An Epidemic Zika Virus Isolate Drives Enhanced T Follicular Helper Cell and B Cell-Mediated Immunity

Abstract

Zika virus (ZIKV) is a mosquito-borne pathogen that recently caused a series of increasingly severe outbreaks. We previously demonstrated that, compared to a pre-epidemic isolate (ZIKV^CDN), a Brazilian ZIKV isolate (ZIKV^BR) possesses a novel capacity to suppress host immunity, resulting in delayed viral clearance. However, whether ZIKV^BR modulates CD4 T cell responses remains unknown. Herein, we show that, in comparison to (ZIKV^CDN) infection, CD4 T cells are less polarized to the Th1 subtype following ZIKV^BR challenge in mice. In contrast, we observed an enhanced accumulation of T follicular helper (Tfh) cells 10, 14, and 21 dpi with ZIKV^BR. This response correlated with an enhanced germinal center B cell response and robust production of higher avidity neutralizing antibodies following ZIKV^BR infection. Together, our data suggest that contemporary ZIKV strains have evolved to differentially induce CD4 T cell, B cell and antibody responses and this could provide a model to further define the signals required for Tfh cell development.

INTRODUCTION

Zika virus (ZIKV) was first isolated in the Zika forest region of Uganda in 1947 (1, 2). Very little was known about the virus during the 20th century and it was linked to mostly innocuous symptoms. Improving our understanding of the immune response to ZIKV infection has become a major research priority following a series of increasingly severe outbreaks in the Federated States of Micronesia (2007), French Polynesia (2013) and South and Central America (with other outbreaks world-wide; 2015) (3–6). These outbreaks, together with smaller outbreaks that have occurred since 2015, have demonstrated the novel capacity of ZIKV to cause wide-spread disease and severe symptoms following infection. In particular, ZIKV infection has been associated with Guillain-Barré syndrome (an autoimmune paralysis) in adults, and fetal microcephaly along with a number of other developmental defects when infection occurs during pregnancy (7, 8). Together, these outbreaks and the symptoms that have become associated with infection raise the question of whether recent, epidemic ZIKV isolates have a distinct capacity to modulate host immunity.

CD4 T cells represent an important part of the host immune response against viral infections with multiple roles during primary infection. Following activation, CD4 T cells can differentiate into various T helper (Th) cell subsets, which serve key functions in supporting the immune response. In the context of viral infection, IL-12 and IFN-γ lead to the generation of Th1 CD4 T cells (9). These cells express the T-box transcription factor T-bet and support the antiviral immune response by secreting effector cytokines such as IFN-γ, TNF-α and IL-2 and by promoting CD8 T cell activation via dendritic cell (DC) licensing (9, 10). T follicular helper (Tfh) cells are an additional CD4 T cell subset that play an important role in promoting antibody responses during viral infections. Tfh cells are defined by expression of CXCR5 and PD-1 on the cell surface, as well as the transcription factor B cell lymphoma-6 (Bcl-6) (11). Canonically, pre-Tfh cells must interact with both DCs and B cells for differentiation, and cytokine-dependent and -independent signals contribute to their development (11). Once differentiated, Tfh cells are able to form or enter germinal centers (GC), distinct structures in secondary lymphoid tissues within which Tfh cells mediate GC B cell affinity maturation and proliferation (11–14).

Several studies have explored CD4 T cell responses during ZIKV infection, in a variety of mouse models (15). In immunocompetent C57BL/6 mice, our group has demonstrated that infection with a pre-epidemic Canadian ZIKV isolate (ZIKV^CDN, PLCal_ZV, Genbank accession KF993678, isolated in 2013 from a Canadian traveler returning from Thailand and exhibiting mild symptoms, https://www.ncbi.nlm.nih.gov/nuccore/KF993678) leads to a robust Th1 CD4 T cell response 7 days post-infection (dpi), characterized by upregulation of T-bet, and production of IFN-γ, TNF-α and IL-2 (16). Experiments using a Cambodian ZIKV isolate (FSS13025) in LysMCre⁺Ifnar^fl/fl mice, which lack IFN-α/β receptor (IFNAR) expression primarily in mature macrophages and granulocytes, revealed a similar Th1 polarization of CD4 T cells, and also identified formation of a Tfh cell response (17). Although CD4 T cells were required for generation of an IgG response, their depletion had no impact on viral burden or CD8 T cell responses (17). In contrast, depleting CD4 T cells from highly susceptible IFNAR knock-out (KO) mice caused higher viral loads, more severe paralysis and reduced survival in 10–12 week old mice infected with a Puerto Rican isolate (PRVABC59), and lethal infection in 3–4 week old mice infected with a Brazilian isolate (PE243) (18, 19). The Tfh cell response to ZIKV was more closely examined in a recent paper by Liang et al., who described a robust Tfh cell response to a Chinese isolate (SZ-WIV01) characterized by IFN-γ production, IgG2c production, and long-lasting antibody responses in C57BL/6 mice treated with an IFNAR blocking antibody (20). Although these studies collectively suggest an important role for CD4 T cells during ZIKV infection, most were carried out in immunodeficient mouse models. Further, none of these studies have compared whether pre-epidemic and epidemic ZIKV isolates differ in their capacity to induce Th1 or Tfh cell responses.

