ZIKA VIRUS CHIMERIC POLYEPITOPE COMPRISING NON-STRUCTURAL PROTEINS AND ITS USE IN AN IMMUNOGENIC COMPOSITION

Abstract

The present invention is directed to a Zika virus (ZIKV) chimeric polyepitope comprising non-structural proteins and its use in an immunogenic composition. The present invention provides means, in particular polynucleotides, vectors and cells expressing said chimeric polyepitope. The present invention also relates to a composition or a vaccine comprising at least one of said polyepitope, polynucleotide, vector or host cell for use in the prevention of a ZIKV infection in a human subject, or for use in the prevention of ZIKV and dengue virus (DENV) infections in a human subject.

Claims

1-24. (canceled)

25. A pharmaceutical composition comprising polynucleotide encoding a chimeric polyepitope an adjuvant and/or a pharmaceutically acceptable vehicle, wherein said chimeric polyepitope comprises: (i) at least the following T-cell epitopes of (a) and (b), or (ii) at least the following T-cell epitopes of (a) and (c), or (iii) at least the following T-cell epitopes of (b) and (c): (a) a T-cell epitope of the non-structural (NS) NS1 protein of a Zika virus (ZIKV) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-12, 14, 15, 17-19, 23, 24 and 78-83, (b) a T-cell epitope of the NS3 protein of a ZIKV comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 29, 31, 33-35, 84 and 85, (c) a T-cell epitope of the NS5 protein of a ZIKV comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48-50, 52-55, 57-60, 62, 64, 67, 69, 72, 73 and 86-91, or a T-cell epitope variant thereof, which differs from the original amino acid sequence of the T-cell epitope of (a), (b) or (c) by point mutation of one or more amino acid residues and which has at least 90% sequence identity or more than 95% sequence identity or 99% sequence identity with said original sequence.

26. The pharmaceutical composition of claim 25, wherein said chimeric polyepitope comprises at least the T-cell epitopes of (a), (b) and (c), or the T-cell epitope variant thereof.

27. The pharmaceutical composition of claim 25, wherein said chimeric polyepitope consists of (i) the T-cell epitopes of (a) and (b), or (ii) the T-cell epitopes of (a) and (c), or (iii) the T-cell epitopes of (b) and (c), or (iv) the T-cell epitopes of (a), (b) and (c), or the T-cell epitope variant thereof.

28. The pharmaceutical composition of claim 25, wherein the T-cell epitope of (a) comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 23 and 78-83, the T-cell epitope of (b) comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 33, 84 and 85, and the T-cell epitope of (c) comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 52, 57, 62, 64, 67 and 86-91.

29. The pharmaceutical composition of claim 25, wherein the T-cell epitope of (a) comprises or consists of the amino acid sequence of SEQ ID NOs: 11, 12, 17-19, 23, 24, 78, 80 and 83, the T-cell epitope of (b) comprises or consists of the amino acid sequence of SEQ ID NOs: 28, 31, 33, 34, 84 and 85, and the T-cell epitope of (c) comprises or consists of the amino acid sequence of SEQ ID NOs: 48-50, 52-55, 57, 58, 60, 62, 67, 88, 89 and 90.

30. The pharmaceutical composition of claim 25, wherein the chimeric polyepitope further comprises at least one T-cell epitope of a ZIKV protein selected from the group consisting of: (i) a T-cell epitope of the C protein of a ZIKV comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4-6 and 75, (ii) a T-cell epitope of the E protein of a ZIKV comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 76 and 77, (iii) a T-cell epitope of the NS2B protein of a ZIKV comprising or consisting of the amino acid sequence of SEQ ID NO: 25, (iv) a T-cell epitope of the NS4A protein of a ZIKV comprising or consisting of the amino acid sequence of SEQ ID NO: 36, and (v) a T-cell epitope of the NS4B protein of a ZIKV comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 40-43.

31. The chimeric polyepitope according to claim 30, wherein said at least one T-cell epitope of a ZIKV protein is selected from the group consisting of: the T-cell epitope of the C protein of a ZIKV comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4-6 and 75, and the T-cell epitope of the NS4B protein of a ZIKV comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 40-43.

32. The pharmaceutical composition of claim 25, wherein the chimeric polyepitope has an amino acid sequence of SEQ ID NO: 99.

33. The pharmaceutical composition of claim 25, which elicits a human leukocyte antigen (HLA)-restricted CD8.sup.+ and/or CD4.sup.+ T cell response (i) against ZIKV, or (ii) against ZIKV and DENV, in particular DENV serotype 1 (DENV1), DENV serotype 2 (DENV2), DENV serotype 3 (DENV3) and DENV serotype 4 (DENV4).

34. The pharmaceutical composition of claim 25, wherein the T-cell epitopes are assembled in a fusion polypeptide.

35. The pharmaceutical composition of claim 25, wherein the ZIKV is from the African lineage, or from the Asian lineage.

36. The pharmaceutical composition of claim 25, wherein said composition is formulated for an administration by a route selected from the group consisting of subcutaneous (s.c.), intradermal (i.d.), intramuscular (i.m.), intraperitoneal (i.p.) and intravenous (i.v.) injection.

37. The pharmaceutical composition of claim 25, formulated for administration in a prime-boost administration regime.

38. A method of (i) preventing a Zika virus (ZIKV) infection in a human subject or (ii) preventing ZIKV and Dengue virus (DENV) infections in a human subject, comprising administering the pharmaceutical composition of claim 25 to the human subject.

39. The method of preventing a ZIKV infection in a human subject according to claim 38, wherein the T-cell epitopes are ZIKV-specific epitopes comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 5, 10, 11, 19, 27, 31, 40-43, 46, 72, 73, 75, 78-80, 82, 84, 85, 87 and 91.

40. The method of preventing ZIKV and DENV infections in a human subject according to claim 38, wherein the T-cell epitopes are ZIKV-DENV cross-reactive epitopes comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 12, 14, 15, 17-19, 23, 24, 27, 28, 33-35, 40, 41, 46, 48-50, 52-55, 57, 59, 60, 62, 64, 67, 69, 72, 73, 84, 85 and 86-91.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0103] FIG. 1A-C. ZIKV-specific response magnitude and frequency of responding donors. Cumulative IFN-γ responses (as spot-forming cells (SFCs) per million cells) for each overlapping peptide spanning the ZIKV proteome is shown for (1A) all donors, (1B) ZIKV donors or (1C) DENV/ZIKV donors. The heat map indicates the number of donors with a positive IFN-γ response to each peptide within each protein (C, capsid; M, membrane; E, envelope, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The numbers below each graph represent percentages of the total response for each protein.

[0104] FIG. 2A-D. ZIKV donors with previous DENV infection reveal a broader T-cell response with a higher magnitude. (2A) Breadth and (2B) magnitude of responses in ZIKV and DENV/ZIKV donors. Each dot represents one donor (open circles, ZIKV donors; filled circles, DENV/ZIKV donors) and the bars represent the median value for each group of donors. The P values were calculated using the nonparametric two-tailed Mann-Whitney test. Frequency of responses against individual peptides, per donor, in ZIKV (2C) and DENV/ZIKV (2D) donors. Each dot represents one peptide. The bars represent the median response for each donor.

[0105] FIG. 3A-B. Comparison of the magnitude of response and sequence identity with DENV in ZIKV and DENV/ZIKV donors. Each dot represents the cumulative response of different donors against one peptide. Percentages represent the mean identity value between the sequences of ZIKV and the 4 DENV serotypes. (3A) peptides inducing a response in ZIKV donors. (3B) peptides inducing a response in DENV/ZIKV donors.

