Innate, T-, and B-Cell Responses in Acute Human Zika Patients

Abstract

Background: There is an urgent need for studies of viral persistence and immunity during human Zika infections to inform planning and conduct of vaccine clinical trials.

Methods: In 5 returned US travelers with acute symptomatic Zika infection, clinical features, viral RNA levels, and immune responses were characterized.

Results: Two pregnant, flavivirus-experienced patients had viral RNA persist in plasma for >44 and >26 days. Three days after symptom onset, transient increases in proinflammatory monocytes began followed at 5 days by transient decreases in myeloid dendritic cells. Anti-Zika virus immunoglobulin M was detected at day 7 after symptom onset, persisted beyond 103 days, and remained equivocal through day 172. Zika virus-specific plasmablasts and neutralizing antibodies developed quickly; dengue virus-specific plasmablasts and neutralizing antibodies at high titers developed only in flavivirus-experienced patients. Zika virus- and dengue virus-specific memory B cells developed in both flavivirus-naive and -experienced patients. CD4+ T cells were moderately activated and produced antiviral cytokines after stimulation with Zika virus C, prM, E, and NS5 peptides in 4/4 patients. In contrast, CD8+ T cells were massively activated, but virus-specific cells that produced cytokines were present in only 2/4 patients assessed.

Conclusions: Acute infections with Zika virus modulated antigen-presenting cell populations early. Flavivirus-experienced patients quickly recalled cross-reactive MBCs to secrete antibodies. Dengue virus-naive patients made little dengue-specific antibody but developed MBCs that cross-reacted against dengue virus. Zika virus-specific functional CD4+ T cells were readily detected, but few CD8+ T cells specific for the tested peptides were found.

Keywords: Zika; flavivirus; immunity; pregnancy; viral persistence.

Figure 1.

Viral loads and phenotypic assays for innate cells, antibody-secreting cells (ASC), and activated T cells. Colored solid symbols and lines identify the 5 acute Zika patients. Open black circles are control data from 5 healthy adults. x-axes for all plots, days post onset of symptoms; y-axes, response magnitudes. Monocytes and dendritic cells (DCs) were gated as in Supplementary Figure S1, and their frequencies are expressed as percentages of total mononuclear cells in blood. ASCs and activated T cells were gated as in Supplementary Figure S2. A, Zika virus (ZIKV) RNA in plasma. B, ZIKV RNA in urine (solid symbols for 4 patients) or whole blood (open diamond; only patient A-23 assessed in this study). C, CD14+CD16+ intermediate monocytes. D, myeloid DC (mDC). Other monocyte subsets, plasmacytoid DC (pDCs), and natural killer (NK) cells showed no clear dynamic trends (not shown). E, ASCs (or plasmablasts). F, Activated CD4+ T cells. G, Activated CD8+ T cells. Abbreviations: ASC, antibody-secreting cells; Ct, cycle threshold for qRT-PCR reactions; DPO, days post onset of symptoms; HLA-DR, human leukocyte antigen-D related; mDC, myeloid dendritic cells; ZIKV, Zika virus.

Figure 2.

Antibody responses. A, Serum anti–Zika virus (ZIKV) immunoglobulin (Ig) M was determined by Zika IgM antibody-capture-enzyme-linked immunosorbent assay (MAC-ELISA) and serum neutralizing antibody (NAb) against ZIKV by focus reduction neutralization test (FRNT). The IgM ELISA values were the ratios of the patients’ serum optical densities to healthy control serum optical densities; <2, negative (lower dashed horizontal line in IgM panel); 2–3, equivocal; >3, positive (upper dashed line). The dashed horizontal line in the NAb panel is the negative cutoff: a titer of <30. The FRNT₅₀ titer is the reciprocal of the serum dilution at which a 50% reduction in viral foci was observed. B, Serum NAb against ZIKV (green symbol and line) or dengue virus-1–4 (black symbols and lines) in the FRNT. Note: subject E-18 also had a past history of extended travel in the tropics. Abbreviations: DENV, dengue virus; DPO, days post onset of symptoms; FRNT, focus reduction neutralization test; Ig, immunoglobulin; NAb, neutralizing antibody; OD, optical density; ZIKV, Zika virus.

Figure 3.

Enzyme-Linked ImmunoSpot (ELISpot) assays for Zika virus (ZIKV)– and dengue virus (DENV)–specific antibody-secreting cells (ASCs) and memory B cells (MBCs). A, Antigen-specific ASCs were quantitated using fresh peripheral blood mononuclear cells (PBMCs). For patient A-23, 3 isotypes of ZIKV-specific immunoglobulin (Ig; M, A, and G) were detected in this flavivirus-naive patient (and for patient B-17, also flavivirus-naive). In contrast, for patient C-16 (flavivirus-experienced), only IgG-secreting ASCs were detected; also true of patient E-18 (also flavivirus-experienced). B, MBCs against ZIKV or DENV were detected in thawed PBMCs. In these assays absolute levels of ZIKV-specific vs DENV-specific ASCs or MBCs cannot be directly compared since the ZIKV antigen was viral lysate and the DENV antigens were recombinant E proteins. Abbreviations: ASC, antibody-secreting cells; DENV, dengue virus; DPO, days post onset of symptoms; Ig, immunoglobulin; LOD, limit of detection; MBC, memory B cell; PBMC, peripheral blood mononuclear cell; ZIKV, Zika virus.

Figure 4.

T-cell intracellular cytokine staining (ICS) assays. CD4+ or CD8+ T cells were stimulated for 6 hours with peptide pools spanning Zika virus (ZIKV) C, prM, E, or NS5 proteins. Thawed peripheral blood mononuclear cells from various days post symptom onset were used in these assays. Antiviral cells expressing intracellular cytokines (interferon [IFN]-g, interleukin [IL]-2, and/or tumor necrosis factor-a) were then detected by ICS and flow cytometry. A and B, ZIKV C-, prM-, and/or E-specific cytokine-producing CD4+ or CD8+ T cells. Summary results are presented here as pooled total cytokine-producing cells analyzed in a Boolean analysis. C, FACS plots demonstrating C-specific CD4+ and CD8+ T cells for patient A-23. CD8+ cells produced primarily IFN-g, whereas CD4+ cells produced both IFN-g and IL-2. D. NS5-specific T cells were assessed with available cells for 3 patients. Closed symbols, CD4+ T cells; open symbols, CD8+ T cells. Patent D-19’s result was negative (as defined in Supplementary Table S1 footnote). E, Polyfunctionality of the CD4+ T cells. The pie charts display the relative proportions of CD4+ T cells producing 1, 2, and 3 cytokines (CTK) (Boolean analysis); 3 CTK (blue), percentage of cells producing all 3 cytokines; 2 CTK (red), cells producing 2 cytokines; and 1 CTK (green), cells producing a single cytokine. Abbreviations: CTK, cytokine; DPO, days post onset of symptoms; FACS, fluorescence activated cell sorter; ICS, intracellular cytokine staining; IFN, interferon; IL, interleukin; ZIKV, Zika virus.