Previously, we have demonstrated that an epidemic ZIKV isolate from Brazil (ZIKV^BR, HS-2015-BA-01, Genbank accession KX520666, isolated in 2015 from a symptomatic patient in Salvador, Bahia, Brazil, https://www.ncbi.nlm.nih.gov/nuccore/KX520666) actively suppresses the CD8 T cell response to infection, which correlates with delayed virus clearance, when compared to rapid control of infection with ZIKV^CDN (21). While virus is detectable by plaque assay in the spleen and kidney at 7 dpi in mice infected with ZIKV^BR, it is cleared from these organs by 14 dpi indicating that while ZIKV^BR establishes a more sustained infection it does not become chronic in mice (21). Herein, we sought to determine whether ZIKV^BR modulates CD4 T cell responses, and to determine potential mechanisms for viral clearance following ZIKV^BR infection. Our data demonstrate that ZIKV^CDN induces a more robust antigen-experienced, Th1-polarized CD4 T cell response than ZIKV^BR 7 dpi. In contrast, ZIKV^BR-infected mice were more efficient at inducing Tfh and GC B cell responses 10 dpi, which persisted through 14 and 21 dpi. These responses correlated with enhanced formation of GCs in the spleen, and production of higher avidity antibodies with greater neutralization potential, likely contributing to the control of ZIKV^BR infection. Conversely, ZIKV^CDN infection favored the induction of extrafollicular B cell responses which correlated with the production of lower quality virus-specific antibodies with reduced neutralization capacity. Together, these data suggest that ZIKV^BR may have evolved to dampen T cell responses that prevent rapid viral clearance, while simultaneously evoking more potent T-dependent humoral immunity that drives a contained, yet more sustained infection.

MATERIALS AND METHODS

Cell Lines

Vero cells (African Green monkey kidney epithelial cells, provided by Steven Varga, The University of Iowa) were cultured in DMEM (Wisent) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Wisent), 1% L-glutamine (Wisent), 1% Penicillin-Streptomycin (Wisent) and 1% non-essential amino acids (Sigma) at 37°C and 5% CO₂.

Viruses

Low passage (p.4) ZIKV^CDN (PLCal_ZV, Genbank accession KF993678, https://www.ncbi.nlm.nih.gov/nuccore/KF993678) derived from a ZIKV-infected traveller returning to Canada who acquired the infection in Thailand in 2013 was provided by Gary Kobinger and David Safronetz (National Microbiology Laboratory and Public Health Agency of Canada) and confirmed via sequencing to be of the Asian lineage (22). Low passage (p.4) ZIKV^BR (HS-2015-BA-01, Genbank accession KX520666, https://www.ncbi.nlm.nih.gov/nuccore/KX520666) isolated in August 2015 in Salvador, Bahia was provided by Mauro Teixeira (Universidade Federal de Minas Gerais). The stock was originally passaged 3 times in C6/36 mosquito cells and once in Vero cells. To propagate ZIKV stocks, 6×10⁶ Vero cells were seeded in 150mmx25mm dishes (Sigma), and 24 hours later infected at a multiplicity of infection (MOI) of 1 for 2 hours in unsupplemented EMEM. After 2 hours, infection media was removed and replaced with 15 mL of DMEM supplemented with 2% heat-inactivated FBS, 1% L-glutamine, 1% non-essential amino acids, 1% Penicillin-Streptomycin and 15mM Hepes. Supernatants were harvested after 48–72 hours post-infection (hpi), centrifuged for 10 minutes at 3000g, aliquoted and stored at −80°C. Viral stocks were titered by plaque assay on Vero cells. Briefly, viral stocks were serially diluted (10-fold) in EMEM (Wisent) and 100 μL of each dilution was used to infect confluent monolayers of Vero cells in 1 mL of unsupplemented EMEM. At 2 hpi, infection media was removed, and cells were overlaid with 1.2% carboxymethyl cellulose (Sigma-Aldrich) and 2% heat-inactivated FBS in EMEM for 4 days prior to fixation for 1 hour with an equal volume of 10% formaldehyde. Monolayers were rinsed gently with distilled water and stained for 30 minutes with 0.1% crystal violet prior to counting plaques. ZIKV^CDN was UV-inactivated by transferring 1 mL of ZIKV into one well of a 6-well plate and exposing it to 3 Joules/cm² of UV irradiation in a UVC 500 Crosslinker (Hoefer) as previously described (16). UV-inactivation was confirmed by plaque assay.

Mouse Experiments

C57BL/6 mice (WT) were purchased from Charles River laboratories, the In Vivo Therapeutic Core at the Indiana University School of Medicine or bred at McGill University. Infected mice were housed in biocontainment level 2 and all animal procedures were carried out in accordance with the Canadian Council on Animal Care and were approved by the McGill University Animal Care Committee or the Indiana University Institutional Animal Care and Use Committee. 6–12-week-old mice of both sexes were used for all experiments.

ZIKV^CDN (p.8 or 9) or ZIKV^BR (p.5 or 6) were injected i.v. at 1×10⁶ PFU. Mice injected with UV-inactivated ZIKV received a dose of inactivated virus equivalent to 1×10⁶ PFU. Mock-infected mice were i.v. injected with an identical volume of mock-infected Vero cell culture supernatant (prepared in parallel to ZIKV stocks).

Flow Cytometry Antibodies

The following antibodies were used in an appropriate combination of fluorochromes: B220 (clone RA3–6B2, BioLegend), Bcl-6 (clone K112–91, BD Biosciences), CD4 (clone GK1.5, BioLegend), CD8α (clone 53–6.7, BioLegend), CD11a (clone M17/4, BioLegend), CD19 (clone 1D3/CD19, BioLegend), CD21 (Clone 7E9, BioLegend), CD23 (Clone B3B4, BioLegend), CD49d (clone R1–2, BioLegend), CD86 (Clone GL-1, BioLegend), CXCR5 (clone L138D7, BioLegend), FoxP3 (clone FJK-16s, eBioscience), GL7 (clone GL7, BioLegend), IFN-γ (clone XMG1.2, BioLegend), IgD (clone 11–26c.2a, BioLegend), IL-2 (clone JES6–5H4, BioLegend), IL-5 (clone TRFK5 BioLegend), IL-17A (clone TC11–18H10.1, BioLegend), PD-1 (clone 29F.1A12, BioLegend), T-bet (clone 4B10, BioLegend), TNF-α (clone MP6-XT22, BioLegend), and appropriate isotype controls.