[0106] FIG. 4. Schematic representation of the 18AAHK3C_pVAX-ZIKV_PolyEpitop_pVAX1 plasmid. The inventors used the pVAX1 plasmid commercialized by Thermo Fisher Scientific. The polynucleotide encoding a chimeric polyepitope of ZIKV as defined in SEQ ID NO: 124 was inserted in said plasmid.

[0107] FIG. 5. HLA-A*2402 transgenic mice were immunized by intradermal injections and in vivo electroporation (prime with 2×50 μg DNA at day 0, and boost with 2×50 μg DNA at day 21) with the plasmid DNA coding for a chimeric polyepitope of ZIKV. Said chimeric polyepitope of ZIKV had the amino acid sequence of SEQ ID NO: 99. The nucleotide sequence of the polynucleotide encoding said chimeric polyepitope was as defined in SEQ ID NO: 124. Fourteen days after the boost, immunized mice were transiently depleted for IFN alpha response by intraperitoneal injection with 2 mg anti-IFNAR antibody (MAR1-5A3) and virus inoculation was performed 24 h after treatment with anti-IFNAR antibody. For virus inoculation, mice received intra-peritoneal injection of the French Guyana strain FG15 of ZIKV, using 10.sup.3 pfu per mouse, and viremia was quantified by qRT-PCR at days 1, 2, 3 and 6 after virus inoculation. Four mice were used as control mice (electroporation with an empty vector) and 5 mice were vaccinated with the pZIKV construct (electroporation with the plasmid DNA coding for the chimeric polyepitope of ZIKV). The electroporation settings, using the AgilePulse apparatus (BTX, Harvard apparatus) consisted of 3 Voltage groups: including the first one with 450V, a pulse length of 50 microseconds, a pulse interval of 0.2 microseconds and 1 pulse, the second one with 450V, a pulse length of 50 microseconds, a pulse interval of 50 microseconds and 1 pulse, and a third one with 110V, a pulse length of 10 milliseconds, a pulse interval of 20 milliseconds and 8 pulses.

EXAMPLES

Ethics Statement

[0108] Human blood samples were obtained from healthy adult donors from the Fundación Hematologica Colombia (Bogota D.C., Colombia) in an anonymous manner. All protocols described in this study were approved by the institutional review board (IRB) of the EL Bosque University (Colombia).

Human Blood Samples

[0109] Donors were of both sexes and between 20 and 60 years of age. A total of 82 samples were obtained from different ZIKV-endemic areas near Bogota D.C. (mainly from Villavicencio, Meta) over a time course of three months between October and December 2016. PBMCs were purified by density gradient centrifugation (Lymphoprep™; Stemcell technologies) and resuspended in FBS (Gibco) containing 10% dimethyl sulfoxide and cryopreserved in liquid nitrogen. Eleven of the 82 blood samples obtained had to be excluded from the study due to poor viability of cells.

Viruses and Cell Lines

[0110] The in vitro assays were conducted using the DENV1 KDH0026A (provided by Dr L. Lambrecht, Institut Pasteur, Paris), DENV2 R0712259 (provided by Dr. A. Failloux, Institut Pasteur, Paris), DENV3 KDH0010A (provided by Dr. L. Lambrecht, Institut Pasteur, Paris), DENV4 CRBIP10.4VIMFH4 (from the Institut Pasteur Collection) and ZIKV KU312312 (provided by Dr. Dominique Rousset, Institut Pasteur. Cayenne). All viruses were grown using the Aedes Albopictus mosquito cell line C6/36 cultured in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum containing 0.1 mM non-essential amino acids and 1× tryptose phosphate broth. Vero-E6 cells and DC-SIGN-expressing U937 were kindly provided by Dr M. Flamand and Dr B. Jacquelin (Institut Pasteur, Paris), respectively.

HLA Typing

[0111] Genomic DNA isolated from PBMCs of the study subjects by standard techniques (QIAmp; Qiagen) was used for HLA typing. High resolution Luminex-based typing for HLA class I (alleles A, B and C) and HLA class II (allele DRB1) was used according to the manufacturer's protocol (Sequence-Specific Oligonucleotides (SSO) typing; Immucor, Lifecodes).

Serology

[0112] ZIKV seropositivity was determined using a recombinant antigen-based (EDIII antigen) indirect ELISA, as previously described (Aubry M, et al. 2017, Emerging infectious diseases 23(4):669-672). Briefly, 96-well plates (Nunc, Life Technologies, Rochester, N.Y.) were coated overnight at 4° C. with 50 ng of antigen in PBS. After washing, 200 μl PBS containing 3% skimmed milk and 0.1% Tween-20 were added for 1 hr at 37°. The blocking solution was replaced by 100 μl of plasma diluted 1:500 in PBS containing 1.5% BSA and 0.1% Tween-20, and plates were incubated at 37° C. for 60 min. After three washes, bound antibodies were detected with a horseradish peroxidase-conjugated goat anti-human IgG immunoglobulin (ROCKLAND). Following incubation at 37° C. for 1 hr and three washes, 100 μl of a substrate solution containing TMB (KPL, Eurobio) were added. After 15 min incubation, the optical density (OD) was determined at 650 nm with an automated plate reader (Tecan infinite 200 pro). Each plasma sample was tested in duplicate. Plasma samples obtained from individuals with positive DENV IgG serology collected before the ZIKV outbreak were used as negative controls. The cut-off was calculated from the negative controls and was 0.196. DENV seropositivity was determined by indirect ELISA for IgGs (Panbio; Alere) and by capture ELISA for IgM (Tecnosuma) following the manufacturer's instructions. For further characterization of seropositive donors, and to confirm the specificity of the ELISA, a flow cytometry-based neutralization assay was performed as described previously (Andreatta M, et al. 2015, Immunogenetics 67(11-12):641-650; Nielsen M & Andreatta M 2016, Genome Med 8(1):33). Briefly, 10-fold serial dilutions of plasma samples were incubated at 37° C. for 1 hour with a dilution of virus inducing 7-15% infection. Virus-antibody mixture was then added to U937-DC-SIGN cells for neutralization of DENV1-4 infection, or to Vero cells for neutralization of ZIKV infection, for 2 hours at 37° C. after which cells were washed 2 times with fresh medium and then incubated for 24 h. The cells were then fixed with 4% paraformaldehyde, stained with 4G2 antibody conjugated to Alexa-488, and the percentage of infected cells was measured by flow cytometry. The neutralization titer of antibodies was expressed as the reciprocal dilution of plasma at which 50% of the virus was inhibited. Plasma samples from donors collected before ZIKV outbreak or from negative samples provided from the Kits to detect anti-DENV antibodies did not reveal any neutralization activity against ZIKV or DENV infection, respectively. Following the ELISA and neutralization assays, from the 71 plasma samples selected for this study, a total of nine samples from ZIKV-seropositive individuals and eleven samples from DENV/ZIKV-seropositive individuals were further selected for ELISPOT analysis. The full list of the twenty blood donors included in this study is listed in Table 1.

Viral Sequences

[0113] The identical amino acid sequence of ZIKV from Colombia (GenBank KX087102 and KU820897) was used as a reference for the set of overlapping 15-mer peptides.

