ZIKV Vaccine Protects NHP & Fetus PDF

npj | vaccines Article Published in partnership with the Sealy Institute for Vaccine Sciences...

npj | vaccines Article Published in partnership with the Sealy Institute for Vaccine Sciences https://doi.org/10.1038/s41541-024-00927-8 Ad26.M.Env ZIKV vaccine protects pregnant rhesus macaques and fetuses against Zika virus infection Check for updates 1,2 3 1 4 5 Amanda J. Martinot , Freek Cox , Peter Abbink , Jonathan L. Hecht , Roderick Bronson , Erica N. Borducchi1, William J. Rinaldi6, Melissa J. Ferguson6, Rafael A. De La Barrera7, Roland Zahn 3 , Leslie van der Fits3 & Dan H. Barouch 1,8 At the start of the Zika virus (ZIKV) epidemic in 2015, ZIKV spread across South and Central America, 1234567890():,; 1234567890():,; and reached parts of the southern United States placing pregnant women at risk for fetal microcephaly, fetal loss, and other adverse pregnancy outcomes associated with congenital ZIKA syndrome (CZS). For this reason, testing of a safe and efficacious ZIKV vaccine remains a global health priority. Here we report that a single immunization with Ad26.M.Env ZIKV vaccine, when administered prior to conception, fully protects pregnant rhesus macaques from ZIKV viral RNA in blood and tissues with no adverse effects in dams and fetuses. Furthermore, vaccination prevents ZIKV distribution to fetal tissues including the brain. ZIKV associated neuropathology was absent in offspring of Ad26.M.Env vaccinated dams, although pathology was limited in fetuses from non-immunized, challenged dams. Vaccine efficacy is associated with induction of ZIKV neutralizing antibodies in pregnant rhesus macaques. These data suggest the feasibility of vaccine prevention of CZS in humans. In late 2015, an epidemic of fetal microcephaly in Brazil associated with high for individuals traveling to endemic areas and for individuals living in areas levels of circulating Zika virus (ZIKV), led to a global race to develop a ZIKV with continued transmission such as the Caribbean where over 15% of vaccine to protect women and unborn children from the potential devas- pregnant women continue to test positive for ZIKV12. CDC guidance still tating effects of congenital ZIKA syndrome (CZS)1–4. ZIKV, a member of the recommends that pregnant women, partners of pregnant women, or those Flaviviridae family, was first identified in Uganda in 1954, and while sharing considering pregnancy to delay travel to areas with ZIKV outbreaks and to a genus with other viruses that cause significant human disease such as consult with medical providers before traveling to ZIKV endemic regions13. dengue, yellow fever, Japanese encephalitis, and West Nile viruses, had only Current low levels of ZIKV world-wide limit the establishment of been associated with asymptomatic to mild flu-like symptoms prior to clinical trial sites and enrollment of participants in Phase III efficacy reports of CZS in Brazil in late 2015. Similar to other medically important studies11,14. Ideally a ZIKV vaccine could be deployed in the event of a ZIKV flaviviruses, ZIKV is primarily acquired by Aedes mosquitos, and can also be resurgence and administered safely to pregnant women. Preclinical studies spread transplacentally in pregnant women5,6, in blood transfusions7, and in mice and non-human primates (NHP) have shown that induction of through sexual contact8. Recently, genetic polymorphisms have been asso- neutralizing antibodies by a number of vaccine platforms is effective in ciated with development of CZS9. preventing ZIKV acquisition15–18). A number of candidate ZIKV vaccines The World Health Organization (WHO) declared an end to the ZIKV have completed safety studies in Phase I and II clinical trials19–22. The epidemic at the end of 201610, and ZIKV transmission is currently at low macaque model has been a useful model for studying dynamics of viral levels world-wide, however vaccine development for emergency deploy- replication and shedding during ZIKV infection16,23–26. We and others have ment remains a high priority by the WHO11. ZIKV also remains a concern shown that the rhesus macaque model consistently reproduces features of 1 Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. 