Flow Cytometry Staining and ex vivo Restimulation

Spleens were isolated and mechanically disrupted to generate single-cell suspensions. Erythrocytes were lysed with Ammonium-Chloride-Potassium (ACK) buffer (0.15 M NH₄Cl, 1 mM KHCO₃, and 0.1mM Na₂EDTA in distilled H₂O, pH 7.2), cells were incubated with TruStain fcX (anti-mouse CD16/CD32, clone 93, BioLegend) and stained with the indicated antibodies, followed by fixation using IC Fixation Buffer (eBioscience). Intracellular staining for transcription factors was performed using the FoxP3/Transcription Factor Staining Buffer Set (eBioscience), as per the manufacturer’s instructions. Samples were analyzed with a BD LSRFortessa flow cytometer (BD Biosciences) and FlowJo software (Tree Star).

For ex vivo restimulation, spleens were harvested and processed as above. Erythrocytes were lysed with ACK buffer and samples were simulated for 5.5 hours at 37°C with 5% CO₂ in the presence of 1 μg per well of plate bound anti-CD3ε antibody (clone 145–2C11, BioLegend) or media alone and brefeldin A (BioLegend). Anti-CD3ε was bound to the plate by incubating overnight in PBS at 4°C, and wells were washed with PBS prior to adding cells. Cells were then stained with surface antibodies and fixed with IC Fixation Buffer (eBioscience), followed by intracellular staining for cytokines in Perm/Wash Buffer (eBioscience). Samples were analyzed with a BD LSRFortessa flow cytometer (BD Biosciences) and FlowJo software (Tree Star).

Confocal Microscopy

Spleens from uninfected, ZIKV^CDN- and ZIKV^BR-infected mice were cut in half, submerged in optimal cutting temperature (OCT) compound, flash frozen with liquid nitrogen vapour and stored at −20°C. The tissues were sectioned into 5 μm slices using a Cryostat. Spleen sections were fixed in an ice-cold acetone solution (75% acetone and 25% ethanol) for 5 minutes. The sections were then placed in PBS and stained in a dark, humid chamber at room temperature. Sections were blocked for 1 hour in 2% BSA with Fc-block (1:100, clone 2.4G2) and then stained in 2% BSA with CD4 (clone GK1.5, eBioscience), GL7 (clone 81E2, BD Pharmingen) and IgD (clone 11–26c, eBioscience) for 1 hour. The slides were then washed in PBS for 7 minutes twice while agitated in the dark and mounted using ProLong Diamond Antifade (Invitrogen). Images were taken using a Zeiss LSM780 laser scanning confocal microscope and analyzed using Fiji software. Using the Zeiss tiling software, 4 by 4 images were taken to count GCs. 5–13 tiled images were taken for each spleen and the average number of GL7⁺ GCs per image for each spleen was graphed.

Plaque Reduction Neutralization Test

To extract serum, blood was collected via cardiac puncture and centrifuged at 7,000 rpm for 10 minutes at 4°C. Serum was diluted 25-, 50-, 100-, 250-, and 500-fold in EMEM, or serially diluted 10-fold in EMEM, and mixed with the corresponding ZIKV isolate. 100 μL of each serum/virus mixture was used to infect confluent monolayers of Vero cells in 1 mL of unsupplemented EMEM. Each 6 well plate included one virus only control well. At 2 hpi, infection media was removed, and cells were overlaid with 1.2% carboxymethyl cellulose (Sigma-Aldrich) and 2% heat-inactivated FBS in EMEM for 4 days prior to fixation for 1 hour with an equal volume of 10% formaldehyde. Monolayers were rinsed gently with distilled water and stained for 30 minutes with 0.1% crystal violet prior to counting plaques. Percent inhibition was calculated as:

$$\%\mathit{\text{Inhibition}}=\frac{\left((\#\mathit{\text{Plaques}}\mathit{\text{in}}\mathit{\text{No}}\mathit{\text{Serum}}\mathit{\text{Well}}-\#\mathit{\text{Plaques}}\mathit{\text{in}}\mathit{\text{Indicated}}\mathit{\text{Dilution}}\mathit{\text{Well}})\right)}{\#\mathit{\text{Plaques}}\mathit{\text{in}}\mathit{\text{No}}\mathit{\text{Serum}}\mathit{\text{Well}}}\times 100\%$$

Standard and antibody avidity ELISA

To measure virus-specific IgG levels, Thermo Scientific Maxisorp plates (Thermo Fisher Scientific) were coated with 1×10⁵ PFU of heat-inactivated (30 minutes at 56°C) ZIKV^CDN or ZIKV^BR overnight at 4°C. Following incubation, plates were washed with 2x with PBS-0.1% Tween and blocked for 2 hours in 5% milk diluted in PBS-0.1% Tween at room temperature. Serum was collected from naïve or infected mice as described above and diluted in 5% milk PBS-0.1% Tween and incubated for 1 hour at room temperature (serum from infected mice was incubated in wells coated with the matching virus isolate). Wells were washed with 2x with PBS-0.1% Tween. For avidity determination, various concentrations of ammonium thiocyanate (4–0.15M range) were added to each well and incubated for 15 minutes prior to washing 3x with PBS-0.1% Tween. Wells were then incubated with HRP-conjugated Donkey anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, 1:20,000 dilution in 5% milk PBS-Tween solution) for 1 hour at room temperature followed by washing 3x in PBS-0.1% Tween. Plates were reacted with TMB substrate and reaction stopped with 4N Sulfuric Acid prior to being read at 450nM using an AccuSkan FC plate reader.