[0114] A total of 50 full length protein coding DENV sequences from Colombia (serotype 1: 14 sequences; serotype 2: 16 sequences; serotype 3: 13 sequences; serotype 4: 7 sequences) were retrieved from GenBank and used for pairwise sequence identity comparisons.

Peptides

[0115] All peptides were synthesized by Mimotopes (Victoria, Australia). A total of 853 15-mer peptides overlapping by 11 amino acids and 197 9-mer peptides overlapping by eight amino acids were tested by ELISPOT assay. For the identification of T-cell epitopes, 15-mer peptides were combined into pools of 12 peptides, and individual peptides from the positive pools were tested in a second ELISPOT assay. Following the identification of the positive 15-mer peptides, and according to their HLA class I or class II restriction potential (predicted or shared between at least two donors), 9-mer peptides were synthesized and tested individually.

Ex Vivo IFN-γ ELISPOT Assay

[0116] PBMCs (2×10.sup.5) were incubated in 96-well flat bottom plates (MSIPS 4510, Millipore, Bedford, Mass.) coated with anti-IFN-γ mAb (clone 1-D1K, Mabtech, Sweden) with 0.2 ml of complete RPMI containing 10% human AB serum with pools of 12 peptides (2 μg/ml, final concentration) or individual peptides (1 μg/ml, final concentration) for 20 hours. Following a 20h-incubation at 37° C., the wells were washed with PBS/0.05% Tween 20 and then incubated with biotinylated anti-IFN-γ mAb (clone 7-B6-1, Mabtech) for 1 h 30 mn. The spots were developed using Streptavidin-alkaline phosphatase (Mabtech) and BCIP/NBT substrate (Promega, France) and counted using an automated ELISPOT reader (Immunospot, Cellular Technology Limited, Germany). The number of IFN-γ-producing cells was expressed as spot forming cells (SFC) relative to 1×10.sup.6 PBMCs. Values were calculated by subtracting the number of spots detected in the non-stimulated control wells. Values were considered positive if they were equal to or greater than 20 spots and at least three times above the means of the unstimulated control wells. As a positive control, cells were stimulated with CEF peptide pool (Mabtech).

Immunogenicity and HLA Restrictions Prediction

[0117] The evaluation of binding possibilities of peptides to MHC class I and class II alleles was analyzed using the NetMHCpan3.0 and NetMHCIIpan3.1 servers, respectively (Andreatta M, et al. 2015, Immunogenetics 67(11-12):641-650; Nielsen M & Andreatta M 2016, Genome Med 8(1):33).

Statistics

[0118] All data were analyzed with Prism software version 7.0 (GraphPad Software). Statistical significance was determined using the nonparametric two-tailed Mann-Whitney test to compare two independent groups. Differences were considered significant at P<0.05.

Results

Identification of Immunodominant Regions of the ZIKV Proteome

[0119] To investigate T-cell immunity induced after ZIKV infection, the inventors examined responses from blood donors living in a ZIKV endemic area in gamma interferon (IFN-γ)-specific enzyme-linked immunosorbent spot (ELISPOT) assays. Blood samples from all study participants were tested for the presence of ZIKV IgG and DENV IgM and IgG by ELISA, and for the presence of virus-specific antibodies by flow cytometry-based neutralization assay against ZIKV and the 4 DENV serotypes, and PBMCs from ZIKV-seropositive individuals were HLA-typed. Details of the blood donors included in this study are listed in Table 1. PBMCs from 20 ZIKV-seropositive donors were screened for T-cell reactivity against pools of 15-mer peptides (overlapping by 11 amino acids) spanning the entire ZIKV proteome. Analysis of the response magnitude (as spot forming cells (SFC) per 10.sup.6 cells) and frequency of responding donors revealed that the non-structural (NS) proteins NS1, NS3 and NS5 were the most vigorously and frequently recognized proteins, and accounted for 69% of the total response (FIG. 1A). Strikingly, these NS1, NS3 and NS5 proteins represented 15%, 19% and 35% of the total response, respectively, in ZIKV donors, whereas the NS3, NS4B and NS5 proteins have been reported to account for 31%, 15% and 22% of the DENV-specific T-cell response, respectively (Simmons C P, et al. 2005, J. Virol. 79(9):5665-5675; Duangchinda T, et al. 2010, Proc Natl Acad Sci USA 107(39):16922-16927; Rivino L, et al. 2013, J. Virol. 87(5):2693-2706; Weiskopf D, et al. 2013, Proc Natl Acad Sci USA 110(22):E2046-2053). As these donors were selected in DENV- and ZIKV-endemic areas, and as these viruses share an overall 43% protein sequence identity (with up to 68% for the non-structural proteins), the inventors sought to distinguish between the ZIKV-specific epitopes and those shared by both viruses. Among the 20 ZIKV-seropositive blood donors, 11 individuals had both anti-DENV and anti-ZIKV IgG antibodies and 9 individuals did not reveal any detectable anti-DENV antibodies (Table 1). The inventors thus analyzed separately T-cell responses from donors having only a history of ZIKV infection (ZIKV donors) and those from donors having a history of DENV and ZIKV infections (DENV/ZIKV donors). As shown in FIGS. 1B and 1C, the NS1, NS3 and NS5 proteins accounted for 13%, 31% and 32% of the responses in ZIKV donors, respectively, whereas they accounted for 15%, 16% and 36% of the responses in DENV/ZIKV donors. These results confirmed that NS1, NS3 and NS5 were the main targets for T cells in ZIKV-infected donors, regardless of a previous infection with DENV, and revealed an increase in the frequency and magnitude of the response against NS5 in donors previously infected with DENV, in comparison with donors infected with ZIKV only.

