferably comprises or consists of EBV surface glycoprotein 350 (EBV gp350), or vice versa.

[0489] In one embodiment of the recombinant virus-based vector encoding a multi-antigen protein nanoparticle, the first infectious disease-associated antigen is from Borrelia, preferably is derived from Borrelia outer surface protein A (OspA), and the second infectious disease- associated antigen is from Borrelia, preferably is derived from Borrelia outer surface protein A (OspA), wherein the first and second disease-associated antigens are different.

[0490] In one embodiment of the recombinant virus-based vector encoding a multi-antigen protein nanoparticle, the first disease-associated antigen is encoded by a nucleic acid encoding an amino acid sequence as depicted in SEQ ID NO: 101 and the second disease-associated antigen is encoded by a nucleic acid encoding an amino acid sequence as depicted in SEQ ID NO: 103, or vice versa.

[0491] In one embodiment of the recombinant virus-based vector encoding a multi-antigen protein nanoparticle, the first and the second heterologous nucleic acids are separated by a nucleic acid encoding a 2A site, preferably a T2A site.

[0492] In one embodiment of the recombinant virus-based vector encoding a multi-antigen protein nanoparticle, each of the first and second heterologous nucleic acid further encodes a signal peptide joined to the fusion protein, preferably the signal peptide is human tissue plasminogen activator (htPA) signal peptide or murine immunoglobulin kappa light chain (IgK LC) signal peptide.

[0493] In one aspect, provided is a process for preparing a recombinant virus-based vector encoding a multi-antigen protein nanoparticle, comprising the steps of:

[0494] (1 ) providing a first heterologous nucleic acid operably linked to a promoter, wherein the first heterologous nucleic acid encodes a first fusion protein comprising a first disease-associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle;

[0495] (2) providing a second heterologous nucleic acid operably linked to a promoter, wherein the second heterologous nucleic acid encodes a second fusion protein comprising a second disease-associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle; wherein the first and second disease-associated antigens are different, and wherein the self-assembling protein nanoparticle in each case is the same;

[0496] (3) preparing a recombinant virus-based vector comprising the first heterologous nucleic acids provided in step (1 ) and the second heterologous nucleic acid provided in step (2) into a virus-based vector;

[0497] (4) obtaining the recombinant virus-based vector.

[0498] In one aspect, provided is a pharmaceutical composition comprising a recombinant virusbased vector encoding a multi-antigen protein nanoparticle, optionally further comprising a pharmaceutically acceptable carrier or excipient.

[0499] In one aspect, provided is a recombinant virus-based vector encoding a multi-antigen protein nanoparticle for use in the prevention or treatment of a disease, preferably an infectious disease or cancer.

[0500] In one embodiment, the recombinant virus-based-vector encoding a multi-antigen protein nanoparticle is for use in the prevention or treatment of two different diseases.

[0501] In one embodiment of the recombinant virus-based vector encoding a multi-antigen protein nanoparticle for use in the prevention or treatment of two different diseases, the first infectious disease-associated antigen is from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and the second infectious disease-associated antigen is from Epstein-Barr Virus (EBV), or vice versa, and the two different infectious diseases are COVID-19 or long COVID and EBV infection or EBV associated cancer. In one embodiment of the recombinant virus-based vector encoding a multi-antigen protein nanoparticle for use in the prevention or treatment of a disease, the first disease-associated antigen is from Borrelia, preferably is derived from Borrelia outer surface protein A (OspA), and the second disease-associated antigen is from Borrelia, preferably is derived from Borrelia outer surface protein A (OspA), wherein the first and second disease-associated antigens are different, and the infectious disease is Lyme disease or Borrelia infection.

[0502] Further aspects and embodiments

[0503] In one aspect, provided is a fusion protein comprising a disease-associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle.

[0504] In one embodiment of the fusion protein, the self-assembling protein nanoparticle is imidazoleglycerol-phosphate dehydratase (HisB), preferably from Mycobacterium tuberculosis; acetyltransferase of pyruvate dehydrogenase (PDH) complex (PdhC), preferably from of Geo-) Bacillus stearothermophilus; and DNA binding protein from starved cells (DPS), preferably from Escherichia coll, preferably is HisB or DPS.

[0505] In one aspect, provided is a nucleic acid encoding the fusion protein.

[0506] In one aspect, provided is a process for preparing the nucleic acid described herein, comprising the steps of:

[0507] (a) providing a first section of the nucleic acid, which first section encodes a disease-associated antigen, or an antigenic part thereof;

[0508] (b) providing a second section of the nucleic acid, which second section encodes a subunit of a self-assembling protein nanoparticle, preferably HisB or DPS;

[0509] (c) joining the first and the second section of the nucleic acid, preferably via a peptide linker;

[0510] (d) obtaining the nucleic acid.

[0511] In one aspect, provided is a use of the nucleic acid described herein for the preparation of the recombinant virus-based vector, the recombinant poxvirus, the recombinant VRP, the RNA molecule or the expression plasmid as described herein.

[0512] In one aspect, provided is a self-assembling protein nanoparticle comprising the fusion protein. Further description

[0513] Modified Vaccinia Virus Ankara (MVA)

[0514] In the past, MVA was generated by 516 serial passages on chicken embryo fibroblasts of the Ankara strain of vaccinia virus (CVA) (for review see Mayr A et al. 1975. Abstammung, Eigenschaften und Verwendung des attenuierten Vaccinia-Stammes MVA. Infection 3:6-14). This virus was renamed from CVA to MVA at passage 570 to account for its substantially altered properties. MVA was subjected to further passages up to a passage number of over 570. As a consequence of these long-term passages, the genome of the resulting MVA virus had about 31 kilobases of its genomic sequence deleted and, therefore, was described as highly host cell restricted for replication to avian cells [7], It was shown in a variety of animal models that the resulting MVA was significantly avirulent compared to the fully replication competent starting material [8].

[0515] An MVA useful in the practice of the present invention includes MVA-572 (deposited as ECACC V94012707 on 27 January 1994); MVA-575 (deposited as ECACC V00120707 on 7 December 2000), MVA-1721 (referenced in [2]), NIH clone 1 (deposited as ATCC® PTA-5095 on 27 March 2003) and MVA-BN (deposited at the European Collection of Cell Cultures (ECACC) under number V00083008 on 30 August 2000).

[0516] More preferably the MVA used in accordance with the present invention includes MVA-BN and MVA-BN derivatives. MVA-BN has been described in WO 02 / 042480. “MVA-BN derivatives” refer to any virus exhibiting essentially the same replication characteristics as MVA-BN, as described herein, but exhibiting differences in one or more parts of their genomes.

[0517] MVA-BN, as well as MVA-BN derivatives, is replication incompetent, meaning a failure to reproductively replicate in vivo and in vitro. More specifically in vitro, MVA-BN or MVA-BN derivatives have been described as being capable of reproductive replication in chicken embryo fibroblasts (CEF), but not capable of reproductive replication in the human keratinocyte cell line HaCaT [9], the human bone osteosarcoma cell line 143B (ECACC Deposit No. 911 12502), the human embryo kidney cell line 293 (ECACC Deposit No. 85120602), and the human cervix adenocarcinoma cell line HeLa (ATCC Deposit No. CCL- 2). Additionally, MVA-BN or MVA-BN derivatives have a virus amplification ratio at least twofold less, more preferably three-fold less than MVA-575 in Hela cells and HaCaT cell lines. Tests and assay for these properties of MVA-BN and MVA-BN derivatives are described in WO 02 / 42480 and WO 03 / 048184. The term “not capable of reproductive replication” in human cell lines in vitro as described above is, for example, described in WO 02 / 42480, which also teaches how to obtain MVA having the desired properties as mentioned above. The term applies to a virus that has a virus amplification ratio in vitro at 4 days after infection of less than 1 using the assays described in WO 02 / 42480 or US 6,761 ,893.

[0518] Exemplary generation of a recombinant MVA virus

[0519] For the generation of recombinant MVA viruses as disclosed herein, different methods may be applicable. The DNA sequence to be inserted into the virus can be placed into an E. coli plasmid construct into which DNA homologous to a section of DNA of the poxvirus has been inserted. Separately, the DNA sequence to be inserted can be ligated to a promoter. The promoter-gene cassette can be positioned in the plasmid construct so that the promoter-gene cassette is flanked on both ends by DNA homologous to DNA sequences flanking a region of poxvirus DNA containing a non-essential locus or not containing any nucleotides. The resulting plasmid construct can be amplified by propagation within E. coli bacteria and isolated. The isolated plasmid containing the DNA gene sequence to be inserted can be transfected into a cell culture, e.g., of chicken embryo fibroblasts (CEFs), at the same time the culture is infected with MVA. Recombination between homologous sequences in the MVA viral genomic DNA and in the plasmid, respectively, can generate an MVA modified by the presence of foreign or heterologous DNA sequences, e.g., nucleotide sequences encoding SARS-CoV-2 or EBV antigens.

[0520] A cell of a suitable (i.e., permissive) cell culture as, e.g., CEF cells, can be infected with an MVA virus. The infected cell can be subsequently transfected with a first plasmid vector comprising a foreign or heterologous gene or genes, such as one or more of the nucleic acids provided herein, preferably under the transcriptional control of a poxvirus expression control element. As explained above, the plasmid vector also comprises sequences capable of directing the insertion of the exogenous sequence into a selected part of the MVA viral genome. Optionally, the plasmid vector also contains a cassette comprising a marker and / or selectable gene operably linked to a poxvirus promoter. The use of selection or marker cassettes facilitates the identification and isolation of the generated recombinant MVA. However, a recombinant poxvirus can also be identified by PCR technology. Subsequently, a further cell can be infected with the recombinant MVA obtained as described above and transfected with a second plasmid vector comprising a second foreign or heterologous gene or genes. In case, this gene shall be introduced into a different insertion site of the poxvirus genome, the second plasmid vector also differs in the poxvirus-homologous sequences directing the integration of the second foreign gene or genes into the genome of the poxvirus. After homologous recombination has occurred, the recombinant virus comprising two or more foreign or heterologous genes can be isolated. For introducing additional foreign genes into the recombinant virus, the steps of infection and transfection can be repeated by using the recombinant virus isolated in previous steps for infection and by using a further vector comprising a further foreign gene or genes for transfection. There are ample of other techniques known to generate recombinant MVA.

[0521] The practice of the invention will employ, if not otherwise specified, conventional techniques of immunology, molecular biology, microbiology, cell biology, and recombinant technology, which are all within the skill of the art. See e.g. Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition, 1989; Current Protocols in Molecular Biology, Ausubel FM, et al., eds, 1987; the series Methods in Enzymology (Academic Press, Inc.); PCR2: A Practical Approach, MacPherson MJ, Flams BD, Taylor GR, eds, 1995; Antibodies: A Laboratory Manual, Harlow and Lane, eds, 1988; Staib et al., Construction and isolation of recombinant MVA, 2004.

[0522] Virus replicon particles (VRP)

[0523] Alphaviruses belong to the Togaviridae family, positive sense, single stranded RNA viruses. Recombinant alphaviruses are capable of inducing high levels of heterologous gene expression, which make them attractive vectors for vaccine development. The alphaviral virion consists of the genomic RNA, which associates with the viral capsid protein (C) to form a an icosahedral nucleocapsid, which is again surrounded by a host cell derived lipid envelope containing the viral envelope protein, the E1 -E2 heterodimer.

[0524] The genomic RNA consists of two regions. The first of these is translated directly from mRNA- like genomic RNA and encodes the genes for the non-structural proteins 1 -4 (NSP1 -4), which form the viral replicase complex. The replicase produces copies of the genomic RNA via a negative strand intermediate. This negative strand intermediate contains a subgenomic promotor, from which the replicase additionally transcribes large quantities of a shorter subgenomic mRNA, containing the second region, and encoding the alphaviral structural proteins C, E3, E2, 6k and E1 . By replacing the genes of the structural proteins with an antigen of choice a recombinant alphaviral replicon can be generated. When supplying an alphaviral replicon with the alphaviral structural proteins in trans, the replicon is packaged, analogous to the packaging of intact alphaviral genomes. This gives rise to propagation defective viral replicon particles (VRPs) which, upon inoculation of VRPs into an animal, induce high levels of antigen expression, giving rise to robust immune responses, but cannot, in the absence of any alphavirus structural proteins, spawn any progeny virus. A number of different alphaviruses have been applied for the generation of VRPs, including the old world alphaviruses Sindbis Virus (SINV) and Semliki Forrest Virus (SFV), as well as the new world Venezuelan Equine Encephalitis Virus (VEEV), the TC-83 vaccine strain of which (GenBank: L01443.1 ) is of particular interest for use in vaccine development.

[0525] Exemplary generation of a recombinant TC-83 derived VRP

[0526] For the generation of recombinant VRPs as disclosed herein, different methods may be applicable. To insert an RNA sequence into a TC-83 replicon where they replace the sequences encoding the TC-83 structural proteins, a recombinant replicon plasmid can be generated. Such a plasmid contains a CMV promotor to launch the generation of the VEEV replicon RNA containing the VEEV TrD (GenBank: L01442.2) 5’ UTR, the coding sequence for the non-structural proteins (nsp) 1 , 2, 3 and 4 of VEEV TC-83 as well as the VEEV TC-83 subgenomic promotor and subgenomic 5’ UTR. Where in the intact TC-83 genome this would be followed by the capsid and envelope gene, the plasmid contains two Sapl restriction sites, which upon digestion remove themselves from the replicon plasmid backbone, leaving overhangs corresponding to the subgenomic start (ATG) and stop (TAA) codons, in between which the desired sequences, e.g., nucleotide sequences encoding SARS-CoV-2, EBV, or Borrelia antigens, can be introduced. The stop codon is followed by the VEEV TC-83 3’UTR, a ploy(A) sequence, the sequence for the HDV antigenomic ribozyme to terminate the replicon RNA. The HDV antigenomic ribozyme is followed by the SV40 poly(A) sequence. The replicon plasmid furthermore contains a ColE1 origin and a Kanamycin resistance cassette for propagation in E. coli.

[0527] VRP stocks were prepared by transfecting cells of a suitable cell culture e.g., a HEK293T suspension culture, see below) with three plasmids: Two pcDNA3.1 derived cytomegalovirus (CMV) promoter driven packaging plasmids, i.e., one encoding the capsid protein and one encoding the envelope polyprotein E3-E2-6k-E1 , and a recombinant replicon plasmid as described above. The replicon plasmid, as well as plasmids encoding the non-structural proteins of VEEV, strain TC83, were obtained as gene synthesis from GeneArt / ThermoFisher.

[0528] HEK293T suspension cells used for preparation of VRP stocks were derived from a HEK293T adhesion cell line (ATCC) by gradual depletion of serum in the growth medium to 1 % and growth on plastic surfaces without surface treatment for adherent growth under shaking conditions on an orbital shaker in a CO2 incubator at 37°C. Cells were seeded in BalanCD- Medium with 4 mM GlutaMAX and 0.1% Pluronic and 1% FCS at 1 x106cells / ml one day prior to transfection. On the day of transfection, cells were counted, and the medium was exchanged for serum-free DMEM. HEK293T suspension cultures (600-1000 ml) were transfected with 1 pg DNA per 1 x106cells at a plasmid molar ratio of 3:3:1 (capsid:envelope:replicon) using PEIpro (Polyplus, Strassbourg, France) according to the manufacturer’s instructions at a PEI:DNA ratio of 3:1 (pil / pig). Transfected cells were grown at 37°C / 125 rpm / 8% CO2 and supernatants were harvested 16-18 hours later. VRPs were harvested by ultracentrifugation of the supernatant at 26,000 rpm in an SW32Ti rotor for 2 hours at 4°C over a 20% sucrose cushion and subsequent resuspension of the pellet in an appropriate volume of resuspension buffer. The VRP stocks were titrated in several dilution steps on Vero cells (ATTC) by transduction. Transduced cells were quantified one day after transduction by staining double stranded RNA (dsRNA) replication intermediates with a dsRNA specific mouse monoclonal antibody (J2, Jena Bioscience) and FACS analysis.

[0529] EXAMPLES

[0530] The following examples serve to further illustrate the disclosure. They should not be understood as limiting the invention, the scope of which is determined by the appended claims.

[0531] EXAMPLE 1 : Materials and methods

[0532] 1 .1 Recombinant viral vectors

[0533] The effect of self-forming protein nanoparticles on antigen immunogenicity was evaluated using viral vectors for delivery of the coding nucleic acids: MVA served as a prototypic DNA viral vector, while virus replicon particles (VRPs) served as RNA vectors.

[0534] Three model antigens were used: The receptor binding domain (RBD) of the SARS-CoV-2 spike protein, the N-terminal domain of Epstein-Barr virus (EBV) surface glycoprotein 350 (gp350) and an artificial Lyme disease antigen based on outer surface protein A (OspA) from Borrelia.

[0535] 1.1.1 Preparation of recombinant MVA

[0536] The recombinant MVAs described herein were derived from MVA-BN® (Modified Vaccinia Virus Ankara of Bavarian Nordic, herein also referred to as “MVA-BN”).

[0537] Bullfrog / H pylori hybrid ferritin (BFF) was used as the self-assembling protein nanoparticle fused to either the SARS-CoV-2 spike RBD or EBV gp350 antigen. 1.1. 1. 1 MVA-RBD monomer and MVA-RBD-BFF

[0538] Recombinant MVA-resO65 and MVA-resO67 (see Fig. 28) encoded the SARS-CoV-2 spike protein receptor binding domain (RBD) as a monomer (see construct-1 in Fig. 1 ) or as a fusion protein of the RBD and Bullfrog / / - / , pylori hybrid ferritin (BFF) (see construct-2 in Fig. 1 ), respectively.

[0539] Herein, MVA-resO65 is also referred to as “MVA-RBD monomer”; MVA-resO67 is also referred to as “MVA-RBD-BFF”.

[0540] For generating MVA-resO65 and MVA-resO67, the original SARS-CoV-2 spike (S) protein sequence (YP 009724390.1 ) containing the RBD (amino acids 331 -524) served as a basis. The amino acid sequence to be expressed contained the RBD amino acid sequence and additional amino acids of the S1 domain (located N- and C-terminally from the RBD), thereby spanning amino acids 320-537 of the original full-length SARS-CoV-2 S1 domain. A secretion tag (signal peptide) from human tissue plasminogen activator (htPA) was added N-terminally to allow efficient secretion of the S1 domain fragment including the RBD.

[0541] MVA-resO67 encoded the htPA-RBD protein fused to BFF for antigen oligomerization. Hybrid BFF protein consisted of amino acids 2-9 of Bullfrog (Rana catesbeiana) ferritin and amino acids 3-167 of the Helicobacter pylori ferritin. Bullfrog ferritin is based on UniProt ID P07797.1 , and H. pylori nonheme ferritin is based on UniProt ID Q9ZLI1.1. A nine amino acid linker (SSGGASVLA) was located between the RBD and the hybrid BFF protein.

[0542] The nucleotide sequences of the inserted transgenes were optimized for human codon usage, and repetitive elements as well as G / C or A / T-rich polynucleotide stretches were removed.

[0543] Expression of the RBD monomer and RBD-BFF fusion protein by MVA was driven by the Pr13.5long promoter (Wennier et al., 2013

[0010] ; WO 2014 / 063832). RBD as well as RBD-BFF expression cassettes were inserted into the intergenic region (IGR) 64 / 65 of MVA-BN (see Fig. 28).

[0544] 1. 1. 1.2 MVA-gp350-GCN4 and MVA-gp350-BFF

[0545] Gene BLLF1 encoding glycoprotein 350 (gp350) of Epstein-Barr Virus (EBV) strain B95-8 was chosen to be inserted in the MVA-BN genome. Recombinant MVA-mBN443B and MVA- mBN510 (see Fig. 28) encoded an EBV strain B95-8 derived gp350 fragment (amino acids 1 - 434) from the extracellular domain of gp350 fused to a GCN4-derived tetramerization domain (see construct-3A in Fig. 8) and an EBV gp350-BFF fusion protein containing amino acids 2- 434 of the extracellular domain of gp350 N-terminally fused to a signal peptide from the murine immunoglobulin kappa light chain and C-terminally fused to BFF (see construct-4 in Fig. 8), respectively.

[0546] Herein, MVA-mBN443B is also referred to as “MVA-gp350-GCN4”, MVA-mBN510 is also referred to as “MVA-gp350-BFF”.

[0547] In MVA-mBN443B, a truncated version (amino acids 1 -434) of the gp350 protein, which gets secreted upon expression based on the naturally contained secretion signal at the N-terminus of the gp350 fragment, was fused to a synthetic multimerization domain derived from yeast GCN4

[0011] , The gp350 protein sequence is based on GenBank entry YP 401667.1 , the yeast derived GCN4 sequence is based on 2IPZ A. A 13 amino acid linker (PKPSTPPGSSCGG) was inserted between gp350 and GCN4.

[0548] In the gp350-BFF fusion protein encoded by MVA-mBN510, the N-terminal part of the gp350 extracellular domain (amino acids 2-434) was fused to BFF for antigen oligomerization. In addition, the signal peptide of the murine Ig kappa light chain (IgK LC) was fused to the N- terminus to allow efficient secretion. The resulting BFF-gp350 fusion protein lacks a transmembrane domain and should therefore be secreted. A ten amino acid linker (PKPSTPPGSS) was located between gp350 and BFF.

[0549] All protein sequences were optimized on nucleotide level for human codon usage, and repetitive elements and nucleotide-stretches were removed.

[0550] Expression of gp350-GCN4 in MVA-mBN443B as well as gp350-BFF in MVA-mBN510 was driven by the Pr13.5long promoter (Wennier et al., 2013

[0010] ; WO 2014 / 063832). For MVA- mBN443B, the gp350-GCN4 tetramer coding sequence was inserted into the intergenic region (IGR) 88 / 89 of the MVA-BN genome; for MVA-mBN510, the gp350-BFF coding sequence was inserted into IGR 64 / 65 (see Fig. 28).

[0551] Both recombinant MVAs encoded additional EBV-derived antigens (i.e., BXLF2 encoding gH, BKRF2 encoding gl_, BRLF1 , BZLF1 , and EBNA3A) (see Fig. 28). In MVA-mBN510, encoded gH and gl_ were separated from each other by a P2A site.

[0552] 1.1.2 Preparation of VRPs

[0553] The virus replicon particles (VRPs) were based on live, attenuated Venezuelan Equine Encephalitis Virus (VEEV), strain TC-83.

[0554] Bullfrog / H pylori hybrid ferritin (BFF) was used as the self-assembling protein nanoparticle fused to either the SARS-CoV-2 spike RBD, EBV gp350 or Lyme disease antigen. Furthermore, nanoparticle candidates other than BFF (see Example 6) were tested. 1.1.2.1 VRP-RBD monomer and VRP-RBD-BFF

[0555] The recombinant VRP-BN035 was a virus replicon particle (VRP) encoding the RBD in monomeric form (see construct-1 in Fig. 1 ), while VRP-BN033 encoded the RBD-BFF fusion protein (see construct-2 in Fig. 1 ).

[0556] Herein, VRP-BN035 is also referred to as “VRP-RBD monomer”; VRP-BN033 is also referred to as “VRP-RBD-BFF”.

[0557] For generating VRP-BN033 and VRP-BN035, construct-1 and construct-2 (see Fig. 1 ) as described in 1.1.1.1 were inserted in a VEEV TC-83 derived replicon, respectively. The sequences were codon optimized and synthesized including homology overhangs from the VEEV subgenomic 5’ and the 3’ UTR on each end, respectively, to facilitate insertion by homology cloning into the VEEV TC-83 replicon plasmid described above (see “Exemplary generation of a recombinant TC-83 derived VRP”).

[0558] 1.1.2.2 VRP-gp350-GCN4 and VRP-gp350-BFF

[0559] The recombinant VRP-BN043 was a virus replicon particle (VRP) encoding gp350 from EBV fused to a GCN4 derived tetramerization domain (see construct-3B in Fig. 8), while VRP- BN052 encoded the gp350-BFF fusion protein (see construct-4 in Fig. 8).

[0560] VRP-BN043 is herein also referred to as “VRP-gp350 GCN4”. VRP-BN052 is herein also referred to as “VRP-gp350-BFF”.

[0561] Sequences for the generation of VRP-BN043 and VRP-BN052 are described in 1.1.1.2 with the exception that the sequence encoding the gp350-GCN4 fusion protein used here is preceded by a sequence encoding the murine Ig Kappa light chain signal peptide (IgK LC SP) to aid secretion. The sequences were codon optimized and synthesized including homology overhangs from the VEEV subgenomic 5’ and the 3’ UTR on each end respectively, to facilitate insertion by homology cloning into the VEEV TC-83 replicon plasmid described above (see “Exemplary generation of a recombinant TC-83 derived VRP”).

[0562] Both VRPs encoded additional EBV antigens ( / .e., BXLF2 encoding gH, BKRF2 encoding gL) separated from each other by P2A sites.

[0563] 1. 1.2.3 VRP-OspAST1 -5-6 and VRP-OspA-ST1-5-6-BFF

[0564] The recombinant VRP-BN083 was a virus replicon particle (VRP) encoding a string of three OspA C-terminal fragments, i.e., a fusion protein of amino acids 126-273 of OspA serotype 1 (ST1 ) from Borrelia burgdorferi sensu stricto (strain B31 ), amino acids 126-273 of OspA ST5 from Borrelia garinii (strain PHei) and amino acids 126-274 of OspA ST6 from Borrelia garinii (DK29). The OspA C-terminal fragments were separated from each other by (GGGGS)4 linkers and fused N-terminally to an htPA signal peptide (see construct-5 in Fig. 12). Furthermore, the C-terminal fragments were stabilized by the introduction of disulfide bridges as described in

[0012] , and all N-X-S / T N-glycosylation sequons were removed by N to Q mutation.

[0565] VRP-BN067 encoded the same fusion protein of OspA C-terminal fragments, but furthermore fused C-terminally to BFF via a nine amino acid SSGGASVLA linker (see construct-6 in Fig. 12).

[0566] VRP-BN083 is herein also referred to as “VRP-OspA ST1-5-6”. VRP-BN067 is herein also referred to as “VRP-OspA ST1-5-6-BFF”.

[0567] Sequences for the generation of VRP-BN083 and VRP-BN067 were codon optimized and synthesized including homology overhangs from the VEEV subgenomic 5’ and the 3’ UTR on each end, respectively, to facilitate insertion by homology cloning into the VEEV TC-83 replicon plasmid described above.

[0568] 1.1.2.4 VRPs encoding RBD fused to nanoparticle candidates

[0569] Imidazoleglycerol-phosphate dehydratase from mycobacterium tuberculosis (HisB, uniprot: P9WML9), Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex from Geobacillus stearothermophilus (PdhC E2, uniprot: P11961 ), DNA protection during starvation protein from Escherichia coll strain K12 (DPS, uniprot: P0ABT2), Sulfur oxygenase / reductase from Acidianus ambivalens (SOR, uniprot: P29082) and Mersacidin decarboxylase from Bacillus sp. strain HIL-Y85 / 54728 (MrsD, uniprot: Q9RC23) were tested as self-assembling nanoparticles capable of displaying antigens. To this end, BFF in the RBD-BFF replicon plasmid (see 1 .1 .2.1 ) was replaced by amino acids 9- 210 of HisB, amino acids 185-428 of PdhC E2, amino acids 11-167 of DPS, amino acids 2- 309 of SOR and amino acids 2-194 of MrsD, respectively (see construct-7 to construct-11 in Fig. 15).

[0570] Additionally, replicon plasmids were generated in which the position of RBD and HisB, DPS, SOR and MrsD were swapped so that the nanoparticle forming subunit was now fused to the RBD N-terminus (see construct-12 to construct-15 in Fig. 17). The sequences were codon optimized and synthesized including homology overhangs from the VEEV subgenomic 5’ and the 3’ UTR on each end respectively, to facilitate insertion by homology cloning into the VEEV TC-83 replicon plasmid described above. The recombinant VRP-BN060 was a virus replicon particle (VRP) encoding the RBD-DPS fusion protein (see construct-9 in Fig. 15). The recombinant VRP-BN070 was a virus replicon particle (VRP) encoding the HisB-RBD fusion protein (see construct-12 in Fig. 17). The recombinant VRP-BN071 was a virus replicon particle (VRP) encoding the DPS-RBD fusion protein (see construct-13 in Fig. 17).

[0571] Herein, VRP-BN060 is also referred to as “VRP-RBD-DPS”, VRP-BN070 is also referred to as “VRP-HisB-RBD” and VRP-BN071 is also referred to as “VRP-DPS-RBD”.

[0572] 1. 1.2.5 VRPs encoding EBV gp350 fused to nanoparticle candidates

[0573] The recombinant VRP-BN096 was a virus replicon particle (VRP) encoding the gp350 sequence form EBV as described in 1.1.1.2 fused to DPS (see construct-17 in Fig. 21), while VRP-BN098 encodes the same gp350 sequence but fused to PdhC (see construct-16 in Fig. 21 ).

[0574] For the generation of VRP-BN096 and VRP-BN098 the sequences coding for construct-14 and construct-15 were codon optimized and synthesized including homology overhangs from the VEEV subgenomic 5’ and the 3’ UTR on each end respectively, to facilitate insertion by homology cloning into the VEEV TC-83 replicon plasmid described above.

[0575] VRP-BN096 is herein also referred to as “VRP-gp350-DPS”. VRP-BN098 is herein also referred to as “VRP-gp350-PdhC”.

[0576] 1.1.2.6 VRPs encoding RBD-BFF / gp350-BFF multi-antigen nanoparticles

[0577] VRP-BN072 was a virus replicon particle (VRP) containing a nucleic acid encoding the gp350- BFF fusion protein (described in 1.1.1.2) followed by a T2A site and the RBD-BFF fusion protein (described in 1.1.1.1 ) (see construct-18 in Fig. 23). For generating VRP-BN072, sequences for construct-2 (see Fig. 1 ) and construct-4 (see Fig. 8) were fused, separated by a T2A site, and inserted in a VEEV TC-83 derived replicon by homology cloning.

[0578] 1.1.2.7 VRPs encoding OspA multi -antigen nanoparticles

[0579] VRP-BN068 was a virus replicon particle (VRP) encoding a string of three OspA C-terminal fragments ( / .e., a fusion protein of amino acids 126-273 of OspA serotype 2 (ST2) from Borrelia afzelii (strain K78), amino acids 126-273 of OspA ST4 from Borrelia bavariensis (strain PBi) and amino acids 126-274 of OspA ST3 from Borrelia garinii (PBr)) fused to BFF via a nine amino acid SSGGASVLA linker. The OspA C-terminal fragments were separated from each other by (GGGGS)4 linkers and fused N-terminally to an htPA signal peptide (see construct-19 in Fig. 26). Furthermore, the C-terminal fragments are stabilized by the introduction of disulfide bridges as described in

[0012] , and all N-X-S / T N-glycosylation sequons were removed by N to Q mutation.

