Adaptive Immune Responses to Zika Virus Are Important for Controlling Virus Infection and Preventing Infection in Brain and Testes

Clayton W. Winkler, Lara M. Myers, Tyson A. Woods, Ronald J. Messer, Aaron B. Carmody, Kristin L. McNally, Dana P. Scott, Kim J. Hasenkrug, Sonja M. Best and Karin E. Peterson
J Immunol May 1, 2017, 198 (9) 3526-3535; DOI: https://doi.org/10.4049/jimmunol.1601949

Abstract

The recent association between Zika virus (ZIKV) and neurologic complications, including Guillain-Barré syndrome in adults and CNS abnormalities in fetuses, highlights the importance in understanding the immunological mechanisms controlling this emerging infection. Studies have indicated that ZIKV evades the human type I IFN response, suggesting a role for the adaptive immune response in resolving infection. However, the inability of ZIKV to antagonize the mouse IFN response renders the virus highly susceptible to circulating IFN in murine models. Thus, as we show in this article, although wild-type C57BL/6 mice mount cell-mediated and humoral adaptive immune responses to ZIKV, these responses were not required to prevent disease. However, when the type I IFN response of mice was suppressed, then the adaptive immune responses became critical. For example, when type I IFN signaling was blocked by Abs in Rag1−/− mice, the mice showed dramatic weight loss and ZIKV infection in the brain and testes. This phenotype was not observed in Ig-treated Rag1−/− mice or wild-type mice treated with anti–type I IFNR alone. Furthermore, we found that the CD8+ T cell responses of pregnant mice to ZIKV infection were diminished compared with nonpregnant mice. It is possible that diminished cell-mediated immunity during pregnancy could increase virus spread to the fetus. These results demonstrate an important role for the adaptive immune response in the control of ZIKV infection and imply that vaccination may prevent ZIKV-related disease, particularly when the type I IFN response is suppressed as it is in humans.

Introduction

Zika virus (ZIKV) is an emerging infection endemic to the forests of Uganda and Southeast Asia that was first discovered in 1947 (1). It has recently spread from these regions to the Pacific and emerged in the Americas in 2015 (2, 3). Infection with ZIKV in humans is often asymptomatic or mild, with clinical signs of a rash, fever, conjunctivitis, and joint pain (4, 5). However, recent outbreaks of ZIKV infection since 2007 have been associated with Guillain-Barré syndrome in adults, as well as an increase in fetal abnormalities, including placental insufficiency, microcephaly, CNS abnormalities, and death resulting from mother-to-fetus transmission (6–8). These neurologic complications of ZIKV infection prompted its declaration as a global health crisis by the World Health Organization in 2016.

ZIKV is primarily transmitted to humans by the bite of an infected mosquito, although sexual transmission also occurs and is predominantly from male to female (9). Following infection by mosquito, ZIKV produces a short viremia in the blood, although it is detectable in other bodily fluids, including saliva, urine, and semen, for a longer period of time. Viral RNA and virus have been detected in the brains of fetal microcephaly cases, indicating that the virus can infect cells of the CNS (6, 10). In vitro studies showed ZIKV infection of human neuroprogenitor cells, fibroblasts, keratinocytes, and dendritic cells (10, 11).

Recent studies demonstrated an important role for type I IFN responses in protection against ZIKV infection. The NS5 protein of ZIKV inhibits human STAT2, suppressing the type I IFN response to ZIKV and allowing for proliferation of virus (12). In contrast, NS5 does not inhibit murine STAT2, allowing for a strong type I IFN response and suppression of virus infection in mice (12). Indeed, wild-type (WT) mice appear to control ZIKV infection, whereas mice deficient in IFNAR1 are susceptible to ZIKV infection, lose weight, and develop neurologic disease (13). Interestingly, WT C57BL/6 mice treated with anti-IFNAR Abs develop viremia but do not lose weight or develop neurologic disease (13). This suggests that when the IFN response is suppressed, but not deficient, other components of the immune system may be able to control ZIKV infection.

Another important component of the antiviral response is the adaptive immune response, including CD4+ and CD8+ T cell and neutralizing Abs (NAbs). Relatively little is known about the adaptive immune responses to ZIKV infection or their effects on viral pathogenesis. In this study, we detected CD4+ and CD8+ T cell proliferation and/or activation, as well as NAb production, in response to ZIKV infection in mice. Furthermore, the effects of these responses on ZIKV pathogenesis were investigated in the presence and absence of strong type I IFN responses. Finally, because maternal transmission of ZIKV to fetuses is one of the major complications of infection, we determined whether pregnancy affected the generation of adaptive immune responses.

Materials and Methods

Ethics statement

All animal work in this study adhered to the U.S. Government principles and applicable humane and ethical policies, in accordance with the Public Health Service Policy, the Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Regulations. Animal work was conducted in compliance with the guidelines of, and under a protocol approved by, the corresponding Institutional Animal Care and Use Committee (Protocol 2016-015). The Rocky Mountain Laboratories facility is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. All animal anesthesia was performed using vaporized isoflurane. Euthanasia, when required, was performed by cervical dislocation only after mice had reached a deep surgical plane of anesthesia.

