ZIKA VIRUS VACCINE AND COMBINATION VACCINE

Abstract

The invention relates to a Zika viral vector vaccine comprising nucleic acid encoding a Zika virus structural antigen, wherein the nucleic acid encoding a Zika virus structural antigen comprises a sequence encoding Zika virus envelope DIII, or part thereof. The invention further relates to a Zika viral vector vaccine in combination with a Chikungunya viral vector vaccine.

Claims

1. A Zika viral vector vaccine comprising nucleic acid encoding a Zika virus structural antigen, wherein the nucleic acid encoding a Zika virus structural antigen comprises a sequence encoding Zika virus envelope DIII, or part thereof.

2. The Zika viral vector vaccine according to claim 1, wherein the nucleic acid encoding the Zika virus structural antigen comprises or consists of the sequence of SEQ ID NO: 7, or part(s) thereof.

3. The Zika viral vector vaccine according to claim 1, wherein the nucleic acid encoding the Zika virus structural antigen comprises or consists of the sequence of SEQ ID NO: 9, or part(s) thereof.

4. The Zika viral vector vaccine according to any preceding claim, wherein the nucleic acid encoding the Zika virus structural antigen comprises or consists of the sequence of SEQ ID NO: 1.

5. The Zika viral vector vaccine according to any preceding claim, wherein the Zika virus envelope comprises the whole Zika virus envelope DIII sequence; or the Zika virus envelope DIII comprises the whole Zika virus envelope DIII sequence of SEQ ID NO: 1.

6. The Zika viral vector vaccine according to any preceding claim, wherein the Zika virus envelope DIII or the nucleic acid encoding the Zika virus envelope DIII is a natural or modified variant thereof.

7. The Zika viral vector vaccine according to any preceding claim, wherein variants of the nucleic acid encoding the Zika virus envelope DIII comprise or consist of a sequence having at least 80% identity with SEQ ID NO: 1.

8. The Zika viral vector vaccine according to any preceding claim, wherein the sequence identity is over at least 50 consecutive nucleotides of SEQ ID NO: 1.

9. The Zika viral vector vaccine according to any preceding claim, wherein variants of Zika virus envelope DIII comprise or consist of a truncated sequence of the Zika virus envelope DIII encoding sequence of SEQ ID NO: 1.

10. The Zika viral vector vaccine according to any preceding claim, wherein the Zika viral vector vaccine does not comprise sequence encoding Zika virus TM (transmembrane) domain or part thereof.

11. The Zika viral vector vaccine according to any preceding claim, wherein the Zika viral vector vaccine does not comprise sequence encoding Zika virus prM domain or part thereof.

12. The Zika viral vector vaccine according to any preceding claim, wherein the Zika viral vector vaccine does not comprise sequence encoding a Zika virus non-structural domain or part(s) thereof.

13. The Zika viral vector vaccine according to any preceding claim, wherein the Zika viral vector vaccine comprises sequence encoding a Zika virus non-structural domain or part(s) thereof.

14. A Zika viral vector vaccine comprising nucleic acid encoding a Zika virus structural antigen, wherein the nucleic acid encoding a Zika virus structural antigen comprises a sequence encoding at least part of the Zika virus prM, and a sequence encoding at least part of the Zika virus envelope protein.

15. The Zika viral vector vaccine according to claim 14, wherein the nucleic acid encoding a Zika virus structural antigen consists essentially of a sequence encoding Zika virus envelope, or a part thereof, and prM, or part thereof.

16. The Zika viral vector vaccine according to claim 14 or 15, wherein the nucleic acid encoding the Zika virus envelope comprises the sequence of SEQ ID NO: 3 (ZENV_noTM), or a part thereof.

17. The Zika viral vector vaccine according to any of claims 14 to 16, wherein the nucleic acid encoding the Zika virus structural antigen comprises or consists of the sequence of SEQ ID NO: 7, or part(s) thereof.

18. The Zika viral vector vaccine according to any of claims 14 to 17, wherein the nucleic acid encoding the Zika virus structural antigen consists of the sequence of SEQ ID NO: 7, or part(s) thereof.

19. The Zika viral vector vaccine according to any of claims 14 to 18, wherein the Zika virus envelope comprises the whole envelope sequence.

20. The Zika viral vector vaccine according to any of claims 14 to 18, wherein the Zika virus envelope comprises at least two of the DI, DII or DII domains, or parts thereof, of the envelope sequence.

21. The Zika viral vector vaccine according to any of claims 14 to 18, wherein the Zika virus envelope may comprise at part of all DI, DII and DII domains of the envelope sequence.

22. The Zika viral vector vaccine according to any of claims 14 to 21, wherein the Zika virus envelope is a natural or modified variant thereof; or the nucleic acid encoding the Zika virus envelope may be a natural or modified variant thereof.

23. The Zika viral vector vaccine according to any of claims 14 to 22, wherein variants of the nucleic acid encoding the Zika virus envelope comprise or consist of a sequence having at least 80% identity with SEQ ID NO: 3.

24. The Zika viral vector vaccine according to claim 23, wherein the sequence identity is over at least 50 consecutive nucleotides of SEQ ID NO: 3.

25. The Zika viral vector vaccine according to any of claims 14 to 24, wherein variants of Zika virus envelope comprise or consist of a truncated sequence of the Zika virus envelope sequence of SEQ ID NO: 3.

26. The Zika viral vector vaccine according to any of claims 14 to 25, wherein the Zika virus prM comprises the whole prM sequence; or the Zika virus prM comprises a sequence encoding the whole prM sequence of SEQ ID NO: 13.

27. The Zika viral vector vaccine according to any of claims 14 to 26, wherein the Zika virus prM is a natural or modified variant thereof; or the nucleic acid encoding the Zika virus prM is a natural or modified variant thereof.

28. The Zika viral vector vaccine according to any of claims 14 to 27, wherein variants of the nucleic acid encoding the Zika virus prM comprise or consist of a sequence having at least 80% identity with SEQ ID NO: 13.

29. The Zika viral vector vaccine according to claim 28, wherein the sequence identity is over at least 50 consecutive nucleotides of SEQ ID NO: 13.

30. The Zika viral vector vaccine according to any of claims 14 to 27, wherein variants of Zika virus prM comprise or consist of a truncated sequence encoding the Zika virus prM sequence of SEQ ID NO: 13.

31. The Zika viral vector vaccine according to any of claims 14 to 30, wherein the Zika viral vector vaccine does not comprise sequence encoding Zika virus TM (transmembrane) domain or part thereof.

32. The Zika viral vector vaccine according to any of claims 14 to 31, wherein the Zika viral vector vaccine does not comprise sequence encoding a Zika virus non-structural domain or part(s) thereof.

33. The Zika viral vector vaccine according to any of claims 14 to 31, wherein the Zika viral vector vaccine comprises sequence encoding a Zika virus non-structural domain or part(s) thereof.

34. The Zika viral vector vaccine according to any preceding claim, wherein the Zika viral vector vaccine further encodes a peptide adjuvant, such as a TPA (tissue plasminogen activator) sequence, or a functional variant thereof.

35. The Zika viral vector vaccine according to any preceding claim, wherein the viral vector comprises nucleic acid encoding non-Zika viral protein, such as adenovirus protein(s) or MVA protein(s).

36. The Zika viral vector vaccine according to any preceding claim, wherein the Zika virus structural antigen is expressed as a non-secreting protein in the cell.

37. The Zika viral vector vaccine according to any preceding claim in combination with another therapeutically or prophylactically active ingredient.

38. The Zika viral vector vaccine according to claim 37, wherein the therapeutically or prophylactically active ingredient comprises a Chikungunya vaccine, optionally wherein the Chikungunya vaccine is a Chikungunya viral vector vaccine.

39. The Zika viral vector vaccine according to claim 38, wherein the Chikungunya viral vector vaccine comprises nucleic acid of SEQ ID NO: 14 or SEQ ID NO: 16; or wherein the Chikungunya viral vector vaccine comprises nucleic acid encoding polypeptides of SEQ ID NO: 15 or SEQ ID NO: 17.

40. The Zika viral vector vaccine according to any preceding claim, wherein the Zika viral vector vaccine is provided in a pharmaceutically acceptable carrier.

41. A nucleic acid encoding the Zika viral vector vaccine according to any preceding claim, or parts thereof.

42. A composition comprising the nucleic acid according to claim 40 or the viral vector according to any of claims 1 to 36.

43. The composition according to claim 42, wherein the composition further comprises another therapeutically or prophylactically active ingredient.

44. The composition according to claim 42 or 43, wherein the composition further comprises a Chikungunya vaccine, optionally wherein the Chikungunya vaccine is a Chikungunya viral vector vaccine.

45. The composition according to claim 44, wherein the Chikungunya viral vector vaccine comprises nucleic acid of SEQ ID NO: 14 or SEQ ID NO: 16; or wherein the Chikungunya viral vector vaccine comprises nucleic acid encoding polypeptides of SEQ ID NO: 15 or SEQ ID NO: 17.