Statistical analyses

Data were analyzed using GraphPad Prism 8 or 9 software. Specific tests for determining statistical significance are indicated in the figure legends. P values of less than 0.05 were considered statistically significant.

RESULTS

ZIKV^CDN drives a more robust antigen-experienced and Th1 CD4 T cell responses

To begin our analysis of the CD4 T cell response to each ZIKV isolate, we compared changes in the total and antigen-experienced CD4 T cell populations in the spleen 7 dpi. Antigen-experienced CD4 T cells were tracked using cell surface expression of CD11a and CD49d, previously established surrogate markers of antigen experience in multiple infection models that we have validated for tracking the global CD4 T cell response to ZIKV infection (16, 21, 23, 24). Consistent with our previous results, the total number of cells in the spleen was significantly higher following infection with ZIKV^CDN than ZIKV^BR (Fig. 1A). Although ZIKV^BR-infected mice had a significantly higher frequency of CD4 T cells, there was no significant difference in the number of CD4 T cells in the spleen between the two infected groups (Fig. 1B–1D). This difference in frequency without a corresponding increase in numbers is likely due to the differential CD8 T cell responses induced by these two infections as we described previously (21). Similar to our previous observations regarding the splenic antigen-experienced CD8 T cell response, we observed a significant reduction in both the frequency and number of CD11a⁺CD49d⁺ CD4 T cells responding to infection with ZIKV^BR in the spleen (Fig. 1B, 1E, 1F), which contrasted to what we previously observed in the blood (21). In addition, the frequency and number of CD11a⁺CD49d⁺ CD4 T cells expressing the Th1 transcription factor T-bet and the expression of T-bet on a per cell basis (as measured by geometric mean fluorescence staining intensity (gMFI)) were significantly lower following ZIKV^BR infection (Fig. 1G–1J). Together, these data demonstrate that ZIKV^CDN induces a stronger CD11a⁺CD49d⁺ antigen-experienced CD4 T cell response than ZIKV^BR, and that those CD4 T cells that are responding to ZIKV^BR infection are less polarized towards the Th1 subset.

Figure 1: The antigen-experienced and Th1 CD4 T cell responses to ZIKV^BR are reduced compared to ZIKV^CDN infection.

(A) Overall spleen cellularity following ZIKV^CDN or ZIKV^BR infection. (B) Gating strategy and representative plots to identify CD4 T cells and antigen-experienced, CD11a⁺CD49d⁺ CD4 T cells 7 dpi in the spleen. (C) Frequency and (D) number of CD4 T cells in the spleen 7 dpi with ZIKV^CDN or ZIKV^BR. (E) Frequency and (F) number of antigen-experienced CD11a⁺CD49d⁺ CD4 T cells in the spleen 7 dpi with ZIKV^CDN or ZIKV^BR. (G) Representative histogram, (H) frequency, (I) number and (J) geometric mean fluorescence intensity (gMFI) of T-bet expression in antigen-experienced CD11a⁺CD49d⁺ CD4 T cells in the spleen 7 dpi with ZIKV^CDN or ZIKV^BR. Shaded grey curve in (G) represents isotype control staining and line on histogram indicates the gating strategy to identify T-bet⁺ cells. Data are representative of three independent experiments with n=3–4 mice per group in each experiment and are shown as mean ± SEM. Data were analyzed with a two-tailed, unpaired Student’s t-test. * p < 0.05; ** p < 0.01.

An important function of Th1-polarized CD4 T cells is the production of effector cytokines such as IFN-γ, TNF-α and IL-2 to support the antiviral immune response (9). Thus, to investigate the functionality of responding Th1 cells 7 dpi with each ZIKV isolate, we analyzed cytokine expression following restimulation with plate bound anti-CD3ε antibody. Similar to our previous observations (16), and consistent with their Th1 polarization, antigen-experienced CD4 T cells produced Th1 cytokines (IFN-γ, TNF-α and IL-2) following restimulation (Fig. 2). The frequency and number of IFN-γ⁺ and TNF-α⁺ antigen-experienced CD4 T cells were significantly higher following infection with ZIKV^CDN compared to infection with ZIKV^BR, although among IFN-γ⁺ or TNF-α⁺ cells, their expression on a per cell basis was similar between the two groups (Fig. 2A–2H). Although the frequency of IL-2⁺ antigen-experienced CD4 T cells and the gMFI of IL-2 staining were similar between the two groups, we observed a significant reduction of IL-2⁺ antigen-experienced CD4 T cells following ZIKV^BR infection (Fig. 2I–2L). These data are consistent with the reduced induction of T-bet expression during ZIKV^BR infection (Fig. 1G–J) and confirm a more robust Th1 response following ZIKV^CDN infection. To determine whether the reduced polarization to the Th1 subset following ZIKV^BR infection was due to polarization towards other Th subsets, we also analyzed expression of IL-5 (Th2) and IL-17A (Th17) following restimulation. Although in some samples we detected small frequencies of IL-5⁺ and IL-17A⁺-producing cells (<1% of CD11a⁺CD49d⁺), we observed no difference between ZIKV^CDN and ZIKV^BR infection (Supplemental Fig. 1). Thus, ZIKV^BR infection induces a CD4 T cell response that is less polarized to the Th1 subset and is less capable of producing Th1 cytokines without inducing polarization towards other Th subsets.