Infection of mice with ZIKV and disease criteria

Rag1−/− mice (purchased from the Jackson Laboratory) and Unc93b1 3D mice (obtained from the Mutant Mouse Regional Resource Center) were maintained on a C57BL/6 background in a breeding colony at the Rocky Mountain Laboratories. Mavs−/− mice were purchased from the Jackson Laboratory on a C57BL/6, 129 mixed background. WT refers to the C57BL/6 strain, unless otherwise noted.

The 2015 ZIKV Paraiba strain is a human microcephaly isolate that was described previously (12) and was kindly provided by S. Whitehead (National Institute of Allergy and Infectious Diseases). At 6–8 (adult) wk of age, mice were inoculated with 104 PFU ZIKV i.p. in a volume of 200 μl per mouse. Virus stocks were diluted to the correct concentration in PBS prior to inoculation. Mock infections consisted of a similar dilution of Aedes albopictus clone C6/36 (American Type Culture Collection) mosquito larval cell culture supernatant diluted into PBS. For experiments characterizing the adaptive immune response to ZIKV infection during pregnancy, adult female mice (8+ wk) were time mated with proven males of the same genotype for 2 d prior to infection. At 7 d postmating, pregnancy was verified via ultrasound and/or abdominal palpation, and mice were infected as described above and housed individually. Mock-infected pregnant mice and age-matched mice that were never mated served as experimental controls.

Infected mice were observed daily for signs of neurologic disease that might include hunched posture, seizures, reluctance or inability to move normally, or paralysis. Mice that received neutralizing or cell-depleting Ab treatment (described below) were regularly monitored for weight loss, which is a known clinical symptom of ZIKV infection in immune-compromised mice (13). Animals with clear clinical signs of disease (including >20% loss of original starting weight) were scored as clinical and euthanized immediately.

Treatment of mice with neutralizing and cell-depleting Abs

For the depletion of T cells, anti-CD8 clone 169.4 and anti-CD4 clone 191.1 hybridomas were grown in RPMI 1640 media containing 10% FBS. Supernatants were harvested and spun at 500 × g for 10 min to remove any cellular debris and then stored at −20°C until use. Infected mice were injected i.p. with 0.5 ml of the supernatant at 1, 3, 5, 12, and 19 d postinfection (dpi). Dual CD8 and CD4 T cell–depleted mice received two injections (a total of 1 ml of supernatant) at each indicated time point. This treatment schedule was shown to deplete ≥95% of either or both T cell populations (14, 15). Control mice were injected on the same schedules with 10% FBS in RPMI 1640.

For neutralization of signaling through the IFN-α receptor, mice were treated i.p. with 1 mg per mouse of the anti-IFNAR1 clone MAR1-5A3 in PBS on 1, 3, 7, 11, and 16 dpi. Control mice were treated with an equivalent amount of normal mouse IgG Ab on the same days. This anti-IFNAR1 treatment protocol has been shown to effectively impair type 1 IFN signaling during ZIKV infection (13).

Surface and intracellular staining Abs and flow cytometry

Splenocytes were isolated from nonpregnant or pregnant WT mice by tissue homogenized through a 100-μm filter to generate a single-cell suspension. At room temperature (rt), RBCs were removed using lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 M EDTA), and cells were washed with phosphate balanced buffered saline. Cells were surface stained with Abs for 30–45 min at 4°C prior to fixation and permeabilization overnight at rt. The Abs used for cell staining were purchased from BD Pharmingen, eBioscience, or BioLegend, unless otherwise noted. The following combinations of Abs were used: CD4–Pacific Blue, CD8–Pacific Blue, CD19-PE/Texas Red, CD11a-FITC, CD69-PE, CD27-allophycocyanin/Cy7, CD43-PerCP/Cy5.5 or -PE/Cy7, CD25-PerCP/Cy5.5, CD62L-PE/Cy7, and CD86-BV605. Intracellular Foxp3 and Ki-67 staining was performed, according to the manufacturer’s recommendation, using Foxp3-allophycocyanin (FJK-16s), Ki-67–Alexa Fluor 700 (SolA15), and a Foxp3 staining kit (eBioscience). For intracellular granzyme B staining, the cells were fixed in 4% paraformaldehyde–PBS and then permeabilized with 0.1% saponin–PBS containing 0.1% sodium azide, 0.5% BSA, and 50 mM glucose. Cells were then stained using anti-human granzyme B–allophycocyanin (GB11) (Molecular Probes). Gates were used to exclude cellular debris and doublets; specific gating strategies are outlined in the figure legends. Data were acquired on an LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo v10 software (TreeStar).

Immunohistochemistry and in situ hybridization

At the experimental end point or clinical time point, some mice were perfused transcardially with heparin saline (100 U/ml), followed by 10% neutral buffered formalin. Whole brain or testes were serially sectioned (5 μm) and mounted on slides. For immunohistochemistry, sections were blocked (5% BSA, 0.05% Triton in PBS) at rt for 1 h and then primary Abs against ZIKV NS5 (1:3000; Aves Labs) and NeuN (1:2500; Abcam) or active caspase 3 (1:300; Promega) or Iba1 (1:250; Dako) were applied and incubated overnight at 4°C in blocking buffer. Secondary Abs were used to label these specific primary Abs (goat anti-chicken Alexa Fluor 488 and donkey anti-rabbit Alexa Fluor 594). Secondary Abs were incubated for 1 h at rt. Slides were coverslipped with ProLong Gold mounting medium containing DAPI (Molecular Probes) prior to imaging.

