Maternal vaccination and protective immunity against Zika virus vertical transmission

Shan, Chao; Xie, Xuping; Luo, Huanle; Muruato, Antonio E.; Liu, Yang; Wakamiya, Maki; La, Jun-Ho; Chung, Jin Mo; Weaver, Scott C.; Wang, Tian; Shi, Pei-Yong

doi:10.1038/s41467-019-13589-1

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Published: 12 December 2019

Maternal vaccination and protective immunity against Zika virus vertical transmission

Chao Shan ORCID: orcid.org/0000-0002-3953-3782^1,2^na1,
Xuping Xie¹^na1,
Huanle Luo³,
Antonio E. Muruato³,
Yang Liu¹,
Maki Wakamiya¹,
Jun-Ho La⁴,
Jin Mo Chung⁴,
Scott C. Weaver^{3,5,6,7,8,9,10},
Tian Wang^3,7,11 &
…
Pei-Yong Shi^{1,5,6,7,8,11,12}

Nature Communications volume 10, Article number: 5677 (2019) Cite this article

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Abstract

An important goal of the Zika virus (ZIKV) vaccine is to prevent a congenital syndrome in fetuses of pregnant women, but studies directly evaluating maternal vaccination for ZIKV are lacking. Here we report maternal vaccination using a live-attenuated ZIKV vaccine (3ʹUTR-∆10-LAV) in a pregnant mouse model. Maternal immunization with 3ʹUTR-∆10-LAV does not cause any adverse effects on pregnancy, fetal development, or offspring behavior. One maternal immunization fully protects dams against ZIKV infection and in utero transmission. Although neutralizing antibody alone is sufficient to prevent in utero transmission, a higher neutralizing titer is required to protect pregnant mice against in utero transmission than that required to protect non-pregnant mice against viral infection. The immunized dams transfer maternal antibodies to pups, which protect neonates against ZIKV infection. Notably, pregnancy weakens maternal T cell response to 3ʹUTR-∆10-LAV vaccination. Our results suggest that, besides vaccinating non-pregnant individuals, 3ʹUTR-∆10-LAV may also be considered for maternal vaccination.

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Introduction

Zika virus (ZIKV) is a mosquito-borne flavivirus that has recently emerged and caused major epidemics¹. The most devastating disease of ZIKV infection is congenital Zika syndrome, including microcephaly, congenital malformations, and demise fetuses from infected pregnant women^2,3. In adults, ZIKV infection is associated with Guillain-Barré syndrome (GBS), an autoimmune disease that can lead to paralysis^4,5. One out of seven infants born to mothers with laboratory-confirmed ZIKV infection during pregnancy has birth defects, most likely due to in utero transmission⁶. Its teratogenic potential and explosive epidemics led the World Health Organization (WHO) to declare ZIKV as a Public Health Emergency of International Concern in February 2016. Since then, various ZIKV vaccine platforms have been developed, with the goal to control future epidemics and to prevent congenital syndrome. These vaccine platforms include inactivated, subunit, and live-attenuated vaccines⁷, some of which have already entered clinical phase I/II trials^8,9,10. We previously developed a live-attenuated ZIKV vaccine with a 10-nucleotide deletion in the 3ʹUTR of viral genome (3ʹUTR-∆10-LAV). A single-dose immunization of 3ʹUTR-∆10-LAV protected against ZIKV infection and in utero transmission in mice and non-human primates^11,12. This vaccine candidate has an excellent safety profile, as evidenced by >1000-fold lower neurovirulence than the two licensed, live-attenuated flavivirus vaccines (yellow fever 17D and Japanese encephalitis SA 14-14-2)¹³.

Despite the above promising progress, a number of important questions about ZIKV vaccine research remain to be addressed. Could maternal vaccination (i.e., vaccination during pregnancy) protect against viral infection and in utero transmission? Does pregnancy affect immune responses to ZIKV vaccination? Does maternal immunity derived from vaccination during pregnancy protect newborns against infection? Do pregnant and non-pregnant individuals require different levels of immunity to prevent ZIKV infections? Is neutralizing antibody alone sufficient to prevent in utero transmission? The answers to these questions are critical to guide the clinical development and regulatory approval of ZIKV vaccines.

In this study, we have addressed the above questions by testing 3ʹUTR-∆10-LAV in the A129 (interferon α/β receptor-deficient) pregnancy mouse model^14,15. Our results show that maternal vaccination of 3ʹUTR-∆10-LAV is safe for pregnant mice and fetuses. A single-dose maternal immunization fully protects dams against ZIKV infection and in utero transmission. Neutralizing antibody alone is enough for protection against in utero transmission. However, a higher neutralizing titer is required to prevent in utero transmission in pregnant mice than that required to prevent viral infection in non-pregnant mice. In addition, pregnancy weakens T cell response to 3ʹUTR-∆10-LAV immunization. Furthermore, maternal neutralizing antibodies are transferred to offspring to prevent ZIKV infection.

