Abstract
Zika virus (ZIKV) is a mosquito-borne arbovirus that can cause severe congenital birth defects. The utmost goal of ZIKV vaccines is to prevent both maternal-fetal infection and congenital Zika syndrome. A Zika purified inactivated virus (ZPIV) was previously shown to be protective in non-pregnant mice and rhesus macaques. In this study, we further examined the efficacy of ZPIV against ZIKV infection during pregnancy in immunocompetent C57BL6 mice and common marmoset monkeys (Callithrix jacchus). We showed that, in C57BL/6 mice, ZPIV significantly reduced ZIKV-induced fetal malformations. Protection of fetuses was positively correlated with virus-neutralizing antibody levels. In marmosets, the vaccine prevented vertical transmission of ZIKV and elicited neutralizing antibodies that remained above a previously determined threshold of protection for up to 18 months. These proof-of-concept studies demonstrate ZPIV’s protective efficacy is both potent and durable and has the potential to prevent the harmful consequence of ZIKV infection during pregnancy.
Introduction
ZIKV is a teratogenic pathogen causing severe fetal developmental defects1,2. The 2015–2016 outbreak of ZIKV infection in the Western Hemisphere, particularly among pregnant women, was associated with a surge in miscarriages and severe cases of congenital abnormalities, including intrauterine growth restriction (IUGR), microcephaly, and other neuro-developmental disorders, referred to as congenital Zika syndrome (CZS)2,3,4,5,6. Consequently, in February 2016, the World Health Organization declared the ZIKV epidemic to be a public health emergency of international concern7,8. As such, the development of a ZIKV vaccine became an urgent public health priority. Despite the greatest need for the vaccine in pregnant women, this population is typically reserved for the final stages of clinical evaluation, given concerns for potential unforeseen adverse effects on the mother and developing fetus9,10,11,12,13. Inactivated virus vaccine platforms, however, have a long track record of safety in both pregnant women and fetuses14. For example, vaccination with inactivated influenza virus vaccines15 is recommended for pregnant women, as the benefits have been shown to outweigh potential risks.
The Zika purified inactivated virus (ZPIV) vaccine is a whole formalin-inactivated ZIKV derived from the Puerto Rico (ZK-PR) strain, PRVABC59, developed by the Walter Reed Army Institute of Research (WRAIR). The vaccine is co-formulated with aluminum hydroxide adjuvant. Preclinical studies in mice and rhesus macaques have shown that ZPIV-induced virus-neutralizing antibodies protected against viremia after ZIKV challenge16,17. Protective immunity persisted for at least 1 year after vaccination in non-human primates (NHPs)18,19. In addition, ZPIV has been shown to be safe and immunogenic in humans in phase 1 clinical trials20,21 and induced cross-protective B cell responses in human against Zika and dengue viruses22. However, the protective efficacy of ZPIV has not been established in the priority population, women of child-bearing age and pregnant women. In this study, we examined proof of concept that a ZIKV vaccine candidate could protect the unborn fetus in two immunocompetent pregnant animal models: C57BL/6 mice and common marmoset monkeys (Callithrix jacchus).
Previously, it was shown that ZIKV infection during pregnancy in wild-type C57BL/6 mice resulted in placental insufficiency and fetal demise23. Thus, pregnant C57BL/6 mice may be a high-fidelity model for non-productive ZIKV infection-induced fetal malformations; a model that may be suitable for an initial evaluation of vaccine efficacy. NHPs, however, are more physiologically relevant models for exploring vaccine efficacy in pregnant women because of the similarities in their placental structure and gestational period24,25,26,27,28. Common marmosets are susceptible to infection with flaviviruses, including dengue virus29,30,31 and ZIKV32,33,34. It previously has been shown that ZIKV infection during pregnancy in marmosets caused trans-placental virus transmission and led to spontaneous abortion within 16 days of infection34. Therefore, marmosets provide clinically relevant models to study the protective efficacy of vaccines against in utero ZIKV infection and trans-placental ZIKV transmission. An additional feature of marmoset biology that enhances their utility as pregnancy models for evaluating prophylactic interventions during pregnancy is their high frequency of multiple births including twins, triplets, and quadruplets35. In this report, we have exploited the relative strengths of these immunocompetent mouse and marmoset models, to test the protective efficacy of ZPIV following ZIKV challenge during pregnancy.
Results
Protection by ZPIV against fetal abnormalities caused by ZIKV infection during pregnancy
We first tested ZPIV’s efficacy in preventing gross morphological defects and fetal abnormalities in C57BL/6 mice following heterologous infection with the Brazilian strain of ZIKV, Brazil SPH2015 (ZK-BR), during pregnancy (Fig. 1a, b). Efficacy was evaluated in terms of reduction of the number of dams (pregnant female mice) bearing fetuses with morphological defects (Fig. 1c) and the percentage of affected fetuses (Fig. 1d). The data show that 100% of ZIKV infected unvaccinated dams bore affected fetuses, whereas a single vaccine dose, 1 ug ZPIV, protected three out of 11 dams (27%) from bearing affected fetuses (Fig. 1c). A single-dose ZPIV was even more pronounced in its efficacy when measuring malformed fetuses-significantly reducing the proportion of affected fetuses from 67% (24 out of 36) in the unvaccinated group to 13.8% (11 out of 80) in the vaccinated group (Fig. 1d, P < 0.0001), resulting in a relative efficacy of ZPIV in preventing fetal malformations of 79.4% (Supplementary Table 1).
An important question was whether a prime-boost regimen would confer additional protection over the single-dose regimen. Interestingly, a second vaccine dose at 28 days after prime reduced the proportion of affected fetuses from 71.1% (54 out of 76) in the unvaccinated group to 13.2% (7 out of 53) after prime-boost vaccination, of which the relative efficacy was slightly improved, compared to the single-dose regimen, to 81.4% (Supplementary Table 1 and Fig. 1f). These results showed that both single-dose and two-dose ZPIV vaccination provided approximately 80% efficacy in the prevention of fetal malformations induced by ZIKV infection. Previously, humans were vaccinated with two doses of ZPIV20,21. In order to maintain consistency with the human clinical trials, we used the prime-boost vaccination regimen to examine the efficacy of ZPIV in pregnant animal models for the rest of the study.
Cross-strain protection by ZPIV in mice
ZPIV is based on the Puerto Rico strain of ZIKV, PRVABC59. We examined the protection of mice against infection with ZK-PR and ZK-BR strains. The purpose of examining protection against both virus strains was two-fold: the first was to make a seamless transition from mouse to marmoset studies because, in the previous study, infection with ZK-BR induced abortion in marmosets34, and the second was to determine whether ZPIV can elicit cross-protection against these two viral strains. Challenge with ZK-PR or ZK-BR strains in two-dose ZPIV vaccinated mice yielded the relative efficacy of 84.9% and 89.7%, respectively (Supplementary Table 2), demonstrating that ZPIV vaccination provided comparable cross-strain protection against two ZIKV strains (Fig. 2).