ZIKV replication was detected in tissue sections by in situ hybridization using probes specific for positive sense ZIKV RNA using a previously described method (16). All slides were then imaged using a Zeiss 710 LSM (Carl Zeiss) with a Plan Apochromat 63× oil-immersion objective (NA 1.40) with a pinhole 1 airy unit to generate confocal images (Fig. 7) or using an Aperio Scanscope FL (fluorescent) slide scanner (Leica Biosystems) to make composite images (Supplemental Fig. 3) with a UPLSAPO 20× objective (NA 0.75). Representative images were exported in TIFF format, and figures were created using Canvas 14 (ACD Systems).

Viremia and NAb detection

For detection of viremia, serially diluted plasma was plated directly onto Vero cells as described below. For quantification of NAbs, serially diluted plasma was mixed with 102 PFU ZIKV in a final volume of 200 μl in DMEM/2% FBS/1% Pen-Strep. The mixture was incubated for 1 h at 37°C for neutralization. After neutralization, the mixture was added to confluent Vero cells in a 24-well plate and incubated again for 1 h at 37°C. After incubation, 500 μl of 1.5% carboxymethyl cellulose in MEM was overlaid onto the cells, and the cells were incubated undisturbed at 37°C for 5 d. Then cells were fixed by adding 10% formaldehyde to each well until full and allowed to sit for 1 h at rt. After fixation, plates were rinsed gently with deionized water and stained with 0.35% Crystal violet for 15 min. Plates were rinsed and allowed to air dry in an inverted position. Viral titer was calculated by dividing the number of plaques per given sample by the plasma dilution factor multiplied by the volume of each well. Neutralizing titer was determined by the dilution that inhibited ≥50% of plaque formation compared with cells infected with 102 PFU ZIKV.

Real-time PCR

Real-time PCR analysis of mRNA expression from brain, spleen, and testes was completed as previously described (17). The primers used included Gapdh.2-152F (5′-TGCACCACCAACTGCTTAGC-3′), Gapdh.2-342R (5′-TGGATGCAGGGATGATGTTC-3′), ZIKVFP8008F (5′-AAGCTGAGATGGTTGGTGGA-3′), and ZIKVFP8121R (5′-TTGAACTTTGCGGATGGTGG-3′). Primers were subjected to BLAST analysis (National Center for Biotechnology Information) to ensure detection of only the specified gene and were tested on positive controls to ensure amplification of a single product. Data for each sample were calculated as the percentage difference in threshold cycle (CT) value (ΔCT = CT for GAPDH gene − CT for specified gene). Gene expression was plotted as the percentage of gene expression relative to that of the GAPDH gene.

Statistical analysis

All statistical analyses were performed using Prism 7.01 software (GraphPad). The statistical test for each experiment is described in the figure legends.

Results

ZIKV infection induces CD4+ T cell proliferation

To examine the adaptive immune response to ZIKV, we infected adult WT (C57BL/6) mice with 104 PFU per mouse i.p. Plasma and spleen samples at 3, 7, 10, 14, or 21 dpi were negative for detectable virus by plaque assay or real-time PCR, respectively (data not shown). Additionally, we were unable to detect viral RNA in brain tissue or lymph nodes, correlating with the ability of WT mice to clear virus infection (13). Approximately one half of each spleen for each time point was used for flow cytometry analysis of CD4+ and CD8+ T cells. CD4+ T cells were gated for CD4+ helper cells (Foxp3−) and regulatory T cells (Foxp3+) (Fig. 1A) and then analyzed for CD69, CD43, Ki67, CD11a, and CD25 expression. Representative flow cytometry plots for CD4 Th cells from naive (Fig. 1B) and ZIKV-infected mice (Fig. 1C) are shown for the 7-dpi time point. Analysis of helper CD4+ T cells (Fig. 1E) and regulatory T cells (Fig. 1D) showed no change in the percentage of the cell population over time. Similarly, increased expression of activation markers CD43, CD69, and CD11a was not significant for any of the analyzed time points during ZIKV infection (Fig. 1F–H), but increased proliferation, as measured by Ki67 staining, was significant at 7 dpi (Fig. 1I). Thus, ZIKV infection resulted in a peak of CD4+ Th cell proliferation around 7 dpi, despite a lack of detectable virus. Analysis of regulatory CD4+ T cells did not show any significant changes in the percentage of CD4+ T cells that were Foxp3+ or in any activation or proliferation markers (Supplemental Fig. 1).

FIGURE 1.
FIGURE 1.