2Departments of Infectious Disease and Global Health and Comparative Pathobiology, Cummings School of Veterinary Medicine, Tufts University, North Grafton, MA, USA. 3Janssen Vaccines & Prevention, Leiden, the Netherlands. 4Division of Anatomic Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA. 5Harvard Medical School, Boston, MA, USA. 6Alphagenesis, Yemassee, SC Alphagenesis, Yemassee, SC, USA. 7Walter Reed Army Institute of Research, Silver Spring, MD, USA. 8Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA. e-mail: [email protected]; [email protected] npj Vaccines | (2024)9:157 1 https://doi.org/10.1038/s41541-024-00927-8 Article ZIKV infection in pregnancy including prolonged ZIKV vRNA in blood vaccinated animals were not challenged to serve as normal pregnant con- and persistence of ZIKV vRNA in lymphoid tissues and the placenta23,26–33. trols. Plasma, sera, cerebral spinal fluid, urine, colorectal, cervical, and saliva Although fetal microcephaly has not been reported as a fetal outcome of samples were collected during pregnancy from all dams as indicated ZIKV infection in monkeys, experimental ZIKV challenge of pregnant non- (Fig. 1a). human primates has recapitulated other adverse outcomes of ZIKV expo- Neutralizing antibody titers were determined by both Immunospot sure observed in human pregnancy including fetal loss, fetal cerebral cal- focus reduction neutralization (FRNT) and by microneutralization assay cifications, gliosis, and long-term developmental alterations in infant (MN50 VNA). Before immunization, animals (OBF, 079 and 437) showed macaques30,34–36. As in humans, these events represent a small proportion of very low neutralization titers, whereas the other six animals showed no overall pregnancy outcomes. While DNA vaccination can protect against neutralization titers when assayed with FRNT (Fig. 1b). Four weeks after ZIKV vRNA in blood, break-through viral replication was seen in a subset of immunization, all animals developed a neutralizing response that was animals16 and DNA vaccination was only partially protective against vRNA maintained or only marginally decreased 8 weeks after immunization and in in blood in pregnant macaques37. Attempts to treat pregnant monkeys with pre-challenge serum which was obtained 10 to 30 weeks after immunization cocktails of neutralizing antibodies also failed to prevent ZIKV vRNA in (Fig. 1b, d). Four weeks after challenge, all animals of the Ad26.M.Env blood and adverse fetal outcomes were observed including fetal loss27. immunized group had increased ZIKV neutralization titers (Fig. 1b). Serum Adenoviruses are a wide-spread cause of common upper respiratory of animals of the non-immunized control group were assayed pre- and post- tract infections that are typically mild and self-limiting. Adenovirus vec- challenge. Pre-challenge, no neutralization titers were detected with FRNT tored vaccines are non-replicating yet self-adjuvanting due to their potent analysis, whereas ZIKV neutralization titers developed after challenge (Fig. induction of innate anti-viral responses that contribute to the induction of 1c). These FRNT results were confirmed by the MN50 neutralization assay broad cellular and humoral immune responses after a single (Supplementary Fig. 2). Vaccinated animals (Group 1) had higher neu- immunization38. Ad26 has emerged as a lead Ad vector platform due to low tralizing Ab four weeks post-vaccination compared to non-vaccinated levels of pre-existing immunity and induction of durable neutralizing Ab animals (p = 0.0010, Mann–Whitney test). Post-challenge neutralizing titers responses39. A common correlate of protection for currently licensed fla- were comparable between Ad26.M.Env and non-vaccinated animals vivirus vaccines including the yellow fever, dengue, and west nile viruses is (Mann–Whitney test), consistent with minimal anamnestic antibody the induction of neutralizing antibodies against the envelope (Env) responses in vaccinated animals and rapid virus neutralization (Fig. 1b, c, protein40. Ad26.M.Env