Viral vectors for enhanced immune responses to multimeric protein particle displayed antigens

Abstract

The present invention relates to viral vectors co-encoding an artificial CD4 T cell epitope (PADRE) for enhanced immune responses to encoded multimeric protein particle displayed vaccine antigens.

Description

[0001] PCT Application

[0002] Bavarian Nordic A / S

[0003] BN126PCT

[0004] VIRAL VECTORS FOR ENHANCED IMMUNE RESPONSES TO MULTIMERIC PROTEIN PARTICLE DISPLAYED ANTIGENS

[0005] Technical Field

[0006] The present invention relates to the field of gene-based vaccines. More specifically, the invention relates to viral vectors, preferably recombinant Modified Vaccinia Virus Ankara (MVA) or alphavirus-derived virus replicon particles (VRPs), encoding a fusion protein comprising a vaccine antigen, a subunit of a self-assembling multimeric protein particle, and an artificial CD4 T cell epitope (PADRE). The invention further relates to pharmaceutical compositions comprising the viral vectors and to medical uses thereof.

[0007] Background

[0008] 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.

[0009] 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 [Meisinger-Henschel etal., 2007], 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® [Suter et al., 2009]. 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 antigen-encoding transgenes for use in vaccination approaches that target diseases with hitherto unmet medical need (e.g., Mvabea® against Ebolavirus disease [Pollard etal., 2021], 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.

[0010] The FDA recently approved MVA-BN® for the prevention of smallpox and mpox disease (JYNNEOS®). In Europe and Canada, MVA-BN® is approved as a vaccine against smallpox and mpox (IMVANEX®). In addition, a recombinant MVA expressing filovirus proteins (Mvabea®) has recently been approved by the European Medicines Agency as a vaccine against Ebola virus disease, as part of a heterologous prime-boost regime in combination with an adenoviral vector.

[0011] 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.

[0012] 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 multimeric particle, also often referred to as “nanoparticle” [Bachmann et al., 1993; Zang et al., 2020]. 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.

[0013] Thus, there is a need for improved viral vector vaccines. Summary of Invention

[0014] It is an objective of the present invention to provide means and method for improving the immunogenicity of viral vector encoded vaccine antigens.

[0015] The objective of the present invention is solved by the provision of viral vectors encoding an antigen displayed on a self-assembling protein particle and co-encoding a pan HLA DR-binding epitope (PADRE).

[0016] In particular, the invention is defined by the appended claims and by the following aspects and embodiments.

[0017] In a first aspect, the invention provides a nucleic acid encoding a fusion protein comprising (i) a disease-associated antigen, or an antigenic part thereof, (ii) a subunit of a self-assembling multimeric protein particle, and (iii) a pan HLA DR-binding epitope (PADRE).

[0018] In a second aspect, the invention provides a fusion protein encoded by a nucleic acid encoding a fusion protein comprising (i) a disease-associated antigen, or an antigenic part thereof, (ii) a subunit of a self-assembling multimeric protein particle, and (iii) a pan HLA DR-binding epitope (PADRE).

[0019] In a third aspect, the invention provides a virus-based vector, preferably a recombinant Modified Vaccinia Virus Ankara (MVA) or a virus replicon particle (VRP), comprising a nucleic acid encoding a fusion protein comprising (i) a disease-associated antigen, or an antigenic part thereof, (ii) a subunit of a self-assembling multimeric protein particle, and (iii) a pan HLA DR-binding epitope (PADRE).

[0020] In a further aspect, the invention provides a use of a nucleic acid as described herein for the preparation of a virus-based vector or a pharmaceutical composition.

[0021] In yet a further aspect, the invention provides a pharmaceutical composition comprising a nucleic acid as described herein, a fusion protein as described herein, or a virus-based vector as described herein, optionally further comprising a pharmaceutically acceptable carrier or excipient.

[0022] In yet a further aspect, the invention provides a virus-based vector as described herein, or a pharmaceutical composition as described herein, for use in the prevention or treatment of a disease, preferably a viral infectious disease or a viral infection associated malignancy. In yet a further aspect, the invention provides a virus-based vector as described herein, or a pharmaceutical composition as described herein, for use in inducing an antibody response to the disease-associated antigen encoded by the virus-based vector.

[0023] In yet a further aspect, the invention provides a process for preparing a recombinant MVA as described herein, the process comprising the steps of:

[0024] (a) providing an acceptor bacterial artificial chromosome (BAC) comprising an MVA genome (MVA-BAC), which MVA genome is mutated such that two essential MVA genes required for MVA replication are defective or lacking, wherein one essential MVA gene is I3L (MVA064L) and the other essential MVA gene is J5L (MVA089L);

[0025] (b) providing a recombination plasmid comprising the essential MVA gene which is defective or lacking in the MVA-BAC of step (a) as a functional gene, the recombination plasmid further comprising a nucleic acid as described herein, operably linked to a poxviral promoter;

[0026] (c) co-transfecting an MVA producer cell with the MVA-BAC of step (a) and the recombination plasmid of step (b), and furthermore infecting the MVA producer cell with a helper virus derived from the family poxviridae, preferably Shope fibroma virus (SFV); (d) allowing reconstitution of MVA from the MVA-BAC and homologous recombination with the recombination plasmid in the MVA producer cell; and

[0027] (e) obtaining the recombinant MVA comprising the functional essential MVA gene and the nucleic acid.

[0028] In yet a further aspect, the invention provides a process for preparing a recombinant VRP as described herein, comprising the steps of:

[0029] (a’) providing a plasmid DNA encoding a self-amplifying replicon RNA, preferably a replicon RNA under the control of a cytomegalovirus (CMV) promoter, encoding a nucleic acid as described herein;

[0030] (b’) transfecting a VRP production cell with the plasmid DNA provided in step (a’), further transfecting the VRP production cell with a first CMV promoter driven packaging plasmid encoding an alphavirus capsid protein and a second CMV promoter driven packaging plasmid encoding an alphavirus envelope protein;

[0031] (c’) culturing the transfected VRP production cell of step (b’);

[0032] (d’) obtaining the recombinant VRP. Description of Drawings Figures

[0033] Figure 1 schematically illustrates recombinant MVA-BN-EBV construct MVA-mBN520. Boxes indicate open reading frames of the inserted EBV-derived antigen genes. Arrows represent poxviral promoters driving antigen expression. IGR 64 / 65 and IGR 88 / 89 are MVA integration sites.

[0034] Figure 2 is a schematic representation of the permanent dominant selection procedure used to generate MVA-mBN520.

[0035] MVA-BN-BAC484 served as starting construct for MVA-mBN520 (“MVA-BN-EBV”). MVA-BN-BAC484 contained two deletions in the coding region of MVA. The first deletion eliminated most of the I3L gene that is essential for MVA replication, leaving only 116 amino acids at the C-terminus that lack a start codon. The second deletion encompassed the complete coding region of the essential J5L gene. MVA-BN-BAC484 was transfected into CCX.2C4 cells together with the two recombination plasmids pDS039 and pDS020 that carried intact versions of the I3L and J5L genes. I3L was coupled to the gp350-PADRE-PDHC (“gp350”) gene, the gL gene and the gH-DPS (“gH”) gene in plasmid pDS039. J5L was coupled to the BRLF1 / BZLF1 fusion gene and the EBNA3Agene in plasmid pDS020. The co-transfected cell culture was additionally infected with the helper virus Shope fibroma virus (SFV), which provides helper functions for reconstitution of the infectious MVA from the MVA-BN-BAC484 and for simultaneous recombination of the MVA genome with the two transfer plasmids. Flanking regions from I2L / I4L and J4R / J6R genes mediated homologous recombination. The result was infectious MVA-mBN520 containing the two repaired essential genes I3L and J5L together with the indicated EBV transgenes in the two intergenic regions (IGRs) 64 / 65 and 88 / 89. Any non-recombinant co-packaged mBN484 genome derived from the input MVA-BN-BAC484 or partially recombinant MVA viruses were counterselected since they were not replication competent due to their lack of one or both essential genes I3L and J5L. Counterselection was facilitated by several rounds of plaque purification.

[0036] Figure 3 shows a schematic overview of MVA-BN-BAC constructs and their generation, (a) Illustration of the MVA-BN-BAC166 genome, which contained the full-length MVA-BN sequence, and the modified genomes of the two acceptor BACs MVA-BN-BAC471 and MVA-BN-BAC484. The three essential genes E4L, I3L, and J5Lare indicated in MVA-BN-BAC166. Inverted terminal repeats (ITRs) at the 5’ and 3’ ends are marked. The BAC cassette (black) contained elements for maintenance and propagation in bacteria, as well as an eGFP-nptll reporter / selection cassette. The locations of deletion sites in acceptor BACs are indicated by black arrows, and the flanking genes are indicated. The BAC genomes were circular but illustrated as linear for simplified presentation purposes, (b) Schematic overview of the multistep cloning process giving rise to the two different acceptor BACs. MVA-BN-BAC471 was derived from parental BAC MVA-BN-BAC166 and gave rise to MVA-BN-BAC484. The dashed arrows indicate that intermediate BACs were involved in the cloning process.

[0037] Figure 4 shows the organization of the gH-DPS fusion protein.

[0038] The extracellular domain of gH (amino acids 1-682) was fused to amino acids 11-167 of DPS via a flexible linker.

[0039] Figure 5 shows the organization of the gp350-PADRE-PdhC fusion protein.

[0040] Amino acids 2-434 of gp350 were fused to amino acids 185-428 of the Geobacillus stearothermophilus (G.st) pyruvate dehydrogenase complex E2 subunit (PdhC E2) via a linker containing the PADRE epitope (amino acid sequence “AKFVAAWTLKAAA”). The PADRE sequence was flanked both N- and C-terminally via short “GS” linker sequences. In addition, the signal peptide (SP) of the murine IgG kappa was fused to the N-terminus to enhance the secretion of the gp350-PADRE-PDHC fusion protein.

[0041] Figure 6 shows the organization (a) and amino acid sequence (b) of the BZLF1-BRLF1 fusion protein.

[0042] (a) The following regions of full-length BRLF1 were removed: dimerization domain (aa 2-23) [Manet et al., 1991], nuclear localization domain (aa 407-421) Hsu et al., 2005]. From full-length BZLF1, the following regions were removed: transactivation domain (aa 33-52 and aa 68-78) [Giot etal., 1991], DNA binding domain (aa 180-187) [Flemington etal., 1990; Giot et al., 1991], dimerization domain (aa 203-208) [Flemington et al., 1990], Ankyrin like Zank domain (aa 237-245) [Dreyfus et al., 2011). In addition, a part of the BZLF1 protein (aa188-202) was shuffled towards the N-terminus between domains (aa1-32 and aa 53-67of BZLF1), and BRLF1 and BZLF1 sequences were fused to result in the BZLF1-BRLF1 fusion protein.

[0043] (b) To prevent the formation of neo-epitopes on junctions between different fragments of the BZLF1-BRLF1 fusion protein, several mutations were introduced (highlighted in bold letters).

[0044] Figure 7 shows modifications introduced to EBNA3A protein on the amino acid sequence level.

[0045] Six potential nuclear localization signals (first six deleted sequences) in EBNA3A were deleted. In addition, binding sites for cellular transcriptional regulators JK (second highlighted sequence starting with “MGY”) [Zhao etal., 1996] and CtBP (two last deleted sequences) [Hickabottom et al., 2002] were eliminated by point mutations and deletions, respectively. A potential glycosylation site (highlighted in black, “NVA”) was modified by mutating threonine to alanine. To avoid formation of potential neoepitopes, deletions were extended beyond the respective motifs. Finally, EBNA3A was modified by addition of an N-terminal secretion tag (first highlighted sequence starting with “MET”) and a C-terminal linker and transmembrane domain (last highlighted sequence starting with “GGG”). Amino acid changes are indicated in bold letters, deleted amino acids by strikethrough letters.

[0046] Figure 8 shows recombination plasmid pDS020.

[0047] Recombination plasmid pDS020 contained the EBV-derived transgenes BZLF1-BRLF1 fusion (under the control of the Pr13.5long promoter) and EBNA3A (under the control of the Pr1328 promoter). In addition, the plasmid also contained MVA-BN DNA sequences flanking the IGR88 / 89 of the MVA-BN genome (IGR88 / 89 F1 DomSel and IGR88 / 89 F2 DomSel) required for recombination, and the essential complementing gene MVA089L (J5L) itself.

[0048] Figure 9 shows recombination plasmid pDS039.

[0049] Recombination plasmid pDS039 contained the EBV-derived transgenes gp350-PADRE-PDHC (under control of the Pr13.5long promoter), gH-DPS (under control of the PrS promoter), and gL (under control of the PrH5m promoter). In addition, the plasmid also contained MVA-BN DNA sequences flanking the IGR 64 / 65 of the MVA-BN genome (IGR64 / 65 F1 DomSel, IGR64 / 65 F2 DomSel) required for recombination, and the essential complementing gene MVA064L (I3L) itself.

[0050] Figure 10 shows a flow-chart of the MVA-mBN520 generation process.

[0051] Figure 11 shows the expression of EBV-derived antigens by MVA-mBN520.

[0052] Expression of the EBV-derived antigens was determined by flow cytometry analysis of MVA-mBN520 infected HeLa cells and the respective controls using antigen-specific antibodies. Data are shown as histogram plots. HeLa cells were seeded in 6-well plates at 5x105cells per well in 1 ml of DMEM containing 10% FCS on the day before infection. Cells were either infected with MVA-mBN520 (black) or MVA-BN (grey, positive control) at a multiplicity of infection (MOI) of 5 for 6 hours. As a negative control, cells were left uninfected (mock, white). Cells were scraped, washed with PBS, fixed with 4% formalin and permeabilized prior to antibody staining to allow detection of both surface as well as intracellular proteins. Homogenous infection of HeLa cells by recombinant MVA-mBN520 or parental MVA-BN was verified by staining with anti-vaccinia virus rabbit polyclonal serum (MVA, upper left panel). Expression of gp350-PADRE-PDHC was determined with the EBV gp350-specific mouse monoclonal antibody clone 10B5 (Santa Cruz, sc56981), gH-DPS / gL with a mouse monoclonal antibody detecting the EBV gH / gL complex (Bioconnect, IT-005-030M8), BRFL1-BZLF1 fusion with a mouse monoclonal antibody directed against EBV BZLF1 (Santa Cruz, sc53904), and EBNA3A was detected using polyclonal sheep anti-EBV EBNA3A (abeam, ab16126). Figure 12 shows a schematic diagram of MVA-mBN520 and control constructs MVA- mBN461 and MVA-mBN516.

[0053] Figure 13 shows the induction of gp350- and gH / gL-specific antibody response.

[0054] Balb / c mice (n=5) were immunized according to a prime-boost regimen (day 0 / 21). Serum was analyzed 40 days after prime immunization for anti-gp350 (A) and anti-gHgL (B) titers by ELISA. Commercially available anti-gp350 (Merck) or anti-gHgL (Immune-tech) antibody was used to establish a standard for anti-gp350 IgG or anti-gHgL IgG serum antibody quantification. Data are shown as Mean ± SEM. Significant differences were found as indicated; * = p < 0.05; *** = p < 0.001; **** = p < 0.0001; ns = non-significant.

[0055] Figure 14 shows the neutralization of EBV infection of Ramos cells.

[0056] Balb / c mice (n=5) were immunized according to a prime-boost regimen (day 0 / 21). Serum was analyzed 40 days after prime immunization for EBV neutralizing antibodies. EBV was incubated with different serum dilutions before infection of Ramos cells. After 30 minutes of incubation with the virus the cells were washed and incubated overnight at 37°C. Next, cells were stained with anti-gp350-AF647 and a viability dye before analysis on a flow cytometer. Live, gp350+Ramos cells were gated using negative controls and analyzed. The infection rate of negative controls was set as 100% infection, and a 4PL-fit curve was calculated for each sample to determine the titer at 50% inhibition. Data are shown as Mean ± SEM. Significant differences were found as indicated; * = p < 0.05; ns = non-significant.

[0057] Figure 15 shows the induction of EBNA3Aand BRLF1 -specific T cell responses.

[0058] Balb / c mice were immunized according to a prime-boost regimen (day 0 / 21). Spleens were isolated 40 days after prime immunization and 0.5 x 106splenocytes per condition were restimulated with BRLF1 PP and EBNA3A PP on anti-IFN-y coated plates overnight. IFN-y-producing cells were stained by HRP-labelled anti-IFN-y antibody and revealed after development with AEC substrate. Spots were counted using an ELISPOT reader (Immunospot). Data are shown as Mean ± SEM. Significant differences were found as indicated; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

[0059] Figure 16 shows a schematic diagram of MVA-mBN520 and control constructs MVA- mBN515 and MVA-mBN520.

[0060] Figure 17 shows the induction of gp350- and gH / gL-specific antibody responses in outbred mice.

[0061] CD-1 mice (n=9) were immunized according to a prime-boost regimen (day 0 / 21). Serum was analyzed 39 days after prime immunization for anti-gp350 (A) and anti-gHgL (B) titers by ELISA. Commercially available anti-gp350 (Merck) or anti-gHgL (Immune-tech) antibody was used to establish a standard for anti-gp350 IgG or anti-gHgL IgG serum antibody quantification. Data are shown as Mean ± SEM. Significant differences were found as indicated; * = p < 0.05; *** = p < 0.001; ““ = p < 0.0001.

[0062] Figure 18 shows the neutralization of EBV infection of Ramos cells.

[0063] CD-1 mice (n=9) were immunized according to a prime-boost regimen (day 0 / 21). Serum was analyzed 39 days after prime immunization for EBV neutralizing antibodies. EBV was incubated with different serum dilutions before infection of Ramos cells. After 30 minutes of incubation with the virus the cells were washed and incubated overnight at 37°C. Next, cells were stained with anti-gp350-AF647 and a viability dye before analysis on a flow cytometer. Live, gp350+Ramos cells were gated using negative controls and analyzed. The infection rate of negative controls was set as 100% infection, and a 4PL-fit curve was calculated for each sample to determine the titer at 50% inhibition. Data are shown as Mean ± SEM. Significant differences were found as indicated; * = p < 0,05; *** = p < 0.001.

[0064] Figure 19 shows the induction of EBV-specific T cell responses in outbred mice.

[0065] CD-1 mice (n=9) were immunized according to a prime-boost regimen (day 0 / 21). Spleens were isolated 40 days after prime immunization and 0.5 x 106splenocytes per condition were restimulated with BRLF1 PP, BZLF1 PP and EBNA3A PP on anti-IFN-y coated plates overnight. IFN-y-producing cells were stained by HRP-labelled anti-IFN-y antibody and revealed after development with AEC substrate. Spots were counted using an ELISPOT reader (Immunospot). Data are shown as Mean ± SEM. Significant differences were found as indicated; *** = p < 0.001; **** = p < 0.0001; ns = non-significant.

[0066] Figure 20 shows a schematic diagram of MVA-mBN520 and control construct MVA- mBN518.

[0067] Figure 21 shows EBV-neutralization in serum of MVA-BN-EBV immunized mice containing PADRE and induction of PADRE-specific T cell responses.

[0068] C57BL / 6 mice (n=5) were immunized according to a prime-boost regimen (day 0 / 21). (A) Serum was analyzed 40 days after prime immunization for EBV neutralizing antibodies. EBV was incubated with different serum dilutions before infection of Ramos cells. After 30 minutes incubation with the virus, the cells were washed and incubated overnight at 37°C. Next, cells were stained with anti-gp350-AF647 and a viability dye before analysis on a flow cytometer. Live, gp350+Ramos cells were gated using negative controls and analyzed. The infection rate of negative controls was set as 100% infection, and a 4PL-fit curve was calculated for each sample to determine the titer at 50% inhibition. Data are shown as Mean ± SEM. (B) Spleens were isolated 40 days after prime immunization and 0.5 x 106splenocytes per condition were restimulated with PADRE peptide (AKFVAAWTLKAAA) on anti-IFN-y coated plates overnight. IFN-v-producing cells were stained by HRP-labelled anti-IFN-y antibody and revealed after development with AEC substrate. Spots were counted using an ELISPOT reader (Immunospot). Data are shown as Mean ± SEM. Significant differences were found as indicated; *** = p < 0.001; **** = p < 0.0001.

[0069] Figure 22 shows a schematic diagram of VRP constructs: VRP-gp350-PADRE-DPS and control constructs VRP-gp350-GCN4 and VRP-gp350-DPS; VRP-RBD-BFF- PADRE and control construct VRP-RBD-BFF.

[0070] Figure 23 shows the induction of gp350-specific antibody and PADRE-specific T cell responses in mice injected with VRPs.

[0071] C57BL / 6 mice were prime-boost immunized intramuscularly with 5 x 107Til of VRP-gp350, VRP-gp350-DPS or VRP-gp350-PADRE-DPS. BB2 buffer was injected as control. (A) Serum was analyzed three weeks after boost immunization for analysis of 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 4PL-fit standard curve calculation in Excel. (B) Splenocytes were isolated three weeks after boost immunization to analyze PADRE-specific T cell responses. For this, splenocytes were restimulated with PADRE peptide overnight on anti-IFNy coated ELISPOT plates. Next, IFNv-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.

[0072] Figure 24 shows the induction of SARS-CoV-2 spike RBD- and Ferritin (BFF)-specific antibody and PADRE-specific T cell responses in mice injected with VRPs. C57BL / 6 mice were prime-boost immunized intramuscularly with 1 x 108TU of VRP-RBD-BFF or VRP-RBD-BFF-PADRE. BB2 buffer was injected as control. (A) Serum was analyzed two weeks after boost immunization to determine anti-RBD IgG titers by ELISA. For this, ELISA plates were coated with RBD and serum was added in different dilutions. Anti-RBD (10500-CV; R& D) was used to establish a standard for serum antibody quantification by a 4PL-fit standard curve calculation in Excel. (B) Splenocytes were isolated two weeks after boost immunization to analyze PADRE-specific T cell responses. For this, splenocytes were restimulated with PADRE peptide overnight on anti-IFNy coated ELISPOT plates. Next, IFNv-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. Brief Description of Sequences

[0073] SEQ ID NO: 1 is a nucleic acid sequence encoding EBV gp350.

[0074] SEQ ID NO: 2 is the amino acid sequence of EBV gp350.

[0075] SEQ ID NO: 3 is a nucleic acid sequence encoding EBV gH.

[0076] SEQ ID NO: 4 is the amino acid sequence of EBV gH.

[0077] SEQ ID NO: 5 is a nucleic acid sequence encoding PdhC.