TABLE-US-00001 TABLE 1 Characteristics of the ZIKV patient cohort used for the epitope reactivity study. Serological test Age HLA Genotyping DENY ZIKV Neutralizing activity (Neut50).sup.c Donor.sup.a (yr) Gender HLA-A HLA-B HLA-C DRB1 IgM IgG IgG DENV1 DENV2 DENV3 DENV4 ZIKV 1 41 Male 02:01:01 35:43:01 01:02:01 04:07:01 − − + 17 23 16 25 311 24:02:01 51:01:01 01:02:01 12:01:01 16 20 Female 01:01:01 15:17:01 07:01:01 13:01:01 − + + 158 79 47 357 2270 03:01:01 38:01:01 12:03:01 13:02:01 20 28 Female 01:01:01 07:02:01 07:02:01 04:11:01 + + + 2049 452 214 438 2470 31:01:02 39:05:01 07:02:01 15:01:01 21 26 Male 31.sup.b 35:01 01.sup.b 04:01:01 − − + 13 13 19 14 566 03:01 18:01 17:02 03:01 26 29 Male 02:01:01 07:02:01 01:02:01 14:02:01 − + + 5397 601 735 235 2476 24:02:01 48:01:01 07:02:57 15:01:01 28 35 Female 02:17:01 40:02:01 03:05 04:11:01 − + + 75 39 24 67 5587 29:02:01 44:03:01 16:01:01 07:01:01 33 32 Female 24:02:01 15:46 01:02:01 04:07:01 − + + 306 114 63 51 829 24:02:01 35:31 03:05 04:07:01 35 39 Female 24:02:01 14:01:01 03:05 01:03 − − + 11 13 10 <10 340 68:01:02 40:02:01  05:129 08:02:01 37 34 Male 02:45 35:01:01 04:01:01 01:03 − − + 18 12 35 12 280 11:01:01 50:01:01 06:02:01 13:01:01 42 40 Male 26:01:01 35:01:01 04:01:01 04:02:01 − − + 11 12 25 12 1689 26:01:01 38:01:01 06:76:02 11:04:01 46 25 Male 32:01:01 39:01:01 06:02:01 04:07:01 − − + 14 11 12 11 903 68:01:02 50:01:01 07:02:01 07:01:01 53 54 Female 23:01:01 40:02:01 01:10 07:01:01 − + + 464 116 29 543 3081 31:01:02 44:03:01 04:01:01 08:02:01 55 23 Male 03:01:01 35:01:01 04:11:01 01:01:01 − + + 2395 612 222 301 3196 11:01:01 51:01:01 15:02:01 07:01:01 56 28 Female 02:01:01 15:17:01 05:01:01 03:01:01 − + + 215 55 <10 156 110 02:01:01 18:01:01 07:01:01 11:01:01 59 26 Male 03:01:01 35:43:01 01:02:01 04:01:01 − − + 31 30 16 24 194 24:02:01 40:01:01 03:04:01 04:07:01 60 20 Female 02:05:01 55:01:01 01:02:01 11:01:01 − − + 11 <10 10 10 514 69:01 58:01:01 07:01:01 13:03:01 63 24 Female 02:01:01 07:02:01 07:02:01 15:01:01 − + + 999 187 126 144 4057 23:01:01 51:08:01 17:02:01 15:03:01 66 21 Female 02:01:01 39:01:01 03:02:01 08:02:01 − + + 2572 471 1386 167 2905 03:01:01 40:02:01 07:29:01 15:01:01 69 25 Male 01:01:01 35:01:01 01:02:01 04:07:01 + + + 749 463 961 92 1205 24:02:01 35:43:01 04:01:01 13:05:01 77 18 Female 02:01:01 40:02:01 04:01:01 13:01:01 − − + 17 12 15 33 110 02:01:01 51:01:01 07:01:01 14:02:01 .sup.aDonors 16, 20, 26, 28, 33, 53, 55, 56, 63, 66, and 69 had previous DENV infection .sup.bAllelic variant was not determined .sup.cThe values in each cell are the 50% neutralization titers determined from two replicates of one experiment. The highest titers for each sample is indicated in boldface

[0120] From the 853 peptides spanning the entire ZIKV proteome, 410 peptides elicited a significant T-cell response, some of which being recognized by multiple donors. For most antigenic peptides, the HLA class I and class II alleles of the responding donors coincided with the alleles predicted to bind to this epitope (Andreatta M, et al. 2015, Immunogenetics 67(11-12):641-650; Nielsen M & Andreatta M 2016, Genome Med 8(1):33). Among the epitopes inducing a strong response in ZIKV and DENV/ZIKV donors, several 15-mer peptides contained short sequences predicted to bind strongly to at least one allele expressed by the responding donors (Table 2). For instance, the NS2B.sub.117-131 peptide (having the amino acid sequence as defined in SEQ ID NO: 25) contained a 10-mer sequence (having the amino acid sequence as defined in SEQ ID NO: 26) predicted to bind strongly to the HLA-A*0301 and -A*1101 molecules expressed by the responding donor 55. In other cases, multiple responding donors expressed at least one common allele with strong potential for binding to the stimulating peptide. This hold for the E.sub.455-469 peptide (having the amino acid sequence as defined in SEQ ID NO: 7) in the envelope that contained the 9-mer (having the amino acid sequence as defined in SEQ ID NO: 9) and the 10-mer (having the amino acid sequence as defined in SEQ ID NO: 8) sequences predicted to bind to the HLA-B*5101 and HLA-A*0201 alleles, both alleles being expressed by the responding donors 1 and 77. This also applied to the NS5.sub.13-27 peptide (having the amino acid sequence as defined in SEQ ID NO: 46), which induced a strong response in donors 55 and 69 that shared the HLA-B*3501 allele, this allele being predicted to bind to the 9-mer peptide MSALEFYSY (having the amino acid sequence as defined in SEQ ID NO: 47) with a high affinity. Interestingly, this epitope was also shown to induce a significant response in transgenic mice carrying the HLA-A*0101 molecule, which was expressed by donor 69 (Wen J, et al. 2017, Nat Microbiol 2:17036). Similarly, a strong T-cell response was observed against the NS5.sub.546-560 peptide (having the amino acid sequence as defined in SEQ ID NO: 67) in donors 28, 53, and 66 that expressed the HLA-B*4002 and -B*4403 alleles and against the NS5.sub.605-619 peptide (having the amino acid sequence as defined in SEQ ID NO: 72) in donors 33 and 59 that shared the predicted HLA-A*2402 allele. Finally, the inventors also identified several 9-mer immunodominant epitopes in the NS4B and NS5 proteins, included in the NS4B.sub.112-126 (having the amino acid sequence as defined in SEQ ID NO: 41), the NS5.sub.293-307 (having the amino acid sequences as defined in SEQ ID NOs: 49 and 50), NS5.sub.297-311 (having the amino acid sequences as defined in SEQ ID NOs: 53-55) and NS5.sub.345-359 (having the amino acid sequence as defined in SEQ ID NO: 58) peptides, which induced substantial T-cell responses in donors that shared one or several alleles with a strong potential for binding to these peptides.

[0121] Remarkably, among the NS3 and NS5 proteins, several epitopes have been already described as immunodominant epitopes, either predicted or validated experimentally after DENV infection or vaccination in humans or after ZIKV infection in mice (Wen J, et al. 2017, Nat Microbiol 2:17036; Dar H, et al. 2016, Asian Pac J Trop Med 9(9):844-850; Weiskopf D, et al. 2015, J. Virol. 89(1):120-128; Dikhit M R, et al. 2016, Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 45:187-197). Indeed, among the 9-mer peptides identified in DENV/ZIKV donors, the NS5.sub.293-307 (having the amino acid sequences as defined in SEQ ID NOs: 49 and 50), NS5.sub.297-311 (having the amino acid sequences as defined in SEQ ID NOs: 53-55) and NS5.sub.345-359 (having the amino acid sequence as defined in SEQ ID NO: 58) have been already detected in PBMCs from HLA-B*3501 individuals, after infection with DENV1, DENV2, or vaccination with DENV live attenuated vaccine (DLAV), with a lysine-to-arginine and a phenylalanine-to-tyrosine amino acid substitution at residues 302 and 350 in the NS5.sub.297-311 (having the amino acid sequences as defined in SEQ ID NOs: 53-55) and NS5.sub.345-359 (having the amino acid sequence as defined in SEQ ID NO: 58) peptides from ZIKV, respectively (Rivino L, et al. 2013, J. Virol. 87(5):2693-2706; Weiskopf D, et al. 2015, J. Virol. 89(1):120-128; Imrie A, et al. 2007, J. Virol. 81(18):10081-10091) (Table 2). These results obtained from DENV/ZIKV donors thus confirmed that these NS5 peptides contained nested epitopes restricted by the HLA-B*3501 molecule. Yet the 15-mer NS3.sub.219-233 peptide (having the amino acid sequence as defined in SEQ ID NO: 28), which contained the APTRVVAAEM epitope (having the amino acid sequence as defined in SEQ ID NO: 29), induced a substantial response in 2 DENV/ZIKV donors that expressed neither HLA-B*0702 nor B*3501, although these alleles were expressed in responding donors vaccinated with DLAV or in ifnar−/− HLA-B*0702 transgenic mice after ZIKV infection (Wen J, et al. 2017, Nat. Microbiol. 2:17036; Weiskopf D, et al. 2015, J. Virol. 89(1):120-128). This suggested that the NS3.sub.219-233 peptide (having the amino acid sequence as defined in SEQ ID NO: 28) contained another epitope or a promiscuous epitope that bound to other HLA alleles, besides HLA-B*0702 or B*3501.