ZIKV infection induces CD4+ T cell proliferation but not an increase in activation markers. Adult WT mice were infected with 104 PFU ZIKV. At 3, 7, 10, 14, and 21 dpi, spleens were removed, and splenocytes were analyzed by flow cytometry, as described in Materials and Methods. (A) CD4+ T cells were separated into helper (Foxp3−) and regulatory (Foxp3+) cells. Examples of splenocytes from mock (B) and ZIKV-infected (C) mice at 7 dpi labeled for CD43/CD69 and CD11a/Ki67. (D and E) CD4+ T cells were separated into helper (Foxp3−) and regulatory (Foxp3+) cells. The overall percentages of Th cells (E) or regulatory T cells (D) did not change over time following ZIKV infection. Average percentage of helper CD4+ T cells expressing CD43 (F), CD69 (G), CD11a (H), and Ki67 (I) are plotted for each time point. Mock-infected mice are shown as 0 dpi. Dotted line represents the average for mock-infected controls. Data are mean ± SD for three to six mice per time point and are the combined data of two experiments. ***p < 0.001 versus mock, one-way ANOVA with a Dunnett multiple-comparison posttest.

ZIKV infection induces CD8+ T cell proliferation and activation

CD8+ T cell responses to ZIKV infection were also analyzed. The gating for CD8+ T cells and representative analyses at 7 dpi are shown in Fig. 2. No difference was observed in the percentage of CD8+ T cells in the spleen over the course of ZIKV infection (Fig. 2D). However, significant proliferation (Fig. 2E) and activation (Fig. 2F, 2G), as well as production of the cytotoxic molecule granzyme B (Fig. 2H), were observed, with peak responses at 7 dpi. At that time point, ∼10% of the CD8+ T cells had the phenotype of activated effectors, as indicated by CD43 expression and granzyme B production (Fig. 2F, 2H). By 10 dpi, these activation markers had returned to baseline (Fig. 2E–H). These results revealed a strong and rapid CD8+ T cell response to ZIKV infection, with most parameters returning to baseline levels by 10 dpi. Thus, WT mice developed a strong, albeit short-lived, CD8+ T cell response to ZIKV infection, despite undetectable virus replication.

FIGURE 2.
FIGURE 2.

ZIKV infection induces CD8+ T cell proliferation and activation. Mice described in Fig. 1 were also analyzed for CD8+ T cell activation. (A) Cells were gated for CD8+ expression. Examples of splenocytes from mock (B) and (C) ZIKV-infected mice at 7 dpi labeled for Ki67/CD43 and CD11a/Granzyme B. (D) The overall percentages of CD8+ T cells did not change over time following ZIKV infection. Average percentage of CD8+ T cells expressing Ki67 (E), CD43 (F), CD11a (G), and granzyme B (H) are plotted for each time point. Mock-infected mice are shown as 0 dpi. Data are mean ± SD for three to six mice per time point and are the combined data of two experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus mock, one-way ANOVA with a Dunnett multiple-comparison posttest.

NAbs are produced in response to ZIKV but are not essential for protection

B cell responses to ZIKV infection were analyzed by expression of activation and proliferation markers, as well as NAb production. Analysis of CD19+ B cell markers by flow cytometry showed no significant changes in response to ZIKV infection with the parameters tested (Supplemental Fig. 2). However, NAb was detected at low levels by 7 dpi and at higher levels by 14 and 21 dpi, indicating a B cell response (Fig. 3). Thus, ZIKV infection elicited humoral and cellular adaptive immune responses in mice, despite limited detection of virus in WT mice. Comparison between sexes did not reveal any difference between males and females in any of the adaptive immune responses (data not shown).

FIGURE 3.
FIGURE 3.

NAb response to ZIKV. Plasma from mice described in Fig. 1 were also analyzed for NAbs. Diluted plasma was mixed with virus prior to plating on Vero cells in a NAb assay described in Materials and Methods. Data are plotted as the highest dilution of plasma that inhibited virus infection by 50%. Data are shown as individual animals at 7, 14, and 21 dpi from two independent experiments. Plasma from mock-infected mice showed no inhibition of virus and were scored as 0 on the graph.

T cell depletion in WT mice results in minor weight loss following ZIKV infection

Because CD4+ and CD8+ T cell proliferation and/or activation were observed following ZIKV infection, we examined whether T cells were essential for protection in mice. We treated WT mice with anti-CD4 and anti-CD8 mAbs, which has been shown to deplete both cell populations (14, 15). Depletion was confirmed by flow cytometry (data not shown). ZIKV-infected mice treated with anti-CD4 and anti-CD8 T cell Abs had a slight, but significant, reduction in body weight beginning at 11 dpi that persisted for the majority of recorded time points until the experimental end point (Fig. 4). No effect was observed as a result of anti-CD4/anti-CD8 treatment in uninfected mice (Fig. 4). Although these findings imply that T cell depletion modestly influenced ZIKV infection, the anti-CD4/anti-CD8 treatment group regained weight during the experiment (Fig. 4), suggesting that depletion did not result in uncontrolled virus replication. Furthermore, ZIKV infection of Rag1−/− mice, which lack both T cell and B cell responses, did not result in significant weight loss or neurologic disease (data not shown). Collectively, these data suggest that the innate immune response is sufficient to control ZIKV infection in mice, even in the absence of adaptive immune responses.

FIGURE 4.
FIGURE 4.

T cell depletion results in ZIKV-induced weight loss in WT mice. WT mice were infected with 104 PFU ZIKV or mock supernatant and treated with anti-CD4 and anti-CD8 Abs, as described in Materials and Methods. Individual mice were weighed every 2 d. Data are plotted as the average increase/decrease in weight over time for six mice per group per strain. *p < 0.05, versus vehicle control, two-way ANOVA with a Tukey multiple-comparison analysis.