[0078] SEQ ID NO: 6 is the amino acid sequence of PdhC.

[0079] SEQ ID NO: 7 is a nucleic acid sequence encoding DPS.

[0080] SEQ ID NO: 8 is the amino acid sequence of DPS.

[0081] SEQ ID NO: 9 is a nucleic acid sequence encoding gp350-PADRE-PdhC.

[0082] SEQ ID NO: 10 is the amino acid sequence of gp350-PADRE-PdhC.

[0083] SEQ ID NO: 11 is a nucleic acid sequence encoding gH-DPS.

[0084] SEQ ID NO: 12 is the amino acid sequence of gH-DPS.

[0085] SEQ ID NO: 13 is a nucleic acid sequence encoding gL.

[0086] SEQ ID NO: 14 is the amino acid sequence of gL.

[0087] SEQ ID NO: 15 is a nucleic acid sequence encoding BZLF1-BRLF1 fusion.

[0088] SEQ ID NO: 16 is the amino acid sequence of BZLF1-BRLF1 fusion.

[0089] SEQ ID NO: 17 is a nucleic acid sequence encoding EBNA3A.

[0090] SEQ ID NO: 18 is the amino acid sequence of EBNA3A.

[0091] SEQ ID NO: 19 is a nucleic acid sequence of Pr13.5-long promoter.

[0092] SEQ ID NO: 20 is a nucleic acid sequence of Pr1328 promoter.

[0093] SEQ ID NO: 21 is a nucleic acid sequence of PrS promoter

[0094] SEQ ID NO: 22 is a nucleic acid sequence of PrH5m promoter.

[0095] SEQ ID NO: 23 is a nucleic acid sequence encoding PADRE.

[0096] SEQ ID NO: 24 is the amino acid sequence of PADRE.

[0097] SEQ ID NO: 25 is a nucleic acid sequence encoding IgK LC signal peptide.

[0098] SEQ ID NO: 26 is the amino acid sequence of IgK LC signal peptide.

[0099] SEQ ID NO: 27 is a nucleic acid sequence of I3L (MVA064L).

[0100] SEQ ID NO: 28 is a nucleic acid sequence of mutated (defective) I3L (MVA064L).

[0101] SEQ ID NO: 29 is a nucleic acid sequence of J5L (MVA089L).

[0102] SEQ ID NO: 30 is a nucleic acid sequence of E4L (MVA051 L).

[0103] SEQ ID NO: 31 is a nucleic acid sequence encoding GCN4.

[0104] SEQ ID NO: 32 is the amino acid sequence of GCN4.

[0105] SEQ ID NO: 33 is a nucleic acid sequence encoding gp350-PADRE-DPS.

[0106] SEQ ID NO: 34 is the amino acid sequence of gp350-PADRE-DPS. SEQ ID NO: 35 is a nucleic acid sequence encoding SARS-CoV 2 spike RBD. SEQ ID NO: 36 is the amino acid sequence of SARS-CoV-2 spike RBD.

[0107] SEQ ID NO: 37 is a nucleic acid sequence encoding Bullfrog / / - / , pylori hybrid ferritin.

[0108] SEQ ID NO: 38 is the amino acid sequence of Bullfrog / / - / , pylori hybrid ferritin. SEQ ID NO: 39 is a nucleic acid sequence encoding SARS-CoV-2 spike RBD-BFF- PADRE.

[0109] SEQ ID NO: 40 is the amino acid sequence of SARS-CoV-2 spike RBD-BFF- PADRE.

[0110] SEQ ID NO: 41 is a nucleic acid sequence encoding htPA signal peptide.

[0111] SEQ ID NO: 42 is the amino acid sequence of htPA signal peptide.

[0112] Detailed Description of Invention

[0113] A serious human disease against which no approved vaccine is available so far is infectious mononucleosis caused by Epstein-Barr virus (EBV) infection. EBV (or human gammaherpesvirus 4, HHV-4) belongs to the Lymphocryptovirus genus within the Herpesviridae family and is an enveloped virus containing a double-stranded DNA genome of about 172,000 bp [Cohen et al., 2000]. EBV is primarily transmitted via saliva and the prevalence in the global adult population is estimated to be 90-95%. Primary infection with EBV can cause infectious mononucleosis (IM), a disease often seen in adolescents and young adults. The disease is characterized by pharyngitis, cervical lymph node enlargement, fatigue, and fever and although generally self-limiting it can last for a period of several weeks. Spleen enlargement is common, and other complications may occur. Occasionally, the disease may persist and result in a chronic infection. This may develop into systemic EBV-positive T cell lymphoma. In addition, infectious mononucleosis is a risk factor for developing multiple sclerosis and a cancer of the lymphatic system called Hodgkin’s lymphoma.

[0114] To develop a vaccine against Epstein-Barr virus (EBV) infection and infectious mononucleosis caused by EBV, we created a recombinant Modified Vaccinia Virus Ankara (MVA) including several EBV-derived transgenes. This recombinant virus referred to as “MVA-BN-EBV” (MVA-mBN520) herein, derived from the attenuated MVA-BN strain of vaccinia virus and included the following EBV-derived transgenes: BZLF1-BRLF1 fusion, EBNA3A, gp350-PADRE-PDHC, gH-DPS, and gL (see Figure 1).

[0115] For the generation of MVA-mBN520, codon-optimized DNA sequences encoding the EBV-derived transgenes controlled by poxviral promoters were inserted into intergenic regions (IGR) 64 / 65 and 88 / 89 of MVA-BN through homologous recombination, using the BAC-based permanent dominant selection method we developed. The mechanism of action of MVA-BN-EBV relies on its multivalent design, which enables potent activation of both humoral and cellular immune responses. By encoding six different EBV antigens (namely: BZLF1, BRLF1, EBNA3A, gp350, gH, and gL), MVA-EBV aims to induce immunity across multiple stages of EBV infection.

[0116] The EBV glycoproteins gp350, gH, and gL are involved in the infection of both B cells and epithelial cells and serve as key targets for neutralizing antibodies [Shannon-Lowe and Rowe, 2014], Therefore, these antigens were included in MVA-BN EBV as primary antibody targets. Furthermore, three T cell targets, i.e., BRLF1, BZLF1, and EBNA3A, which are expressed during distinct phases of the EBV replication cycle were introduced into MVA-BN-EBV. Both, BZLF1 and BRLF1 are transcription factors controlling the switch from latent to lytic replication. EBNA3A is one of the latent antigens expressed during latency type III in EBV-infected cells. BRLF1, BZLF1 and EBNA3Aare immunodominant T cell targets in natural EBV infection, and EBV infection is naturally controlled by the immune system by EBV-specific T cells. In addition, antibodies against gp350, gH, and gL have been shown to be protective in animal models and a gp350 antigen was protective in humans against infectious mononucleosis. Thus, these antigens play a critical role in inducing protective immune responses, particularly by activation T cells that target EBV-infected cells at various stages of viral persistence [Damania et al.

[0117] 2022].

[0118] An important distinguishing feature of MVA-BN-EBV is its unique mode of antigen presentation: gp350 as well as gH / gL expressed by MVA-BN-EBV is capable of forming selfassembling multimeric antigen particles (MAPs). For that purpose, MVA-EBV encodes gp350 as a fusion protein in which the antigen is fused to a subunit of self-assembling multimeric protein particle PdhC (acetyltransferase of pyruvate dehydrogenase (PDH) complex (PdhC) of Geo-) Bacillus stearothermophilus) and gH as a fusion protein in which the antigen is fused to self-assembling multimeric protein particle DPS (DNA binding protein from starved cells of E. coll). This antigen multimerization results in a high-density and unidirectional antigen display which increases immunogenicity of the presented antigen and promotes strong antigen-specific antibody responses [Bachmann et al., 1993]. Furthermore, larger particles (e.g., MAPs) enter the draining lymph node more efficiently compared to small monomeric antigens and accumulate over time, resulting in a significantly prolonged half-life of the antigen [McLennan etal., 2005].

[0119] Taken together, the data presented here demonstrates the multilayered mechanism of action of MVA-BN-EBV. First, it induces neutralizing antibody responses against the key EBV glycoproteins gp350 and gH / IL, which effectively block EBV infection of B cells and epithelial cells, and the activation of other antibody-mediated immune mechanisms. Second, MVA-BN- EBV triggers potent cellular immune responses that are critical for recognition of EBV infected cells during various stages of the viral life cycle.

[0120] Notably, MVA-mBN520 encodes the artificial CD4 T cell epitope pan HLA DR-binding epitope (PADRE) as a linker between the gp350 antigen and PdhC. PADRE is a synthetic adjuvant epitope with high-affinity to a broad HLA-haplotype spectrum that increases CD4-mediated T cell responses [Alexander etal., 1994).

[0121] CD4 T cells are essential for generating effective antibody responses. They activate B cells through direct interaction and cytokine secretion, promoting B cell proliferation, differentiation, and isotype switching. Additionally, CD4 T cells facilitate affinity maturation and contribute to the formation of memory B cells, ensuring a quicker and stronger response upon re-exposure to pathogens. Overall, their role is crucial for coordinating the adaptive immune response and enhancing vaccine effectiveness. For antigens that lack effective CD4 T cell epitopes, either generally or for specific HLA haplotypes, the addition of a CD4 T cell epitope can rescue antigen-specific antibody responses.

[0122] Here, it was shown that MVA-mBN520 encoding the gp350-PADRE-PdhC fusion protein induced significantly more neutralizing antibodies than recombinant MVA encoding the corresponding fusion protein without PADRE (gp350-PdhC). PADRE-specific T cells were also induced by MVA-mBN520.

[0123] Moreover, the immunogenicity increasing effect of co-encoded PADRE was confirmed with DPS instead of PdhC and viral replicon particles (VRPs) as the viral vector encoding the fusion protein (gp350-PADRE-DPS). The immunogenicity increasing effect was also observed with VRPs encoding Bullfrog / / - / , pylori hybrid ferritin as the self-assembling multimeric protein particle and a SARS-CoV-2 antigen ( / .e., SARS-CoV-2 RBD-BFF-PADRE fusion protein). Additionally, it was shown that PADRE peptide can be encoded as a linker between proteins and also at the C-terminal end of a protein and the positive immunogenic effect is unaffected. In summary, the findings presented here regarding PADRE demonstrate that incorporating a promiscuous CD4 T-cell epitope into an antigen displaying self-assembling multimeric protein particle enhances humoral responses to viral vector vaccines. Due to PADRE's ability to bind to a diverse array of haplotypes, it has the potential to facilitate a more robust vaccine-induced antibody response in human, regardless of the given haplotype. Definitions

[0124] 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.

[0125] 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.”

[0126] 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.

[0127] 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. 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.

[0128] 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.

[0129] 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.

[0130] The term “transgene” as used herein refers to a foreign or heterologous gene that has been inserted into a poxvirus, e.g., an MVA genome, by genetic engineering.

[0131] The term “open-reading frame” or “ORF” as used herein, e.g., an ORF of a transgene, means a nucleotide sequence located between a start codon and a stop codon.

[0132] The term “operably linked” as used herein in respect of a promoter means that the promoter is placed in a position where it can direct transcription of the coding sequence of a gene of interest, e.g., a transgene.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] The term “self-assembling multimeric particle” as used herein refers to a polymeric assembly of monomeric polypeptides referred to as “subunits” that are capable of directing their selfassembly into the protein particle. Sometimes, as in scientific literature (see, e.g., Zang etal., 2020), such protein nanoparticles are referred to as “nanoparticles”, “self-assembling nanoparticles” or “self-assembling protein nanoparticles”.

[0137] The term “multimeric antigen particle” or “MAP” as used herein refers to a self-assembling multimeric protein particle displaying or presenting antigens or antigenic determinants on its surface.

[0138] By “antigenic part thereof” as used herein is intended a portion or fragment of a protein that can induce for example the production of an antibody that will bind to it.

[0139] The term “bacterial artificial chromosome” or “BAC” as used herein describes a large, circular DNA molecule derived from bacterial plasmids, engineered to carry and replicate large fragments of foreign DNA within bacterial cells.

[0140] The term “reconstitution” refers to the process of assembling a functional viral genome from individual genetic segments or fragments, enabling the recovery of infectious viral particles from modified or engineered DNA.

[0141] The term “recombination plasmid” (also known as donor plasmid) as used herein describes a circular DNA vector designed to introduce specific genetic sequences into a target genome through homologous recombination.

[0142] The term “homology-directed recombination” as used herein describes the process where genetic exchange occurs between similar or identical sequences in two viral genomes, allowing for the repair of damaged genomes.

[0143] The term “dominant selection” as used describes a genetic selection method in which only viral genomes carrying a dominant allele or trait - typically conferred by an introduced gene -can successfully replicate under selective conditions, thereby enabling the isolation and propagation of these specific viral genomes.

[0144] Abbreviations

[0145] BAC bacterial artificial chromosome

[0146] BFF Bullfrog / / - / , pylori hybrid ferritin

[0147] CCX.2C4 continuous adherent Coturnix japonica (Japanese quail) cell line,

[0148] clone 2C4

[0149] CEF chicken embryo fibroblast

[0150] CVA chorioallantois vaccinia virus Ankara DPS DNA-binding protein from starved cells (also known as DNA protection during starvation protein)

[0151] EBV Epstein-Barr virus

[0152] eGFP-nptll fusion of enhanced green fluorescent protein and neomycin phosphotransferase II enzyme

[0153] htPA SP signal peptide of human tissue plasminogen activator

[0154] IgK LC signal peptide of murine Ig kappa light chain

[0155] IGR intergenic region

[0156] IGR64 / 65 IGR between genes MVA064L (I3L) and MVA065L (I4L)

[0157] IGR88 / 89 IGR between genes MVA088R (J4RL) and MVA089L (J5L)

[0158] i.m. intramuscularly

[0159] InfU infectious units

[0160] ITR inverted terminal repeat

[0161] HLA human leucocyte antigen

[0162] MAP multimeric antigen particle

[0163] MOI multiplicity of infection

[0164] MVA Modified Vaccinia Virus Ankara

[0165] MVA-BN MVA-BN® of Bavarian Nordic

[0166] MVA-BN-EBV MVA-mBN520

[0167] MVA-mBN520 recombinant MVA-BN encoding EBV-derived antigens gH-DPS, gL, gp350- PADRE-PdhC, BZLF1-BRLF1 fusion, and EBNA3A

[0168] ORF open reading frame

[0169] PADRE Pan HLA DR-binding epitope

[0170] PdhC pyruvate dehydrogenase (PDH) complex dihydrolipoamide acetyl transferase (also known as acetyltransferase of pyruvate dehydrogenase (PDH) complex (PdhC)

[0171] pDS recombinant plasmid used for permanent dominant selection

[0172] PP plaque purification, e.g., PP4 means 4thround of single plaque isolation PreMaster amplified seed stock of the respective virus

[0173] RBD receptor-binding domain of SARS-CoV-2 spike (S) protein

[0174] SARS-CoV-2 Severe Acute Respiratory Syndrome-related Coronavirus 2

[0175] SFV Shope (rabbit) fibroma virus

[0176] SP signal peptide

[0177] VACV vaccinia virus

[0178] VRP virus replicon particle or virus-derived replicon particle

[0179] VEEV Venezuelan Equine Encephalitis Virus Embodiments and further aspects

[0180] 1.1 Aspects and embodiments relating to recombinant MVA encoding EBV antigens In one aspect, provided is a recombinant poxvirus comprising a heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes a fusion protein comprising an Epstein-Barr virus (EBV) antigen, or an antigenic part thereof, joined to a subunit of a self-assembling multimeric protein particle which particle is either acetyltransferase of pyruvate dehydrogenase (PDH) complex (PdhC), preferably from Geo-) Bacillus stearothermophilus, or DNA binding protein from starved cells (DPS), preferably from Escherichia coll.

[0181] In one embodiment, the recombinant poxvirus comprises:

[0182] (i) a heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes a fusion protein comprising an EBV antigen, or an antigenic part thereof, joined to a subunit of self-assembling multimeric protein particle PdhC, preferably from Geo-) Bacillus stearothermophilus; and / or

[0183] (ii) a heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes a fusion protein comprising an EBV antigen, or an antigenic part thereof, joined to a subunit of self-assembling multimeric protein particle DPS, preferably from Escherichia coll.

[0184] In one embodiment, the recombinant poxvirus comprises:

[0185] (i) a first heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes a fusion protein comprising an EBV antigen, or an antigenic part thereof, joined to a subunit of self-assembling multimeric protein particle PdhC, preferably from {Geo-) Bacillus stearothermophilus; and

[0186] (ii) a second heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes a fusion protein comprising an EBV antigen, or an antigenic part thereof, joined to a subunit of self-assembling multimeric protein particle DPS, preferably from Escherichia coll.

[0187] In one embodiment, the N-terminus of the subunit of self-assembling multimeric protein particle PdhC and / or DPS is joined to the C-terminus of the EBV antigen.

[0188] 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. In one embodiment, the EBV antigen is selected from the group consisting of gp350 (encoded by BLLF1), Rta (encoded by BRLF1), Zta (encoded by BZLF1), EBNA3A (encoded by BLRF3 and BERF1), gH (encoded by BXLF2), and gL (encoded by BKRF2).

[0189] In one embodiment, in which the self-assembling multimeric protein particle is PdhC, the EBV antigen is surface glycoprotein 350 (EBV gp350).

[0190] In one embodiment, EBV gp350 and the subunit of PdhC are joined via a linker.

[0191] In one embodiment, EBV gp350 and the subunit of PdhC are joined via a linker comprising or consisting of a pan HLA DR-binding epitope (PADRE). Preferably, the linker comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 24. More preferably, the amino acid sequence as depicted in SEQ ID NO: 24 is flanked both N- and C-terminally via short GS linker sequences. Preferably, the amino acid sequence of PADRE is as depicted in SEQ ID NO: 24. Preferably, the amino acid sequence of PADRE is encoded by a nucleic acid sequence as depicted in SEQ ID NO. 23.

[0192] In one embodiment, the fusion protein comprises EBV gp350 which comprises or consists of amino acids 2-434 of a EBV gp350. Preferably, the amino acid sequence of EBV gp350 is as depicted in SEQ ID NO: 2. Preferably, the amino acid sequence of EBV gp350 is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 1

[0193] In one embodiment, the fusion protein comprises a subunit of PdhC which comprises or consists of amino acids 185-428 of a subunit of PdhC. Preferably, the amino acid sequence of the subunit of PdhC is as depicted in SEQ ID NO: 6. Preferably, the amino acid sequence of the subunit of PdhC is encoded by a nucleic acid as depicted in SEQ ID NO: 5.

[0194] In one embodiment, the heterologous nucleic acid that encodes EBV gp350 joined to a subunit of PdhC via a linker, preferably comprising or consisting of PADRE, further encodes a signal peptide joined N-terminally to the fusion protein.

[0195] In one embodiment, the signal peptide (SP) is murine immunoglobulin kappa light chain (IgK LC) signal peptide. Preferably, the amino acid sequence of IgK LC is as depicted in SEQ ID NO: 26. Preferably, the amino acid sequence of IgK LC is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 25.

[0196] In one embodiment, the heterologous nucleic acid that encodes EBV gp350 joined to a subunit of PdhC via a linker, preferably comprising or consisting of PADRE, is operably linked to poxviral promoter Pr13.5 long. In one embodiment, the heterologous nucleic acid that encodes EBV gp350 joined to a subunit of PdhC via a linker comprising PADRE comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO: 9.

[0197] In one embodiment, the heterologous nucleic acid that encodes EBV gp350 joined to a subunit of PdhC via a linker comprising PADRE encodes an amino acid sequence as depicted in SEQ ID NO: 10.

[0198] In one embodiment, in which the self-assembling multimeric protein particle is DPS, the EBV antigen is glycoprotein gH (EBV gH).

[0199] In one embodiment, EBV gH and the subunit of DPS are joined via a linker.

[0200] In one embodiment, the fusion protein comprises EBV gH which comprises or consists of the extracellular domain or amino acids 1-682 of EBV gH. Preferably, the amino acid sequence of EBV gH is as depicted in SEQ ID NO: 4. Preferably, the amino acid sequence of EBV gH is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 3.

[0201] In one embodiment, the fusion protein comprises a subunit of DPS which comprises or consists of amino acids 11-167 of a subunit of DPS. Preferably, the amino acid sequence of the subunit of DPS is depicted in SEQ ID NO: 8. Preferably, the amino acid sequence of the subunit of DPS is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 7.

[0202] In one embodiment, the heterologous nucleic acid that encodes EBV gH joined to the subunit of DPS is operably linked to poxviral promoter PrS.

[0203] In one embodiment, the heterologous nucleic acid that encodes EBV gH joined to a subunit of DPS via a linker comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO: 11.

[0204] In one embodiment, the heterologous nucleic acid that encodes EBV gH joined to a subunit of DPS encodes an amino acid sequence as depicted in SEQ ID NO: 12.

[0205] In one embodiment, preferably in which the self-assembling multimeric protein particle is DPS and the EBV antigen is EBV gH, the recombinant poxvirus further comprises a heterologous nucleic acid operably linked to a poxviral promoter, wherein the further heterologous nucleic acid encodes EBV antigen glycoprotein gL (EBV gl_), or an antigenic part thereof.

[0206] In one embodiment, the recombinant poxvirus comprises:

[0207] (i) a first heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes a fusion protein comprising an EBV antigen, or an antigenic part thereof, joined to a subunit of self-assembling protein particle PdhC, preferably from Geo-) Bacillus stearothermophilus;

[0208] (ii) a second heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes a fusion protein comprising an EBV antigen, or an antigenic part thereof, joined to a subunit of self-assembling protein particle DPS, preferably from Escherichia coll; and

[0209] (iii) a third heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes EBV antigen glycoprotein gL (EBV gl_), or an antigenic part thereof.

[0210] In one embodiment, the heterologous nucleic acid that encodes EBV gL is operably linked to poxviral promoter PrH5m.

[0211] In one embodiment, the amino acid sequence of EBV gL is as depicted in SEQ ID NO: 14. Preferably, the amino acid sequence of EBV gL is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 13.

[0212] In one embodiment, the recombinant poxvirus comprises yet a further heterologous nucleic acid operably linked to a poxviral promoter, wherein the yet further heterologous nucleic acid encodes a fusion protein of EBV antigens encoded by BZLF1 and BRLF1 (EBV BZLF1-BRLF1 fusion), or an antigenic part thereof.

[0213] In one embodiment, the recombinant poxvirus comprises yet a further heterologous nucleic acid operably linked to a poxviral promoter, wherein the yet further heterologous nucleic acid encodes EBV antigen EBNA3A (EBV EBNA3A), or an antigenic part thereof.