Results

Rapid immunity and long-lasting neutralizing antibodies

We chose 3ʹUTR-∆10-LAV in the current study because it elicits rapid and long-lasting protective immunity after one immunization. A single immunization of ten-week-old A129 mice with 10³ FFU of 3ʹUTR-∆10-LAV elicits neutralizing antibody titers (NT₅₀, defined as a sera dilution that inhibits 50% viral infection, data represents 1/NT₅₀ ± standard deviations) of 1/1300 ± 310, 1/6800 ± 900, and 1/13,900 ± 3000 on days 6, 10, and 14 post-immunization, respectively (Supplementary Fig. 1). The NT₅₀ values remain above 1/20,000 at week 55 post-immunization (Supplementary Fig. 2). Given the short gestation period of 20 days in mice, a rapid development of immunity after 3ʹUTR-∆10-LAV immunization is required to study maternal vaccination in a mouse pregnancy model. Other vaccine platforms that require multiple doses (e.g., inactivated vaccine or DNA subunit vaccine) are not suitable for studying maternal vaccination in mice because they cannot elicit protective immunity within the short gestation period in the mouse model. Thus, we chose 3ʹUTR-∆10-LAV to evaluate maternal vaccination in a pregnant A129 mouse model.

Safety analysis of 3ʹUTR-∆10-LAV for maternal vaccination

Safety is paramount for vaccine development. We performed three experiments to test the safety of 3ʹUTR-∆10-LAV in A129 pregnant mice. The first experiment examined potential adverse effects when mice were immunized during early pregnancy. Ten- to twelve-week-old A129 female mice were mated and closely observed for vaginal plugs [that define embryonic day 0.5 (E0.5)]. At E0.5, the pregnant mice were subcutaneously infected with 10⁵ FFU wild-type (WT) ZIKV or 3ʹUTR-∆10-LAV. At E18.5, the animals were sacrificed and analyzed for fetal development, viral loads, and neutralizing antibody titers (Fig. 1a). In the WT virus group, 54% of the fetuses were resorbed (Fig. 1b) and the weights of non-resorbed fetuses were significantly lower than those from the PBS-challenged placebo group (Fig. 1c). In contrast, 3ʹUTR-∆10-LAV infection did not affect fetal resorption or fetal body weight (Fig. 1b–d). No infectious virus was detected in maternal spleen (Fig. 1e), brain (Fig. 1f), placenta (Fig. 1g), or fetal head (Fig. 1h) from both WT- and 3ʹUTR-∆10-LAV-infected groups at E18.5, suggesting that viral infections had been cleared by this time point. To increase the detection sensitivity, we also measured viral RNA loads in placenta and fetal head using quantitative RT-PCR (qRT-PCR). No viral RNA was detected in placenta (Fig. 1i) or fetal head (Fig. 1j) from the 3ʹUTR-∆10-LAV-infected mice at E18.5; however, the WT virus-infected animals developed viral RNA in every placenta (100%), but none in fetal head from the non-absorbed fetus (0%; Fig. 1i–j). To further demonstrate that 3ʹUTR-∆10-LAV does not infect fetuses at earlier gestation timepoints, we performed qRT-PCR on the placenta and fetus/fetal head harvested at E10.5 and E14.5 (Supplementary Fig. 3a); the results showed that 34.6% (9/26) and 7.4% (2/27) of the placenta developed viral RNA, respectively; no viral RNA was detected from any of the fetus/fetal head (Supplementary Fig. 3b, c). At E18.5, robust neutralizing antibody titers were detected in dams (~1/15,300 ± 7000; Fig. 1k) and fetuses (~1/3700 ± 1800; Fig. 1l) from both the WT and 3ʹUTR-∆10-LAV groups. These results suggest that maternal vaccination of 3ʹUTR-∆10-LAV does not cause fetal infection, and that maternal-to-fetal antibody transfer may contribute to the neutralizing activity in fetuses.

**Fig. 1: The safety of 3′UTR-Δ10-LAV in pregnant A129 mice when vaccinated at E0.5.**

The second safety experiment examined potential adverse effects when mice were immunized at E10.5 (Supplementary Fig. 4a). We chose E10.5 to vaccinate the mice after placenta has been formed at E9.5 (Supplementary Fig. 5). Infection of pregnant mice at this time point with WT or 3ʹUTR-∆10-LAV did not increase fetal resorption (Supplementary Fig. 4b). However, WT virus, but not 3ʹUTR-∆10-LAV, caused significant fetal weight loss (Supplementary Fig. 4c). Corroboratively, infectious virus was detected in maternal spleen (50%), brain (100%), and placenta (100%) from the WT ZIKV group; in contrast, no infectious virus was found in any organs from the 3ʹUTR-∆10-LAV group (Supplementary Fig. 4d, f). No infectious virus was detected in the fetal brains from WT and 3ʹUTR-∆10-LAV groups under this experimental condition (Supplementary Fig. 4g). Corroboratively, qRT-PCR showed that the WT virus-infected animals developed viral RNA in placenta (100%; Supplementary Fig. 4h) and fetal head (100%; Supplementary Fig. 4i). Although the 3ʹUTR-∆10-LAV-infected mice also developed viral RNA in placenta, the viral RNA load was 673-fold lower than that in the WT virus-infected placenta (Supplementary Fig. 4h). Furthermore, no viral RNA was detected in the fetal head from the 3ʹUTR-∆10-LAV-infected animals (Supplementary Fig. 4i). At E18.5 or E21, comparable neutralizing antibody titers were detected in dams from the WT and 3ʹUTR-∆10-LAV groups (Supplementary Fig. 4j). In contrast, no neutralizing activities were detected from the fetuses collected at E18.5, but low neutralizing antibody levels (~1/130) were found in the pups born at E21 (Supplementary Fig. 4k), suggesting that vaccination at E10.5 may not allow sufficient time to develop maternal IgG antibodies that are transferrable to fetuses¹⁶.