Figure 2: Antigen-experienced CD4 T cells produce less IFN-γ and TNF-α following ZIKV^BR infection.

Mice were infected with ZIKV^CDN or ZIKV^BR and spleens were harvested 7 dpi. Splenocytes were restimulated with anti-CD3ε antibody or media alone prior to intracellular staining for cytokines. (A) Representative plots, (B) frequency, (C) number and (D) gMFI of IFN-γ production by antigen-experienced CD11a⁺CD49d⁺ CD4 T cells. (E-H) Same as (A-D) but for TNF-α production. (I-L) Same as (A-D) but for IL-2 production. Data are representative of three independent experiments with n=3 mice per group in each experiment and are shown as mean ± SEM. Data were analyzed with a two-tailed, unpaired Student’s t-test. * p < 0.05; ** p < 0.01.

ZIKV^BR infection drives a more robust Tfh cell response than ZIKV^CDN

CD4 T cell priming events involve a fate decision-making process directing CD4 T cells toward either effector (e.g. Th1) differentiation or Tfh cell differentiation (11). While the signals that influence this fate decision remain incompletely understood, which subset is favoured may depend on the balance of signals that are present. For example, while IL-2 or IL-12 signaling and T-bet expression have been shown to favour Th1 differentiation, IL-6 stimulation and strong TCR signaling promotes Tfh cell responses (11, 25, 26). To address whether differences exist between the Tfh cell response to ZIKV^CDN and ZIKV^BR, mice were infected with either isolate, or injected with UV-inactivated ZIKV^CDN (UV-ZIKV) as a control, and the Tfh cell response was analyzed in the spleen 10, 14 and 21 dpi. We observed a small population of PD-1^hiCXCR5⁺ Tfh cells that remained comparable across all time points in mice injected with UV-ZIKV or ZIKV^CDN-infected mice (Fig. 3A–3I). However, following ZIKV^BR infection we observed a significantly higher frequency and number of PD-1^hiCXCR5⁺ Tfh cells across all time points tested (Fig. 3A–3I). To confirm that these cells represented a bona fide Tfh cell response, we analyzed the PD-1^hiCXCR5⁺ Tfh cells for expression of the transcription factor Bcl-6, a master regulator of Tfh function (11). As expected, Tfh cells from ZIKV^BR-infected mice expressed significantly more Bcl-6 on a per cell basis than non-Tfh CD4 T cells from the same mouse at all time points tested (Fig. 3J–3L), confirming that ZIKV^BR induces a prototypical Tfh cell response. We conducted the same analysis within ZIKV^CDN-infected mice and found that the small frequency of Tfh cells present within these mice expressed significantly more Bcl-6 than their non-Tfh CD4 T cell counterparts (Supplemental Fig. 2). Nonetheless, at 10 dpi the expression of Bcl-6 (as measured by gMFI) was higher among Tfh cells from ZIKV^BR-infected mice than ZIKV^CDN-infected mice (Fig. 3J and Supplemental Fig. 2). These data indicate that infection with ZIKV^BR promotes a more robust Tfh response than infection with ZIKV^CDN.

Figure 3: ZIKV^BR infection induces enhanced and sustained Tfh cell responses.

Mice were injected with UV-ZIKV or infected with ZIKV^CDN or ZIKV^BR, and PD-1^hiCXCR5⁺ Tfh cell responses were analyzed. (A) Representative histograms, (B) frequency and (C) number of PD-1^hiCXCR5⁺ Tfh cells in the spleen 10 dpi. (D-F) Same as (A-D) but at 14 dpi. (G-I) Same as (A-D) but at 21 dpi. (J) Representative histogram and gMFI of Bcl-6 expression in PD-1^hiCXCR5⁺ Tfh cells and CXCR5⁻ non-Tfh cells 10 dpi with ZIKV^BR. (K) and (L) Same as (J) but for 14 dpi and 21 dpi, respectively. Data are representative of two independent experiments with n=3 mice per group in each experiment and are shown as mean ± SEM. Data in (B-C), (E-F) and (H-I) were analyzed by one-way ANOVA with Tukey’s post-test of multiple comparisons. Data in (J-L) were analyzed by two-tailed, paired Student’s t-test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Within the Tfh cell population, a subset of FoxP3⁺ T follicular regulatory (Tfr) cells, which develop from T regulatory cells, may also be induced following infection to regulate the Tfh and GC B cell responses (27). To determine whether each ZIKV isolate differentially induced Tfr cell responses, we analyzed FoxP3 expression among the Tfh subset, 10, 14 and 21 dpi with either ZIKV isolate. Although we observed a trend toward a higher number of Tfr cells at 10 and 21 dpi, and significantly more Tfr cells 14 dpi with ZIKV^BR, this population represented a much smaller proportion of the total Tfh cell population within the ZIKV^BR-infected mice due to the larger increase in overall Tfh cell numbers in those mice (Supplemental Fig. 3). In contrast, the frequency of Tfr cells among CXCR5⁺PD-1⁺ cells in ZIKV^CDN-infected mice showed a trending increase 10 dpi and was significantly higher 14 and 21 dpi (Supplemental Fig. 3). Thus, the enhanced Tfh cell response to ZIKV^BR infection is not associated with a significant change in Tfr numbers at most of the time points examined suggesting that changes in the Tfr population are unlikely to be responsible for the modulation of Tfh populations in this model.