is a monovalent recombinant Ad26-based ZIKV Supplementary Fig. 2). Next, antibody responses against ZIKV NS1 protein vaccine candidate that encodes for ZIKV membrane (M) protein, lacking were measured by ELISA. Ad26.M.Env does not contain a NS1 antigen. In the peptide precursor, and envelope (Env) antigens (amino acids 216–794 of accordance, Ad26.M.Env vaccinated or non-vaccinated animals had low the polyprotein) derived from the ZIKV strain BeH815744 that was shown (079) or undetectable NS1 binding antibody responses in pre-challenge to be immunogenic in animal models41). This construct has also been tested samples. Four weeks after challenge, all 5 non-vaccinated dams developed in a human Phase I study (Ad26.ZIKV.001) where it was shown to be well- high NS1-specific antibody titers (mean titer of 3.69 log10). Eight out of 9 tolerated and immunogenic22. In addition, it was shown to be protective dams that received Ad26.M.Env also developed NS1-specific titers after against fetal demise in pregnant interferon alpha/beta receptor knock-out challenge although the group mean NS1 titer (1.75log10) was approximately mice42), a highly susceptible model where ZIKV infection leads to high levels 100-fold (2 log10) lower compared to the group mean NS1-titer in the non- of ZIKV plasma vRNA levels and placental ZIKV vRNA and fetal loss43. We immunized animals (p = 0.005, Mann–Whitney test; Supplementary Fig. 3). opted to test this vaccine for safety and efficacy in pregnant rhesus macaques that were ZIKV challenged during the critical equivalent of human first Ad26.M.Env vaccination induced anti-Env cellular immune trimester pregnancy. Here we show that Ad26.M.Env prevents peripheral responses in macaques blood ZIKV vRNA and tissue vRNA in pregnant macaques and fetuses with Env and prM directed cellular immune responses were measured by IFNγ no evidence of ZIKV-associated fetal pathology in rhesus monkeys. ELISPOT on frozen PBMC’s isolated pre- immunization, post-immuniza- tion, pre-challenge, and post-challenge. Ad26.M.Env vaccination resulted in Results induction of ZIKV specific cellular responses (Fig. 2). The geometric mean Ad26.M.Env vaccination induced potent anti-ZIKV neutralizing Env-specific cellular immune responses in the group that received antibodies and prior vaccination did not impact conception in Ad26.M.Env was above the LOD of the assay, determined at 50 SFU per 106 female macaques PBMCs, at week 4 and 8 after immunization, and at the pre-challenge Thirteen female macaques were immunized with 1011vp of Ad26.M.Env timepoint (geomean SFU 55.03, 55.92, and 97.41, respectively). The Env- expressing both the ZIKV M protein transmembrane domain without the specific cellular immune responses after immunization were higher when peptide precursor, and the envelope (Env) antigens (Ad26.M.Env) and were compared to the Env-specific cellular immune responses pre-immuniza- returned to the breeding colony 17 days post-vaccination (Fig. 1a). Dams tion, or in non-immunized animals which were both below the limit of were vaccinated a minimum of 2 months and a maximum of 7 months prior detection (Fig. 2a, b). Vaccinated animals had significantly higher anti-Env to challenge. An additional 13 females were selected that received no vac- cellular immune responses pre-challenge (p = 0.0106) and post-challenge cination that were placed in the breeding colony at the same time as the (p = 0.0470) compared to non-vaccinated animals (Mann–Whitney test; vaccine group. No adverse events were reported in either vaccinated or non- with data points below the LOD of 50 SFU set on LOD). The prM-specific vaccinated animals prior to ZIKV challenge. Some animals in both the cellular responses were generally low and geometric mean responses do not vaccinated and non-vaccinated groups had reports of weight loss, diarrhea, exceed the cut-off of 50 SFU per 106 PBMCs (Fig. 2c, d). Notably, Env and and trauma that required clinical intervention, but need for clinical inter- prM cellular responses did not increase after challenge as compared to the vention was not linked to vaccination or ZIKV challenge. Vaccinated