A single-dose live-attenuated vaccine prevents Zika virus pregnancy transmission and testis damage

Abstract

Zika virus infection during pregnancy can cause congenital abnormities or fetal demise. The persistence of Zika virus in the male reproductive system poses a risk of sexual transmission. Here we demonstrate that live-attenuated Zika virus vaccine candidates containing deletions in the 3' untranslated region of the Zika virus genome (ZIKV-3'UTR-LAV) prevent viral transmission during pregnancy and testis damage in mice, as well as infection of nonhuman primates. After a single-dose vaccination, pregnant mice challenged with Zika virus at embryonic day 6 and evaluated at embryonic day 13 show markedly diminished levels of viral RNA in maternal, placental, and fetal tissues. Vaccinated male mice challenged with Zika virus were protected against testis infection, injury, and oligospermia. A single immunization of rhesus macaques elicited a rapid and robust antibody response, conferring complete protection upon challenge. Furthermore, the ZIKV-3'UTR-LAV vaccine candidates have a desirable safety profile. These results suggest that further development of ZIKV-3'UTR-LAV is warranted for humans.Zika virus infection can result in congenital disorders and cause disease in adults, and there is currently no approved vaccine. Here Shan et al. show that a single dose of a live-attenuated Zika vaccine prevents infection, testis damage and transmission to the fetus during pregnancy in different animal models.

Fig. 1

ZIKV-3′UTR-∆10-LAV protects pregnant C57BL/6 mice and their developing fetuses. a Scheme of immunization of wild-type (WT) C57BL/6 female mice with 10⁵ FFU of ZIKV-3′UTR-∆10-LAV (∆10; n = 12) or PBS sham (n = 16). b Serum was collected at day 28 post immunization and analyzed for neutralizing activity using an mCherry infectious ZIKV. Representative neutralization curves are shown. Error bars denote the SD of duplicate technical replicates. c NT₅₀ values of neutralizing antibodies were measured for individual animals. The dashed lines indicate the limit of detection (LOD) of the assay. d–g At day 35 post immunization, vaccinated female mice were mated with WT C57BL/6 males. A subset of the female mice developed vaginal plugs. Pregnant mice (n = 8 pooled from two independent experiments) were administered 2 mg of anti-Ifnar1 blocking antibody on E5, and 1 day later (E6), challenged with 10⁵ FFU of a pathogenic, mouse adapted ZIKV Dakar 41519 strain. On E13, animals were euthanized; maternal spleen (d), maternal brain (e), placenta (f), and fetal heads (g) were harvested and quantified for viral RNA levels. Median viral RNA levels are indicated for each group. Asterisks indicate significant differences (Mann–Whitney test: ****P-value < 0.0001). All negative samples are plotted at the half value of LOD. The results in the Figure are pooled from two independent experiments

Fig. 2

ZIKV-3′UTR-∆10-LAV protects young A129 male mice against testis infection and injury. a Scheme of immunization of 3-week-old A129 male mice with 10⁴ FFU of ZIKV-3′UTR-∆10-LAV (∆10; n = 6) or PBS sham (n = 4 or 6). At day 28 post immunization, mice were measured for neutralization antibody titers. On the same day, mice from one sham group and mice from ∆10-immunized group were challenged with 10⁶ FFU of ZIKV-PRVABC59, and viremia was measured at day 2 post challenge (day 30 post immunization). At day 49 post immunization, mice were analyzed for sperm counts and viral load in the testis. b Viremia after immunization with ∆10 vaccine candidate. c NT₅₀ values of antibody neutralization at day 28 post immunization were measured for individual animals in each group. The dashed lines indicate the limit of detection (LOD) of the assay. d Viremia at day 2 post challenge (day 30 post immunization) with ZIKV PRVABC59. e Viral load in the testis at day 21 post challenge (day 49 post immunization). f, g Total (f) and motile (g) sperm counts at day 21 post challenge. h, i Testis weight (h) and representative images of testis (i) from animals from sham, sham with challenge, and ∆10-immunized and challenged groups at day 21 post challenge. Scale bar, 1 mm. Asterisks indicate significant differences (one-way ANOVA: ****P-value < 0.0001; ***P-value < 0.001). Nonsignificant (n.s.), P-value > 0.05. All negative samples are plotted at the half value of LOD. Error bars represent SDs

Fig. 3

ZIKV-3′UTR-∆10-LAV and ZIKV-3′UTR-∆20-LAV protect rhesus macaques (RMs) from ZIKV infection. a Scheme of immunization of RMs with 10³ FFU of WT ZIKV strain FSS13025 (n = 4), ZIKV-3′UTR-∆10-LAV (∆10; n = 4), ZIKV-3′UTR-∆20-LAV (∆20; n = 3), ZIKV-NS1-LAV or PBS sham (n = 2) via the subcutaneous route. b Viremia was measured at days 2, 3, 4, 5, 7, and 10 post immunization by qRT-PCR. Each colored line represents data from a different animal in each group. The dashed line indicates the limit of detection (LOD) of the assay. c Pre- and post-challenge antibody neutralization titers. On various days post immunization, sera were measured for neutralizing titers using an mCherry ZIKV infection assay. Red arrows indicate challenge with 10³ FFU of epidemic ZIKV strain PRVABC59 via the subcutaneous route at day 56 post immunization. The number of animals whose antibody neutralization titers increased by ≥ 4-fold after challenge is indicated by symbol “↑” for each experimental group. d Post-challenge viremia. Viremia was measured by qRT-PCR at days 2, 3, 4, 5, 7, and 10 post challenge. All negative samples are plotted at the half value of LOD. Error bars represent SDs

Fig. 4

Safety evaluation of ZIKV-3′UTR-∆20-LAV (∆20) vaccine candidate. a Viral loads in organs of infected A129 mice. Three-week-old A129 mice (n = 7) were subcutaneously immunized with 10³ FFU of WT ZIKV FSS13025 (left panel) and its derivative ∆20 vaccine candidate (right panel). Organs from infected mice were collected and homogenized at days 6 and 10 post infection. The amounts of viruses were quantified on Vero cells using a focus-forming assay. The mean results from seven animals are presented. Bars denote standard errors. The dashed lines indicate the limit of detection (LOD) of the assay. b–f Effect of ∆20 vaccination on the testis. Three-week-old A129 mice (n = 5) were subcutaneously infected with 1 × 10³ FFU of WT ZIKV FSS13025 or ∆20 vaccine candidate. At day 28 post infection, animals from each group were analyzed for testis weight (b), testis size (c), total sperm counts (d), motile sperm counts (e), and viral RNA load (f). Scale bar, 1 mm. g Comparison of neurovirulence of WT ZIKV FSS13025 and ∆20 vaccine candidate in outbred CD-1 mice. One-day-old CD-1 mice (n = 7–8/group) were injected intracranially with 10–10⁴ FFU of WT ZIKV or 10³–10⁴ FFU of ∆20 vaccine candidate. Surviving mice and total infected animals are indicated. h Analysis of vector competency. Aedes aegypti were fed on artificial blood-meals spiked with 10⁶ FFU/ml of WT ZIKV FSS13025 or ∆20 vaccine virus. At day 7 post feeding, individual engorged mosquitoes were assayed for infection by immunostaining of viral protein expression on inoculated Vero cells. The number of infected mosquitos and total number of engorged mosquitoes are indicated. Asterisks indicate significant differences (one-way ANOVA: ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05). Nonsignificant (n.s.) with P-value > 0.05. All negative samples are plotted at the half value of LOD. Error bars represent SDs