The third safety experiment examined the effect of maternal vaccination on pups’ development and behavior. Behavior tests are believed to provide a more sensitive readout for potential adverse effects than the parameters measured in the two preceding safety experiments. Pups delivered from maternally vaccinated dams (subcutaneously immunized with 3ʹUTR-∆10-LAV at E0.5) were subjected to three behavior tests (including rotarod, grip strength, and tail pressure tests) when they reached 6-week-old of age (Supplementary Fig. 6a). Maternal vaccination did not change pup’s body weight, body length, or behavior scores (Supplementary Fig. 6b–f).

Taken together, the above safety results showed that maternal vaccination with 3ʹUTR-∆10-LAV did not yield any detectable adverse effects on pregnancy, fetal development, or offspring behavior. We previously showed that a single immunization with 10 FFU of 3’UTR-∆10-LAV elicited sterilizing immunity (defined by no increase of neutralizing antibody titers after WT ZIKV challenge) in A129 mice¹². Thus, the dose of 10⁵ FFU that we used in the above safety experiments is 10,000-fold higher than the 10-FFU sterilizing dose, underscoring the excellent safety profile of 3ʹUTR-∆10-LAV.

Efficacy of 3ʹUTR-∆10-LAV for maternal vaccination

We tested the efficacy of 3ʹUTR-∆10-LAV in maternal vaccination in A129 mice. Ten- to twelve-week-old pregnant A129 mice were subcutaneously immunized with 10³ FFU 3ʹUTR-∆10-LAV at E0.5 (Fig. 2a). High neutralizing antibody titers of ~1/13,200 ± 1700 were detected at E10.5 (Fig. 2b). After subcutaneously challenged with 10⁶ PFU Puerto Rico ZIKV strain (PRVABC59) at E10.5, the PBS placebo group developed 8.9 × 10⁵ PFU/ml viremia at E12.5, whereas no viremia was detected from the 3ʹUTR-∆10-LAV group (Fig. 2c). At E18.5, infectious virus was recovered from maternal spleen (50%), brain (83%), placenta (100%), and fetal brain (8%) from the placebo group; whereas no virus was detected in any organs from the 3ʹUTR-∆10-LAV-vaccinated animals (Fig. 2d-g). In addition, 3ʹUTR-∆10-LAV vaccination also prevented fetal weight loss (Fig. 2h-i). Compared with the neutralizing antibody titers detected at E10.5 (Fig. 2b), ZIKV challenge did not increase the maternal neutralizing titers at E18.5 (Fig. 2j), indicating that the pregnant mice had achieved sterilizing immunity after vaccination. In addition, high maternal neutralizing antibody titers (1/2,600 ± 600) were detected in the fetuses from the vaccinated dams (Fig. 2k).

**Fig. 2: The efficacy of 3′UTR-Δ10-LAV in pregnant A129 mice when immunized at E0.5.**

Besides vaccination at E0.5, we also tested the efficacy of vaccination at E4.5 (Supplementary Fig. 7). In the mouse pregnancy model, E4.5 is the latest time that could be rationalized for maternal vaccination because (i) six days are needed to allow 3ʹUTR-∆10-LAV to elicit protective immunity (Supplementary Fig. 1) and (ii) E10.5 is an optimal infection time to achieve analyzable fetal viral loads and developmental defects¹⁵. Similar to vaccination at E0.5 (Fig. 2), immunization at E4.5 elicited robust neutralizing antibody titers (~1/5325 ± 2148; Supplementary Fig. 7b) that fully prevented maternal and fetal infections as well as fetal weight loss (Supplementary Fig. 7c-h). Challenge with ZIKV PRVABC59 did not significantly boost maternal neutralizing antibody titers in the 3ʹUTR-∆10-LAV-vaccinated dams (Compare Supplementary Fig. 7b, i). Maternal antibody titers of ~1/980 ± 528 were detected in the fetuses collected at E18.5 (Supplementary Fig. 7j). Altogether, these results demonstrate that a single maternal vaccination of 10³ FFU 3ʹUTR-∆10-LAV prevents in utero transmission during pregnancy. Under both vaccination schemes at E0.5 and E4.5, maternal neutralizing antibodies (1/2600 ± 600 and 1/980 ± 530, respectively) were transferred to pups (Fig. 2k and Supplementary Fig. 7j).

The effect of maternal vaccination on offspring protection

It is important to characterize the effect of maternal vaccination on offspring protection against ZIKV infection. We performed three experiments to address this question (Fig. 3a). The first experiment examined the protection of maternally transferred immunity against ZIKV infection in pups. Dams subcutaneously immunized with 10³ FFU 3ʹUTR-∆10-LAV at E0.5 developed comparable neutralizing antibody titers of ~1/10,000 at E10.5 and E21 (Fig. 3b). One-day-old pups born to the vaccinated dams showed an average neutralizing titer of ~1/3000 ± 1100 (Fig. 3c). The milk recovered from the pups’ milk spot exhibited an average neutralizing titer of ~1/2500 ± 800 (Fig. 3c). These one-day-old pups survived a subcutaneous challenge of 100 PFU PRVABC59 (Fig. 3d), whereas naïve pups succumbed to challenges as low as 1 PFU ZIKV (Supplementary Fig. 8). The results demonstrate that maternal antibodies (transferred to pups through placenta and milk) can protect neonates against ZIKV infection.