and pre-challenge timepoint indicating a lack of anamnestic cellular responses in non-vaccinated females were monitored clinically by complete red blood Ad26.M.Env vaccinated animals (Fig. 2a, c). cell count (CBC). Animals generally maintained normal reference ranges for white blood cell count, red blood cell count, and total lymphocyte counts Ad26.M.Env vaccinated pregnant females were completely pro- through-out the study period (Supplementary Fig. 1). tected against ZIKV vRNA in blood and tissues Females in all groups were monitored bi-weekly by ultrasound for Pregnant females in the Group 1 (Ad26.M.Env vaccinated) and Group 2 pregnancy. Pregnant females in the Ad26.M.Env vaccine and no vaccine (non-vaccinated) were infected with 1x103 PFU (1×106 vp) Zika virus from groups were infected with 1×106 vp of ZIKV via the subcutaneous route 6 the 2015 Brazilian epidemic at 6 weeks post-conception (being 10 to weeks post-conception (10–30 weeks post-vaccination based on timing of 30 weeks post vaccination). Vaccinated dams had no detectable virus in confirmed pregnancy; Supplementary Table 1). Three pregnant non- plasma post-ZIKV challenge even though animals were challenged from 10 npj Vaccines | (2024)9:157 2 https://doi.org/10.1038/s41541-024-00927-8 Article Fig. 1 | Ad26.M.Env induces robust neutralizing antibodies in sera in female the log10 of one dilution below the start dilution of the samples (0.70 log10). breeding macaques. a Macaques were vaccinated 17 days prior to introduction into Individual animals of group 1 (Ad26.M.Env immunized) are color coded to repre- breeding groups, monitored for pregnancy every two weeks and challenged with sent number of weeks between immunization and challenge; green (10 weeks); ZIKV-BR six weeks post-conception. ZIKV-PR neutralizing antibody responses in purple (12 weeks); blue (14 weeks); gray (18 weeks); red (22 weeks); brown sera of dams (n = 9) immunized with 1011 vp Ad26.M.Env (b, d) or non-immunized (30 weeks). Black arrow indicates time of vaccination; colored arrows indicate time control animals (n = 5) (c) determined by FRNT. d Colored connective lines of challenge matched to inter-immunization and challenge interval color in figure represent the neutralization response in time. Neutralizing antibody titers are legend. Amino amniocentesis, CSF cerebral spinal fluid, CR colorectal swab, CV reported as the log10 of the inverse of the serum dilution that reduce the number of cervical swab, PO oropharyngeal swab. b Friedman one-way ANOVA, followed by input virus by 50% (IC50). The mean responses per group are indicated with a Dunn’s test for multiple comparisons (c) Mann–Whitney test; *p = /< 0.05; **p = /< horizontal line. The dashed line shows the lower limit of detection (LOD) defined as 0.01; ***p = < 0.001. Samples below LOD were set to 0.70. to 30 weeks after vaccination, depending on the timepoint of conceiving of 7 to 56 days consistent with previous reports that pregnancy prolongs (Fig. 3a). In contrast, non-vaccinated, challenged pregnant macaques all had ZIKV viremia in rhesus monkeys. All colorectal, vaginal, saliva, and detectable viral load, with a mean peak vRNA of 5.5 log10 on day 7 post- amniocentesis samples were negative for ZIKV vRNA for all ZIKV- challenge (Fig. 3b) consistent with peak vRNA reported for ZIKV infected challenged dams irrespective of vaccination status. non-pregnant and pregnant macaques23,25,26,28,32,36,37,44,45. On average, non- After confirmation of pregnancy, dams were monitored monthly for vaccinated, challenged animals had detectable virus for 42 days with a range fetal biometric analyses including measurements of biparietal diameter, npj Vaccines | (2024)9:157 3 https://doi.org/10.1038/s41541-024-00927-8 Article Env specific a Cellular IFN- response b Env specific Cellular IFN- response SFU per 106 plasma derived cells 1000 1000 100 100 10 10 1 0.1 1 0BF 07G e e ng k k e e 4w 8w ng ng ng ng si le le le le do 07M al al al al e- ch ch ch ch Pr e- e- st st 437 Pr Pr po po k k 0C1 4w 4w c 079 0BE d prM specific prM specific Cellular IFN response Cellular IFN response 539 SFU per 