CD4 and CD8 depletion affects ZIKV-induced weight loss in Mavs−/− mice

The ZIKV NS5 protein antagonizes STAT2 in human cells to suppress the type I IFN response to ZIKV and allow a more productive infection (12). However, STAT2 is not inhibited by NS5 in mouse cells, allowing for a strong type I IFN response and suppression of ZIKV replication. The type I IFN response to flaviviruses is primarily initiated following activation of cytosolic RIG-I–like receptors (RLRs) signaling to the adaptor MAVS, as well as endosomal TLRs signaling through MyD88 (18, 19). We compared viral RNA levels in Mavs−/− mice (deficient in RLR responses) or in Unc93b1 3D mice (with a mutation in Unc93b1 that prevents trafficking of TLR3, TLR7, and TLR9 to the endosome). Mice with the Unc93b1 3D mutation were similar to WT mice, with low to undetectable levels of virus present in the spleen (Fig. 5A). However, ZIKV replicated 100-fold higher in the spleen of Mavs−/− mice at 3 dpi, followed by the rapid control of replication by 7 dpi (Fig. 5A). Thus, Mavs−/− mice develop an acute infection reminiscent of that seen in humans, suggesting that these mice could provide a model in which to examine the role of the adaptive immune response to ZIKV.

FIGURE 5.
FIGURE 5.

T cell depletion results in ZIKV-induced weight loss in Mavs−/− mice. WT C57BL/6, Unc93b1 3D, and Mavs−/− mice were infected with 104 PFU ZIKV and analyzed for viral RNA (A), weight loss (B), or NAb production (C). (A) At 3 and 7 dpi, spleens were removed and analyzed for viral RNA levels. Data are from five or six mice per group per time point. ***p < 0.001 versus WT control at each time point. (B) Mock- and ZIKV-infected Mavs−/− mice were treated with anti-CD4 and anti-CD8 Abs, as described in Materials and Methods. Individual mice were weighed every 2 d. Data are plotted as the average increase/decrease in weight over time for six mice per group per strain. *p < 0.05 versus vehicle control, two-way ANOVA with a Tukey multiple-comparison analysis. (C) Plasma from Mavs−/− mice at 7 dpi or at the end of the weight-loss experiment (25 dpi) were analyzed for NAb, as described in Materials and Methods. Data are plotted for individual mice on a log2 scale. No inhibition was observed in mock-infected mice, which are indicated on the graph as a dotted line at the undiluted fraction.

To examine whether T cell responses were necessary for control of the increased virus replication in Mavs−/− mice, we performed a T cell–depletion study in these mice. Mavs−/− mice that were mock infected and T cell depleted or ZIKV infected and vehicle-control treated did not exhibit significant weight loss over time (Fig. 5B). In contrast, ZIKV infection of T cell–depleted Mavs−/− mice resulted in ∼10% weight loss starting at 8–9 dpi that was sustained until the end of the experiment (24 dpi) (Fig. 5B, ▪). This weight loss did not increase over time, and animals did not develop clinical signs of neurologic disease (Fig. 5B). Thus, T cell responses were necessary for prevention of weight loss induced by ZIKV infection. Interestingly, no viral RNA was observed in the brain or spleen tissue at the end of the experiment by real-time PCR analysis or immunohistochemistry (data not shown), suggesting that infection was controlled by a mechanism not involving MAVS signaling or T cells. Analysis of plasma from ZIKV-infected Mavs−/− mice demonstrated high levels of NAb, independent of T cell depletion (Fig. 5C) Interestingly, Mavs−/− mice had higher levels of NAb at both time points analyzed (Fig. 5C) compared with WT mice (Fig. 3), correlating with the higher level of early virus infection in Mavs−/− mice (Fig. 5A).

Suppression of type I IFN responses in the absence of humoral and cellular immune responses leads to CNS disease and widespread virus infection

To further examine the role of the adaptive immune response, we used Rag1−/− mice, which lack functional T and B cell responses. We treated WT or Rag1−/− mice with anti-IFNAR Abs at −1 dpi and again on 1, 3, 7, 11, and 15 dpi to mimic conditions of an insufficient type I IFN response. Previous studies showed that anti-IFNAR treatment in mice increased virus replication but did not result in disease, as indicated by body weight loss or neurologic signs (13). Similarly, in our study, WT mice treated with anti-IFNAR did not lose weight or develop neurologic disease, although increased virus replication occurred in the spleen compared with Ig-treated controls (Fig. 6A, 6B). However, anti-IFNAR–treated Rag1−/− mice lost significant weight by 9 dpi and reached criteria for euthanasia (≥20% weight loss) at 17 dpi (Fig. 6A). Tissue analysis showed a five-log10 increase in viral RNA in spleen compared with controls (Fig. 5B), as well as high levels of viral RNA in the lymph nodes (Fig. 6C) and brain (Fig. 6D). Thus, adaptive immune responses were required to control ZIKV replication and spread in anti-IFNAR–treated mice.

