Zika virus (ZIKV) is an emerging virus of the Flaviviridae family, transmitted by Aedes species mosquitoes. The high level of dengue virus (DENV) seroprevalence in areas where ZIKV is circulating and the immunological cross reactivity between both viruses have raised concerns on the risk of increased disease severity for patients with a history of previous DENV infection. To determine the role of DENV pre-immunity in ZIKV infection and disease outcome, the aim of this project was to analyze the specificity and strengths of T and B cell responses against ZIKV. Using PBMC from blood donors with previous history of DENV/ZIKV or ZIKV infection, we have identified ZIKV epitopes by screening T-cell responses against 15-mer overlapping peptides spanning the ZIKV proteome by IFN-gamma enzyme-linked immunospot (ELISPOT) analysis. Our results show that the non-structural proteins NS1, NS3 and NS5 contain most of the immunodominant peptides that induce a strong T-cell response. Interestingly, in donors with a history of DENV infection, specific peptides were also identified as DENV CD8+ T-cell epitopes and the strongest T-cell responses observed in these donors corresponded to sequences with a high level of amino acid identity with the four serotypes of DENV. These results strongly support the activation of cross-reactive T-cells in this context. We also developed a flow cytometry-based assay to quantify the neutralizing and enhancing activities of antibodies against DENV and ZIKV infections. Using plasma samples from the same donors, we have shown that DENV-induced antibodies can enhance ZIKV infection. In addition, all samples from ZIKV-infected donors exhibited neutralizing activity only against ZIKV, and one donor showed enhancing activity for DENV4 infection. The highest neutralizing activity against ZIKV infection was observed in samples from donors with previous DENV and ZIKV infection, strongly suggesting the induction of cross-reacting antibodies induced upon sequential DENV and ZIKV infection. These data have crucial implications for future ZIKV and DENV vaccines and provide new opportunities to study the role of subsets of DENV- or ZIKV-specific T cells in the induction of broadly neutralizing antibodies in the context of sequential flavivirus infections, which could modulate disease severity.
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