106 plasma derived cells 1000 1000 032 100 100 10 10 1 0.1 1 e e ng k k e e 4w 8w ng ng ng ng si le le le le do al al al al e- ch ch ch ch Pr e- e- st st Pr Pr po po k k 4w 4w Fig. 2 | Ad26.M.Env vaccine induces low level cellular immune anti-Env considered positive. Individual animals of group 1 (Ad26.M.Env immunized) are responses in pregnant macaques. IFNγ ELISPOT responses in PBMCs of dams color coded to represent number of weeks between immunization and challenge; (n = 9) immunized with 1011 vp Ad26.M.Env (a, c) or non-immunized control green (10wk); purple (12 weeks); blue (14 weeks); gray (18 weeks); red (22 weeks); animals (n = 5) (b, d). a, b Env-specific responses and (c, d) PrM specific responses brown (30 weeks). a Friedman one-way ANOVA, followed by Dunn’s test for are shown. The geometric mean response per group is indicated with a horizontal multiple comparisons; *p = /< 0.05; **p = /< 0.01; ***p = < 0.001. Samples below line. Responses above 50 SFU per 106 PBMCs indicated by the dotted line are LOD were set to 50. occipitofrontal length, head circumference, and femur length by ultra- Neonates born to Ad26.M.Env vaccinated dams were negative sonography. No abnormalities were noted in fetal biometric parameters in for ZIKV vRNA and had no ZIKV-associated histopathological all study groups (Supplementary Fig. 4) and fetal brain weights and abnormalities brain:fetal body weight ratios at necropsy were similar across groups Following C-section and euthanasia, fetuses were inspected for gross (Supplementary Fig. 5). abnormalities and fetal tissues collected for histopathology and eva- Dams had scheduled Cesarian sections (C-section) and eutha- luation of vRNA. Fetuses of vaccinated dams had no evidence of viral nasia when fetuses were term, approximately 2 weeks prior to esti- replication in tissues (Fig. 4a, upper) while 2/5 fetuses from non-vacci- mated delivery date. Maternal tissues previously shown to have nated, challenged dams had detectable virus in tissues, one of which detectable virus throughout pregnancy were collected for evaluation (Fetus 560) had extensive detection of ZIKV in the brain (Fig. 4a, lower). by RT-PCR for ZIKV vRNA33). None of the nine Ad26.M.Env vac- Histopathologic evaluation of brain from fetuses born to Ad26.M.Env cinated dams had detectable vRNA in any tissues surveyed (Fig. 3c). vaccinated dams showed no evidence of previously reported ZIKV All non-vaccinated, challenged dams had detectable vRNA in at least neuropathology including microcalcifications and perivascular edema one of the analyzed tissues. 4/5 animals showed positive vRNA in (Fig. 4b–e, Fig. 5). Fetuses from non-vaccinated, challenged dams had a maternal spleen, consistent with previous reports23,33,34), and one dam myriad of abnormal findings (Table 1) including a gross cerebellar had vRNA detected in the axillary LN. One dam had detectable malformation (Fig. 4f), asymmetry of the left parietal lobe (Supple- vRNA in the uterus, and 3/5 dams had virus detectable in the pla- mentary Fig.6, Table 1), and a gross dystrophic calcification on the liver centa (Fig. 3d). Placental pathology was evaluated for dams in all (Table 1), focal edema (Fig. 4g), microcalcification (Fig. 4h), and groups by both a veterinary pathologist and a human gynecological meningeal proliferation (Fig. 4i). However, the fetuses from the non- pathologist specializing in placental histopathology. Histopathologi- vaccinated, challenged dams overall had fewer histopathological find- cal placental findings in all groups were typical of near-term/term ings than previously reported (Fig.5)33. Tissues that were positive for placentas in macaques with evidence of maternal thrombosis and ZIKV by qPCR were evaluated by IHC and RNAscope ISH, but virus was infarction in all groups (Supplementary Fig. 5, Table 1)46. Fetal: not detected within lesions therefore gross and histological abnormal- placental ratios were within the expected limits for term fetuses and ities in fetuses from non-vaccinated, challenged dams could not be did not vary significantly between groups (Supplementary Fig. 5). definitively linked