**Fig. 3: Protection of offspring born to dams immunized with 3′UTR-Δ10-LAV at E0.5.**

The second experiment examined the longevity of maternal immunity in offspring. The neutralizing antibody titers in the offspring gradually waned from ~1/4400 ± 1200 on day 21 to ~1/90 ± 60 on day 70 (Fig. 3e). This is in contrast with no decrease in neutralizing antibody titers in adult animals immunized with 3ʹUTR-∆10-LAV (Supplementary Fig. 2). After challenging the 70-day-old pups with 10⁶ PFU ZIKV PRVABC59, 32% of the animals were protected against viremia (Fig. 3f). Correlation analysis between NT₅₀ titers and viremia development suggested that a minimal NT₅₀ of 1/120 is required to prevent ZIKV infection accompanied by viremia (Fig. 3g).

The third experiment determined the minimal NT₅₀ level of maternally transferred immunity that was required to block in utero transmission when the female offspring became pregnant (Supplementary Fig. 9). Six- to seven-week-old female offspring born to maternally vaccinated dams (subcutaneously immunized with 10³ FFU 3ʹUTR-∆10-LAV at E0.5) were mated with male mice. At E10.5, the pregnant offspring were bled for NT₅₀ measurement and subcutaneously challenged with 10⁶ PFU ZIKV PRVABC59. The pregnant offspring were quantified for viremia at E12.5 as well as for organ viral loads at E18.5 (Supplementary Fig. 9). Correlation analysis suggested that different minimal NT₅₀ titers are required to protect different organs against ZIKV infection during pregnancy: 1/200, 1/290, 1/290, and 1/430 for viremia, maternal brain, spleen, and placenta, respectively (Supplementary Fig. 9b–i). Since IgG is the main trans-placenta antibody^17,18, these results suggest that antibody immunity alone may be sufficient to prevent in utero transmission.

Neutralizing antibodies for pregnant and non-pregnant mice

To validate that antibody alone is sufficient to prevent vertical transmission, we performed passive transfer experiments (Fig. 4a). Ten- to twelve-week-old pregnant mice were passively transferred at E9.5 with different amounts of neutralizing antibodies (derived from ZIKV-immunized mice). At E10.5, the pregnant mice were measured for NT₅₀ levels and subcutaneously challenged with 10⁶ PFU ZIKV PRVABC59. The animals were assayed for viremia at E12.5 and organ viral loads at E18.5 (Fig. 4a). The passively transferred antibodies at E9.5 led to detectable neutralizing activities at E10.5, and conferred protection against maternal viremia and viral organ loads at E18.5 in a dose-responsive manner (Fig. 4b–i). Correlation analysis showed that distinct minimal NT₅₀ titers were required to protect different tissues/organs against viral infection: ≥1/100 for viremia (Fig. 4b, c); ≥1/460 for maternal brain (Fig. 4d, e), spleen (Fig. 4f, g), and placenta (Fig. 4h, i). In addition, a minimal NT₅₀ titer of ≥1/460 was required to prevent fetal weight loss during pregnancy infection (Fig. 4j). Notably, pregnant mice transfused with low levels of neutralizing antibodies (i.e., NT₅₀ titers <1/100) developed higher viral load in maternal spleen than the animals transfused with placebo control (Fig. 4g), possibly due to antibody-mediated enhancement. However, no such enhancement was observed for viremia (Fig. 4c), maternal brain viral load (Fig. 4e), or placenta viral load (Fig. 4i) in the antibody-transfused animals.

**Fig. 4: The minimal neutralizing antibody titers (NT₅₀) required for protection of pregnant mice.**

The above results prompted us to examine if pregnant and non-pregnant mice require distinct levels of neutralizing antibodies to prevent apparent ZIKV infection. Ten- to twelve-week-old non-pregnant mice were passively transferred with different amounts of neutralizing antibodies (Supplementary Fig. 10a). On the next day, the mice were bled for measuring NT₅₀ titers and subcutaneously challenged with 10⁶ PFU ZIKV PRVABC59. Viremia was assayed on day 2 (peak viremia time) and organ viral loads on day 9 (Supplementary Fig. 10a). Correlation analysis suggested that a minimal NT₅₀ titer of ≥1/130 was required to prevent non-pregnant mice from viremia and viral loads in brain and spleen (Supplementary Fig. 10b–g). Thus, the minimal NT₅₀ titer required to protect pregnant mice (≥1/460) was about 3.5-fold higher than that required to protect non-pregnant mice (≥1/130).

Weaker T cell response to vaccination during pregnancy

Besides neutralizing antibodies, T cells play an important role in preventing ZIKV infection and disease^19,20,21. Thus, we examined T cell response to maternal vaccination and compared the T cell responses between maternal and non-maternal vaccinations. For maternal vaccination, 12-week-old female mice were mated, and subcutaneously immunized with 10³ FFU 3ʹUTR-∆10-LAV at E0.5. At E18.5, mouse spleens were harvested for T cell analysis by culturing splenocytes, stimulating them with ZIKV or a viral envelope peptide¹⁹, and analysis by an intracellular cytokine staining (ICS) assay and a Bio-Plex immunoassay. For non-maternal vaccination, age-matched non-pregnant female mice were vaccinated and analyzed in the same manner (Fig. 5a). In both maternal and non-maternal groups, 3ʹUTR-∆10-LAV elicited robust ZIKV-specific IFN-γ⁺ and IFN-γ⁺TNF-α⁺ CD4⁺ and CD8⁺ T cell responses (Fig. 5b–d). Consistently, the splenocytes from 3ʹUTR-∆10-LAV-immunized animals produced significantly higher levels of interleukin-2 (IL-2) and IFN-γ than those from the mock-vaccinated animals (Fig. 5e, f). Notably, compared with non-pregnant vaccination, maternal vaccination elicited significantly lower numbers of IFN-γ⁺ CD4⁺ T cells, IFN-γ⁺ CD8⁺ T cells, and IFN-γ⁺TNF-α⁺ CD8⁺ T cells. It also induced lower levels of IL2 and IFN-γ production (Fig. 5e, f). The results indicate that pregnancy has a lower T cell response to 3ʹUTR-∆10-LAV vaccination.