[0580] VRP-BN069 encoded both the OspA-BFF fusion proteins encoded in VRP-BN067 (see 1 .1 .2.3) and VRP-BN068, separated by a T2A site (see construct-20 in Fig. 26).

[0581] VRP-BN068 is herein also referred to as “VRP-OspA ST2-4-3-BFF”. VRP-BN069 is herein also referred to as “VRP-OspA ST1 -5-6-BFF / OspA ST2-4-3-BFF”.

[0582] The sequences for the generation of VRP-BN068 were codon optimized and synthesized including homology overhangs from the VEEV subgenomic 5’ and the 3’ UTR on each end, respectively, to facilitate insertion by homology cloning into the VEEV TC-83 replicon plasmid described above. For generating VRP-BN069 sequences for construct-6 (Fig. 12) and construct-19 (Fig. 26) were fused, separated by a T2A site, and inserted in a VEEV TC-83 derived replicon by homology cloning.

[0583] 1 .2 mRNA, saRNA and plasmid DNA

[0584] Nucleic acids encoding the SARS-CoV-2 spike receptor binding domain (RBD) as a monomer (see construct-1 in Fig. 1 ) or as a fusion protein of the RBD and Bullfrog / / - / , pylori hybrid ferritin (BFF) (see construct-2 in Fig. 1 ) were generated in the form of (i) an expression plasmid containing the respective gene under the control of a CMV promotor, (ii) a self-amplifying RNA (saRNA) based on the VEEV replicon, and (iii) a capped and polyadenylated mRNA.

[0585] Both mRNA and saRNA were generated by in vitro transcription (IVT) from DNA templates using HiScribe® T7 High Yield RNA Synthesis Kit (NEB, E2040S). CleanCap AU (TriLink, N- 7114-1 ) was added into the IVT reaction mix to generate capped RNA. After IVT, RNAs were purified using Monarch® RNA Cleanup Kit (NEB, T2050S).

[0586] Plasmids containing templates for mRNA or saRNA were cloned. The two plasmids containing templates for saRNA were generated by using the plasmid pCR-Blunt ll-TOPO (Invitrogen) as vector. The two templates shared identical sequences with the replicon plasmids used for the preparation of VRPs (see Example 1.1.2.1 ), but with a T7 promoter instead of the CMV promoter before the replicon sequence. After the replicon sequence, a poly(A) tail with about 120 adenines was added. Additionally, PaqCI restriction sites were incorporated after the poly(A) tail for linearization of the template plasmids. After IVT, the two resulting saRNA molecules contained the identical replicon sequence as VRP-BN033 and VRP-BN035, but with a pseudo capping structure (CleanCap AU) and a long poly(A) tail of about 120 adenines. The two plasmids containing templates for mRNA were generated by using the plasmid pcDNA4 / TO / myc-His A (ThermoFisher) as vector. The two mRNA templates contained the identical coding sequences for monomeric SARS-CoV-2 Spike RBD protein or the fusion protein of the SARS-CoV-2 Spike RBD and BFF as the saRNA. The coding sequences were flanked by the 5’ and 3’ untranslated regions (UTRs). The 5’ and 3’ UTRs were derived from the human beta globin (HBG) gene. A T7 promoter was introduced before the 5’ UTR for launching the IVT. Additionally, an AT sequence was inserted between the T7 promoter and the 5’ UTR for adding the CleanCap-AU capping analog. Following the 3’ UTR, a poly(A) tail consisting of about 120 adenine residues was added. A Bael restriction site was introduced immediately following the poly(A) tail for linearization of transcription template.

[0587] For plasmid DNA, two expression plasmids encoding construct-1 or construct-2 (Fig. 1 ) were prepared by subcloning the coding sequences into the pcDNA3.1 vector at the Hindlll and EcoRI restriction sites. The inserted coding sequences were under control of the CMV promotor, allowing gene expression in eukaryotic cells.

[0588] 1.3 Analysis of antigen expression and nanoparticle assembly

[0589] 1.3.1 In vitro protein expression

[0590] Cellular expression and secretion of antigens as well as nanoparticle assembly was examined in cell cultures.

[0591] For infection with MVA, 0.5x106HeLa cells / well were seeded in 6-well plates and infected on the next day with 1 .0x107InfU of the relevant MVA. Cells and supernatant were harvested for analysis 20-24 hours later.

[0592] Instead of complete VRPs, VEEV derived replicon plasmids able to launch the replicon upon transfection into cells were used. For transfection, 1.3x106HEK293T cells / well were seeded in 6-well plates and transfected on the next day with 1 pg of the relevant replicon launching plasmid using PEIpro transfection reagent. Cells and supernatant were harvested for analysis 16-24 hours later.

[0593] VEEV TC-83 derived recombinant VRPs were used for analysis of protein expression. For infection, 5x105Vero cells / well were seeded in 6-well plates and infected on the next day with 5x106TU of the relevant VRP. Cells and supernatant were harvested for analysis 16-24 hours later. For transfection with mRNA, saRNA or plasmid DNA, 1.3x106HEK293T cells / well were seeded in 6-well plates and infected on the next day with 1 pg of the relevant nucleic acid. Cells and supernatant were harvested for analysis 16-48 hours later.

[0594] 1.3.2 Western blot

[0595] Lysates and supernatants of cells infected with recombinant MVA, transfected with plasmid DNA encoding the CMV launched recombinant replicon, or transfected with mRNA, saRNA, or plasmid DNA were analyzed for expressed antigens.

[0596] Cell lysates and supernatants were collected and analyzed by Western blot. Samples of lysates or supernatants were separated by electrophoresis on 10% poly-acrylamide gels (PAGE) under denaturing conditions, and supernatants were additionally run on 3-12% polyacrylamide gels under native conditions in the presence of G-250 dye. After electrophoresis, gels were blotted onto nitrocellulose membranes, which were blocked in 10% skim milk powder in Tris-buffered saline with 0.1 % Tween 20 (TBS-T).

[0597] Antigens were detected using antigen-specific antibodies: rabbit anti-SARS-CoV-2 spike antibody (Sino Biological #40592-T62); anti-EBV gp350 (72A1 , Sigma #MAB10219); rabbit anti-ftnA (Cusabio #CSB-PA009053ZA01 HUV); horseradish peroxidase (HRP) conjugated goat anti-rabbit antibody (Promega #W401 B); and goat anti-rabbit antibody (Promega #W402B); respectively. All antibodies were diluted in 5% skim milk powder in Tris-buffered saline with 0.1 % Tween 20 (TBS-T). Detected bands were visualized using chemiluminescent HRP substrate (Amersham ECL Detection Reagents).

[0598] 1.4 Immunogenicity studies in vivo

[0599] 1.4.1 Mice experiments

[0600] Female Balb / c mice (6-8 weeks old, Janvier) were immunized intramuscularly on day 0 (prime immunization) and day 21 (boost immunization) with 1 x108or 1 x107InfU of recombinant MVA, 1 x108TU of VRPs, or 2.5 pg saRNA. Similarly, CD-1 mice were immunized intramuscularly on day 0 (prime immunization) and day 21 (boost immunization) with 1 x108InfU of recombinant MVA or 1 x108TU of VRPs. Blood for serum isolation was drawn between day 34 or day 42 after prime immunization. Total anti-antigen IgG titers in the serum were analyzed by ELISA (see 1 .4.2), the ePass technology was used to assess antigen-binding antibody titers (as 50% inhibition) (see 1 .4.3). For T cell analysis, mice were sacrificed on day 35 or 42 after prime immunization and single-cell suspensions from splenocytes were analyzed by EliSpot (see Example 1.4.4). 1.4.2 ELISA

[0601] Anti-RBD Ig titers in the serum were analyzed by enzyme-linked immunosorbent assay (ELISA) on RBD-coated plates with a starting dilution of 1 :300 to determine total anti-RBD Ig titers using anti-mouse IgG (H+L) antibodies. Anti-OspA-ST1 or anti-OspA-ST2 Ig titers in the serum were analyzed by ELISA on OspA-ST1 or OspA-ST2-coated plates with a starting dilution of 1 :300 to determine total anti-OspA-ST 1 or anti-OspA-ST2 Ig titers using anti-mouse IgG (Fc) antibody conjugated to HRP. Arbitrary titers (AU = arbitrary units) were calculated via a 4PL-fit curve with an intercept at OD = 0.3. Additionally, anti-RBD and anti-gp350 IgG titers were analyzed by a standard based ELISA. For this, serum was diluted in 1 :5 dilution steps starting at 1 :100 or 1 :200 and commercially available anti-RBD (R&D Systems; MAB105808) or anti-gp350 (Merck; MAB8183) antibody was used to establish a standard for anti-RBD or anti-gp350 IgG serum antibody quantification. Data are shown as Mean ± SEM.

[0602] 1 .4.3 Neutralization assay (ePass) ePass is a validated surrogate virus neutralization test that measures the ability of immune sera to interfere with the binding of an antigen to its receptor on human cells, i.e., binding of SARS-CoV-2 Spike RBD to angiotensin converting enzyme-2 (ACE2).

[0603] In detail, serum samples were serially diluted in 3-fold dilutions steps starting at a dilution of 1 :100. 1 :1000 diluted RBD-HRP was mixed with serum samples in 1 :1 ratio and incubated for 30 min at 37°C. After incubation, serum-RBD-HRP mixture was added to the ACE2-coated plate and incubated for 15 min at 37°C. After washing, TMB solution was added to each well and the plate was incubated in dark at 25°C for 15 min. After this incubation time, the reaction was stopped, and the absorbance was read at 450 nm immediately using a microtiter plate reader. Titers were calculated at 50% RBD binding inhibition compared to the negative control.

[0604] 1 .4.4 ELISpot

[0605] IFN-y producing T cells were detected using an Enzyme Linked Immuno Spot (ELISpot) assay. For this, 0.5x106splenocytes were seeded in anti-IFN-y coated ELISpot plates and restimulated with Spike Peptide Pool A (containing the SARS-CoV-2 Spike RBD region) (Genscript) or gp350 peptide pool (JPT Peptide Technologies or Peptides&Elephants), or with control medium. ELISpot plates were incubated overnight at 37°C before development. IFN- y-producing cells were revealed with biotin-conjugated anti-IFN-y antibody in combination with streptavidin-horseradish peroxidase (HRP) and 3-amino-9-ethylcarbazole (AEC) substrate. Spots were counted using an ELISpot reader (Immunospot S6 Universal Analyzer). 1.4.5 FACS

[0606] RBD-specific memory B cells and germinal center B cells were revealed with a combination of fluorescently labelled RBD protein and different antibodies before analysis by flow cytometry. Briefly, single cells were stained for 20 min at 4°C with Zombie Aqua in PBS (Biolegend) viability dye in 96-well V-bottom plates. After incubation the cells were washed twice with fluorescence-activated cell sorting (FACS) buffer (PBS supplemented with 2 % FCS and 0.02 % sodium azide) and then stained for 10 minutes at 4°C with AF488 or AF647- labelled RBD protein to stain RBD-specific B cells. Labelled RBD protein was acquired through R&D Systems. 50 pL antibody mix containing anti-GL7-PerCp-Cy5.5, anti-CD38-PE- eFLuor610, anti-lgG1 -BV711 , anti-lgG2a / b-BV650, anti-lgM-BV605, anti-CD3-BV510, anti- lgD-APC-Cy7 and anti-CD45R-AF700 was added to each well. All antibodies were acquired through BD Biosciences, Biolegend or ThermoFisher Scientific. Cells were stained for an additional 20 minutes before being washed twice with FACS buffer. Finally, cells were filtered through a 100 pm membrane and analyzed on an LSR-II flow cytometer (BD Biosciences).

[0607] EXAMPLE 2: Effect of Bullfrog / H. pylori hybrid ferritin (BFF) nanoparticle on the expression and immunogenicity of SARS-CoV-2 Spike receptor binding domain (RBD) antigen delivered by MVA or VRPs

[0608] The receptor binding domain (RBD) of the SARS-CoV-2 spike protein was used as a first model antigen. The RBD is the target for most neutralizing antibodies against SARS-CoV-2, and high anti-RBD antibody titers are known to correlate with protection from disease.

[0609] 2.1 SARS-CoV-2 Spike RBD antigen constructs with and without BFF nanoparticle

[0610] Two antigen constructs were designed: A first one containing the RBD of SARS-CoV-2 spike protein and a signal peptide of human tissue plasminogen activator (htPA) fused thereto (see construct-1 in Fig. 1 ); and a second one containing the RBD and the htPA signal peptide like in construct-1 , but with the RBD being additionally fused to Bullfrog / / - / , pylori hybrid ferritin (BFF), (see construct-2 in Fig. 1 ). During synthesis of the RBD monomer or RBD-BFF fusion protein, the signal peptide is cleaved off and is thus not present in the mature protein anymore.

[0611] Recombinant MVA and VRPs encoding either construct-1 or construct-2 were prepared as described in Example 1.1.1 and 1.1.2, respectively. 2.2 Western blot analysis of RBD and RBD-BFF proteins encoded by recombinant MVA

[0612] HeLa cells were infected with recombinant MVA (MOI of 10) encoding the monomeric RBD protein or RBD-BFF fusion protein. The next day, cell lysates and supernatants were collected and analyzed by Western blot (see Example 1 .3.2).

[0613] Results are shown in Fig. 2. Protein bands of the expected sizes were indicative of expression (lysate) and secretion (supernatants) of RBD and RBD-BFF, and of nanoparticle assembly (supernatant, native conditions).

[0614] The monomeric SARS-CoV-2 Spike RBD has a calculated molecular weight of 24.48 kDa, which is further increased through the presence of two N-glycans that are added to the protein during processing within the secretory pathway (resulting in an apparent molecular weight in Western blot of about 30 kDa). The RBD-BFF fusion protein has a calculated molecular weight of 45.29 kDa, which is likewise increased through the presence of the same two N-glycans. The assembled 24mer RBD-BFF nanoparticle has a molecular weight of 1 .09 mDa (24 x 45.29 kDa) plus additional weight from the glycan.

[0615] Under denaturing electrophoresis conditions, a band of around 30 kDa was detected in lysates and supernatants of MVA-RBD monomer infected cells (lane 1 in Fig. 2A, 2B), indicating successful expression and secretion of the RBD monomer. A band of around 45 kDa was detected in lysates and supernatants of MVA-RBD-BFF infected cells (lane 2 in Fig. 2A, 2B), indicating successful expression and secretion also of the RBD-BFF fusion protein.

[0616] When cell supernatants underwent electrophoresis under native conditions, a faint band at a molecular weight of around 60 kDa was observed for MVA-RBD infected cells (lane 1 in Fig. 2C), suggesting some dimerization of the monomeric RBD in solution. Most notably, however, a clearly defined high molecular weight band was detected in supernatants of MVA- RBD-BFF infected cells (lane 2 in Fig. 2C), indicating an assembly of RBD-BFF fusion protein into nanoparticles.

[0617] Thus, recombinant MVA as an example of DNA viral vectors was shown to deliver selfassembling RBD-BFF nanoparticles.

[0618] 2.3 Western blot analysis of RBD and RBD-BFF proteins encoded by VRPs

[0619] HEK293T cells were transfected with plasmid DNA containing either the CMV launched recombinant replicon encoding the monomeric RBD protein (construct-1 in Fig. 1 ) or the CMV launched recombinant replicon encoding the RBD-BFF fusion protein (construct-2 in Fig. 1 ). The next day, cell lysates and supernatants were collected and analyzed by Western blot.

[0620] Results are shown in Fig. 3. For comments on the molecular weights of RBD and RBD-BFF proteins and respective sizes of their protein bands, see Example 2.2 above.

[0621] Under denaturing electrophoresis conditions, monomeric RBD was detected in lysates and supernatants of cells transfected with VRP-RBD (lane 1 in Fig. 3A, 3B). Similarly, RBD-BFF was detected in lysates and supernatants of cells transfected with VRP-RBD-BFF (lane 2 in Fig. 3A, 3B). Thus, VRP encoded RBD and RBD-BFF proteins were successfully expressed and secreted.

[0622] After native electrophoresis, the faint band at a molecular weight of around 60 kDa observed for VRP-RBD infected cells (lane 1 in Fig. 3C) suggested dimerization of the monomeric RBD in solution, while the clearly defined high molecular weight band detected in supernatants of VRP-RBD-BFF infected cells (lane 2 in Fig. 3C) indicated assembly of RBD-BFF fusion protein into nanoparticles.

[0623] Thus, VRPs as an example of RNA viral vectors were shown to deliver self-assembled BFF- RBD nanoparticles.

[0624] 2.4 Immune responses to RBD or RBD-BFF delivered by recombinant MVA or VRPs

[0625] To assess the immunogenicity of the RBD antigen, either presented on a BFF nanoparticle or not, Balbc mice were prime / boost immunized on day 0 and 21 with the respective recombinant MVA or VRPs.

[0626] Recombinant MVA encoding monomeric RBD (MVA-RBD monomer) was compared to recombinant MVA expressing the BFF-RBD fusion protein (MVA-RBD-BFF) (Fig. 4A-C), and VRPs encoding monomeric RBD (VRP-RBD-monomer) was compared to VRP encoding BFF- RBD fusion (VRP-RBD-BFF) (Fig. 4D-F).

[0627] The results presented in Fig. 4 show strong induction of humoral immune responses when RBD was presented on self-forming nanoparticle BFF (see “MVA-RBD-BFF” and “VRP-RBD- BFF” in Fig. 4). Total anti-RBD IgG were strongly elevated compared to non-particulate antigen (see “MVA-RBD-monomer” and “VRP-RBD-monomer” in Fig. 4A, 4D). Furthermore, neutralizing RBD-binding antibody titers were measured using serum samples. To that aim, a commercially available surrogate virus neutralization assay (Genscript-cPass) was used and by generating serial dilution curves, we determined the 50% inhibition titer. This is the titer at which 50% of RBD-ACE2 binding is inhibited by the serum antibodies. Similar to total IgG titers, neutralizing RBD antibodies were strongly increased compared to non-particulate antigen (Fig. 4B, 4E).

[0628] In addition to humoral immune responses, T cell responses were assessed. Splenocytes were isolated on day 34 (14 days after boost immunization) and IFN-y ELISpot analyses were performed (see Example 1.4.4). IFN-y spots were counted and are presented in Fig. 4C, 4F. In all animal test groups strong SARS-CoV-2-Spike specific T cell responses were induced, and no difference was detected between the RBD-monomer and RBD-BFF.

[0629] Taken together, the data demonstrated that BFF-mediated antigen presentation induced solid T cell responses, but most importantly strongly elevated antibody responses irrespective of whether the antigen was delivered by recombinant MVA or VRP.

[0630] 2.5 Induction of germinal center and memory B cells by RBD or RBD-BFF delivered by recombinant MVA

[0631] After intramuscular immunization, antigen quickly reaches the draining lymph nodes, and an immune response is initialized. Recognition of antigen by B cells and T cells leads to cell activation and the generation of germinal centers (GC) in the lymph nodes. Germinal centers are highly specialized clusters of B and T cells in which antigen-specific B cells are maturing and may eventually become highly specific memory B cells or antibody-producing plasma cells. Germinal centers are therefore a prerequisite for strong antibody responses and B cell memory.

[0632] To examine whether nanoparticle-associated antigen expressed by recombinant MVA is a potent inducer of germinal centers reactions, Balb / c mice were immunized intramuscularly with 1 x108Infll of MVA-RBD, MVA-RBD-BFF or an equal volume of T ris-buffered saline (TBS) as buffer control. Inguinal and popliteal lymph nodes, the draining lymph nodes after intramuscular injection, were isolated 7 or 14 days after immunization to analyze the capacity of the recombinant MVAs to induce GC B cells and memory B cells. Lymph nodes were pooled and processed, and single cells were stained with fluorochrome-labelled antibodies and fluorochrome-labelled RBD protein for flow cytometric analysis (see Example 1.4.5). The results are shown in Fig. 5.

[0633] Analysis of B cells in the lymph nodes showed that MVA-RBD-BFF induced high percentage of GC B cells by day 7 (Fig. 5A) resulting in high abundance of memory B cells on day 14 (Fig. 5B). These results indicate that MVA-RBD-BFF is a better inducer of GC and memory B cell responses than MVA-RBD. EXAMPLE 3: Effect of BFF nanoparticle on RBD antigen expression and immunogenicity when delivered by mRNA, saRNA or plasmid DNA

[0634] It was investigated whether nanoparticle assembly and increased antibody induction would also take place when the antigen was delivered by “naked” nucleic acid, i.e., mRNA, selfamplifying RNA (saRNA) or plasmid DNA.

[0635] 3.1 mRNA, saRNA or plasmid DNA encoding RBD antigen constructs with and without BFF nanoparticle

[0636] Each type of the “naked” nucleic acids encoded either construct-1 or construct-2 (see Fig. 1 ). The mRNA, saRNA and plasmid DNA were prepared as described in Example 1 .2.

[0637] 3.2 Western blot analysis of RBD and RBD-BFF proteins encoded by mRNA, saRNA or plasmid DNA

[0638] HEK293T cells were transfected with mRNA, saRNA or plasmid DNA encoding construct-1 or construct-2. For this purpose, HEK293T cells seeded in 6-well plates were transfected with 1 pg mRNA or saRNA per well by using the jetMESSENGER RNA transfection reagent (PolyPlus). For transfection of plasmid DNA, HEK293T cells seeded in 6-well plates were transfected with 1 pg DNA per well by using the PEIpro transfection reagent (PolyPlus). At 24 hours post transfection cell lysates and supernatants were collected and analyzed by Western blot.

[0639] Results are shown in Fig. 6. For comments on the molecular weights of the RBD-monomer and RBD-BFF fusion protein and respective sizes of their protein bands, see Example 2.2.

[0640] Under denaturing electrophoresis conditions, a band at around 30 kDa {i.e., the apparent molecular weight of RBD) was detected in blots of lysates and supernatants of cells transfected with mRNA, saRNA or plasmid DNA encoding the monomeric RBD (see lanes 1 in Fig. 6A, 6B), while a band at around 45 kDa (indicating RBD-BFF fusion protein) was detected in blots of lysates and supernatants of cells transfected with mRNA, saRNA or plasmid DNA encoding the RBD-BFF fusion protein (see lanes 2 in Fig. 6A, 6B). This indicated that both construct-1 and construct-2 were expressed upon delivery by all three types of “naked” nucleic acids. The level of expression varied though, which was lowest for mRNA transfected cells and equivalent for cells transfected with saRNA or with plasmid DNA.

[0641] The clearly defined high molecular weight band detected in blots of supernatants run under native electrophoresis conditions (see lanes 2 in Fig. 6C) showed that assembly of RBD-BFF into nanoparticle took place, irrespective of the type of nucleic acid used for delivery. 3.3 Immune responses to RBD or RBD-BFF antigens delivered by saRNA

[0642] To assess the immunogenicity of saRNA expressing BFF nanoparticle-forming proteins fused to the antigen RBD, mice were prime / boost immunized on days 0 and 21 with either saRNA encoding monomeric RBD (saRNA-RBD) or saRNA encoding RBD fused to BFF (saRNA- RBD-BFF) mixed with invivaJET PEIpro transfection reagent (PolyPlus). Buffer immunization was used as control.

[0643] The results presented in Fig. 7-1 A showed strong induction of humoral immune responses when RBD was presented on self-forming nanoparticle BFF and expressed by saRNA.

[0644] In addition, T cell responses were assessed. Splenocytes were isolated on day 42 and IFN-y ELISpot analysis was performed (see Example 1.4.4). For this, 0.5x106splenocytes were restimulated with SARS-CoV-2-Spike peptide pool A (Genscript) containing the RBD region. IFN-y spots were counted and are presented in Fig. 7-1 B. In both saRNA vaccination groups strong SARS-CoV-2-Spike specific T cell responses were induced, and no difference was detected between the RBD-monomer and RBD-BFF expressing saRNA.

[0645] Taken together, these data demonstrated that BFF-mediated antigen presentation induced T cell responses and strongly elevated antibody responses when the RBD-BFF fusion protein was encoded by saRNA.

[0646] 3.4 Immune responses to RBD or RBD-BFF antigens delivered by saRNA or mRNA packaged in LNPs

[0647] To assess the immunogenicity of saRNA and pseudouridin-modified mRNA expressing BFF nanoparticle-forming proteins fused to the antigen RBD, all RNAs were packed in comparable lipid nanoparticles (LNPs) with SM-102 as ionizable lipid. LNPs were used with a size between 75-125 nm as determined by DLS and an encapsulation efficiency <85% as determined by Ribogreen assay (Genscript).

[0648] Mice were prime / boost immunized on days 0 and 22 with either LNP-packaged saRNA encoding for monomeric RBD (saRNA-RBD), or LNP-packaged saRNA or mRNA encoding RBD fused to BFF (saRNA-RBD-BFF, mRNA-RBD-BFF) at two different doses (0.1 or 1 pg). Buffer, VRP-RBD and VRP-RBD-BFF immunizations were used as control.

[0649] The results presented in Figure 7-2A show strong induction of humoral immune responses when RBD was presented on a self-forming nanoparticle and expressed by VRP, saRNA or mRNA compared to RBD-specific antibody titers induced by monomeric RBD expressed by VRP or saRNA. The lower dose of saRNA-RBD-BFF or mRNA still induced comparable or higher antibody titers compared to mice immunized with VRP-RBD-BFF.

[0650] In addition, T cell responses were assessed. Splenocytes were isolated on day 34 and IFN-y ELISpot analyses were performed. For this, 0.5 x 106splenocytes were restimulated with SARS-CoV-2-Spike peptide pool A (Genscript) containing the RBD region. IFN-y spots were counted and are presented in Figure 7-2B. In all saRNA vaccinated or VRP vaccinated groups strong SARS-CoV-2-Spike specific T cell responses were induced, and no difference was detected between the RBD-monomer and RBD-BFF expressing saRNA or VRP. Only saRNA or VRP expressing RBD-BFF induced anti-ferritin (BFF) T cell responses. A lower amount of saRNA used in immunization did not change SARS-CoV-2-Spike or ferritin-specific responses. mRNA expressing RBD-BFF induced SARS-CoV-2-Spike- and ferritin-specific T cell responses, although to a lower level than VRP or saRNA. Lower amounts of mRNA also led to lower responses in the ELISpot.

[0651] Together, these data demonstrated that self-assembling nanoparticle-mediated antigen presentation induced solid T cell responses and most importantly, strongly elevated antigenspecific antibody responses when it was encoded as VRP, saRNA or mRNA.

[0652] EXAMPLE 4: Effect of BFF nanoparticle on the expression and immunogenicity of Epstein-Barr virus (EBV) gp350 antigen delivered by MVA or VRP

[0653] The results obtained with the RBD monomer and RBD-BFF fusion protein (see Example 2) were reexamined with another model antigen, namely the N-terminal domain of Epstein-Barr virus (EBV) surface glycoprotein 350 (gp350). EBV gp350 is, amongst other EBV proteins (such as gH and gL), required for entry of the virus into B-cells and epithelial cells. They are also major targets of antibody responses.

[0654] 4.1 EBV gp350 antigen constructs with and without BFF nanoparticle

[0655] Two antigen constructs were designed: A first one containing EBV gp350 fused to a GCN4 derived tetramerization domain and to murine immunoglobulin kappa light chain (IgK LC) signal peptide (see Fig. 8, construct-3A, 3B); and a second one also containing EBV gp350 fused to the IgK LC signal peptide, but with EBV gp350 fused to BFF instead of the GCN4 derived tetramerization domain (see Fig. 8, construct-4).

[0656] Recombinant MVA and VRPs encoding either construct-3A, 3B or construct-4 were prepared as described in Example 1 .1 .1 .2 and 1 .1 .2.2, respectively. 4.2 Western blot analysis of gp350-GCN4 and gp350-BFF proteins encoded by recombinant MVA

[0657] HeLa cells were infected with recombinant MVA (MOI of 10) encoding the tetrameric gp350- GCN4 protein (construct-3A in Fig. 8) or the gp350-BFF fusion protein (construct-4 in Fig. 8). The next day, cell lysates and supernatants were collected and analyzed by Western blot. Results are shown in Fig. 9.

[0658] The polypeptide backbone of a single subunit of the tetrameric gp350-GCN4 glycoprotein has a molecular weight of 50.5 kDa, which is increased by around 30 kDa through the addition of multiple N-glycans during protein synthesis and translocation into the cellular secretory pathway. The GCN4-tetramer disassembles during denaturation for electrophoresis into two covalently linked dimers, that do not further disassemble in the absence of a reducing reagent, resulting in an apparent molecular weight of approximately 160 kDa. The polypeptide backbone of the gp350-BFF fusion glycoprotein has a molecular weight of 70.4 kDa, which is increased to around 100 kDa by N-glycan addition. Consequently, the assembled gp350-BFF nanoparticle with its 24 subunits has a molecular weight of around 2.4 mDa.

[0659] Expression of gp350-GCN4 by recombinant MVA was lower than expression of gp350-BFF, making the protein hardly visible by Western blot in cell lysates separated under denaturing electrophoresis conditions (see lane 3A in Fig. 9A). A band of the expected size of approximately 160 kDa in the supernatant, however, confirmed expression and secretion of gp350-GCN4 (see lane 3A in Fig. 9B).

[0660] After native electrophoresis, gp350-GCN4 produced a clearly defined band between 242 and 480 kDa (see lane 3 in Fig. 9C), and a high molecular weight band was observed for gp350- BFF (see lane 4 in Fig. 9C), demonstrating assembly of gp350-GCN4 into tetramers and of gp350-BFF into nanoparticles, respectively.

[0661] Thus, recombinant MVA was shown to deliver not only the SARS-CoV-2 RBD antigen on BFF nanoparticles (see Example 2.2), but also the EBV gp350 antigen.

[0662] 4.3 Immune responses to gp350 or gp350-BFF induced by recombinant MVA or VRP

[0663] The in vivo immunogenicity of tetrameric gp350 (MVA-gp350-GCN4) encoded by recombinant MVA was compared to that of MVA-encoded gp350-BFF fusion protein (MVA-gp350-BFF) (Fig 10A, 10B), and tetrameric gp350 encoded by VRPs (VRP-gp350-GCN4) were compared to VRP-encoded gp350-BFF fusion protein (VRP-gp350-BFF) (Fig 10C, 10D). To this end, Balb / c mice were prime / boost immunized on day 0 and 21 with the respective recombinant MVAs or VRPs. The results presented In Fig. 10A, 10C showed that total anti-gp350 IgG was strongly elevated when gp350 was presented on self-forming nanoparticle BBF (“MVA-gp350-BFF” and “VRP- gp350-BFF” in Fig. 10) as compared to tetrameric gp350 (“MVA-gp350-GCN4” and “VRP- gp350-GCN4” in Fig. 10).