[0214] In one embodiment, the recombinant poxvirus comprises:

[0215] (i) a first heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes a fusion protein comprising an EBV antigen, or an antigenic part thereof, joined to a subunit of self-assembling protein particle PdhC, preferably from Geo-) Bacillus stearothermophilus;

[0216] (ii) a second heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes a fusion protein comprising an EBV antigen, or an antigenic part thereof, joined to a subunit of self-assembling protein particle DPS, preferably from Escherichia coli;

[0217] (iii) a third heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes an EBV antigen glycoprotein gL (EBV gL), or an antigenic part thereof; (iv) a fourth heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes a fusion protein of EBV antigens encoded by BZLF1 and BRLF1 (EBV BZLF1-BRLF1 fusion), or an antigenic part thereof; and

[0218] (v) a fifth heterologous nucleic acid operably linked to a poxviral promoter, wherein the heterologous nucleic acid encodes EBV antigen EBNA3A (EBV EBNA3A), or an antigenic part thereof.

[0219] In one embodiment, the heterologous nucleic acid that encodes EBV BZLF1-BRLF1 fusion is operably linked to poxviral promoter Pr13.5-long.

[0220] In one embodiment, the heterologous nucleic acid that encodes EBV BZLF1-BRLF1 fusion comprises or consists of a nucleic acid sequence as is as depicted in SEQ ID NO: 15.

[0221] In one embodiment, the heterologous nucleic acid that encodes EBV BZLF1-BRLF1 fusion encodes an amino acid sequence as depicted in SEQ ID NO: 16.

[0222] In one embodiment, the heterologous nucleic acid that encodes EBV EBNA3A is operably linked to poxviral promoter Pr1328.

[0223] In one embodiment, the heterologous nucleic acid that encodes EBV EBNA3A comprises or consist of a nucleic acid sequence as depicted in SEQ ID NO: 17.

[0224] In one embodiment, the heterologous nucleic acid that encodes EBV EBNA3A fusion encodes an amino acid sequence as depicted in SEQ ID NO: 18.

[0225] 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®.

[0226] In one embodiment, the recombinant poxvirus is derived from a member of the Avipoxvirus, Orthopoxvirus or Parapoxvirus genus.

[0227] 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.

[0228] 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.

[0229] 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). In one embodiment, the recombinant poxvirus is a recombinant vaccinia virus.

[0230] 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).

[0231] 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.

[0232] In one embodiment, the recombinant poxvirus is recombinant MVA.

[0233] 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.

[0234] 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.

[0235] In one embodiment, the recombinant poxvirus is recombinant MVA MVA-BN®.

[0236] 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.

[0237] In one embodiment, the recombinant poxvirus is recombinant MVA, and the (first) heterologous nucleic acid encoding EBV gp350-PADRE-PdhC, the (second) heterologous nucleic acid encoding EBV gH-DPS, and / or the (third) heterologous nucleic acid encoding EBV gL is inserted at IGR 64 / 65.

[0238] In one embodiment, the recombinant poxvirus is recombinant MVA, and the (fourth) heterologous nucleic acid encoding EBV BZLF1-BRLF1 fusion, and / or the (fifth) heterologous nucleic acid encoding EBV EBNA3A is inserted into the MVA genome at IGR 88 / 89. i.2 Aspects and embodiments relating to transcription units encoding EBV antigens In one aspect, provided is a transcription unit comprising a nucleic acid operably linked to a poxviral promoter, wherein the nucleic acid encodes a fusion protein comprising an EBV antigen, or an antigenic part thereof, joined to a subunit of a self-assembling multimeric protein particle which particle is either acetyltransferase of pyruvate dehydrogenase (PDH) complex (PdhC), preferably from Geo-) Bacillus stearothermophilus, or DNA binding protein from starved cells (DPS), preferably from Escherichia coll.

[0239] In one embodiment, the N-terminus of the subunit of self-assembling multimeric protein particle PdhC and / or DPS is joined to the C-terminus of the EBV antigen.

[0240] 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. In one embodiment, the EBV antigen is selected from the group consisting of gp350 (encoded by BLLF1), Rta (encoded by BRLF1), Zta (encoded by BZLF1), EBNA3A (encoded by BLRF3 and BERF1), gH (encoded by BXLF2), and gL (encoded by BKRF2).

[0241] In one embodiment, in which the self-assembling multimeric protein particle is PdhC, the EBV antigen is surface glycoprotein 350 (EBV gp350).

[0242] In one embodiment, EBV gp350 and the subunit of PdhC are joined via a linker.

[0243] In one embodiment, EBV gp350 and the subunit of PdhC are joined via linker comprising or consisting of a pan HLA DR-binding epitope (PADRE). Preferably, the linker comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 24. More preferably, the amino acid sequence as depicted in SEQ ID NO: 24 is flanked both N- and C-terminally via short GS linker sequences. Preferably, the amino acid sequence of PADRE is as depicted in SEQ ID NO: 24. Preferably, the amino acid sequence of PADRE is encoded by a nucleic acid sequence as depicted in SEQ ID NO. 23.

[0244] In one embodiment, the fusion protein comprises EBV gp350 which comprises or consists of amino acids 2-434 of a EBV gp350. Preferably, the amino acid sequence of EBV gp350 is as depicted in SEQ ID NO: 2. Preferably, the amino acid sequence of EBV gp350 is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 1

[0245] In one embodiment, the fusion protein comprises a subunit of PdhC which comprises or consists of amino acids 185-428 of a subunit of PdhC. Preferably, the amino acid sequence of the subunit of PdhC is as depicted in SEQ ID NO: 6. Preferably, the amino acid sequence of the subunit of PdhC is encoded by a nucleic acid as depicted in SEQ ID NO: 5.

[0246] In one embodiment, the nucleic acid that encodes EBV gp350 joined to a subunit of PdhC via a linker, preferably comprising or consisting of PADRE, further encodes a signal peptide joined N-terminally to the fusion protein.

[0247] In one embodiment, the signal peptide (SP) is murine immunoglobulin kappa light chain (IgK LC) signal peptide. Preferably, the amino acid sequence of IgK LC is as depicted in SEQ ID NO: 26. Preferably, the amino acid sequence of IgK LC is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 25.

[0248] In one embodiment, the nucleic acid that encodes EBV gp350 joined to a subunit of PdhC via a linker, preferably comprising or consisting of PADRE, is operably linked to poxviral promoter Pr13.5 long.

[0249] In one embodiment, the nucleic acid that encodes EBV gp350 joined to a subunit of PdhC via a linker comprising PADRE comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO: 9.

[0250] In one embodiment, the nucleic acid that encodes EBV gp350 joined to a subunit of PdhC via a linker comprising PADRE encodes an amino acid sequence as depicted in SEQ ID NO: 10. In one embodiment, in which the self-assembling multimeric protein particle is DPS, the EBV antigen is glycoprotein gH (EBV gH).

[0251] In one embodiment, EBV gH and the subunit of DPS are joined via a linker.

[0252] In one embodiment, the fusion protein comprises EBV gH which comprises or consists of the extracellular domain or amino acids 1-682 of EBV gH. Preferably, the amino acid sequence of EBV gH is as depicted in SEQ ID NO: 4. Preferably, the amino acid sequence of EBV gH is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 3.

[0253] In one embodiment, the fusion protein comprises a subunit of DPS which comprises or consists of amino acids 11-167 of a subunit of DPS. Preferably, the amino acid sequence of the subunit of DPS is depicted in SEQ ID NO: 8. Preferably, the amino acid sequence of the subunit of DPS is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 7.

[0254] In one embodiment, the nucleic acid that encodes EBV gH joined to the subunit of DPS is operably linked to poxviral promoter PrS. In one embodiment, the nucleic acid that encodes EBV gH joined to a subunit of DPS via a linker comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO: 11.

[0255] In one embodiment, the nucleic acid that encodes EBV gH joined to a subunit of DPS encodes an amino acid sequence as depicted in SEQ ID NO: 12.

[0256] In one aspect, provided is a transcription unit for the preparation of a recombinant poxvirus, preferably a recombinant vaccinia virus, more preferably a recombinant MVA, or for the preparation of a vaccine.

[0257] 1.3 Aspects and embodiments relating to nucleic acids encoding EBV antigens

[0258] In one aspect, provided is a nucleic acid encoding a fusion protein comprising an EBV antigen, or an antigenic part thereof, joined to a subunit of a self-assembling multimeric protein particle, which is either PdhC, preferably from Geo-) Bacillus stearothermophilus or DPS, preferably from Escherichia coli.

[0259] In one embodiment, the N-terminus of the subunit of self-assembling multimeric protein particle PdhC and / or DPS is joined to the C-terminus of the EBV antigen.

[0260] In one embodiment, the EBV antigen is selected from the group consisting of gp350 (encoded by BLLF1), Rta (encoded by BRLF1), Zta (encoded by BZLF1), EBNA3A (encoded by BLRF3 and BERF1), gH (encoded by BXLF2), and gL (encoded by BKRF2).

[0261] In one embodiment, in which the self-assembling multimeric protein particle is PdhC, the EBV antigen is surface glycoprotein 350 (EBV gp350).

[0262] In one embodiment, EBV gp350 and the subunit of PdhC are joined via a linker.

[0263] In one embodiment, EBV gp350 and the subunit of PdhC are joined via linker comprising or consisting of a pan HLA DR-binding epitope (PADRE). Preferably, the linker comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 24. More preferably, the amino acid sequence as depicted in SEQ ID NO: 24 is flanked both N- and C-terminally via short GS linker sequences. Preferably, the amino acid sequence of PADRE is as depicted in SEQ ID NO: 24. Preferably, the amino acid sequence of PADRE is encoded by a nucleic acid sequence as depicted in SEQ ID NO. 23.

[0264] In one embodiment, the fusion protein comprises EBV gp350 which comprises or consists of amino acids 2-434 of a EBV gp350. Preferably, the amino acid sequence of EBV gp350 is as depicted in SEQ ID NO: 2. Preferably, the amino acid sequence of EBV gp350 is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 1 In one embodiment, the fusion protein comprises a subunit of PdhC which comprises or consists of amino acids 185-428 of a subunit of PdhC. Preferably, the amino acid sequence of the subunit of PdhC is as depicted in SEQ ID NO: 6. Preferably, the amino acid sequence of the subunit of PdhC is encoded by a nucleic acid as depicted in SEQ ID NO: 5.

[0265] In one embodiment, the nucleic acid that encodes EBV gp350 joined to a subunit of PdhC via a linker, preferably comprising or consisting of PADRE, further encodes a signal peptide joined N-terminally to the fusion protein.

[0266] In one embodiment, the signal peptide (SP) is murine immunoglobulin kappa light chain (IgK LC) signal peptide. Preferably, the amino acid sequence of IgK LC is as depicted in SEQ ID NO: 26. Preferably, the amino acid sequence of IgK LC is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 25.

[0267] In one embodiment, the nucleic acid that encodes EBV gp350 joined to a subunit of PdhC via a linker comprising PADRE comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO: 9.

[0268] In one embodiment, the nucleic acid that encodes EBV gp350 joined to a subunit of PdhC via a linker comprising PADRE encodes an amino acid sequence as depicted in SEQ ID NO: 10. In one embodiment, in which the self-assembling multimeric protein particle is DPS, the EBV antigen is glycoprotein gH (EBV gH).

[0269] In one embodiment, EBV gH and the subunit of DPS are joined via a linker.

[0270] In one embodiment, the fusion protein comprises EBV gH which comprises or consists of the extracellular domain or amino acids 1-682 of EBV gH. Preferably, the amino acid sequence of EBV gH is as depicted in SEQ ID NO: 4. Preferably, the amino acid sequence of EBV gH is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 3.

[0271] In one embodiment, the fusion protein comprises a subunit of DPS which comprises or consists of amino acids 11-167 of a subunit of DPS. Preferably, the amino acid sequence of the subunit of DPS is depicted in SEQ ID NO: 8. Preferably, the amino acid sequence of the subunit of DPS is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 7.

[0272] In one embodiment, the nucleic acid that encodes EBV gH joined to a subunit of DPS via a linker comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO: 11.

[0273] In one embodiment, the nucleic acid that encodes EBV gH joined to a subunit of DPS encodes an amino acid sequence as depicted in SEQ ID NO: 12. In one aspect, provided is a use of the nucleic acid for the preparation of a recombinant poxvirus, preferably a recombinant vaccinia virus, more preferably a recombinant MVA, or for the preparation of a vaccine.

[0274] 1.4 Aspects and embodiments relating to fusion proteins comprising EBV antigens

[0275] In one aspect, provided is a fusion protein comprising an EBV antigen, or an antigenic part thereof, joined to a subunit of a self-assembling multimeric protein particle, which is either PdhC, preferably from Geo-) Bacillus stearothermophilus or DPS, preferably from Escherichia coll.

[0276] In one embodiment, the N-terminus of the subunit of self-assembling multimeric protein particle PdhC and / or DPS is joined to the C-terminus of the EBV antigen.

[0277] In one embodiment, the EBV antigen is selected from the group consisting of gp350 (encoded by BLLF1), Rta (encoded by BRLF1), Zta (encoded by BZLF1), EBNA3A (encoded by BLRF3 and BERF1), gH (encoded by BXLF2), and gL (encoded by BKRF2).

[0278] In one embodiment, in which the self-assembling multimeric protein particle is PdhC, the EBV antigen is surface glycoprotein 350 (EBV gp350).

[0279] In one embodiment, EBV gp350 and the subunit of PdhC are joined via a linker.

[0280] In one embodiment, EBV gp350 and the subunit of PdhC are joined via linker comprising or consisting of a pan HLA DR-binding epitope (PADRE). Preferably, the linker comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 24. More preferably, the amino acid sequence as depicted in SEQ ID NO: 24 is flanked both N- and C-terminally via short GS linker sequences. Preferably, the amino acid sequence of PADRE is as depicted in SEQ ID NO: 24. Preferably, the amino acid sequence of PADRE is encoded by a nucleic acid sequence as depicted in SEQ ID NO. 23.

[0281] In one embodiment, the fusion protein comprises EBV gp350 which comprises or consists of amino acids 2-434 of a EBV gp350. Preferably, the amino acid sequence of EBV gp350 is as depicted in SEQ ID NO: 2. Preferably, the amino acid sequence of EBV gp350 is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 1

[0282] In one embodiment, the fusion protein comprises a subunit of PdhC which comprises or consists of amino acids 185-428 of a subunit of PdhC. Preferably, the amino acid sequence of the subunit of PdhC is as depicted in SEQ ID NO: 6. Preferably, the amino acid sequence of the subunit of PdhC is encoded by a nucleic acid as depicted in SEQ ID NO: 5. In one embodiment of the fusion protein, the nucleic acid that encodes EBV gp350 joined to a subunit of PdhC via a linker comprising PADRE comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO: 9.

[0283] In one embodiment of the fusion protein, the nucleic acid that encodes EBV gp350 joined to a subunit of PdhC via a linker comprising PADRE encodes an amino acid sequence as depicted in SEQ ID NO: 10.

[0284] In one embodiment, in which the self-assembling multimeric protein particle is DPS, the EBV antigen is glycoprotein gH (EBV gH).

[0285] In one embodiment, EBV gH and the subunit of DPS are joined via a linker.

[0286] In one embodiment, the fusion protein comprises EBV gH which comprises or consists of the extracellular domain or amino acids 1-682 of EBV gH. Preferably, the amino acid sequence of EBV gH is as depicted in SEQ ID NO: 4. Preferably, the amino acid sequence of EBV gH is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 3.

[0287] In one embodiment, the fusion protein comprises a subunit of DPS which comprises or consists of amino acids 11-167 of a subunit of DPS. Preferably, the amino acid sequence of the subunit of DPS is depicted in SEQ ID NO: 8. Preferably, the amino acid sequence of the subunit of DPS is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 7.

[0288] In one embodiment of the fusion protein the nucleic acid that encodes EBV gH joined to a subunit of DPS via a linker comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO: 11.

[0289] In one embodiment of the fusion protein, the nucleic acid that encodes EBV gH joined to a subunit of DPS encodes an amino acid sequence as depicted in SEQ ID NO: 12.

[0290] In one aspect, provided is a self-assembling multimeric protein particle comprising a fusion protein as described herein.

[0291] 1.5 Aspects and embodiments relating to pharmaceutical compositions, kits, medical uses, and medical treatments

[0292] In one aspect, provided is a pharmaceutical composition comprising the recombinant poxvirus as described herein.

[0293] In one embodiment, the pharmaceutical composition is a vaccine. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0294] In another aspect, provided is a process for preparing a pharmaceutical composition as described herein.

[0295] 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, preferably, the kit or kit of parts is designed for use in a prime-boost regimen.

[0296] In one aspect, provided is a recombinant poxvirus as described herein for use in the prevention or treatment of EBV infection, infectious mononucleosis caused by EBV, or an EBV associated malignancy.

[0297] In one embodiment, the pharmaceutical composition is administered to a subject intramuscularly, subcutaneously or intranasally, preferably intramuscularly.

[0298] In one embodiment, the pharmaceutical composition is administered to a subject once, twice, three times or more often.

[0299] In one embodiment, the pharmaceutical composition is administered to a subject as part of a prime-boost regimen.

[0300] In one embodiment, the subject is a mammal, preferably a human.

[0301] In one aspect, provided is a method of prevention or treatment of EBV infection, infectious mononucleosis caused by EBV, or an EBV associated malignancy, the method comprising the step of administering to a subject the recombinant MVA or pharmaceutical composition as described herein.

[0302] In one aspect, provided is a method for inducing an immune response comprising the step of administering to a subject the recombinant MVA or pharmaceutical composition as described herein.

[0303] In some embodiments, the step of administering the recombinant MVA or pharmaceutical composition as described herein results in an immune response in the subject such as, for example, the production of antibodies (e.g., neutralizing antibodies) and / or T cell activation. In this manner, the invention provides a method of stimulating an immune response in a subject comprising the step of administering a recombinant MVA or pharmaceutical composition as described herein 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 an antigen encoded by the recombinant MVA are present in the subject following administration of the recombinant MVA or pharmaceutical composition. For example, an immune response is said to be produced in a subject following administration of the recombinant poxvirus or pharmaceutical composition if antibodies are produced in the subject that recognize the encoded antigen full-length, or a part or an antigenic determinant thereof an antigenic determinant of an encoded antigen. Measurement of antibodies in a subject can be any of a variety of methods well-known in the art.

[0304] In some embodiments, the step of administering the recombinant MVA 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 encoded antigen full-length, 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.

[0305] In one embodiment, the step of administering to a subject a recombinant MVA or pharmaceutical composition comprises intramuscular, subcutaneous or intranasal, preferably intramuscular injection of the composition.

[0306] II. Aspects and embodiments relating to the BAC-based permanent dominant selection method

[0307] In one aspect, provided is a process for preparing a recombinant poxvirus, preferably a recombinant vaccinia virus, more preferably a recombinant MVA comprising one or more transgenes, the process comprising the steps of:

[0308] (a) providing an acceptor bacterial artificial chromosome (BAC) comprising an MVA genome (MVA-BAC), which MVA genome is mutated such that an essential MVA gene required for MVA replication is defective or lacking;

[0309] (b) providing a recombination plasmid comprising the essential MVA gene which is defective or lacking in the MVA-BAC of step (a) as a functional gene, the recombination plasmid further comprising a transgene operably linked to a poxviral promoter;

[0310] (c) co-transfecting an MVA producer cell with the MVA-BAC of step (a) and the recombination plasmid of step (b), and furthermore infecting the MVA producer cell with a helper virus derived from the family poxviridae, preferably Shope fibroma virus (SFV); (d) allowing reconstitution of MVA from the MVA-BAC and homologous recombination with the recombination plasmid in the MVA producer cell; and (e) obtaining the recombinant MVA comprising the functional essential MVA gene and the transgene.

[0311] In one embodiment, the essential MVA gene is selected from the group consisting of I3L (MVA064L), J5L (MVA089L), and E4L (MVA051 L).

[0312] In one embodiment, the process comprises the steps of:

[0313] (a) providing an acceptor bacterial artificial chromosome (BAC) comprising an MVA genome (MVA-BAC), which MVA genome is mutated such that two essential MVA genes required for MVA replication are defective or lacking, preferably wherein one essential MVA gene is I3L (MVA064L) and the other essential MVA gene is J5L (MVA089L);

[0314] (b) providing:

[0315] (i) a first recombination plasmid comprising an essential MVA gene, preferably I3L (MVA064L), which is defective or lacking in the MVA-BAC of step (a) as a functional gene, the recombination plasmid further comprising a first transgene operably linked to a poxviral promoter; and

[0316] (ii) a second recombination plasmid comprising another essential MVA gene, preferably J5L, which is defective or lacking in the MVA-BAC of step (a) as a functional gene, the recombination plasmid further comprising a second transgene operably linked to a poxvirus promoter;

[0317] (c) co-transfecting an MVA producer cell with the MVA-BAC of step (a) and the first and second recombination plasmids of step (b), and furthermore infecting the MVA producer cell with a helper virus derived from the family poxviridae, preferably Shope fibroma virus (SFV);

[0318] (d) allowing reconstitution of MVA from the MVA-BAC and homologous recombination with the recombination plasmids in the MVA producer cell; and

[0319] (e) obtaining the recombinant MVA comprising the functional essential MVA gene and the transgenes.

[0320] In one embodiment, the I3L gene is defective in that the gene is partly deleted.

[0321] In one embodiment, the defective I3L gene encodes 105 to 125 amino acids, preferably 110 to 120 amino acids, more preferably 114 to 118 amino acids, most preferably 116 amino acids at the C-terminus of a wild-type I3L. In one embodiment, the wild-type I3L is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 27.

[0322] In one embodiment, the defective I3L gene is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 28. In one embodiment, the J5L gene is lacking in that the complete coding region is deleted. In one embodiment, the J5L gene is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 29.

[0323] In one embodiment, the E4L gene is lacking in that the complete coding region is deleted. In one embodiment, the E4L gene is encoded by a nucleic acid as depicted in SEQ ID NO: 30.

[0324] In one embodiment, the first transgene encodes EBV gp350 joined to a subunit of PdhC via a linker comprising PADRE, preferably comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO: 9.

[0325] In one embodiment, the first transgene encodes EBV gH joined to a subunit of DPS via a linker, preferably comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO: 11.

[0326] In one embodiment, the first transgene encodes EBV gL, preferably comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO: 13.