ZIKV^BR infection induces enhanced GC B cell and higher avidity neutralizing antibody responses

One of the primary functions of Tfh cells is to promote GC reactions in secondary lymphoid organs, which function to mediate GC B cell isotype class switching and affinity maturation (13, 14). Together, these processes generate B cells with higher affinity for cognate antigen, which in turn leads to production of higher affinity antibodies against the pathogen (13, 14). Since we observed a more robust Tfh cell response following ZIKV^BR infection, we next analyzed the GC B cell response to UV-ZIKV, ZIKV^CDN or ZIKV^BR, 10, 14 and 21 dpi. Similar to the Tfh cell response, we detected only small frequencies of IgD^loGL7⁺ GC B cells at all time points following injection with UV-ZIKV or infection with ZIKV^CDN (Fig. 4A–4I). Both the frequency and number of IgD^loGL7⁺ GC B cells were significantly increased following infection with ZIKV^BR across all time points analyzed (Fig. 4A–4I). In addition, we analyzed splenic tissue sections for the presence of GL7⁺ GCs by confocal microscopy. We observed similar numbers of GCs in uninfected and ZIKV^CDN-infected mice, while the average number of GCs per spleen section was significantly higher in ZIKV^BR-infected mice (Fig. 4J–4K). Together, these data demonstrate that infection with ZIKV^BR leads to enhanced generation of GCs in the spleen and a more robust GC B cell response than ZIKV^CDN.

Figure 4: GC formation and GC B cell response are enhanced following ZIKV^BR infection.

Mice were injected with UV-ZIKV or infected with ZIKV^CDN or ZIKV^BR, and IgD^loGL7⁺ GC B cell responses were analyzed. (A) Representative histograms, (B) frequency and (C) number of IgD^loGL7⁺ GC B cells in the spleen 10 dpi. (D-F) Same as (A-D) but at 14 dpi. (G-I) Same as (A-D) but at 21 dpi. (J) Representative confocal microscopy images of spleen sections from uninfected, ZIKV^CDN- and ZIKV^BR-infected mice 10 dpi. CD4 (CD4 T cells) is in red, IgD (naïve B cells) is in green and GL7 (GC B cells) is in blue. (K) Average number of GL7⁺ GCs counted per section. Data in (A-I) are representative of two independent experiments with n=3 mice per group in each experiment and are shown as mean ± SEM. Data in (J-K) are pooled from three independent experiments with n=1 uninfected mouse per experiment and n=2 mice per infection group in each experiment, and are shown as mean ± SEM. Data in (B-C), (E-F), (H-I) and (K) were analyzed by one-way ANOVA with Tukey’s post-test of multiple comparisons. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

One of the primary functions of antibodies is to neutralize virus particles, thereby preventing further spread to uninfected cells. Thus, we sought to determine whether the generation of neutralizing antibodies differed between infection with each ZIKV isolate. To address this question, we conducted plaque reduction neutralization tests (PRNTs) to analyze the capacity of serum from UV-ZIKV, ZIKV^CDN and ZIKV^BR-infected mice 10, and 21 dpi to neutralize virus and prevent infection of Vero cell monolayers. Serum was serially diluted and incubated with the same isolate as the mice had been infected with (i.e. serum from ZIKV^CDN-infected mice was incubated with ZIKV^CDN virus and serum from ZIKV^BR-infected mice was incubated with ZIKV^BR virus) prior to incubation with Vero cells for plaque assay. Each serum dilution was compared to a no serum control well to calculate the percent neutralization. Serum from UV-ZIKV-injected mice was unable to neutralize ZIKV^CDN at any serum dilution (data not shown), indicating that there was no non-specific neutralization within mouse serum. Serum from ZIKV^CDN-infected mice neutralized virus only at low dilutions 10 dpi, which was quickly lost at dilutions of 100-fold or higher (Fig. 5A). Strikingly, serum from ZIKV^BR-infected mice 10 dpi showed high neutralization across the same range of dilutions, and our data indicates that neutralization capacity was only lost after being diluted 10,000-fold (Fig. 5A and Supplemental Fig. 4). This pattern was maintained at 21 dpi, with serum from ZIKV^CDN-infected mice showing only minor increases in neutralization capacity, and serum from ZIKV^BR-infected mice retaining robust neutralization capacity (Fig. 5B). Thus, ZIKV^BR induces an antibody response that potently neutralizes viral infection in cell culture.

Figure 5: ZIKV^BR infection induces a more potent neutralizing antibody response.

(A,B) Plaque reduction neutralization tests (PRNTs) were performed using serially diluted (25-fold to 500-fold) serum from ZIKV^CDN- and ZIKV^BR-infected mice (A) 10 dpi and (B) 21 dpi to test each sample’s capacity to neutralize infection of Vero cell monolayers by the same ZIKV isolate with which the mice had been infected. Data are expressed as the percent neutralization when compared to a no serum control well. Data were pooled from two independent experiments with n=3 mice per group and are shown as mean ± SEM. (C) ELISA analysis of virus specific IgG. Serum was isolated from ZIKV^CDN or ZIKV^BR on 21 dpi or from naïve mice and reacted with plate bound heat-killed corresponding virus isolate. Data are representative of 2 experiments with 5 infected mice per group in each experiment. Naïve serum was pooled from >5 uninfected mice and are shown as mean ± SEM. (D) IC₅₀ of ammonium thiocyanate needed to remove IgG binding. Diluted serum from ZIKV infected mice were incubated with increasing concentration of ammonium thiocyanate and % binding when compared to untreated samples was used to calculate IC₅₀ for each individual mouse. Data are representative of 2 experiments with 5 infected mice per group in each experiment and are shown as mean ± SEM. Data in (A, B and D) were analyzed by two-tailed, unpaired student’s t-test. *** p < 0.001; **** p < 0.0001.