46. A method of treatment or prophylaxis of Zika viral infection comprising the administration to a subject of: the nucleic acid according to claim 41; the composition according to any of claims 42 to 45; or the viral vector vaccine according to any of claims 1 to 40.

47. A method of treatment or prophylaxis of Zika and/or Chikungunya viral infection comprising the administration to a subject of: the composition according to any of claim 44 or 45; or the viral vector vaccine according to any of claims 38 to 40.

48. An agent for use in the prophylaxis or treatment of Zika viral infection in a subject, the agent comprising or consisting of: the nucleic acid according to claim 41; the composition according to any of claims 42 to 45; or the viral vector vaccine according to any of claims 1 to 40.

49. An agent for use in the prophylaxis or treatment of Zika and/or Chikungunya viral infection in a subject, the agent comprising or consisting of: the composition according to any of claim 44 or 45; or the viral vector vaccine according to any of claims 38 to 40.

50. The nucleic acid according to claim 41; the composition according to any one of claims 42 to 45; or the viral vector vaccine according to any of claims 1 to 40; for use in, or as, a vaccine.

51. A prime boost vaccination kit comprising a prime vaccination comprising: the nucleic acid according to claim 41; the composition according to any one of claims 42 to 45; or the viral vector according to any of claims 1 to 40; and optionally a boost vaccination comprising: the nucleic acid according to claim 41; the composition according to any one of claims 42 to 45; or the viral vector according to any of claims 1 to 40.

52. The kit according to claim 51, wherein the prime and boost vaccinations are different.

53. The kit according to claim 52, wherein the prime and boost vaccination comprise different viral vectors from different viral species.

54. A combination vaccine composition comprising the Zika viral vector vaccine according to any one of claims 1 to 40 and a Chikungunya vaccine.

55. The combination vaccine composition according to claim 54, wherein the Chikungunya vaccine comprises or consists of a Chikungunya viral vector vaccine.

56. The combination vaccine composition according to claim 55, wherein the Chikungunya viral vector vaccine comprises nucleic acid of SEQ ID NO: 14 or SEQ ID NO: 16; or wherein the Chikungunya viral vector vaccine comprises nucleic acid encoding polypeptides of SEQ ID NO: 15 or SEQ ID NO: 17.

Description

[0085] Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.

[0086] FIG. 1. Construction of a Zika Virus consensus sequence. (A) Available sequences from Zika Virus were gathered; sequences were curated and only full genome sequences were used for further analysis. Sequences belonging to both African and Asian lineages were identified, sorted by host species and geographical locations. Special attention was given to human isolates (African, Asian and Imported cases). Initial phylogenetic tree was produced with the sequences available as in 27/Nov/2015/ (A, left). As in April 2016 an updated phylogenetic tree was also produced, and the amount of available genetic sequences increased by 6-fold (A, right). Nucleotide and protein alignment was performed; by the 10th of December 2015, Zika Virus sequences available in the gene-bank were not annotated. Annotation was performed by sequence similarity with Dengue Virus and Yellow Fever Virus genomes/proteins. Two types of consensus sequences were built: a consensus that covered only the Asian lineage and a consensus sequence covering both Asian and African linages. We have produced for both a whole genome consensus sequence that covered structural (Capsid, prM, Envelope) and non-structural (NS1, NS2, NS3, NS4 and NS5). For the Zika virus structural genes, we focused in the prM and Envelope consensus sequence that was built from the Zika Asian Lineage as this lineage is the one that has spread across the Americas (C). Conservation between African and Asian sequences was about 92% (data analysis not shown). Consensus sequences were as close as 95-100% similarity between both lineages when compared to all available genome sequences as in April 2016 (D). Therefore, a prM and Envelope structural DNA cassette was requested to Geneart (Thermofisher) for synthesis. The Zika prM-Envelope sequence was codon-optimised and designed to allow sub-cloning to pMono and MVA plasmids, recombination with ChAdOx1 (adenovirus), as well as restriction sites used for cloning to Phlsec (protein production)

[0087] FIG. 2. ZIKV versions produced from consensus transgene. (A) A synthetic genetic consensus sequence encoding the Zika virus prM and Envelope (Env) protein was produced (geneart?) as in FIG. 1 (construct 1). Polymerase Chain Reaction (PCR)-cloning was performed using construct 1 as a template and using specific primers to produce the following DNA constructs: a prM and Env lacking the transmembrane domain (TMD) (construct 2); a full Env lacking prM (construct 3); an Env lacking prM and TMD (construct 4) and an Env-domain III only (DIII) (construct 5). (B, left and right) All DNA constructs were sub-cloned by restriction and DNA-ligation into an expression plasmid under the CMV promoter activity, denominated pMono. Restriction analysis released specific DNA band-sizes, corresponding to all constructs and empty pmono plasmids. Alternatively, all DNA pmono plasmids were confirmed by DNA sequencing. (C) As a proof of plasmid expression, Vero cells were transfected with construct 1 and construct 2, respectively. After transfection, cells were subjected to immunofluorescence (IF) against a mouse anti-Flavivirus Envelope antibody and later stained with an Alexa 488 conjugated goat anti-mouse Ig antibody. Green cells showed that those plasmids are able to induce expression of Zika Envelope. (D) prM and Env DNA was sub-cloned into a Phlsec His-tagged plasmid for protein expression in HEK293 cells; restriction analysis (left) showed specific DNA sizes for both prM and Env. HEK293 cells were transfected with Phlsec-prM and Phlsec-Env and a western blot was performed using an HRP-conjugated anti-mouse His tag antibody (right panel). * shows specific His-tag recognition in both total cell and soluble fractions.

[0088] FIG. 3. Assessment of mice immunogenicity after Zika-DNA vaccination. Groups of BalbC mice were immunised (prime) with the Zika DNA vaccines shown in FIG. 2A and 2B. Two weeks after the prime, mice were bled to isolate peripheral blood mononuclear cells (PBMCs) and sera. Same groups were subjected to a second immunisation two weeks after the prime (boost). PBMCs and sera were also recovered two weeks after boost. For T-cell responses, a pool of 20mer peptides (10aa overlap) comprising the full Env was prepared. (A) Elispot analysis showed that all DNA vaccinations elicited T-cell responses. Importantly, responses for INF-g varied within groups in both prime (grey) or boost (red) regimes. T-cell responses were modulated based on having or not the prM and/or the TMD. For example, DNA vaccine encoding the Envelope with no TMD gave the highest responses. Full version of ENV elicited 3-fold down INFg producing PBMCs in comparison with Env no TMD. Combination of prM and TMD also impacted the breath of T-cell INFg responses in all groups. (B) Intracellular Cytokine Staining (ICS) and flow cytometry analysis was performed on the Prime-Boost mice groups. Based on the analysis it can be concluded that all vaccines elicited both CD8 (top panels) and CD4 (bottom panels) T-Cells. Further analysis on those samples confirmed that the DNA vaccine carrying the Envelope with no TMD elicited the highest CD8 T-cell responses shown in the ELispot data (FIG. 2A), whereas the DNA vaccine carrying the Env DIII only elicited the highest CD4 T-Cells responses. Again, modulation of both CD8 and CD4 T-Cell response were achieved by absence or presence of TMD and/or prM. Note Env with TMD sample was lost during processing. (C) BalbC spleenocytes from mice vaccinated in a Prime-Boost DNA vaccine regime were subjected to epitope T-Cell mapping by stimulating T-Cells with every single 20-mer peptide spanning the whole Zika virus envelope. 50 peptides where used to screen and identify the most immunogenic peptides. We have identified two immunodominant peptides: peptide number 7 (YEASISDMASDSRCP) and peptide number 36 (VGRLITANPVITESTEN). Further conservation analysis (C, right) shows the degree of peptide homology between other flaviviruses such as Dengue (Alignments and figures modified from Science 22 Apr. 2016: Vol. 352, Issue 6284, pp. 467-470). (D) Enzyme Linked Immuno-Sorbent Assay (ELISA) revealed that DNA vaccines were able to produce antibodies against the Zika Envelope protein as measured by OD405 colorimetric levels. However, Prime-Boost regime (right) seemed to maintain almost the same antibody levels of that reached by a single DNA vaccination (left). DNA vaccine carrying the full Envelope protein elicited the most detectable antibodies in comparison with a DNA vaccine control. Other vaccines elicited very modest responses right above the background (dotted line representing the average of control OD405 background plus 2 times their SD).