FIGURE 6.
FIGURE 6.

Treatment with anti-IFNAR in Rag1−/− mice results in clinical disease and high levels of virus. WT and Rag1−/− mice were infected with 104 PFU ZIKV and followed for weight loss (A) or measured for viral RNA in the spleen, lymph nodes, or brain (B–D). Mice were treated with anti-IFNAR1 (MAR1-5A3) on −1, 1, 3, 7, 11, and 15 dpi. Control mice were treated with an equivalent amount of normal mouse IgG Ab. Individual mice were weighed every 2 d. Data are plotted as the average increase/decrease in weight over time for nine mice per group per strain. *p < 0.05 versus WT C57BL/6 mouse IgG, two-way ANOVA with a Tukey multiple-comparison analysis. Rag1−/− mice treated with anti-IFNAR1 were euthanized at 17 dpi due to a 20% loss in weight (τ). At 17 dpi for anti-IFNAR1–treated Rag1−/− mice or 21 dpi for the other three groups, spleen (B), lymph nodes (C), and brain tissue (D) were removed and analyzed for viral RNA by real-time PCR. Individual mice are plotted using the same symbols as for (A), with the mean average shown as a horizontal line. ● and ○, Ig-treated mice; ▴ and △, anti-IFNAR1–treated mice. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with a Tukey multiple-comparison posttest.

Virus infection of neurons, but not astrocytes or microglia, in the brains of anti-IFNAR–treated Rag1−/− mice

We analyzed virus infection in the brain and testes of anti-IFNAR–treated Rag1−/− mice by immunohistochemistry. Virus, as detected by ZIKV NS5 staining (green fluorescence), was readily detected in the hippocampus and cortex (Supplemental Fig. 3) in NeuN+ neurons (red fluorescence) of the CA1, CA3, and dentate gyrus regions (Fig. 7A–D). Staining of astrocytes with GFAP (Fig. 7E–H) or staining of microglia/macrophages with Iba1 (Fig. 7I–L) indicated that these cells were not productively infected. However, astrocytes and microglia displayed morphological characteristics consistent with activation, such as cell body enlargement, process ramification, and, in the case of microglia, cellular engulfment of infected neurons (Fig. 7I–L, insets). Costaining of infected neurons for active caspase 3 demonstrated that some of the infected neurons, particularly those in the CA1 and CA3 regions, were undergoing apoptosis (Fig. 7M–P, arrows). Thus, ZIKV infection of the CNS of anti-IFNAR–treated Rag1−/− mice was associated with the infection and apoptosis of neurons and the activation of glial cells.

FIGURE 7.
FIGURE 7.

ZIKV infection of neurons, but not astrocytes or microglia, in brain tissue from anti-IFNAR–treated Rag1−/− mice. (A–P) Brain tissue from anti-IFNAR–treated Rag1−/− mice described in Fig. 6 was processed for histology and stained for ZIKV NS5 protein (green fluorescence). Samples were costained with red (pseudo-colored magenta) fluorescent label for NeuN to detect neurons (A–D), GFAP to detect astrocytes (E–H), Iba1 to detect microglia/macrophages (I–L), and active caspase 3 to detect apoptotic cells (M–P). Representative sections from the CA1 (A, E, I, and M), CA3 (B, F, J, and N), and dentate gyrus regions (C, G, K, and O) of the hippocampus and layers II/III of the cortex (D, H, L, and P). Samples with active caspase 3 costaining were also counterstained with DAPI (blue fluorescence) to indicate nuclei. (M–P) Arrows demonstrate active caspase 3, NS5 dual-positive cells. The scale bar is the same for all images.

ZIKV infection of polygonal cells in the testes

Because ZIKV has also been detected in the testes, we also analyzed tissues from anti-IFNAR–treated Rag1−/− mice by immunohistochemistry. Virus (green fluorescence) was not detected in Ig-treated WT control mice (Fig. 8A) but was readily detected in polygonal cells in anti-IFNAR–treated Rag1−/− mice (Fig. 8B). The location and structure of infected cells indicate that they are likely germinal spermatogonia or primary spermatocytes, and the lack of colocalization with vimentin staining suggested that they were not stromal or Sertoli cells (Fig. 8C). Colocalization with active caspase 3 (red fluorescence) indicated apoptosis of infected and uninfected cells (Fig. 8C–E) that was not observed in control mice (Fig. 8A). These immunohistochemistry results demonstrated ZIKV infection in two cell types in different tissues of anti-IFNAR–treated Rag1−/− mice. Furthermore, ZIKV infection was associated with cellular apoptosis in both tissues.

FIGURE 8.
FIGURE 8.