[0664] In addition, T cell responses were assessed. Splenocytes were isolated on day 35 (MVA) or day 42 (VRP) after prime immunization and IFN-y ELISpot analyses were performed. For this, 0.5x106splenocytes were restimulated with a gp350 peptide pool. IFN-y spots were counted and are presented in Fig. 10B, 10D, showing that in all animal test groups receiving either recombinant MVA or VRPs, gp350-specific T cell responses were induced. No difference was detected between the gp350 tetramer and RBD-BFF expressing vectors.

[0665] 4.4 gp350 or gp350-BFF delivered by recombinant MVA - Immune responses in

[0666] CD-1 mice

[0667] Next, immunogenicity of MVA-gp350-BFF was tested in an outbred mouse model, namely CD-1 mice, which is characterized by a diverse range of MHC haplotype combinations, thus presenting a scenario more analogous to the human condition. Balb / c mice (see, e.g., Example 4.3), in contrast, are an inbred mouse strain characterized by one haplotype.

[0668] CD-1 mice were prime / boost immunized intramuscularly on days 0 and 21 with MVA-gp350- GCN4 or MVA-gp350-BFF, or TBS buffer. Gp350-specific humoral responses were analyzed on day 40 after prime immunization.

[0669] As shown in Fig. 11 , MVA-gp350-BFF encoding BFF-fused gp350 induced significantly higher anti-gp350 IgG titers than MVA-gp350-GCN4 encoding tetrameric gp350 (Fig. 1 1 A). No gp350-specific antibodies were found in control mice injected with TBS (Figure 11 A).

[0670] In addition, antigen-specific T cell responses after immunization of CD-1 mice were analyzed. Spleens were isolated on day 41 after prime immunization for preparation of a single cell suspension. Splenocytes were then restimulated overnight with gp350 Peptide Pool (PP). IFNy-producing antigen-specific T cells were counted using ELISpot assay. Analysis of antigen-specific T cells after immunization of CD-1 mice showed that both MVA-gp350-GCN4 and MVA-gp350-BFF induced comparable levels of gp350-specific T cell responses (Fig. 1 1 B).

[0671] Summing up, it was shown that recombinant MVA encoding gp350 fused to the nanoparticle BFF (MVA-gp350-BFF) induced significantly higher anti-gp350 IgG titers in outbred CD-1 mice compared to recombinant MVA encoding tetrameric gp350-GCN4. At the same time, antigenspecific T cell responses to gp350 were preserved. This demonstrates that the increase of antigen-specific antibodies by fusing an antigen to a BFF backbone can be translated to a mouse cohort containing various MHC haplotypes. Since human MHC genes are highly polymorphic and there is a large variety of different HLA haplotypes in the human population, the fact that the nanoparticle effect is not specific to a certain mouse strain or MHC haplotype is an important observation.

[0672] EXAMPLE 5: Effect of BFF nanoparticle on the expression and immunogenicity of a Lyme disease antigen

[0673] The results obtained with the SARS-CoV-2 RBD (see Example 2) and EBV gp350 (see Example 4) antigens were reexamined with a bacterial model antigen, namely OspA of Borrelia burgdorferi sensu latu. OspA is an outer surface protein of Borrelia burgdorferi sensu latu, the causative agent of Lyme disease, where it plays a role in the traversal of the bacteria from the tick midgut to the salivary glands. Previous work by others showed that vaccination with OspA can induce protective immune responses against Lyme disease.

[0674] 5.1 Lyme disease antigen constructs with and without BFF nanoparticle

[0675] Two antigen constructs were designed: A first one containing the htPA signal peptide followed by a string of the C-terminal fragments (CF) of OspA of three serotypes (see construct-5 in Fig. 12) and a second one containing the same OspA CF string fused to BFF (see construct-6 in Fig. 12).

[0676] Recombinant VRPs encoding either construct-5 or construct-6 were prepared as described in Example 1 .1.2.3.

[0677] 5.2 Immune responses to OspA-ST1-5-6 or OspA-ST1-5-6-BFF antigens delivered by VRPs

[0678] The in vivo immunogenicity of monomeric OspA-ST 1 -5-6 encoded by VRP (“VRP-OspA-ST 1 - 5-6”) was compared to that of VRP-encoded OspA-ST1 -5-6-BFF fusion protein (“VRP-OspA- ST1 -5-6-BFF”) (Fig 13). To this end, Balb / c mice were prime / boost immunized on day 0 and 21 with the respective VRPs.

[0679] The results presented in Fig. 13 show strong induction of humoral immune responses when OspA-ST1 -5-6 was presented on self-forming nanoparticle BFF (see “VRP-OspA-ST1 -5-6- BFF”). Total anti-OspA-ST1 IgG were strongly elevated compared to non-particulate antigen (see “VRP-OspA-1 -5-6”). Together, similar to the results obtained with the SARS-CoV-2 RBD and EBV gp350 antigens, also the results obtained with the OspA-ST1 -5-6 antigen demonstrated that BFF-mediated antigen presentation strongly elevated antibody responses when the antigen-BFF fusion protein was encoded by VRPs.

[0680] EXAMPLE 6: Nanoparticle candidates as alternatives to BFF - Effect on the expression and immunogenicity of SARS-CoV-2 Spike RBD antigen

[0681] To expand the repertoire of available nanoparticles beyond BFF, the Protein Database (PDB) was screened for structures of bacterial or archaeal homo-oligomeric, self-assembling symmetric multimers. Based on their size, their oligomeric state, and the surface exposition of their termini, five candidates (one 60mer, two 24mers, two 12mers) were selected to be screened as potential nanoparticulate antigen presentation platforms.

[0682] The nanoparticles selected were as follows (see Fig. 14):

[0683] HisB Imidazoleglycerol-phosphate dehydratase, an enzyme of histidine biosynthesis, from Mycobacterium tuberculosis;

[0684] PdhC Acetyltransferase of pyruvate dehydrogenase (PDH) complex from (Geo-) Bacillus stearothermophilus;

[0685] DPS DNA binding protein from starved cells from E. coll;

[0686] SOR Sulfur oxygenase / reductase from Acidianus ambivalens;

[0687] MrsD Mersacidin decarboxylase from Bacillus sp. strain HIL-Y85 / 54728.

[0688] 6.1 RBD antigen constructs with nanoparticle candidates

[0689] Five antigen constructs were designed containing the RBD of SARS-CoV-2 Spike protein which was N-terminally fused to the signal peptide of htPA and C-terminally fused to a subunit of one of the five nanoparticle candidates (see Fig. 15; construct-7 to construct-11 ).

[0690] The nucleic acids encoding construct-7 to construct-1 1 were cloned into the VEEV replicon plasmid to generate recombinant VRPs, as described in Example 1 .1.2.4.

[0691] 6.2 Western blot analysis of RBD-nanoparticle candidate fusion proteins encoded by VRP

[0692] Expression and nanoparticle assembly was analyzed by transfecting HEK293T cells with plasmid DNA containing the recombinant CMV launched replicons encoding the SARS-CoV-2 Spike RBD-nanoparticle fusion proteins. The next day, cell lysates and supernatants were collected and analyzed by Western blot (see Example 1.3.2). Results are shown in Fig. 16.

[0693] In the lysate of HEK293T cells transfected with the VEEV replicon plasmid encoding SARS- CoV-2 Spike RBD-HisB fusion protein (calculated MW = 47.1 kDa without glycosylation) a band of just under 50 kDa was detected (Fig. 16A, lane 7), but no band was detectable in the supernatant (Fig. 16B, lane 7).

[0694] In the lysate of cells transfected with the VEEV replicon plasmid encoding SARS-CoV-2 Spike RBD-PdhC fusion protein (calculated MW = 51.8 kDa without glycosylation) a band of just over 50 kDa was detected (Fig. 16A, lane 8). A band of the same size was detected in the cell supernatant when the electrophoresis gel was is run under denaturing conditions (Fig. 16B, lane 8), but shifts to a size that is bigger than the largest marker band of 1 ,236 kDa when the gel was run under native conditions (Fig. 16C, lane 8). Thus, the RBD-PdhC fusion protein was expressed, secreted, and assembled into a nanoparticle.

[0695] In the lysate of cells transfected with the VEEV replicon plasmid encoding SARS-CoV-2 Spike RBD-DPS fusion protein (calculated MW = 42.8 kDa without glycosylation) a band of around 40 kDa was detected (Fig. 16A, lane 9). A similarly sized band was detected in the cell supernatant under denaturing conditions (Fig. 16B, lane 9), while a band of >720 kDa was detected in the native blot (Fig. 16C, lane 9). Thus, the RBD-DPS fusion protein was expressed, secreted and assembled into a nanoparticle.

[0696] A band of around 60 kDa was detected in the lysates of cells transfected with the VEEV replicon plasmid encoding SARS-CoV-2 Spike RBD-SOR (calculated MW = 60.4 kDa without glycosylation) (Fig. 16A, lane 10), and a band of around 50 kDa was detected in the lysates of cells transfected with the VEEV replicon plasmid encoding SARS-CoV-2 Spike RBD-MrsD (calculated MW = 52.4 kDa without glycosylation) (Fig. 16A, lane 11 ). For both constructs, no band was detected in the cell supernatants (Fig. 16B, 16C, lanes 10 and 511 , indicating a failure of protein secretion and nanoparticle assembly.

[0697] These results suggest that, while all the SARS-CoV-2 Spike RBD-nanoparticle fusion proteins were expressed in replicon transfected HEK293T cells, only SARS-CoV-2 Spike RBD-PdhC and SARS-CoV-2 Spike RBD-DPS were secreted efficiently. PdhC normally exists as a 60mer (see Fig. 14), so the assembled SARS-CoV-2 Spike RBD-PdhC nanoparticle should have a size of around >3,000 kDa (60 * (51 .8 kDa + MWgiyCan)). As this size lies beyond the size range covered by the used size standard (NativeMark™ Unstained Protein Standard) a more precise size estimate was not possible, but the sharp edges and overall shape of the detected band (Fig. 16C, lane 8) suggested successful nanoparticle assembly of the SARS-CoV-2 Spike RBD-PdhC nanoparticle. DPS normally exists as a 12mer (see Fig. 14). Hence a molecular weight of >500 kDa (12 * (42.8 kDa + MWgiyCan)) was expected for the assembled SARS-CoV- 2 Spike RBD-DPS nanoparticle, which is lower than the band at >720 kDa detected in the native gel (Fig. 16C, lane 9). However, migration behavior on native gels is heavily dependent on protein folding and assembly. The clear definition of the band shown in Fig. 16C, lane 9 suggested successful particle assembly rather than unspecific multimerization such as aggregation being responsible for the increased apparent size when comparing the bands detected in denaturing and native gels.

[0698] Taken together these results suggested that both PdhC and DPS could be useful for nanoparticulate antigen presentation.

[0699] 6.3 RBD antigen constructs with nanoparticle candidates in which the RBD and nanoparticle are fused in reverse orientation

[0700] In the SARS-CoV-2 Spike RBD-nanoparticle fusion proteins described so far, the nanoparticle ( / .e., BFF and all nanoparticle candidates tested) had been fused to SARS-CoV-2 Spike RBD via their N-terminus. However, some antigens are normally anchored to the pathogen at their N-terminus so that they would be displayed in an “upside-down” orientation if their C-terminus was fused to the nanoparticle’s N-terminus.

[0701] While BFF can only be fused to antigens at their C-terminus, as the N-terminus is positioned inside the assembled nanoparticle, and PdhC also has only its N-terminus exposed on the particle surface, both termini are exposed on HisB, DPS, SOR and MrsD nanoparticles (see Fig. 14). To assess whether these nanoparticles are suitable for C-terminal display of the SARS-CoV-2 Spike RBD, four additional constructs were designed (see Fig. 17) containing HisB (construct- 12), DPS (construct-13), SOR (construct-14), or MrsD (construct-15) fused at their respective C-terminus to the N-terminus of the RBD. In these constructs, each nanoparticle candidate was N-terminally fused to an htPA signal peptide (see Fig. 17).

[0702] Recombinant VRPs encoding one of construct-12 to construct-15 were prepared as described in Example 1 .1 .2.4.

[0703] 6.4 Western blot analysis of reverse RBD-nanoparticle candidate fusion proteins encoded by VRP

[0704] HEK293T cells were transfected with replicon plasmids encoding one of construct-12 to construct-15 as described in Example 1.3.1 above. Cell lysates and supernatants were collected and analyzed by Western blot (see Example 1 .3.2). Results are shown in Fig. 18. The calculated molecular weights of the reverse fusion proteins were identical to those of corresponding N-terminal SARS-CoV-2 Spike RBD fusion proteins (see Example 6.2). Bands of the expected sizes were detected in lysates of cells transfected with each of the replicon plasmids (Fig. 18A).

[0705] However, while all reverse fusion proteins were expressed in transfected cells in detectable amounts (Fig. 18A), only HisB-RBD and DPS-RBD fusion proteins were secreted, as indicated by a band of the expected size in the respective supernatants (Fig. 18B, lanes 12 and 13). Bands of high molecular weight detectable under native conditions furthermore suggested successful nanoparticle assembly of both the secreted fusion proteins.

[0706] Thus, both HisB and DPS could be useful for sterically correct presentation of membrane antigens originally anchored in the membrane via their N-terminus.

[0707] 6.5 Immune responses to RBD-nanoparticle candidate fusion proteins delivered by VRPs

[0708] Mice were immunized intramuscularly on day 0 and day 21 with VRP-RBD monomer or VRP- RBD-DPS, or with VRP encoding reverse fusion proteins, i.e., VRP-DPS-RBD and VRP-HisB- RBD, respectively. Serum was isolated from blood drawn on days 14 and 35 and used to perform ELISA to detect RBD-specific total antibody titers or to perform ePass to determine RBD-neutralizing antibody titers. Results are shown in Fig. 19.

[0709] Confirming the previous findings with RBD-BFF (see Fig. 4), the VRPs launching DPS or HisB nanoparticles N-terminally fused to the RBD antigen also induced a stronger antibody response to RBD on day 35 compared to VRP-RBD delivering the RBD antigen as a monomer (Fig. 19A). VRP-DPS-RBD and the VRP encoding the reverse fusion protein, i.e., VRP-RBD- DPS, induced similar total anti-RBD IgG titers, while the titers induced by VRP-HisB-RBD were even higher (Fig. 19A).

[0710] Furthermore, neutralizing RBD-binding antibody titers were measured using serum samples from day 35. A significant increase of RBD-binding neutralizing antibodies was observed in all VRP-nanoparticle immunization groups as compared to VRP-RBD monomer (Fig. 19B).

[0711] In addition, T cell responses were assessed. Splenocytes were isolated on day 35 (14 days after boost immunization) and IFN-y ELISpot analyses were performed by restimulation with SARS-CoV-2-spike peptide pool A (Genscript). In all vaccination groups strong SARS-CoV-2- Spike specific T cell responses were induced and no difference was detected between the RBD monomer and RBD-nanoparticle expressing VRPs (Fig. 19C). Taken together, VRPs delivering the SARS-CoV-2 RBD fused to nanoparticle candidate DPS or HisB were shown to be highly efficient in eliciting anti-RBD antibody as well as T cell response after i.m. immunizations. The orientation of the RBD antigen within the fusion protein, i.e., whether N-terminally or C-terminally fused to DPS, did not alter the efficiency of neutralizing RBD antibody induction. Thus, DPS as well as HisB turned out to be a useful alternative to BFF.

[0712] 6.6 RBD-nanoparticle candidate fusion proteins delivered by VRPs - Immune responses in CD-1 mice

[0713] The immunogenicity of VRPs expressing the RBD antigen either as monomer or fused to a self-forming nanoparticle was tested in an outbred mouse model, which is characterized by a diverse range of MHC haplotype combinations, thus presenting a scenario more analogous to the human condition. CD-1 mice were prime / boost immunized intramuscularly on days 0 and 21 with VRP RBD monomer, VRP-HisB-RBD, VRP-DPS-RBD, or VRP RBD-BFF. RBD- specific humoral responses were analyzed on day 42 after prime immunization. Results are shown in Fig. 20.

[0714] VRP-HisB-RBD, VRP-DPS-RBD and VRP-RBD-BFF induced higher anti-RBD IgG titers than VRP-RBD monomer (Fig. 20A). No RBD-specific antibodies were found in buffer immunized control mice (Fig. 20A).

[0715] In addition, antigen-specific T cell responses after immunization of CD-1 mice were analyzed. Spleens were isolated on day 42 after prime immunization for preparation of a single cell suspension. Splenocytes were then restimulated overnight with RBD Peptide Pool. IFNy- producing antigen-specific T cells were counted using ELISpot assay. Analysis of antigenspecific T cells after immunization of CD-1 mice showed that all VRP-immunized groups induced comparable levels of RBD- specific T cell responses (Fig. 20B).

[0716] Summing up, it was shown that VRP expressing RBD fused to the nanoparticles BFF, HisB and DPS, respectively, induced higher anti-RBD IgG titers in outbred CD-1 mice while preserving antigen-specific T cell responses. This demonstrates that the increase of antibodies by fusing antigen to a nanoparticle backbone can be translated to a mouse cohort containing various MHC haplotypes. Since human MHC genes are highly polymorphic and there is a large variety of different HLA haplotypes in the human population, the fact that the nanoparticle effect is not specific to a certain mouse strain is an important observation. 6.7 gp350 antigen constructs with nanoparticle candidates

[0717] To test immunogenicity of different nanoparticle candidates presenting gp350, two novel VRP constructs were generated (Fig. 21 ). Specifically, VRP-DPS fused to gp350 as well as VRP- PdhC fused to gp350 were examined. PdhC is a 60-mer (see Fig. 14) and offers higher antigen valency compared to DPS.

[0718] 6.8 Immune responses to gp350-nanoparticle candidate fusion proteins delivered by VRPs

[0719] Mice were prime / boost immunized on days 0 and 21 with VRP-gp350, VRP-gp350-BFF, VRP- DPS-gp350, and VRP- PdhC-gp350, respectively. Antibody responses against gp350 induced by VRPs expressing tetrameric gp350 (VRP-gp350-GCN4) were compared to those induced by VRPs expressing gp350 fused to BFF, DPS or PdhC. The results presented in Fig. 22 showed strong induction of humoral immune responses when gp350 was presented on one of the self-assembling nanoparticles. Antibody responses were comparable between the different nanoparticles, regardless of their size.

[0720] EXAMPLE 7: Multi-antigen nanoparticle - Expression and immunogenicity of two distinct antigens when both displayed on the same BFF nanoparticle

[0721] Furthermore, it was asked whether it would be possible to induce immune responses against two non-related antigens presented on the same nanoparticle. Such dual-antigen nanoparticles, also referred to as “mosaic” nanoparticles, would allow to induce immune- responses against two different antigens from a single vaccine vector, where both antigens would profit from the increased immunogenicity afforded by their display on a nanoparticle.

[0722] 7.1 Multi-antigen nanoparticle construct for the display of SARS-CoV-2 spike RBD and EBV gp350 on BFF

[0723] A recombinant VRP encoding gp350-BBFF and RBD-BFF separated by a T2A site (see construct-18 in Fig. 23) was prepared as described in Example 1 .1.2.6.

[0724] 7.2 Western blot analysis of RBD-BFF / gp350 multi-antigen nanoparticle proteins

[0725] HEK293T cells were transfected with replicon plasmids encoding either construct-18 (Fig. 23), construct -2 (Fig. 1 ) or construct-4 (Fig. 8) for comparison, as described in Example 1.3.1 above. Cell supernatants were collected and analyzed by native Western blot (see Example 1 .3.2). Results are shown in Fig. 24. When using an anti-RBD antibody (Fig. 24A), bands were detected in the supernatants of cells expressing construct-18 or construct-2. When using an anti-gp350 antibody (Fig. 24B), bands were detected in the supernatants of cells expressing construct- 18 or construct-4. When using an anti-ferritin antibody, bands were detected in the supernatants of cells expression all three constructs (see Fig. 24C).

[0726] These findings confirm that both antigens, i.e.. SARS CoV-2 RBD and EBV gp350, were expressed by cells transfected with the replicon plasmid encoding construct-18. As described above (Example 2.2), the assembled 24mer RBD-BFF nanoparticle has a molecular weight of > 1.1 mDa (24 x 45.29 kDa plus additional weight from added glycans), while the heavily glycosylated gp350-BFF has a molecular weight of weight of around 2.4 mDa when assembled. This corresponds well with the position of bands detected in the supernatants of cells transfected with replicon plasmids encoding construct-2 or construct-4. The band detected in the supernatants of cells transfected with the replicon plasmid encoding construct- 18 was detected at a position indicating a molecular weight between that of the assembled RBD-BFF 24mer and the gp350-BFF 24mer, indicating successful assembly of the dualantigen nanoparticles.

[0727] 7.3 Immune responses to RBD-BFF / gp350-BFF multi-antigen nanoparticles encoded by VRPs

[0728] Mice were prime / boost immunized intramuscularly on days 0 and 21 with the bi-valent nanoparticle encoding VRPs or buffer as control. Serum was taken on day 35 and anti-gp350 and anti-RBD antibody responses were determined. Results are shown in Fig. 25.

[0729] VRP-RBD-BFF-gp350-BFF induced antigen-specific antibody titers against both antigens (Fig. 25A, 25B). However, a decrease of antibody titers compared to single-antigen VRPs was detected for anti-RBD IgG titers (Fig. 25A). Anti-gp350 IgG titers seemed to be less affected by mixed-nanoparticle presentation (Fig. 25B).

[0730] In addition, antigen-specific T cell responses were analyzed. Spleens were isolated on day 35 after prime immunization for preparation of a single cell suspension. Splenocytes were then restimulated overnight with RBD Peptide Pool (Fig. 25C) or gp350 peptide pool (Fig. 25D). IFN-y-producing antigen-specific T cells were counted using ELISpot assay. Analysis of antigen-specific T cells after prime / boost immunization showed that VRP-RBD-BFF-gp350- BFF elicited both RBD- as well as gp350-specific T cell responses. VRP-immunized groups induced comparable levels of RBD-specific T cell responses, whereas gp350-specific T cell responses were slightly reduced compared to VRPs expressing the respective single antigen on BFF (Fig. 25C, 25D). In conclusion, it was shown that BFF nanoparticles were useful for the induction of antibody as well as T cell responses against two non-related antigens, i.e., antigens from different viruses (here: SARS-CoV-2 and EBV). This would allow the development of vaccines that target different diseases at the same time.

[0731] 7.4 Multi-antigen nanoparticle constructs for the display of two distinct Lyme disease antigens on BFF

[0732] To further evaluate the feasibility of multi-antigen nanoparticles, and to generate a single recombinant VRP encoding OspA fragments from the six most disease relevant serotypes, a VEEV TC83 replicon plasmid encoding OspA-ST 1 -5-6-BFF (construct-6 in Fig. 12) and OspA- ST2-4-3-BFF (construct-19 in Fig. 26), separated from each other by a 2A site, i.e., construct- 20 in Fig. 26, was prepared as described in Example 1 .1.2.7.

[0733] 7.5 Immune responses to OspA-ST1-5-6-BFF / OspA-ST2-4-3-BFF multi-antigen nanoparticles encoded by VRPs

[0734] Immunogenicity of VRP-OspA-ST1 -5-6-BFF / OspA-ST2-4-3-BFF encoding construct-19 (Fig. 26) was compared to that of a mixture of VRP-OspA-ST1 -5-6-BFF and OspA-ST2-4-3-BFF, i.e., the two recombinant VRPs each encoding only one of the two fusion proteins.

[0735] Mice were prime / boost immunized intramuscularly on days 0 and 21 with VRP-OspA-ST1 -5- 6-BFF-OspA-ST2-4-3-BFF or with a 1 :1 mix of VRPs encoding the respective single antigen constructs (VRP-OspA-ST1 -5-6-BFF and VRP-OspA-ST2-4-3-BFF, respectively). Serum was taken on day 35 and anti-OspAST1 and anti-OspAST2 antibody responses were determined.

[0736] As shown in Fig. 27, VRP-OspA-ST1 -5-6-BFF-OspA-ST2-4-3-BFF induced antigen-specific antibody titers against both OspA-ST1 (Fig. 27A) and OspA-ST2 (Fig. 27B) antigens. Importantly, antibody titers against both antigens were comparable to those detected when mice were immunized with the mix of VRPs encoding antigens singly, i.e., VRP-OspA-ST1 -5- 6-BFF and VRP-OspA-ST2-4-3-BFF.

[0737] In conclusion, it was shown that VRPs expressing BFF nanoparticles that presented two different strings of OspA fragments from Borrelia on the surface were as efficient as a mix of two VRPs each expressing BFF that presented only one string on the surface. This would allow for a more efficient vaccine manufacturing process in that only one type of VRPs needs to be produced instead of two. Final remark: Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer’s specifications, instructions, etc.) are hereby incorporated by reference in their entirety. To the extent, the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0738] References

[0739] 1 . Meisinger-Henschel C, Schmidt M, Lukassen S, Linke B, Krause L, Konietzny S, Goesmann A, Howley P, Chaplin P, Suter M, Hausmann J. 2007. Genomic sequence of chorioallantois vaccinia virus Ankara, the ancestor of modified vaccinia virus Ankara. J Gen Virol 88:3249-3259.

[0740] 2. Suter M, Meisinger-Henschel C, Tzatzaris M, Hulsemann V, Lukassen S, Wulff NH, Hausmann J, Howley P, Chaplin P. 2009. Modified vaccinia Ankara strains with identical coding sequences actually represent complex mixtures of viruses that determine the biological properties of each strain. Vaccine 27:7442-7450.

[0741] 3. Pollard AJ, Launay O, Lelievre JD, Lacabaratz C, Grande S, Goldstein N, Robinson C, Gaddah A, Bockstal V, Wiedemann A, Leyssen M, Luhn K, Richert L, Betard C, Gibani MM, Clutterbuck EA, Snape MD, Levy Y, Douoguih M, Thiebaut R, group EEs. 2021 . Safety and immunogenicity of a two-dose heterologous Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen in adults in Europe (EBOVAC2): a randomised, observer-blind, participant-blind, placebo-controlled, phase 2 trial. Lancet Infect Dis 21 :493-506.

[0742] 4. Bachmann, M.F., et al., The influence of antigen organization on B cell responsiveness. Science, 1993. 262(5138): p. 1448-51.

[0743] 5. Zhang, B., et al., A platform incorporating trimeric antigens into self-assembling nanoparticles reveals SARS-CoV-2-spike nanoparticles to elicit substantially higher neutralizing responses than spike alone. Sci Rep, 2020. 10(1 ): p. 18149.

[0744] 6. Kanekiyo, M., et al., Rational Design of an Epstein-Barr Virus Vaccine Targeting the Receptor-Binding Site. Cell, 2015. 162(5): p. 1090-100.

[0745] 7. Meyer H, Sutter G, Mayr A. 1991 . Mapping of deletions in the genome of the highly attenuated vaccinia virus MVA and their influence on virulence. J Gen Virol 72:1031 - 1038.

[0746] 8. Mayr A, Danner K. 1978. Vaccination against pox diseases under immunosuppressive conditions. Dev Biol Stand 41 :225-234.

[0747] 9. Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE. 1988. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 106:761-771.

[0748] 10. Wennier ST, Brinkmann K, SteinhauBer C, Maylander N, Mnich C, Wielert U, Dirmeier U, Hausmann J, Chaplin P, Steigerwald R. 2013. A novel naturally occurring tandem promoter in modified vaccinia virus Ankara drives very early gene expression and potent immune responses. PLoS ONE 8:e73511 .

[0749] 11 . Harbury, P.B., et al., A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. Science, 1993. 262(5138): p. 1401-7.

[0750] 12. Comstedt, P., et al. 2014. Design and development of a novel vaccine for protection against Lyme borreliosis. PLOS One 9(11 ) :e113294. Sequences

[0751] SEQ ID NO: 1 Nucleic acid sequence encoding SARS-CoV-2 spike RBD.

[0752] >

[0753] GTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAATCTGTGTCCTTTTGG

[0754] CGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATC

[0755] AGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTTCTCCACCTTCAA

[0756] GTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCTG

[0757] ACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGGACAGACAGGCAA

[0758] GATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCTGTGTGATTGCCTGGA

[0759] ACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTGTACAGGCTGTTT

[0760] CGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGATCTATCAGGCAG

[0761] GCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGTCCTAC

[0762] GGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAGAGTGGTGGTGCTGAGCT

[0763] TCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCACCAACCTGGT

[0764] GAAGAACAAA

[0765] SEQ ID NO: 2 Amino acid sequence of SARS-CoV-2 spike RBD.

[0766] VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYG

[0767] VSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDS

[0768] KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG

[0769] YQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

[0770] SEQ ID NO: 3 Nucleic acid sequence encoding EBV gp350.

[0771] ATGGAAGCAGCTCTGCTCGTGTGCCAGTACACCATCCAGAGCCTCATCCACCTGACAG

[0772] GAGAGGATCCTGGCTTCTTCAACGTGGAAATTCCAGAGTTTCCCTTCTACCCTACCTGC

[0773] AATGTGTGCACAGCCGACGTGAACGTGACCATCAACTTCGACGTTGGAGGCAAGAAGC

[0774] ACCAGCTGGACCTGGATTTCGGACAACTGACACCTCACACCAAGGCTGTGTATCAGCCT

[0775] AGAGGAGCCTTTGGTGGTTCTGAGAATGCCACCAACCTGTTTCTCCTGGAGCTGCTTG

[0776] GAGCTGGCGAGCTCGCACTGACCATGAGAAGCAAGAAACTGCCCATCAATGTGACCAC

[0777] AGGCGAGGAACAGCAGGTGTCCCTGGAAAGCGTGGACGTGTACTTTCAAGACGTGTTC

[0778] GGCACCATGTGGTGCCACCATGCCGAGATGCAGAACCCTGTGTACCTGATCCCAGAGA

[0779] CAGTGCCCTACATCAAGTGGGACAACTGCAACAGCACAAACATCACAGCCGTCGTGAG

[0780] GGCTCAGGGACTGGATGTGACACTGCCTCTGTCTCTGCCAACCAGTGCCCAGGACAGC

[0781] AACTTCAGCGTGAAGACCGAGATGCTGGGAAACGAGATCGACATCGAGTGCATCATGG

[0782] AAGATGGCGAGATCAGCCAGGTACTGCCTGGCGACAACAAGTTCAACATCACATGCAGT

[0783] GGCTACGAGAGCCACGTGCCATCTGGAGGCATCCTGACAAGCACAAGCCCAGTGGCCA

[0784] CACCGATCCCTGGCACAGGCTACGCCTACAGCCTGAGACTGACACCCAGACCCGTGTC

[0785] CAGATTCCTGGGCAACAACAGCATCCTGTACGTGTTCTACTCTGGCAACGGACCCAAGG

[0786] CCTCTGGTGGCGATTACTGTATCCAGAGCAACATTGTGTTCAGCGATGAGATCCCTGCC

[0787] AGCCAGGACATGCCAACCAATACCACCGATATCACCTACGTGGGAGACAATGCCACCTA

[0788] CAGCGTGCCCATGGTCACCTCCGAGGACGCCAACAGCCCTAATGTGACCGTGACAGCC

[0789] TTCTGGGCATGGCCTAACAACACCGAGACAGACTTCAAGTGCAAGTGGACCCTGACCT

[0790] CTGGCACACCTAGTGGCTGCGAGAATATCAGCGGAGCCTTCGCCAGCAACCGGACCTT

[0791] CGACATTACCGTGTCTGGCCTTGGCACAGCTCCCAAGACCCTGATCATCACCAGGACTG

[0792] CCACCAATGCCACAACCACAACCCACAAAGTGATCTTCAGCAAGGCTCCTGAAAGCACC

[0793] ACAACTAGTCCTACACTG

[0794] SEQ ID NO: 4 Amino acid sequence of EBV gp350.