[0089] FIG. 4. Mice immunogenicity after a single dose of the Adenoviral-vectored ChAdOx1-Zika vaccine. (A) Na?ve BalbC and C57BL6 mice strains were immunised with 10e8 IU of a ChAdOx1-Zika vaccine carrying the prM and full Envelope genes. ELispot assay was performed 2 weeks after prime, using Zika Envelope peptides. Higher INFg responses were found in C57BL6 (black dots) than BalbC mice (green), being both responses abundantly higher (4 an 8-fold increase) in comparison with the responses elicited in a prime-boost DNA vaccination regime that carries the same antigens (red dots). Unrelated ChAdOx1 was used as a control (purple and blue dots). Modest INFg responses against prM were detected. (B) IFNg responses from C57BL6 mice were followed for 2 weeks (black dots) and 4 weeks (green dots) after prime with ChAdOx1-Zika immunisation. T-cell responses maintained the immunogenic profile seen in standard adenoviral vectored vaccines. (C) Immunodominant peptides detected in BalbC mice DNA vaccination as in FIG. 3 were confirmed in mice vaccinated with ChAdOx1-Zika (right panel). For C57BL6 mice, those peptides were not immunogenic but a single peptide ID:AC6 (left panel). The peptide corresponded to the starting N-terminal region of Zlka envelope (IRCIGVSNRDFVEGMSGGTW) and that share low homology to other known flaviviruses (D top and bottom figure). (Alignments and figures modified from Science 22 Apr. 2016: Vol. 352, Issue 6284, pp. 467-470) (E) Enzyme Linked Immuno-Sorbent Assay (ELISA) revealed that ChAdOx1-Zika vaccine carrying the prM and full Envelope genes were able to produce antibodies against the Zika Envelope protein as measured by OD405 colorimetric levels in comparison with a unrelated ChAdOX1 vaccine control. (Background is represented as a dotted line, which is the average of control OD405, plus 3 times their SD). (F) Further dilution of sera from vaccinated mice were plotted against OD405 showing the increase of OD405 in ChAdOx1-Zika vaccinated mice (squares) in comparison with control (triangles).

[0090] FIG. 5 shows (A) Cellular immune responses, which were quantified in BALB/c mice following an immunisation with the ChAdOx1-Zika vaccines. 14 days post-vaccination, peripheral blood mononuclear cells (PBMCs) were obtained by tail bleeding. Cells were resuspended using EDTA anticoagulant. PBMCs were further purified by eliminating or lysing red blood cells and were suspended in DMEM media, plated in ELISpot plates with PDVF membranes. PBMCs were incubated during 18 hours in presence of peptide pools spanning the whole structural region of the zika virus. Peptide pools consisted on 20-mers overlapping by 10 and were used at a final concentration of bug per peptide. Results are expressed as spot-forming colonies per million PBMCs and the responses indicated are ex vivo, which means no further incubation to expand cells and increase responses was made, and all the frequencies reported are from cells tested immediately after bleeding.

[0091] DIII resulted in the lowest T cell responses and this perhaps indicates that these are CD4s, which would be confirmed by flow cytometry. The rest of the constructs induced robust T cell responses in averages between 3,000 and 5,000 SFU/million PBMCs.

[0092] (B) FIG. 5B indicates antibody responses elicited after immunisation with the various versions of ChAdOx1-Zika vaccines, as indicated in the figure. Immunisations were made as described in A.

[0093] FIG. 6. Zika vaccine design. (A) A phylogenetic tree for ZIKV genomes up to October 2016; blue, red and green labels represent the Asian and African lineages of ZIKV and other Flaviviruses (such as DENV), respectively. (B) Conservation homology of Asian (top) and African/Asian (bottom) consensus sequences versus all genomic sequences depicted in A; circle represents the ZIKV-BR strain used for the challenge experiment. (C) Schematic representation of the Zika immunogen versions used in this study; cross-hatch block represents the TPA leading sequence. (D) Restriction enzyme analysis of the plasmid DNA vaccines constructed, a 3.3 Kb band size represents the Pmono plasmid back bone. (E) HEK293 expression of the plasmid DNA encoding the Zika immunogens using a generated anti-ZIKV Envelope antibody. (F) Immunofluorescence analysis of Vero cells transfected with plasmid DNA encoding the ZIKV immunogens as depicted in D; using a commercial anti-flavivirus antibody.

[0094] FIG. 7. Immune Responses Elicited by DNA vs ChAdOx1 vaccines. (A) For ZIKV DNA vaccines, BALB/c mice (n=6 per group) were immunised intramuscularly (i.m.) with a dose of 100 ?g/mice, followed by a DNA Boost two weeks thereafter. For ChAdOx1 Zika vaccines, a single dose of 10.sup.8 IU/mice was i.m. administered. Blood samples were obtained at depicted time points for ELISA and ELISPOT assays. (B) Humoral responses elicited by DNA Prime-Boost after two weeks (left graph) and by a single immunisation of ChAdOx1 Zika vaccines at two weeks (right) and four months (bottom). Antibody responses were quantified by ELISA plates coated with ZIKV envelope protein. Error bar and bars represent the mean with SD. (C) PBMCs-INF? producing cells from DNA Prime, DNA Prime-Boost after two weeks (left graph) and ChAdOx1 Zika vaccines at two weeks (right) and three months (bottom) after single immunisation were quantified by ELISPOT. 20mer peptides spanning the ZIKV envelope protein (10 ?g/ml) were used for stimulation.

[0095] FIG. 8. Assessment of Protective Efficacy induced by ChAdOx1 Zika vaccines. (A) Balb/C mice (n=5) were immunised with a single i.m. shot of ChAdOx1 Zika vaccines and a ChAdOx1 unrelated vector were intravenously challenged with 105 VP of ZIKA-BR at week four after prime. (B) Viral load in vaccinated groups was monitored followed 7 days in sera to follow the onset of viraemia. (C) ELISA endpoint OD titers and (D) reciprocal ELISA titers of 4 weeks pre-challenge sera from vaccinated mice were calculated. (E) Vaccine efficacy scenarios observed in groups vaccinated with ChAdOx1 Zika vaccines

[0096] FIG. 9. T-Cell epitope mapping for NS3 and Envelope Zika (A) Peptide stimulation of peptides spanning all the proteins involved in the development of Zika vaccines. PBMCs from mice immunised with ChAdOx1 prME and CprME/NS were used for comparison. (B) DENV2 NS3 pools and Zika NS3 pools were assayed in ELISPOT to determine the immunodominant peptide and its homology with other flaviviruses (C), which was mapped in the Helicase domain I (alpha-helix (see arrow)) of Zika NS3 (D). (E) Zika envelope pools were also assayed to determine the immunodominant peptides along with their homology with other flaviviruses (F), which were mapped in the domain II (DII ribbon) and domain III (DIII loop) of Zika envelope (G).

[0097] FIG. 10. Comparative immunogenicity against Zika and Chikungunya structural antigens, elicited by a bivalent vaccine. Antibody titers were compared between a single-component vaccine and a bivalent vaccine delivered as a mixture of two ChAdOx1-Zika/ChAdOx1-Chikungunya delivered in the same leg or a co-administration of both ChAdOx1-Zika+Chik applied in different legs. No statistical differences were observed.

[0098] FIG. 11. Antibody responses against Zika virus envelope upon vaccination with a ChAdOx1-Zika vaccine alone or in combination of a ChAdOx1-Chikungunya vaccine as a mixture or co-vaccination in different legs.

[0099] FIG. 12. Antibody responses against Chikungunya virus envelope 2 protein upon vaccination with a ChAdOx1-Chikungunya vaccine alone or in combination of a ChAdOx1-Zika vaccine as a mixture or co-vaccination in different legs.

EXAMPLE 1

Zika Virus Vaccine Development

[0100] Vaccine development is a lengthy process that requires careful selection of the best candidates to provide the best protection. Every pathogen's genetic sequence inserted into a new viral vectored vaccine will produce proteins that will follow various pathways of secretion depending on the leading sequences and presence of transmembrane regions. Thus the recombinant viruses described herein contain various versions of the ZIKV structural antigens with or without anchoring regions. This has a profound effect on immunogenicity and ultimately in protective efficacy. Therefore, it is important to study and carefully select all these variables in order to find the most efficacious vaccine, supported by the use of functional assays.

[0101] The Zika virus structural antigens have been carefully designed and consist on a consensus sequence derived from all Asian ZIKV genetic sequences reported in the literature. We obtained an immunogen with 98% homology to the ZIKV causing the current epidemics in the Americas. An antigen based on a consensus sequence will maximise coverage, yielding a vaccine that will be useful not only in endemic countries like Brazil but also in other affected regions in Asia and the potential to cover African Zika lineages. To minimise future issues of low immunogenicity in humans, we have constructed 5 variants of a ZIKV antigen to be used in 10 vaccines and we aim to apply functional assays to find the most immunogenic and protective vaccine, suitable for the clinic.

[0102] Zika vaccine candidates were constructed using a cassette expressing the Zika structural antigens, which contain the following regions: Pre-membrane (PrM) and Envelope (Env). All cassettes contained a 5 leading sequence known as tPA, used in the Jenner ChAdOx vaccines to support secretion of the proteins once they are produced within cells. Two cassettes expressed the PrM structural antigen and three cassettes did not express the PrM.