to previous or on-going ZIKV viral replication. npj Vaccines | (2024)9:157 4 https://doi.org/10.1038/s41541-024-00927-8 Article a b Ad26.M.Env 07G Non-vaccinated 8 0BF 8 Log ZIKV copies/mL Log ZIKV copies/mL 07M 05L 0C1 02X 6 6 05K 437 560 558 4 0BE 4 539 032 2 2 0 20 40 60 80 100 0 20 40 60 80 100 Days Post Infection Days Post Infection c Ad26.M.Env d Non-vaccinated 2 07G 2 Log ZIKV copies/Pg RNA Log ZIKV copies/Pg RNA 0BF 07M 05L 0C1 02X 437 05K 1 1 560 0BE 558 539 032 0 0 P1 P2 P3 P4 P5 P6 P7 P8 Spleen Ax LN Ing LN Uterus Ovary Umblicus P1 P2 P3 P4 P5 P6 P7 P8 Spleen Ax LN Ing LN Uterus Ovary Umblicus Mammary Mammary Placenta Placenta Lymphoid/Repro Lymphoid/Repro Fig. 3 | Ad26.M.Env vaccination prevents viral replication in blood and dis- represent number of weeks between immunization and challenge; green (10wk); semination of ZIKV to tissues in pregnant dams. Pregnant Ad26.M.Env vacci- purple (12 weeks); blue (14 weeks); gray (18 weeks); red (22 weeks); brown nated (n = 9) and non- vaccinated (n = 5) macaques were challenged with 1×106 vp (30 weeks). Lymphoid and reproductive tissues were collected from dams at time of of ZIKV at 6 weeks following conception and sera was monitored longitudinally for Cesarian section (C-section). C-sections were performed 10–14 days prior to esti- 100 days following challenge. ZIKV log10 copies per mL of sera from (a) mated full-term delivery dates (~21–23 weeks pregnancy) at approximately 16 weeks Ad26.M.Env vaccinated dams and (b) non-vaccinated dams were determined by following ZIKV challenge. Viral RNA was determined by RT-PCR for (c) qRT-PCR and depicted as log10 ZIKV copies/mL sera at days 0, 7, 14, 28, 42, 56, 70, Ad26.M.Env vaccinated and (d) non-vaccinated dams on tissues collected at 84, and 98. The limit of detection of this assay was 100 copies/mL sera (2Log10). necropsy. AxLN axillary lymph node, IngLN inguinal LN. a Individual animals of group 1 (Ad26.M.Env immunized) are color coded to Discussion limited. The Ad26.CoV2.S vaccine construct was widely deployed during ZIKV viral infections world-wide resulted in a pandemic of fetal mal- the COVID-19 pandemic and was protective against severe disease and formations and fetal loss in pregnant women in 2016. The repercussions of variant infection after a single or prime-boost immunization47,48. In addition, this devasting disease will continue to manifest itself as cohorts of exposed Ad26.CoV2.S showed durable neutralizing antibody responses which may women and children continue to be followed. Although massive ZIKV offer some advantages over similar mRNA constructs in resource-limited exposure across endemic regions of South America has likely resulted in settings or during outbreak scenarios49. Currently an Ad26 Ebola vaccine elevated neutralizing antibodies in exposed populations, waning of immu- (Ad26.ZEBOV) is licensed and recommended as a childhood vaccine in nity over time will likely lead to cyclical re-emergence of ZIKV as seen with Ebola endemic regions50. Although, adenoviral based vaccines used to previous outbreaks in Malaysia. The United States is at risk for the re- combat COVID-19 were associated with immune-mediated thrombocy- emergence of a future ZIKV pandemic since the Aedes aegypti mosquito topenia and thrombosis, the mechanisms for these AE have not been fully vector is endemic to the southern United States. The lack of exposure of the elucidated, and more work is required to determine if these AE are related to US population to ZIKV during the 2016 epidemic makes US individuals the interplay between the Adenovirus based vaccine and the COVID-19 vulnerable to future ZIKV epidemics. Development of a well-tolerated and spike protein, or to the Adenovirus vaccine platform itself and can be safe vaccine that can be used pre- and perinatally is critical to protecting avoided by improvements in vaccine platform. mRNA based vaccines naïve individuals during future ZIKV outbreaks. against ZIKV have also been shown to be immunogenic and protective in The Ad26.M.Env (Ad26.ZIKV.001) vaccine was tested in a Phase I NHP challenge studies, with variable efficacy in