TABLE-US-00002 TABLE 2 Characteristics of antigenic peptides from ZIKV (having the amino acid sequences as   defined in SEQ ID NOs: 1-75) identified in this study. SFC/million Pre- PBMC.sup.d Predicted  dicted Score Peptide.sup.a Sequence.sup.b Donors HLA.sup.c 15-mer 9-mer epitope HLA (rank).sup.e C.sub.13-27 IVNM PF 28 A ,29; B40,44;  170 <20 MLKRGVARV A0217 1.9 C03,16; DRB104,07 60 A ,69; B55,58;  50 60 A0205 1.3 C01,07; DRB111,13 C.sub.85-99 KKDL RK 26 A ,24; B07,48;  100 <20 AAMLRIINA A0201 4.5 C01,07; DRB114,15 60 A ,69; B55,58;  <20 75 KDLAAMLRI B5501 1.4 C01,07; DRB111,13 K INARK 28 A ,29; B40,44;  230 65 B4002 2.0 C03,16; DRB104,07 E.sub.455-469 GMSWFSQILIGTLLM 1 A ,24; B35, ;  120 NT GMSWFSQILI A0201 0.9 C01,01; DRB104,12 77 A ; B40,51;  35 NT MSWFSQILI B5101 0.12 C04,07; DRB113,14 NS1.sub.63-77 MEN LNA 21 A31,03; B35,18;  65 50 MENIMWRSVE DRB10405 50 C01,17; DRB104,03 GELNA MENIM A 28 A ,29; B40,44;  245 58 IMWRSVEGEL A0217 0.5 C03,16; DRB104,07 56 A ; B15,18;  70 148 A0201 1.2 C05,07; DRB103,11 NS1.sub.83-97 G VKNPM 26 A ,24; B07,48;  145 23 VQLTVVVGSV A0201 1.7 C01,07; DRB114,15 28 A ,29; B40,44;  165 35 A0217 3 C03,16; DRB104,07 NS1.sub.163-177 FHTS SL 28 A02,29; B40,44;  230 75 HTSVWLKVRE A0101 0.4 C03,16; DRB104,07 DY FHTSV L 55 A03,11; B35,51;  110 125 HTSVWLKVR A3101 0.4 C04,15; DRB101,07 46 A32,68; B39,50;  50 32 A6801 0.6 C06,07; DRB104,07 20 A01,31; B07,39;  310 105 VWLKVREDY A2902 1.3 C07,07; DRB104,15 FHTSVWLKV B3905 0.4 B3901 0.4 NS1.sub.275-289 IRFEE E 33 A24,24; B15, ;  215 55 CPGTKVHVE B3501 8.5 C01,03; DRB104,04 55 A03,11; B ,51;  130 115 B3531 6.5 C04,15; DRB101,07 NS2B.sub.117-131 AAGAWYVYVKTGKRS 55 A03,11; B35,51;  445 NT AAGAWYVYVK A0301 0.6 C04,15; DRB101,07 A1101 0.12 YVYVKTGKR A0301 1.8 NS3.sub.219-233 TVILAPTRVVAAEME 53 A23,31; B40,44;  100 NT TVILAPTRVV DRB10802 1.5 C01,04; DRB107, AAEME 66 A02,03; B39,40;  65 NT ILAPTRVVAA A0201 1.6 C03,07; DRB1 ,15 NS3.sub.271-285 LQPIRVPNYNLYIMD 42 A26,26; B35,38;  165 NT VPNYNLYIM B3501 0.06 C05,06; DRB104,11 NS3.sub.311-325 AAI RDA 28 A02,29; B40,44;  255 85 AAIFMTATPP DRB10401 4 C03,16; DRB104,07 GTRDA FMTATPPGT A0217 5.5 AA TRDA 33 A24,24; B15,35;  215 30 IFMTATPPG A2402 5 C01,03; DRB104,04 NS4A.sub.86-100 VTLGASAWLMWLSEI 55 A03,11; B35,51;  178 NT SAWLMWLSEI B5101 0.9 C04,15; DRB101,07 60 A02,69; B55,58;  125 NT VTLGASAWL A6901 1.3 C01,07; DRB111,13 LGASAWLMW B5801 0.07 NS4B.sub.112-126 MYLIPG 28 A02,29; B40,44;  60 58 AIILLVAHY A2902 0.6 C03,16; DRB104,07 37 A02,11; B35,50;  75 30 A1101 3.5 C04,06; DRB101,13 AII PG 60 A02,69; B55,58;  100 68 LLVAHYMYL A0205 0.3 C01,07; DRB111,13 AIIL IPG 60 A02,69; B55,58;  100 35 LVAHYMYLI A6901 0.15 C01,07; DRB111,13 A0205 0.2 NS5.sub.13-27 KARLNQMSALEFYSY 55 A03,11; B ,51;  260 NT MSALEFYSY B3501 0.15 C04,15; DRB101,07 69 A01,24; B ;  145 NT A0101 0.09 C01,04; DRB104,13 NS5.sub.293-307 WFFDEN 55 A03,11; B ,51;  1580 308 HPYRTWAYH B3501 0.4 C04,15; DRB101,07 W TWAYH 69 A01,24; B ;  40 218 FFDENHPY A0101 1.6 C01,04; DRB104,13 NS5.sub.297-311 E HGSYE 55 A03,11; B ,51;  1280 358 NHPYRTWAY B3501 3 C04,15; DRB101,07 ENHP YE 69 A01,24; B ;  75 188 YRTWAYHGSY B3501 1.7 C01,04; DRB104,13 A0101 0.3 ENHPY E 69 A01,24; B35,35;  75 205 RTWAYHGSY A0101 0.5 C01,04; DRB104,13 NS5.sub.345-359 TDT KEK 33 A24,24; B15, ;  1315 395 TPYGQQRVF B3531 0.7 C01,03; DRB104,04 55 A03,11; B ,51;  2095 523 B3501 0.3 C04,15; DRB101,07 69 A01,24; B ,35;  785 763 C01,04; DRB104,13 NS5.sub.425-439 ALVDKE 28 A02,29; B40,44;  150 100 AVNDPRFWAL A0301 1.1 C03,16; DRB104,07 VDK 55 A03,11; B35,51;  120 125 A1101 0.6 C04,15; DRB101,07 56 A02,02; B15,18;  90 240 C05,07; DRB103,11 NS5.sub.461-475 KKQGEFGKAKGSRAI 28 A02,29; B ,44;  300 NT KKQGEFGKAK DRB10701 32 C03,16; DRB104, GSRAI 53 A23,31; B ,44;  105 NT GEFGKAKGSR B4002 0.7 C01,04; DRB1 ,08 AI NS5.sub.473-487 RAIWYMWLGARFLEF 28 A02,29; B40,44;  210 NT YMWLGARFL A0217 0.03 C03,16; DRB104, 55 A03,11; B35,51;  295 NT AIWYMWLGAR A0301 1.3 C04,15; DRB101, RAIWYMWLGA DRB10701 16 RFLEF NS5.sub.546-560 RFDLENEALITNQME 28 A02,29; B ,44;  245 NT NEALITNQM B4002 0.8 C03,16; DRB104,07 53 A23,31; B ,44;  190 NT B4403 0.6 C01,04; DRB107,08 66 A02,03; B39, ;  80 NT B3901 1.8 C03,07; DRB108,15 NS5.sub.565-579 LALAIIKYTYQNKVV 28 A02,29; B ,44;  240 NT LALAIIKYTY A2902 0.5 C03,16; DRB104,07 53 A23,31; B ,44;  120 NT ALAIIKYTY A2902 0.25 C01,04; DRB107,08 56 A02,02; B15,18;  150 NT LALAIIKYTY B1517 1.2 C05,07; DRB103,11 NS5.sub.605-619 QVVTYALNTFTNLVV 33 A ; B15,35;  240 NT TYALNTFTNL A24:02 0.09 C01,03; DRB104,04 59 A03, ; B35,40;  50 42 YALNTFTNL B35:43 0.4 C01,03; DRB104,04 B35:31 0.25 * The position of peptides were determined according to NCBI Reference Sequence YP_002790881.1; † The underlined and in bold sequence correspond to the 9-mer peptide tested; ‡ The common alleles between donors are underlined and in bold; § Cumulative SFC/million PBMC; NT, not tested; ¶ Calculated using NetMHCpan 3.0 and NetMHCIIpan3.1 servers: for MHC class I, strong binders <0.5, weak binders <2.
Broader Responses with a Higher Magnitude in Donors with Previous DENV Infection