[0327] In embodiment, the first recombination plasmid comprises a transgene encoding EBV gp350 joined to a subunit of PdhC via a linker comprising PADRE, a transgene encoding EBV gH joined to a subunit of DPS via a linker, and a transgene encoding EBV gL.

[0328] In one embodiment, the second transgene encodes EBV BZLF1-BRLF1 fusion, preferably comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO: 15.

[0329] In one embodiment, the second transgene encodes EBV EBNA3A, preferably comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO:17.

[0330] In one embodiment, the second recombination plasmid comprises a transgene encoding EBV BZLF1-BRLF1 fusion and a transgene encoding EBV EBNA3A.

[0331] In one embodiment, the process further comprises the step of:

[0332] (f) passaging the recombinant MVA of step (e) and subjecting it to single or multiple rounds of plaque purification.

[0333] In one embodiment, the MVA producer cell is an MVA permissive avian cell, preferably a chicken DF-1 cell or a cell of a quail cell line, more preferably a cell of CCX.2C4 or CCX. E10 cell line, most preferably the MVA producer cell is a CCX.2C4 cell. III. Aspects and embodiments relating to co-encoded PADRE

[0334] Nucleic acid molecule

[0335] In one aspect, provided is a nucleic acid, or a nucleic acid molecule, encoding a fusion protein comprising (i) a disease-associated antigen, or an antigenic part thereof, (ii) a subunit of a self-assembling multimeric protein particle, and (iii) a pan HLA DR-binding epitope (PADRE).

[0336] In one embodiment, the nucleic acid is DNA, RNA or mRNA.

[0337] In one embodiment, the disease-associated antigen is a protein selected from the group consisting of a viral, bacterial, fungal, plant, parasite, non-human animal, and human protein. In one embodiment, the disease-associated antigen is an infectious disease-associated antigen.

[0338] 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.

[0339] In one embodiment, the disease-associated antigen is from Epstein-Barr virus (EBV), preferably selected from the group consisting of EBV proteins BLLF1a / 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.

[0340] In one embodiment, the disease-associated antigen is EBV gp350, preferably comprising or consisting of amino acids 2-434 of EBV gp350. Preferably, the amino acid sequence of EBV gp350 comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 2. Preferably, EBV gp350 is encoded by a nucleic acid comprising or consisting of a nucleic acid sequence as depicted in SEQ ID NO: 1.

[0341] In one embodiment, the disease-associated antigen is from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), preferably comprising or consisting of a receptor binding domain (RBD) of SARS-CoV-2 spike protein (SARS-CoV-2 spike RBD). Preferably, the disease-associated antigen comprises or consists of amino acids 331-524 of SARS-CoV-2 spike protein. More preferably, the disease-associated antigen comprises or consists of amino acids 320-537 of the full-length SARS-CoV-2 S1 domain. Preferably, the amino acid sequence of SARS-CoV-2 spike RBD comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 36. Preferably, SARS-CoV-2 spike RBD is encoded by a nucleic acid comprising or consisting of a nucleic acid sequenced as depicted in SEQ ID NO: 35.

[0342] In one embodiment, the self-assembling multimeric protein particle is selected from the group consisting of hybrid protein Bullfrog / H pylori hybrid ferritin (BFF), imidazoleglycerol-phosphate dehydratase (HisB), preferably from Mycobacterium tuberculosis, acetyltransferase of pyruvate dehydrogenase (PDH) complex (PdhC), and DNA binding protein from starved cells (DPS).

[0343] In one embodiment, the self-assembling multimeric protein particle is hybrid protein BFF. Preferably, a BFF subunit comprises or consists of amino acids 2-9 of Bullfrog Rana catesbeiana) ferritin and amino acids 3-167 of the Helicobacter pylori ferritin. Preferably, the amino acid sequence of the BFF subunit comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 38. Preferably, the BFF subunit is encoded by a nucleic acid comprising or consisting of a nucleic acid sequence as depicted in SEQ ID NO: 37.

[0344] In one embodiment, the self-assembling multimeric protein particle is PdhC, preferably from Geo-) Bacillus stearothermophilus. Preferably, a subunit comprises or consists of amino acids 185-428 of a PdhC subunit. Preferably, the amino acid sequence of the PdhC subunit comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 6. Preferably, the PdhC subunit is encoded by a nucleic acid comprising or consisting of a nucleic acid sequence as depicted in SEQ ID NO: 5.

[0345] In one embodiment, the self-assembling multimeric protein particle is DPS, preferably from Escherichia coli. Preferably, a subunit comprises or consists of amino acids 11-167 of a DPS subunit. Preferably, the amino acid sequence of the DPS subunit comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 8. Preferably, the DPS subunit is encoded by a nucleic acid comprising or consisting of a nucleic acid sequence as depicted in SEQ ID NO: 7.

[0346] In one embodiment, in the encoded fusion protein, the subunit of a self-assembling multimeric protein particle is joined to PADRE, preferably either directly or via a short amino acid linker ( / .e., a linker of about 2-6 amino acids), more preferably via a GS linker.

[0347] In one embodiment, in the encoded fusion protein, the N-terminus of the subunit of a selfassembling multimeric protein particle is joined to the C-terminus of PADRE. Preferably, the self-assembling multimeric protein particle is PdhC or DPS.

[0348] In one embodiment, in the encoded fusion protein, the disease associated antigen, or an antigenic part thereof, and the subunit of a self-assembling multimeric protein particle are joined via a linker, preferably an amino acid linker, comprising or consisting of PADRE. Preferably, the self-assembling multimeric protein particle is PdhC or DPS.

[0349] In one embodiment, in the encoded fusion protein, the C-terminus of the subunit of a selfassembling multimeric protein particle is joined to the N-terminus of PADRE. Preferably, the self-assembling multimeric protein particle is BFF.

[0350] In one embodiment, in the encoded fusion protein, the N-terminus of PADRE is joined to the C-terminus of the disease-associated antigen, or the antigenic part thereof, and the C-terminus of PADRE is joined to the N-terminus of the subunit of a self-assembling multimeric protein particle. Preferably, the disease-associated antigen is EBV gp350 and / or the selfassembling multimeric protein particle is PdhC or DPS. More preferably, EBV gp350 and PdhC are joined via linker comprising or consisting of PADRE.

[0351] In one embodiment, in the encoded fusion protein, the N-terminus of PADRE is joined to the C-terminus of the subunit of a self-assembling multimeric protein particle, and the N-terminus of the subunit of a self-assembling multimeric protein particle is joined to the C-terminus of the disease-associated antigen, or the antigenic part thereof. Preferably, the disease-associated antigen is SARS-CoV-2 spike RBD and / or the self-assembling multimeric protein particle is BFF.

[0352] In one embodiment, PADRE is encoded by a section of the nucleic acid comprising or consisting of a nucleic acid sequence as depicted in SEQ ID NO: 23 or is encoded by a section of the nucleic acid comprising or consisting of a nucleic acid encoding an amino acid sequence as depicted in SEQ ID NO: 24. In one embodiment, the amino acid sequence of PADRE comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 24. Preferably, the amino acid sequence as depicted in SEQ ID NO: 24 is flanked N-terminally and / or C-terminally by a short amino acid linker ( / .e., a linker of about 2-6 amino acids), more preferably via a GS linker.

[0353] In one embodiment, the nucleic acid comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO: 9. In one embodiment, the amino acid sequence encoded by the nucleic acid sequence is as depicted in SEQ ID NO: 10.

[0354] In one embodiment, the nucleic acid comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO: 33. In one embodiment, the amino acid sequence encoded by the nucleic acid sequence is as depicted in SEQ ID NO: 34.

[0355] In one embodiment, the nucleic acid comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO: 39. In one embodiment, the amino acid sequence encoded by the nucleic acid sequence is as depicted in SEQ ID NO: 40.

[0356] In one embodiment, the nucleic acid further encodes a signal peptide joined N-terminally to the encoded fusion protein. Preferably, the signal peptide is murine immunoglobulin kappa light chain (IgK LC) or human tissue plasminogen activator (htPA) signal peptide. Preferably, the IgK LC signal peptide is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 25. Preferably, the amino acid sequence of IgK LC signal peptide is as depicted in SEQ IIC NO: 26. Preferably, the htPA signal peptide is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 41. Preferably, the amino acid sequence of htPA signal peptide is as depicted in SEQ IIC NO: 42.

[0357] In one aspect, provided is a use of the nucleic acid as described herein for the preparation of a virus-based vector, a pharmaceutical composition or a vaccine, preferably a virus-based vector vaccine.

[0358] Fusion protein

[0359] In one aspect, provided is a fusion protein encoded by the nucleic acid encoding a fusion protein comprising (i) a disease-associated antigen, or an antigenic part thereof, (ii) a subunit of a self-assembling multimeric protein particle, and (iii) a pan HLA DR-binding epitope (PADRE) as described herein.

[0360] Virus-based vector

[0361] In one aspect, provided is a virus-based vector comprising the nucleic acid encoding a fusion protein comprising (i) a disease-associated antigen, or an antigenic part thereof, (ii) a subunit of a self-assembling multimeric protein particle, and (iii) a pan HLA DR-binding epitope (PADRE) as described herein.

[0362] In one embodiment, the virus-based vector is derived from a DNA virus or an RNA virus, preferably the virus-based vector is a recombinant DNA virus or a recombinant RNA virus. In one embodiment, the virus based-vector is a recombinant poxvirus or a virus-replicon particle (VRP).

[0363] In one aspect, provided is a recombinant poxvirus comprising the nucleic acid encoding a fusion protein comprising (i) a disease-associated antigen, or an antigenic part thereof, (ii) a subunit of a self-assembling multimeric protein particle, and (iii) a pan HLA DR-binding epitope (PADRE) as described herein, wherein the nucleic acid is operably linked to a poxviral promoter.

[0364] 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®.

[0365] In one embodiment, the recombinant poxvirus is derived from a member of the Avipoxvirus, Orthopoxvirus or Parapoxvirus genus.

[0366] In one embodiment, the member of the Avipoxvirus genus is selected from the group consisting of canarypox virus, fowlpox virus, mynahpox virus, pigeonpox virus, and quailpox virus.

[0367] 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.

[0368] 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).

[0369] In one embodiment, the recombinant poxvirus is a recombinant vaccinia virus 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). 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.

[0370] In one embodiment, the recombinant poxvirus is recombinant MVA 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.

[0371] 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.

[0372] 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.

[0373] In one embodiment, the recombinant poxvirus is recombinant MVA, and the 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 IGR 64 / 65.

[0374] In one embodiment, the recombinant RNA virus is a virus replicon particle (VRP) derived from alphavirus, preferably from Venezuelan Equine Encephalitis Virus (VEEV), more preferably derived from VEEV strain TC83 and / or TrD.

[0375] In one embodiment, the VRP comprises a VEEV TC83 genomic promoter and a VEEV TC83 subgenomic promoter.

[0376] In one aspect, the invention provides a use of a virus-based vector as described herein for the preparation of a vaccine.

[0377] In one aspect, provided is a process for preparing a recombinant MVA, the process comprising the steps of:

[0378] (a) providing an acceptor bacterial artificial chromosome (BAC) comprising an MVA genome (MVA-BAC), which MVA genome is mutated such that two essential MVA genes required for MVA replication are defective or lacking, wherein one essential MVA gene is I3L (MVA064L) and the other essential MVA gene is J5L (MVA089L);

[0379] (b) providing a recombination plasmid comprising the essential MVA gene which is defective or lacking in the MVA-BAC of step (a) as a functional gene, the recombination plasmid further comprising a nucleic acid as described herein operably linked to a poxviral promoter;

[0380] (c) co-transfecting an MVA producer cell with the MVA-BAC of step (a) and the recombination plasmid of step (b), and furthermore infecting the MVA producer cell with a helper virus derived from the family poxviridae, preferably Shope fibroma virus (SFV); (d) allowing reconstitution of MVA from the MVA-BAC and homologous recombination with the recombination plasmid in the MVA producer cell; and

[0381] (e) obtaining the recombinant MVA comprising the functional essential MVA gene and the nucleic acid molecule.

[0382] In one aspect, provided is a process for preparing a recombinant VRP, comprising the steps of:

[0383] (a’) providing a plasmid DNA encoding a self-amplifying replicon RNA, preferably a replicon RNA under the control of a cytomegalovirus (CMV) promoter, encoding a nucleic acid as described herein;

[0384] (b’) transfecting a VRP production cell with the plasmid DNA provided in step (a’), further transfecting the VRP production cell with a first CMV promoter driven packaging plasmid encoding an alphavirus capsid protein and a second CMV promoter driven packaging plasmid encoding an alphavirus envelope protein;

[0385] (c’) culturing the transfected VRP production cell of step (b’);

[0386] (d’) obtaining the recombinant VRP.

[0387] Pharmaceutical compositions, medical uses, and medical treatments

[0388] In one aspect, provided is a pharmaceutical composition comprising a nucleic acid as described herein, a fusion protein as described herein, or a virus-based vector as described herein, optionally further comprising a pharmaceutically acceptable carrier or excipient.

[0389] In one embodiment, the pharmaceutical composition is a vaccine.

[0390] In one aspect, provided is a virus-based vector as described herein, or a pharmaceutical composition as described herein for use in the prevention or treatment of a disease.

[0391] In one embodiment of the medical use, the disease is an infectious disease, preferably a viral infectious disease or a viral infection-associated malignancy.

[0392] In one embodiment of the medical use, the virus-based vector comprises a nucleic acid encoding a fusion protein comprising (i) EBV gp350, or an antigenic part thereof, (ii) a subunit of a self-assembling multimeric protein particle, and (iii) a pan HLA DR-binding epitope (PADRE) as described herein, and the virus-based vector is for use in the prevention or treatment of EBV infection, infectious mononucleosis caused by EBV, or an EBV associated malignancy.

[0393] In one aspect, the invention provides a virus-based vector as described herein for use in inducing an antibody response to the disease-associated antigen encoded by the virus-based vector.

[0394] In one embodiment of the medical use, the antibody response induced by the virus-based vector comprising a nucleic acid encoding a fusion protein comprising (i) a disease-associated antigen, or an antigenic part thereof, (ii) a subunit of a self-assembling multimeric protein particle, and (iii) a pan HLA DR-binding epitope (PADRE) is increased as compared to the antibody response induced by a virus-based vector comprising a nucleic acid encoding a fusion protein comprising (i) a disease-associated antigen, or an antigenic part thereof, and (ii) a subunit of a self-assembling multimeric protein particle, but not (iii) a pan HLA DR-binding epitope (PADRE).

[0395] In one aspect, provided is a method of prevention or treatment of a disease comprising the step of administering to a subject a pharmaceutical composition as described herein.

[0396] In embodiment of the method of prevention or treatment, the disease is an infectious disease, preferably a viral infectious disease or a viral infection-associated malignancy.

[0397] In one embodiment of the method of prevention or treatment, the disease is EBV infection, infectious mononucleosis caused by EBV, or an EBV associated malignancy, and the method comprises administering to a subject a pharmaceutical composition comprising a virus-based vector comprising a nucleic acid encoding a fusion protein comprising (i) EBV gp350, or an antigenic part thereof, (ii) a subunit of a self-assembling multimeric protein particle, and (iii) a pan HLA DR-binding epitope (PADRE).

[0398] In one aspect, provided is a method for inducing an antibody response to a disease-associated antigen encoded by a virus-based vector as described herein, comprising administering to a subject a pharmaceutical composition comprising the virus-based vector.

[0399] In one embodiment of the method, the antibody response induced by a virus-based vector comprising a nucleic acid encoding a fusion protein comprising (i) a disease-associated antigen, or an antigenic part thereof, (ii) a subunit of a self-assembling multimeric protein particle, and (iii) a pan HLA DR-binding epitope (PADRE) is increased as compared to the antibody response induced by a virus-based vector comprising a nucleic acid encoding a fusion protein comprising (i) a disease-associated antigen, or an antigenic part thereof, and (ii) a subunit of a self-assembling multimeric protein particle, but not (iii) a pan HLA DR-binding epitope (PADRE).

[0400] IV. Embodiments relating to SEQ ID NOs

[0401] Regarding SEQ ID NOs, the disclosure considers certain sequence identities.

[0402] In one embodiment, a nucleic acid sequence has at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the nucleic acid sequence as depicted in any nucleic acid SEQ ID NO. In one embodiment, an amino acid sequence has at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence as depicted in any amino acid SEQ ID NO.

[0403] Further description

[0404] Modified Vaccinia Virus Ankara (MVA)

[0405] In the past, MVA was generated by 516 serial passages on chicken embryo fibroblasts (CEFs) of the Ankara strain of vaccinia virus (CVA) (for review see Mayr A etal. 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 multiple 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 [Meyer et al., 1991]. It was shown in a variety of animal models that the resulting MVA was significantly avirulent compared to the fully replication competent starting material [Mayr and Danner, 1978].

[0406] 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 Suter et al., 2009), 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).

[0407] 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. 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 [Boukamp et al., 1988], the human bone osteosarcoma cell line 143B (ECACC Deposit No. 91112502), 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 two-fold 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.

[0408] 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.

[0409] Exemplary generation of a recombinant MVA virus

[0410] 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. Recombinant MVA with additional transgenes encoding vaccine antigens or proteins in general can also be generated by a process that we termed permanent dominant selection, which is described herein.

[0411] 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.

[0412] 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.

[0413] Virus replicon particles (VRP)

[0414] 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 an icosahedral nucleocapsid, which is again surrounded by a host cell derived lipid envelope containing the viral envelope protein, the E1-E2 heterodimer. 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.

[0415] 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.

[0416] Exemplary generation of a recombinant TC-83 derived VRP

[0417] 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 or EBV antigens, can be introduced. The stop codon is followed by the VEEV TC-833’UTR, a poly(A) sequence and 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. 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. 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.

[0418] 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 (pl / pg). 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.

[0419] EXAMPLES

[0420] 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.

[0421] EXAMPLE 1: Preparation of MVA-mBN520 by permanent dominant selection A method for the generation of MVA recombinants, termed BAC-based permanent dominant selection, was developed. This method employs a modified MVA genome that lacks one or more essential MVA genes cloned as a bacterial artificial chromosome (BAC). Infectious recombinant MVA can be reconstituted from this BAC DNA in permissive avian cells, such as cells of the CCX.2C4 quail cell line, when using a helper virus, such as Shope fibroma virus (SFV). During reconstitution, the essential gene(s) together with the desired transgene(s) are introduced into the modified MVA-BAC genome (acceptor BAC) via homology-directed recombination. Selection is dominant because a replication-competent recombinant MVA can only be produced if the missing essential genes are reinserted by recombination. This selective advantage over the acceptor BAC-derived virus also remains permanently present. Genes I3L and J5L are both essential for vaccinia virus replication and we determined that they are also essential for MVA and are suitable genes to be used as selection genes in the permanent dominant selection procedure. Using the developed permanent dominant selection method, a recombinant MVA-BN-EBV termed MVA-mBN520 (see Figure 1) was prepared. A schematic representation of the procedure is shown in Figure 2.

[0422] More specifically, an acceptor BAC (MVA-BN-BAC484) containing the MVA-BN genome with complete or partial deletion of essential MVA genes I3L and J5L was co-transfected together with recombination plasmids into CCX.2C4 quail cells. In transfected cells, reactivation of MVA-BN and homologous recombination take place in the cytoplasm. The recombination plasmid contained the foreign genes encoding EBV-derived antigens to be inserted into the MVA-BN genome adjacent to a functional full-length version of that particular essential gene which was deleted in the acceptor BAC. Virus reconstitution was launched in SFV-infected cells, which provided the essential helper functions for reactivation of the BAC plasmid to an infectious recombinant MVA. Recombinant MVA viruses were passaged and plaque purified to separate genetically correct clones from replication-defective parental BAC genomes and SFV. SFV is unable to effectively replicate in avian CCX.2C4 cells and was thus eliminated from the cultures upon passaging and plaque purification.

[0423] 1.1 Modified Vaccinia Virus Ankara Bavarian Nordic (MVA-BN)

[0424] The recombinant MVAs described herein were derived from MVA-BN® (Modified Vaccinia Virus Ankara of Bavarian Nordic, herein also referred to as “MVA-BN”).

[0425] MVA-BN is a well-characterized virus isolated from a modified vaccinia virus Ankara (MVA) virus stock. MVA originates from the dermal vaccinia virus strain chorioallantois vaccinia virus Ankara (CVA). MVA was obtained by serial propagation (more than 570 passages) of CVA on primary chicken embryo fibroblasts (CEFs). This MVA was further passaged by Bavarian Nordic (BN) and designated MVA-BN, corresponding to passage 583. MVA-BN lacks approximately 15% of the genome compared to the ancestral CVA virus (loss of 31 kb resulting in six major deletion sites and many minor deletions, insertions, and base substitutions). The major deletions and the other mutations affect several virulence and host range genes, as well as the gene for A-type inclusion bodies. MVA-BN can attach to enter and efficiently express virally encoded genes in human cells. However, assembly and release of progeny virus does not occur in human cells.

[0426] 1.2 Acceptor BAC DNA

[0427] Different acceptor BACs were generated, containing deletions of either one or two essential MVA genes ( / .e., I3L or MVA064L, J5L or MVA089L). Acceptor BACs were all based on the parental MVA-BN-BAC166 containing the full unmodified MVA-BN genome [Meisinger-Henschel et al., 2010].

[0428] The acceptor BAC MVA-BN-BAC484 was generated in a multistep cloning process including various intermediate BAC constructs from parental MVA-BN-BAC166 (Figure 3). Most of the open reading frame (ORF) of the essential MVA gene I3L and the entire ORF of J5L were deleted to give rise to MVA-BN-BAC484. In the permanent dominant selection procedure, the BAC cassette inserted in IGR64 / 65 present in all acceptor BACs was removed during reconstitution by homologous recombination using I3L as a selection marker. Reconstituted recombinant MVAs generated by this method therefore never contain the BAC cassette. Nextgeneration sequencing of the entire MVA-BN coding regions of acceptor BAC MVA-BN-BAC484 confirmed that no unintended changes were introduced into the MVA-BN coding region contained within the BAC.

[0429] 1.3 Shope fibroma virus (SFV)

[0430] Shope or rabbit fibroma virus (SFV), belongs to the Leporipoxvirus genus within the Poxviridae family, primarily affecting rabbits, hares, and squirrels [Wilier et al., 1999]. The virus was originally isolated from a wild rabbit in Ohio showing symptoms of the disease and was obtained from ATCC (VR-364). At Bavarian Nordic, SFV has been passaged six times in Statens Seruminstitut Rabbit Cornea (SIRC) cells obtained from ATCC (CCL-60). The crude cell lysate from passage 6 (P6) was harvested, aliquoted, and its titer determined.