[0103] Regarding the Env, two cassettes expressed the whole Env protein, which includes the domain I, II and III of Env and a transmembrane region(TM) located at the C-terminus region of the Env protein. Three cassettes did not contain such TM region, in order to further promote secretion of the protein to the extracellular milieu and stimulate antibody responses. The reasoning behind this is that the TM region could anchor a protein to cell membranes, preventing secretion. Finally, one cassette contained only the DIII region, which is part of the Env and the aim of this construct was to stimulate antibody responses only against the DIII, which is the domain used by the Zika virus to attach to cells. Anti-DIII antibodies may block and neutralise the virus and prevent attachment and entry, while at the same time, no induction of antibodies would take place against the rest of the protein, which has been involved in the antibody dependent enhancement (ADE), whereby antibodies against PrM, DI and DII enhance entry of virus rather than neutralisation, provoking higher viraemias and severity of the Zika or Dengue diseases (Zika could promote dengue ADE and vice versa).

Sequences

[0104] Sequences, or encoded sequences, of the potential Zika viral vector components are described below. The Zika viral vector of the invention may comprise any one of the nucleic acid sequences provided below, or variants thereof. Alternatively, or additionally, the Zika viral vector of the invention may comprise nucleic acid encoding any one of the amino acid sequences provided below, or variants thereof.

[0105] The sequence of the antigenic component of the Zika viral vector of the invention (i.e. not including the viral vector backbone such as ChAdOx sequence) may consist essentially of one of the following sequences, or variants thereof (or sequences encoding the amino acid sequences, or variants thereof, where appropriate).

TABLE-US-00001 ZIKAEnvelopeDomainIII(ZDIII)(AlsoknownasDIII) (SEQIDNO:1) ATGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTT CACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGT ACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACC CTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAA CAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCG TGGGAGAGAAGAAGATCACCCACCACTGGCACAGAAGCGGCAGCACCATCGGCAAG Protein (SEQIDNO:2) MKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMA VDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHW HRSGSTIGK ZIKAEnvelopewithnoTransmembranedomain(ZENV_noTM) (AlsoknownasEnvnoTM) (SEQIDNO:3) ATGCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATG GGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGACAAGCCCA CCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTAC TGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGA GGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATA GAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAG TTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCG GATCATGCTGAGCGTGCACGGCAGCCAGCACTCCGGCATGATCGTGAACGACACCGGCC ACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAG GCCACCCTGGGCGGCTTTGGATCTCTGGGACTGGACTGCGAGCCCAGAACCGGCCTGGA CTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGT GGTTCCACGACATCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGA ACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTG GTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGA AATGGATGGCGCCAAAGGCAGACTGTCCAGCGGCCACCTGAAGTGCCGGCTGAAGATGG ACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACC AAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCAC CGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCG TGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATG ATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAA GAAGATCACCCACCACTGGCACAGAAGCGGCAGCACCATCGGCAAGGCCTTTGAGGCTA CAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGAGATACCGCCTGGGACTTTGGCTCT GTGGGCGGAGCCCTGAACTCTCTG Protein (SEQIDNO:4) MRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMA EVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLF GKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDE NRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKE WFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALA GALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHG TVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLEL DPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG SVGGALNSL ZIKAEnvelopewithTransmembranedomain(ZENV_TM) (AlsoknownasEnv) (SEQIDNO:5) ATGCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATG GGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGACAAGCCCA CCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTAC TGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGA GGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATA GAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAG TTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCG GATCATGCTGAGCGTGCACGGCAGCCAGCACTCCGGCATGATCGTGAACGACACCGGCC ACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAG GCCACCCTGGGCGGCTTTGGATCTCTGGGACTGGACTGCGAGCCCAGAACCGGCCTGGA CTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGT GGTTCCACGACATCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGA ACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTG GTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGA AATGGATGGCGCCAAAGGCAGACTGTCCAGCGGCCACCTGAAGTGCCGGCTGAAGATGG ACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACC AAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCAC CGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCG TGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATG ATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAA GAAGATCACCCACCACTGGCACAGAAGCGGCAGCACCATCGGCAAGGCCTTTGAGGCTA CAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGAGATACCGCCTGGGACTTTGGCTCT GTGGGCGGAGCCCTGAACTCTCTGGGCAAGGGAATCCACCAGATCTTCGGCGCTGCCTT CAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTGA TGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGATGTGCCTGGCTCTGGGA GGCGTGCTGATCTTCCTGAGCACAGCCGTGTCCGCC Protein (SEQIDNO:6) MRCIGVSNRDFVEGM8GGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMA EVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLF GKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDE NRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKE WFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALA GALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHG TVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLEL DPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG SVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISL MCLALGGVLIFLSTAVSA ZIKAprMEnvelopewithnoTransmembranedomain(ZprMENV_noTM) (AlsoknownasprMEnoTM) (SEQIDNO:7) ACAAGACGGGGCAGCGCCTACTACATGTACCTGGACAGAAACGACGCCGGCGAGGCCAT CAGCTTCCCTACCACACTGGGCATGAACAAGTGCTACATCCAGATCATGGACCTGGGCC ACATGTGCGACGCCACAATGAGCTACGAGTGCCCCATGCTGGACGAGGGCGTGGAACCC GACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGTGTACGGCACCTGTCA CCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGCCACAGCA CCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCAC CTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCCGCTGCTGC TATTGCTTGGCTGCTGGGCAGCTCCACCTCCCAGAAAGTGATCTACCTCGTGATGATCC TGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTG GAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGAC AGTGATGGCCCAGGACAAGCCCACCGTGGACATCGAGCTCGTGACCACCACCGTGTCCA ATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGAC AGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGT GTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGG GCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATC CAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAGCACTCCGG CATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCA CCCCCAACAGCCCTAGAGCCGAGGCCACCCTGGGCGGCTTTGGATCTCTGGGACTGGAC TGCGAGCCCAGAACCGGCCTGGACTTCAGCGACCTGTACTACCTGACCATGAACAACAA GCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCCGGCG CTGATACAGGCACACCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCC CACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGC TCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCAGCGGCC ACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTG TGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACT GTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGT GGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCG AGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTAC ATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGAAGCGGCAGCAC CATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGAG ATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACTCTCTG Protein (SEQIDNO:8) TRRGSAVYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMSYECPMLD EGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQT WLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAP AYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGSVTVMAQDKPTVDIELVTTTVS NMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGC GLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHE TDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLV HKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHT ALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAET LHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMM LELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAW DFGSVGGALNSL (PrMsequenceisunderlined) ZIKAprMEnvelopewithTransmembranedomain(ZprMENV_TM) (AlsoknownasprME) (SEQIDNO:9) ACAAGACGGGGCAGCGCCTACTACATGTACCTGGACAGAAACGACGCCGGCGAGGCCAT CAGCTTCCCTACCACACTGGGCATGAACAAGTGCTACATCCAGATCATGGACCTGGGCC ACATGTGCGACGCCACAATGAGCTACGAGTGCCCCATGCTGGACGAGGGCGTGGAACCC GACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGTGTACGGCACCTGTCA CCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGCCACAGCA CCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCAC CTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCCGCTGCTGC TATTGCTTGGCTGCTGGGCAGCTCCACCTCCCAGAAAGTGATCTACCTCGTGATGATCC TGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTG GAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGAC AGTGATGGCCCAGGACAAGCCCACCGTGGACATCGAGCTCGTGACCACCACCGTGTCCA ATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGAC AGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGT GTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGG GCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATC CAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAGCACTCCGG CATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCA CCCCCAACAGCCCTAGAGCCGAGGCCACCCTGGGCGGCTTTGGATCTCTGGGACTGGAC TGCGAGCCCAGAACCGGCCTGGACTTCAGCGACCTGTACTACCTGACCATGAACAACAA GCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCCGGCG CTGATACAGGCACACCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCC CACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGC TCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCAGCGGCC ACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTG TGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACT GTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGT GGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCG AGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTAC ATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGAAGCGGCAGCAC CATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGAG ATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACTCTCTGGGCAAGGGAATC CACCAGATCTTCGGCGCTGCCTTCAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCA GATCCTGATCGGCACCCTGCTGATGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCT CCCTGATGTGCCTGGCTCTGGGAGGCGTGCTGATCTTCCTGAGCACAGCCGTGTCCGCC Protein (SEQIDNO:10) TRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMSYECPMLD EGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQT WLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAP AYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVS NMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGC GLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHE TDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLV HKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHT ALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAET LHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMM LELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAW DFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGS ISLMCLALGGVLIFLSTAVSA TPA5 leadersequence: (SEQIDNO:11) MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRR Sharkinvariantchainsequence (SEQIDNO:12) SLLWGGVTVLAAMLIAGQVASSVVFLV pRMaminoacidsequence: (SEQIDNO:13) TRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMSYECPMLD EGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQT WLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAP AYS

CprME/NS (Comprises Capsid, prM and Envelope Directly Followed by NS2b (a Cofactor of NS3), and NS3 (with Enzymatic Activity to cleave Capsid from prME)

[0106] This construct is more similar to the African Lineage, but still provides good protection in a challenge model with Asian Zika isolate (Brazilian)