[0122] Given the ZIKV-specific antibody response against NS1 and the low level of CD4 T-cell cross-reactivity between DENV and ZIKV against the E and NS1 proteins (Stettler K, et al. 2016, Science 353(6301):823-826), the inventors compared, among the immunodominant epitopes, the T-cell responses in PBMCs from ZIKV donors with those from DENV/ZIKV donors. First, comparison of the frequency of responding T cells in ZIKV and DENV/ZIKV donors underlined the higher magnitude of response in DENV/ZIKV donors, relative to ZIKV donors (FIGS. 1B and 1C). The number of stimulating peptides per donor, as well as the average response per donor differed in these two groups, with a significantly broader response and a higher magnitude of response in donors with previous DENV infection (FIG. 2A, left and right panels). To determine whether this difference concerned only a small number of peptides that elicited a stronger response in each donor, or if it concerned the majority of the peptides, the inventors plotted the frequency of responses against the different peptides, per donor, in the two different groups. As shown in FIG. 2B, two out of nine individuals among the ZIKV donors revealed a median response higher than 100 SFC/million cells, whereas six out of eleven DENV/ZIKV donors developed this strong response, which was also directed against a higher number of peptides. This result revealed the activation of a higher frequency of T cells against ZIKV peptides, with a higher magnitude of response, in donors previously infected with DENV, in comparison with naïve donors. This strongly argued for the existence of cross-reactive T cells, these T cells being primed during the initial infection with DENV and expanded thereafter during the following infection with ZIKV, as shown recently in mice after sequential infection with DENV and ZIKV (Wen J, et al. 2017, Nat Microbiol 2:17036).

DENV/ZIKV-Cross-Reactive T Cells Mainly Target the NS5 Protein

[0123] To identify more specifically ZIKV-specific peptides and DENV/ZIKV cross-reactive peptides, the inventors compared the sequences of the most immunodominant epitopes recognized by both types of donors. As shown in FIG. 2A and Table 3, NS1 and NS3 proteins contained a high proportion of peptides that elicited strong responses in both ZIKV and DENV/ZIKV donors, whereas the E protein and to a higher extent the NS5 protein contained a majority of peptides inducing a strong response only in DENV/ZIKV donors. This suggested that the NS1 and NS3 proteins contained more ZIKV-specific epitopes, whereas the NS5 protein contained more epitopes shared by DENV and ZIKV and recognized by cross-reactive T cells. Strikingly, most of the peptides recognized only by DENV/ZIKV donors exhibited high degree of identity with the four DENV serotypes. For instance, in the NS1 protein, two out of the five epitopes that induced a response in ZIKV donors revealed a sequence identity higher than 60% with the four DENV serotypes, whereas eight out of the eleven epitopes in the NS5 protein that induced a strong response in DENV/ZIKV donors showed a sequence identity higher than 66.7% with the four DENV serotypes (Table 3). To determine whether the increased magnitude of response was correlated with the recognition of peptides having a higher sequence identity with DENV, the inventors plotted the cumulative responses for each peptide against the percentage of identity between DENV and ZIKV sequences. Among the ZIKV donors, only four ZIKV peptides with about 60% identity with DENV could elicit a response higher than 300 SFC per million cells, whereas twenty-one ZIKV peptides with at least 70% identity with DENV induced this strong response in DENV/ZIKV donors (FIG. 3); the four peptides inducing the strongest T-cell response in these donors shared the highest sequence identity with DENV. Altogether, these data strongly supported the activation of cross-reactive T cells induced after DENV and ZIKV infections, which recognized common epitopes between DENV and ZIKV, and dominated the T-cell response against ZIKV.

TABLE-US-00003 TABLE 3 Immunodominant epitopes in ZIKV and DENV/ZIKV donors (having the amino acid sequences as  defined in SEQ ID NOs: 17, 25, 46, 48, 52, 57, 62, 64, 67 and 76-93) ZIKV DENV/ZIKV SFC/ SFC/ % Identity million million Peptide.sup.a Sequence Donors PBMC.sup.b Donors PBMC.sup.b DENV1 DENV2 DENV3 DENV4 C.sub.49-63 AILAFLRFTAIKPSL 60 60 28,63 365 60.0% 53.3% 60.0% 40.0% E.sub.67-81 DMASDSRCPTQGEAY 33 465 66.7% 53.3% 66.7% 53.3% E.sub.87-101 DTQYVCKRTLVDRGW 56 505 66.7% 53.8% 73.3% 66.7% NS1.sub.19-33 VFVYNDVEAWRDRYK 21, 46, 60 195 28, 56 380 46.7% 33.3% 46.7% 40.0% NS1.sub.55-69 CGISSVSRMENIMWR 35, 46 125 56 275 67.1% 66.3% 60.0% 60.0% NS1.sub.91-105 GSVKNPMWRGPQRLP 21, 35, 46,  275 28 165 13.3% 33.3% 20.0% 33.3% 60 NS1.sub.107-121 PVNELPHGWKAWGKS 28, 53 430 40.0% 46.7% 46.7% 50.5% NS1.sub.147-161 HRAWNSFLVEDHGFG 46 40 33, 53 445 66.7% 73.3% 66.7% 76.2% NS1.sub.163-177 FHTSVWLKVREDYSL 46 35 20, 28, 55 450 46.7% 46.3% 53.3% 46.7% NS1.sub.195-209 HSDLGYWIESEKNDT 28, 33 615 80.0% 73.3% 66.2% 73.3% NS2B.sub.117-131 AAGAWYVYVKTGKRS 55 445 33.3% 33.3% 26.7% 26.7% NS3.sub.131-145 PAGTSGSPILDKCGR 21, 42 405 26, 55, 63 495 53.3% 60.8% 53.3% 54.3% NS3.sub.143-157 CGRVIGLYGNGVVIK 21 350 20, 55, 63,  550 60.0% 66.7% 72.3% 80.0% 66 NS3.sub.311-325 AAIFMTATPPGTRDA 28, 33 470 80.0% 80.0% 93.3% 80.0% NS5.sub.13-27 KARLNQMSALEFYSY 55, 69 405 53.3% 46.7% 53.3% 40.0% NS5.sub.293-307 WFFDENHPYRTWAYH 55, 69 1620 66.7% 66.7% 60.0% 66.7% NS5.sub.297-311 ENHPYRTWAYHGSYE 55, 69 1330 80.0% 80.0% 73.3% 80.0% NS5.sub.325-339 VVRLLSKPWDVVTGV 28, 55, 66 495 73.3% 80.0% 73.3% 66.7% NS5.sub.345-359 TDTTPYGQQRVFKEK 33, 55, 69 4195 93.3% 93.3% 93.3% 93.3% NS5.sub.373-387 QVMSMVSSWLWKELG 60 130 55, 66, 69 340 40.0% 53.3% 46.7% 46.7% NS5.sub.461-475 KKQGEFGKAKGSRAI 28, 53 405 93.3% 93.3% 93.3% 86.7% NS5.sub.465-479 EFGKAKGSRAIWYMW 28, 53, 55,  1085 100.0% 100.0% 100.0% 93.3% 56 NS5.sub.473-487 RAIWYMWLGARFLEF 28, 55 505 100.0% 100.0% 93.3% 100.0% NS5.sub.481-495 GARFLEFEALGFLNE 28, 53, 56,  1870 93.3% 100.0% 93.3% 100.0% 63 NS5.sub.546-560 RFDLENEALITNQME 28, 53, 66 515 60.0% 47.1% 53.3% 60.0% NS5.sub.573-586 TYQNKVVKVLRPAEK 28, 53, 56 615 72.9% 66.7% 73.3% 80.0% NS5.sub.849-863 CGSLIGHRPRTTWAE 60 90 33, 55 340 66.7% 66.7% 66.7% 66.7% † Cumulative SFC/million PBMC * The position of peptides were determined according to NCBI Reference Sequence YP_002790881.1