[0795] MEAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCTADVNVTINFDVGGKKHQLDL

[0796] DFGQLTPHTKAVYQPRGAFGGSENATNLFLLELLGAGELALTMRSKKLPINVTTGEEQQVSL

[0797] ESVDVYFQDVFGTMWCHHAEMQNPVYLIPETVPYIKWDNCNSTNITAVVRAQGLDVTLPLS LPTSAQDSNFSVKTEMLGNEIDIECIMEDGEISQVLPGDNKFNITCSGYESHVPSGGILTSTS PVATPIPGTGYAYSLRLTPRPVSRFLGNNSILYVFYSGNGPKASGGDYCIQSNIVFSDEIPASQ DMPTNTTDITYVGDNATYSVPMVTSEDANSPNVTVTAFWAWPNNTETDFKCKWTLTSGTP SGCENISGAFASNRTFDITVSGLGTAPKTLIITRTATNATTTTHKVIFSKAPESTTTSPTL

[0798] SEQ ID NO: 5 Nucleic acid sequence of Bullfrog / / - / , pylori hybrid ferritin.

[0799] GAATCTCAAGTTCGGCAGCAGTTCAGCAAGGACATCGAGAAGCTGCTGAACGAGCAAG

[0800] TGAACAAAGAGATGCAGAGCAGCAACCTGTACATGAGCATGAGCAGCTGGTGCTACACC

[0801] CACAGCCTTGATGGCGCCGGACTGTTCCTGTTTGATCACGCCGCCGAGGAATACGAGC

[0802] ACGCCAAGAAGCTGATCATCTTCCTGAACGAGAACAACGTGCCCGTGCAGCTGACCAG

[0803] CATTTCTGCCCCTGAGCACAAGTTCGAGGGCCTGACACAGATCTTCCAGAAGGCCTAC

[0804] GAACACGAGCAGCACATTAGCGAGAGCATCAACAACATCGTGGACCACGCCATTAAGAG

[0805] CAAGGATCACGCCACCTTCAACTTTCTGCAGTGGTACGTGGCCGAACAGCACGAGGAA

[0806] GAGGTGCTGTTCAAGGACATCCTGGACAAGATCGAGCTGATCGGCAACGAGAACCACG

[0807] GCCTGTATCTGGCCGACCAGTACGTGAAGGGAATCGCCAAGAGCAGAAAGAGC

[0808] SEQ ID NO: 6 Amino acid sequence of Bullfrog / / - / , pylori hybrid ferritin.

[0809] ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHA

[0810] KKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFL

[0811] QWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

[0812] SEQ ID NO: 7 Nucleic acid sequence encoding HisB.

[0813] GCATCTAGAAGGGCCAGAATCGAGAGAAGAACCCGCGAGAGCGACATCGTGATCGAGC

[0814] TGGATCTGGATGGCACAGGACAGGTGGCAGTGGATACAGGCGTGCCCTTCTACGACCA

[0815] CATGCTGACAGCTCTGGGCAGCCACGCCTCTTTCGACCTGACAGTTAGAGCCACCGGC

[0816] GACGTGGAAATCGAGGCCCACCACACAATCGAGGACACCGCTATCGCTCTGGGAACAG

[0817] CTCTTGGACAGGCCCTGGGCGATAAGAGAGGCATCAGAAGATTCGGCGACGCTTTCAT

[0818] CCCCATGGACGAGACACTGGCTCACGCTGCCGTTGACCTTAGCGGCAGACCTTACTGT

[0819] GTGCATACCGGCGAGCCTGACCATCTGCAGCACACAACAATCGCCGGCAGCAGCGTGC

[0820] CATACCACACCGTGATCAACAGACACGTGTTCGAGAGCCTGGCCGCCAACGCTAGAATC

[0821] GCTCTGCATGTGCGGGTGCTGTACGGCAGAGATCCCCACCACATCACAGAGGCCCAGT

[0822] ACAAGGCTGTGGCCAGGGCTCTGAGACAGGCCGTTGAACCTGATCCTAGAGTGTCCGG

[0823] CGTGCCATCTACAAAGGGTGCTCTG

[0824] SEQ ID NO: 8 Amino acid sequence of HisB.

[0825] ASRRARIERRTRESDIVIELDLDGTGQVAVDTGVPFYDHMLTALGSHASFDLTVRATGDVEIE

[0826] AHHTIEDTAIALGTALGQALGDKRGIRRFGDAFIPMDETLAHAAVDLSGRPYCVHTGEPDHL

[0827] QHTTIAGSSVPYHTVINRHVFESLAANARIALHVRVLYGRDPHHITEAQYKAVARALRQAVEP

[0828] DPRVSGVPSTKGAL

[0829] SEQ ID NO: 9 Nucleic acid sequence encoding PdhC.

[0830] GCTGCTGCTAAACCTGCTACAACAGAGGGCGAGTTCCCCGAGACACGCGAGAAGATGT

[0831] CTGGCATCAGAAGGGCTATCGCCAAGGCCATGGTGCACAGCAAGCACACAGCTCCTCA

[0832] CGTGACCCTGATGGACGAGGCCGATGTGACAAAGCTGGTGGCCCACAGAAAGAAGTTC

[0833] AAGGCCATTGCCGCCGAGAAGGGAATCAAGCTGACCTTCCTGCCTTACGTGGTCAAGG

[0834] CCCTGGTTTCTGCCCTGAGAGAATACCCCGTGCTGAACACCAGCATCGACGACGAGAC

[0835] AGAGGAAATCATCCAGAAGCACTACTACAACATCGGAATCGCCGCCGACACCGACAGAG

[0836] GACTGCTGGTGCCTGTGATCAAGCACGCCGACAGAAAGCCCATCTTCGCCCTGGCTCA

[0837] AGAGATCAACGAGCTGGCTGAGAAGGCCAGAGATGGCAAGCTGACACCCGGCGAAAT

[0838] GAAGGGCGCCAGCTGTACCATCACCAACATCGGTTCTGCTGGCGGCCAGTGGTTCACC CCAGTGATCAATCACCCTGAGGTGGCCATCCTCGGCATCGGCAGAATCGCTGAGAAGC

[0839] CCATCGTCCGCGACGGCGAAATTGTGGCTGCTCCTATGCTGGCCCTGAGCCTGAGCTT

[0840] CGACCACAGAATGATCGACGGCGCCACAGCTCAGAAGGCCCTGAACCACATCAAGAGA

[0841] CTGCTGAGCGACCCCGAGCTGCTGCTGATGGAAGCT

[0842] SEQ ID NO: 10 Amino acid sequence of PdhC.

[0843] AAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIA

[0844] AEKGIKLTFLPYVVKALVSALREYPVLNTSIDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHAD

[0845] RKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIA

[0846] EKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLMEA

[0847] SEQ ID NO: 11 Nucleic acid sequence encoding DPS.

[0848] GCTACAAATCTGCTGTACACCAGAAACGACGTGTCCGACAGCGAGAAGAAGGCCACAG

[0849] TCGAGCTGCTGAACAGACAAGTGATCCAGTTCATCGACCTGAGCCTGATCACCAAGCAG

[0850] GCCCACTGGAACATGAGAGGCGCCAACTTTATCGCCGTGCACGAGATGCTGGACGGCT

[0851] TCAGAACAGCCCTGATCGACCACCTGGACACCATGGCTGAAAGAGCTGTGCAGCTTGG

[0852] CGGAGTGGCTCTGGGCACAACCCAAGTGATCAACAGCAAGACCCCTCTGAAGTCTTAC

[0853] CCTCTGGACATCCACAACGTGCAGGACCACCTGAAAGAACTGGCCGACAGATACGCCA

[0854] TCGTGGCCAATGATGTGCGGAAGGCTATCGGCGAGGCCAAGGACGATGATACCGCCGA

[0855] TATCCTGACAGCCGCCAGCAGAGATCTGGACAAGTTCCTGTGGTTCATCGAGAGCAACA TCGAG

[0856] SEQ ID NO: 12 Amino acid sequence of DPS.

[0857] ATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWNMRGANFIAVHEMLDGFRTALI

[0858] DHLDTMAERAVQLGGVALGTTQVINSKTPLKSYPLDIHNVQDHLKELADRYAIVANDVRKAIG

[0859] EAKDDDTADILTAASRDLDKFLWFIESNIE

[0860] SEQ ID NO: 13 Nucleic acid sequence encoding SOR.

[0861] CCTAAACCTTACGTGGCCATCAACATGGCCGAGCTGAAGAACGAGCCCAAGACCTTCG

[0862] AGATGTTCGCCAGCGTGGGCCCCAAAGTGTGCATGGTCACAGCTAGACACCCTGGCTT

[0863] CGTGGGCTTCCAGAACCACATCCAGATCGGCATCCTGCCTTTCGGCAACAGATACGGC

[0864] GGAGCTAAGATGGACATGACCAAAGAGTCCAGCACCGTCAGAGTGCTGCAGTACACCT

[0865] TCTGGAAGGATTGGAAGGACCACGAGGAAATGCACAGACAGAACTGGTCCTACCTGTT

[0866] CAGACTGTGCTACAGCTGCGCCAGCCAGATGATCTGGGGACCTTGGGAGCCCATCTAC

[0867] GAGATCATCTACGCCAACATGCCCATCAACACCGAGATGACCGACTTCACAGCCGTCGT

[0868] GGGCAAGAAGTTCGCTGAGGGCAAGCCTCTGGACATCCCCGTGATCTCTCAGCCCTAC

[0869] GGCAAGAGAGTGGTGGCCTTCGCTGAGCACTCTGTGATCCCCGGCAAAGAGAAGCAG

[0870] TTCGAGGACGCCATCGTGCGGACCCTGGAAATGCTGAAGAAGGCCCCTGGCTTTCTGG

[0871] GCGCCATGGTGCTGAAAGAAATCGGCGTGTCCGGCATCGGCAGCATGCAGTTTGGCGC

[0872] TAAGGGCTTCCACCAGGTGCTGGAAAACCCCGGCTCTCTGGAACCCGATCCTAACAAC

[0873] GTGATGTACAGCGTGCCCGAGGCCAAGAACACCCCTCAGCAGTATATCGTGCACGTGG

[0874] AATGGGCCAACACAGACGCCCTGATGTTCGGCATGGGACGCGTGCTGCTGTACCCTGA

[0875] ACTGAGACAGGTGCACGACGAAGTGCTGGACACACTGGTGTACGGCCCCTACATCAGA

[0876] ATCCTGAATCCTATGATGGAAGGCACGTTCTGGCGCGAGTACCTGAACGAACAA

[0877] SEQ ID NO: 14 Amino acid sequence of SOR.

[0878] PKPYVAINMAELKNEPKTFEMFASVGPKVCMVTARHPGFVGFQNHIQIGILPFGNRYGGAK

[0879] MDMTKESSTVRVLQYTFWKDWKDHEEMHRQNWSYLFRLCYSCASQMIWGPWEPIYEIIYA

[0880] NMPINTEMTDFTAVVGKKFAEGKPLDIPVISQPYGKRVVAFAEHSVIPGKEKQFEDAIVRTLE

[0881] MLKKAPGFLGAMVLKEIGVSGIGSMQFGAKGFHQVLENPGSLEPDPNNVMYSVPEAKNTP QQYIVHVEWANTDALMFGMGRVLLYPELRQVHDEVLDTLVYGPYIRILNPMMEGTFWREYL NEQ

[0882] SEQ ID NO: 15 Nucleic acid sequence encoding MrsD.

[0883] TCTATCAGCATCCTGAAGGACAAGAAGCTGCTGATCGGCATCTGCGGCAGCATCAGCTC

[0884] TGTGGGCATCTCCAGCTACCTGCTGTACTTCAAGAGCTTCTTCAAAGAAATCCGGGTCG

[0885] TGATGACCAAGACCGCCGAGGATCTGATCCCTGCTCACACCGTGTCCTACTTTTGCGAC

[0886] CACGTGTACAGCGAGCACGGCGAGAATGGCAAGAGACACAGCAACGTGGAAATCGGC

[0887] AGATGGGCCGACATCTACTGCATCATCCCTGCCACCGCTAACATCCTGGGCCAGACAGC

[0888] TAATGGCGTGGCCATGAACCTGGTGGCCACAACCGTTCTGGCTCACCCTCACAACACC

[0889] ATCTTTTTCCCCAACATGAACGACCTGATGTGGAACAAGACCGTGGTGTCCAGAAACAT

[0890] CGAGCAGCTGAGAAAGGACGGCCACATCGTGATCGAGCCTGTGGAAATCATGGCCTTC

[0891] GAGATCGCCACCGGCACCAGAAAGCCTAACAGAGGCCTGATCACCCCTGACAAGGCCC

[0892] TGCTGGCTATCGAGAAGGGCTTCAAAGAGAGAACAAAGCACCCCAGCCTGACC

[0893] SEQ ID NO: 16 Amino acid sequence of MrsD.

[0894] SISILKDKKLLIGICGSISSVGISSYLLYFKSFFKEIRVVMTKTAEDLIPAHTVSYFCDHVYSEHG

[0895] ENGKRHSNVEIGRWADIYCIIPATANILGQTANGVAMNLVATTVLAHPHNTIFFPNMNDLMWN

[0896] KTVVSRNIEQLRKDGHIVIEPVEIMAFEIATGTRKPNRGLITPDKALLAIEKGFKERTKHPSLT

[0897] SEQ ID NO: 17 Nucleic acid sequence encoding SARS-CoV-2 spike RBD with htPA signal peptide (“construct-1 ”) in MVA.

[0898] AIGGATGCCATGAAGCGTGGACTGTGCTGTGTGCTGCTCCTGTGCGGAGCTGTGTTCG TTAGTGCCTCTGTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAATCTGT GCCCTTTTGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCG GAAGCGGATCAGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTTCT CCACCTTCAAGTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCAAC GTGTACGCTGACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGGAC AGACAGGCAAGATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCTGTGTG ATTGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTGTA CAGGCTGTTTCGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGATC TATCAGGCAGGCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCACT GCAGTCCTACGGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAGAGTGGTG GTGCTGAGCTTCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCA CCAACCTGGTGAAGAACAAA

[0899] SEQ ID NO: 18 Nucleic acid sequence encoding SARS-CoV-2 Spike RBD with htPA signal peptide (“construct-1 ”) in VRPs.

[0900] ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCG TCTCGGCTAGCGTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAATCTG TGTCCTTTTGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACC GGAAGCGGATCAGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTT CTCCACCTTCAAGTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCA ACGTGTACGCTGACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGG ACAGACAGGCAAGATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCTGTG TGATTGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTG TACAGGCTGTTTCGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGAT CTATCAGGCAGGCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCAC TGCAGTCCTACGGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAGAGTGGTG GTGCTGAGCTTCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCA

[0901] CCAACCTGGTGAAGAACAAA

[0902] SEQ ID NO: 19 Nucleic acid sequence encoding SARS-CoV-2 Spike RBD with htPA signal peptide (“construct- 1 ”) in mRNA, saRNA, and plasmid

[0903] DNA (with LITR sequence underlined).

[0904] AUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCAUGGA

[0905] UGCAAUGAAGAGAGGGCUCUGCUGUGUGCUGCUGCUGUGUGGAGCAGUCUUCGUC UCGGCUAGCGUGCAGCCCACAGAGUCUAUCGUGCGGUUCCCUAACAUCACCAAUCU GUGUCCUUUUGGCGAGGUGUUCAACGCCACCAGAUUCGCCUCUGUGUACGCCUGGA ACCGGAAGCGGAUCAGCAAUUGCGUUGCCGACUACAGCGUGCUGUACAACUCUGCC

[0906] AGCUUCUCCACCUUCAAGUGCUAUGGCGUGUCUCCUACCAAGCUGAACGACCUGUG CUUCACCAACGUGUACGCUGACAGCUUCGUGAUCAGAGGCGACGAAGUGAGACAGA UUGCUCCUGGACAGACAGGCAAGAUUGCCGAUUACAACUACAAGCUCCCUGACGACU UCACAGGCUGUGUGAUUGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGAGGU

[0907] AACUACAACUACCUGUACAGGCUGUUUCGGAAGUCCAACCUGAAGCCUUUCGAGAGA GACAUCAGCACCGAGAUCUAUCAGGCAGGCAGCACACCUUGCAAUGGCGUGGAAGG CUUCAACUGCUACUUCCCACUGCAGUCCUACGGCUUCCAGCCUACAAAUGGAGUGG GCUACCAGCCUUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCAUGCUCCUGCC

[0908] ACAGUGUGCGGACCUAAGAAAAGCACCAACCUGGUGAAGAACAAAUGAUAAAGCUCG CUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUA AACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAU UUAUUUUCAUUGCGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGU

[0909] UCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGA U UCUGCCU AAU AAAAAACAU U U AU U U UC AU UGCAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0910] SEQ ID NO: 20 Amino acid sequence of SARS-CoV-2 Spike RBD with htPA signal peptide (“construct-1 ”).

[0911] MDAMKRGLCCVLLLCGAVFVSASVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCN GVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

[0912] SEQ ID NO: 21 Nucleic acid sequence encoding SARS-CoV-2 Spike RBD-

[0913] Bullfrog / H. pylori hybrid ferritin fusion protein with htPA signal peptide (“construct-2”) in MVA.

[0914] ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCG TCTCGGCTAGCGTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAATCTG TGTCCTTTTGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACC GGAAGCGGATCAGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTT CTCCACCTTCAAGTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCA ACGTGTACGCTGACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGG ACAGACAGGCAAGATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCTGTG TGATTGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTG TACAGGCTGTTTCGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGAT CTATCAGGCAGGCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCAC TGCAGTCCTACGGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAGAGTGGTG GTGCTGAGCTTCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCA

[0915] CCAACCTGGTGAAGAACAAGAGCAGCGGCGGAGCCTCTGTGCTGGCCGAATCTCAAGT

[0916] TCGGCAGCAGTTCAGCAAGGACATCGAGAAGCTGCTGAACGAGCAAGTGAACAAAGAG

[0917] ATGCAGAGCAGCAACCTGTACATGAGCATGAGCAGCTGGTGCTACACCCACAGCCTTGA

[0918] TGGCGCCGGACTGTTCCTGTTTGATCACGCCGCCGAGGAATACGAGCACGCCAAGAAG

[0919] CTGATCATCTTCCTGAACGAGAACAACGTGCCCGTGCAGCTGACCAGCATTTCTGCCCC

[0920] TGAGCACAAGTTCGAGGGCCTGACACAGATCTTCCAGAAGGCCTACGAACACGAGCAG

[0921] CACATTAGCGAGAGCATCAACAACATCGTGGACCACGCCATTAAGAGCAAGGATCACGC

[0922] CACCTTCAACTTTCTGCAGTGGTACGTGGCCGAACAGCACGAGGAAGAGGTGCTGTTC

[0923] AAGGACATCCTGGACAAGATCGAGCTGATCGGCAACGAGAACCACGGCCTGTATCTGG

[0924] CCGACCAGTACGTGAAGGGAATCGCCAAGAGCAGAAAGAGC

[0925] SEQ ID NO: 22 Nucleic acid sequence encoding SARS-CoV-2 Spike RBD-

[0926] Bullfrog / H. pylori hybrid ferritin fusion protein with htPA signal peptide (“construct-2”) in VRPs.

[0927] AIGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCG TCTCGGCTAGCGTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAATCTG TGTCCTTTTGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACC GGAAGCGGATCAGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTT CTCCACCTTCAAGTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCA ACGTGTACGCTGACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGG ACAGACAGGCAAGATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCTGTG TGATTGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTG TACAGGCTGTTTCGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGAT CTATCAGGCAGGCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCAC TGCAGTCCTACGGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAGAGTGGTG GTGCTGAGCTTCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCA CCAACCTGGTGAAGAACAAGAGCAGCGGCGGAGCCTCTGTGCTGGCCGAATCTCAAGT TCGGCAGCAGTTCAGCAAGGACATCGAGAAGCTGCTGAACGAGCAAGTGAACAAAGAG ATGCAGAGCAGCAACCTGTACATGAGCATGAGCAGCTGGTGCTACACCCACAGCCTTGA TGGCGCCGGACTGTTCCTGTTTGATCACGCCGCCGAGGAATACGAGCACGCCAAGAAG CTGATCATCTTCCTGAACGAGAACAACGTGCCCGTGCAGCTGACCAGCATTTCTGCCCC TGAGCACAAGTTCGAGGGCCTGACACAGATCTTCCAGAAGGCCTACGAACACGAGCAG CACATTAGCGAGAGCATCAACAACATCGTGGACCACGCCATTAAGAGCAAGGATCACGC CACCTTCAACTTTCTGCAGTGGTACGTGGCCGAACAGCACGAGGAAGAGGTGCTGTTC AAGGACATCCTGGACAAGATCGAGCTGATCGGCAACGAGAACCACGGCCTGTATCTGG CCGACCAGTACGTGAAGGGAATCGCCAAGAGCAGAAAGAGC

[0928] SEQ ID NO: 23 Nucleic acid sequence encoding SARS-CoV-2 Spike RBD-

[0929] Bullfrog / H. pylori hybrid ferritin fusion protein with htPA signal peptide (“construct-2”) in mRNA, saRNA, and plasmid DNA (with UTR sequence underlined).

[0930] AUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCAUGGA UGCAAUGAAGAGAGGGCUCUGCUGUGUGCUGCUGCUGUGUGGAGCAGUCUUCGUC UCGGCUAGCGUGCAGCCCACAGAGUCUAUCGUGCGGUUCCCUAACAUCACCAAUCU GUGUCCUUUUGGCGAGGUGUUCAACGCCACCAGAUUCGCCUCUGUGUACGCCUGGA ACCGGAAGCGGAUCAGCAAUUGCGUUGCCGACUACAGCGUGCUGUACAACUCUGCC AGCUUCUCCACCUUCAAGUGCUAUGGCGUGUCUCCUACCAAGCUGAACGACCUGUG CUUCACCAACGUGUACGCUGACAGCUUCGUGAUCAGAGGCGACGAAGUGAGACAGA UUGCUCCUGGACAGACAGGCAAGAUUGCCGAUUACAACUACAAGCUCCCUGACGACU UCACAGGCUGUGUGAUUGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGAGGU

[0931] AACUACAACUACCUGUACAGGCUGUUUCGGAAGUCCAACCUGAAGCCUUUCGAGAGA

[0932] GACAUCAGCACCGAGAUCUAUCAGGCAGGCAGCACACCUUGCAAUGGCGUGGAAGG

[0933] CUUCAACUGCUACUUCCCACUGCAGUCCUACGGCUUCCAGCCUACAAAUGGAGUGG

[0934] GCUACCAGCCUUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCAUGCUCCUGCC

[0935] ACAGUGUGCGGACCUAAGAAAAGCACCAACCUGGUGAAGAACAAGAGCAGCGGCGGA

[0936] GCCUCUGUGCUGGCCGAAUCUCAAGUUCGGCAGCAGUUCAGCAAGGACAUCGAGAA

[0937] GCUGCUGAACGAGCAAGUGAACAAAGAGAUGCAGAGCAGCAACCUGUACAUGAGCAU

[0938] GAGCAGCUGGUGCUACACCCACAGCCUUGAUGGCGCCGGACUGUUCCUGUUUGAUC

[0939] ACGCCGCCGAGGAAUACGAGCACGCCAAGAAGCUGAUCAUCUUCCUGAACGAGAACA

[0940] ACGUGCCCGUGCAGCUGACCAGCAUUUCUGCCCCUGAGCACAAGUUCGAGGGCCUG

[0941] ACACAGAUCUUCCAGAAGGCCUACGAACACGAGCAGCACAUUAGCGAGAGCAUCAAC

[0942] AACAUCGUGGACCACGCCAUUAAGAGCAAGGAUCACGCCACCUUCAACUUUCUGCAG

[0943] UGGUACGUGGCCGAACAGCACGAGGAAGAGGUGCUGUUCAAGGACAUCCUGGACAA

[0944] GAUCGAGCUGAUCGGCAACGAGAACCACGGCCUGUAUCUGGCCGACCAGUACGUGA

[0945] AGGGAAUCGCCAAGAGCAGAAAGAGCUGAUAAGCUCGCUUUCUUGCUGUCCAAUUU

[0946] CUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAA

[0947] GGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCGCUCGC

[0948] UUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAA

[0949] ACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUU

[0950] U AU U U UC AU UGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0951] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0952] AAAAAAAAAAAAAAAAAAAAA

[0953] SEQ ID NO: 24 Amino acid sequence of SARS-CoV-2 Spike RBD-Bullfrog / H pylori hybrid ferritin fusion protein with htPA signal peptide(“construct-2”).

[0954] MDAMKRGLCCVLLLCGAVFVSASVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCN GVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSSGG ASVLAESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAE EYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDH ATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

[0955] SEQ ID NO: 25 Nucleic acid sequence encoding EBV gp350-GCN4 fusion protein

[0956] (“construct-3A”) in MVA.

[0957] ATGGAAGCAGCTCTGCTCGTGTGCCAGTACACCATCCAGAGCCTGATCCACCTGACAG GAGAGGATCCTGGCTTCTTCAACGTGGAAATCCCAGAGTTTCCCTTCTACCCTACCTGC AACGTGTGCACAGCCGACGTGAACGTGACCATCAACTTCGACGTTGGAGGCAAGAAGC ACCAGCTGGACCTGGATTTCGGACAGCTGACACCTCACACCAAGGCTGTGTATCAGCC TAGAGGAGCCTTTGGTGGCAGCGAGAACGCCACCAACCTGTTTCTGCTGGAACTGCTT GGAGCTGGCGAGCTCGCACTGACCATGAGAAGCAAGAAACTGCCCATCAATGTGACCA CAGGCGAGGAACAGCAGGTGTCCCTGGAAAGCGTGGACGTGTACTTTCAAGACGTGTT CGGCACCATGTGGTGCCACCACGCCGAGATGCAGAACCCTGTGTACCTGATCCCAGA GACAGTGCCCTACATCAAGTGGGACAACTGCAACAGCACCAACATCACAGCCGTCGTG AGAGCTCAGGGACTGGATGTGACACTGCCTCTGAGCCTGCCTACCAGTGCCCAGGACA GCAACTTCAGCGTGAAGACCGAGATGCTGGGCAACGAGATCGACATCGAGTGCATCAT GGAAGATGGCGAGATCAGCCAGGTGCTGCCTGGCGACAACAAGTTCAACATCACATGC AGTGGCTACGAGAGCCACGTGCCATCTGGAGGCATCCTGACCAGCACAAGCCCAGTG GCCACACCCATCCCTGGCACAGGCTACGCCTACAGCCTGAGACTGACACCCAGACCC GTGTCCAGATTCCTGGGCAACAACAGCATCCTGTACGTGTTCTACTCTGGCAACGGAC CCAAGGCCTCTGGTGGCGATTACTGTATCCAGAGCAACATCGTGTTCAGCGACGAGAT CCCTGCCAGCCAGGACATGCCAACCAATACCACCGACATCACCTACGTGGGAGACAAT

[0958] GCCACCTACAGCGTGCCCATGGTCACCTCCGAGGACGCCAACAGCCCTAATGTGACC

[0959] GTGACAGCCTTCTGGGCATGGCCTAACAACACCGAGACAGACTTCAAGTGCAAGTGGA

[0960] CCCTGACCTCTGGCACACCTAGTGGCTGCGAGAATATCAGCGGAGCCTTCGCCAGCAA

[0961] CCGGACCTTCGATATCACCGTGTCTGGCCTTGGCACAGCTCCCAAGACCCTGATCATC

[0962] ACCAGGACTGCCACCAATGCCACAACCACAACCCACAAAGTGATCTTCAGCAAGGCTC

[0963] CTGAGAGCACCACAACTAGTCCTACACTGCCTAAGCCCAGCACACCTCCTGGCAGCTC

[0964] TTGTGGAGGCATGAAAGTGAAGCAGCTGGTGGACAAGGTGGAAGAACTGCTGAGCAA

[0965] GAACTACCACCTCGTGAATGAGGTGGCACGGCTCGTGAAGCTCGTGGGAGAAAGAGG TGGC

[0966] SEQ ID NO: 26 Nucleic acid sequence encoding EBV gp350-GCN4 fusion protein with IgK LC signal peptide (“construct-3B”) in VRPs.