**Fig. 5: Pregnancy affects T cell response to 3′UTR-Δ10-LAV vaccination in A129 mice.**

Discussion

The feasibility of a safe and efficacious ZIKV vaccine is supported by the availability of clinically licensed vaccines for four flaviviruses (yellow fever, tick-borne encephalitis, Japanese encephalitis, and dengue). Compared with the licensed flavivirus vaccines, a successful ZIKV vaccine has to overcome the unique challenge of protecting pregnant women against in utero viral transmission. Thus, it is essential to determine the correlations of protection against ZIKV infection in non-pregnant individuals as well as against in utero transmission in pregnant women. Given the decline of ZIKV human cases, identifying these parameters has become even more critical to guide the ongoing clinical trials²². Our study has demonstrated that neutralizing antibody alone is sufficient to protect against apparent ZIKV infection as well as in utero transmission in mice. However, a higher neutralizing antibody titer is required to prevent in utero transmission (NT₅₀ ≥ 1/460) than that required to prevent apparent ZIKV infection in non-pregnant mice (NT₅₀ ≥ 1/130). The requirement of NT₅₀ ≥ 1/460 for prevention of in utero transmission was supported by two different types of experiments: pregnant mice with waned antibody levels (Supplementary Fig. 9) and pregnant mice with passively transferred antibodies (Fig. 4). The requirement of NT₅₀ ≥ 1/130 for prevention of apparent ZIKV infection in non-pregnant mice is in agreement with previous studies^{10,23,24,25,26}. Besides the different requirements for protective NT₅₀ titers between pregnancy and non-pregnancy, we also found that pregnancy has a lower T cell response and cytokine production after 3ʹUTR-∆10-LAV vaccination. This is not surprising as pregnancy has been well documented to alter systemic immunity^27,28,29. Future studies are needed to define the mechanism and impact of such pregnancy-mediated T cell attenuation on the development of humoral immunity.

Besides prevention of in utero transmission, ZIKV vaccine development also has to overcome the challenge of antibody-dependent enhancement (ADE) of closely related dengue virus and vice versa^30,31,32,33. Recent studies reported that passively transferred DENV-specific antibodies or previous DENV infection could increase ZIKV pathogenesis in pregnant mice through increasing damage to the placenta and developing fetus^34,35,36. However, some epidemiologic data do not support the presence of immune enhancement in naturally infected humans^37,38. More epidemiologic studies are needed to further determine if pre-immunity derived from natural DENV or other flavivirus infection enhances ZIKV infection and congenital syndromes³⁹. Vaccination with 3ʹUTR-∆10-LAV may mitigate the risk of ADE because it can elicit both humoral and cellular immunity against viral structural and non-structural proteins⁴⁰. This concept has been proposed to explain the undesirable outcome for the licensed Dengvaxia⁴¹.

We explored the possibility of maternal vaccination to prevent in utero transmission during pregnancy. Our results indicate that 3ʹUTR-∆10-LAV has an excellent safety profile for maternal vaccination: no detectable infectious virus or viral RNA in fetal heads after vaccination, and no detectable adverse effects on murine pregnancy, fetal development, or offspring behavior. Despite these promising results, we could not exclude the possibility that maternal vaccination may cause subtle adverse effect on fetal development that could not be detected by the current assays. Infection of placenta with WT ZIKV was recently shown to mediate pregnancy complications^42,43, suggesting that even a very low level of 3ʹUTR-∆10-LAV replication in maternal organs might cause potential immunopathology in developing fetuses. Nevertheless, we showed that a single maternal vaccination of 10³ FFU 3ʹUTR-∆10-LAV probably conferred sterilizing immunity, prevented apparent in utero transmission, transferred maternal antibodies to pups, and protected neonates against ZIKV infection. However, the protection of offspring declined as the maternal neutralizing antibody titers waned. Altogether, the results suggest that 3ʹUTR-∆10-LAV may be considered for maternal vaccination. In support of this idea, maternal vaccination is recommended by the WHO for yellow fever and Japanese encephalitis vaccines during outbreaks in endemic regions or when the risk of viral infection is high^44,45. Other successful maternal vaccines, such as Tetanus-Diphtheria-Pertussis (Tdap) vaccine and inactivated influenza vaccine (IIV), are recommended by the Center for Disease Control and Prevention (CDC) to all pregnant women⁴⁶. After all, it should be emphasized that an ideal vaccination strategy for ZIKV is to immunize children before they reach child-bearing age.

The current study took the advantage of rapid immune response to 3ʹUTR-∆10-LAV immunization to study maternal vaccination and protective immunity for vertical transmission in a ZIKV pregnant mouse model. Compared with LAVs, other vaccine platforms (particularly inactivated ZIKV vaccine or DNA subunit vaccine) may be more appropriate for immunocompromised individuals and pregnant women because of their non-infective nature. Although both inactivated ZIKV vaccine or DNA subunit vaccine require multiple doses, the gestation period of human pregnancy is long enough for using them for maternal vaccination. Considering the strengths and weaknesses of different vaccine technologies, it is important to develop multiple vaccine platforms in parallel to provide complementary options for preventing ZIKV infection and disease. Finally, caution should be taken when extrapolating the mouse results to humans because placental biology and antibody transfer are fundamentally different between mouse and human pregnancy.