ZIKV infection of polygonal cells of the testes from anti-IFNAR–treated Rag1−/− mice. Testes from mock-treated WT mice (A and G) and anti-IFNAR–treated Rag1−/− mice (B–F and H) described in Fig. 6 were processed for histology and stained for ZIKV NS5 protein (green fluorescence) and active caspase 3 (A, B, and D–F) or vimentin (red fluorescence, pseudo-colored magenta) (C). (G and H) Sections were also stained by in situ hybridization for ZIKV sense RNA. ZIKV+ cells localized primarily to regions of polygonal cells in the testes (B) and, to a lesser extent, stromal and Sertoli cells (C) and induced high levels of apoptosis in anti-IFNAR–treated Rag1−/− mice (B) but not in mock-treated WT mice (A). (D–F) Higher-resolution image of square in (B) showing some colocalization (arrowheads) between ZIKV NS5 protein and active caspase 3+ cells. Images are representative between mice. Detection of ZIKV RNA is undetectable in control animals (G), but the polygonal area is heavily positive for viral RNA in anti-IFNAR–treated Rag1−/− mice (H), suggesting that a large number of cells are infected with virus. The scale bar in (A) also applies to images in (B)–(F), and the scale bar in (G) also applies to (H).

Pregnancy reduces CD4 and CD8+ T cells responses but not NAb

One the most concerning pathologies associated with ZIKV infection is microcephaly in the fetus following infection of a pregnant individual. Pregnant women are at increased risk for certain infectious diseases, a phenomenon linked to a unique immunological condition associated with pregnancy (20). To investigate whether the adaptive immune response was influenced by pregnancy, CD4+ and CD8+ T cell responses were measured at 7 dpi in pregnant WT mice, representing the peak of response for both of these cell types (Figs. 1, 2). Because gestation in a C57BL/6 mouse is ∼18 d, we infected mice at 7 d of gestation, during the early-mid stages of pregnancy. Analysis of CD4+ and CD8+ T cell responses revealed demonstrable reductions in the responses of both subsets in pregnant mice compared with nonpregnant controls (Fig. 9A–F). In pregnant mice, the percentages of proliferating (Ki67+) CD4+ and CD8+ T cells were reduced (Fig. 9A, 9B), and significantly fewer CD8+ T cells appeared activated (CD11a+ and CD62L−, CD43+) (Fig. 9C–E). In addition, there were significantly fewer granzyme B+ CD8+ T cells in pregnant mice infected with ZIKV (Fig. 9F), suggesting a reduction in cytolytic effector function. These results indicated that pregnancy influenced the cellular arm of the adaptive immune response. In contrast, no significant difference in NAb responses in pregnant mice was observed compared with nonpregnant females (Supplemental Fig. 4), suggesting that pregnancy may not significantly affect the humoral immune response to ZIKV.

FIGURE 9.
FIGURE 9.

Pregnancy suppresses CD4+ and CD8+ T cell responses to ZIKV infection. Nonpregnant and pregnant mice were infected with 104 PFU ZIKV at 7 d postmating, as described in Materials and Methods. At 7 dpi, spleens were removed and analyzed for flow cytometry. Cells were gated for CD4+ (A) or CD8+ expression (B–F) and then analyzed for Ki67 (A and B), CD11a (C), CD62L (D), CD43 (E), and granzyme B (F). Symbols indicate individual animals, and horizontal lines denote the mean per group. Data are the combined results of two experiments. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey multiple-comparison posttest.

Discussion

In this study, we examined the adaptive immune response to ZIKV infection and its potential role in regulating virus infection and preventing disease. We found a detectable T cell response and NAb response in WT mice, despite undetectable levels of virus in multiple tissues. These adaptive immune responses were not necessary to control virus in mice with fully competent type I IFN responses, because Rag1−/− mice did not lose weight or show any signs of clinical disease following ZIKV infection. However, when Rag1−/− mice were treated with anti-IFNAR Abs, to more closely recapitulate the suppressed anti-IFN response in human ZIKV infection, the mice developed severe disease, with ≥20% weight loss and high levels of virus in the brain and testes. Thus, when the type I IFN response is not fully competent, the adaptive immune response has an important role in regulating ZIKV infection and preventing the spread of virus to the brain and testes.

Staining for ZIKV in the testes of anti-IFNAR–treated Rag1−/− mice indicated that the primary cell type infected in the testes is polygonal cells, most likely spermatogonia or primary spermatocytes. This is similar to a recent study by Govero et al. (21), which found that ZIKV infected spermatogonia, primary spermatocytes, and Sertoli cells in the testes and induced apoptosis of these cells. This was observed in anti-IFNAR–treated WT or Rag1−/− mice, most notably when using a mouse-adapted strain of ZIKV, Dakar. The Dakar strain of ZIKV induced more apoptosis of ZIKV than did the Asian ZIKV strain (H/PF/2013). Analysis with caspase 3 in our studies with the Paraiba strain of ZIKV demonstrated that the majority of infected cells were not undergoing apoptosis, suggesting that spermatogonia function as a cellular reservoir of ZIKV. Because ZIKV is sexually transmitted (9), it is possible that one of the mechanisms of sexual transmission is through the transfer of infected spermatozoa (21).

ZIKV infection of anti-IFNAR–treated Rag1−/− mice also showed high levels of virus present in the CNS (Fig. 6), with the focal areas of ZIKV infection being neurons in the cortex and the hippocampal region (Supplemental Fig. 3). The primary cells infected by ZIKV were neurons, whereas astrocytes and microglia did not show obvious signs of infection (Fig. 7). However, astrocytes and microglia displayed morphological characteristics consistent with activation, such as cell body enlargement, process ramification, and, in the case of microglia, cellular engulfment. Microglia were recently shown to contribute to West Nile virus–mediated neuronal damage through a mechanism of complement-driven synapse loss (22). Astrocytes can also contribute to CNS damage through recruitment of inflammatory cells and breakdown of the blood–brain barrier (23). Thus, activation of these glial cells in areas of virus infection may contribute to CNS damage caused by ZIKV infection.