[0967] AIGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTG GTGACGAAGCAGCTCTGCTCGTGTGCCAGTACACCATCCAGAGCCTGATCCACCTGAC

[0968] AGGAGAGGATCCTGGCTTCTTCAACGTGGAAATCCCAGAGTTTCCCTTCTACCCTACCT GCAACGTGTGCACAGCCGACGTGAACGTGACCATCAACTTCGACGTTGGAGGCAAGAA GCACCAGCTGGACCTGGATTTCGGACAGCTGACACCTCACACCAAGGCTGTGTATCAG CCTAGAGGAGCCTTTGGTGGCAGCGAGAACGCCACCAACCTGTTTCTGCTGGAACTGC TTGGAGCTGGCGAGCTCGCACTGACCATGAGAAGCAAGAAACTGCCCATCAATGTGAC CACAGGCGAGGAACAGCAGGTGTCCCTGGAAAGCGTGGACGTGTACTTTCAAGACGT GTTCGGCACCATGTGGTGCCACCACGCCGAGATGCAGAACCCTGTGTACCTGATCCCA GAGACAGTGCCCTACATCAAGTGGGACAACTGCAACAGCACCAACATCACAGCCGTCG TGAGAGCTCAGGGACTGGATGTGACACTGCCTCTGAGCCTGCCTACCAGTGCCCAGGA CAGCAACTTCAGCGTGAAGACCGAGATGCTGGGCAACGAGATCGACATCGAGTGCATC ATGGAAGATGGCGAGATCAGCCAGGTGCTGCCTGGCGACAACAAGTTCAACATCACAT GCAGTGGCTACGAGAGCCACGTGCCATCTGGAGGCATCCTGACCAGCACAAGCCCAG TGGCCACACCCATCCCTGGCACAGGCTACGCCTACAGCCTGAGACTGACACCCAGACC CGTGTCCAGATTCCTGGGCAACAACAGCATCCTGTACGTGTTCTACTCTGGCAACGGA CCCAAGGCCTCTGGTGGCGATTACTGTATCCAGAGCAACATCGTGTTCAGCGACGAGA TCCCTGCCAGCCAGGACATGCCAACCAATACCACCGACATCACCTACGTGGGAGACAA TGCCACCTACAGCGTGCCCATGGTCACCTCCGAGGACGCCAACAGCCCTAATGTGACC GTGACAGCCTTCTGGGCATGGCCTAACAACACCGAGACAGACTTCAAGTGCAAGTGGA CCCTGACCTCTGGCACACCTAGTGGCTGCGAGAATATCAGCGGAGCCTTCGCCAGCAA CCGGACCTTCGATATCACCGTGTCTGGCCTTGGCACAGCTCCCAAGACCCTGATCATC ACCAGGACTGCCACCAATGCCACAACCACAACCCACAAAGTGATCTTCAGCAAGGCTC CTGAGAGCACCACAACTAGTCCTACACTGCCTAAGCCCAGCACACCTCCTGGCAGCTC TTGTGGAGGCATGAAAGTGAAGCAGCTGGTGGACAAGGTGGAAGAACTGCTGAGCAA GAACTACCACCTCGTGAATGAGGTGGCACGGCTCGTGAAGCTCGTGGGAGAAAGAGG TGGC

[0969] SEQ ID NO: 27 Amino acid sequence EBV gp350-GCN4 fusion protein

[0970] (“construct-3”).

[0971] MEAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCTADVNVTINFDVGGKKHQLDL DFGQLTPHTKAVYQPRGAFGGSENATNLFLLELLGAGELALTMRSKKLPINVTTGEEQQVSL ESVDVYFQDVFGTMWCHHAEMQNPVYLIPETVPYIKWDNCNSTNITAVVRAQGLDVTLPLS LPTSAQDSNFSVKTEMLGNEIDIECIMEDGEISQVLPGDNKFNITCSGYESHVPSGGILTSTS PVATPIPGTGYAYSLRLTPRPVSRFLGNNSILYVFYSGNGPKASGGDYCIQSNIVFSDEIPASQ DMPTNTTDITYVGDNATYSVPMVTSEDANSPNVTVTAFWAWPNNTETDFKCKWTLTSGTP SGCENISGAFASNRTFDITVSGLGTAPKTLIITRTATNATTTTHKVIFSKAPESTTTSPTLPKPS TPPGSSCGGMKVKQLVDKVEELLSKNYHLVNEVARLVKLVGERGG SEQ ID NO: 28 Nucleic acid sequence encoding EBV gp350-Bullfrog / / - / . pylori hybrid ferritin fusion protein with IgK LC signal peptide (“construct-4”) in MVA.

[0972] AIGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTG GTGACGAAGCAGCTCTGCTCGTGTGCCAGTACACCATCCAGAGCCTCATCCACCTGAC AGGAGAGGATCCTGGCTTCTTCAACGTGGAAATTCCAGAGTTTCCCTTCTACCCTACCT GCAATGTGTGCACAGCCGACGTGAACGTGACCATCAACTTCGACGTTGGAGGCAAGAA GCACCAGCTGGACCTGGATTTCGGACAACTGACACCTCACACCAAGGCTGTGTATCAG CCTAGAGGAGCCTTTGGTGGTTCTGAGAATGCCACCAACCTGTTTCTCCTGGAGCTGCT TGGAGCTGGCGAGCTCGCACTGACCATGAGAAGCAAGAAACTGCCCATCAATGTGACC ACAGGCGAGGAACAGCAGGTGTCCCTGGAAAGCGTGGACGTGTACTTTCAAGACGTGT TCGGCACCATGTGGTGCCACCATGCCGAGATGCAGAACCCTGTGTACCTGATCCCAGA GACAGTGCCCTACATCAAGTGGGACAACTGCAACAGCACAAACATCACAGCCGTCGTG AGGGCTCAGGGACTGGATGTGACACTGCCTCTGTCTCTGCCAACCAGTGCCCAGGACA GCAACTTCAGCGTGAAGACCGAGATGCTGGGAAACGAGATCGACATCGAGTGCATCAT GGAAGATGGCGAGATCAGCCAGGTACTGCCTGGCGACAACAAGTTCAACATCACATGC AGTGGCTACGAGAGCCACGTGCCATCTGGAGGCATCCTGACAAGCACAAGCCCAGTGG CCACACCGATCCCTGGCACAGGCTACGCCTACAGCCTGAGACTGACACCCAGACCCGT GTCCAGATTCCTGGGCAACAACAGCATCCTGTACGTGTTCTACTCTGGCAACGGACCCA AGGCCTCTGGTGGCGATTACTGTATCCAGAGCAACATTGTGTTCAGCGATGAGATCCCT GCCAGCCAGGACATGCCAACCAATACCACCGATATCACCTACGTGGGAGACAATGCCAC CTACAGCGTGCCCATGGTCACCTCCGAGGACGCCAACAGCCCTAATGTGACCGTGACA GCCTTCTGGGCATGGCCTAACAACACCGAGACAGACTTCAAGTGCAAGTGGACCCTGA CCTCTGGCACACCTAGTGGCTGCGAGAATATCAGCGGAGCCTTCGCCAGCAACCGGAC CTTCGACATTACCGTGTCTGGCCTTGGCACAGCTCCCAAGACCCTGATCATCACCAGGA CTGCCACCAATGCCACAACCACAACCCACAAAGTGATCTTCAGCAAGGCTCCTGAAAGC ACCACAACTAGTCCTACACTGCCTAAGCCCAGCACACCTCCTGGCAGCTCTGAATCTCA AGTTCGGCAGCAGTTCAGCAAAGACATCGAGAAGCTCCTCAACGAGCAAGTGAACAAG GAAATGCAGAGCAGCAACCTGTACATGAGCATGAGCAGCTGGTGCTACACCCACAGCC TTGATGGAGCTGGACTGTTCCTGTTTGATCACGCTGCCGAGGAATACGAGCACGCCAA GAAGCTGATCATCTTCCTGAACGAGAACAACGTGCCTGTGCAGCTGACCAGCATTTCTG CACCTGAGCACAAGTTCGAAGGCCTGACACAGATCTTCCAGAAGGCCTACGAACACGA GCAGCACATTAGCGAGAGCATCAACAACATCGTGGACCACGCCATTAAGAGCAAGGATC ACGCCACCTTCAACTTTCTGCAGTGGTACGTTGCCGAACAGCACGAGGAAGAGGTGCT GTTCAAGGACATCCTGGACAAGATCGAGCTGATCGGCAACGAAAACCATGGCCTGTATC TTGCCGACCAGTACGTGAAGGGAATCGCCAAGAGCAGAAAGAGC

[0973] SEQ ID NO: 29 Nucleic acid sequence encoding EBV gp350-Bullfrog / / - / . pylori hybrid ferritin fusion protein with IgK LC signal peptide (“construct-4”) in VRPs.

[0974] ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTG GTGACGAAGCAGCTCTGCTCGTGTGCCAGTACACCATCCAGAGCCTGATCCACCTGAC AGGAGAGGATCCTGGCTTCTTCAACGTGGAAATCCCAGAGTTTCCCTTCTACCCTACCT GCAACGTGTGCACAGCCGACGTGAACGTGACCATCAACTTCGACGTTGGAGGCAAGAA GCACCAGCTGGACCTGGATTTCGGACAGCTGACACCTCACACCAAGGCTGTGTATCAG CCTAGAGGAGCCTTTGGTGGCAGCGAGAACGCCACCAACCTGTTTCTGCTGGAACTGC TTGGAGCTGGCGAGCTCGCACTGACCATGAGAAGCAAGAAACTGCCCATCAATGTGAC CACAGGCGAGGAACAGCAGGTGTCCCTGGAAAGCGTGGACGTGTACTTTCAAGACGT GTTCGGCACCATGTGGTGCCACCACGCCGAGATGCAGAACCCTGTGTACCTGATCCCA GAGACAGTGCCCTACATCAAGTGGGACAACTGCAACAGCACCAACATCACAGCCGTCG TGAGAGCTCAGGGACTGGATGTGACACTGCCTCTGAGCCTGCCTACCAGTGCCCAGGA CAGCAACTTCAGCGTGAAGACCGAGATGCTGGGCAACGAGATCGACATCGAGTGCATC

[0975] ATGGAAGATGGCGAGATCAGCCAGGTGCTGCCTGGCGACAACAAGTTCAACATCACAT

[0976] GCAGTGGCTACGAGAGCCACGTGCCATCTGGAGGCATCCTGACCAGCACAAGCCCAG

[0977] TGGCCACACCCATCCCTGGCACAGGCTACGCCTACAGCCTGAGACTGACACCCAGACC

[0978] CGTGTCCAGATTCCTGGGCAACAACAGCATCCTGTACGTGTTCTACTCTGGCAACGGA

[0979] CCCAAGGCCTCTGGTGGCGATTACTGTATCCAGAGCAACATCGTGTTCAGCGACGAGA

[0980] TCCCTGCCAGCCAGGACATGCCAACCAATACCACCGACATCACCTACGTGGGAGACAA

[0981] TGCCACCTACAGCGTGCCCATGGTCACCTCCGAGGACGCCAACAGCCCTAATGTGACC

[0982] GTGACAGCCTTCTGGGCATGGCCTAACAACACCGAGACAGACTTCAAGTGCAAGTGGA

[0983] CCCTGACCTCTGGCACACCTAGTGGCTGCGAGAATATCAGCGGAGCCTTCGCCAGCAA

[0984] CCGGACCTTCGATATCACCGTGTCTGGCCTTGGCACAGCTCCCAAGACCCTGATCATC

[0985] ACCAGGACTGCCACCAATGCCACAACCACAACCCACAAAGTGATCTTCAGCAAGGCTC

[0986] CTGAGAGCACCACAACTAGTCCTACACTGCCTAAGCCCAGCACACCTCCTGGCAGCTC

[0987] TGAATCTCAAGTTCGGCAGCAGTTCAGCAAGGACATCGAGAAGCTGCTGAACGAGCAA

[0988] GTGAACAAAGAGATGCAGAGCAGCAACCTGTACATGAGCATGAGCAGCTGGTGCTACA

[0989] CCCACAGCCTTGATGGCGCCGGACTGTTCCTGTTTGATCACGCCGCCGAGGAATACGA

[0990] GCACGCCAAGAAGCTGATCATCTTCCTGAACGAGAACAACGTGCCCGTGCAGCTGACC

[0991] AGCATTTCTGCCCCTGAGCACAAGTTCGAGGGCCTGACACAGATCTTCCAGAAGGCCT

[0992] ACGAACACGAGCAGCACATTAGCGAGAGCATCAACAACATCGTGGACCACGCCATTAA

[0993] GAGCAAGGATCACGCCACCTTCAACTTTCTGCAGTGGTACGTGGCCGAACAGCACGAG

[0994] GAAGAGGTGCTGTTCAAGGACATCCTGGACAAGATCGAGCTGATCGGCAACGAGAACC

[0995] ACGGCCTGTATCTGGCCGACCAGTACGTGAAGGGAATCGCCAAGAGCAGAAAGAGC

[0996] SEQ ID NO: 30 Amino acid sequence of EBV gp350-Bullfrog / / - / . pylori hybrid ferritin fusion protein with IgK LC signal peptide (“construct-4”).

[0997] METDTLLLWVLLLWVPGSTGDEAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCT ADVNVTINFDVGGKKHQLDLDFGQLTPHTKAVYQPRGAFGGSENATNLFLLELLGAGELALT MRSKKLPINVTTGEEQQVSLESVDVYFQDVFGTMWCHHAEMQNPVYLIPETVPYIKWDNC NSTNITAVVRAQGLDVTLPLSLPTSAQDSNFSVKTEMLGNEIDIECIMEDGEISQVLPGDNKF NITCSGYESHVPSGGILTSTSPVATPIPGTGYAYSLRLTPRPVSRFLGNNSILYVFYSGNGPKA SGGDYCIQSNIVFSDEIPASQDMPTNTTDITYVGDNATYSVPMVTSEDANSPNVTVTAFWA WPNNTETDFKCKWTLTSGTPSGCENISGAFASNRTFDITVSGLGTAPKTLIITRTATNATTTT HKVIFSKAPESTTTSPTLPKPSTPPGSSESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSM SSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKA YEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLA DQYVKGIAKSRKS

[0998] SEQ ID NO: 31 Nucleic acid sequence encoding an OspA serotypes 1 , 5 and 6 C- terminal fragment string (OspA ST1 -5-6) with htPA signal peptide (“construct-5”) in VRPs.

[0999] ATGGATGCAATGAAGAGAGGGCTCTGTTGCGTGTTGCTTTTGTGTGGAGCAGTCTTCGT CTCGGCTAGCTTCAACGAGAAGGGCGAAGTGTCCGAGAAGATCATCACCAGAGCCGAC GGCACCAGACTCGAGTACACAGGCATCAAGTCTGACGGCAGCGGCAAGGCCAAAGAG GTGCTGAAGCAGTTCACCCTGGAAGGCAAGGTGGCCAACGACAAGACCACACTGGTG GTCAAGTGCGGCACCGTGACACTGAGCAAGCAGATCAGCAAGAGCGGCGAGGTGTCC GTGGAACTGCAGGACACAGATAGCAGCGCCGCCACCAAGAAAACCGCCGCCTGGAAT AGCGGCACCAGCACACTGACCATCACCGTGAACAGCAAAAAGACCAAGGACCTGGTGT TCACCAAAGAAAACACCATCACAGTGCAGCAGTACGACAGCCAGGGCACCAAGCTGGA AGGCTCCGCCGTGGAAATCACAAAGCTGGACGAGATCTGCAACGCCCTGAAAGGCGG TGGCGGCTCTGGCGGAGGTGGAAGCGGAGGAGGAGGAAGTGGCGGAGGCGGTTCTT TTAATGAGAAAGGCGAGATCAGCGAGAAAACCATCGTCAGAGCCCAAGGCACACGGCT CGAGTATACCGACATCAAGAGCGATAAGACCGGGAAAGCCAAAGAAGTCCTCAAGGAT

[1000] TTCACACTCGAGGGCACCCTGGCCGCCGATGGAAAGACAACCCTGAAAGTGACCTGTG

[1001] GCACAGTGACCCTGTCCAAGCAAATCTCCAAGAGCGGAGAGATCACAGTGGCCCTGGA

[1002] CGATACCGATAGCTCCGGCAACAAGAAGTCCGGCACCTGGGATTCTGGCACCTCCACT

[1003] CTGACCATCTCTAAGCAGCGGACCAAGACAAAGCAGCTGGTCTTTACCAAAGAGGACA

[1004] CAATCACCGTCCAGAACTACGACTCCGCCGGCACAAACCTCGAGGGAAAAGCCGTCGA

[1005] GATCACCACACTGAAAGAGCTGTGTAACGCTCTCAAAGGTGGTGGCGGTTCCGGTGGC

[1006] GGTGGCAGTGGTGGTGGTGGATCAGGCGGTGGCGGAAGCTTTAATGGCAAGGGCGAG

[1007] ACATCCGAAAAGACGATCGTTCGCGCCCAAGGAACCCGGCTTGAGTACACCGATATTA

[1008] AGTCCGACGGCTCTGGAAAGGCTAAAGAAGTTCTGAAAGACTTTACCCTCGAAGGGAC

[1009] TCTCGCCGCTGACGGCAAGACCACTCTGAAAGTTACATGCGGAACCGTGGTGCTGTCT

[1010] AAGAACATCCTGAAGTCCGGGGAGATCACTGCCGCTCTGGACGACAGCGATACCACAC

[1011] GGGCCACCAAAAAGACAGGCAAGTGGGACAGCAAGACAAGCACCCTGACAATCAGCG

[1012] TGAACTCCCAGAAAACGAAGAATCTCGTGTTTACGAAAGAAGATACCATTACGGTCCAG

[1013] CGCTACGACAGCGCTGGGACAAATCTGGAAGGGAAAGCTGTGGAAATTACGACCCTCA

[1014] AAGAACTGTGCAATGCCCTCAAG

[1015] SEQ ID NO: 32 Amino acid sequence of an OspA serotypes 1 , 5 and 6 C-terminal fragment string (OspA ST1 -5-6) with htPA signal peptide (“construct-5”) in VRPs.

[1016] MDAMKRGLCCVLLLCGAVFVSASFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKQ FTLEGKVANDKTTLVVKCGTVTLSKQISKSGEVSVELQDTDSSAATKKTAAWNSGTSTLTIT VNSKKTKDLVFTKENTITVQQYDSQGTKLEGSAVEITKLDEICNALKGGGGSGGGGSGGGG SGGGGSFNEKGEISEKTIVRAQGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKV TCGTVTLSKQISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKQRTKTKQLVFTKEDTIT VQNYDSAGTNLEGKAVEITTLKELCNALKGGGGSGGGGSGGGGSGGGGSFNGKGETSEK TIVRAQGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEI TAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKA VEITTLKELCNALK

[1017] SEQ ID NO: 33 Nucleic acid sequence encoding an OspA serotypes 1 , 5 and 6 C- terminal fragment string (OspA ST1 -5-6)-Bullfrog / / - / . pylori hybrid ferritin fusion protein with htPA signal peptide (“construct-6”) in VRPs.

[1018] ATGGATGCAATGAAGAGAGGGCTCTGTTGCGTGTTGCTTTTGTGTGGAGCAGTCTTCGT CTCGGCTAGCTTCAACGAGAAGGGCGAAGTGTCCGAGAAGATCATCACCAGAGCCGAC GGCACCAGACTCGAGTACACAGGCATCAAGTCTGACGGCAGCGGCAAGGCCAAAGAG GTGCTGAAGCAGTTCACCCTGGAAGGCAAGGTGGCCAACGACAAGACCACACTGGTG GTCAAGTGCGGCACCGTGACACTGAGCAAGCAGATCAGCAAGAGCGGCGAGGTGTCC GTGGAACTGCAGGACACAGATAGCAGCGCCGCCACCAAGAAAACCGCCGCCTGGAATA GCGGCACCAGCACACTGACCATCACCGTGAACAGCAAAAAGACCAAGGACCTGGTGTT CACCAAAGAAAACACCATCACAGTGCAGCAGTACGACAGCCAGGGCACCAAGCTGGAA GGCTCCGCCGTGGAAATCACAAAGCTGGACGAGATCTGCAACGCCCTGAAAGGCGGT GGCGGCTCTGGCGGAGGTGGAAGCGGAGGAGGAGGAAGTGGCGGAGGCGGTTCTTT TAATGAGAAAGGCGAGATCAGCGAGAAAACCATCGTCAGAGCCCAAGGCACACGGCTC GAGTATACCGACATCAAGAGCGATAAGACCGGGAAAGCCAAAGAAGTCCTCAAGGATTT CACACTCGAGGGCACCCTGGCCGCCGATGGAAAGACAACCCTGAAAGTGACCTGTGG CACAGTGACCCTGTCCAAGCAAATCTCCAAGAGCGGAGAGATCACAGTGGCCCTGGAC GATACCGATAGCTCCGGCAACAAGAAGTCCGGCACCTGGGATTCTGGCACCTCCACTCT GACCATCTCTAAGCAGCGGACCAAGACAAAGCAGCTGGTCTTTACCAAAGAGGACACAA TCACCGTCCAGAACTACGACTCCGCCGGCACAAACCTCGAGGGAAAAGCCGTCGAGAT

[1019] CACCACACTGAAAGAGCTGTGTAACGCTCTCAAAGGTGGTGGCGGTTCCGGTGGCGGT

[1020] GGCAGTGGTGGTGGTGGATCAGGCGGTGGCGGAAGCTTTAATGGCAAGGGCGAGACA

[1021] TCCGAAAAGACGATCGTTCGCGCCCAAGGAACCCGGCTTGAGTACACCGATATTAAGTC

[1022] CGACGGCTCTGGAAAGGCTAAAGAAGTTCTGAAAGACTTTACCCTCGAAGGGACTCTC

[1023] GCCGCTGACGGCAAGACCACTCTGAAAGTTACATGCGGAACCGTGGTGCTGTCTAAGA

[1024] ACATCCTGAAGTCCGGGGAGATCACTGCCGCTCTGGACGACAGCGATACCACACGGGC

[1025] CACCAAAAAGACAGGCAAGTGGGACAGCAAGACAAGCACCCTGACAATCAGCGTGAAC

[1026] TCCCAGAAAACGAAGAATCTCGTGTTTACGAAAGAAGATACCATTACGGTCCAGCGCTAC

[1027] GACAGCGCTGGGACAAATCTGGAAGGGAAAGCTGTGGAAATTACGACCCTCAAAGAAC

[1028] TGTGCAATGCCCTCAAGAGCAGCGGCGGAGCCTCTGTGCTGGCCGAATCTCAAGTTCG

[1029] GCAGCAGTTCAGCAAGGACATCGAGAAGCTGCTGAACGAGCAAGTGAACAAAGAGATG

[1030] CAGAGCAGCAACCTGTACATGAGCATGAGCAGCTGGTGCTACACCCACAGCCTTGATG

[1031] GCGCCGGACTGTTCCTGTTTGATCACGCCGCCGAGGAATACGAGCACGCCAAGAAGCT

[1032] GATCATCTTCCTGAACGAGAACAACGTGCCCGTGCAGCTGACCAGCATTTCTGCCCCTG

[1033] AGCACAAGTTCGAGGGCCTGACACAGATCTTCCAGAAGGCCTACGAACACGAGCAGCA

[1034] CATTAGCGAGAGCATCAACAACATCGTGGACCACGCCATTAAGAGCAAGGATCACGCCA

[1035] CCTTCAACTTTCTGCAGTGGTACGTGGCCGAACAGCACGAGGAAGAGGTGCTGTTCAA

[1036] GGACATCCTGGACAAGATCGAGCTGATCGGCAACGAGAACCACGGCCTGTATCTGGCC

[1037] GACCAGTACGTGAAGGGAATCGCCAAGAGCAGAAAGAGC

[1038] SEQ ID NO: 34 Amino acid sequence of an OspA serotypes 1 , 5 and 6 C-terminal fragment string (OspA ST1 -5-6) -Bullfrog / / - / , pylori hybrid ferritin fusion protein with htPA signal peptide (“construct-6”) in VRPs.

[1039] MDAMKRGLCCVLLLCGAVFVSASFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKQ FTLEGKVANDKTTLVVKCGTVTLSKQISKSGEVSVELQDTDSSAATKKTAAWNSGTSTLTIT VNSKKTKDLVFTKENTITVQQYDSQGTKLEGSAVEITKLDEICNALKGGGGSGGGGSGGGG SGGGGSFNEKGEISEKTIVRAQGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKV TCGTVTLSKQISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKQRTKTKQLVFTKEDTIT VQNYDSAGTNLEGKAVEITTLKELCNALKGGGGSGGGGSGGGGSGGGGSFNGKGETSEK TIVRAQGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEI TAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKA VEITTLKELCNALKSSGGASVLAESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCY THSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQ HISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVK GIAKSRKS

[1040] SEQ ID NO: 35 Nucleic acid sequence encoding SARS-CoV-2 Spike RBD-HisB fusion protein with htPA signal peptide (“construct-7”) in VRPs.

[1041] AIGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCG TCTCGGCTAGCGTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAATCTG TGTCCTTTTGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACC GGAAGCGGATCAGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTT CTCCACCTTCAAGTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCA ACGTGTACGCTGACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGG ACAGACAGGCAAGATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCTGTG TGATTGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTG TACAGGCTGTTTCGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGAT CTATCAGGCAGGCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCAC TGCAGTCCTACGGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAGAGTGGTG GTGCTGAGCTTCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCA CCAACCTGGTGAAGAACAAGAGCAGCGGCGGAGCCTCTGTGCTGGCCGCATCTAGAA

[1042] GGGCCAGAATCGAGAGAAGAACCCGCGAGAGCGACATCGTGATCGAGCTGGATCTGG

[1043] ATGGCACAGGACAGGTGGCAGTGGATACAGGCGTGCCCTTCTACGACCACATGCTGAC

[1044] AGCTCTGGGCAGCCACGCCTCTTTCGACCTGACAGTTAGAGCCACCGGCGACGTGGAA

[1045] ATCGAGGCCCACCACACAATCGAGGACACCGCTATCGCTCTGGGAACAGCTCTTGGAC

[1046] AGGCCCTGGGCGATAAGAGAGGCATCAGAAGATTCGGCGACGCTTTCATCCCCATGGA

[1047] CGAGACACTGGCTCACGCTGCCGTTGACCTTAGCGGCAGACCTTACTGTGTGCATACC

[1048] GGCGAGCCTGACCATCTGCAGCACACAACAATCGCCGGCAGCAGCGTGCCATACCACA

[1049] CCGTGATCAACAGACACGTGTTCGAGAGCCTGGCCGCCAACGCTAGAATCGCTCTGCA

[1050] TGTGCGGGTGCTGTACGGCAGAGATCCCCACCACATCACAGAGGCCCAGTACAAGGCT

[1051] GTGGCCAGGGCTCTGAGACAGGCCGTTGAACCTGATCCTAGAGTGTCCGGCGTGCCAT

[1052] CTACAAAGGGTGCTCTG

[1053] SEQ ID NO: 36 Amino acid sequence of SARS-CoV-2 Spike RBD-HisB fusion protein with htPA signal peptide (“construct-7”).

[1054] MDAMKRGLCCVLLLCGAVFVSASVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD

[1055] YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCN GVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSSGG

[1056] ASVLAASRRARIERRTRESDIVIELDLDGTGQVAVDTGVPFYDHMLTALGSHASFDLTVRATG DVEIEAHHTIEDTAIALGTALGQALGDKRGIRRFGDAFIPMDETLAHAAVDLSGRPYCVHTGE PDHLQHTTIAGSSVPYHTVINRHVFESLAANARIALHVRVLYGRDPHHITEAQYKAVARALRQ AVEPDPRVSGVPSTKGAL

[1057] SEQ ID NO: 37 Nucleic acid sequence encoding SARS-CoV-2 Spike RBD-PdhC fusion protein with htPA signal peptide (“construct-8”) in VRPs.

[1058] ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCG TCTCGGCTAGCGTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAATCTG TGTCCTTTTGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACC GGAAGCGGATCAGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTT CTCCACCTTCAAGTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCA ACGTGTACGCTGACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGG ACAGACAGGCAAGATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCTGTG TGATTGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTG TACAGGCTGTTTCGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGAT CTATCAGGCAGGCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCAC TGCAGTCCTACGGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAGAGTGGTG GTGCTGAGCTTCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCA CCAACCTGGTGAAGAACAAGAGCAGCGGCGGAGCCTCTGTGCTGGCCGCTGCTGCTA AACCTGCTACAACAGAGGGCGAGTTCCCCGAGACACGCGAGAAGATGTCTGGCATCAG AAGGGCTATCGCCAAGGCCATGGTGCACAGCAAGCACACAGCTCCTCACGTGACCCTG ATGGACGAGGCCGATGTGACAAAGCTGGTGGCCCACAGAAAGAAGTTCAAGGCCATTG CCGCCGAGAAGGGAATCAAGCTGACCTTCCTGCCTTACGTGGTCAAGGCCCTGGTTTC TGCCCTGAGAGAATACCCCGTGCTGAACACCAGCATCGACGACGAGACAGAGGAAATC ATCCAGAAGCACTACTACAACATCGGAATCGCCGCCGACACCGACAGAGGACTGCTGG TGCCTGTGATCAAGCACGCCGACAGAAAGCCCATCTTCGCCCTGGCTCAAGAGATCAA CGAGCTGGCTGAGAAGGCCAGAGATGGCAAGCTGACACCCGGCGAAATGAAGGGCGC CAGCTGTACCATCACCAACATCGGTTCTGCTGGCGGCCAGTGGTTCACCCCAGTGATCA ATCACCCTGAGGTGGCCATCCTCGGCATCGGCAGAATCGCTGAGAAGCCCATCGTCCG CGACGGCGAAATTGTGGCTGCTCCTATGCTGGCCCTGAGCCTGAGCTTCGACCACAGA ATGATCGACGGCGCCACAGCTCAGAAGGCCCTGAACCACATCAAGAGACTGCTGAGCG ACCCCGAGCTGCTGCTGATGGAAGCT SEQ ID NO: 38 Amino acid sequence of SARS-CoV-2 Spike RBD-PdhC fusion protein with htPA signal peptide (“construct-8”).

[1059] MDAMKRGLCCVLLLCGAVFVSASVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCN GVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSSGG ASVLAAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRK KFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNTSIDDETEEIIQKHYYNIGIAADTDRGLLVP VIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAI LGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLMEA

[1060] SEQ ID NO: 39 Nucleic acid sequence encoding SARS-CoV-2 Spike RBD-DPS fusion protein with htPA signal peptide (“construct-9”) in VRPs.

[1061] AIGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCG TCTCGGCTAGCGTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAATCTG TGTCCTTTTGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACC GGAAGCGGATCAGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTT CTCCACCTTCAAGTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCA ACGTGTACGCTGACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGG ACAGACAGGCAAGATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCTGTG TGATTGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTG TACAGGCTGTTTCGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGAT CTATCAGGCAGGCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCAC TGCAGTCCTACGGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAGAGTGGTG GTGCTGAGCTTCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCA CCAACCTGGTGAAGAACAAGAGCAGCGGCGGAGCCTCTGTGCTGGCCGCTACAAATCT GCTGTACACCAGAAACGACGTGTCCGACAGCGAGAAGAAGGCCACAGTCGAGCTGCT GAACAGACAAGTGATCCAGTTCATCGACCTGAGCCTGATCACCAAGCAGGCCCACTGG AACATGAGAGGCGCCAACTTTATCGCCGTGCACGAGATGCTGGACGGCTTCAGAACAG CCCTGATCGACCACCTGGACACCATGGCTGAAAGAGCTGTGCAGCTTGGCGGAGTGGC TCTGGGCACAACCCAAGTGATCAACAGCAAGACCCCTCTGAAGTCTTACCCTCTGGACA TCCACAACGTGCAGGACCACCTGAAAGAACTGGCCGACAGATACGCCATCGTGGCCAA TGATGTGCGGAAGGCTATCGGCGAGGCCAAGGACGATGATACCGCCGATATCCTGACA GCCGCCAGCAGAGATCTGGACAAGTTCCTGTGGTTCATCGAGAGCAACATCGAG

[1062] SEQ ID NO: 40 Amino acid sequence of SARS-CoV-2 Spike RBD-DPS fusion protein with htPA signal peptide (“construct-9”).