[0124] In this study, using PBMCs from ZIKV-infected human blood donors, the inventors identified numerous T-cell epitopes that were specific to ZIKV or shared between DENV and ZIKV. While the DENV-specific T-cell responses are predominantly directed against NS3, NS4B and NS5, the response against ZIKV mainly targeted epitopes in the NS1, NS3 and NS5 proteins. The stronger and broader IFN-γ response against peptides from the NS5 protein, observed in donors previously infected with DENV, led the inventors to postulate that this region contained more peptides recognized by cross-reactive T cells, whereas the NS1 protein was preferentially targeted by ZIKV-specific T cells. These data were consistent with the higher percentage of identity observed between ZIKV and DENV sequences in the NS5 protein, in comparison with the NS1 protein. In addition to its sequence identity, the high NS1 secretability observed with the Asian lineages of ZIKV (Liu Y, et al. 2017, Nature 545 (7655): 482-486) could also explain the higher frequency of NS1-specific T cells induced in ZIKV-infected donors, in comparison with the frequency of NS1-specific T cells observed in DENV-infected donors (Weiskopf D, et al. 2013, Proc Natl Acad Sci USA 110(22):E2046-2053).

[0125] For several epitopes, the 15-mer or 9-mer peptides matched epitopes recently identified in transgenic mice expressing human HLA molecules, thus confirming the class I allele restriction for this peptide. This was the case for 15-mer peptide VARVSPFGGLKRLPA (having the amino acid sequence as defined in SEQ ID NO: 92) inducing a response in a donor expressing the HLA-B*0702 allele (data not shown), which contained the C25-35 peptide SPFGGLKRLPA (having the amino acid sequence as defined in SEQ ID NO: 93) shown to elicit a significant response in HLA-B*0702 transgenic mice infected with ZIKV (Wen J, et al. 2017, Nat Microbiol 2:17036). The same correlations were established with NS3 (FPDSNSPIM, having the amino acid sequence as defined in SEQ ID NO: 94), NS4B (RGSYLAGASLIYTVT, having the amino acid sequence as defined in SEQ ID NO: 95) and NS5 (NQMSALEFYSY, having the amino acid sequence as defined in SEQ ID NO: 96) peptides that induced a strong response in human donors expressing the HLA-B*0702 and HLA-A*0101 alleles, respectively (data not shown and Table 2), and in transgenic mice expressing these alleles (Wen J, et al. 2017, Nat Microbiol 2:17036). In other cases, the epitopes identified in HLA-B*0702 and HLA-A*0101 transgenic mice were also identified in responding donors that nevertheless did not express these alleles, such as the NS3.sub.219-233 peptide (having the amino acid sequence as defined in SEQ ID NO: 28) (Table 2) and the NS1.sub.19-33 (having the amino acid sequence as defined in SEQ ID NO: 78) or the NS5.sub.13-27 (having the amino acid sequence as defined in SEQ ID NO: 46) peptides (Table 3), which elicited a response in donors that expressed neither of the two alleles, HLA-B*0702 or HLA-A*0101. For these donors, one possibility could be that the epitope identified in transgenic mice had a higher affinity for a human HLA allele different from the allele expressed by the transgenic mice, or that the 15-mer peptide contained another epitope that bound to a different allele. Binding studies with 9-mer epitopes and HLA class I stabilization assays using TAP-deficient cells should discriminate between these possibilities.

