Pre-existing Immunity to Japanese Encephalitis Virus Alters CD4 T Cell Responses to Zika Virus Inactivated Vaccine

Abstract

The epidemic spread of Zika virus (ZIKV), associated with devastating neurologic syndromes, has driven the development of multiple ZIKV vaccines candidates. An effective vaccine should induce ZIKV-specific T cell responses, which are shown to improve the establishment of humoral immunity and contribute to viral clearance. Here we investigated how previous immunization against Japanese encephalitis virus (JEV) and yellow fever virus (YFV) influences T cell responses elicited by a Zika purified-inactivated virus (ZPIV) vaccine. We demonstrate that three doses of ZPIV vaccine elicited robust CD4 T cell responses to ZIKV structural proteins, while ZIKV-specific CD4 T cells in pre-immunized individuals with JEV vaccine, but not YFV vaccine, were more durable and directed predominantly toward conserved epitopes, which elicited Th1 and Th2 cytokine production. In addition, T cell receptor repertoire analysis revealed preferential expansion of cross-reactive clonotypes between JEV and ZIKV, suggesting that pre-existing immunity against JEV may prime the establishment of stronger CD4 T cell responses to ZPIV vaccination. These CD4 T cell responses correlated with titers of ZIKV-neutralizing antibodies in the JEV pre-vaccinated group, but not in flavivirus-naïve or YFV pre-vaccinated individuals, suggesting a stronger contribution of CD4 T cells in the generation of neutralizing antibodies in the context of JEV-ZIKV cross-reactivity.

Keywords: CD4 T cell; TCR repertoire; cross-reactivity; flavivirus; vaccine; zika virus.

Figure 1

Methodology for T cell response analysis. (A) Vaccination timeline showing the analysis performed for each time-point; (B) Representative flow cytometry plots showing CD4 and CD8 T cell proliferation analysis based on CellTrace CFSE staining.

Figure 2

Pre-vaccination with JEV vaccine, but not YFV vaccine, alters duration of CD4 T cell memory to ZIKV. CD4 T cell responses against non-conserved (A) and flavivirus conserved (B) peptides from ZIKV structural proteins. (C) CD4 T cell responses against whole viral particle (ZPIV) at week 52. (D) Comparison of magnitudes of CD4 T cell responses at the peak time-point (week 30) between stimulations with different ZIKV peptide pools. (E) CD4 T cell responses against peptides from JEV structural proteins. (F) CD4 T cell responses against peptides from YFV structural and non-structural proteins. Statistical significance was determined by Kruskal-Wallis followed by Dunn's multiple comparison test, except for (D), which Wilcoxon matched-pairs signed rank test was used. P values are represented as *P < 0.05, **P < 0.005, ***P < 0.0005, and ***P < 0.00005.

Figure 3

Greater cytokine production in PBMC from individuals pre-vaccinated with JEV vaccine. Cytokine concentrations were measured from the supernatant of PBMC samples from week 30 after 6 days of stimulation with peptide pools or DMSO (negative control). Statistical significance was determined by Kruskal-Wallis followed by Dunn's multiple comparison test. NC, non-conserved; C, conserved peptides. P values are represented as *P < 0.05 and **P < 0.005.

Figure 4

Cross-reactive CD4 T cell clonotypes elicited by heterologous vaccinations. (A) Morisita-Horn index was calculated to assess the similarity of TCR repertoire of CD4 T cells from before (week 0) and after 3 doses of ZPIV vaccination (week 52) responding to stimulation with peptides from ZIKV proteins (non-conserved and flavivirus-conserved peptides) and peptides from correspondent pre-vaccination (JEV—left panel or YFV—right panel). The heatmap shows the level of similarity between samples in each group compared against each other plotted as columns and rows, hierarchically clustered based on their similarity distances. (B) Circos plots show CD4 T cell clonotypes from participants with highest TCR repertoire similarity in groups pre-vaccinated with JEV (left circos plots) and YFV (right circos plots), in which the links represent identical clonotypes detected at different time-points and stimulations within each individual.

Figure 5

Richness and diversity changes in TCR repertoire of CFSE^low CD4 T cells after ZPIV vaccination. (A) Normalized Shannon Diversity index and (B) Richness index of TCR repertoire after stimulation with non-conserved (blue) or conserved (yellow) peptides from ZIKV proteins in flavivirus-naïve individuals vaccinated with ZPIV after 2 weeks post-1st dose (Week 2), 2 weeks post-2nd dose (Week 6) and 24 weeks post-3rd dose (week 52) of ZPIV vaccine. Normalized Shannon Diversity index of TCR repertoire after stimulation with non-conserved (C) or flavivirus-conserved (D) peptides from ZIKV proteins in individuals pre-vaccinated with JEV (red) or YFV (green). Richness index of TCR repertoire after stimulation with non-conserved (E) or flavivirus-conserved (F) peptides from ZIKV proteins in individuals pre-vaccinated with JEV (red) or YFV (green). (G,H) Richness index comparison between different vaccination groups at week 52. Statistical significance was determined by Wilcoxon matched-pairs signed rank test for (C–F), or by Kruskal-Wallis followed by Dunn's multiple comparison test for (G,H).

Figure 6

Correlation of ZIKV neutralizing antibody titers with CD4 T cell responses. (A) ZIKV microneutralization titers at week 30 (peak) and 52 (last time-point). (B) Correlation of microneutralization titers and CD4 T cell responses against non-conserved epitopes at week 30. (C) Correlation of microneutralization titers and CD4 T cell responses against conserved epitopes at week 30. In (B,C), P-values for each group are shown with same color code from (A).