[0431] SFV acts as a helper virus to initiate the MVA replication cycle, facilitating the reconstitution of recombinant MVA from MVA-BN-BAC by supplying essential poxviral factors to initiate genome reconstitution and replication of the newly generated recombinant MVA as well as intermediate and late transcription. The host range of SFV is limited, as it can only infect rabbit cells and a few monkey cell lines [Yao etal., 2003]. human infection is highly unlikely.

[0432] While SFV can infect certain cells and provide helper functions, it cannot replicate in non-permissive avian cells like quail-derived CCX.2C4 cells due to its abortive infection cycle. As a result, SFV is counterselected during subsequent passages in CCX.2C4 cells and becomes undetectable after several passages or plaque purifications.

[0433] 1.4 CCX.2C4 quail cell line

[0434] Monolayers of the adherent Japanese quail (Coturnix japonica) cell line CCX.2C4 master or working cell bank have been tested free of mycoplasma and microbial contamination. CCX.2C4 cells used for passaging and plaque purification were thawed, passaged, and seeded for virus propagation and selection for a maximum of 20 passages after thawing. CCX.2C4 cells were cultured in OptiPRO™ SFM serum-free culture medium supplemented with 4 mM GlutaMAX.

[0435] 1.5 Origin and properties of EBV-derived transgenes

[0436] For all EBV-derived transgenes expressed from MVA-mBN520, the full-length sequences of naturally occurring genes from EBV isolate B95-8 (GenBank accession number V01555.2) served as reference. Six EBV genes were chosen to be inserted into the MVA-BN genome: gp350 encoded by BLLF1, Rta encoded by BRLF1, Zta encoded by BZLF1, EBNA3A encoded by BLRF3 and BERF1, gH encoded by BXLF2, and gL encoded by BKRF2 (Figure 1).

[0437] The EBV glycoproteins gp350, gH, and gL are essential for infecting B cells and epithelial cells [Connolly et al., 2011]. They are known major targets of antibody responses [Cohen, 2015]. The gH and gL proteins need to be co-expressed and assemble as a hetero-dimer for correct transport to the cell membrane. In MVA-mBN520, gL and gH were expressed independently from each other via different poxviral promotors. The accessory gL protein was expressed as a full-length protein.

[0438] For expression of gH, the N-terminal extracellular domain (amino acids 1-682) was fused to DPS (DNA-binding protein from starved cells) of Escherichia coli (Figure 4). E. coli DPS is a very compact and stable multifunctional protein (about 80-90 A in diameter) forming particle complexes consisting of 12 identical subunits with a flexible and lysine-rich N-end protruding from the dodecamer. The DPS monomer has essentially the same three-dimensional structure as ferritin, which forms a 24-mer with 432 symmetry, a hollow core and pores at the three-fold axes. DPS forms a 12-mer with 23 (tetrahedral) point group symmetry which also has a hollow core and pores at the three-folds [Grant et al., 1998]. The resulting gH-DPS fusion protein lacked a transmembrane domain and should therefore be secreted as a multimeric antigen particle (MAP).

[0439] Full-length gp350 is 907 amino acids long and consists of an extracellular domain, a transmembrane domain, as well as a cytoplasmic domain. In MVA-mBN520, the N-terminal part of the gp350 extracellular domain (amino acids 2-434) was fused to PdhC (pyruvate dehydrogenase (PDH) complex dihydrolipoamide acetyltransferase) subunit E2 of Geobacillus stearothermophilus. The PdhC E2 protein is formed by 60 subunits that selfassemble into a hollow structure with a cubic core or an icosahedron with twelve 5-nm openings, respectively [Milne et al., 2006]. Gp350 is fused to PdhC via a PADRE-containing linker sequence (Figure 5). PADRE (pan HLA DR-binding epitope) is a synthetic adjuvant epitope with high-affinity to a broad HLA-haplotype spectrum that increases CD4-mediated T cell responses. Hence, there is no HLA-dependency [Alexander et al. 1994], The resulting gp350-PADRE-PDHC fusion protein lacked a transmembrane domain and should therefore be secreted as a MAP.

[0440] Both, BZLF1 and BRLF1 are transcription factors controlling the switch from latent to lytic replication [O’Connor etal., 1989]. Peptides of both proteins are expected to serve as strong T-cell antigens. In MVA-mBN520, several biologically active regions of BRLF1 and BZLF1 were removed, a part of the BZLF1 protein shuffled, and both proteins fused to result in a BZLF1-BRLF1 fusion protein (Figure 6).

[0441] EBNA3A is a nuclear protein with the ability to bind cellular transcriptional regulators Kang and Kieff, 2015. In MVA-mBN520, all six potential nuclear localization signals of EBNA3A have been eliminated. In addition, several protein-protein interaction sites were deleted. Finally, EBNA3A was further modified with an N-terminal secretion tag (murine Ig kappa-chain V-J2-C signal peptide) and a C-terminal glycine linker and transmembrane domain (derived from human platelet-derived growth factor receptor beta isoform 2) (Figure 7).

[0442] Gp350-PADRE-PDHC and gH-DPS was inserted into the intergenic region (IGR) 64 / 65 of the MVA-BN genome, and EBNA3A and BZLF1-BRLF1 fusion was inserted into the IGR88 / 89 of the MVA-BN genome (Figure 1).

[0443] Both gH and gL showed 100% identity to GenBank entries YP 401700.1 and YP 401678.1, respectively. Escherichia coll DPS was derived from Uniprot ID P0ABT2. The gp350 protein sequence was based on GenBank entry YP 401667.1 and Geobacillus stearothermophilus PdhC E2 was based on UniProt ID P11961. The BZLF1-BRLF1 fusion protein was based on GenBank sequences YP_401674.1 (BRLF1) and YP_401673.1 (BZLF1). The EBNA3A sequence was based on GenBank entry YP 401669.1. The nucleotide sequences of all inserted transgenes were optimized for human codon usage, and repetitive elements as well as nt-stretches were removed. 1.6 Description of inserted poxviral promoters

[0444] The Pr13.5-long promoter comprises 124 bp of the intergenic region between genes 14L and 13.5L, driving the expression of the native MVA13.5L gene [Wennier etal., 2013]. The Pr13.5-long promoter was described as an immediate early promoter and exhibits a very strong early expression caused by two early promoter core sequences.

[0445] Pr1328 is a native immediate early promoter of 100 bp driving the expression of the WR184 gene of vaccinia virus strain Western Reserve (B2R gene in vaccinia virus strain Copenhagen nomenclature). The sequence is highly similar to the promoter of MVA168R (B2R ortholog in MVA) with only one nucleotide difference.

[0446] The promoter PrS is a synthetic promoter designed from consensus sequences of early and late elements of vaccinia virus promoters [Chakrabarti etal., 1997],

[0447] The PrH5m promoter is a modified (m) version of the native vaccinia virus early late promoter driving expression of the H5R gene.

[0448] 1.7 Construction of recombinant plasmids

[0449] 1.7.1 Construction of pDS020

[0450] The DNA fragment encoding BZLF1-BRLF1 fusion under the control of the Pr13.5-long promoter was generated by gene synthesis (plasmid pMISC502). The fragment was subcloned into the multiple cloning site of precursor plasmid pDSX004, containing the flanking regions of IGR88 / 89 and the essential gene J5L (MVA089L) of MVA, resulting in plasmid pDS017. The DNA fragment encoding EBNA3A under control of the Pr1328 promoter was generated by gene synthesis (plasmid pMISC502), and subcloned into the pDS017 vector downstream of the BZLF1-BRLF1 fusion gene, giving rise to plasmid pDS020 (Figure 8). 1.7.2 Construction of pDS039

[0451] The DNA fragment encoding gH-DPS under the control of PrS promoter and gL under control of the PrH5m promoter (plasmid pMISC1306) as well as the DNA fragment encoding gp350-PADRE-PDHC under control of the Pr13.5-long promoter (plasmid pMISC1282) were generated by gene synthesis. Fragment gp350-PADRE-PDHC together with the Pr13.5-long promoter had been previously subcloned into recombination plasmid pDS034, which already contains the flanking regions of IGR 64 / 65 and the essential gene I3L (MVA064L) of MVA. Both gH-DPS, as well as gL with their corresponding promoters (PrS and PrH5m), were cloned into pDS034, resulting in the final plasmid pDS039 (Figure 9). 1.8 Generation of recombinant MVA-mBN520

[0452] To create a recombinant MVA expressing EBV-derived transgenes, the recombination plasmids pDS020 and pDS039 were constructed as described above (see 1.6.1 and 1.6.2) and the acceptor MVA-BN-BAC484 was generated as described above (see 1.1.). CCX.2C4 quail cells were co-transfected with MVA-BN-BAC484, pDS020, and pDS039, and were subsequently infected with the helper virus SFV. The helper virus provides the machinery for initiating recombinant MVA replication since the MVA-BN-BAC484 DNA is not infectious by itself. By homologous recombination using the respective IGR flanking regions, the essential genes (I3L, J5L) are repaired and simultaneously the EBV-derived transgenes are inserted while at the same time, the BAC backbone sequences are released that are inserted in IGR64 / 65 of the acceptor BAC. CCX.2C4 quail cells are permissive for the reconstituted recombinant MVA but not for the SFV helper virus. Thus, initial reconstitution and further passaging on CCX.2C4 cells result in MVA-mBN520 amplification while SFV is eliminated by the passaging and plaque purification steps.

[0453] After amplification and plaque purification of the recombinant MVA-mBN520 (thirteen passages; five of them as plaque purification) the recombinant MVA-mBN520 PreMaster P13PP5 clone #26 (P13PP5 #26) was obtained.

[0454] Homologous recombination, reconstitution, as well as further passaging and plaque purifications by limiting dilution, were performed with serum-free OptiPRO SFM medium on CCX.2C4 cells. The generation process to obtain MVA-mBN520 is summarized in Figure 10. Characterization of MVA-mBN520 through insertion site-specific PCR confirmed that the transgenes were properly integrated into the intended genomic target sites. Sanger sequencing further validated the accuracy of sequences of all the transgenes and their corresponding poxviral promoters. The absence of SFV was proven by repeatedly negative results in an SFV-specific PCR assay of samples from three consecutive recombinant MVA passages and on the final PreMaster virus stock. An additional PCR test showed that the MVA-mBN520 PreMaster stock was free of DNA from the input MVA-BN-BAC484 that was used in the initial stages of recombinant MVA generation by BAC-based permanent dominant selection. EXAMPLE 2: Analysis of EBV-derived antigen expression by MVA-mBN520 Expression of all transgenes was determined at the protein level using flow cytometry.

[0455] Expression of BZLF1-BRLF1 fusion, EBNA3A, gp350-PADRE-PDHC, and gH-DPS complexed with gL was determined by total staining using permeabilized HeLa cells and antibodies directed against BZLF1, EBNA3A, gp350, and the gH / gL complex, respectively. All proteins were detected in the test sample of MVA-mBN520 PreMaster P13PP5 #26, but not detected in the MVA-BN negative control (Figure 11).

[0456] Thus, flow cytometry confirmed successful expression of the EBV-derived proteins from the inserted transgenes.

[0457] EXAMPLE 3: Immunogenicity studies

[0458] 3.1 Methods

[0459] 3.1.1. Mouse experiments

[0460] Female Balb / c or CD1 mice (6-8 weeks old, Janvier) were immunized intramuscularly on day 0 (prime immunization) and day 21 (boost immunization) with 1 x107InfU of recombinant MVA. Blood for serum isolation was drawn between day 40-41 after prime immunization. Total antiantigen IgG titers in the serum were analyzed by ELISA (see 3.1.2.), EBV neutralizing assay was used to determine virus neutralization titers (reported as 50% inhibition) (see 3.1.3). For T cell analysis, mice were sacrificed on day 41-42 after prime immunization and single-cell suspensions from splenocytes were analyzed by EliSpot (see Example 3.1.4).

[0461] 3.1.2 ELISA

[0462] Anti-gp350 IgG titers and anti-gHgL 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-gHgL (Immune-tech; IT-005-030M8) 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.

[0463] 3.1.3 Neutralization assay

[0464] EBV was produced by purifying and concentrating the virus from an EBV-producing cell line (B95-8). Next, the virus productions were tested, pooled and aliquoted to create a master stock for future analyses. For the assay, a fixed amount of EBV from the pooled production is mixed with different dilutions of serum before infection of 2x105Ramos cells for 30 minutes. After infection, the cells are washed and incubated overnight at 37°C. Next, the cells are stained for cell viability and gp350 with an in-house labelled anti-gp350 antibody (Merck) before fixation with Cytofix (BD) and analysis on a flow cytometer. Live, gp350+ Ramos cells were gated using negative controls and analyzed. The infection rate of negative controls was set as 100% infection, and a 4PL-fit curve was calculated for each sample to determine the serum titer at 50% inhibition.

[0465] 3.1.4 ELISpot

[0466] 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 EBNA3A, BRLF1 or BZLF1 peptide pool (JPT Peptide Technologies), PADRE peptide (AKFVAAWTLKAAA; 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).

[0467] 3.2 Immunogenicity of MVA-mBN520 in inbred Balb / c mice

[0468] To investigate potent immune activation against all encoded EBV antigens, a mouse study was performed. In addition to MVA-BN-EBV, two control constructs were included, i.e., MVA-mBN461 and MVA-mBN516 (see Figure 12), to examine the benefit of MAP-mediated B cell activation / immune activation. MVA-mBN461 encoded tetrameric gp350 and served as a control for MAP-gp350 presentation; MVA-mBN516 expressed membrane bound gH / gL which served as control for MAP-gH / gL presentation.

[0469] BALB / c mice were vaccinated intramuscularly (i.m.) with MVA-BN-EBV (MVA-mBN520) or one of the control MVA constructs (dose: 1 x 107InfU each) in a prime-boost regimen three weeks apart (prime: day 0; boost: day 21).

[0470] Serum was assessed for EBV-specific antibody responses and neutralizing activity three weeks after the second vaccination. T cell responses were evaluated in the spleen three weeks following booster vaccination.

[0471] ELISA analysis of serum showed that prime-boost immunization with MVA-BN-EBV (MVA-mBN520) induced high antibody titers against both EBV glycoproteins, gp350 as well as gH / gL (Figure 13). Furthermore, MAP-mediated gp350 and gH / gL presentation was clearly superior to tetrameric gp350 and the membrane bound gH / gL expression, respectively (Figure 13A, compare MVA-mBN516 and MVA-mBN520 to MVA-mBN461; Figure 13B, compare MVA-mBN520 to MVA-mBN516).

[0472] An EBV neutralization assay was conducted to assess the ability of MVA-BN-EBV to induce antibodies that protect B cells from EBV infection (Figure 14). EBV-neutralizing titers induced by MVA-BN-EBV (MVA-mBN520) were more than 5-fold higher than titers obtained by immunization of mice with MVA-mBN461 expressing tetrameric gp350 (Figure 14). These results demonstrated that MVA-BN-EBV elicited robust antibody responses against the encoded antigens gp350 and gH / gL, which also possessed neutralizing activity that effectively blocked EBV infection of B cells.

[0473] Finally, T cell responses elicited by i.m. MVA-BN-EBV immunization were analyzed using ELISPOT. Splenocytes were restimulated with peptide pools (PP) of BRLF1 and EBNA3A, and IFN-y-production was measured. T cell responses against both EBNA3Aand BZLF1 were detected, with stronger responses to BZLF1 (Figure 15B) than to EBNA3A (Figure 15A) in Balb / c mice. As expected, MVA-mBN461 expressing only gp350-GCN4 did not show T cell responses against these antigens because they were not included in the construct (Figure 12). In summary, MVA-BN-EBV is a potent inducer of both humoral and cellular immune responses against multiple disease-relevant EBV antigens.

[0474] 3.3 Immunogenicity of MVA-mBN520 in CD-1 outbred mice

[0475] While the above presented study was performed in the inbred mouse strain Balb / c we analyzed whether MAP-mediated gp350 and gH / gL presentation could induce improved antibody titers in a more genetically divers environment. Therefore, CD-1 outbred mice were immunized with MVA-BN-EBV (MVA-mBN520) or control constructs that expressed only a tetrameric gp350 (MVA-mBN461 ) or membrane-bound gH / gL (MVA-mBN515) (see Figure 16). Mice were prime / boost (prime: day 0; boost: day 21) immunized intramuscularly with MVA-BN-EBV (MVA-mBN520) or one of control MVA constructs (dose: 1 x 107InfU each). Three weeks after boost immunization serum was isolated for antigen-specific antibody analysis and spleens were isolated to determine antigen-specific T cell responses via ELISPOT.

[0476] First, antibody responses against the two encoded EBV surface molecules gp350 and gHgL were analyzed. The results clearly showed that MAP-mediated presentation of gp350, i.e., on PDHC and DPS, led to highly efficient gp350-specific antibody induction compared to trimeric gp350 (MVA-mBN461) (Figure 17A). Similarly, gH / gL presentation on DPS in MVA-mBN520 immunized mice improved antibody responses relative to membrane bound gH / gL (MVA-mBN515) (Figure 17B). In addition to total IgG titers, we analyzed the ability of MVA-BN-EBV to induce antibodies in outbred mice that protect B cells from EBV infection via an EBV neutralization assay. As previously shown for total IgG titers, EBV neutralizing titers induced by MVA-mBN461 expressing tetrameric gp350 were significantly lower than EBV-neutralizing titers obtained by immunization of mice with MVA expressing gp350-PDHC MAP (MVA-mBN520) (Figure 18). Finally, to assess EBV-specific T cell responses, 0.5 x 106splenocytes per condition were restimulated with BRLF1, BZLF1, and EBNA3A peptide pools. Given the outbred nature and haplotype diversity of CD-1 mice, we anticipated more variable T cell responses to the different antigens. However, on average, the mice exhibited comparable EBV-specific T cell responses across all tested peptide pools (Figure 19). As expected, no responses were detected in MVA-mBN461 -immunized mice, which lacked the tested antigens (Figure 19). While low and variable responses were observed against the BZLF1 peptide pool, robust T cell responses against EBNA3A and BRLF1 were detected in CD-1 mice immunized with MVA-mBN520 (Figure 19).

[0477] EXAMPLE 4: Enhancing immunogenicity by MVA-BN-EBV encoded CD4 helper epitope PADRE

[0478] Another important element of MVA-BN-EBV is the integrated promiscuous CD4 T helper epitope PADRE (pan HLA DR-binding epitope). CD4 T cells are essential for generating effective antibody responses. They activate B cells through direct interaction and cytokine secretion, promoting B cell proliferation, differentiation, and isotype switching. Additionally, CD4 T cells facilitate affinity maturation and contribute to the formation of memory B cells, ensuring a quicker and stronger response upon re-exposure to pathogens. Overall, their role is crucial for coordinating the adaptive immune response and enhancing vaccine effectiveness. For antigens that lack effective CD4 T cell epitopes, either generally or for specific HLA haplotypes, the addition of a CD4 T cell epitope can rescue antigen-specific antibody responses. Therefore, the artificial CD4T cell epitope PADRE has been included into MVA-BN-EBV as a linker between gp350 and PDHC. PADRE is a synthetic adjuvant epitope with high-affinity to a broad HLA-haplotype spectrum that increases CD4-mediated T cell responses [Alexander etal., 1994],

[0479] PADRE was originally designed as a promiscuous CD4 T cell epitope capable of being presented on multiple human HLAs. However, it was also shown to be efficiently presented on murine l-Ab, the MHCII haplotype of C57BL / 6 mice. Therefore, a mouse study was conducted in this mouse strain to demonstrate the contribution of PADRE to the immunogenicity of MVA-BN-EBV. C57BL / 6 mice were vaccinated i.m. on day 0 and day 21 (prime / boost) with MVA- BN-EBV (MVA-mBN520) or a control construct (MVA-mBN518; see Figure 20) that contains all the antigens present in MVA-BN-EBV but lacks PADRE (dose: 1 x 107Infll each). Serum was assessed for neutralizing antibody titers three weeks after the second vaccination. PADRE-specific T cell responses were evaluated in the spleen three weeks following booster vaccination.

[0480] In line with published data, MVA-BN-EBV immunization resulted in potent activation of PADRE-specific T cells in C57BL / 6 mice (Figure 21 B). Importantly, a significant drop of EBV neutralizing antibody titers was observed when the helper epitope PADRE was absent in the vaccine (Figure 21 A, compare MVA-mBN520 to MVA-mBN518).

[0481] EXAMPLE 5: Enhancing immunogenicity of VRP encoded antigens by co-encoded PADRE

[0482] In addition to MVA-BN-EBV (see Example 4) as an example of recombinant DNA viral vectors, another type of viral vector was tested, namely virus replicon particles (VRPs) as an example of recombinant RNA viral vectors.

[0483] The VRPs were based on live, attenuated Venezuelan Equine Encephalitis Virus (VEEV), strain TC-83.

[0484] VRPs were prepared that encoded EBV gp350 joined to subunits of a self-assembling multimeric protein particle, here DPS, either via PADRE as a linker (VRP-gp350-PADRE-DPS) or via a flexible ten amino acid (PKPSTPPGSS) linker (VRP-gp350-DPS). As for MVA-BN-EBV, gene BLLF1 of EBV strain B95-8 was chosen for encoding EBV gp350.

[0485] Furthermore, VRPs were prepared that encoded another antigen, the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein, joined to Bullfrog / / - / , pylori hybrid ferritin (BFF) as the self-assembling multimeric protein particle (VRP-RBD-BFF), and VRPs co-encoding PADRE C-terminally of BFF (VRP-RBD-BFF-PADRE).

[0486] 5.1 VRPs encoding gp350-DPS or gp350-PADRE-DPS fusion protein

[0487] 5.1.1 Generation of VRPs

[0488] VRP-gp350-GCN4 (VRP-BN102) served as control for gp350 expression without being displayed on a self-assembling multimeric protein particle. VRP-gp350-GCN4 encoded an EBV strain B95-8 derived gp350 fragment (amino acids 2-434) from the extracellular domain of gp350 fused at its C-terminus to the tetramerization domain GCN4 and at its N-terminus to the secretion signal peptide of murine immunoglobulin kappa light chain (IgK LC) (Fig. 22). GCN4 is a synthetic tetramerization domain derived from yeast [Harbury et al., 1993]. The yeast derived GCN4 sequence is based on 2IPZ A. A 13 amino acid linker (PKPSTPPGSSCGG) was inserted between gp350 and GCN4.