TABLE-US-00002 DNAsequence (SEQIDNO:18) ACCAT GAAGAACCCCAAGAAGAAGTCCGGCGGCTTCCGGATCGTGAACATGCTGA AACGGGGCGTGGCCAGAGTGAACCCTCTGGGCGGACTGAAGAGACTGCCT GCCGGACTGCTGCTGGGCCACGGCCCTATTAGAATGGTGCTGGCCATCCT GGCCTTTCTGCGGTTCACCGCCATCAAGCCTAGCCTGGGCCTGATCAACA GATGGGGCAGCGTGGGCAAGAAAGAAGCCATGGAAATCATCAAGAAGTTC AAGAAAGACCTGGCCGCCATGCTGCGGATCATCAACGCCCGGAAAGAGCG GAAGCGGAGAGGCGCCGATACCAGCATCGGCATCATTGGCCTGCTGCTGA CCACAGCCATGGCCGCCGAGATCACCAGAAGAGGCAGCGCCTACTACATG TACCTGGACAGAAGCGACGCCGGCAAGGCCATCAGCTTTGCCACAACCCT GGGCGTGAACAAGTGCCACGTGCAGATCATGGACCTGGGCCACATGTGCG ACGCCACAATGAGCTACGAGTGCCCCATGCTGGACGAGGGCGTGGAACCC GACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGTGTACGG CACCTGTCACCACAAGAAGGGCGAGGCCAGACGGTCTAGAAGGGCCGTGA CACTGCCTAGCCACAGCACCCGGAAGCTGCAGACCAGAAGCCAGACCTGG CTGGAAAGCAGAGAGTACACCAAGCACCTGATCAAGGTGGAAAACTGGAT CTTCCGGAACCCCGGCTTCGCCCTGGCTGCCGTGGCTATTGCTTGGCTGC TGGGAAGCAGCACCAGCCAGAAAGTGATCTACCTCGTGATGATCCTGCTG ATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTT CGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATG GCGGCTGCGTGACAGTGATGGCCCAGGACAAGCCCACCGTGGACATCGAG CTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTA CGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGG GGGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGG ACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGG CAGCCTCGTGACCTGCGCCAAGTTCACCTGTAGCAAGAAGATGACCGGCA AGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCAC GGCTCCCAGCACAGCGGCATGATCGTGAATGACATCGGCCACGAGACAGA CGAGAACCGGGCCAAAGTGGAAGTGACCCCCAACAGCCCTAGAGCCGAGG CCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACC GGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAACACTG GCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCTG GCGCTGATACAGGCACCCCCCACTGGAACAACAAAGAGGCCCTGGTGGAG TTCAAGGACGCCCACGCCAAGAGGCAGACCGTGGTGGTGCTGGGATCTCA GGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGG ATGGCGCTAAGGGCCGGCTGTTTAGCGGCCACCTGAAGTGCCGGCTGAAG ATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGC CTTCACCTTCACCAAGGTGCCCGCCGAAACCCTGCACGGCACAGTGACTG TGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAG ATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGC CAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAAC TGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGACAAG AAGATCACCCACCACTGGCACCGCAGCGGCAGCACAATCGGAAAGGCCTT CGAAGCCACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGCGATACCG CCTGGGATTTTGGCTCTGTGGGCGGCGTGTTCAACTCCCTGGGCAAGGGA ATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGGCATGAG CTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGACTGA ACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGGGGAGTG ATGATCTTCCTGAGCACCGCCGTGTCCGCCCCTAGCGAAGTGCTGACAGC CGTGGGACTGATCTGCGCTCTGGCAGGCGGATTCGCCAAGGCCGACATTG AGATGGCCGGACCCATGGCTGCTGTGGGACTGCTGATTGTGTCCTACGTG GTGTCCGGCAAGTCTGTGGACATGTACATCGAGAGAGCCGGCGACATCAC CTGGGAGAAGGACGCCGAAGTGACAGGCAACAGCCCCAGACTGGACGTGG CCCTGGATGAGAGCGGCGATTTCAGTCTGGTGGAAGAGGACGGCCCTCCC ATGCGCGAGATCATTCTGAAAGTGGTGCTGATGGCAATCTGCGGGATGAA CCCTATCGCCATCCCCTTCGCTGCCGGCGCTTGGTACGTGTACGTGAAAA CAGGCAAGCGGAGCGGAGCCCTGTGGGATGTGCCTGCCCCCAAAGAAGTG AAGAAAGGCGAGACAACCGACGGCGTGTACAGAGTGATGACCCGCAGACT GCTGGGCAGCACACAAGTGGGAGTGGGCGTGATGCAGGAAGGGGTGTTCC ACACCATGTGGCACGTGACCAAAGGCGCCGCTCTGAGATCTGGCGAGGGC AGGCTGGATCCTTACTGGGGCGACGTGAAGCAGGACCTGGTGTCCTATTG CGGCCCTTGGAAGCTGGACGCCGCTTGGGATGGACTGAGCGAGGTGCAGC TGCTGGCTGTGCCTCCTGGCGAGAGGGCCAGAAACATCCAGACCCTGCCA GGCATCTTCAAGACCAAGGACGGGGACATCGGCGCCGTGGCTCTGGATTA TCCTGCCGGCACAAGCGGCTCCCCCATCCTGGACAAGTGTGGCAGAGTGA TCGGCCTGTACGGCAACGGCGTCGTGATCAAGAATGGCAGCTATGTGTCC GCCATCACCCAGGGCAAGCGGGAAGAGGAAACCCCTGTGGAATGCTTCGA GCCCTCCATGCTGAAGAAAAAGCAGCTGACCGTGCTGGACCTGCACCCTG GCGCCGGAAAAACCAGAAGGGTGCTGCCTGAGATCGTGCGGGAAGCCATC AAGAAACGGCTGAGAACCGTGATCCTGGCCCCCACCAGAGTGGTGGCTGC CGAGATGGAAGAAGCCCTGAGAGGACTGCCCGTGCGGTACATGACAACCG CCGTGAACGTGACCCACTCTGGCACCGAGATCGTGGATCTGATGTGTCAC GCCACCTTCACAAGCCGGCTGCTGCAGCCCATCCGGGTGCCCAACTACAA CCTGTACATCATGGACGAGGCCCACTTCACCGACCCCAGCTCCATTGCCG CCAGAGGCTACATCAGCACACGGGTGGAAATGGGCGAAGCTGCCGCCATC TTCATGACCGCCACACCTCCCGGAACCAGGGACGCCTTCCCCGACAGCAA CTCCCCTATCATGGACACCGAGGTGGAAGTGCCCGAGAGAGCCTGGTCCA GCGGCTTCGACTGGGTCACAGATCACTCCGGCAAGACCGTGTGGTTCGTG CCCTCTGTGCGGAACGGCAATGAGATCGCCGCCTGTCTGACAAAGGCCGG GAAGAGAGTGATCCAGCTGAGCCGCAAGACCTTCGAGACAGAGTTCCAGA AAACAAAGAACCAGGAATGGGATTTCGTGATCACCACAGACATCTCCGAG ATGGGCGCCAACTTCAAGGCCGATCGCGTGATCGACAGCCGGCGGTGTCT GAAGCCCGTGATTCTGGACGGCGAAAGAGTGATTCTGGCCGGACCTATGC CCGTGACCCATGCCTCTGCCGCTCAGAGAAGAGGCCGGATCGGCAGAAAC CCCAACAAGCCCGGCGACGAGTATATGTACGGCGGAGGCTGCGCCGAGAC TGACGAGGATCATGCCCATTGGCTGGAAGCCAGAATGCTGCTGGACAACA TATACCTGCAGGACGGCCTGATCGCCTCCCTGTACAGACCCGAGGCTGAC AAAGTGGCTGCCATCGAGGGCGAGTTCAAGCTGAGGACCGAGCAGAGAAA GACATTTGTGGAACTGATGAAGCGGGGCGACCTGCCTGTGTGGCTGGCCT ATCAGGTGGCATCTGCCGGCATCACCTACACCGACAGACGGTGGTGCTTC GACGGCACCACCAACAACACCATCATGGAAGATAGCGTGCCAGCCGAAGT GTGGACCAAATACGGCGAGAAGCGCGTGCTGAAGCCCCGGTGGATGGACG CCAGAGTGTGTTCTGATCACGCCGCACTGAAGTCCTTCAAAGAGTTCGCC GCTGGCAAGTGATGAGCGGCCGCTCGAGTACGTCTG ProteinsequenceofCprME/NS (SEQIDNO:19) MKNPKKKSGGFRIVNMLKRGVARVNPLGGLKRLP AGLLLGHGPIRMVLAILAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKF KKDLAAMLRIINARKERKRRGADTSIGilGLLLTTAMAAEITRRGSAYYM YLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEP DDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTW LESREYTKHLIKVENWIFRNPGFALAAVAIAWLLGSSTSQKVIYLVMILL IAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIE LVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKR TLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVH GSQHSGMIVNDIGHETDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRT GLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVE FKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLK MDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQ MAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDK KITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDEGSVGGVENSLGKG IHQIFGAAFKSLEGGMSWESQILIGTLLVWLGLNTKNGSISLTCLALGGV MIFLSTAVSAPSEVLTAVGLICALAGGFAKADIEMAGPMAAVGLLIVSYV VSGKSVDMYIERAGDITWEKDAEVTGNSPRLDVALDESGDFSLVEEDGPP MREIILKVVLMAICGMNPTATPFAAGAWYVYVKTGKRSGALWDVPAPKEV KKGETTDGVYRVMTRRLLGSTQVGVGVMQEGVFHTMWHVTKGAALRSGEG RLDPYWGDVKQDLVSYCGPWKLDAAWDGLSEVQLLAVPPGERARNIQTLP GIFKTKDGDIGAVALDYPAGTSGSP1LDKCGRVIGLYGNGVVIKNGSYVS AITQGKREEETPVECFEPSMLKKKQLTVLDLHPGAGKTRRVLPEIVREAI KKRLRTVILAPTRVVAAEMEEALRGLPVRYMTTAVNVTHSGTEIVDLMCH ATFTSRLLQPIRVPNYNLYIMDEAHFTDPSSIAARGYISTRVEMGEAAAI FMTATPPGTRDAFPDSNSPIMDTEVEVPERAWSSGFDWVTDHSGKTVWFV PSVRNGNEIAACLTKAGKRVIQLSRKTFETEFQKTKNQEWDFVITTDISE MGANFKADRVIDSRRCLKPVILDGERVILAGPMPVTHASAAQRRGRIGRN PNKPGDEYMYGGGCAETDEDHAHWLEARMLLDNIYLQDGLIASLYRPEAD KVAAIEGEFKLRTEQRKTFVELMKRGDLPVWLAYQVASAGITYTDRRWCF DGTTNNTIMEDSVPAEVWTKYGEKRVLKPRWMDARVCSDHAALKSFKEFA AGK

