Impact of pre-existing dengue immunity on human antibody and memory B cell responses to Zika

Abstract

Little is known about enduring memory B cell (MBC) responses to Zika virus (ZIKV) and their relationship with circulating antibodies. Here we comprehensively assess MBC frequency and specificity alongside serum binding and neutralizing antibody responses to ZIKV ~2 weeks and ~8 months postinfection in 31 pediatric subjects with 0, 1 or >1 prior infections with the related dengue virus (DENV). ZIKV infection elicits a robust type-specific MBC response, and the majority of late convalescent anti-ZIKV serum neutralizing activity is attributable to ZIKV-specific antibodies. The number of prior DENV infections does not influence type-specific or cross-reactive MBC responses, although ZIKV has the highest cross-reactivity with DENV3. DENV cross-reactive MBCs expanded by ZIKV infection decline in number and proportion by late convalescence. Finally, ZIKV induces greater cross-reactivity in the MBC pool than in serum antibodies. Our data suggest immunity to DENV only modestly shapes breadth and magnitude of enduring ZIKV antibody responses.

Fig. 1

Type-specific and cross-reactive IgG-secreting MBC in ZIKV+ patients. MBC responses in patients with RT-PCR-confirmed ZIKV infection and no (DENV-naïve/ZIKV), one (DENV-ZIKV), or more than one (+DENV-ZIKV) prior DENV infection(s) were analyzed by the Multi-Color FluoroSpot assay at ~14 days and ~8 months postinfection. a Representative images of activated MBCs from patient 6188 (DENV-naïve/ZIKV) after ~14 days postinfection. Antigen specificity of MBC responses to ZIKV and the four DENV serotypes are shown as spots represented in each filter. The total ASC of activated MBCs was analyzed in parallel (Total IgG). b Proportion of antigen-reactive MBCs to ZIKV, ZIKV and DENV, and DENV in representative patients from the DENV-naïve/ZIKV, DENV-ZIKV, and +DENV-ZIKV groups at ~14 days and ~8 months postinfection. ZIKV-TS, MBCs that react with ZIKV and not with any DENV serotype; ZIKV&DENV-CR, MBCs that react with ZIKV and at least with one DENV serotype; DENV, MBCs that react with one or more DENV serotypes but not with ZIKV

Fig. 2

ZIKV type-specific MBC response is maintained over time in ZIKV+ patients. ZIKV type-specific MBC cell responses in ZIKV-infected patients with no (DENV-naïve/ZIKV), one (DENV-ZIKV), or more than one (+DENV-ZIKV) prior DENV infection. a, b ZIKV type-specific IgG-secreting MBC response over total antigen response at ~14 days (a) and ~8 months (b) postinfection. c, d ZIKV type-specific IgG-secreting MBC response over total ASC response at ~14 days (c) and ~8 months (d) postinfection. The median and interquartile range (IQR) are shown for all graphs. Significance was determined by Kruskal–Wallis test and Dunn’s test; *p < 0.05, **p < 0.01, ***p < 0.001. DENV-naïve/ZIKV, n = 11; DENV-ZIKV, n = 12; +DENV-ZIKV, n = 7 at ~14 days postinfection (one PBMC sample was not viable) and n = 8 at ~8 months postinfection

Fig. 3

ZIKV and DENV cross-reactive MBCs decrease over time in ZIKV+ patients. ZIKV and DENV cross-reactive B cell responses in ZIKV-infected patients with no (DENV-naïve/ZIKV), one (DENV-ZIKV), or more than one (+DENV-ZIKV) prior DENV infection are shown. a, b ZIKV and DENV cross-reactive IgG-secreting MBC responses over total antigen response at ~14 days (a) and ~8 months (b) postinfection. c, d ZIKV and DENV cross-reactive IgG-secreting MBC response over total ASC response at ~14 days (c) and ~8 months (d) postinfection. Median and IQR are shown for all graphs. Significance was determined by Kruskal–Wallis test and Dunn’s test; *p < 0.05, **p < 0.01, ***p < 0.001. DENV-naïve, n = 11; DENV-ZIKV, n = 12; +DENV-ZIKV, n = 7 at ~14 days postinfection (one PBMC sample was not viable) and n = 8 at ~8 months postinfection