[0489] VRP-gp350-DPS (VRP-BN096) encoded the amino acids 2-434 fragment of the extracellular domain of gp350 N-terminally fused to the IgK LC secretion signal and C-terminally fused to DPS (Fig. 22). A 10 amino acid linker (PKPSTPPGSS) was inserted between gp350 and DPS. The gp350 protein sequence is based on GenBank entry YP 401667.1.

[0490] VRP-gp350-PADRE-DPS (VRP-BN101) encoded the amino acids 2-434 fragment of the extracellular domain of gp350 N-terminally fused to the IgK LC secretion signal. PADRE (AKFVAAWTLKAAA) was fused N-terminally to gp350 via a 2 amino acid (GS) sequence and C-terminally to DPS via a 2 amino acid (GS) sequence.

[0491] The fusion protein constructs as illustrated in Fig. 22 were inserted in a VEEV TC-83 derived replicon. 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. All protein sequences were optimized on nucleotide level for human codon usage and repetitive elements as well as G / C or A / T-rich polynucleotide stretches were removed.

[0492] 5.1.2 Immunogenicity

[0493] To assess immunogenicity, mice were prime / boost immunized i.m. on days 0 and 21 with VRP-gp350-GCN4, VRP-gp350-DPS or VRP-gp350-PADRE-DPS. Three weeks after boost immunization, antibody titers in the serum were analyzed. Additionally, PADRE-specific T cell responses were analyzed by ELISPOT after restimulation of splenocytes with PADRE peptide. As shown in Fig. 23A, gp350-specific antibody responses were increased upon immunization of mice with VRPs expressing gp350-PADRE-DPS as compared to VRPs expressing gp350-DPS without PADRE. Additionally, VRP-gp350-PADRE-DPS induced PADRE-specific T cell responses (Fig. 23B).

[0494] Thus, the results shown in Fig. 23 substantiate the findings described in Example 4, i.e. viral vector co-encoded PADRE (VRP-gp350-PADRE-DPS) further increases the immunogenicity already increased by self-assembling multimeric protein particles (VRP-gp350-GCN4 vs. VRP-gp350-DPS). 5.2 VRPs encoding RBD-BFF or RBD-BFF-PADRE fusion protein

[0495] 5.2.1 Generation of VRPs

[0496] VRP-RBD-BFF (VRP-BN041) encoded the RBD of SARS-CoV-2 spike protein fused at its C-terminus to BFF and at its N-terminus to the htPA signal peptide. A nine amino acid linker (SSGGASVLA) was located between the RBD and the BFF subunit.

[0497] VRP-RBD-BFF-PADRE (VRP-BN081) encoded the SARS-CoV-2 spike RBD fused at its C-terminus to BFF via a 9 amino acid linker and at its N-terminus to the htPA signal peptide, and BFF fused at its C-terminus to PADRE (AKFVAAWTLKAAA) via HRLK cathepsin cleavage site.

[0498] The original SARS-CoV-2 spike 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.

[0499] 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. The fusion protein constructs as illustrated in Fig. 22 were inserted in a VEEV TC-83 derived replicon. As described for the VRPs in Example 5.1.1., 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. All protein sequences were optimized on nucleotide level for human codon usage, and repetitive elements as well as G / C or A / T-rich polynucleotide stretches were removed.

[0500] 5.2.2 Immunogenicity

[0501] To assess immunogenicity, C57BL / 6 mice were prime / boost immunized i.m. on days 0 and 21 with VRP-RBD-BFF or VRP-RBD-BFF-PADRE. Two weeks after boost immunization, RBD-specific and ferritin (BFF)-specific antibody titers in the serum were analyzed, respectively. Additionally, PADRE-specific T cell responses were analyzed by ELISPOT after restimulation of splenocytes with PADRE peptide. As shown in Fig. 24A, RBD-specific and ferritin-specific antibody responses were enhanced when BFF was fused to PADRE. Additionally, VRP-RBD-BFF-PADRE induced PADRE-specific T cell responses (Fig. 24B). These findings further support the results described in Examples 4 and 5.2, here using a different antigen and self-assembling multimeric protein particle.

[0502] 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.

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[0534] Yao X-D, Evans DH. High-frequency genetic recombination and reactivation of orthopoxviruses from DNA fragments transfected into leporipoxvirus-infected cells. J Virol. 2003;77(13):7281 -7290.

[0535] 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.

[0536] Zhao B, Marshall DR, Sample CE. A conserved domain of the Epstein-Barr virus nuclear antigens 3A and 3C binds to a discrete domain of Jkappa. J Virol. 1996;70(7):4228- 36. Sequences

[0537] Remark: Start and stop codons are highlighted by underscoring.

[0538] SEQ ID NO: 1 Nucleic acid sequence encoding EBV gp350.

[0539] ATGGAAGCAGCTCTGCTCGTGTGCCAGTACACCATCCAGAGCCTCATCCACCTGACAG GAGAGGATCCTGGCTTCTTCAACGTGGAAATTCCAGAGTTTCCCTTCTACCCTACCTGC AATGTGTGCACAGCCGACGTGAACGTGACCATCAACTTCGACGTTGGAGGCAAGAAGC ACCAGCTGGACCTGGATTTCGGACAACTGACACCTCACACCAAGGCTGTGTATCAGCCT AGAGGAGCCTTTGGTGGTTCTGAGAATGCCACCAACCTGTTTCTCCTGGAGCTGCTTG GAGCTGGCGAGCTCGCACTGACCATGAGAAGCAAGAAACTGCCCATCAATGTGACCAC AGGCGAGGAACAGCAGGTGTCCCTGGAAAGCGTGGACGTGTACTTTCAAGACGTGTTC GGCACCATGTGGTGCCACCATGCCGAGATGCAGAACCCTGTGTACCTGATCCCAGAGA CAGTGCCCTACATCAAGTGGGACAACTGCAACAGCACAAACATCACAGCCGTCGTGAG GGCTCAGGGACTGGATGTGACACTGCCTCTGTCTCTGCCAACCAGTGCCCAGGACAGC AACTTCAGCGTGAAGACCGAGATGCTGGGAAACGAGATCGACATCGAGTGCATCATGG AAGATGGCGAGATCAGCCAGGTACTGCCTGGCGACAACAAGTTCAACATCACATGCAGT GGCTACGAGAGCCACGTGCCATCTGGAGGCATCCTGACAAGCACAAGCCCAGTGGCCA CACCGATCCCTGGCACAGGCTACGCCTACAGCCTGAGACTGACACCCAGACCCGTGTC CAGATTCCTGGGCAACAACAGCATCCTGTACGTGTTCTACTCTGGCAACGGACCCAAGG CCTCTGGTGGCGATTACTGTATCCAGAGCAACATTGTGTTCAGCGATGAGATCCCTGCC AGCCAGGACATGCCAACCAATACCACCGATATCACCTACGTGGGAGACAATGCCACCTA CAGCGTGCCCATGGTCACCTCCGAGGACGCCAACAGCCCTAATGTGACCGTGACAGCC TTCTGGGCATGGCCTAACAACACCGAGACAGACTTCAAGTGCAAGTGGACCCTGACCT CTGGCACACCTAGTGGCTGCGAGAATATCAGCGGAGCCTTCGCCAGCAACCGGACCTT CGACATTACCGTGTCTGGCCTTGGCACAGCTCCCAAGACCCTGATCATCACCAGGACTG CCACCAATGCCACAACCACAACCCACAAAGTGATCTTCAGCAAGGCTCCTGAAAGCACC ACAACTAGTCCTACACTG

[0540] SEQ ID NO: 2 Amino acid sequence of EBV gp350.

[0541] MEAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCTADVNVTINFDVGGKKHQLDL DFGQLTPHTKAVYQPRGAFGGSENATNLFLLELLGAGELALTMRSKKLPINVTTGEEQQVSL ESVDVYFQDVFGTMWCHHAEMQNPVYLIPETVPYIKWDNCNSTNITAVVRAQGLDVTLPLS LPTSAQDSNFSVKTEMLGNEIDIECIMEDGEISQVLPGDNKFNITCSGYESHVPSGGILTSTS PVATPIPGTGYAYSLRLTPRPVSRFLGNNSILYVFYSGNGPKASGGDYCIQSNIVFSDEIPASQ DMPTNTTDITYVGDNATYSVPMVTSEDANSPNVTVTAFWAWPNNTETDFKCKWTLTSGTP SGCENISGAFASNRTFDITVSGLGTAPKTLIITRTATNATTTTHKVIFSKAPESTTTSPTL

[0542] SEQ ID NO: 3 Nucleic acid sequence encoding EBV gH.

[0543] ATGCAGCTGCTGTGCGTGTTCTGCCTGGTGCTGCTGTGGGAAGTGGGAGCTGCAAGC CTGAGCGAAGTGAAGCTGCACCTGGACATCGAGGGTCACGCCAGCCACTACACCATCC CTTGGACAGAGCTGATGGCCAAGGTGCCTGGACTGTCTCCTGAAGCTCTGTGGCGAGA AGCCAACGTGACCGAGGATCTGGCTTCCATGCTGAACCGGTACAAGCTGATCTACAAGA CCAGCGGAACCCTGGGAATCGCTCTGGCAGAACCTGTGGATATCCCTGCTGTGTCTGA GGGCAGCATGCAGGTGGACGCCAGCAAAGTGCACCCAGGAGTGATCAGCGGACTGAA CAGTCCTGCCTGTATGCTGAGCGCTCCTCTGGAAAAGCAGCTGTTCTACTACATCGGTA CCATGCTGCCTAACACCAGACCTCACAGCTACGTCTTCTACCAGCTGCGGTGCCATCTG AGCTACGTCGCTCTGAGCATCAACGGAGACAAGTTCCAGTACACCGGAGCTATGACCAG CAAGTTCCTGATGGGTACCTACAAGAGAGTGACCGAGAAGGGAGACGAACACGTGCTG AGCCTGGTGTTCGGCAAGACCAAGGACCTGCCTGACCTGAGAGGACCCTTCAGCTACC CTTCCCTCACCAGCGCTCAGAGCGGAGACTACAGCCTCGTGATCGTGACCACATTCGT GCACTACGCCAACTTCCACAACTACTTCGTGCCCAACCTGAAGGACATGTTCAGCAGAG CCGTGACCATGACAGCTGCAAGCTACGCCAGATACGTGCTGCAGAAACTGGTCCTGCT GGAAATGAAGGGAGGATGCAGAGAGCCTGAGCTGGACACAGAGACACTGACAACCATG TTCGAGGTGTCCGTGGCCTTCTTCAAAGTGGGACACGCTGTGGGAGAGACAGGCAATG GCTGTGTGGACCTGAGATGGCTGGCCAAGAGTTTCTTCGAGCTGACCGTGCTGAAAGA TATCATTGGCATTTGCTACGGAGCCACCGTGAAAGGCATGCAGAGCTACGGACTAGAAA GACTGGCAGCTATGCTGATGGCTACCGTGAAGATGGAAGAACTGGGACACCTCACCAC AGAGAAGCAGGAATACGCTCTGAGACTGGCCACCGTGGGCTATCCTAAAGCTGGAGTG TACTCCGGACTGATCGGTGGAGCTACAAGCGTGCTGCTGAGTGCCTACAACCGACACC CTCTGTTCCAGCCTCTGCACACCGTGATGAGAGAGACCCTGTTCATCGGAAGCCATGTC GTGCTGCGAGAGCTGAGACTGAATGTGACAACCCAGGGACCTAACCTGGCTCTCTATC AGCTGCTGAGCACCGCTCTGTGTTCCGCTCTCGAGATCGGAGAAGTGCTAAGAGGACT CGCACTGGGCACAGAGAGCGGACTGTTCAGCCCTTGCTACCTGTCCCTGAGATTCGAC CTGACCAGAGACAAGCTGCTGTCCATGGCACCTCAGGAAGCCACACTGGATCAGGCAG CCGTGTCCAACGCTGTGGATGGCTTTCTGGGACGACTGTCACTGGAAAGAGAGGACAG GGACGCCTGGCATCTGCCTGCCTATAAGTGCGTGGACCGACTGGACAAGGTGCTGATG ATCATTCCACTGATCAACGTGACCTTCATCATCAGCTCCGACCGAGAGGTGCGAGGCAG TGCCCTGTATGAAGCCAGCACCACATACCTGAGCTCCAGCCTGTTTCTGAGCCCTGTGA TCATGAACAAGTGTAGCCAGGGTGCTGTGGCTGGAGAGCCTAGACAGATCCCTAAGATC CAGAACTTCACCCGAACCCAGAAGTCCTGCATCTTCTGTGGCTTTGCTCTGCTGTCCTA CGACGAGAAGGAGGGACTGGAAACCACCACCTACATCACCAGCCAGGAAGTCCAGAAT AGCATCCTGTCCAGCAATTACTTCGACTTCGACAACCTGCATGTGCACTACCTGCTCCTG ACCACAAACGGCACAGTGATGGAAATCGCTGGACTGTACGAGGAACGAGCTCATGTGG TCCTGGCCATTATCCTCTATTTTATCGCCTTCGCCCTGGGTATTTTTTTGGTACATAAGATC GTGATGTTTTTTCTGTAA

[0544] SEQ ID NO: 4 Amino acid sequence of EBV gH.

[0545] MQLLCVFCLVLLWEVGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVT EDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAP LEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTE KGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDM FSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGN GCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQ EYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLN VTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQE ATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVR GSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDE KEGLETTTYITSQEVQNSILSSNYFDFDNLHVHYLLLTT

[0546] SEQ ID NO: 5 Nucleic acid sequence encoding PdhC.

[0547] GCTGCTGCTAAACCTGCTACAACAGAGGGCGAGTTCCCCGAGACACGCGAGAAGATGT CTGGCATCAGAAGGGCTATCGCCAAGGCCATGGTGCACAGCAAGCACACAGCTCCTCA CGTGACCCTGATGGACGAGGCCGATGTGACAAAGCTGGTGGCCCACAGAAAGAAGTTC AAGGCCATTGCCGCCGAGAAGGGAATCAAGCTGACCTTCCTGCCTTACGTGGTCAAGG CCCTGGTTTCTGCCCTGAGAGAATACCCCGTGCTGAACACCAGCATCGACGACGAGAC AGAGGAAATCATCCAGAAGCACTACTACAACATCGGAATCGCCGCCGACACCGACAGAG GACTGCTGGTGCCTGTGATCAAGCACGCCGACAGAAAGCCCATCTTCGCCCTGGCTCA AGAGATCAACGAGCTGGCTGAGAAGGCCAGAGATGGCAAGCTGACACCCGGCGAAAT GAAGGGCGCCAGCTGTACCATCACCAACATCGGTTCTGCTGGCGGCCAGTGGTTCACC CCAGTGATCAATCACCCTGAGGTGGCCATCCTCGGCATCGGCAGAATCGCTGAGAAGC CCATCGTCCGCGACGGCGAAATTGTGGCTGCTCCTATGCTGGCCCTGAGCCTGAGCTT CGACCACAGAATGATCGACGGCGCCACAGCTCAGAAGGCCCTGAACCACATCAAGAGA CTGCTGAGCGACCCCGAGCTGCTGCTGATGGAAGCT

[0548] SEQ ID NO: 6 Amino acid sequence of PdhC.

[0549] AAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIA AEKGIKLTFLPYVVKALVSALREYPVLNTSIDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHAD RKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIA EKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLMEA

[0550] SEQ ID NO: 7 Nucleic acid sequence encoding DPS.

[0551] GCTACAAATCTGCTGTACACCAGAAACGACGTGTCCGACAGCGAGAAGAAGGCCACAG TCGAGCTGCTGAACAGACAAGTGATCCAGTTCATCGACCTGAGCCTGATCACCAAGCAG GCCCACTGGAACATGAGAGGCGCCAACTTTATCGCCGTGCACGAGATGCTGGACGGCT TCAGAACAGCCCTGATCGACCACCTGGACACCATGGCTGAAAGAGCTGTGCAGCTTGG CGGAGTGGCTCTGGGCACAACCCAAGTGATCAACAGCAAGACCCCTCTGAAGTCTTAC CCTCTGGACATCCACAACGTGCAGGACCACCTGAAAGAACTGGCCGACAGATACGCCA TCGTGGCCAATGATGTGCGGAAGGCTATCGGCGAGGCCAAGGACGATGATACCGCCGA TATCCTGACAGCCGCCAGCAGAGATCTGGACAAGTTCCTGTGGTTCATCGAGAGCAACA TCGAG

[0552] SEQ ID NO: 8 Amino acid sequence of DPS.

[0553] ATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWNMRGANFIAVHEMLDGFRTALI DHLDTMAERAVQLGGVALGTTQVINSKTPLKSYPLDIHNVQDHLKELADRYAIVANDVRKAIG EAKDDDTADILTAASRDLDKFLWFIESNIE

[0554] SEQ ID NO: 9 Nucleic acid sequence encoding gp350-PADRE-PdhC.

[0555] ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTG GTGACGAAGCAGCTCTGCTCGTGTGCCAGTACACCATCCAGAGCCTCATCCACCTGAC AGGAGAGGATCCTGGCTTCTTCAACGTGGAAATTCCAGAGTTTCCCTTCTACCCTACCT GCAATGTGTGCACAGCCGACGTGAACGTGACCATCAACTTCGACGTTGGAGGCAAGAA GCACCAGCTGGACCTGGATTTCGGACAACTGACACCTCACACCAAGGCTGTGTATCAG CCTAGAGGAGCCTTTGGTGGTTCTGAGAATGCCACCAACCTGTTTCTCCTGGAGCTGC TTGGAGCTGGCGAGCTCGCACTGACCATGAGAAGCAAGAAACTGCCCATCAATGTGAC CACAGGCGAGGAACAGCAGGTGTCCCTGGAAAGCGTGGACGTGTACTTTCAAGACGT GTTCGGCACCATGTGGTGCCACCATGCCGAGATGCAGAACCCTGTGTACCTGATCCCA GAGACAGTGCCCTACATCAAGTGGGACAACTGCAACAGCACAAACATCACAGCCGTCG TGAGGGCTCAGGGACTGGATGTGACACTGCCTCTGTCTCTGCCAACCAGTGCCCAGGA CAGCAACTTCAGCGTGAAGACCGAGATGCTGGGAAACGAGATCGACATCGAGTGCATC ATGGAAGATGGCGAGATCAGCCAGGTACTGCCTGGCGACAACAAGTTCAACATCACAT GCAGTGGCTACGAGAGCCACGTGCCATCTGGAGGCATCCTGACAAGCACAAGCCCAG TGGCCACACCGATCCCTGGCACAGGCTACGCCTACAGCCTGAGACTGACACCCAGAC CCGTGTCCAGATTCCTGGGCAACAACAGCATCCTGTACGTGTTCTACTCTGGCAACGG ACCCAAGGCCTCTGGTGGCGATTACTGTATCCAGAGCAACATTGTGTTCAGCGATGAG ATCCCTGCCAGCCAGGACATGCCAACCAATACCACCGATATCACCTACGTGGGAGACA ATGCCACCTACAGCGTGCCCATGGTCACCTCCGAGGACGCCAACAGCCCTAATGTGAC CGTGACAGCCTTCTGGGCATGGCCTAACAACACCGAGACAGACTTCAAGTGCAAGTGG ACCCTGACCTCTGGCACACCTAGTGGCTGCGAGAATATCAGCGGAGCCTTCGCCAGCA ACCGGACCTTCGACATTACCGTGTCTGGCCTTGGCACAGCTCCCAAGACCCTGATCAT CACCAGGACTGCCACCAATGCCACAACCACAACCCACAAAGTGATCTTCAGCAAGGCT CCTGAAAGCACCACAACTAGTCCTACACTGGGCTCTGCCAAGTTTGTGGCTGCCTGGA CACTGAAAGCTGCAGCTGGCAGCGCTGCTGCTAAACCTGCTACAACAGAAGGCGAGTT TCCCGAGACACGCGAGAAGATGTCTGGCATCAGAAGGGCTATCGCCAAGGCCATGGT GCACAGCAAGCACACAGCTCCTCACGTGACCCTGATGGACGAGGCTGATGTGACAAAG CTGGTGGCACACAGAAAGAAGTTCAAGGCCATTGCTGCCGAGAAAGGAATCAAGCTGA CCTTCCTGCCTTACGTGGTCAAAGCCCTGGTTTCTGCCCTGAGAGAATATCCCGTGCTG AACACCAGCATCGACGACGAGACAGAGGAAATCATCCAGAAGCACTACTACAACATCG GAATCGCAGCCGACACCGACAGAGGACTGCTGGTGCCTGTGATCAAGCATGCCGACA GAAAGCCCATCTTCGCTCTGGCTCAAGAGATCAACGAGCTGGCTGAGAAGGCCAGAGA TGGCAAGCTGACACCTGGCGAGATGAAGGGAGCCAGCTGTACCATCACCAACATCGGT TCTGCTGGTGGACAGTGGTTCACACCAGTGATCAATCACCCTGAGGTGGCCATCCTCG GCATCGGCAGAATCGCTGAGAAGCCCATCGTCAGAGACGGTGAAATTGTGGCTGCTCC TATGCTTGCCCTGAGCCTAAGCTTCGACCACAGAATGATCGACGGTGCCACAGCTCAG AAAGCCCTGAACCACATCAAGAGACTGCTGAGCGACCCTGAGCTGCTGCTGATGGAAG CTTAATGA

[0556] SEQ ID NO: 10 Amino acid sequence of gp350-PADRE-PdhC.

[0557] METDTLLLWVLLLWVPGSTGDEAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCT ADVNVTINFDVGGKKHQLDLDFGQLTPHTKAVYQPRGAFGGSENATNLFLLELLGAGELALT MRSKKLPINVTTGEEQQVSLESVDVYFQDVFGTMWCHHAEMQNPVYLIPETVPYIKWDNC NSTNITAVVRAQGLDVTLPLSLPTSAQDSNFSVKTEMLGNEIDIECIMEDGEISQVLPGDNKF NITCSGYESHVPSGGILTSTSPVATPIPGTGYAYSLRLTPRPVSRFLGNNSILYVFYSGNGPKA SGGDYCIQSNIVFSDEIPASQDMPTNTTDITYVGDNATYSVPMVTSEDANSPNVTVTAFWA WPNNTETDFKCKWTLTSGTPSGCENISGAFASNRTFDITVSGLGTAPKTLIITRTATNATTTT HKVIFSKAPESTTTSPTLGSAKFVAAWTLKAAAGSAAAKPATTEGEFPETREKMSGIRRAIAK AMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNT SIDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMK GASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDG ATAQKALNHIKRLLSDPELLLMEA

[0558] SEQ ID NO: 11 Nucleic acid sequence encoding gH-DPS.