Methods

Ethics statement

Mouse studies were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the University of Texas Medical Branch (UTMB). The protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at UTMB (Protocol Numbers 1708051 and 1412070). Subcutaneous and intraperitoneal injections were performed under anesthesia that was induced and maintained with isoflurane. All efforts were made to minimize animal suffering.

Viruses, antibodies, and cells

The ZIKV Cambodian strain FSS13025 (GenBank number KU955593.1, referred as WT in text) was produced from an infectious cDNA clone⁴⁷. The ZIKV 3’UTR-∆10-LAV strain was generated from the FSS13025 infectious cDNA clone¹². The Zika Puerto Rico strain PRVABC59 (GenBank number KU501215) used for challenge was obtained originally from Dr. Robert Tesh from the World Reference Center of Emerging Virus and Arboviruses (WRCEVA) at UTMB. ZIKV-specific HMAF (hyper-immune ascitic fluid, dilution 1:2,000) was also obtained from Dr. Robert Tesh from WRCEVA at UTMB.

Anti-mouse IgG antibody labeled with horseradish peroxidase (KPL, Gaithersburg, MD) was purchased from SeraCare (Cat. No,: 5220-0286, dilution 1:2,000). Vero cells were purchased from the American Type Culture Collection (ATCC CCL-81; Bethesda, MD), and maintained at 37C with 5% CO₂ in high glucose Dulbecco modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA). The cell line was tested negative for mycoplasma.

Quantification of viral load in sera and organs from A129 mice

Viremia was quantified by plaque assay as described previously⁴⁷. Briefly, Serum was prepared after coagulation and centrifugation. Then 15 µl of sera was used for 10-fold serial dilutions. For each dilution, 100 µl of diluent was added to 24-well plate with 90% confluency Vero cells for infection of 1 h with swirling every 15 min. After 1 h incubation, 0.5 ml of overlay containing 0.8% methyl cellulose, 2% FBS, and 1% Penicillin–Streptomycin was added to each well. The plates were incubated 4 days at 37 °C with 5% CO₂ followed with fixing and staining. The maternal and fetal tissues (harvested at E18.5 from pregnant A129 mice) were homogenized by Qiagen TissueLyser II. The supernatant was used for plaque assay to measure the viral load in tissues after centrifuge. To quantify the 3ʹUTR-∆10-LAV virus from tissues or blood, immunostaining was performed as previous report¹². Briefly, after 4 days of incubation, the methyl cellulose overlay was removed, 0.5 ml methanol-acetone (1:1) fixation solution was added per well, and the plate was incubated for 15 min at room temperature. After removing the fixation solution, the plates were washed three times with PBS, incubated in PBS with 3% FBS for 1 h, and reacted with ZIKV-specific HMAF for 1 h. The plates were washed three times with PBS, incubated for 1 h with the horseradish peroxidase-conjugated secondary antibody (KPL, Gaithersburg, MD), and detected for immunostaining by addition of amino ethylcarbazole substrate following the manufacturer’s instructions (ENZO Life sciences, Farmingdale, MA).

Antibody response from A129 female mice

Ten-week-old female A129 were immunized with 10³ FFU ZIKV-3ʹUTR-∆10-LAV by subcutaneous route. The blood was collected by retro orbital sinus (R.O.) bleeding on days 6, 10, and 14 post-immunization. The sera were collected and used to measure the neutralizing antibody titer using an mCherry ZIKV reporter virus¹¹. Supplementary Fig. 11 depicts the construction of the cDNA clone of mCherry ZIKV. Briefly, sera were heat-inactivated at 56 °C for 30 min, then sera were 2-fold serially diluted starting at 1:50 in DMEM containing 2% FBS and 1% penicillin/streptomycin and then incubated with an equal volume of mCherry ZIKV reporter virus at 37 °C for 1 h. Antibody-virus complexes were added to Vero cell monolayers in a 96-well plate which was seeded one day before. At 48 h post-infection (p.i.), mCherry fluorescence-positive cells were quantified by Cytation 5 Cell Imaging Multi-Mode Reader (Biotek). The fluorescence-positive cells from serum-treated wells were normalized to those of non-treatment controls (set as 100%). A dose-response curve was plotted using GraphPad Prism 7 software (La Jolla, CA), and the effective dilution of sera to reduce 50% of mCherry-positive cells (NT₅₀) was calculated using nonlinear regression analysis.

A129 mouse pregnancy safety experiment

A129 mice were bred and housed in pathogen-free mouse facilities at UTMB. Ten- to twelve-week-old female A129 mice were paired with ten-week-old male mice. The vaginal plug was checked daily. Once the plug was identified, the mice were defined at starting embryotic stage (E0.5). The mice were infected with 10⁵ FFU WT ZIKV or 3ʹUTR-∆10-LAV by subcutaneous route at E0.5 or E10.5. At E18.5, mice were euthanized and necropsied. Maternal blood was obtained by cardiac puncture. Maternal spleen, brain, and placenta were harvest for measuring viral loads. Fetal body weight was measured immediately after necropsy. After decapitation, fetal head and blood were collected for viral load by plaque assay and neutralizing antibody measurement, respectively. All sera were heat-inactivated at 56 °C for 30 min prior to measuring neutralizing antibodies by the mCherry ZIKV reporter virus as described above.