[1063] MDAMKRGLCCVLLLCGAVFVSASVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCN GVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSSGG ASVLAATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWNMRGANFIAVHEMLD GFRTALIDHLDTMAERAVQLGGVALGTTQVINSKTPLKSYPLDIHNVQDHLKELADRYAIVAN DVRKAIGEAKDDDTADILTAASRDLDKFLWFIESNIE

[1064] SEQ ID NO: 41 Nucleic acid sequence encoding SARS-CoV-2 Spike RBD-SOR fusion protein with htPA signal peptide (“construct- 10”) in VRPs. AIGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCG

[1065] TCTCGGCTAGCGTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAATCTG

[1066] TGTCCTTTTGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACC

[1067] GGAAGCGGATCAGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTT

[1068] CTCCACCTTCAAGTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCA

[1069] ACGTGTACGCTGACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGG

[1070] ACAGACAGGCAAGATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCTGTG

[1071] TGATTGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTG

[1072] TACAGGCTGTTTCGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGAT

[1073] CTATCAGGCAGGCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCAC

[1074] TGCAGTCCTACGGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAGAGTGGTG

[1075] GTGCTGAGCTTCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCA

[1076] CCAACCTGGTGAAGAACAAGAGCAGCGGCGGAGCCTCTGTGCTGGCCCCTAAACCTTA

[1077] CGTGGCCATCAACATGGCCGAGCTGAAGAACGAGCCCAAGACCTTCGAGATGTTCGCC

[1078] AGCGTGGGCCCCAAAGTGTGCATGGTCACAGCTAGACACCCTGGCTTCGTGGGCTTCC

[1079] AGAACCACATCCAGATCGGCATCCTGCCTTTCGGCAACAGATACGGCGGAGCTAAGATG

[1080] GACATGACCAAAGAGTCCAGCACCGTCAGAGTGCTGCAGTACACCTTCTGGAAGGATT

[1081] GGAAGGACCACGAGGAAATGCACAGACAGAACTGGTCCTACCTGTTCAGACTGTGCTA

[1082] CAGCTGCGCCAGCCAGATGATCTGGGGACCTTGGGAGCCCATCTACGAGATCATCTAC

[1083] GCCAACATGCCCATCAACACCGAGATGACCGACTTCACAGCCGTCGTGGGCAAGAAGT

[1084] TCGCTGAGGGCAAGCCTCTGGACATCCCCGTGATCTCTCAGCCCTACGGCAAGAGAGT

[1085] GGTGGCCTTCGCTGAGCACTCTGTGATCCCCGGCAAAGAGAAGCAGTTCGAGGACGC

[1086] CATCGTGCGGACCCTGGAAATGCTGAAGAAGGCCCCTGGCTTTCTGGGCGCCATGGTG

[1087] CTGAAAGAAATCGGCGTGTCCGGCATCGGCAGCATGCAGTTTGGCGCTAAGGGCTTCC

[1088] ACCAGGTGCTGGAAAACCCCGGCTCTCTGGAACCCGATCCTAACAACGTGATGTACAG

[1089] CGTGCCCGAGGCCAAGAACACCCCTCAGCAGTATATCGTGCACGTGGAATGGGCCAAC

[1090] ACAGACGCCCTGATGTTCGGCATGGGACGCGTGCTGCTGTACCCTGAACTGAGACAGG

[1091] TGCACGACGAAGTGCTGGACACACTGGTGTACGGCCCCTACATCAGAATCCTGAATCCT

[1092] ATGATGGAAGGCACGTTCTGGCGCGAGTACCTGAACGAACAA

[1093] SEQ ID NO: 42 Amino acid sequence of SARS-CoV-2 Spike RBD-SOR fusion protein with htPA signal peptide (“construct-10”).

[1094] MDAMKRGLCCVLLLCGAVFVSASVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD

[1095] YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCN GVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSSGG

[1096] ASVLAPKPYVAINMAELKNEPKTFEMFASVGPKVCMVTARHPGFVGFQNHIQIGILPFGNRY GGAKMDMTKESSTVRVLQYTFWKDWKDHEEMHRQNWSYLFRLCYSCASQMIWGPWEPI

[1097] YEIIYANMPINTEMTDFTAVVGKKFAEGKPLDIPVISQPYGKRVVAFAEHSVIPGKEKQFEDAI VRTLEMLKKAPGFLGAMVLKEIGVSGIGSMQFGAKGFHQVLENPGSLEPDPNNVMYSVPE AKNTPQQYIVHVEWANTDALMFGMGRVLLYPELRQVHDEVLDTLVYGPYIRILNPMMEGTF WREYLNEQ

[1098] SEQ ID NO: 43 Nucleic acid sequence encoding SARS-CoV-2 Spike RBD-MrsD fusion protein with htPA signal peptide (“construct- 11 ”) in VRPs.

[1099] AIGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCG TCTCGGCTAGCGTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAATCTG TGTCCTTTTGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACC GGAAGCGGATCAGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTT CTCCACCTTCAAGTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCA ACGTGTACGCTGACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGG ACAGACAGGCAAGATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCTGTG TGATTGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTG

[1100] TACAGGCTGTTTCGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGAT

[1101] CTATCAGGCAGGCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCAC

[1102] TGCAGTCCTACGGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAGAGTGGTG

[1103] GTGCTGAGCTTCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCA

[1104] CCAACCTGGTGAAGAACAAGAGCAGCGGCGGAGCCTCTGTGCTGGCCTCTATCAGCAT

[1105] CCTGAAGGACAAGAAGCTGCTGATCGGCATCTGCGGCAGCATCAGCTCTGTGGGCATC

[1106] TCCAGCTACCTGCTGTACTTCAAGAGCTTCTTCAAAGAAATCCGGGTCGTGATGACCAA

[1107] GACCGCCGAGGATCTGATCCCTGCTCACACCGTGTCCTACTTTTGCGACCACGTGTACA

[1108] GCGAGCACGGCGAGAATGGCAAGAGACACAGCAACGTGGAAATCGGCAGATGGGCCG

[1109] ACATCTACTGCATCATCCCTGCCACCGCTAACATCCTGGGCCAGACAGCTAATGGCGTG

[1110] GCCATGAACCTGGTGGCCACAACCGTTCTGGCTCACCCTCACAACACCATCTTTTTCCC

[1111] CAACATGAACGACCTGATGTGGAACAAGACCGTGGTGTCCAGAAACATCGAGCAGCTG

[1112] AGAAAGGACGGCCACATCGTGATCGAGCCTGTGGAAATCATGGCCTTCGAGATCGCCA

[1113] CCGGCACCAGAAAGCCTAACAGAGGCCTGATCACCCCTGACAAGGCCCTGCTGGCTAT

[1114] CGAGAAGGGCTTCAAAGAGAGAACAAAGCACCCCAGCCTGACC

[1115] SEQ ID NO: 44 Amino acid sequence of SARS-CoV-2 Spike RBD-MrsD fusion protein with htPA signal peptide (“construct-1 1 ”).

[1116] MDAMKRGLCCVLLLCGAVFVSASVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD

[1117] YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCN GVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSSGG

[1118] ASVLASISILKDKKLLIGICGSISSVGISSYLLYFKSFFKEIRVVMTKTAEDLIPAHTVSYFCDHV YSEHGENGKRHSNVEIGRWADIYCIIPATANILGQTANGVAMNLVATTVLAHPHNTIFFPNMN DLMWNKTVVSRNIEQLRKDGHIVIEPVEIMAFEIATGTRKPNRGLITPDKALLAIEKGFKERTK HPSLT

[1119] SEQ ID NO: 45 Nucleic acid sequence encoding HisB-SARS-CoV-2 Spike RBD fusion protein with htPA signal peptide (“construct- 12”) in VRPs.

[1120] AIGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCG TCTCGGCTAGCGCATCTAGAAGGGCCAGAATCGAGAGAAGAACCCGCGAGAGCGACA TCGTGATCGAGCTGGATCTGGATGGCACAGGACAGGTGGCAGTGGATACAGGCGTGC CCTTCTACGACCACATGCTGACAGCTCTGGGCAGCCACGCCTCTTTCGACCTGACAGT TAGAGCCACCGGCGACGTGGAAATCGAGGCCCACCACACAATCGAGGACACCGCTAT CGCTCTGGGAACAGCTCTTGGACAGGCCCTGGGCGATAAGAGAGGCATCAGAAGATTC GGCGACGCTTTCATCCCCATGGACGAGACACTGGCTCACGCTGCCGTTGACCTTAGCG GCAGACCTTACTGTGTGCATACCGGCGAGCCTGACCATCTGCAGCACACAACAATCGC CGGCAGCAGCGTGCCATACCACACCGTGATCAACAGACACGTGTTCGAGAGCCTGGC CGCCAACGCTAGAATCGCTCTGCATGTGCGGGTGCTGTACGGCAGAGATCCCCACCAC ATCACAGAGGCCCAGTACAAGGCTGTGGCCAGGGCTCTGAGACAGGCCGTTGAACCT GATCCTAGAGTGTCCGGCGTGCCATCTACAAAGGGTGCTCTGAGCAGCGGCGGAGCC TCTGTGCTGGCCGTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAATC TGTGTCCTTTTGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAA CCGGAAGCGGATCAGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGC TTCTCCACCTTCAAGTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCAC CAACGTGTACGCTGACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCT GGACAGACAGGCAAGATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCT GTGTGATTGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTA CCTGTACAGGCTGTTTCGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACC GAGATCTATCAGGCAGGCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACT TCCCACTGCAGTCCTACGGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAG AGTGGTGGTGCTGAGCTTCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAG

[1121] AAAAGCACCAACCTGGTGAAGAACAAA

[1122] SEQ ID NO: 46 Amino acid sequence of HisB-SARS-CoV-2 Spike RBD fusion protein with htPA signal peptide (“construct-12”).

[1123] MDAMKRGLCCVLLLCGAVFVSASASRRARIERRTRESDIVIELDLDGTGQVAVDTGVPFYDH MLTALGSHASFDLTVRATGDVEIEAHHTIEDTAIALGTALGQALGDKRGIRRFGDAFIPMDETL AHAAVDLSGRPYCVHTGEPDHLQHTTIAGSSVPYHTVINRHVFESLAANARIALHVRVLYGR DPHHITEAQYKAVARALRQAVEPDPRVSGVPSTKGALSSGGASVLAVQPTESIVRFPNITNL CPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYA DSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRK SNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLH APATVCGPKKSTNLVKNK

[1124] SEQ ID NO: 47 Nucleic acid sequence encoding DPS-SARS-CoV-2 Spike RBD fusion protein with htPA signal peptide (“construct- 13”) in VRPs.

[1125] ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCG TCTCGGCTAGCGCTACAAATCTGCTGTACACCAGAAACGACGTGTCCGACAGCGAGAA GAAGGCCACAGTCGAGCTGCTGAACAGACAAGTGATCCAGTTCATCGACCTGAGCCTG ATCACCAAGCAGGCCCACTGGAACATGAGAGGCGCCAACTTTATCGCCGTGCACGAGA TGCTGGACGGCTTCAGAACAGCCCTGATCGACCACCTGGACACCATGGCTGAAAGAGC TGTGCAGCTTGGCGGAGTGGCTCTGGGCACAACCCAAGTGATCAACAGCAAGACCCCT CTGAAGTCTTACCCTCTGGACATCCACAACGTGCAGGACCACCTGAAAGAACTGGCCG ACAGATACGCCATCGTGGCCAATGATGTGCGGAAGGCTATCGGCGAGGCCAAGGACGA TGATACCGCCGATATCCTGACAGCCGCCAGCAGAGATCTGGACAAGTTCCTGTGGTTCA TCGAGAGCAACATCGAGAGCAGCGGCGGAGCCTCTGTGCTGGCCGTGCAGCCCACAG AGTCTATCGTGCGGTTCCCTAACATCACCAATCTGTGTCCTTTTGGCGAGGTGTTCAACG CCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTTGC CGACTACAGCGTGCTGTACAACTCTGCCAGCTTCTCCACCTTCAAGTGCTATGGCGTGT CTCCTACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCTGACAGCTTCGTGATC AGAGGCGACGAAGTGAGACAGATTGCTCCTGGACAGACAGGCAAGATTGCCGATTACA ACTACAAGCTCCCTGACGACTTCACAGGCTGTGTGATTGCCTGGAACAGCAACAACCTG GACAGCAAAGTCGGAGGTAACTACAACTACCTGTACAGGCTGTTTCGGAAGTCCAACCT GAAGCCTTTCGAGAGAGACATCAGCACCGAGATCTATCAGGCAGGCAGCACACCTTGC AATGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGTCCTACGGCTTCCAGCCTAC AAATGGAGTGGGCTACCAGCCTTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCAT GCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCACCAACCTGGTGAAGAACAAA

[1126] SEQ ID NO: 48 Amino acid sequence of DPS-SARS-CoV-2 Spike RBD fusion protein with htPA signal peptide (“construct-13”).

[1127] MDAMKRGLCCVLLLCGAVFVSASATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQA HWNMRGANFIAVHEMLDGFRTALIDHLDTMAERAVQLGGVALGTTQVINSKTPLKSYPLDIH

[1128] NVQDHLKELADRYAIVANDVRKAIGEAKDDDTADILTAASRDLDKFLWFIESNIESSGGASVLA VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYG

[1129] VSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDS KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG

[1130] YQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK SEQ ID NO: 49 Nucleic acid sequence encoding SOR-SARS-CoV-2 Spike RBD fusion protein with htPA signal peptide (“construct- 14”) in VRPs.

[1131] AIGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCG TCTCGGCTAGCCCTAAACCTTACGTGGCCATCAACATGGCCGAGCTGAAGAACGAGCC CAAGACCTTCGAGATGTTCGCCAGCGTGGGCCCCAAAGTGTGCATGGTCACAGCTAGA CACCCTGGCTTCGTGGGCTTCCAGAACCACATCCAGATCGGCATCCTGCCTTTCGGCA ACAGATACGGCGGAGCTAAGATGGACATGACCAAAGAGTCCAGCACCGTCAGAGTGCT GCAGTACACCTTCTGGAAGGATTGGAAGGACCACGAGGAAATGCACAGACAGAACTGG TCCTACCTGTTCAGACTGTGCTACAGCTGCGCCAGCCAGATGATCTGGGGACCTTGGG AGCCCATCTACGAGATCATCTACGCCAACATGCCCATCAACACCGAGATGACCGACTTC ACAGCCGTCGTGGGCAAGAAGTTCGCTGAGGGCAAGCCTCTGGACATCCCCGTGATCT CTCAGCCCTACGGCAAGAGAGTGGTGGCCTTCGCTGAGCACTCTGTGATCCCCGGCAA AGAGAAGCAGTTCGAGGACGCCATCGTGCGGACCCTGGAAATGCTGAAGAAGGCCCC TGGCTTTCTGGGCGCCATGGTGCTGAAAGAAATCGGCGTGTCCGGCATCGGCAGCAT GCAGTTTGGCGCTAAGGGCTTCCACCAGGTGCTGGAAAACCCCGGCTCTCTGGAACCC GATCCTAACAACGTGATGTACAGCGTGCCCGAGGCCAAGAACACCCCTCAGCAGTATA TCGTGCACGTGGAATGGGCCAACACAGACGCCCTGATGTTCGGCATGGGACGCGTGC TGCTGTACCCTGAACTGAGACAGGTGCACGACGAAGTGCTGGACACACTGGTGTACGG CCCCTACATCAGAATCCTGAATCCTATGATGGAAGGCACGTTCTGGCGCGAGTACCTG AACGAACAAAGCAGCGGCGGAGCCTCTGTGCTGGCCGTGCAGCCCACAGAGTCTATC GTGCGGTTCCCTAACATCACCAATCTGTGTCCTTTTGGCGAGGTGTTCAACGCCACCAG ATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTTGCCGACTAC AGCGTGCTGTACAACTCTGCCAGCTTCTCCACCTTCAAGTGCTATGGCGTGTCTCCTAC CAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCTGACAGCTTCGTGATCAGAGGC GACGAAGTGAGACAGATTGCTCCTGGACAGACAGGCAAGATTGCCGATTACAACTACA AGCTCCCTGACGACTTCACAGGCTGTGTGATTGCCTGGAACAGCAACAACCTGGACAG CAAAGTCGGAGGTAACTACAACTACCTGTACAGGCTGTTTCGGAAGTCCAACCTGAAG CCTTTCGAGAGAGACATCAGCACCGAGATCTATCAGGCAGGCAGCACACCTTGCAATG GCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGTCCTACGGCTTCCAGCCTACAAAT GGAGTGGGCTACCAGCCTTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCATGCTC CTGCCACAGTGTGCGGACCTAAGAAAAGCACCAACCTGGTGAAGAACAAA

[1132] SEQ ID NO: 50 Amino acid sequence of SOR-SARS-CoV-2 Spike RBD fusion protein with htPA signal peptide (“construct-14”).

[1133] MDAMKRGLCCVLLLCGAVFVSASPKPYVAINMAELKNEPKTFEMFASVGPKVCMVTARHPG FVGFQNHIQIGILPFGNRYGGAKMDMTKESSTVRVLQYTFWKDWKDHEEMHRQNWSYLF RLCYSCASQMIWGPWEPIYEIIYANMPINTEMTDFTAVVGKKFAEGKPLDIPVISQPYGKRVV AFAEHSVIPGKEKQFEDAIVRTLEMLKKAPGFLGAMVLKEIGVSGIGSMQFGAKGFHQVLEN PGSLEPDPNNVMYSVPEAKNTPQQYIVHVEWANTDALMFGMGRVLLYPELRQVHDEVLDT LVYGPYIRILNPMMEGTFWREYLNEQSSGGASVLAVQPTESIVRFPNITNLCPFGEVFNATR FASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVR QIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDIST EIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKST NLVKNK

[1134] SEQ ID NO: 51 Nucleic acid sequence encoding MrsD-SARS-CoV-2 Spike RBD fusion protein with htPA signal peptide (“construct- 15”) in VRPs.

[1135] AIGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCG TCTCGGCTAGCTCTATCAGCATCCTGAAGGACAAGAAGCTGCTGATCGGCATCTGCGGC AGCATCAGCTCTGTGGGCATCTCCAGCTACCTGCTGTACTTCAAGAGCTTCTTCAAAGA AATCCGGGTCGTGATGACCAAGACCGCCGAGGATCTGATCCCTGCTCACACCGTGTCC TACTTTTGCGACCACGTGTACAGCGAGCACGGCGAGAATGGCAAGAGACACAGCAACG

[1136] TGGAAATCGGCAGATGGGCCGACATCTACTGCATCATCCCTGCCACCGCTAACATCCTG

[1137] GGCCAGACAGCTAATGGCGTGGCCATGAACCTGGTGGCCACAACCGTTCTGGCTCACC

[1138] CTCACAACACCATCTTTTTCCCCAACATGAACGACCTGATGTGGAACAAGACCGTGGTG

[1139] TCCAGAAACATCGAGCAGCTGAGAAAGGACGGCCACATCGTGATCGAGCCTGTGGAAA

[1140] TCATGGCCTTCGAGATCGCCACCGGCACCAGAAAGCCTAACAGAGGCCTGATCACCCC

[1141] TGACAAGGCCCTGCTGGCTATCGAGAAGGGCTTCAAAGAGAGAACAAAGCACCCCAGC

[1142] CTGACCAGCAGCGGCGGAGCCTCTGTGCTGGCCGTGCAGCCCACAGAGTCTATCGTG

[1143] CGGTTCCCTAACATCACCAATCTGTGTCCTTTTGGCGAGGTGTTCAACGCCACCAGATT

[1144] CGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTTGCCGACTACAGC

[1145] GTGCTGTACAACTCTGCCAGCTTCTCCACCTTCAAGTGCTATGGCGTGTCTCCTACCAA

[1146] GCTGAACGACCTGTGCTTCACCAACGTGTACGCTGACAGCTTCGTGATCAGAGGCGAC

[1147] GAAGTGAGACAGATTGCTCCTGGACAGACAGGCAAGATTGCCGATTACAACTACAAGCT

[1148] CCCTGACGACTTCACAGGCTGTGTGATTGCCTGGAACAGCAACAACCTGGACAGCAAA

[1149] GTCGGAGGTAACTACAACTACCTGTACAGGCTGTTTCGGAAGTCCAACCTGAAGCCTTT

[1150] CGAGAGAGACATCAGCACCGAGATCTATCAGGCAGGCAGCACACCTTGCAATGGCGTG

[1151] GAAGGCTTCAACTGCTACTTCCCACTGCAGTCCTACGGCTTCCAGCCTACAAATGGAGT

[1152] GGGCTACCAGCCTTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCATGCTCCTGCC

[1153] ACAGTGTGCGGACCTAAGAAAAGCACCAACCTGGTGAAGAACAAA

[1154] SEQ ID NO: 52 Amino acid sequence of MrsD-SARS-CoV-2 Spike RBD fusion protein with htPA signal peptide (“construct-15”).

[1155] MDAMKRGLCCVLLLCGAVFVSASSISILKDKKLLIGICGSISSVGISSYLLYFKSFFKEIRVVMT KTAEDLIPAHTVSYFCDHVYSEHGENGKRHSNVEIGRWADIYCIIPATANILGQTANGVAMN

[1156] LVATTVLAHPHNTIFFPNMNDLMWNKTVVSRNIEQLRKDGHIVIEPVEIMAFEIATGTRKPNR GLITPDKALLAIEKGFKERTKHPSLTSSGGASVLAVQPTESIVRFPNITNLCPFGEVFNATRFA

[1157] SVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQI APGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEI

[1158] YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTN LVKNK

[1159] SEQ ID NO: 53 Nucleic acid sequence encoding EBV gp350-PdhC fusion protein with IgK LC signal peptide (“construct-16”) in VRPs.

[1160] ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGG TGACGAAGCAGCTCTGCTCGTGTGCCAGTACACCATCCAGAGCCTCATCCACCTGACA GGAGAGGATCCTGGCTTCTTCAACGTGGAAATTCCAGAGTTTCCCTTCTACCCTACCTG CAATGTGTGCACAGCCGACGTGAACGTGACCATCAACTTCGACGTTGGAGGCAAGAAG CACCAGCTGGACCTGGATTTCGGACAACTGACACCTCACACCAAGGCTGTGTATCAGC CTAGAGGAGCCTTTGGTGGTTCTGAGAATGCCACCAACCTGTTTCTCCTGGAGCTGCTT GGAGCTGGCGAGCTCGCACTGACCATGAGAAGCAAGAAACTGCCCATCAATGTGACCA CAGGCGAGGAACAGCAGGTGTCCCTGGAAAGCGTGGACGTGTACTTTCAAGACGTGTT CGGCACCATGTGGTGCCACCATGCCGAGATGCAGAACCCTGTGTACCTGATCCCAGAG ACAGTGCCCTACATCAAGTGGGACAACTGCAACAGCACAAACATCACAGCCGTCGTGA GGGCTCAGGGACTGGATGTGACACTGCCTCTGTCTCTGCCAACCAGTGCCCAGGACAG CAACTTCAGCGTGAAGACCGAGATGCTGGGAAACGAGATCGACATCGAGTGCATCATG GAAGATGGCGAGATCAGCCAGGTACTGCCTGGCGACAACAAGTTCAACATCACATGCA GTGGCTACGAGAGCCACGTGCCATCTGGAGGCATCCTGACAAGCACAAGCCCAGTGGC CACACCGATCCCTGGCACAGGCTACGCCTACAGCCTGAGACTGACACCCAGACCCGTG TCCAGATTCCTGGGCAACAACAGCATCCTGTACGTGTTCTACTCTGGCAACGGACCCAA GGCCTCTGGTGGCGATTACTGTATCCAGAGCAACATTGTGTTCAGCGATGAGATCCCTG CCAGCCAGGACATGCCAACCAATACCACCGATATCACCTACGTGGGAGACAATGCCACC TACAGCGTGCCCATGGTCACCTCCGAGGACGCCAACAGCCCTAATGTGACCGTGACAG CCTTCTGGGCATGGCCTAACAACACCGAGACAGACTTCAAGTGCAAGTGGACCCTGAC

[1161] CTCTGGCACACCTAGTGGCTGCGAGAATATCAGCGGAGCCTTCGCCAGCAACCGGACC

[1162] TTCGACATTACCGTGTCTGGCCTTGGCACAGCTCCCAAGACCCTGATCATCACCAGGAC

[1163] TGCCACCAATGCCACAACCACAACCCACAAAGTGATCTTCAGCAAGGCTCCTGAAAGCA

[1164] CCACAACTAGTCCTACACTGCCTAAGCCCAGCACACCTCCTGGCAGCAGCGCTGCTGC

[1165] TAAACCTGCTACAACAGAGGGCGAGTTCCCCGAGACACGCGAGAAGATGTCTGGCATC

[1166] AGAAGGGCTATCGCCAAGGCCATGGTGCACAGCAAGCACACAGCTCCTCACGTGACCC

[1167] TGATGGACGAGGCCGATGTGACAAAGCTGGTGGCCCACAGAAAGAAGTTCAAGGCCAT

[1168] TGCCGCCGAGAAGGGAATCAAGCTGACCTTCCTGCCTTACGTGGTCAAGGCCCTGGTT

[1169] TCTGCCCTGAGAGAATACCCCGTGCTGAACACCAGCATCGACGACGAGACAGAGGAAA

[1170] TCATCCAGAAGCACTACTACAACATCGGAATCGCCGCCGACACCGACAGAGGACTGCT

[1171] GGTGCCTGTGATCAAGCACGCCGACAGAAAGCCCATCTTCGCCCTGGCTCAAGAGATC

[1172] AACGAGCTGGCTGAGAAGGCCAGAGATGGCAAGCTGACACCCGGCGAAATGAAGGGC

[1173] GCCAGCTGTACCATCACCAACATCGGTTCTGCTGGCGGCCAGTGGTTCACCCCAGTGA

[1174] TCAATCACCCTGAGGTGGCCATCCTCGGCATCGGCAGAATCGCTGAGAAGCCCATCGT

[1175] CCGCGACGGCGAAATTGTGGCTGCTCCTATGCTGGCCCTGAGCCTGAGCTTCGACCAC

[1176] AGAATGATCGACGGCGCCACAGCTCAGAAGGCCCTGAACCACATCAAGAGACTGCTGA

[1177] GCGACCCCGAGCTGCTGCTGATGGAAGCT

[1178] SEQ ID NO: 54 Amino acid sequence of EBV gp350-PdhC fusion protein with IgK

[1179] LC signal peptide (“construct-16”) in VRPs.

[1180] METDTLLLWVLLLWVPGSTGDEAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCT ADVNVTINFDVGGKKHQLDLDFGQLTPHTKAVYQPRGAFGGSENATNLFLLELLGAGELALT MRSKKLPINVTTGEEQQVSLESVDVYFQDVFGTMWCHHAEMQNPVYLIPETVPYIKWDNC NSTNITAVVRAQGLDVTLPLSLPTSAQDSNFSVKTEMLGNEIDIECIMEDGEISQVLPGDNKF NITCSGYESHVPSGGILTSTSPVATPIPGTGYAYSLRLTPRPVSRFLGNNSILYVFYSGNGPKA SGGDYCIQSNIVFSDEIPASQDMPTNTTDITYVGDNATYSVPMVTSEDANSPNVTVTAFWA WPNNTETDFKCKWTLTSGTPSGCENISGAFASNRTFDITVSGLGTAPKTLIITRTATNATTTT HKVIFSKAPESTTTSPTLPKPSTPPGSSAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSK HTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNTSIDDETE EIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTIT NIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKAL NHIKRLLSDPELLLMEA

[1181] SEQ ID NO: 55 Nucleic acid sequence encoding EBVgp350-DPS fusion protein with IgK LC signal peptide (“construct-17”) in VRPs.

[1182] ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTG GTGACGAAGCAGCTCTGCTCGTGTGCCAGTACACCATCCAGAGCCTCATCCACCTGAC

[1183] AGGAGAGGATCCTGGCTTCTTCAACGTGGAAATTCCAGAGTTTCCCTTCTACCCTACCT GCAATGTGTGCACAGCCGACGTGAACGTGACCATCAACTTCGACGTTGGAGGCAAGAA GCACCAGCTGGACCTGGATTTCGGACAACTGACACCTCACACCAAGGCTGTGTATCAG CCTAGAGGAGCCTTTGGTGGTTCTGAGAATGCCACCAACCTGTTTCTCCTGGAGCTGC TTGGAGCTGGCGAGCTCGCACTGACCATGAGAAGCAAGAAACTGCCCATCAATGTGAC CACAGGCGAGGAACAGCAGGTTTCCCTGGAAAGCGTGGACGTGTACTTTCAAGACGTG TTCGGCACCATGTGGTGCCACCATGCCGAGATGCAGAACCCTGTGTACCTGATCCCAG AGACAGTGCCCTACATCAAGTGGGACAACTGCAACAGCACAAACATCACAGCCGTCGT GAGGGCTCAGGGACTGGATGTGACACTGCCTCTGTCTCTGCCAACCAGTGCCCAGGA CAGCAACTTCAGCGTGAAGACCGAGATGCTGGGAAACGAGATCGACATCGAGTGCATC ATGGAAGATGGCGAGATCAGCCAGGTACTGCCTGGCGACAACAAGTTCAACATCACAT GCAGTGGCTACGAGAGCCACGTGCCATCTGGAGGCATCCTGACAAGCACAAGCCCAG TGGCCACACCGATCCCTGGCACAGGCTACGCCTACAGCCTGAGACTGACACCCAGAC CCGTGTCCAGATTCCTGGGCAACAACAGCATCCTGTACGTGTTCTACTCTGGCAACGG ACCCAAGGCCTCTGGTGGCGATTACTGTATCCAGAGCAACATTGTGTTCAGCGATGAG

[1184] ATCCCTGCCAGCCAGGACATGCCAACCAATACCACCGATATCACCTACGTGGGAGACA

[1185] ATGCCACCTACAGCGTGCCCATGGTCACCTCCGAGGACGCCAACAGCCCTAATGTGAC

[1186] CGTGACAGCCTTCTGGGCATGGCCTAACAACACCGAGACAGACTTCAAGTGCAAGTGG

[1187] ACCCTGACCTCTGGCACACCTAGTGGCTGCGAGAATATCAGCGGAGCCTTCGCCAGCA

[1188] ACCGGACCTTCGACATTACCGTGTCTGGCCTTGGCACAGCTCCCAAGACCCTGATCAT

[1189] CACCAGGACTGCCACCAATGCCACAACCACAACCCACAAAGTGATCTTCAGCAAGGCT

[1190] CCTGAAAGCACCACAACTAGTCCTACACTGCCTAAGCCCAGCACACCTCCTGGCAGCA

[1191] GCGCTACCAATCTGCTGTACACCAGAAACGACGTGTCCGACAGCGAGAAGAAAGCCAC

[1192] CGTGGAACTGCTGAACCGGCAAGTGATCCAGTTCATCGACCTGAGCCTGATCACCAAG

[1193] CAGGCCCACTGGAATATGAGAGGCGCCAACTTTATCGCCGTGCACGAAATGCTGGACG

[1194] GCTTCAGAACAGCCCTGATCGACCACCTGGATACCATGGCCGAAAGAGCCGTTCAGCT

[1195] TGGCGGAGTGGCTCTGGGCACAACCCAAGTGATCAACAGCAAGACCCCTCTGAAGTCT

[1196] TACCCTCTGGACATCCACAACGTGCAGGACCACCTGAAAGAGCTGGCCGACAGATACG

[1197] CCATCGTGGCCAATGATGTGCGGAAGGCCATTGGCGAGGCCAAGGATGATGACACCG

[1198] CCGATATTCTGACCGCCGCCAGCAGAGATCTGGACAAGTTCCTGTGGTTCATCGAGTC

[1199] CAATATCGAG

[1200] SEQ ID NO: 56 Amino acid sequence of EBVgp350-DPS fusion protein with IgK LC signal peptide (“construct-17”) in VRPs.