[0126] The inventors also reported the identification of several peptides that shared common sequences with DENV and were preferentially targeted by cross-reactive T cells, after DENV and ZIKV infection. Among these peptides, the NS5.sub.293-307 (having the amino acid sequence as defined in SEQ ID NO: 48) and NS5.sub.297-311 (having the amino acid sequence as defined in SEQ ID NO: 52) peptides contained the amino acid sequence HPYRTWAYH (having the amino acid sequence as defined in SEQ ID NO: 49), which shared seven amino acids with an epitope previously identified in Pacific Islanders infected with DENV1 (Imrie A, et al. 2007, J. Virol. 81(18):10081-10091). Similarly, the NS5325-339 (having the amino acid sequence as defined in SEQ ID NO: 86) peptide contained the amino acid sequence KPWDWTGV (having the amino acid sequence as defined in SEQ ID NO: 97), which was also 66.7% identical to the epitope KPWDVIPMV (having the amino acid sequence as defined in SEQ ID NO: 98) identified in these individuals infected with DENV1 (Imrie A, et al. 2007 J. Virol. 81(18):10081-10091). Finally, the NS5.sub.345-359 (having the amino acid sequence as defined in SEQ ID NO: 58), NS5.sub.465-479 (having the amino acid sequence as defined in SEQ ID NO: 88) and NS5.sub.481-495 (having the amino acid sequence as defined in SEQ ID NO: 89) peptides inducing the strongest response in DENV/ZIKV donors (Table 3) also contained 9-mer epitopes that were previously identified in DENV-infected individuals (Weiskopf D, et al. 2015, J. Virol. 89(1):120-128). Altogether, these data revealed the activation of DENV/ZIKV cross-reactive T cells that dominated the response following sequential DENV and ZIKV infections. Notably, although these cross-reactive peptides exhibited a high degree of sequence identity with DENV and could stimulate a T-cell response after DENV infection, these peptides did not induce a response after primary infection with ZIKV, suggesting that these peptides were immunodominant in the context of DENV but not in the context of ZIKV infection. This result was expected, as the immunodominance of an epitope or its relative abundance depends on the other epitopes expressed by the protein. This was also in agreement with previous observations showing that epitope production correlated with cleavability of flanking residues expressed in the protein sequence (Zhang S C, et al. 2012, J. Immunol. 188(12):5924-5934). Importantly, for these cross-reactive epitopes, the absence of a T-cell response in ZIKV-infected donors was not simply due to the absence of the presenting HLA allele in this population, as most of the alleles expressed in responding DENV/ZIKV donors were also expressed in ZIKV donors (Table 1). This is what the inventors observed for the NS5.sub.13-27 (having the amino acid sequence as defined in SEQ ID NO: 46), NS5.sub.293-307 (having the amino acid sequence as defined in SEQ ID NO: 48), NS5.sub.345-359 (having the amino acid sequence as defined in SEQ ID NO: 57) and NS5.sub.546-560 (having the amino acid sequence as defined in SEQ ID NO: 67) epitopes, predicted to be strong binders to the HLA-B*3501 and HLA-B*4002 alleles, respectively, that were frequently expressed by ZIKV donors (Table 2 and FIG. 3). Altogether, these results showed that, in the case of initial ZIKV infection, there was a preferential recognition of ZIKV-specific epitopes, whereas there was a more frequent and stronger T-cell response against cross-reactive epitopes after heterologous DENV/ZIKV infection. Interestingly, the strong T-cell response observed in DENV/ZIKV donors against these NS5 epitopes relied primarily on donors that expressed the HLA-B*3501 allele, an allele associated with high magnitude responses against DENV, and a stronger protection against DENV infection and disease (Weiskopf D, et al. 2013, Proc Natl Acad Sci USA 110(22):E2046-2053). As all blood samples were obtained from donors with asymptomatic ZIKV infection history, the inventors could not relate the strength of the ZIKV-specific T-cell response obtained in HLA-B*3501 donors to the protection against the disease. Further studies with more subjects with a higher susceptibility to disease following primary ZIKV infection are required to determine whether, as for DENV, there is an HLA-linked protective role for T cells in ZIKV infection. Likewise, it would also be important to compare disease severity in donors having or not experienced a previous DENV infection, to determine whether cross-reactive T cells induced after DENV infection could mediate a better protection against ZIKV infection and disease, as recently suggested in mice (Wen J, et al. 2017, Nat Microbiol 2:17036; Elong Ngono A, et al. 2017, Cell host & microbe 21(1):35-46). As both CD4+ and CD8+ T cells were shown to contribute to protection against DENV infection, a comprehensive analysis of MHC class II-restricted response is needed to determine the role of CD4 in ZIKV infection and disease protection. Finally, further phenotypic analyses of ZIKV-specific T cells, in asymptomatic or symptomatic donors will help in defining correlates of protection in natural immunity and vaccination against ZIKV infection and disease. It will be particularly important to determine whether, as for DENV-specific T cells, strong responses against ZIKV-specific peptides are more frequent in specific HLA alleles and are associated with multifunctionality (Weiskopf D, et al. 2013, Proc Natl Acad Sci USA 110(22):E2046-2053).

[0127] In conclusion, while many studies have focused on the antibody response against ZIKV, more specifically the identification of B cell epitopes shared between ZIKV and DENV, little is known regarding the role of T cells in the control of ZIKV infection. Using PBMCs from blood donors with recent history of ZIKV infection, seropositive or not for DENV, the inventors established the first map of the distribution of ZIKV T-cell epitopes by screening the complete proteome by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assay. The inventors showed that the non-structural proteins NS1, NS3 and NS5 contained most of the immunodominant peptides that induced a strong T-cell response. The inventors also showed that the NS5 protein contained many epitopes shared by both viruses, and which induced the highest response following DENV and ZIKV infections. Strikingly, donors with a history of DENV infection revealed a substantial response against peptides previously identified as DENV CD8+ T-cell epitopes. The strongest T-cell responses observed in these donors corresponded to sequences with a high level of amino acid identity with the four DENV serotypes, suggesting the activation of cross-reactive T cells. These results have crucial implications for future ZIKV and DENV vaccines and provide new opportunities to study the role of ZIKV-specific and DENV/ZIKV shared T-cell epitopes in the induction of long-term immunity against these viruses.

Poly-ZIKV DNA Vaccination in Mice

[0128] DNA immunization will be performed using plasmid coding for the chimeric polyepitope and electroporation, with 2×50 μg DNA, at 3 weeks interval and challenge 15 days after the boost with the virus (intraperitoneal injection of ZIKV with 10.sup.3 pfu/mouse).

[0129] DNA vaccination with plasmids and electroporation (EP) will be performed as follows:

[0130] For vaccination, two injections of 25 μl each of DNA at 2 mg/ml will be performed by intradermal inoculation in the back, followed immediately by electroporation using AgilePulse apparatus (BTX Harvard Apparatus).

[0131] The electroporation procedure will consist of 3 voltage groups:

Group 1: 450V, pulse length 50 μsec, pulse interval of 0.2 μsec, Nb pulses: 1;
Group 2: 450V, pulse length 50 μsec, pulse interval of 50 μsec, Nb pulses: 1;
Group 3: 110V, pulse length 10 msec, pulse interval of 20 msec, Nb pulses: 8.

[0132] At day −1 before the challenge with ZIKV, intraperitoneal injection with 2 mg anti-IFNAR antibody (MAR1-5A3) will be performed to transiently block the IFN type I response.

[0133] The viremia will be quantified by qRT-PCR in plasma samples from day 1 to day 6 after the challenge.

DNA Vaccination in Non-Human Primates (Indian Rhesus Macaques Monkeys)

[0134] DNA vaccination will be performed by 2 intramuscular injections with 1 mg plasmid coding for the chimeric polyepitope of ZIKV, at 0 and 4 weeks, followed by a challenge 4 weeks later by inoculating subcutaneously 10.sup.4 pfu ZIKV, as previously described (Dowd, K. A., et al. Science 2016, 354, 237-240).

[0135] PBMC will be collected at day 0 (before the prime), day 7 after the prime, then at day 35 (one week after the boost) and day 60 (before challenge with ZIKV), to analyse the T cell response (IFN-γ and TNF-α) by ELISpot against overlapping peptides covering the whole chimeric polyepitope sequence. The number and phenotype of Monocytes CD14, DCs, T cells, B cells, and NK cells, and the cytokine profile of T cells (CD8 T cells) will be analysed by intracellular staining.

[0136] Blood samples will also be collected at day 0 (prior to immunization), day 14, day 28 (prior to boost) and day 56 (prior to challenge) to determine neutralizing Ab titres by Focus Reduction Neutralization Titres (FRNT).

[0137] Plasma samples will be tested for quantification of viremia by qRT-PCR, daily from day 56 (prior to challenge) until day 66.

Alternate Protocol Using DNA Vaccination with Plasmids Expressing Cytokines, as Genetic Adjuvants to Remove the Electroporation

[0138] Recent studies have shown that co-administration of plasmids expressing T cell epitopes with plasmids expressing either IL-12, or GM-CSF, or a combination of both IL-12 and GM-CSF improves the T cell response, sufficiently to remove the need for electroporation (EP) (Boyer, J. D., et al. J Med Primatol 2005, 34, 262-270; Suschak, J. J., et al. The Journal of infectious diseases 2018; Suschak, J. J., et al. Antiviral research 2018, 159, 113-121).

[0139] The inventors will thus assess the effect of IL-12 and GM-CSF DNA immunization combined with Poly-ZIKV DNA immunization on the magnitude of T cell response against ZIKV peptides and the immune protection against ZIKV infection in Rhesus monkeys.