A129 mouse pregnancy efficacy experiment

Ten- to twelve-week-old female A129 mice were paired with ten-week-old male mice. The pregnant mice were immunized with 10³ FFU of 3ʹUTR-∆10-LAV (∆10) by subcutaneous route at E0.5 or E4.5. At E10.5, the mice were bled by retro orbital sinus (R.O.), and immediately challenged with 10⁶ PFU ZIKV PRVABC59 via subcutaneous route. At E12.5, mice were anesthetized and bled via the R.O. for viremia testing. At E18.5, mice were euthanized and necropsied. Maternal blood was obtained by cardiac puncture. Maternal spleen, brain, and placenta were harvest for measuring viral loads by plaque assay as described above. Fetal body weight was measured immediately after necropsy. After decapitation, fetal head and blood were collected for viral load by plaque assay and neutralizing antibody measurement as mentioned above, respectively.

Neutralizing antibody from fetal blood and maternal milk

The blood was collected from decapitated fetuses at E18.5 or from new born pups or one-day-old pups. The maternal milk was harvested from one-day-old pup’s milk spot. After centrifugation of the harvested milk at 12,000 rpm, the supernatant was collected and inactivated at 56 °C for 30 min prior to neutralizing antibody test. All antibody neutralization titers were determined using an mCherry ZIKV as described above.

Behavioral assays

Ten- to twelve-week-old A129 mice were immunized with 10³ FFU 3′UTR-Δ10-LAV or PBS (placebo group) at E0.5. All the pregnant mice gave birth at full term. The following behavioral experiments were performed on 6-week-old mice. (i) Rotarod test: The motor ability of mice was assessed by determining their performance on a rotarod (AccuScan EzRod, Omnitech Electronics, Inc., Columbus, OH). After three days of training, mice were placed on a rotarod in a ramp acceleration mode (20 rpm/min), and the latency to fall was measured. The values obtained from three trials were averaged on each test day. (ii) Grip strength test: this test was used as another measurement of motor functions. Mice were individually placed on a metal grid attached to a force transducer in a grip strength meter (GT3, Bioseb, Pinellas Park, FL). The mouse was pulled until its grip onto the metal grid is released, and the maximum grip force developed during this procedure was recorded. The values obtained from two trials were averaged. (iii) Tail pressure test: the mechanical nociceptive sensory function of mice was assessed by using a custom-made Randall Selitto device. The tail base of mouse was pressed until the mouse withdraws its tail, and the force evoking such withdrawal was recorded.

Virulence of ZIKV in one-day-old A129 pups

To determine the lethal dose of ZIKV PRVABC59 on one-day-old A129 pups, pups were injected with 1, 10, or 100 PFU ZIKV PRVABC59 via subcutaneous route and monitored for morbidity and mortality. To test the maternal antibody protection against ZIKV infection, one-day-old pups from 3’UTR-∆10-LAV- or PBS-immunized mice were inoculated subcutaneously with 100 PFU ZIKV PRVABC59. The infected pups were monitored for morbidity and mortality for 21 days.

Passive transfer of antibody to non-pregnant A129 mice

Mouse immune sera from 3’UTR-∆10-LAV immunized A129 mice were pooled and passively transferred into 12-week-old female A129 mice via the intravenous (I.V.) route. One day after serum transfer, the mice were bled and immediately challenged with 10⁶ PFU ZIKV PRVABC59 via subcutaneous route. On day 2 post-challenge, the mice were bled for measuring viremia. All mice were sacrificed on day 8 post-challenge and organs were harvested for measuring viral loads by plaque assay as described above.

Passive transfer antibody to pregnant A129 mice

Ten- to twelve-week-old female mice were paired with 12-week-old male mice. Mouse immune sera from 3’UTR-∆10-LAV-immunized A129 mice were pooled and passively transferred into pregnant A129 mouse via I.V. route at E9.5. At E10.5, the mice were bled and immediately challenged with 10⁶ PFU ZIKV PRVABC59 via subcutaneous route. The mice were bled for measuring viremia at E12.5. At E18.5, dams were euthanized and blood were collected by cardiac puncture. Maternal organs (brain, spleen, and placenta) and fetuses were harvested. Fetal weight was measured immediately after necropsy. After decapitation, fetal head and blood were collected. The viral loads in maternal brain, spleen, and placenta and fetal head were determined by plaque assay on Vero cells⁴⁸ as mentioned above. Neutralization antibodies in fetal blood were measured using mCherry ZIKV reporter virus infection assay as described above.