Increased neutralization by antibodies from ZIKV^BR infected mice could be due to a difference in either antibody quantity, quality or both. To address this question, we harvested serum from naïve mice (pooled serum from naïve mice was used for these experiments) or on 21 dpi with either ZIKV^CDN or ZIKV^BR and measured ZIKV-specific IgG levels via ELISA. We observed that despite a reduced neutralization capacity, ZIKV^CDN infection induces a stronger virus-specific IgG response at day 21 post-infection (Fig. 5C). Next, we tested the avidity of the antibody response via ELISA and thiocyanate elution (28). We observed that the concentration of thiocyanate required to remove 50% of IgG binding (IC₅₀ also referred to as affinity index) was significantly greater for ZIKV^BR-infection induced antibodies, establishing that these antibodies are of higher avidity (Fig. 5D). Together, this suggests that the stronger Tfh and GC B cell responses induced following ZIKV^BR infection leads to a higher avidity antibody response that is correlated with a greatly enhanced neutralization capacity despite lower overall production of virus-specific IgG.

ZIKV^CDN infection induces an extrafollicular antibody response

The production of higher levels of low avidity virus-specific antibodies following ZIKV^CDN infection in the absence of a robust Tfh response suggests that this viral isolate may favor the induction of an extrafollicular response. This response involves the early production of lower avidity antibodies and the activation of non-GC B cell subsets such as the activation of marginal zone B (MZB) cells, which can rapidly produce IgM and IgG antibodies in response to pathogens, including viruses (29–33). At 7 dpi, we observed that infection with ZIKV^CDN lead to a significant increase in the activation of MZB cells, as measured by their surface expression of the costimulatory molecule CD86, compared to mock-infected or ZIKV^BR infected mice (Fig. 6A–B). In addition, we observed that ZIKV^CDN infection, but not ZIKV^BR infection, led to the induction of a virus-specific IgG response at this early time point (Fig. 6C). Together, this suggests that infection with ZIKV^CDN primarily drives an extrafollicular B cell response which is associated with greater production of virus-specific IgG antibodies with lower avidity and less capacity to neutralize infection.

Figure 6: ZIKV^CDN infection induces enhanced activation of MZB cells and early virus-specific antibody production.

(A) Representative histogram and (B) gMFI of CD86 expression on MZB cells (gated on B220⁺ CD19⁺ CD21⁺ CD23^lo cells) at 7 dpi with either ZIKV^CDN, ZIKV^BR or mock-infection. Data are representative of 2 experiments with 5 infected mice per group in each experiment and are shown as mean ± SEM. (C) ELISA analysis of virus specific IgG. Serum was isolated from ZIKV^CDN or ZIKV^BR on 7 dpi or from naïve mice and reacted with plate bound heat-killed corresponding virus isolate. Data are representative of 2 experiments with 5 infected mice per group in each experiment. Naïve serum was pooled from >5 uninfected mice and are shown as mean ± SEM. Data in (B) were analyzed by one-way ANOVA with Tukey’s post-test of multiple comparisons. **** p < 0.0001.

DISCUSSION

Since the South and Central American outbreak, many studies have sought to improve our understanding of the immune response to ZIKV infection. However, few of these studies have directly compared the immune response induced by distinct ZIKV isolates, an approach that may help further our understanding of how ZIKV has changed in its interactions with the host immune system as it acquired epidemic capacity. Here, we have conducted an analysis of CD4 T cell differentiation following infection with a pre-epidemic isolate (ZIKV^CDN) or an epidemic isolate (ZIKV^BR). Our data demonstrate that while ZIKV^CDN induces more robust Th1 and global antigen-experienced CD4 T cell responses than ZIKV^BR 7 dpi, there is a stronger Tfh cell response to ZIKV^BR at 10 dpi, which persists through to 21 dpi. This Tfh cell response correlates with increased GC formation and increased generation of GC B cells, which in turn leads to a more potent neutralizing antibody response in the serum and correlates with the induction of a higher avidity virus-specific antibody response. Conversely, ZIKV^CDN induces the production of more virus-specific IgG at various time-points post-infection, even as early as 7dpi. However, these antibodies are of lower avidity, have poor neutralization capacity and correlate with the stronger activation of extrafollicular B cells such as MZB cells. Together, these data suggest that although ZIKV^BR may have evolved to subvert antiviral Th1 CD4 T cell responses and CD8 T cell responses that develop earlier following infection (21), this ultimately results in stronger GC B cell-mediated immunity that develops later in the infection and likely prevents the establishment of chronic infection. One caveat that remains is that our study centered on investigating the total antigen-experienced CD4 T cells using surrogate markers rather than defined antigen-specific CD4 T cells using ZIKV specific peptides or tetramers. Our findings regarding a change of polarization within the total pool of virus-activated CD4 T cells provide an overview of the virus-specific CD4 T cell response to these two ZIKV isolates. However, it will be of interest to determine whether these two isolates induce CD4 T cell that target different viral epitopes, as we have recently observed regarding the CD8 T cell response following infection with these ZIKV isolates, and this will be the focus of future investigation (21).

Impaired CD4 T cell immunity following ZIKV^BR infection could have a broad impact on the host’s capacity to respond to infection. The cytokines produced by Th1 CD4 T cells play an important role in activating other immune cells and enhancing the antiviral immune response (9). If the reduced presence of Th1 CD4 T cells impacted DC licensing as well, it could in turn impact the generation of protective CD8 T cell immunity, which would be consistent with our previous observations of an impaired CD8 T cell response to ZIKV^BR infection, which correlated with delayed viral clearance in some tissues (21). CD4 T cells clearly play an important role in protection against ZIKV infection in susceptible mouse strains, since their depletion leads to more severe paralysis, higher viral loads and increased mortality (18, 19). In addition, transferring memory CD4 T cells from ZIKV-immune to naïve IFNAR KO mice protected these mice from lethal ZIKV challenge (18, 19). The fact that this protection was lost in the absence of IFN-γ receptor signaling or B cells implies an important role for both Th1 and Tfh-dependent B cell responses in controlling ZIKV infection (19).