Example 2

Antigen Combination in Single Viral Vectors

[0107] The aim of this study is provide a new bivalent vaccine to induce simultaneous immunity against Zika and Chikungunya, simplifying future vaccination campaigns for countries where both diseases co-circulate in the same regions, and where protection against both diseases is needed. Infection by ZIKV is a major concern worldwide due to the neurologic conditions, such as Guillain-Barr? syndrome and a concurrent 20-fold increase in the incidence of microcephaly during the ZIKV outbreak in Brazil between 2014 and 2015 and in Mexico, where microcephaly caused by ZIKV has been confirmed. Aedes mosquitoes transmit Chikungunya virus

[0108] (CHIKV), ZIKV and Dengue in the same geographical regions. CHIKV produces symptomatic disease in approximately ? of infected people, leading in many cases to long-term sequelae in people of all ages. Persistent arthritis cause disability for several years, contributing to poverty as young adults are unable to perform their physical activities required for work. Costs for families and governments are augmented due to the need to administer anti-inflammatory drugs to provide a short-term relief in patients. No vaccine is yet licensed for the prevention of CHIKV or ZIKV infections.

[0109] The sudden presence of Zika and Chikungunya in the same geographical regions have overwhelmed health systems that were already challenged by Dengue, thus increasing the failure to provide treatment and preventive measures to their populations during the outbreak, while posing new challenges for treatment of both Zika and Chikungunya due to the long-term sequelae of more than 6 years for these diseases. These diseases are transitioning from an epidemic nature towards endemic diseases due to enabling drivers such as poor socioeconomic conditions, climate change and migration. A major breakthrough will be to provide governments with tools to simultaneously fight these highly prevalent arbovirus diseases and a multivalent vaccine able to protect against both Zika and Chikungunya would be an ideal preventive solution. This proposal has various aims:

[0110] Provided is a bivalent vaccine to provide simultaneous protection against Zika and Chikungunya, caused by two arboviruses co-circulating in the same geographical regions. Both, Zika and Chikunguna vaccines will be applied concurrently in a single administration without the need of adjuvants, taking into advantage that they are based on the same ChAdOx1 platform. This approach is simple and has the potential to stimulate fast induction of antibodies in only 10 days after the administration to provide long-lasting immunity in humans.

[0111] A multi-valent vaccine to protect against Zika and Chikungunya viruses can be highly attractive for vaccination campaigns in regions where both viruses co-circulate. This would be an efficient strategy to reduce costs and prevent arbovirus diseases that would rely on a concurrent delivery of the multivalent vaccine.

[0112] For a number of years, approaches have been pursued to develop viral vectors expressing multiple antigens to provide better protection against infection by increasing the breadth of both, T-cell and antibody responses to multiple antigens (Ported, D. W. et al. Vaccine 2011; Prieur, D. et al. PNAS, 2013; Bauza, K et al; Inf and Immun, 2016). Nevertheless, performance of the vaccine upon a challenge is difficult to predict, both in mice and human challenges with pathogens. Porter et al.

[0113] reported two poxviral vectors expressing various malaria vaccine candidates. The polyprotein vaccine insert known as L3SEPTL contained pre-erythrocytic malaria vaccine antigens linked together, including liver stage antigen 3 (LSA3), sporozoite threonine and asparagine rich protein (STARP), exported protein-1 (Exp1), Pfs16, thrombospondin-related adhesion protein (TRAP) and liver stage antigen-1 (LSA1). Surprisingly, T-cell immunogenicity against the antigens in the L3SEPTL vaccine was lower than viral vectors expressing individually some of the antigens. Protection against a challenge was negative and the vaccine was not further developed.

[0114] Bauza et al. (Inf and Immun, 2016) reported that a combination of the vaccine candidates Circumsporozoite Protein (CSP) and Thrombospondin Related Anonymous Protein (TRAP) from Plasmodium berghei failed to significantly enhance protective efficacy when expressed by viral vectors, but this was improved when CSP was used as a protein and TRAP as a viral vector. Similar observations were made by Salman et al. (Sci. Reports, 2017) when assessing a chimeric P. vivax CSP antigen expressed by chimpanzee adenoviruses, whereby protective efficacy against a sporozoite challenge was low compared to the antigens presented in Rv21, a virus-like particle currently developed for clinical trials.

[0115] Nevertheless, the present invention finds that a combination of viral vectors expressing the structural proteins of the Chikungunya and Zika viruses could have a potential to induce strong antibody responses, at least similar to responses elicited by individual viral vectors.

Chikungunya and Zika Viruses Bivalent Formulation

[0116] To determine if both vaccines can be injected as a bivalent formulation without compromising immunogenicity, a combination of the ChAdOx1-Zika with the ChAdOx1-Chikungunya vaccines was administered into mice, either as a mixed single component or co-administered in different legs (FIG. 10). Results indicated that immune responses against Zika and Chikungunya proteins were similar (no statistical differences) when vaccines were administered alone or combined. These preliminary results support their use as a bivalent vaccine.

[0117] An analysis using a single time point may not reflect if memory responses, the goal of vaccination, are sustained at high levels and therefore, antibody responses in mice were assessed at various time points to investigate if the kinetics of the antibody responses is affected positively or negatively by a vaccine combination. Surprisingly, it was observed that a mixture in the same syringe of the two vaccines or a co-vaccination in different legs induced similar antibody responses to those induced individually by a ChAdOx1-Chikungunya vaccine or a ChAdOx1-Zika for over 20 weeks after a single vaccination or one week post MVA boost (FIGS. 11 and 12)

Chikungunya Vaccine Sequences

Structural Genes Encoded by ChAdOx1 Spol (ChAdOx1 Chik) Vaccine

[0118]