[1201] METDTLLLWVLLLWVPGSTGDEAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCT ADVNVTINFDVGGKKHQLDLDFGQLTPHTKAVYQPRGAFGGSENATNLFLLELLGAGELALT MRSKKLPINVTTGEEQQVSLESVDVYFQDVFGTMWCHHAEMQNPVYLIPETVPYIKWDNC NSTNITAVVRAQGLDVTLPLSLPTSAQDSNFSVKTEMLGNEIDIECIMEDGEISQVLPGDNKF NITCSGYESHVPSGGILTSTSPVATPIPGTGYAYSLRLTPRPVSRFLGNNSILYVFYSGNGPKA SGGDYCIQSNIVFSDEIPASQDMPTNTTDITYVGDNATYSVPMVTSEDANSPNVTVTAFWA WPNNTETDFKCKWTLTSGTPSGCENISGAFASNRTFDITVSGLGTAPKTLIITRTATNATTTT HKVIFSKAPESTTTSPTLPKPSTPPGSSATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLIT KQAHWNMRGANFIAVHEMLDGFRTALIDHLDTMAERAVQLGGVALGTTQVINSKTPLKSYPL DIHNVQDHLKELADRYAIVANDVRKAIGEAKDDDTADILTAASRDLDKFLWFIESNIE

[1202] SEQ ID NO: 57 Nucleic acid sequence encoding EBV gp350-Bullfrog / / - / . pylori hybrid ferritin fusion protein with IgK LC signal peptide and SARS- CoV-2 Spike RBD-Bullfrog / H pylori hybrid ferritin fusion protein with htPA signal peptide (“construct- 18”) in VRPs.

[1203] ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTG GTGACGAAGCAGCTCTGCTCGTGTGCCAGTACACCATCCAGAGCCTGATCCACCTGAC AGGAGAGGATCCTGGCTTCTTCAACGTGGAAATCCCAGAGTTTCCCTTCTACCCTACCT GCAACGTGTGCACAGCCGACGTGAACGTGACCATCAACTTCGACGTTGGAGGCAAGAA GCACCAGCTGGACCTGGATTTCGGACAGCTGACACCTCACACCAAGGCTGTGTATCAG CCTAGAGGAGCCTTTGGTGGCAGCGAGAACGCCACCAACCTGTTTCTGCTGGAACTGC TTGGAGCTGGCGAGCTCGCACTGACCATGAGAAGCAAGAAACTGCCCATCAATGTGAC CACAGGCGAGGAACAGCAGGTGTCCCTGGAAAGCGTGGACGTGTACTTTCAAGACGT GTTCGGCACCATGTGGTGCCACCACGCCGAGATGCAGAACCCTGTGTACCTGATCCCA GAGACAGTGCCCTACATCAAGTGGGACAACTGCAACAGCACCAACATCACAGCCGTCG TGAGAGCTCAGGGACTGGATGTGACACTGCCTCTGAGCCTGCCTACCAGTGCCCAGGA CAGCAACTTCAGCGTGAAGACCGAGATGCTGGGCAACGAGATCGACATCGAGTGCATC ATGGAAGATGGCGAGATCAGCCAGGTGCTGCCTGGCGACAACAAGTTCAACATCACAT GCAGTGGCTACGAGAGCCACGTGCCATCTGGAGGCATCCTGACCAGCACAAGCCCAG TGGCCACACCCATCCCTGGCACAGGCTACGCCTACAGCCTGAGACTGACACCCAGACC CGTGTCCAGATTCCTGGGCAACAACAGCATCCTGTACGTGTTCTACTCTGGCAACGGA CCCAAGGCCTCTGGTGGCGATTACTGTATCCAGAGCAACATCGTGTTCAGCGACGAGA

[1204] TCCCTGCCAGCCAGGACATGCCAACCAATACCACCGACATCACCTACGTGGGAGACAA

[1205] TGCCACCTACAGCGTGCCCATGGTCACCTCCGAGGACGCCAACAGCCCTAATGTGACC

[1206] GTGACAGCCTTCTGGGCATGGCCTAACAACACCGAGACAGACTTCAAGTGCAAGTGGA

[1207] CCCTGACCTCTGGCACACCTAGTGGCTGCGAGAATATCAGCGGAGCCTTCGCCAGCAA

[1208] CCGGACCTTCGATATCACCGTGTCTGGCCTTGGCACAGCTCCCAAGACCCTGATCATC

[1209] ACCAGGACTGCCACCAATGCCACAACCACAACCCACAAAGTGATCTTCAGCAAGGCTC

[1210] CTGAGAGCACCACAACTAGTCCTACACTGCCTAAGCCCAGCACACCTCCTGGCAGCTC

[1211] TGAATCTCAAGTTCGGCAGCAGTTCAGCAAGGACATCGAGAAGCTGCTGAACGAGCAA

[1212] GTGAACAAAGAGATGCAGAGCAGCAACCTGTACATGAGCATGAGCAGCTGGTGCTACA

[1213] CCCACAGCCTTGATGGCGCCGGACTGTTCCTGTTTGATCACGCCGCCGAGGAATACGA

[1214] GCACGCCAAGAAGCTGATCATCTTCCTGAACGAGAACAACGTGCCCGTGCAGCTGACC

[1215] AGCATTTCTGCCCCTGAGCACAAGTTCGAGGGCCTGACACAGATCTTCCAGAAGGCCT

[1216] ACGAACACGAGCAGCACATTAGCGAGAGCATCAACAACATCGTGGACCACGCCATTAA

[1217] GAGCAAGGATCACGCCACCTTCAACTTTCTGCAGTGGTACGTGGCCGAACAGCACGAG

[1218] GAAGAGGTGCTGTTCAAGGACATCCTGGACAAGATCGAGCTGATCGGCAACGAGAACC

[1219] ACGGCCTGTATCTGGCCGACCAGTACGTGAAGGGAATCGCCAAGAGCAGAAAGAGCA

[1220] GCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGAC

[1221] CTATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTT

[1222] CGTCTCGGCTAGCGTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAAT

[1223] CTGTGTCCTTTTGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGA

[1224] ACCGGAAGCGGATCAGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAG

[1225] CTTCTCCACCTTCAAGTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCA

[1226] CCAACGTGTACGCTGACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCC

[1227] TGGACAGACAGGCAAGATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGC

[1228] TGTGTGATTGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACT

[1229] ACCTGTACAGGCTGTTTCGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCAC

[1230] CGAGATCTATCAGGCAGGCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTAC

[1231] TTCCCACTGCAGTCCTACGGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACA

[1232] GAGTGGTGGTGCTGAGCTTCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAA

[1233] GAAAAGCACCAACCTGGTGAAGAACAAGAGCAGCGGCGGAGCCTCTGTGCTGGCCGA

[1234] GTCCCAGGTGCGCCAGCAGTTCTCCAAGGATATCGAGAAACTGCTCAATGAGCAGGTG

[1235] AACAAGGAGATGCAGTCCTCCAATCTGTACATGTCCATGTCCTCCTGGTGCTATACCCA

[1236] CTCCCTGGACGGCGCCGGCCTGTTCCTGTTCGACCACGCCGCTGAGGAGTACGAGCA

[1237] CGCTAAGAAACTGATTATCTTCCTGAATGAGAATAACGTGCCTGTGCAGCTGACCTCCA

[1238] TCTCCGCTCCCGAGCACAAATTCGAAGGCCTGACCCAGATCTTTCAGAAAGCCTATGA

[1239] GCATGAGCAGCACATTTCCGAGTCCATCAATAACATCGTCGACCACGCCATCAAGTCCA

[1240] AAGACCACGCCACCTTTAACTTCCTGCAGTGGTATGTGGCCGAGCAGCACGAGGAGGA

[1241] AGTGCTGTTTAAGGATATCCTGGATAAGATTGAGCTGATCGGAAACGAGAACCATGGCC

[1242] TGTACCTGGCCGATCAGTATGTGAAGGGCATCGCCAAGTCCCGCAAGTCC

[1243] SEQ ID NO: 58 Amino acid sequence of EBV gp350-Bullfrog / / - / . pylori hybrid ferritin fusion protein with IgK LC signal peptide and SARS-CoV-2 Spike RBD-Bullfrog / / - / . pylori hybrid ferritin fusion protein with htPA signal peptide separated by a T2A site (“construct-18”) (T2A site indicated in bold).

[1244] METDTLLLWVLLLWVPGSTGDEAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCT ADVNVTINFDVGGKKHQLDLDFGQLTPHTKAVYQPRGAFGGSENATNLFLLELLGAGELAL TMRSKKLPINVTTGEEQQVSLESVDVYFQDVFGTMWCHHAEMQNPVYLIPETVPYIKWDN CNSTNITAVVRAQGLDVTLPLSLPTSAQDSNFSVKTEMLGNEIDIECIMEDGEISQVLPGDNK FNITCSGYESHVPSGGILTSTSPVATPIPGTGYAYSLRLTPRPVSRFLGNNSILYVFYSGNGP KASGGDYCIQSNIVFSDEIPASQDMPTNTTDITYVGDNATYSVPMVTSEDANSPNVTVTAF WAWPNNTETDFKCKWTLTSGTPSGCENISGAFASNRTFDITVSGLGTAPKTLIITRTATNAT

[1245] TTTHKVIFSKAPESTTTSPTLPKPSTPPGSSESQVRQQFSKDIEKLLNEQVNKEMQSSNLYM SMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQ KAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLY LADQYVKG IAKSRKSSGEGRGSLLTCGDVEENPGPM DAM KRGLCCVLLLCGAVFVSASVQ

[1246] PTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVS

[1247] PTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKV

[1248] GGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ PYRVVVLSFELLHAPATVCGPKKSTNLVKNKSSGGASVLAESQVRQQFSKDIEKLLNEQVN

[1249] KEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPE HKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKI

[1250] ELIGNENHGLYLADQYVKGIAKSRKS

[1251] SEQ ID NO: 59 Nucleic acid sequence encoding an OspA serotypes 2, 4 and 3 C- terminal fragment string (OspA ST2 4 3) -Bullfrog / H pylori hybrid ferritin fusion protein with htPA signal peptide (“construct-19”) in VRPs.

[1252] ATGGACGCTATGAAGAGGGGCCTGTGCTGTGTGCTGCTGCTGTGCGGAGCTGTGTTTG TGTCCGCCAGCTTTAATGAGAAAGGCGAGCTGAGCGCCAAGACCATGACCAGAGAGCA GGGCACAAAGCTCGAGTATACCGAGATGAAGTCCGATGGCACAGGGAAAGCTAAAGAG GTCCTCAAACAGTTCACGCTCGAGGGCAAAGTCGCCAATGACAAAGTGACACTGGAAG TGAAGTGTGGCACAGTGACCCTGTCCAAAGAGATCGCCAAGTCCGGCGAAGTGACAGT GGCCCTGCAAGATACCCAAACCACACAGGCCACCAAAAAGACTGGCGCCTGGGACAGC AAGACAAGCACCCTGACAATCTCTGTGAATAGCAAGAAAACGACCCAGCTCGTGTTTAC CAAGCAGGACACAATCACCGTCCAGAAGTACGATAGCGCCGGAACAAACCTGGAAGGG ACCGCCGTCGAGATCAAGACCCTGGACGAACTGTGTAATGCCCTGAAGGGTGGCGGAG GATCTGGTGGCGGTGGCAGTGGCGGAGGCGGTTCTGGTGGTGGCGGATCTTTCAATG CCAAGGGCGAGCTGAGCGAGAAAACCATCCTGAGAGCCCAAGGCACCCGGCTCGAGT ACACAGAGATCAAGTCTGACGGCACCGGCAAGGCCAAAGAGGTGCTGAAGGATTTCGC CCTGGAAGGCACACTGGCCGCCGATAAGACCACACTGAAAGTGACCTGTGGCACCGTG GTGCTGAGCAAGCACATCCCTAATAGCGGCGAGATCACCGTGGAACTGCAGGACAGCC AAAGCACCCAGGCCACAAAGAAAACCGGCAAGTGGGACAGCCAGACCAGTACACTGAC CATCAGCGTGAACAGCAAAAAGACCAAGAACATCGTGTTCACCAAAGAGGACACCATCA CCGTGCAAAAGTATGACAGCGCCGGCACCAACCTCGAAGGGAATGCCGTGGAAATCAA GACACTGGATGAGCTGTGCAACGCCCTTAAAGGCGGTGGCGGCTCTGGAGGCGGTGG TTCAGGCGGAGGAGGAAGCGGAGGCGGAGGCAGCTTTAACGATAAGGGCAAGCTGTC CGAGAAGGTGGTCACCAGAGCCCAGGGAACAAGACTCGAGTATACAGAAATTAAGAAC GATGGCAGCGGGAAAGCCAAAGAAGTTCTGAAAGGCTTCGCTCTCGAGGGCACCCTTA CAGATGGCGGCGAGACAAAGCTGACCGTGACATGCGGAACCGTGACACTGAGCAAGC AGATCAGCAAGTCCGGGGAGATCACAGTGGCCCTCCAGGACACCGAAACAACCCCTGC CGACAAGAAAACAGGCGAATGGAAGTCCGACACAAGCACCCTCACCATTTCCAAGAAC AGCCAGAAACCTAAGCAGCTGGTCTTTACCAAAGAAAACACGATTACCGTGCAGAACTA CAATAGAGCCGGCAACGCTCTGGAAGGCTCCCCAGCCGAAATCAAGGACCTGGCTGAA CTGTGCGCCGCTCTGAAAAGCAGCGGCGGAGCCTCTGTGCTGGCCGAATCTCAAGTTC GGCAGCAGTTCAGCAAGGACATCGAGAAGCTGCTGAACGAGCAAGTGAACAAAGAGAT GCAGAGCAGCAACCTGTACATGAGCATGAGCAGCTGGTGCTACACCCACAGCCTTGAT GGCGCCGGACTGTTCCTGTTTGATCACGCCGCCGAGGAATACGAGCACGCCAAGAAGC TGATCATCTTCCTGAACGAGAACAACGTGCCCGTGCAGCTGACCAGCATTTCTGCCCCT GAGCACAAGTTCGAGGGCCTGACACAGATCTTCCAGAAGGCCTACGAACACGAGCAGC ACATTAGCGAGAGCATCAACAACATCGTGGACCACGCCATTAAGAGCAAGGATCACGCC ACCTTCAACTTTCTGCAGTGGTACGTGGCCGAACAGCACGAGGAAGAGGTGCTGTTCA AGGACATCCTGGACAAGATCGAGCTGATCGGCAACGAGAACCACGGCCTGTATCTGGC

[1253] CGACCAGTACGTGAAGGGAATCGCCAAGAGCAGAAAGAGC

[1254] SEQ ID NO: 60 Amino acid sequence of an OspA serotypes 2, 4 and 3 C-terminal fragment string (OspA ST2-4-3) -Bullfrog / / - / , pylori hybrid ferritin fusion protein with htPA signal peptide (“construct- 19”) in VRPs.

[1255] MDAMKRGLCCVLLLCGAVFVSASFNEKGELSAKTMTREQGTKLEYTEMKSDGTGKAKEVL KQFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALQDTQTTQATKKTGAWDSKTSTLTI SVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGGGGSGGGGSGGG GSGGGGSFNAKGELSEKTILRAQGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKV TCGTVVLSKHIPNSGEITVELQDSQSTQATKKTGKWDSQTSTLTISVNSKKTKNIVFTKEDTIT VQKYDSAGTNLEGNAVEIKTLDELCNALKGGGGSGGGGSGGGGSGGGGSFNDKGKLSEK VVTRAQGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKQISKSGEI TVALQDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSP AEIKDLAELCAALKSSGGASVLAESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCY THSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQ HISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVK GIAKSRKS

[1256] SEQ ID NO: 61 Nucleic acid sequence encoding an OspA serotypes 1 , 5 and 6 C- terminal fragment string (OspA ST1 -5-6) -Bullfrog / / - / , pylori hybrid ferritin fusion protein with htPA signal peptide and an OspA serotypes 2, 4 and 3 C-terminal fragment string (OspA ST243) - Bullfrog / / - / , pylori hybrid ferritin fusion protein with htPA signal peptide separated by a P2A site (“construct-20”) in VRPs.

[1257] ATGGATGCAATGAAGAGAGGGCTCTGTTGCGTGTTGCTTTTGTGTGGAGCAGTCTTCGT CTCGGCTAGCTTCAACGAGAAGGGCGAAGTGTCCGAGAAGATCATCACCAGAGCCGAC GGCACCAGACTCGAGTACACAGGCATCAAGTCTGACGGCAGCGGCAAGGCCAAAGAG GTGCTGAAGCAGTTCACCCTGGAAGGCAAGGTGGCCAACGACAAGACCACACTGGTG GTCAAGTGCGGCACCGTGACACTGAGCAAGCAGATCAGCAAGAGCGGCGAGGTGTCC GTGGAACTGCAGGACACAGATAGCAGCGCCGCCACCAAGAAAACCGCCGCCTGGAATA GCGGCACCAGCACACTGACCATCACCGTGAACAGCAAAAAGACCAAGGACCTGGTGTT CACCAAAGAAAACACCATCACAGTGCAGCAGTACGACAGCCAGGGCACCAAGCTGGAA GGCTCCGCCGTGGAAATCACAAAGCTGGACGAGATCTGCAACGCCCTGAAAGGCGGT GGCGGCTCTGGCGGAGGTGGAAGCGGAGGAGGAGGAAGTGGCGGAGGCGGTTCTTT TAATGAGAAAGGCGAGATCAGCGAGAAAACCATCGTCAGAGCCCAAGGCACACGGCTC GAGTATACCGACATCAAGAGCGATAAGACCGGGAAAGCCAAAGAAGTCCTCAAGGATTT CACACTCGAGGGCACCCTGGCCGCCGATGGAAAGACAACCCTGAAAGTGACCTGTGG CACAGTGACCCTGTCCAAGCAAATCTCCAAGAGCGGAGAGATCACAGTGGCCCTGGAC GATACCGATAGCTCCGGCAACAAGAAGTCCGGCACCTGGGATTCTGGCACCTCCACTCT GACCATCTCTAAGCAGCGGACCAAGACAAAGCAGCTGGTCTTTACCAAAGAGGACACAA TCACCGTCCAGAACTACGACTCCGCCGGCACAAACCTCGAGGGAAAAGCCGTCGAGAT CACCACACTGAAAGAGCTGTGTAACGCTCTCAAAGGTGGTGGCGGTTCCGGTGGCGGT GGCAGTGGTGGTGGTGGATCAGGCGGTGGCGGAAGCTTTAATGGCAAGGGCGAGACA TCCGAAAAGACGATCGTTCGCGCCCAAGGAACCCGGCTTGAGTACACCGATATTAAGTC CGACGGCTCTGGAAAGGCTAAAGAAGTTCTGAAAGACTTTACCCTCGAAGGGACTCTC GCCGCTGACGGCAAGACCACTCTGAAAGTTACATGCGGAACCGTGGTGCTGTCTAAGA ACATCCTGAAGTCCGGGGAGATCACTGCCGCTCTGGACGACAGCGATACCACACGGGC CACCAAAAAGACAGGCAAGTGGGACAGCAAGACAAGCACCCTGACAATCAGCGTGAAC TCCCAGAAAACGAAGAATCTCGTGTTTACGAAAGAAGATACCATTACGGTCCAGCGCTAC

[1258] GACAGCGCTGGGACAAATCTGGAAGGGAAAGCTGTGGAAATTACGACCCTCAAAGAAC

[1259] TGTGCAATGCCCTCAAGAGCAGCGGCGGAGCCTCTGTGCTGGCCGAATCTCAAGTTCG

[1260] GCAGCAGTTCAGCAAGGACATCGAGAAGCTGCTGAACGAGCAAGTGAACAAAGAGATG

[1261] CAGAGCAGCAACCTGTACATGAGCATGAGCAGCTGGTGCTACACCCACAGCCTTGATG

[1262] GCGCCGGACTGTTCCTGTTTGATCACGCCGCCGAGGAATACGAGCACGCCAAGAAGCT

[1263] GATCATCTTCCTGAACGAGAACAACGTGCCCGTGCAGCTGACCAGCATTTCTGCCCCTG

[1264] AGCACAAGTTCGAGGGCCTGACACAGATCTTCCAGAAGGCCTACGAACACGAGCAGCA

[1265] CATTAGCGAGAGCATCAACAACATCGTGGACCACGCCATTAAGAGCAAGGATCACGCCA

[1266] CCTTCAACTTTCTGCAGTGGTACGTGGCCGAACAGCACGAGGAAGAGGTGCTGTTCAA

[1267] GGACATCCTGGACAAGATCGAGCTGATCGGCAACGAGAACCACGGCCTGTATCTGGCC

[1268] GACCAGTACGTGAAGGGAATCGCCAAGAGCAGAAAGAGCAGCGGAGAGGGCAGAGGA

[1269] AGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGACCTATGGACGCTATGAAGAG

[1270] GGGCCTGTGCTGTGTGCTGCTGCTGTGCGGAGCTGTGTTTGTGTCCGCCAGCTTTAAT

[1271] GAGAAAGGCGAGCTGAGCGCCAAGACCATGACCAGAGAGCAGGGCACAAAGCTCGAG

[1272] TATACCGAGATGAAGTCCGATGGCACAGGGAAAGCTAAAGAGGTCCTCAAACAGTTCAC

[1273] GCTCGAGGGCAAAGTCGCCAATGACAAAGTGACACTGGAAGTGAAGTGTGGCACAGTG

[1274] ACCCTGTCCAAAGAGATCGCCAAGTCCGGCGAAGTGACAGTGGCCCTGCAAGATACCC

[1275] AAACCACACAGGCCACCAAAAAGACTGGCGCCTGGGACAGCAAGACAAGCACCCTGA

[1276] CAATCTCTGTGAATAGCAAGAAAACGACCCAGCTCGTGTTTACCAAGCAGGACACAATC

[1277] ACCGTCCAGAAGTACGATAGCGCCGGAACAAACCTGGAAGGGACCGCCGTCGAGATCA

[1278] AGACCCTGGACGAACTGTGTAATGCCCTGAAGGGTGGCGGAGGATCTGGTGGCGGTG

[1279] GCAGTGGCGGAGGCGGTTCTGGTGGTGGCGGATCTTTCAATGCCAAGGGCGAGCTGA

[1280] GCGAGAAAACCATCCTGAGAGCCCAAGGCACCCGGCTCGAGTACACAGAGATCAAGTC

[1281] TGACGGCACCGGCAAGGCCAAAGAGGTGCTGAAGGATTTCGCCCTGGAAGGCACACT

[1282] GGCCGCCGATAAGACCACACTGAAAGTGACCTGTGGCACCGTGGTGCTGAGCAAGCAC

[1283] ATCCCTAATAGCGGCGAGATCACCGTGGAACTGCAGGACAGCCAAAGCACCCAGGCCA

[1284] CAAAGAAAACCGGCAAGTGGGACAGCCAGACCAGTACACTGACCATCAGCGTGAACAG

[1285] CAAAAAGACCAAGAACATCGTGTTCACCAAAGAGGACACCATCACCGTGCAAAAGTATG

[1286] ACAGCGCCGGCACCAACCTCGAAGGGAATGCCGTGGAAATCAAGACACTGGATGAGCT

[1287] GTGCAACGCCCTTAAAGGCGGTGGCGGCTCTGGAGGCGGTGGTTCAGGCGGAGGAG

[1288] GAAGCGGAGGCGGAGGCAGCTTTAACGATAAGGGCAAGCTGTCCGAGAAGGTGGTCA

[1289] CCAGAGCCCAGGGAACAAGACTCGAGTATACAGAAATTAAGAACGATGGCAGCGGGAA

[1290] AGCCAAAGAAGTTCTGAAAGGCTTCGCTCTCGAGGGCACCCTTACAGATGGCGGCGAG

[1291] ACAAAGCTGACCGTGACATGCGGAACCGTGACACTGAGCAAGCAGATCAGCAAGTCCG

[1292] GGGAGATCACAGTGGCCCTCCAGGACACCGAAACAACCCCTGCCGACAAGAAAACAG

[1293] GCGAATGGAAGTCCGACACAAGCACCCTCACCATTTCCAAGAACAGCCAGAAACCTAAG

[1294] CAGCTGGTCTTTACCAAAGAAAACACGATTACCGTGCAGAACTACAATAGAGCCGGCAA

[1295] CGCTCTGGAAGGCTCCCCAGCCGAAATCAAGGACCTGGCTGAACTGTGCGCCGCTCT

[1296] GAAAAGCAGCGGCGGAGCCTCTGTGCTGGCCGAGTCCCAGGTGCGCCAGCAGTTCTC

[1297] CAAGGATATCGAGAAACTGCTCAATGAGCAGGTGAACAAGGAGATGCAGTCCTCCAATC

[1298] TGTACATGTCCATGTCCTCCTGGTGCTATACCCACTCCCTGGACGGCGCCGGCCTGTTC

[1299] CTGTTCGACCACGCCGCTGAGGAGTACGAGCACGCTAAGAAACTGATTATCTTCCTGAA

[1300] TGAGAATAACGTGCCTGTGCAGCTGACCTCCATCTCCGCTCCCGAGCACAAATTCGAAG

[1301] GCCTGACCCAGATCTTTCAGAAAGCCTATGAGCATGAGCAGCACATTTCCGAGTCCATC

[1302] AATAACATCGTCGACCACGCCATCAAGTCCAAAGACCACGCCACCTTTAACTTCCTGCA

[1303] GTGGTATGTGGCCGAGCAGCACGAGGAGGAAGTGCTGTTTAAGGATATCCTGGATAAGA

[1304] TTGAGCTGATCGGAAACGAGAACCATGGCCTGTACCTGGCCGATCAGTATGTGAAGGG

[1305] CATCGCCAAGTCCCGCAAGTCC

[1306] SEQ ID NO: 62 Amino acid sequence of an OspA serotypes 1 , 5 and 6 C-terminal fragment string (OspA ST1 -5-6) -Bullfrog / / - / , pylori hybrid ferritin fusion protein with htPA signal peptide and an OspA serotypes 2, 4 and 3 C-terminal fragment string (OspA ST243) -Bullfrog / / - / , pylori hybrid ferritin fusion protein with htPA signal peptide separated by a P2A site (“construct-20z”) in VRPs (T2A site indicated in bold).

[1307] MDAMKRGLCCVLLLCGAVFVSASFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKQ FTLEGKVANDKTTLVVKCGTVTLSKQISKSGEVSVELQDTDSSAATKKTAAWNSGTSTLTIT VNSKKTKDLVFTKENTITVQQYDSQGTKLEGSAVEITKLDEICNALKGGGGSGGGGSGGGG SGGGGSFNEKGEISEKTIVRAQGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKV TCGTVTLSKQISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKQRTKTKQLVFTKEDTIT VQNYDSAGTNLEGKAVEITTLKELCNALKGGGGSGGGGSGGGGSGGGGSFNGKGETSEK TIVRAQGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEI TAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKA VEITTLKELCNALKSSGGASVLAESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCY THSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQ HISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVK GIAKSRKSSGEGRGSLLTCGDVEENPGPMDAMKRGLCCVLLLCGAVFVSASFNEKGELSA KTMTREQGTKLEYTEMKSDGTGKAKEVLKQFTLEGKVANDKVTLEVKCGTVTLSKEIAKSG EVTVALQDTQTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEG TAVEIKTLDELCNALKGGGGSGGGGSGGGGSGGGGSFNAKGELSEKTILRAQGTRLEYTEI KSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELQDSQSTQATKK TGKWDSQTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGG GGSGGGGSGGGGSGGGGSFNDKGKLSEKVVTRAQGTRLEYTEIKNDGSGKAKEVLKGFA LEGTLTDGGETKLTVTCGTVTLSKQISKSGEITVALQDTETTPADKKTGEWKSDTSTLTISKN SQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKSSGGASVLAESQVRQQ FSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNE NNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAE QHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

[1308] SEQ ID NO: 63 Nucleic acid sequence encoding SARS-CoV-2 Spike RBD-

[1309] Bullfrog / / - / . pylori hybrid ferritin fusion protein.

[1310] GTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAATCTGTGTCCTTTTGG CGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATC

[1311] AGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTTCTCCACCTTCAA GTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCTG ACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGGACAGACAGGCAA GATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCTGTGTGATTGCCTGGA ACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTGTACAGGCTGTTT CGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGATCTATCAGGCAG GCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGTCCTAC GGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAGAGTGGTGGTGCTGAGCT TCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCACCAACCTGGT GAAGAACAAGAGCAGCGGCGGAGCCTCTGTGCTGGCCGAATCTCAAGTTCGGCAGCA GTTCAGCAAGGACATCGAGAAGCTGCTGAACGAGCAAGTGAACAAAGAGATGCAGAGC AGCAACCTGTACATGAGCATGAGCAGCTGGTGCTACACCCACAGCCTTGATGGCGCCG GACTGTTCCTGTTTGATCACGCCGCCGAGGAATACGAGCACGCCAAGAAGCTGATCATC TTCCTGAACGAGAACAACGTGCCCGTGCAGCTGACCAGCATTTCTGCCCCTGAGCACA AGTTCGAGGGCCTGACACAGATCTTCCAGAAGGCCTACGAACACGAGCAGCACATTAG CGAGAGCATCAACAACATCGTGGACCACGCCATTAAGAGCAAGGATCACGCCACCTTCA ACTTTCTGCAGTGGTACGTGGCCGAACAGCACGAGGAAGAGGTGCTGTTCAAGGACAT CCTGGACAAGATCGAGCTGATCGGCAACGAGAACCACGGCCTGTATCTGGCCGACCAG TACGTGAAGGGAATCGCCAAGAGCAGAAAGAGC SEQ ID NO: 64 Amino acid sequence of SARS-CoV-2 Spike RBD-Bullfrog / H pylori hybrid ferritin fusion protein.