Intracellular cytokine staining (ICS)

The pregnant mice were immunized with 10³ FFU of 3’UTR-∆10-LAV or PBS by subcutaneous route at E0.5. Age-matched non-pregnant mice were immunized at the same time to serve as controls. Eighteen days after immunization, the mice were sacrificed and splenocytes were harvested for analyses. Approximately 2.5 × 10⁶ splenocytes were stimulated with 10⁵ FFU live ZIKV (strain FSS13025) for 24 h or 10 mg/mL E peptide (amino acids 294–302 from ZIKV polyprotein) for 5 h. Live ZIKV was used as a stimulant to measure CD4⁺ T cell response⁴⁹. The E peptide was used as a stimulant to measure CD8⁺ T cell response¹⁹. During the final 5 h of stimulation, BD GolgiPlug (BD Bioscience, Cat. no.: 555029; dilution 1:1,000) was added to block protein transport. Cells were stained with antibodies for CD3 (APC-conjugated; Invitrogen, Cat. no.: 17003182; dilution 1:10), CD4 (FITC-conjugated; BD Biosciences, Cat. no.: 557307; dilution 1:10), or CD8 (FITC-conjugated; BD Biosciences, Cat. no.: 11008182; dilution 1:10). Afterward, cells were fixed in 2% paraformaldehyde and permeabilized with 0.5% saponin. Cells were then incubated with PE-conjugated anti-IFN-γ (Invitrogen, Cat. no.: 12731182; dilution: 1:10) and PE-Cy7-conjugated anti-TNF-α (Invitrogen, Cat. no.: 25732180; dilution: 1:10) antibodies or control PE-conjugated rat IgG1. Samples were processed with a BD Accuri C6 Flow Cytometer instrument. Dead cells were excluded on the basis of forward and side light scatter. Data were analyzed with a Cflow Plus Flow Cytometer (BD Biosciences).

Bio-Plex immunoassay

Approximately 3 × 10⁵ splenocytes were plated in 96-well plates and stimulated with 1.2 × 10⁴ FFU ZIKV FSS13025 or 10 mg/mL E peptide¹⁹ for 3 days. Culture supernatants were harvested and frozen at −80 °C. Cytokines IL-2 and IFN-γ in the culture supernatants were measured using a Bio-Plex Pro Mouse Cytokine Assay (Bio-Rad) according to the manufacturer’s instructions.

Data analysis

All data were analyzed with GraphPad Prism v7.02 software. Data are expressed as the mean ± standard deviation (SD). Comparisons of groups were performed using Mann–Whitney test or one-way ANOVA with a multiple comparisons correction. A p value of <0.05 indicates statistically significant.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information files, or are available from the authors upon request. The source data underlying Figures and Supplementary Figures are provided as a Source Data file.

Code availability

There in no custom code generated in this study.

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Acknowledgements

The authors thank all members of the Shi lab and colleagues at University of Texas Medical Branch (UTMB) for helpful discussions during the course of this study. The authors thank Dr. Robert Tesh at the World Reference Center of Emerging Virus and Arboviruses (WRCEVA) from UTMB for providing ZIKV PRVABC59 strain and ZIKV-specific hyper-immune ascitic fluid. P.-Y.S. lab was supported by NIH grants U19AI142759, AI127744, Clinical and Translational Science Award UL1TR001439, the Kleberg Foundation Award, John S. Dunn Foundation, Amon G. Carter Foundation, Summerfield Robert Foundation, and CDC grant U01CK0000512 for the Western Gulf Center of Excellence for Vector-Borne Diseases. This research was also supported by the World Reference Center for Emerging Viruses and Arboviruses, NIH grant AI120942 to S.C.W. T.W. was supported by NIH grants R01 AI099123 and R01 AI27744, and a grant from Sealy Institute for Vaccine Sciences at UTMB.

Author information

These authors contributed equally: Chao Shan, Xuping Xie.

Authors and Affiliations

Department of Biochemistry & Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA
Chao Shan, Xuping Xie, Yang Liu, Maki Wakamiya & Pei-Yong Shi
Wuhan National Biosafety Laboratory, Mega-Science Center for Bio-Safety Research, Chinese Academy of Sciences, Wuhan, Hubei, China
Chao Shan
Department of Microbiology & Immunology, University of Texas Medical Branch, Galveston, Texas, USA
Huanle Luo, Antonio E. Muruato, Scott C. Weaver & Tian Wang
Department of Neuroscience, Cell Biology, & Anatomy, University of Texas Medical Branch, Galveston, Texas, USA
Jun-Ho La & Jin Mo Chung
Institute for Human Infections & Immunity, University of Texas Medical Branch, Galveston, Texas, USA
Scott C. Weaver & Pei-Yong Shi
Institute for Translational Sciences, University of Texas Medical Branch, Galveston, Texas, USA
Scott C. Weaver & Pei-Yong Shi
Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Galveston, Texas, USA
Scott C. Weaver, Tian Wang & Pei-Yong Shi
Sealy Center for Structural Biology & Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas, USA
Scott C. Weaver & Pei-Yong Shi
Center for Biodefense & Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas, USA
Scott C. Weaver
World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, Texas, USA
Scott C. Weaver
Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA
Tian Wang & Pei-Yong Shi
Department of Phamarcology & Toxicology, University of Texas Medical Branch, Galveston, Texas, USA
Pei-Yong Shi

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Chao Shan
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Contributions

C.S., X.X., H.L., A.E.M., Y.L., M.W., and J-H.L. performed experiments and data analysis. C.S., X.X., H.L., J-H.L, J.M.C, S.C.W., T.W., and P.-Y.S. designed the experiments and interpreted the results. C.S., X.X., H.L., J-H.L, J.M.C, S.C.W., T.W., and P.-Y.S. wrote the manuscript.

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Correspondence to Pei-Yong Shi.

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Shan, C., Xie, X., Luo, H. et al. Maternal vaccination and protective immunity against Zika virus vertical transmission. Nat Commun 10, 5677 (2019). https://doi.org/10.1038/s41467-019-13589-1

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Received: 15 July 2019
Accepted: 14 November 2019
Published: 12 December 2019
DOI: https://doi.org/10.1038/s41467-019-13589-1

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