TABLE-US-00003 NUCLEOTIDESEQUENCE (SEQIDNO:14) ATGGAATTCATCCCCACCCAGACCTTCTACAACCGCAGATACCAGCCCAG ACCCTGGACCCCCAGACCCACCATCCAAGTGATCAGACCCCGGCCTAGAC CCCAGAGACAGGCTGGACAGCTGGCTCAGCTGATCTCCGCCGTGAACAAG CTGACCATGAGAGCCGTGCCCCAGCAGAAGCCCAGAAAGAACCGGAAGAA CAAGAAGCAGAAACAGAAGCAGCAGGCCCCCCAGAACGACCCCAAGCAGA AGAAGCAGCCTCCTCAGAAGAAACCCGCCCAGAAGAAGAAAAAGCCCGGC AGACGCGAGCGGATGTGCATGAAGATCGAGAACGACTGCATCTTCGAAGT GAAGCACGAGGGCAAAGTGATGGGCTACGCCTGCCTCGTGGGCGACAAAG TGATGAAGCCCGCCCACGTGAAGGGCACCATCGACAATGCCGACCTGGCC AAGCTGGCCTTCAAGCGGAGCAGCAAATACGACCTGGAATGCGCCCAGAT CCCCGTGCACATGAAGTCCGACGCCAGCAAGTTCACCCACGAGAAGCCCG AGGGCTACTACAACTGGCACCATGGCGCCGTGCAGTACAGCGGCGGCAGA TTCACAATCCCCACCGGCGCTGGAAAGCCTGGCGATAGCGGCAGACCCAT CTTCGACAACAAGGGCCGGGTGGTGGCCATCGTGCTGGGCGGAGCTAATG AGGGCGCCAGAACAGCCCTGAGCGTCGTGACCTGGAACAAGGACATCGTG ACCAAGATCACCCCCGAGGGCGCCGAGGAATGGTCCCTGGCTATCCCTGT GATGTGCCTGCTGGCCAACACCACCTTCCCATGCAGCCAGCCCCCTTGCA CCCCTTGCTGCTACGAGAAAGAGCCCGAGAGCACCCTGCGGATGCTGGAA GATAACGTGATGAGGCCCGGCTACTACCAGCTGCTGAAGGCCTCCCTGAC CTGCAGCCCTCACCGGCAGAGAAGATCCACCAAGGACAACTTCAACGTGT ACAAGGCCACCAGACCCTACCTGGCCCACTGCCCTGATTGTGGCGAGGGC CACTCTTGCCACTCTCCCGTGGCCCTGGAACGGATCAGAAACGAGGCCAC CGACGGCACCCTGAAGATCCAGGTGTCCCTGCAGATCGGCATCAAGACCG ACGACAGCCACGACTGGACCAAGCTGCGGTACATGGACAACCACATGCCC GCCGATGCCGAGAGGGCAGGACTGCTCGTGCGGACATCTGCCCCCTGTAC CATCACCGGCACAATGGGCCACTTCATCCTGGCCAGATGCCCCAAGGGCG AGACACTGACCGTGGGCTTCACCGATGGCCGGAAGATCAGCCACAGCTGC ACCCACCCCTTCCACCACGATCCTCCCGTGATCGGCAGAGAGAAGTTCCA CAGCAGACCCCAGCACGGCAAAGAGCTGCCCTGCAGCACATACGTGCAGA GCACAGCCGCCACCGCCGAAGAGATCGAGGTGCACATGCCTCCCGACACC CCCGACAGAACCCTGATGTCTCAGCAGAGCGGCAACGTGAAGATCACCGT GAACGGCCAGACCGTGCGGTACAAGTGCAACTGCGGCGGCTCCAATGAGG GCCTGACCACCACAGACAAAGTGATCAACAACTGCAAGATCGACCAGTGC CACGCCGCCGTGACCAACCACAAGAAGTGGCAGTACAACAGCCCCCTGGT GCCCAGAAATGCCGAGCTGGGCGACCGGAAGGGCAAGATCCACATCCCTT TCCCCCTGGCCAACGTGACCTGCCGGGTGCCCAAAGCCAGAAACCCCACC GTGACCTACGGCAAGAACCAAGTGATTATGCTGCTGTACCCCGACCACCC CACCCTGCTGAGCTACAGAAACATGGGCGAGGAACCCAACTACCACGAAG AGTGGGTCACCCACAAGAAAGAAGTGCGGCTGACCGTGCCCACCGAGGGC CTGGAAGTGACCTGGGGCAACAACGAGCCCTACAAGTACTGGCCCCAGCT GAGCACCAATGGCACAGCCCACGGACACCCCCACGAGATCATCCTGTACT ACTACGAGCTGTACCCTACCATGACCGTCGTGATCGTGTCTGTGGCCAGC TTCGTGCTGCTGAGCATGGTGGGAACAGCCGTGGGCATGTGTATGTGCGC CAGACGGCGGTGCATCACCCCTTACGAACTGACCCCTGGCGCCACCGTGC CCTTTCTGCTGAGCCTGATCTGCTGCATCCGGACCGCCAAGGCCGCCACC TATTATGAGGCCGCTGCCTACCTGTGGAACGAGCAGCAGCCCCTGTTTTG GCTGCAAGCCCTGATTCCTCTGGCCGCCCTGATCGTGCTGTGCAACTGCC TGAGACTGCTGCCCTGCTGCTGCAAGACCCTGGCCTTTCTGGCCGTGATG AGCATCGGAGCCCACACCGTGTCTGCCTACGAGCACGTGACCGTGATCCC CAACACAGTGGGCGTGCCCTACAAAACCCTCGTGAACAGACCCGGCTACA GCCCTATGGTGCTGGAAATGGAACTGCTGAGCGTGACCCTGGAACCCACC CTGAGCCTGGACTACATCACATGCGAGTACAAGACAGTGATCCCTAGCCC CTACGTGAAGTGCTGCGGCACCGCCGAGTGCAAGGACAAGAGCCTGCCCG ACTACAGCTGCAAGGTGTTCACCGGCGTGTACCCCTTCATGTGGGGCGGA GCCTACTGCTTTTGCGACGCCGAGAACACACAGCTGAGCGAGGCCCACGT GGAAAAGAGCGAGAGCTGCAAAACCGAGTTCGCCAGCGCCTACAGGGCCC ACACAGCCTCTGCCTCTGCCAAGCTGAGAGTGCTGTACCAGGGCAACAAT ATCACCGTGGCCGCCTACGCCAACGGCGACCATGCCGTGACAGTGAAGGA CGCCAAGTTCATCGTGGGCCCCATGAGCAGCGCCTGGACACCCTTCGATA ACAAGATTGTGGTGTATAAGGGGGATGTGTACAACATGGACTACCCCCCC TTTGGCGCCGGACGGCCTGGACAGTTTGGCGACATCCAGAGCAGAACCCC TGAGAGCAAGGACGTGTACGCCAACACCCAGCTGGTGCTGCAGAGGCCTG CAGCCGGAACAGTGCACGTGCCATACTCTCAGGCCCCCAGCGGCTTCAAG TATTGGCTGAAAGAGAGAGGCGCCAGCCTGCAGCATACCGCCCCTTTCGG CTGTCAGATCGCCACCAATCCTGTGCGGGCCGTGAATTGCGCCGTGGGAA ACATCCCCATCAGCATCGACATCCCCGACGCCGCCTTCACCAGAGTGGTG GATGCCCCTAGCCTGACCGACATGAGCTGCGAAGTGCCCGCCTGCACACA CAGCAGCGATTTTGGCGGAGTGGCCATCATTAAGTACGCCGCCTCCAAGA AAGGCAAGTGTGCCGTGCACAGCATGACCAACGCCGTGACAATCCGCGAG GCCGAGATTGAGGTGGAAGGCAACAGCCAGCTGCAGATCAGCTTCTCCAC AGCCCTGGCCAGCGCCGAGTTCAGAGTGCAAGTGTGCAGCACCCAGGTGC ACTGCGCTGCCGCTTGTCACCCCCCCAAGGACCACATCGTGAACTACCCT GCCAGCCACACCACCCTGGGCGTGCAGGATATCAGCACCACCGCCATGTC CTGGGTGCAGAAAATCACAGGGGGCGTGGGACTGATCGTGGCCGTGGCTG CTCTGATTCTGATTGTGGTGCTGTGCGTGTCCTTCAGCCGGCACTGATGA PROTEINSEQUENCE(StructuralPolyprotein) (SEQIDNO:15) MEFIPTQTFYNRRYQPRPWTPRPTIQVIRPRPRPQRQAGQLAQLISAVNK LTMRAVPQQKPRKNRKNKKQKQKQQAPQNDPKQKKQPPQKKPAQKKKKPG RRERMCMKIENDCIFEVKHEGKVMGYACLVGDKVMKPAHVKGTIDNADLA KLAFKRSSKYDLECAQIPVHMKSDASKFTHEKPEGYYNWHHGAVQYSGGR FTIPTGAGKPGDSGRPIFDNKGRVVAIVLGGANEGARTALSVVTWNKDIV TKITPEGAEEWSLAIPVMCLLANTTFPCSQPPCTPCCYEKEPESTLRMLE DNVMRPGYYQLLKASLTCSPHRQRRSTKDNFNVYKATRPYLAHCPDCGEG HSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHMP ADAERAGLLVRTSAPCTITGTMGHFILARCPKGETLTVGFTDGRKISHSC THPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATAEEIEVHMPPDT PDRTLMSQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKIDQC HAAVTNHKKWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPT VTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYHEEWVTHKKEVRLTVPTEG LEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVIVSVAS FVLLSMVGTAVGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKAAT YYEAAAYLWNEQQPLFWLQALIPLAALIVLCNCLRLLPCCCKTLAFLAVM SIGAHTVSAYEHVTVIPNTVGVPYKTLVNRPGYSPMVLEMELLSVTLEPT LSLDYITCEYKTVIPSPYVKCCGTAECKDKSLPDYSCKVFTGVYPFMWGG AYCFCDAENTQLSEAHVEKSESCKTEFASAYRAHTASASAKLRVLYQGNN ITVAAYANGDHAVTVKDAKFIVGPMSSAWTPFDNKIVVYKGDVYNMDYPP FGAGRPGQFGDIQSRTPESKDVYANTQLVLQRPAAGTVHVPYSQAPSGFK YWLKERGASLQHTAPFGCQIATNPVRAVNCAVGNIPISIDIPDAAFTRVV DAPSLTDMSCEVPACTHSSDFGGVAIIKYAASKKGKCAVHSMTNAVTIRE AEIEVEGNSQLQISFSTALASAEFRVQVCSTQVHCAAACHPPKDHIVNYP ASHTTLGVQDISTTAMSWVQKITGGVGLIVAVAALILIVVLCVSFSRH**