Fig. 4

The number of previous DENV exposures does not affect type-specific and cross-reactive MBC responses. Comparison of ZIKV type-specific and ZIKV and DENV cross-reactive responses in ZIKV-infected patients with one (DENV-ZIKV) or more than one (+DENV-ZIKV) prior DENV exposure at ~14 days and ~8 months postinfection. a Proportion of ZIKV type-specificity (TS) of DENV-ZIKV and+DENV-ZIKV subjects over total antigen response. b Proportion of ZIKV & DENV cross-reactivity (CR) of DENV-ZIKV and +DENV-ZIKV subjects over total antigen response. c Frequency of type-specific responses of DENV-ZIKV and+ DENV-ZIKV subjects over total activated MBCs (ASC). d Frequency of cross-reactive responses of DENV-ZIKV and +DENV-ZIKV subjects over total ASC. The median and IQR are shown for all graphs. Statistical analysis was performed by Mann–Whitney test comparing DENV-ZIKV and +DENV-ZIKV at each time point, but no significant differences were found among different groups. DENV-naïve, n = 11; DENV-ZIKV, n = 12; +DENV-ZIKV, n = 7 at ~14 days postinfection (one PBMC sample was not viable), and n = 8 at ~8 months postinfection

Fig. 5

ZIKV infection elicits a strong type-specific binding response in serum antibodies. Analysis of the total ZIKV response in MBCs and serum from ZIKV-infected patients with no (DENV-naïve/ZIKV), one (DENV-ZIKV), or more than one (+DENV-ZIKV) prior DENV infection. a Proportion of ZIKV type-specific (TS) and cross-reactive (CR) responses over total ZIKV MBC response at ~8 months post-ZIKV infection. b Proportion of ZIKV type-specific (TS) and cross-reactive (CR) responses over total ZIKV serum antibody-binding response at ~8 months post-ZIKV infection. DENV-reactive antibodies were depleted from patient sera to obtain the proportion of the ZIKV response that is due to only ZIKV type-specific antibodies. c Correlation of the proportion of ZIKV TS responses in MBCs and serum. The median and IQR are shown for a and b. Significance was determined by Mann–Whitney test for a and b, and correlation analysis in c was performed using Spearman r test. *p < 0.05, **p < 0.01, ***p < 0.001. The sample size for each group is as follows: DENV-naïve/ZIKV MBC, n = 13 and serum n = 10; DENV-ZIKV, MBC n = 12 and serum n = 11; +DENV-ZIKV MBC n = 8 and serum n = 7

Fig. 6

Depletion of DENV antibodies in serum does not have a significant effect on ZIKV neutralization. Neutralization analysis of DENV-depleted serum samples from ZIKV-infected patients with no (DENV-naïve/ZIKV), one (DENV-ZIKV), or more than one (+DENV-ZIKV) prior DENV infection. a ZIKV neutralizing antibody titer (FRNT₅₀) in DENV-depleted versus control (BSA)-depleted samples for each group of ZIKV-infected patients at ~8 months postinfection. Lines connect results from each individual. b Statistical analysis of ZIKV neutralizing titers before and after depletion of DENV-reactive antibodies at ~8 months postinfection. Statistical analysis was performed by Wilcoxon test, but no significance differences were found among nondepleted and DENV-depleted ZIKV neutralizing antibody titers. c ZIKV neutralization antibody titers before and after depletion of DENV-reactive antibodies from three representative subjects analyzed in a. d Analysis of ZIKV type-specificity (TS) and ZIKV and DENV cross-reactivity (CR) in total ZIKV neutralizing response. Median and IQR is shown for d, and significance was determined by Mann–Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001. Neutralizing antibody titers were log₁₀ transformed for the analysis of a–c. DENV-naïve/ZIKV, n = 10; DENV-ZIKV, n = 10; +DENV-ZIKV, n = 8

Fig. 7

ZIKV infection displays highest cross-reactivity to DENV3. The MBC cross-reactivity between ZIKV and DENV was analyzed for each DENV serotype individually. The analysis was performed in the DENV-naïve/ZIKV+ and DENV-infected/ZIKV+groups. a, b Proportion of ZIKV cross-reactivity to DENV serotypes in DENV-infected/ZIKV + individuals at ~14 days (a) and ~8 months (b) post-ZIKV infection. c, d Proportion of ZIKV cross-reactivity to DENV serotypes in DENV-naïve/ZIKV + patients at ~14 days (c) and ~8 months (d) post-ZIKV infection. e Venn diagrams of type-specific and cross-reactive responses to ZIKV and DENV in MBCs of activated PBMCs from patient 4006 at ~8 months post-ZIKV infection. f DENV-depleted and nondepleted (BSA control) serum responses from patient 4006 at ~8 months post-ZIKV infection. The median and IQR are shown for a–d. Significance was determined by ordinary one-way Anova Multiple Comparison. *p < 0.05, **p < 0.01, ***p < 0.001. DENV-naïve/ZIKV+, n = 12; DENV-infected/ZIKV+, n = 17