[0559] ATGCAGCTGCTGTGCGTGTTCTGCCTGGTGCTGCTGTGGGAAGTGGGAGCTGCAAGC CTGAGCGAAGTGAAGCTGCACCTGGACATCGAGGGTCACGCCAGCCACTACACCATCC CTTGGACAGAGCTGATGGCCAAGGTGCCTGGACTGTCTCCTGAAGCTCTGTGGCGAGA AGCCAACGTGACCGAGGATCTGGCTTCCATGCTGAACCGGTACAAGCTGATCTACAAG ACCAGCGGAACCCTGGGAATCGCTCTGGCAGAACCTGTGGATATCCCTGCTGTGTCTG AGGGCAGCATGCAGGTGGACGCCAGCAAAGTGCACCCAGGAGTGATCAGCGGACTGA ACAGTCCTGCCTGTATGCTGAGCGCTCCTCTGGAAAAGCAGCTGTTCTACTACATCGGT ACCATGCTGCCTAACACCAGACCTCACAGCTACGTCTTCTACCAGCTGCGGTGCCATCT GAGCTACGTCGCTCTGAGCATCAACGGAGACAAGTTCCAGTACACCGGAGCTATGACC AGCAAGTTCCTGATGGGTACCTACAAGAGAGTGACCGAGAAGGGAGACGAACACGTGC TGAGCCTGGTGTTCGGCAAGACCAAGGACCTGCCTGACCTGAGAGGACCCTTCAGCTA CCCTTCCCTCACCAGCGCTCAGAGCGGAGACTACAGCCTCGTGATCGTGACCACATTC GTGCACTACGCCAACTTCCACAACTACTTCGTGCCCAACCTGAAGGACATGTTCAGCAG AGCCGTGACCATGACAGCTGCAAGCTACGCCAGATACGTGCTGCAGAAACTGGTCCTG CTGGAAATGAAGGGAGGATGCAGAGAGCCTGAGCTGGACACAGAGACACTGACAACC ATGTTCGAGGTGTCCGTGGCCTTCTTCAAAGTGGGACACGCTGTGGGAGAGACAGGCA ATGGCTGTGTGGACCTGAGATGGCTGGCCAAGAGTTTCTTCGAGCTGACCGTGCTGAA AGATATCATTGGCATTTGCTACGGAGCCACCGTGAAAGGCATGCAGAGCTACGGACTA GAAAGACTGGCAGCTATGCTGATGGCTACCGTGAAGATGGAAGAACTGGGACACCTCA CCACAGAGAAGCAGGAATACGCTCTGAGACTGGCCACCGTGGGCTATCCTAAAGCTGG AGTGTACTCCGGACTGATCGGTGGAGCTACAAGCGTGCTGCTGAGTGCCTACAACCGA CACCCTCTGTTCCAGCCTCTGCACACCGTGATGAGAGAGACCCTGTTCATCGGAAGCC ATGTCGTGCTGCGAGAGCTGAGACTGAATGTGACAACCCAGGGACCTAACCTGGCTCT CTATCAGCTGCTGAGCACCGCTCTGTGTTCCGCTCTCGAGATCGGAGAAGTGCTAAGA GGACTCGCACTGGGCACAGAGAGCGGACTGTTCAGCCCTTGCTACCTGTCCCTGAGAT TCGACCTGACCAGAGACAAGCTGCTGTCCATGGCACCTCAGGAAGCCACACTGGATCA GGCAGCCGTGTCCAACGCTGTGGATGGCTTTCTGGGACGACTGTCACTGGAAAGAGA GGACAGGGACGCCTGGCATCTGCCTGCCTATAAGTGCGTGGACCGACTGGACAAGGT GCTGATGATCATTCCACTGATCAACGTGACCTTCATCATCAGCTCCGACCGAGAGGTGC GAGGCAGTGCCCTGTATGAAGCCAGCACCACATACCTGAGCTCCAGCCTGTTTCTGAG CCCTGTGATCATGAACAAGTGTAGCCAGGGTGCTGTGGCTGGAGAGCCTAGACAGATC CCTAAGATCCAGAACTTCACCCGAACCCAGAAGTCCTGCATCTTCTGTGGCTTTGCTCT GCTGTCCTACGACGAGAAGGAGGGACTGGAAACCACCACCTACATCACCAGCCAGGAA GTCCAGAATAGCATCCTGTCCAGCAATTACTTCGACTTCGACAACCTGCATGTGCACTA CCTGCTCCTGACCACAAACGGCACAGTGATGGAAATCGCTGGACTGTACGAGGAACGA GCTCATGTGGTCCTGCCTAAGCCCAGCACACCTCCTGGCAGCTCCGCTACCAATCTGC TGTACACCAGAAACGACGTGTCCGACAGCGAGAAGAAAGCCACCGTGGAACTGCTGAA CAGACAAGTGATCCAGTTCATCGACCTGAGCCTGATCACCAAGCAGGCTCACTGGAAT ATGAGAGGAGCAAACTTTATCGCTGTGCACGAAATGCTGGACGGCTTCAGAACAGCCC TGATCGACCACCTGGATACCATGGCAGAAAGAGCCGTTCAGCTTGGAGGCGTGGCTCT GGGCACAACCCAAGTGATCAACAGCAAGACACCTCTGAAGTCTTACCCTCTGGACATC CACAACGTGCAGGACCACCTGAAAGAGCTCGCTGACAGATACGCCATCGTGGCCAATG ATGTGCGGAAGGCCATTGGCGAGGCCAAGGATGATGACACTGCCGATATTCTGACCGC TGCCAGCAGAGATCTGGACAAGTTCCTGTGGTTCATCGAGTCCAATATCGAGTGATAA

[0560] SEQ ID NO: 12 Amino acid sequence of gH-DPS.

[0561] MQLLCVFCLVLLWEVGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVT EDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAP LEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTE KGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDM FSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGN GCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQ EYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLN VTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQE ATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVR GSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDE KEGLETTTYITSQEVQNSILSSNYFDFDNLHVHYLLLTTNGTVMEIAGLYEERAHVVLPKPST PPGSSATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWNMRGANFIAVHEMLD GFRTALIDHLDTMAERAVQLGGVALGTTQVINSKTPLKSYPLDIHNVQDHLKELADRYAIVAN DVRKAIGEAKDDDTADILTAASRDLDKFLWFIESNIE

[0562] SEQ ID NO: 13 Nucleic acid sequence encoding gL.

[0563] ATGAGAGCCGTGGGAGTGTTCCTGGCCATCTGCCTCGTGACCATCTTCGTGCTGCCCA CCTGGGGTAACTGGGCTTACCCTTGTTGCCATGTGACCCAGCTGCGAGCCCAGCATCT GCTCGCTCTGGAGAACATCAGCGACATCTACCTGGTGTCCAACCAGACCTGCGACGGC TTTAGCCTCGCATCCCTGAATAGTCCCAAGAACGGCAGCAATCAGCTCGTCATCTCCAG ATGTGCCAACGGACTCAATGTGGTGTCCTTCTTCATCTCCATCCTGAAGCGGAGCAGTA GCGCTCTGACAGGCCACCTGAGAGAGCTGCTGACCACCCTGGAAACCCTGTACGGCA GCTTCAGCGTAGAAGATCTGTTCGGAGCCAATCTGAACAGATACGCATGGCATAGAGG AGGCTAGTAA

[0564] SEQ ID NO: 14 Amino acid sequence of gL.

[0565] MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRAQHLLALENISDIYLVSNQTCDGFSLAS LNSPKNGSNQLVISRCANGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGAN LNRYAWHRGG SEQ ID NO: 15 Nucleic acid sequence encoding BZLF1-BRLF1 fusion.

[0566] ATGAGCCTGGTGTCCGACTACTGCAACGTGCTGAACAAAGAGTTCACAGCTGGCAGCG TGGAAATCACTCTGCGGAGCTACAAGATCTGCAAGGCCTTCATCAACGAGGCCAAGGC TCATGGCAGAGAATGGGGTGGACTGATGGCCACCCTGAACATCTGCAATTTCTGGGCT ATCCTGCGGAACAACAGAGTGAGACGGAGAGCCGAGAACGCTGGCAATGATGCCTGC TCTATCGCCTGTCCTATCGTGATGAGATACGTGCTGGACCACCTGATCGTCGTGACCG ACCGGTTCTTCATCCAAGCTCCCAGCAATAGAGTGATGATTCCTGCCACCATCGGCACA GCCATGTACAAGCTGCTGAAGCACAGTAGAGTGAGAGCCTACACCTACAGCAAGGTGC TGGGAGTGGACAGAGCAGCCATCATGGCTAGTGGCAAACAGGTGGTGGAACACCTGA ACCGGATGGAGAAAGAGGGACTGCTGAGCAGCAAGTTCAAGGCCTTCTGCAAGTGGG TGTTCACCTACCCTGTGCTGGAAGAGATGTTCCAGACCATGGTGTCCAGCAAGACAGG ACACCTGACCGACGACGTGAAAGATGTGAGAGCTCTGATCAAGACACTGCCCAGAGCC AGCTACAGCTCTCACGCAGGTCAGAGAAGCTACGTGTCAGGCGTGCTGCCTGCATGTC TGCTGTCCACCAAGAGCAAGGCTGTGGAAACACCCATCCTGGTGTCTGGAGCCGACAG AATGGACGAAGAACTGATGGGCAACGACGGTGGAGCCAGCCATACAGAGGCCAGATA CTCTGAGTCTGGCCAGTTCCACGCCTTCACCGACGAGCTGGAAAGCCTGCCTAGCCCT ACCATGCCTCTGAAACCTGGAGCCCAGTCTGCCGACTGTGGCGATAGCTCCTCTTCAA GCAGTGACAGTGGCAACAGCGATACCGAGCAGAGCGAGAGAGAAGAGGCTAGAGCCG AAGCTCCTAGACTGAGAGCACCCAAGAGCAGAAGAACCAGCAGACCCAACAGAGGACA GACACCCTGTCCTTCTAACGCTGCAGAGCCTGAGCAGCCTTGGATTGCTGCCGTGCAC CAGGAAAGCGACGAGAGACCTATCTTCCCACATCCCAGCAAGCCAACCTTCCTGATGT TCGATCCTGCTCCTGAGGCAGGCTCTGCCATCTCCGATGTGTTCGAGGGACGGGAAGT GTGCCAGCCCAAGCGGATCAGACCCTTCCATCCTCCTGGAAGCCCTTGGGCTAACAGA CCTCTGCCAGCCTCTCTTGCTCCAACACCTACAGGACCTGTGCACGAGCCTGTGGGCA GCCTGACACCAGCTCCAGTGCCTCAGCCTCTGGATCCAGCTCCTGCCGTGACACCTGA GGCCAGCCATCTGCTGGAAGATCCCGACGAAGAGACAAGCCAGGCAGTGAAGGCCCT GAGAGAGATGGCTGATACAGTGATCCCACAGAAAGAAGAGGCAGCCATTTGTGGCCAG ATGGACCTGTCTCACCCTCCACCTAGAGGCCACCTGGATGAGCTGACCACAACCCTGG AATCCATGACCGAGGACCTGAACCTGGACAGCCCTCTGACTCCCGAGCTGAACGAGAT CCTGGACACCTTTCTGAACGACGAGTGCCTGCTGCACGCCATGCACATCAGCACCGGA GACAGCATCTTCGACACCAGCCTGTTCATGATGGACCCTAACAGCACCAGCGAGGACG TGAAGTTCACTCCCGACCCTTACCAGGTGCCCTTCGTGCAGGCCTTCGATCAGGCCAC CGAGAATGCCTGCAGAAGTGCCTACAAGCAGGACGACCAGCACTACAGAGAGCCTGA GCCTCTGCCTCAGGGACAGCTGACAGCCTACCACGTGTCACAGCCTGCACCCGAGAA CGCCTACCAGGCCTATGCTGCACCTCAGCTGTTTCCCGTGTCCGACATCACCCAGAAC CAACAGACCAACCAGGCTGGAGGCGAAGCTCCTCAGCCTGGCGATAATAGCACCGTG CAGACAGCTGCAGCTGTGGTGTTTGCTTGCCCTGGAGCTAATCAGGGTCAGCAGCTGG CAGATATTGGCGTGCCACAGCCAGCACCTGTGGCTGCTCCTGCCAGAAGGACCAGAAA GCCTCAGCAACCCGAGAGCCTGGAAGAGTGCGACAGCGAACTGGAAATCAAGCGGAG CGAGAACGACAGACTGGAACTGCTGCTGAAACAGATGTGTCCCAGCCTGGACGTGGAC TCCATCATCCCTAGAACACCCGACTGATAA

[0567] SEQ ID NO: 16 Amino acid sequence of BZLF1-BRLF1 fusion.

[0568] MSLVSDYCNVLNKEFTAGSVEITLRSYKICKAFINEAKAHGREWGGLMATLNICNFWAILRNN RVRRRAENAGNDACSIACPIVMRYVLDHLIVVTDRFFIQAPSNRVMIPATIGTAMYKLLKHSR VRAYTYSKVLGVDRAAIMASGKQVVEHLNRMEKEGLLSSKFKAFCKWVFTYPVLEEMFQT MVSSKTGHLTDDVKDVRALIKTLPRASYSSHAGQRSYVSGVLPACLLSTKSKAVETPILVSG ADRMDEELMGNDGGASHTEARYSESGQFHAFTDELESLPSPTMPLKPGAQSADCGDSSS SSSDSGNSDTEQSEREEARAEAPRLRAPKSRRTSRPNRGQTPCPSNAAEPEQPWIAAVH QESDERPIFPHPSKPTFLMFDPAPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWANRPLP ASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKALREMADTV IPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILDTFLNDECLLH AMHISTGDSIFDTSLFMMDPNSTSEDVKFTPDPYQVPFVQAFDQATENACRSAYKQDDQHY REPEPLPQGQLTAYHVSQPAPENAYQAYAAPQLFPVSDITQNQQTNQAGGEAPQPGDNST VQTAAAVVFACPGANQGQQLADIGVPQPAPVAAPARRTRKPQQPESLEECDSELEIKRSEN DRLELLLKQMCPSLDVDSIIPRTPD

[0569] SEQ ID NO: 17 Nucleic acid sequence encoding EBNA3A.

[0570] ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTG GTGACGACAAGGACAGACCTGGACCACCTGCCCTGGACGACAACATGGAAGAGGAAG TTCCCAGCACCAGCGTGGTGCAGGAACAGGTGTCAGCTGGCGACTGGGAGAACGTGC TGATCGAGCTGAGCGACAGCAGCAGCGAGAAAGAGGCTGAGGACGCACATCTGGAAC CTGCTCAGAAAGGCACCGTGGACCATGATGCTGGAGGCTCTGCTCCAGCCAGACCTAT GCTGCCTCCTCAGCCTGATCTGCCTGGCAGAGAGGCCATCCTGAGAAGATTCCCACTG GACCTGCGGACCCTGCTGCAGGCTATTGGAGCAGCTGCCACACGGATCGACACCAGA GCCATCGACCAGTTCTTCGGCAGCCAGATCAGCAACACCGAGATGTACATTATGTACG CCATGGCCATCAGACAGGCCATTAGGGATCAGGCCAAATGGAGGCTGCAGACACTGG CTGCAGGCTGGCCGATGGGTTACCAGGCCTACAGCAGCTGGATGTACAGCTACACCG ACCACCAGACCACACCCACCTTCGTGCATCTGCAAGCAGCAGCTGGAGCTACTGGAGG GAGAAGATGCCACGTGACATTCAGTGCTGGCACCTTCAAGCTGCCCAGATGCACACCT GGAGACAGACAGTGGCTGTACGTGCAGTCTAGCGTGGGCAACATCGTGCAGAGCTGC AACCCTCGGTACAGCATCTTCTTCGACTACATGGCCATTCACCGGTCTCTGACCAAGAT CTGGGAAGAGGTGCTGACACCAGACCAGAGAGTGTCCTTTATGGAATTCCTGGGCTTC CTGCAGCGGACCGACCTGAGCTACATCAAGAGCTTCGTGTCCGACGCTCTGGGCACCA CCAGCATCCAGACTCCCTGGATCGACGACAACCCTAGCACAGAAACAGCTCAGGCTTG GAACGCAGGCTTCCTGAGAGGCAGAGCCTACGGCATCGACCTGCTGAGAACAGAGGG AGAGCATGTGGAAGGAGCTACCGGTGAAACCAGAGAGGAAAGCGAGGACACCGAGAG CGACGGAGACGACAGACTGCTGCTGATGACCGAGCAAGGCAAAGAAGTGCTGGAGAA GGCCAGAGGCTCCACCTACGGCACACCTAGACCTCCTGTGCCCAAGCCTAGACCTGA GGTGCCACAGAGCGACGAGACAGCCACATCTCACGGCTCTGCCCAGGTGCCTGAGCC ACCTACAATTCATCTGGCAGCTCAGGGCATGGCCTACCCACTGCATGAACAGCACGGC ATGGCTCCTTGTCCTGTGGCTCAGGCACCTCCTACACCTCTGCCTCCTGTGTCTCCTG GCGATCAGCTGCCTGGCGTGTTCAGCGACGGAAGAGTGGCCTGTGCTCCTGTTCCTG CACCTGCAGGACCAATTGTGAGACCTTGGGAGCCTAGCCTGACACAGGCTGCAGGACA GGCCTTTGCTCCGGTGAGACCTCAGCACATGCCTGTGGAACCTGTGCCAGTGCCTACC GTTGCCCTGGAAAGACCCGTGTACCCTAAGCCTGTGAGGCCAGCTCCACCCAAGATTG CCATGCAGGGACCTGGTGAGACAAGTGGCATTTGGAGGCCTGCTCCTTGGACACCCAA TCCACCTAGAAGCCCTTCGCAGATGAGCGTGCTGAGAGCCGAGGCACAAGTGAAGCA GGCCAGCGTGGAAGTGCAGCCACCTCAGCTGACTCAGGTGTCACCTCAGCAGCCCAT GGAGGGACCTCTGGTACCTGAGCAGCAGATGTTTCCTGGTGCTCCTTTCAGCCAGGTG GCTGATGTCGTGAGGGCTCCTGGCGTGCCAGCTATGCAGCCACAGTACTTCGACCTGC CTCTGATCCAGCCCATCAGCCAGGGAGCACCAGTGGCTCCTCTGAGAGCCTCTATGGG ACCTGTGCCTCCAGTGCCAGCAACCCAGCCTCAGTATTTCGATATCCCTCTGACCGAG CCTATCAATCAGGGAGCCTCTGCAGCACACTTCCTGCCACAGCAGCCTATGGAGGGAC CACTGGTGCCTGAACAATGGATGTTCCCAGGAGCTGCTCTGAGCCAGTCTGTGAGACC AGGCGTGGCACAGAGCCAGTACTTTGATCTGCCTCTGACACAGCCAATCAACCACGGA GCACCTGCTGCTCACTTTCTGCACCAACCTCCAATGGAAGGTCCTTGGGTACCAGAGC AGTGGATGTTTCAGGGAGCTCCTCCTAGCCAGGGCACCGATGTGGTGCAGCATCAGCT GGACGCTCTGGGCTACACACTGCACGGACTGAATCATCCAGGTGTGCCAGTGTCTCCA GCCGTTAATCAGTACCACCTGAGCCAGGCTGCCTTCGGCCTGCCCATTGATGAGGATG AGAGCGGAGAGGGCAGCGACACATCTGAGCCTTGCGAGATCCACGGCAGACCCTGTC CTCAGGCACCAGAATGGCCAGTTCAGGAAGAAGGAGGCCAGGACGCCACCGAGATTC ACGGAAGGCCTAGACCCAGAACTCCTGAGTGGCCAGTGCAGGGAGAGGGTGGACAGA ATGTGGCTGGTCCTGAGACTAGACGGGTGGTGGTGTCTGCTGTGGTGCACATGTGTCA GGACGACGAGTTCCCTGACCTGCAGGATCCTCCTGATGAGGCCGGAGGGGGTGGCTC TGGTGGGGGAGGGTCCGGCGGAGGCGGTTCAGCTGTGGGCCAGGACACGCAGGAGG TCATCGTGGTGCCACACTCCTTGCCCTTTAAGGTGGTGGTGATCTCAGCCATCCTGGC CCTGGTGGTGCTCACCATCATCTCCCTTATCATCCTCATCATGCTTTGGCAGAAGAAGC CACGTTGATAA

[0571] SEQ ID NO: 18 Amino acid sequence of EBNA3A.

[0572] METDTLLLWVLLLWVPGSTGDDKDRPGPPALDDNMEEEVPSTSVVQEQVSAGDWENVLIE LSDSSSEKEAEDAHLEPAQKGTVDHDAGGSAPARPMLPPQPDLPGREAILRRFPLDLRTLL QAIGAAATRIDTRAIDQFFGSQISNTEMYIMYAMAIRQAIRDQAKWRLQTLAAGWPMGYQAY SSWMYSYTDHQTTPTFVHLQAAAGATGGRRCHVTFSAGTFKLPRCTPGDRQWLYVQSSV GNIVQSCNPRYSIFFDYMAIHRSLTKIWEEVLTPDQRVSFMEFLGFLQRTDLSYIKSFVSDAL GTTSIQTPWIDDNPSTETAQAWNAGFLRGRAYGIDLLRTEGEHVEGATGETREESEDTESD GDDRLLLMTEQGKEVLEKARGSTYGTPRPPVPKPRPEVPQSDETATSHGSAQVPEPPTIHL AAQGMAYPLHEQHGMAPCPVAQAPPTPLPPVSPGDQLPGVFSDGRVACAPVPAPAGPIV RPWEPSLTQAAGQAFAPVRPQHMPVEPVPVPTVALERPVYPKPVRPAPPKIAMQGPGETS GIWRPAPWTPNPPRSPSQMSVLRAEAQVKQASVEVQPPQLTQVSPQQPMEGPLVPEQQ MFPGAPFSQVADVVRAPGVPAMQPQYFDLPLIQPISQGAPVAPLRASMGPVPPVPATQPQ YFDIPLTEPINQGASAAHFLPQQPMEGPLVPEQWMFPGAALSQSVRPGVAQSQYFDLPLT QPINHGAPAAHFLHQPPMEGPWVPEQWMFQGAPPSQGTDVVQHQLDALGYTLHGLNHP GVPVSPAVNQYHLSQAAFGLPIDEDESGEGSDTSEPCEIHGRPCPQAPEWPVQEEGGQD ATEIHGRPRPRTPEWPVQGEGGQNVAGPETRRVVVSAVVHMCQDDEFPDLQDPPDEAG GGGSGGGGSGGGGSAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0573] SEQ ID NO: 19 Nucleic acid sequence of Pr13.5-long promoter.