[1312] VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYG

[1313] VSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDS KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG YQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSSGGASVLAESQVRQQFSKDIEKLLNEQ VNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISA PEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILD KIELIGNENHGLYLADQYVKGIAKSRKS

[1314] SEQ ID NO: 65 Nucleic acid sequence encoding EBV gp350-Bullf rog / H. pylori hybrid ferritin fusion protein in MVA.

[1315] GAAGCAGCTCTGCTCGTGTGCCAGTACACCATCCAGAGCCTCATCCACCTGACAGGAG

[1316] AGGATCCTGGCTTCTTCAACGTGGAAATTCCAGAGTTTCCCTTCTACCCTACCTGCAATG TGTGCACAGCCGACGTGAACGTGACCATCAACTTCGACGTTGGAGGCAAGAAGCACCA GCTGGACCTGGATTTCGGACAACTGACACCTCACACCAAGGCTGTGTATCAGCCTAGAG GAGCCTTTGGTGGTTCTGAGAATGCCACCAACCTGTTTCTCCTGGAGCTGCTTGGAGCT GGCGAGCTCGCACTGACCATGAGAAGCAAGAAACTGCCCATCAATGTGACCACAGGCG AGGAACAGCAGGTGTCCCTGGAAAGCGTGGACGTGTACTTTCAAGACGTGTTCGGCAC CATGTGGTGCCACCATGCCGAGATGCAGAACCCTGTGTACCTGATCCCAGAGACAGTG CCCTACATCAAGTGGGACAACTGCAACAGCACAAACATCACAGCCGTCGTGAGGGCTC AGGGACTGGATGTGACACTGCCTCTGTCTCTGCCAACCAGTGCCCAGGACAGCAACTT CAGCGTGAAGACCGAGATGCTGGGAAACGAGATCGACATCGAGTGCATCATGGAAGAT GGCGAGATCAGCCAGGTACTGCCTGGCGACAACAAGTTCAACATCACATGCAGTGGCT ACGAGAGCCACGTGCCATCTGGAGGCATCCTGACAAGCACAAGCCCAGTGGCCACACC GATCCCTGGCACAGGCTACGCCTACAGCCTGAGACTGACACCCAGACCCGTGTCCAGA TTCCTGGGCAACAACAGCATCCTGTACGTGTTCTACTCTGGCAACGGACCCAAGGCCTC TGGTGGCGATTACTGTATCCAGAGCAACATTGTGTTCAGCGATGAGATCCCTGCCAGCC AGGACATGCCAACCAATACCACCGATATCACCTACGTGGGAGACAATGCCACCTACAGC GTGCCCATGGTCACCTCCGAGGACGCCAACAGCCCTAATGTGACCGTGACAGCCTTCT GGGCATGGCCTAACAACACCGAGACAGACTTCAAGTGCAAGTGGACCCTGACCTCTGG CACACCTAGTGGCTGCGAGAATATCAGCGGAGCCTTCGCCAGCAACCGGACCTTCGAC ATTACCGTGTCTGGCCTTGGCACAGCTCCCAAGACCCTGATCATCACCAGGACTGCCAC CAATGCCACAACCACAACCCACAAAGTGATCTTCAGCAAGGCTCCTGAAAGCACCACAA CTAGTCCTACACTGCCTAAGCCCAGCACACCTCCTGGCAGCTCTGAATCTCAAGTTCGG CAGCAGTTCAGCAAAGACATCGAGAAGCTCCTCAACGAGCAAGTGAACAAGGAAATGC AGAGCAGCAACCTGTACATGAGCATGAGCAGCTGGTGCTACACCCACAGCCTTGATGG AGCTGGACTGTTCCTGTTTGATCACGCTGCCGAGGAATACGAGCACGCCAAGAAGCTG ATCATCTTCCTGAACGAGAACAACGTGCCTGTGCAGCTGACCAGCATTTCTGCACCTGA GCACAAGTTCGAAGGCCTGACACAGATCTTCCAGAAGGCCTACGAACACGAGCAGCAC ATTAGCGAGAGCATCAACAACATCGTGGACCACGCCATTAAGAGCAAGGATCACGCCAC CTTCAACTTTCTGCAGTGGTACGTTGCCGAACAGCACGAGGAAGAGGTGCTGTTCAAG GACATCCTGGACAAGATCGAGCTGATCGGCAACGAAAACCATGGCCTGTATCTTGCCGA CCAGTACGTGAAGGGAATCGCCAAGAGCAGAAAGAGC

[1317] SEQ ID NO: 66 Amino acid sequence of EBV gp350-Bullfrog / / - / . pylori hybrid ferritin fusion protein.

[1318] EAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCTADVNVTINFDVGGKKHQLDLD FGQLTPHTKAVYQPRGAFGGSENATNLFLLELLGAGELALTMRSKKLPINVTTGEEQQVSLE SVDVYFQDVFGTMWCHHAEMQNPVYLIPETVPYIKWDNCNSTNITAVVRAQGLDVTLPLSL PTSAQDSNFSVKTEMLGNEIDIECIMEDGEISQVLPGDNKFNITCSGYESHVPSGGILTSTSP VATPIPGTGYAYSLRLTPRPVSRFLGNNSILYVFYSGNGPKASGGDYCIQSNIVFSDEIPASQ

[1319] DMPTNTTDITYVGDNATYSVPMVTSEDANSPNVTVTAFWAWPNNTETDFKC

[1320] SEQ ID NO: 67 Nucleic acid sequence encoding an OspA serotypes 1 , 5 and 6 C- terminal fragment string (OspA ST1 -5-6)-Bullfrog / / - / . pylori hybrid ferritin fusion protein.

[1321] TTCAACGAGAAGGGCGAAGTGTCCGAGAAGATCATCACCAGAGCCGACGGCACCAGAC TCGAGTACACAGGCATCAAGTCTGACGGCAGCGGCAAGGCCAAAGAGGTGCTGAAGCA GTTCACCCTGGAAGGCAAGGTGGCCAACGACAAGACCACACTGGTGGTCAAGTGCGG CACCGTGACACTGAGCAAGCAGATCAGCAAGAGCGGCGAGGTGTCCGTGGAACTGCA GGACACAGATAGCAGCGCCGCCACCAAGAAAACCGCCGCCTGGAATAGCGGCACCAG CACACTGACCATCACCGTGAACAGCAAAAAGACCAAGGACCTGGTGTTCACCAAAGAA AACACCATCACAGTGCAGCAGTACGACAGCCAGGGCACCAAGCTGGAAGGCTCCGCC GTGGAAATCACAAAGCTGGACGAGATCTGCAACGCCCTGAAAGGCGGTGGCGGCTCTG GCGGAGGTGGAAGCGGAGGAGGAGGAAGTGGCGGAGGCGGTTCTTTTAATGAGAAAG GCGAGATCAGCGAGAAAACCATCGTCAGAGCCCAAGGCACACGGCTCGAGTATACCGA CATCAAGAGCGATAAGACCGGGAAAGCCAAAGAAGTCCTCAAGGATTTCACACTCGAG GGCACCCTGGCCGCCGATGGAAAGACAACCCTGAAAGTGACCTGTGGCACAGTGACC CTGTCCAAGCAAATCTCCAAGAGCGGAGAGATCACAGTGGCCCTGGACGATACCGATA GCTCCGGCAACAAGAAGTCCGGCACCTGGGATTCTGGCACCTCCACTCTGACCATCTC TAAGCAGCGGACCAAGACAAAGCAGCTGGTCTTTACCAAAGAGGACACAATCACCGTC CAGAACTACGACTCCGCCGGCACAAACCTCGAGGGAAAAGCCGTCGAGATCACCACAC TGAAAGAGCTGTGTAACGCTCTCAAAGGTGGTGGCGGTTCCGGTGGCGGTGGCAGTG GTGGTGGTGGATCAGGCGGTGGCGGAAGCTTTAATGGCAAGGGCGAGACATCCGAAAA GACGATCGTTCGCGCCCAAGGAACCCGGCTTGAGTACACCGATATTAAGTCCGACGGC TCTGGAAAGGCTAAAGAAGTTCTGAAAGACTTTACCCTCGAAGGGACTCTCGCCGCTGA CGGCAAGACCACTCTGAAAGTTACATGCGGAACCGTGGTGCTGTCTAAGAACATCCTGA AGTCCGGGGAGATCACTGCCGCTCTGGACGACAGCGATACCACACGGGCCACCAAAAA GACAGGCAAGTGGGACAGCAAGACAAGCACCCTGACAATCAGCGTGAACTCCCAGAAA ACGAAGAATCTCGTGTTTACGAAAGAAGATACCATTACGGTCCAGCGCTACGACAGCGC TGGGACAAATCTGGAAGGGAAAGCTGTGGAAATTACGACCCTCAAAGAACTGTGCAATG CCCTCAAGAGCAGCGGCGGAGCCTCTGTGCTGGCCGAATCTCAAGTTCGGCAGCAGTT CAGCAAGGACATCGAGAAGCTGCTGAACGAGCAAGTGAACAAAGAGATGCAGAGCAGC AACCTGTACATGAGCATGAGCAGCTGGTGCTACACCCACAGCCTTGATGGCGCCGGAC TGTTCCTGTTTGATCACGCCGCCGAGGAATACGAGCACGCCAAGAAGCTGATCATCTTC CTGAACGAGAACAACGTGCCCGTGCAGCTGACCAGCATTTCTGCCCCTGAGCACAAGT TCGAGGGCCTGACACAGATCTTCCAGAAGGCCTACGAACACGAGCAGCACATTAGCGA GAGCATCAACAACATCGTGGACCACGCCATTAAGAGCAAGGATCACGCCACCTTCAACT TTCTGCAGTGGTACGTGGCCGAACAGCACGAGGAAGAGGTGCTGTTCAAGGACATCCT GGACAAGATCGAGCTGATCGGCAACGAGAACCACGGCCTGTATCTGGCCGACCAGTAC GTGAAGGGAATCGCCAAGAGCAGAAAGAGC

[1322] SEQ ID NO: 68 Amino acid sequence of an OspA serotypes 1 , 5 and 6 C-terminal fragment string (OspA ST 1 -5-6)-Bullf rog / H. pylori hybrid ferritin fusion protein.

[1323] FNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKQFTLEGKVANDKTTLVVKCGTVTLS KQISKSGEVSVELQDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSQ GTKLEGSAVEITKLDEICNALKGGGGSGGGGSGGGGSGGGGSFNEKGEISEKTIVRAQGT RLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKQISKSGEITVALDDTD SSGNKKSGTWDSGTSTLTISKQRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELC NALKGGGGSGGGGSGGGGSGGGGSFNGKGETSEKTIVRAQGTRLEYTDIKSDGSGKAKE VLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTST

[1324] LTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKSSGGASVLAESQ

[1325] VRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLI

[1326] IFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQW

[1327] YVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

[1328] SEQ ID NO: 69 Nucleic acid sequence encoding SARS-CoV-2 Spike RBD-PdhC fusion protein.

[1329] GTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAATCTGTGTCCTTTTGG CGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATC AGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTTCTCCACCTTCAA GTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCTG ACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGGACAGACAGGCAA GATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCTGTGTGATTGCCTGGA ACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTGTACAGGCTGTTT CGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGATCTATCAGGCAG GCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGTCCTAC GGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAGAGTGGTGGTGCTGAGCT TCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCACCAACCTGGT GAAGAACAAGAGCAGCGGCGGAGCCTCTGTGCTGGCCGCTGCTGCTAAACCTGCTACA ACAGAGGGCGAGTTCCCCGAGACACGCGAGAAGATGTCTGGCATCAGAAGGGCTATCG CCAAGGCCATGGTGCACAGCAAGCACACAGCTCCTCACGTGACCCTGATGGACGAGGC CGATGTGACAAAGCTGGTGGCCCACAGAAAGAAGTTCAAGGCCATTGCCGCCGAGAAG GGAATCAAGCTGACCTTCCTGCCTTACGTGGTCAAGGCCCTGGTTTCTGCCCTGAGAG AATACCCCGTGCTGAACACCAGCATCGACGACGAGACAGAGGAAATCATCCAGAAGCA CTACTACAACATCGGAATCGCCGCCGACACCGACAGAGGACTGCTGGTGCCTGTGATC AAGCACGCCGACAGAAAGCCCATCTTCGCCCTGGCTCAAGAGATCAACGAGCTGGCTG AGAAGGCCAGAGATGGCAAGCTGACACCCGGCGAAATGAAGGGCGCCAGCTGTACCAT CACCAACATCGGTTCTGCTGGCGGCCAGTGGTTCACCCCAGTGATCAATCACCCTGAG GTGGCCATCCTCGGCATCGGCAGAATCGCTGAGAAGCCCATCGTCCGCGACGGCGAAA TTGTGGCTGCTCCTATGCTGGCCCTGAGCCTGAGCTTCGACCACAGAATGATCGACGG CGCCACAGCTCAGAAGGCCCTGAACCACATCAAGAGACTGCTGAGCGACCCCGAGCT GCTGCTGATGGAAGCT

[1330] SEQ ID NO: 70 Amino acid sequence of SARS-CoV-2 Spike RBD-PdhC fusion protein.

[1331] VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYG VSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDS KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG YQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSSGGASVLAAAAKPATTEGEFPETREKM SGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSAL REYPVLNTSIDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDG KLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLS FDHRMIDGATAQKALNHIKRLLSDPELLLMEA

[1332] SEQ ID NO: 71 Nucleic acid sequence encoding SARS-CoV-2 Spike RBD-DPS fusion protein.

[1333] GTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAATCTGTGTCCTTTTGG CGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATC AGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTTCTCCACCTTCAA GTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCTG

[1334] ACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGGACAGACAGGCAA

[1335] GATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCTGTGTGATTGCCTGGA

[1336] ACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTGTACAGGCTGTTT

[1337] CGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGATCTATCAGGCAG

[1338] GCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGTCCTAC

[1339] GGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAGAGTGGTGGTGCTGAGCT

[1340] TCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCACCAACCTGGT

[1341] GAAGAACAAGAGCAGCGGCGGAGCCTCTGTGCTGGCCGCTACAAATCTGCTGTACACC

[1342] AGAAACGACGTGTCCGACAGCGAGAAGAAGGCCACAGTCGAGCTGCTGAACAGACAA

[1343] GTGATCCAGTTCATCGACCTGAGCCTGATCACCAAGCAGGCCCACTGGAACATGAGAG

[1344] GCGCCAACTTTATCGCCGTGCACGAGATGCTGGACGGCTTCAGAACAGCCCTGATCGA

[1345] CCACCTGGACACCATGGCTGAAAGAGCTGTGCAGCTTGGCGGAGTGGCTCTGGGCAC

[1346] AACCCAAGTGATCAACAGCAAGACCCCTCTGAAGTCTTACCCTCTGGACATCCACAACG

[1347] TGCAGGACCACCTGAAAGAACTGGCCGACAGATACGCCATCGTGGCCAATGATGTGCG

[1348] GAAGGCTATCGGCGAGGCCAAGGACGATGATACCGCCGATATCCTGACAGCCGCCAGC

[1349] AGAGATCTGGACAAGTTCCTGTGGTTCATCGAGAGCAACATCGAG

[1350] SEQ ID NO: 72 Amino acid sequence of SARS-CoV-2 Spike RBD-DPS fusion protein.

[1351] VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYG VSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDS KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG YQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSSGGASVLAATNLLYTRNDVSDSEKKATV ELLNRQVIQFIDLSLITKQAHWNMRGANFIAVHEMLDGFRTALIDHLDTMAERAVQLGGVALG TTQVINSKTPLKSYPLDIHNVQDHLKELADRYAIVANDVRKAIGEAKDDDTADILTAASRDLDK FLWFIESNIE

[1352] SEQ ID NO: 73 Nucleic acid sequence encoding HisB-SARS-CoV-2 Spike RBD fusion protein.

[1353] GCATCTAGAAGGGCCAGAATCGAGAGAAGAACCCGCGAGAGCGACATCGTGATCGAGC TGGATCTGGATGGCACAGGACAGGTGGCAGTGGATACAGGCGTGCCCTTCTACGACCA CATGCTGACAGCTCTGGGCAGCCACGCCTCTTTCGACCTGACAGTTAGAGCCACCGGC GACGTGGAAATCGAGGCCCACCACACAATCGAGGACACCGCTATCGCTCTGGGAACAG CTCTTGGACAGGCCCTGGGCGATAAGAGAGGCATCAGAAGATTCGGCGACGCTTTCAT CCCCATGGACGAGACACTGGCTCACGCTGCCGTTGACCTTAGCGGCAGACCTTACTGT GTGCATACCGGCGAGCCTGACCATCTGCAGCACACAACAATCGCCGGCAGCAGCGTGC CATACCACACCGTGATCAACAGACACGTGTTCGAGAGCCTGGCCGCCAACGCTAGAATC GCTCTGCATGTGCGGGTGCTGTACGGCAGAGATCCCCACCACATCACAGAGGCCCAGT ACAAGGCTGTGGCCAGGGCTCTGAGACAGGCCGTTGAACCTGATCCTAGAGTGTCCGG CGTGCCATCTACAAAGGGTGCTCTGAGCAGCGGCGGAGCCTCTGTGCTGGCCGTGCA GCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAATCTGTGTCCTTTTGGCGAGG TGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAA TTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTTCTCCACCTTCAAGTGCT ATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCTGACAGC TTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGGACAGACAGGCAAGATTG CCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCTGTGTGATTGCCTGGAACAGC AACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTGTACAGGCTGTTTCGGAA GTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGATCTATCAGGCAGGCAGC ACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGTCCTACGGCTT CCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAGAGTGGTGGTGCTGAGCTTCGAG CTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCACCAACCTGGTGAAGA

[1354] ACAAA

[1355] SEQ ID NO: 74 Amino acid sequence of HisB-SARS-CoV-2 Spike RBD fusion protein.

[1356] ASRRARIERRTRESDIVIELDLDGTGQVAVDTGVPFYDHMLTALGSHASFDLTVRATGDVEIE AHHTIEDTAIALGTALGQALGDKRGIRRFGDAFIPMDETLAHAAVDLSGRPYCVHTGEPDHL QHTTIAGSSVPYHTVINRHVFESLAANARIALHVRVLYGRDPHHITEAQYKAVARALRQAVEP DPRVSGVPSTKGALSSGGASVLAVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCN GVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

[1357] SEQ ID NO: 75 Nucleic acid sequence encoding DPS-SARS-CoV-2 Spike RBD fusion protein.

[1358] GCTACAAATCTGCTGTACACCAGAAACGACGTGTCCGACAGCGAGAAGAAGGCCACAG TCGAGCTGCTGAACAGACAAGTGATCCAGTTCATCGACCTGAGCCTGATCACCAAGCAG GCCCACTGGAACATGAGAGGCGCCAACTTTATCGCCGTGCACGAGATGCTGGACGGCT TCAGAACAGCCCTGATCGACCACCTGGACACCATGGCTGAAAGAGCTGTGCAGCTTGG CGGAGTGGCTCTGGGCACAACCCAAGTGATCAACAGCAAGACCCCTCTGAAGTCTTAC CCTCTGGACATCCACAACGTGCAGGACCACCTGAAAGAACTGGCCGACAGATACGCCA TCGTGGCCAATGATGTGCGGAAGGCTATCGGCGAGGCCAAGGACGATGATACCGCCGA TATCCTGACAGCCGCCAGCAGAGATCTGGACAAGTTCCTGTGGTTCATCGAGAGCAACA TCGAGAGCAGCGGCGGAGCCTCTGTGCTGGCCGTGCAGCCCACAGAGTCTATCGTGC GGTTCCCTAACATCACCAATCTGTGTCCTTTTGGCGAGGTGTTCAACGCCACCAGATTC GCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTTGCCGACTACAGCG TGCTGTACAACTCTGCCAGCTTCTCCACCTTCAAGTGCTATGGCGTGTCTCCTACCAAG CTGAACGACCTGTGCTTCACCAACGTGTACGCTGACAGCTTCGTGATCAGAGGCGACG AAGTGAGACAGATTGCTCCTGGACAGACAGGCAAGATTGCCGATTACAACTACAAGCTC CCTGACGACTTCACAGGCTGTGTGATTGCCTGGAACAGCAACAACCTGGACAGCAAAG TCGGAGGTAACTACAACTACCTGTACAGGCTGTTTCGGAAGTCCAACCTGAAGCCTTTC GAGAGAGACATCAGCACCGAGATCTATCAGGCAGGCAGCACACCTTGCAATGGCGTGG AAGGCTTCAACTGCTACTTCCCACTGCAGTCCTACGGCTTCCAGCCTACAAATGGAGTG GGCTACCAGCCTTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCATGCTCCTGCCA CAGTGTGCGGACCTAAGAAAAGCACCAACCTGGTGAAGAACAAA

[1359] SEQ ID NO: 76 Amino acid sequence of DPS-SARS-CoV-2 Spike RBD fusion protein.

[1360] ATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWNMRGANFIAVHEMLDGFRTALI DHLDTMAERAVQLGGVALGTTQVINSKTPLKSYPLDIHNVQDHLKELADRYAIVANDVRKAIG EAKDDDTADILTAASRDLDKFLWFIESNIESSGGASVLAVQPTESIVRFPNITNLCPFGEVFNA TRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDE VRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDI STEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKK STNLVKNK

[1361] SEQ ID NO: 77 Nucleic acid sequence encoding EBV gp350-PdhC fusion protein.

[1362] GAAGCAGCTCTGCTCGTGTGCCAGTACACCATCCAGAGCCTCATCCACCTGACAGGAG

[1363] AGGATCCTGGCTTCTTCAACGTGGAAATTCCAGAGTTTCCCTTCTACCCTACCTGCAATG TGTGCACAGCCGACGTGAACGTGACCATCAACTTCGACGTTGGAGGCAAGAAGCACCA

[1364] GCTGGACCTGGATTTCGGACAACTGACACCTCACACCAAGGCTGTGTATCAGCCTAGAG

[1365] GAGCCTTTGGTGGTTCTGAGAATGCCACCAACCTGTTTCTCCTGGAGCTGCTTGGAGCT

[1366] GGCGAGCTCGCACTGACCATGAGAAGCAAGAAACTGCCCATCAATGTGACCACAGGCG

[1367] AGGAACAGCAGGTGTCCCTGGAAAGCGTGGACGTGTACTTTCAAGACGTGTTCGGCAC

[1368] CATGTGGTGCCACCATGCCGAGATGCAGAACCCTGTGTACCTGATCCCAGAGACAGTG

[1369] CCCTACATCAAGTGGGACAACTGCAACAGCACAAACATCACAGCCGTCGTGAGGGCTC

[1370] AGGGACTGGATGTGACACTGCCTCTGTCTCTGCCAACCAGTGCCCAGGACAGCAACTT

[1371] CAGCGTGAAGACCGAGATGCTGGGAAACGAGATCGACATCGAGTGCATCATGGAAGAT

[1372] GGCGAGATCAGCCAGGTACTGCCTGGCGACAACAAGTTCAACATCACATGCAGTGGCT

[1373] ACGAGAGCCACGTGCCATCTGGAGGCATCCTGACAAGCACAAGCCCAGTGGCCACACC

[1374] GATCCCTGGCACAGGCTACGCCTACAGCCTGAGACTGACACCCAGACCCGTGTCCAGA

[1375] TTCCTGGGCAACAACAGCATCCTGTACGTGTTCTACTCTGGCAACGGACCCAAGGCCTC

[1376] TGGTGGCGATTACTGTATCCAGAGCAACATTGTGTTCAGCGATGAGATCCCTGCCAGCC

[1377] AGGACATGCCAACCAATACCACCGATATCACCTACGTGGGAGACAATGCCACCTACAGC

[1378] GTGCCCATGGTCACCTCCGAGGACGCCAACAGCCCTAATGTGACCGTGACAGCCTTCT

[1379] GGGCATGGCCTAACAACACCGAGACAGACTTCAAGTGCAAGTGGACCCTGACCTCTGG

[1380] CACACCTAGTGGCTGCGAGAATATCAGCGGAGCCTTCGCCAGCAACCGGACCTTCGAC

[1381] ATTACCGTGTCTGGCCTTGGCACAGCTCCCAAGACCCTGATCATCACCAGGACTGCCAC

[1382] CAATGCCACAACCACAACCCACAAAGTGATCTTCAGCAAGGCTCCTGAAAGCACCACAA

[1383] CTAGTCCTACACTGCCTAAGCCCAGCACACCTCCTGGCAGCAGCGCTGCTGCTAAACCT

[1384] GCTACAACAGAGGGCGAGTTCCCCGAGACACGCGAGAAGATGTCTGGCATCAGAAGG

[1385] GCTATCGCCAAGGCCATGGTGCACAGCAAGCACACAGCTCCTCACGTGACCCTGATGG

[1386] ACGAGGCCGATGTGACAAAGCTGGTGGCCCACAGAAAGAAGTTCAAGGCCATTGCCGC

[1387] CGAGAAGGGAATCAAGCTGACCTTCCTGCCTTACGTGGTCAAGGCCCTGGTTTCTGCC

[1388] CTGAGAGAATACCCCGTGCTGAACACCAGCATCGACGACGAGACAGAGGAAATCATCC

[1389] AGAAGCACTACTACAACATCGGAATCGCCGCCGACACCGACAGAGGACTGCTGGTGCC

[1390] TGTGATCAAGCACGCCGACAGAAAGCCCATCTTCGCCCTGGCTCAAGAGATCAACGAG

[1391] CTGGCTGAGAAGGCCAGAGATGGCAAGCTGACACCCGGCGAAATGAAGGGCGCCAGC

[1392] TGTACCATCACCAACATCGGTTCTGCTGGCGGCCAGTGGTTCACCCCAGTGATCAATCA

[1393] CCCTGAGGTGGCCATCCTCGGCATCGGCAGAATCGCTGAGAAGCCCATCGTCCGCGAC

[1394] GGCGAAATTGTGGCTGCTCCTATGCTGGCCCTGAGCCTGAGCTTCGACCACAGAATGAT

[1395] CGACGGCGCCACAGCTCAGAAGGCCCTGAACCACATCAAGAGACTGCTGAGCGACCC

[1396] CGAGCTGCTGCTGATGGAAGCT

[1397] SEQ ID NO: 78 Amino acid sequence of EBV gp350-PdhC fusion protein.

[1398] EAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCTADVNVTINFDVGGKKHQLDLD

[1399] FGQLTPHTKAVYQPRGAFGGSENATNLFLLELLGAGELALTMRSKKLPINVTTGEEQQVSLE

[1400] SVDVYFQDVFGTMWCHHAEMQNPVYLIPETVPYIKWDNCNSTNITAVVRAQGLDVTLPLSL

[1401] PTSAQDSNFSVKTEMLGNEIDIECIMEDGEISQVLPGDNKFNITCSGYESHVPSGGILTSTSP

[1402] VATPIPGTGYAYSLRLTPRPVSRFLGNNSILYVFYSGNGPKASGGDYCIQSNIVFSDEIPASQ

[1403] DMPTNTTDITYVGDNATYSVPMVTSEDANSPNVTVTAFWAWPNNTETDFKCKWTLTSG...

Claims

Claims1 . A pharmaceutical composition comprising an RNA or DNA molecule, the RNA or DNA molecule comprising a nucleic acid section encoding a fusion protein comprising a disease-associated antigen, or an antigenic part thereof, joined to a subunit of a selfassembling protein nanoparticle.

2. The pharmaceutical composition of claim 1 , wherein the RNA molecule is a messenger RNA (mRNA) or a self-amplifying RNA (saRNA).

3. The pharmaceutical composition of claim 1 , wherein the DNA molecule is an expression plasmid.

4. The pharmaceutical composition of anyone of claims 1 to 3, wherein the selfassembling protein nanoparticle is selected from the group consisting of hybrid protein Bullfrog / / - / , pylori hybrid ferritin (BFF); imidazoleglycerol-phosphate dehydratase (HisB), preferably from Mycobacterium tuberculosis; acetyltransferase of pyruvate dehydrogenase (PDH) complex (PdhC), preferably from Geo-) Bacillus stearothermophilus; and DNA binding protein from starved cells (DPS), preferably from Escherichia coli.

5. The pharmaceutical composition of claim 4, wherein the self-assembling protein nanoparticle is HisB, PdhC or DPS.

6. The pharmaceutical composition of claim 4, wherein the self-assembling protein nanoparticle is selected from the group consisting of BFF, PdhC and DPS, and wherein in each case the N-terminus of the subunit of the self-assembling nanoparticle is joined to the C-terminus of the disease-associated antigen.

7. The pharmaceutical composition or vaccine of claim 4, wherein the self-assembling protein nanoparticle is HisB or DPS, and wherein in each case the C-terminus of the subunit of the self-assembling nanoparticle is joined to the N-terminus of the disease- associated antigen.

8. The pharmaceutical composition of anyone of claims 1 to 7, wherein the disease- associated antigen is an infectious disease-associated antigen, preferably a viral or bacterial antigen.

9. The pharmaceutical composition of claim 8, wherein the infectious disease-associated antigen is from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), preferably comprises or consists of receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein (SARS-CoV-2 S RBD).

10. The pharmaceutical composition of anyone of claims 1 to 9, wherein the nucleic acid section further encodes a signal peptide joined to the fusion protein, preferably the signal peptide is human tissue plasminogen activator (htPA) signal peptide.1 1 . The pharmaceutical composition of anyone of claims 1 to 10, wherein the RNA or DNA molecule further comprises another nucleic acid section encoding a fusion protein comprising another disease-associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle, wherein the disease-associated antigen and the other disease-associated antigen are different, and wherein the selfassembling protein nanoparticle is the same.

12. The pharmaceutical composition of anyone of claims 1 to 11 , further comprising an adjuvant and a pharmaceutically acceptable nucleic acid transfection reagent.

13. A process for preparing a pharmaceutical composition of anyone of claims 1 to 12, comprising the steps of:(1 ) providing a nucleic acid encoding a fusion protein comprising a disease- associated antigen, or an antigenic part thereof, joined to a subunit of a selfassembling protein nanoparticle;(2) generating an RNA or DNA molecule comprising the nucleic acid provided in step (1 );(3) obtaining the RNA or DNA molecule generated in step (2);(4) combining the RNA or DNA molecule obtained in step (3) with a pharmaceutically acceptable carrier or excipient.

14. A pharmaceutical composition of anyone of claims 1 to 12 for use in the prevention or treatment of a disease, preferably an infectious disease or cancer.

15. The pharmaceutical composition for use of claim 14, wherein the infectious disease- associated antigen is from SARS-CoV-2 and the disease is COVID-19 or long COVID.

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