Only a few studies have directly analyzed Tfh and GC B cell responses to ZIKV infection. In LysMCre⁺Ifnar^fl/fl mice, ZIKV infection induces Tfh and GC B cell responses that are important for IgG isotype class switching (17). Liang et al. similarly detected a robust “Th1-like” (IFN-γ⁺) Tfh cell response following ZIKV infection in mice that had been treated with an IFNAR-blocking antibody prior to infection (20). Similar to our observations herein, this Tfh response was readily detectable at 10 and 14 dpi, and correlated with generation of GC B cells, IgG2a production, and neutralizing antibody responses (20). Our previous work has identified that type I IFN production is reduced following ZIKV^BR infection, consistent with the role Liang et al. identified for IFNAR signaling in modulating the Tfh cell response to ZIKV (20, 21). Whether IFNAR signaling directly or indirectly influences Tfh cell differentiation following ZIKV infection remains an intriguing question for future study. We also demonstrated previously that ZIKV^BR remains detectable by plaque assay in the spleen and kidney 7 dpi but is cleared by 14 dpi (21). Thus, the enhanced Tfh and GC B cell responses we observe, and the resulting increase in antibody avidity and neutralization capacity following ZIKV^BR infection could provide a potential mechanism for the eventual clearance of this virus.

Our observation of a balance between the induction of Th1- or Tfh-mediated immunity following infection with ZIKV^CDN or ZIKV^BR is similar to observations that have been made following S. enterica and T. gondii infection (25, 26). Following S. enterica infection, IL-12 enhances Th1 differentiation at the expense of the Tfh cell response, which is dependent on IL-12 receptor signaling and T-bet (26). Further, this contributes to the inhibition of GC formation observed following S. enterica infection. During T. gondii infection, although IL-12 initially induces genes that may contribute to both Th1 and Tfh cell phenotypes, T-bet and IFN-γ antagonize Tfh cell responses and promote Th1 differentiation, which in turn limits GC formation and antibody responses (25). Thus, the robust Th1 response to ZIKV^CDN could be limiting the development of Tfh cell and GC B cell responses, while the lack of a Th1 response and Th1 cytokines may have the opposite impact following ZIKV^BR infection. However, Tfh cell differentiation does not represent a default differentiation pathway and requires more than an absence of conditions or cytokines that promote Th1 differentiation for their generation (11). For example, Tfh cell responses are promoted by strong TCR signals, such as those that occur following high affinity TCR:peptide-MHC-II interactions, or by repeated stimulation (11). Fahey et al. demonstrated that while both LCMV Armstrong and Clone 13 initially drive comparable Th1 responses, over time as LCMV Clone 13 persists the CD4 T cell response becomes increasingly Tfh cell dominant (34). This suggests that delayed viral clearance (longer antigen exposure) following ZIKV^BR infection could contribute to the more robust Tfh cell response we observe. Interestingly, infection with ZIKV^CDN yielded the production of higher levels of virus-specific antibodies but these antibodies had significantly lower avidity for ZIKV antigens and lower neutralization capacity. This was associated with stronger activation of MZB cells suggesting that this virus isolate primarily activates extrafollicular B cell responses. The signals that determine the activation of extrafollicular and follicular B cell responses following ZIKV infection remain unknown and will be the focus of future investigation. In addition, future studies will seek to determine the cytokine-dependent and -independent signals that impact the Th1/Tfh cell balance following ZIKV infection. Nonetheless, the ability to compare the Th1 and Tfh cell responses between two isolates of the same virus suggests ZIKV could provide a useful model for dissecting other signals that promote or restrain Tfh cell differentiation.

In conclusion, our data demonstrate that while ZIKV^BR infection induced a weaker Th1 response than infection with ZIKV^CDN, this contrasts with more robust Tfh and GC B cell responses. As a result, ZIKV^BR infection generates a potent neutralizing antibody response, which persists as late as 21 dpi. In contrast, infection with ZIKV^CDN induces only a minor neutralizing antibody response despite producing high levels of virus-specific IgG antibodies. Together, these data suggest that ZIKV^BR may have evolved to subvert Th1 CD4 T cell responses, but at the cost of promoting B cell-mediated immunity, which develops later in the infection. However, the difference in timing of these responses could provide a crucial window of viremia within which ZIKV^BR continues to circulate, which may have important implications during ZIKV infection, including potentially increasing the transmission window of the virus and increased risk of pathogenesis in the context of pregnancy.

Key points:

1. Infection with ZIKV^BR induces a dampened Th1 response

2. Infection with ZIKV^BR drives an enhanced Tfh/GC B cell response

3. Antibodies induced by ZIKV^BR are of higher avidity and neutralize infection better

ABBREVIATIONS

Bcl-6

B cell lymphoma-6

dpi

days post-infection

DC

dendritic cell

GC

germinal center

IFNAR

IFN-α/β receptor

KO

knockout

MZB cell

marginal zone B cell

MOI

multiplicity of infection

OCT

optimal cutting temperature

PRNTs

plaque reduction neutralization tests

T-bet

T-box transcription factor

Tfh

T follicular helper

Tfr

T follicular regulatory

UV-ZIKV

UV-inactivated ZIKV^CDN

WT

wild-type

ZIKV

Zika virus