[0574] TAAAAATAGAAACTATAATCATATAATAGTGTAGGTTGGTAGTATTGCTCTTGTGACTAG AGACTTTAGTTAAGGTACTGTAAAAATAGAAACTATAATCATATAATAGTGTAGGTTGGT AGTA

[0575] SEQ ID NO: 20 Nucleic acid sequence of Pr1328 promoter.

[0576] TATATTATTAAGTGTGGTGTTTGGTCGATGTAAAATTTTTGTCGATAAAAATTAAAAAATA ACTTAATTTATTATTGATCTCGTGTGTACAACCGAAATC

[0577] SEQ ID NO: 21 Nucleic acid sequence of PrS promoter.

[0578] AAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAA

[0579] SEQ ID NO: 22 Nucleic acid sequence of PrH5m promoter.

[0580] TAAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAAT AATCATAAATAATTTCATTATCGCGATATCCGTTAAGTTTGTATCGTA

[0581] SEQ ID NO: 23 Nucleic acid sequence of PADRE.

[0582] GCCAAGTTTGTGGCTGCCTGGACACTGAAAGCTGCAGCT

[0583] SEQ ID NO: 24 Amino acid sequence of PADRE.

[0584] AKFVAAWTLKAAA

[0585] SEQ ID NO: 25 Nucleic acid sequence encoding IgK LC signal peptide.

[0586] ATGGATGCCATGAAGCGTGGACTGTGCTGTGTGCTGCTCCTGTGCGGAGCTGTGTTCG TTAGTGCCTCT SEQ ID NO: 26 Amino acid sequence of IgK LC signal peptide.

[0587] METDTLLLWVLLLWVPGSTGD

[0588] SEQ ID NO: 27 Nucleic acid sequence of I3L (MVA064L).

[0589] ATGAGTAAGGTAATCAAGAAGAGAGTTGAAACTTCACCAAGACCTACTGCATCTAGCGAT TCTCTACAGACTTGTGCGGGTGTTATAGAGTATGCAAAATCGATTAGTAAATCTAATGCAA AATGTATCGAATACGTTACACTAAATGCTTCTCAATACGCTAATTGTTCGTCTATCTCTATAA AACTTACTGATAGTTTATCTAGTCAAATGACTTCCACTTTTATTATGTTGGAAGGAGAGACT AAACTTTATAAAAATAAATCTAAACAAGATAGAAGCGATGGATACTTTCTAAAAATAAAAGT TACCGCGGCTAGTCCTATGTTGTATCAACTTCTAGAAGCCGTCTATGGAAACATTAAGCA CAAGGAACGCATTCCAAATTCTTTGCATAGTCTTTCGGTGGAAACTATTACAGAGAAAAC ATTTAAGGATGAATCCATCTTCATCAACAAATTAAACGGATCCATGGTAGAATATGTTTCGA CTGGAGAATCATCCATTCTCAGATCTATAGAAGGTGAACTAGAATCACTCAGTAAAAGAG AAAGACAATTGGCCAAGGCAATTATCACACCTATCGTCTTCTATAGATCCGGAACGGAAA CAAAAATTACATTCGCACTCAAGAAACTAATCATTGATAGAGAAGTGGTGGCTAACGTTAT CGGACTCTCTGGAGATAGTGAACGTGTATCAATGACTGAAAATGTAGAAGAAGATCTGGC TCGTAATCTGGGACTTGTTGATATTGATGATGAATATGATGAAGATAGCGATAAAGAAAAG CCAATATTCAATGTATAA

[0590] SEQ ID NO: 28 Nucleic acid sequence of mutated (defective) I3L (MVA064L). TTAAACGGATCCATGGTAGAATATGTTTCGACTGGAGAATCATCCATTCTCAGATCTATA GAAGGTGAACTAGAATCACTCAGTAAAAGAGAAAGACAATTGGCCAAGGCAATTATCAC ACCTATCGTCTTCTATAGATCCGGAACGGAAACAAAAATTACATTCGCACTCAAGAAACT AATCATTGATAGAGAAGTGGTGGCTAACGTTATCGGACTCTCTGGAGATAGTGAACGTG TATCAATGACTGAAAATGTAGAAGAAGATCTGGCTCGTAATCTGGGACTTGTTGATATTG ATGATGAATATGATGAAGATAGCGATAAAGAAAAGCCAATATTCAATGTATAA

[0591] SEQ ID NO: 29 Nucleic acid sequence of J5L (MVA089L).

[0592] ATGACGGACGAACAAATTTATGCATTCTGTGATGCTAACAAAGACGATATACGATGTAAAT GTATTTATCCTGATAAAAGCATAGTACGGATAGGAATAGATACAAGATTACCCTATTATTGTT GGTACGAGCCATGTAAACGAAGCGATGCGTTGTTACCAGCCTCTTTAAAAAAAAATATAA CAAAATGCAATGTATCGGATTGTACCATTTCATTGGGAAACGTTTCCATTACAGATAGTAA ATTAGATGTAAATAATGTTTGTGATTCCAAACGAGTAGCTACCGAGAATATAGCTGTCCGC TATCTGAATCAGGAAATTAGATACCCTATTATAGATATCAAATGGCTTCCGATTGGATTACTA GCGTTAGCTATTTTAATATTAGCATTTTTCTAA

[0593] SEQ ID NO: 30 Nucleic acid sequence of E4L (MVA051 L).

[0594] ATGGAAAATGTATACATTAGTAGTTACTCATCCAATGAACAAACATCAATGGCGGTAGCCG CTACTGATATCCGAGAATTACTATCACAATATGTGGATGATGCCAACTTGGAAGACTTAATA GAATGGGCCATGGAAAAATCATCAAAGTACTACATCAAGAATATAGGTAATACAAAATCTA ATATCGAAGAAACTAAATTCGAATCAAAGAATAATATTGGTATAGAATACTCAAAGGATTCC AGAAACAAACTATCGTATAGAAATAAACCGTCTATTGCCACAAATTTGGAATATAAAACACT ATGTGATATGATTAAGGGTACTAGCGGCACCGAAAAAGAATTCCTTCGCTATCTCTTATTC GGTATAAAATGCATTAAGAAAGGAGTAGAATACAATATAGATAAAATAAAGGATGTGAGTTA CAACGATTATTTTAACGTTCTCGACGAGAAATACAATACACCGTGTCCTAACTGTAAAAGT AGGAATACTACGCCGATGATGATTCAAACTAGAGCCGCTGACGAACCTCCACTAGTTAGA CATGCGTGTAGAGATTGCAAACAACACTTTAAGCCTCCCAAATTTAGAGCATTTCGCAAT CTTAATGTTACAACGCAATCGATACATGAAAACAAGGAAATAACAGAGATTCTTCCAGATA ATAATCCATCTCCTCCAGAATCTCCAGAACCAGCATCACCTATAGATGACGGGTTAATCAG ATCCACATTCGATAGAAACGACGAACCACCAGAGGATGATGAATAA SEQ ID NO: 31 Nucleic acid sequence of GCN4 multimerization domain.

[0595] ATGAAAGTGAAGCAGCTGGTGGACAAGGTGGAAGAACTGCTGAGCAAGAACTACCACC TCGTGAATGAGGTGGCACGGCTCGTGAAGCTCGTGGGAGAAAGAGGTGGC

[0596] SEQ ID NO: 32 Amino acid sequence of GCN4 multimerization domain.

[0597] MKVKQLVDKVEELLSKNYHLVNEVARLVKLVGERGG

[0598] SEQ ID NO: 33 Nucleic acid sequence encoding gp350-PADRE-DPS.

[0599] ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTGTGGGTGCCAGGATCTACAG GGGATGAAGCCGCTCTGCTCGTGTGCCAGTACACCATCCAGAGCCTGATCCACCTGAC CGGCGAGGATCCCGGCTTCTTCAACGTGGAAATCCCTGAGTTCCCTTTCTACCCTACCT GCAACGTGTGCACCGCCGACGTGAACGTGACCATCAACTTTGATGTCGGCGGCAAGAA GCACCAGCTGGACCTGGATTTTGGCCAGCTGACCCCTCACACCAAGGCCGTGTATCAA CCTAGAGGCGCCTTTGGCGGCAGCGAGAACGCCACCAATCTGTTTCTGCTGGAACTGC TCGGAGCCGGCGAACTGGCTCTGACCATGAGAAGCAAGAAACTGCCCATCAATGTGAC CACAGGCGAGGAACAGCAGGTTTCCCTGGAAAGCGTGGACGTGTACTTCCAAGACGTG TTCGGCACCATGTGGTGCCACCACGCCGAGATGCAGAACCCCGTGTATCTGATCCCCG AGACAGTGCCCTACATCAAGTGGGACAACTGCAACAGCACCAACATCACCGCCGTCGT CAGAGCCCAAGGCCTGGATGTTACACTGCCTCTGAGCCTGCCTACAAGCGCCCAGGAC AGCAACTTCAGCGTGAAAACCGAGATGCTGGGCAACGAGATCGACATCGAGTGCATCAT GGAAGATGGCGAGATCAGCCAGGTGCTGCCCGGCGACAACAAGTTCAACATCACATGC AGCGGCTACGAGAGCCACGTGCCATCTGGCGGAATCCTGACAAGCACAAGCCCAGTG GCCACACCTATTCCAGGCACCGGCTACGCCTATAGCCTGAGACTGACACCCAGACCTGT GTCCAGATTCCTGGGCAACAACAGCATCCTGTACGTGTTCTACAGCGGCAACGGCCCTA AAGCCTCTGGCGGCGATTACTGCATCCAGTCCAACATCGTGTTCAGCGACGAGATCCCC GCCAGCCAGGACATGCCTACCAACACCACCGATATCACCTACGTGGGCGACAATGCCA CCTACAGCGTGCCAATGGTCACCAGCGAGGATGCCAACTCTCCCAACGTGACCGTGAC AGCCTTTTGGGCCTGGCCTAACAACACCGAGACAGACTTCAAGTGCAAGTGGACCCTG ACCAGCGGCACACCTTCTGGCTGCGAGAATATCAGCGGAGCCTTCGCCAGCAACCGGA CCTTTGATATCACCGTGTCTGGCCTGGGCACAGCCCCTAAGACACTGATCATCACCAGG ACCGCCACCAACGCCACAACCACCACACACAAAGTGATCTTCAGCAAGGCCCCTGAGA GCACCACCACCTCTCCAACACTGGGCAGCGCCAAATTTGTGGCCGCCTGGACACTGAA AGCCGCCGCTGGATCTGCCACAAATCTGCTGTACACCCGGAACGACGTGTCCGACAGC GAGAAGAAAGCCACCGTCGAGCTGCTGAACCGGCAAGTGATCCAGTTCATCGATCTGT CCCTGATCACCAAGCAGGCCCACTGGAATATGAGAGGCGCCAACTTTATCGCCGTGCAC GAAATGCTGGACGGCTTCAGAACAGCCCTGATCGACCACCTGGATACCATGGCCGAAA GAGCCGTTCAGCTTGGCGGAGTGGCTCTGGGAACAACCCAAGTGATCAACAGCAAGAC ACCCCTGAAGTCTTACCCTCTGGACATCCACAACGTGCAGGACCACCTGAAAGAGCTG GCCGACAGATACGCCATCGTGGCCAATGATGTGCGGAAGGCCATTGGCGAGGCCAAGG ATGATGACACAGCCGACATTCTGACCGCCGCCAGCAGAGATCTGGACAAGTTCCTGTG GTTCATCGAGTCCAATATCGAG

[0600] SEQ ID NO: 34 Amino acid sequence of gp350-PADRE-DPS.

[0601] METDTLLLWVLLLWVPGSTGDEAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCT ADVNVTINFDVGGKKHQLDLDFGQLTPHTKAVYQPRGAFGGSENATNLFLLELLGAGELALT MRSKKLPINVTTGEEQQVSLESVDVYFQDVFGTMWCHHAEMQNPVYLIPETVPYIKWDNC NSTNITAVVRAQGLDVTLPLSLPTSAQDSNFSVKTEMLGNEIDIECIMEDGEISQVLPGDNKF NITCSGYESHVPSGGILTSTSPVATPIPGTGYAYSLRLTPRPVSRFLGNNSILYVFYSGNGPKA SGGDYCIQSNIVFSDEIPASQDMPTNTTDITYVGDNATYSVPMVTSEDANSPNVTVTAFWA WPNNTETDFKCKWTLTSGTPSGCENISGAFASNRTFDITVSGLGTAPKTLIITRTATNATTTT HKVIFSKAPESTTTSPTLGSAKFVAAWTLKAAAGSATNLLYTRNDVSDSEKKATVELLNRQVI QFIDLSLITKQAHWNMRGANFIAVHEMLDGFRTALIDHLDTMAERAVQLGGVALGTTQVINSK TPLKSYPLDIHNVQDHLKELADRYAIVANDVRKAIGEAKDDDTADILTAASRDLDKFLWFIESNI E

[0602] SEQ ID NO: 35 Nucleic acid sequence encoding SARS-CoV 2 spike RBD.

[0603] GTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAATCTGTGTCCTTTTGG CGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATC AGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTTCTCCACCTTCAA GTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCTG ACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGGACAGACAGGCAA GATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCTGTGTGATTGCCTGGA ACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTGTACAGGCTGTTT CGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGATCTATCAGGCAG GCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGTCCTAC GGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAGAGTGGTGGTGCTGAGCT TCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCACCAACCTGGT GAAGAACAAA

[0604] SEQ ID NO: 36 Amino acid sequence of SARS-CoV 2 spike RBD.

[0605] VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYG VSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDS KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG YQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

[0606] SEQ ID NO: 37 Nucleic acid sequence encoding Bullfrog / / - / . pylori hybrid ferritin. GAATCTCAAGTTCGGCAGCAGTTCAGCAAGGACATCGAGAAGCTGCTGAACGAGCAAG TGAACAAAGAGATGCAGAGCAGCAACCTGTACATGAGCATGAGCAGCTGGTGCTACACC CACAGCCTTGATGGCGCCGGACTGTTCCTGTTTGATCACGCCGCCGAGGAATACGAGC ACGCCAAGAAGCTGATCATCTTCCTGAACGAGAACAACGTGCCCGTGCAGCTGACCAG CATTTCTGCCCCTGAGCACAAGTTCGAGGGCCTGACACAGATCTTCCAGAAGGCCTAC GAACACGAGCAGCACATTAGCGAGAGCATCAACAACATCGTGGACCACGCCATTAAGAG CAAGGATCACGCCACCTTCAACTTTCTGCAGTGGTACGTGGCCGAACAGCACGAGGAA GAGGTGCTGTTCAAGGACATCCTGGACAAGATCGAGCTGATCGGCAACGAGAACCACG GCCTGTATCTGGCCGACCAGTACGTGAAGGGAATCGCCAAGAGCAGAAAGAGC

[0607] SEQ ID NO: 38 Amino acid sequence of Bullfrog / / - / , pylori hybrid ferritin.

[0608] ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHA KKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFL QWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

[0609] SEQ ID NO: 39 Nucleic acid sequence of SARS-CoV-2 spike RBD-BFF-PADRE. ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCG TCTCGGCTAGCGTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTAACATCACCAATCTG TGTCCTTTTGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACC GGAAGCGGATCAGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTT CTCCACCTTCAAGTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCA ACGTGTACGCTGACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGG ACAGACAGGCAAGATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCTGTG TGATTGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTG TACAGGCTGTTTCGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGAT CTATCAGGCAGGCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCAC TGCAGTCCTACGGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAGAGTGGTG GTGCTGAGCTTCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCA CCAACCTGGTGAAGAACAAGAGCAGCGGCGGAGCCTCTGTGCTGGCCGAATCTCAAGT TCGGCAGCAGTTCAGCAAGGACATCGAGAAGCTGCTGAACGAGCAAGTGAACAAAGAG ATGCAGAGCAGCAACCTGTACATGAGCATGAGCAGCTGGTGCTACACCCACAGCCTTGA TGGCGCCGGACTGTTCCTGTTTGATCACGCCGCCGAGGAATACGAGCACGCCAAGAAG CTGATCATCTTCCTGAACGAGAACAACGTGCCCGTGCAGCTGACCAGCATTTCTGCCCC TGAGCACAAGTTCGAGGGCCTGACACAGATCTTCCAGAAGGCCTACGAACACGAGCAG CACATTAGCGAGAGCATCAACAACATCGTGGACCACGCCATTAAGAGCAAGGATCACGC CACCTTCAACTTTCTGCAGTGGTACGTGGCCGAACAGCACGAGGAAGAGGTGCTGTTC AAGGACATCCTGGACAAGATCGAGCTGATCGGCAACGAGAACCACGGCCTGTATCTGG CCGACCAGTACGTGAAGGGAATCGCCAAGAGCAGAAAGAGCCACCGGCTGAAGGCCA AGTTTGTGGCCGCCTGGACACTGAAAGCTGCCGCT

[0610] SEQ ID NO: 40 Amino acid sequence of SARS-CoV-2 spike RBD-BFF-PADRE. MDAMKRGLCCVLLLCGAVFVSASVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCN GVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSSGG ASVLAESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAE EYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDH ATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSHRLKAKFVAA WTLKAAA

[0611] SEQ ID NO: 41 Nucleic acid sequence of htPA signal peptide.

[0612] ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCG TCTCGGCTAGC

[0613] SEQ ID NO: 42 Amino acid sequence of htPA signal peptide.

[0614] MDAMKRGLCCVLLLCGAVFVSAS

Claims

PCT ApplicationBavarian Nordic A / SBN126PCTClaims1. A nucleic acid encoding a fusion protein comprising (i) a disease-associated antigen, or an antigenic part thereof, (ii) a subunit of a self-assembling multimeric protein particle, and (iii) a pan HLA DR-binding epitope (PADRE).

2. The nucleic acid of claim 1, wherein the self-assembling multimeric 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).

3. The nucleic acid of claim 1 or 2, wherein the (ii) subunit of a self-assembling multimeric protein particle is joined to (iii) PADRE, preferably via a short amino acid linker.

4. The nucleic acid of claim 3, wherein the N-terminus of the (ii) subunit of a selfassembling multimeric protein particle is joined to the C-terminus of (iii) PADRE.

5. The nucleic acid of claim 3 or 4, wherein the (i) disease-associated antigen, or an antigenic part thereof, and the (ii) subunit of a self-assembling multimeric protein particle are joined via an amino acid linker comprising or consisting of (iii) PADRE.

6. The nucleic acid of claim 3, wherein the C-terminus of the (ii) subunit of a selfassembling multimeric protein particle is joined to the N-terminus of PADRE.

7. The nucleic acid of anyone of claims 1 to 6, wherein PADRE is encoded by a section of the nucleic acid comprising or consisting of a nucleic acid as depicted in SEQ ID NO: 23, or wherein PADRE is encoded by a section of the nucleic acid comprising or consisting of a nucleic acid encoding an amino acid sequence as depicted in SEQ ID NO: 24.

8. The nucleic acid of anyone of claims 1 to 7, wherein the antigen is a viral antigen, preferably is Epstein-Barr virus (EBV) surface glycoprotein 350 (gp350) or receptor binding domain (RBD) of SARS-CoV-2 spike protein (SARS-CoV-2 spike RBD).

9. Use of the nucleic acid of anyone of claims 1 to 8 for the preparation of a virus-based vector or a pharmaceutical composition.

10. A fusion protein encoded by the nucleic acid of anyone of claims 1 to 8.

11. A virus-based vector, comprising the nucleic acid of anyone of claim 1 to 8.

12. The virus-based vector of claim 11, which is a recombinant poxvirus, preferably a recombinant vaccinia virus, more preferably a recombinant Modified Vaccinia Virus Ankara (MVA), or which is a virus replicon particle (VRP).

13. A pharmaceutical composition comprising a nucleic acid of anyone of claims 1 to 8, a fusion protein of claim 10, or a virus-based vector of claim 11 or 12, optionally further comprising a pharmaceutically acceptable carrier or excipient.

14. A virus-based vector of claim 11 or 12, or a pharmaceutical composition of claim 13 for use in the prevention or treatment of a disease, preferably a viral infectious disease or a viral infection-associated malignancy.

15. A virus-based vector of claim 11 or 12, or a pharmaceutical composition of claim 13 for use in inducing an antibody response to the disease-associated antigen encoded by the virus-based vector.

16. A process for preparing a recombinant poxvirus of claim 12, which is a recombinant MVA, the process comprising the steps of:(a) providing an acceptor bacterial artificial chromosome (BAC) comprising an MVA genome (MVA-BAC), which MVA genome is mutated such that two essential MVA genes required for MVA replication are defective or lacking, wherein one essential MVA gene is I3L (MVA064L) and the other essential MVA gene is J5L (MVA089L);(b) providing a recombination plasmid comprising the essential MVA gene which is defective or lacking in the MVA-BAC of step (a) as a functional gene, the recombination plasmid further comprising a nucleic acid of anyone of claims 1 to 8 operably linked to a poxviral promoter;(c) co-transfecting an MVA producer cell with the MVA-BAC of step (a) and the recombination plasmid of step (b), and furthermore infecting the MVA producer cell with a helper virus derived from the family poxviridae, preferably Shope fibroma virus (SFV);(d) allowing reconstitution of MVA from the MVA-BAC and homologous recombination with the recombination plasmid in the MVA producer cell; and (e) obtaining the recombinant MVA comprising the functional essential MVA gene and the nucleic acid.

7. A process for preparing a recombinant VRP of claim 12, comprising the steps of: (a’) providing a plasmid DNA encoding a self-amplifying replicon RNA, preferably a replicon RNA under the control of a cytomegalovirus (CMV) promoter, encoding a nucleic acid of anyone of claims 1 to 8;(b’) transfecting a VRP production cell with the plasmid DNA provided in step (a’), further transfecting the VRP production cell with a first CMV promoter driven packaging plasmid encoding an alphavirus capsid protein and a second CMV promoter driven packaging plasmid encoding an alphavirus envelope protein; (c’) culturing the transfected VRP production cell of step (b’);(d’) obtaining the recombinant VRP.

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