Neuronal infection was primarily found in the hippocampus, including the dentate gyrus, and in the cortex of Rag1−/− anti-IFNAR–treated mice (Supplemental Fig. 3). Analysis of these cells with active caspase 3 and ZIKV NS5 staining showed areas of heavy neuronal loss, particularly in the CA3 region, but also in the CA1 and cortex regions, suggesting that these regions may be particularly susceptible to ZIKV infection and neuronal damage. Infection of the hippocampus and/or dentate gyrus was noted for two other flaviviruses: West Nile virus and tick-borne encephalitis virus (24–27). Thus, in adults, ZIKV may have similar neuronal cellular tropism as other flaviviruses. These data indicate that ZIKV can readily infect neurons in the adults if peripheral responses are suppressed sufficiently to allow virus entry into the CNS and suggest that immunocompromised individuals may be susceptible to ZIKV-induced encephalitis, even as adults.

One condition that may lead to reduced immune responses to ZIKV is pregnancy. Studies with other viruses suggested that pregnancy can reduce the immune response to pathogen infections, including viruses (20). This study demonstrates reduced CD4+ and CD8+ T activation and proliferation in infected pregnant dams compared with nonpregnant controls (Fig. 9). Interestingly, although the cellular response to ZIKV was suppressed by pregnancy, the production of NAbs was not. We did not see an increase in virus RNA in the spleens of pregnant versus nonpregnant mice, indicating that decreased T cell responses were not sufficient to allow uncontrolled virus replication (data not shown). This is similar to the anti-CD4/anti-CD8 studies in WT and Mavs−/− mice in which weight loss was observed, but viral RNA or viremia remained undetectable. Possibly, early innate responses coupled with the NAb responses in both cases is sufficient to control virus replication, even in the absence of CD4 or CD8 T cell responses.

The antiviral type I IFN response in human cells is antagonized by ZIKV. In our effort to understand the contribution of adaptive responses in this context, we examined peripheral viral replication in the type I IFN–inhibited Mavs−/− and Unc93b1 3D mouse strains. We found that ZIKV RNA was 100-fold higher in the spleen of Mavs−/− mice relative to WT controls, whereas viral RNA was unchanged in mice deficient in Unc93b1 3D (Fig. 5A). This finding is consistent with an earlier study in a ZIKV vaginal infection model that showed higher viral replication in Mavs−/− mice but not in TLR7−/− mice (28). NAb responses were also higher in Mavs−/− mice (Fig. 5C) compared with WT mice (Fig. 3) at 1 and 3 wk postinfection, suggesting a more active infection in Mavs−/− mice. Furthermore, depletion of T cells in Mavs−/− mice (Fig. 5B) resulted in significant weight loss that was not observed in T cell–depleted WT mice (Fig. 4). Collectively, these findings indicate that RLR–MAVS signaling may contribute to the early type I IFN–mediated suppression of ZIKV replication. However, this MAVS-dependent antiviral response is clearly not solely responsible for early viral clearance because Mavs−/− mice alone did not lose weight or succumb to infection (Fig. 5B) (13) as did our anti-IFNAR1–treated Rag1−/− mice (Fig. 6). We found that T cell–depleted Mavs−/− mice generated a robust NAb response that is likely involved in controlling viral replication (Fig. 5C); however, we cannot exclude the possibility that the remaining type I IFN response is also involved. Thus, RLR–MAVS–dependent and -independent antiviral signaling is important for early control of ZIKV infection, but it requires a robust adaptive response to prevent disease if it is suppressed.

In conclusion, we found that ZIKV infection induces a strong adaptive immune response that provides protection against ZIKV-induced weight loss, as well as virus spread to the brain and testes. These results support the development of vaccines that drive a strong T cell and humoral Ab response, which could protect against ZIKV spread, even in the case of weakened or suppressed type I IFN responses. Future studies on how pregnancy affects the development of these responses and whether pregnancy suppresses already formed anti-ZIKV responses will be important in determining how to best generate strong adaptive immune responses in terms of protecting pregnant mothers from ZIKV infection.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Suzette Priola, Dr. Kerry Lavender, Dr. Lydia Roberts, Dr. Audrey Chong, and Dr. Kerry Long for critical readings of the manuscript. We thank Dan Long and Rebecca Rosenske for assistance with histological staining of tissue sections and Morgan Weidow for processing of tissue sections. Finally, we thank the Rocky Mountain Veterinary Branch and, in particular, Donna Norton, Shelby Malingo, and Maarit von Kutzleben for outstanding care and assistance with the immunocompromised mice used in this study.

Footnotes

  • This work was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases.

  • The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    CT
    cycle threshold
    dpi
    day postinfection
    NAb
    neutralizing Ab
    RLR
    RIG-I–like receptor
    rt
    room temperature
    WT
    wild-type
    ZIKV
    Zika virus.

  • Received November 17, 2016.
  • Accepted February 22, 2017.

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