Structural Genes Encoded by ChAdOx1 Chik-Non Capsid Vaccine

[0119]

TABLE-US-00004 NUCLEOTIDESEQUENCE (SEQIDNO:16) GAGGAATGGTCCCTGGCTATCCCTGTGATGTGCCTGCTGGCCAACACCAC CTTCCCATGCAGCCAGCCCCCTTGCACCCCTTGCTGCTACGAGAAAGAGC CCGAGAGCACCCTGCGGATGCTGGAAGATAACGTGATGAGGCCCGGCTAC TACCAGCTGCTGAAGGCCTCCCTGACCTGCAGCCCTCACCGGCAGAGAAG ATCCACCAAGGACAACTTCAACGTGTACAAGGCCACCAGACCCTACCTGG CCCACTGCCCTGATTGTGGCGAGGGCCACTCTTGCCACTCTCCCGTGGCC CTGGAACGGATCAGAAACGAGGCCACCGACGGCACCCTGAAGATCCAGGT GTCCCTGCAGATCGGCATCAAGACCGACGACAGCCACGACTGGACCAAGC TGCGGTACATGGACAACCACATGCCCGCCGATGCCGAGAGGGCAGGACTG CTCGTGCGGACATCTGCCCCCTGTACCATCACCGGCACAATGGGCCACTT CATCCTGGCCAGATGCCCCAAGGGCGAGACACTGACCGTGGGCTTCACCG ATGGCCGGAAGATCAGCCACAGCTGCACCCACCCCTTCCACCACGATCCT CCCGTGATCGGCAGAGAGAAGTTCCACAGCAGACCCCAGCACGGCAAAGA GCTGCCCTGCAGCACATACGTGCAGAGCACAGCCGCCACCGCCGAAGAGA TCGAGGTGCACATGCCTCCCGACACCCCCGACAGAACCCTGATGTCTCAG CAGAGCGGCAACGTGAAGATCACCGTGAACGGCCAGACCGTGCGGTACAA GTGCAACTGCGGCGGCTCCAATGAGGGCCTGACCACCACAGACAAAGTGA TCAACAACTGCAAGATCGACCAGTGCCACGCCGCCGTGACCAACCACAAG AAGTGGCAGTACAACAGCCCCCTGGTGCCCAGAAATGCCGAGCTGGGCGA CCGGAAGGGCAAGATCCACATCCCTTTCCCCCTGGCCAACGTGACCTGCC GGGTGCCCAAAGCCAGAAACCCCACCGTGACCTACGGCAAGAACCAAGTG ATTATGCTGCTGTACCCCGACCACCCCACCCTGCTGAGCTACAGAAACAT GGGCGAGGAACCCAACTACCACGAAGAGTGGGTCACCCACAAGAAAGAAG TGCGGCTGACCGTGCCCACCGAGGGCCTGGAAGTGACCTGGGGCAACAAC GAGCCCTACAAGTACTGGCCCCAGCTGAGCACCAATGGCACAGCCCACGG ACACCCCCACGAGATCATCCTGTACTACTACGAGCTGTACCCTACCATGA CCGTCGTGATCGTGTCTGTGGCCAGCTTCGTGCTGCTGAGCATGGTGGGA ACAGCCGTGGGCATGTGTATGTGCGCCAGACGGCGGTGCATCACCCCTTA CGAACTGACCCCTGGCGCCACCGTGCCCTTTCTGCTGAGCCTGATCTGCT GCATCCGGACCGCCAAGGCCGCCACCTATTATGAGGCCGCTGCCTACCTG TGGAACGAGCAGCAGCCCCTGTTTTGGCTGCAAGCCCTGATTCCTCTGGC CGCCCTGATCGTGCTGTGCAACTGCCTGAGACTGCTGCCCTGCTGCTGCA AGACCCTGGCCTTTCTGGCCGTGATGAGCATCGGAGCCCACACCGTGTCT GCCTACGAGCACGTGACCGTGATCCCCAACACAGTGGGCGTGCCCTACAA AACCCTCGTGAACAGACCCGGCTACAGCCCTATGGTGCTGGAAATGGAAC TGCTGAGCGTGACCCTGGAACCCACCCTGAGCCTGGACTACATCACATGC GAGTACAAGACAGTGATCCCTAGCCCCTACGTGAAGTGCTGCGGCACCGC CGAGTGCAAGGACAAGAGCCTGCCCGACTACAGCTGCAAGGTGTTCACCG GCGTGTACCCCTTCATGTGGGGCGGAGCCTACTGCTTTTGCGACGCCGAG AACACACAGCTGAGCGAGGCCCACGTGGAAAAGAGCGAGAGCTGCAAAAC CGAGTTCGCCAGCGCCTACAGGGCCCACACAGCCTCTGCCTCTGCCAAGC TGAGAGTGCTGTACCAGGGCAACAATATCACCGTGGCCGCCTACGCCAAC GGCGACCATGCCGTGACAGTGAAGGACGCCAAGTTCATCGTGGGCCCCAT GAGCAGCGCCTGGACACCCTTCGATAACAAGATTGTGGTGTATAAGGGGG ATGTGTACAACATGGACTACCCCCCCTTTGGCGCCGGACGGCCTGGACAG TTTGGCGACATCCAGAGCAGAACCCCTGAGAGCAAGGACGTGTACGCCAA CACCCAGCTGGTGCTGCAGAGGCCTGCAGCCGGAACAGTGCACGTGCCAT ACTCTCAGGCCCCCAGCGGCTTCAAGTATTGGCTGAAAGAGAGAGGCGCC AGCCTGCAGCATACCGCCCCTTTCGGCTGTCAGATCGCCACCAATCCTGT GCGGGCCGTGAATTGCGCCGTGGGAAACATCCCCATCAGCATCGACATCC CCGACGCCGCCTTCACCAGAGTGGTGGATGCCCCTAGCCTGACCGACATG AGCTGCGAAGTGCCCGCCTGCACACACAGCAGCGATTTTGGCGGAGTGGC CATCATTAAGTACGCCGCCTCCAAGAAAGGCAAGTGTGCCGTGCACAGCA TGACCAACGCCGTGACAATCCGCGAGGCCGAGATTGAGGTGGAAGGCAAC AGCCAGCTGCAGATCAGCTTCTCCACAGCCCTGGCCAGCGCCGAGTTCAG AGTGCAAGTGTGCAGCACCCAGGTGCACTGCGCTGCCGCTTGTCACCCCC CCAAGGACCACATCGTGAACTACCCTGCCAGCCACACCACCCTGGGCGTG CAGGATATCAGCACCACCGCCATGTCCTGGGTGCAGAAAATCACAGGGGG CGTGGGACTGATCGTGGCCGTGGCTGCTCTGATTCTGATTGTGGTGCTGT GCGTGTCCTTCAGCCGGCACTGATGA PROTEINSEQUENCE(StructuralPolyproteinwithno Capsidincluded) (SEQIDNO:17) MEEWSLAIPVMCLLANTTFPCSQPPCTPCCYEKEPESTLRMLEDNVMRPG YYQLLKASLTCSPHRQRRSTKDNFNVYKATRPYLAHCPDCGEGHSCHSPV ALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHMPADAERAG LLVRTSAPCTITGTMGHFILARCPKGETLTVGFTDGRKISHSCTHPFHHD PPVIGREKFHSRPQHGKELPCSTYVQSTAATAEEIEVHMPPDTPDRTLMS QQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKIDQCHAAVTNH KKWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQ VIMLLYPDHPTLLSYRNMGEEPNYHEEWVTHKKEVRLTVPTEGLEVTWGN NEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVIVSVASFVLLSMV GTAVGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKAATYYEAAAY LWNEQQPLFWLQALIPLAALIVLCNCLRLLPCCCKTLAFLAVMSIGAHTV SAYEHVTVIPNTVGVPYKTLVNRPGYSPMVLEMELLSVTLEPTLSLDYIT CEYKTVIPSPYVKCCGTAECKDKSLPDYSCKVFTGVYPFMWGGAYCFCDA ENTQLSEAHVEKSESCKTEFASAYRAHTASASAKLRVLYQGNNITVAAYA NGDHAVTVKDAKFIVGPMSSAWTPFDNKIVVYKGDVYNMDYPPFGAGRPG QFGDIQSRTPESKDVYANTQLVLQRPAAGTVHVPYSQAPSGFKYWLKERG ASLQHTAPFGCQIATNPVRAVNCAVGNIPISIDIPDAAFTRVVDAPSLTD MSCEVPACTHSSDFGGVAIIKYAASKKGKCAVHSMTNAVTIREAEIEVEG NSQLQISFSTALASAEFRVQVCSTQVHCAAACHPPKDHIVNYPASHTTLG VQDISTTAMSWVQKITGGVGLIVAVAALILIVVLCVSFSRH**