inducing neutralizing clinical trial and all regimens tested were well tolerated, with no safety antibody responses in people18,20. Nevertheless, adenoviral constructs concerns identified. Vaccination induced robust ZIKV neutralizing titers remain one of the most studied vaccine vectors and will likely continue to be that were of similar magnitude to those induced in macaques in this study22. an important construct for global use. In addition, transfer of immune sera from vaccinated study participants to Here we show that an Ad26.M.Env vaccine is safe and efficacious mice protected mice against ZIKV vRNA in blood22. Viral vectored vaccines against preventing ZIKV vRNA in sera and tissue of pregnant rhesus that are immunogenic with a single immunization remain an attractive macaques. Antibody titers induced by A26.M.Env ZIKV vaccination in option for use in resource-poor settings where vaccine access may be macaques were of similar magnitude as those induced by vaccination in a npj Vaccines | (2024)9:157 5 https://doi.org/10.1038/s41541-024-00927-8 Article Fig. 4 | Ad26.M.Env vaccination prevents ZIKV dissemination and tissue vasculature within cerebellum (c), midbrain progenitor cells (d), and meninges (e) pathology in fetuses following ZIKV challenge of pregnant dams. Necropsy and as compared to cerebellar dysplasia (f, dotted white line), perivascular edema of collection of fetal tissues were performed on fetuses following euthanasia after term cerebellar vessel (g), microcalcification within midbrain progenitor cells (h), and Cesarian delivery. Each dam had only a single fetus. a RT-PCR was performed on unusual thickening of the meninges (i) in macaque infants from non-vaccinated fetal tissues in fetuses from Ad26.M.Env vaccinated and ZIKV-challenged dams dams. AxLN axillary lymph node, IngLN inguinal LN, Mes LN mesenteric lymph (upper) and non-vaccinated and ZIKV-challenged dams (lower). Formalin-fixed node, PFC prefrontal cortex, FC frontal cortex, BG1 basal ganglia section 1, HC/TH gross specimens and histopathological changes in fetuses from Ad26.M.Env vac- hippocampus, thalamus, OC occipital lobe; CB cerebellum, BS brain stem, CSC cinated and ZIKV challenged dams (b–e) as compared to non-vaccinated and ZIKV cervical spinal cord, TSC thoracic spinal cord, UC umbilical cord. Scale bars = challenged dams (f–i) showing normal cerebellar folio (b; dotted white line), 200 uM (d, h); 500 uM (c, e, g, i). Phase I clinical trial in people22 and to titers induced by the inactivated Zika during pregnancy. In both this study and the Phase I clinical trial with virus vaccine (ZPIV)19. While current low levels of ZIKV worldwide will Ad26.ZIKV.00122) no anti-PrM cellular immune responses were observed. impede the ability to conduct prevention of infection studies in people, This may be the consequence of limited antigen size or immunodominance studies of ZIKV vaccine protection in non-human primates provide critical of the Env epitope. data that can be extrapolated for immunobridging studies as has been done This study had several limitations that prevented us from assessing the with Ebola vaccines51,52. Vaccine induced neutralizing antibodies against full potential of the ZIKV Ad26.M.Env in protecting pregnant dams. In our ZIKV have been shown to be sufficient for protection in mice that have effort to capture fetal neuropathology, we allowed the pregnancies to con- undergone CD4+ and CD8 + T cell depletions15. Similar studies have not tinue to term which limited our ability to study ZIKV associated placental been performed to confirm that Ab titers alone are sufficient for maternal pathology which has been well-described in the macaque and marmoset protection during pregnancy. Notably, Ad26.M.Env also induced robust models of ZIKV infection during pregnancy53. Similarly, we did not sacrifice anti-Env CD4+ and CD8 + T cell responses. Further work is necessary to fetuses at timepoints where unvaccinated dams remained viremic in plasma, determine if cellular immune responses offer improved quality of protection which limited our ability to more rigorously determine if vaccination npj Vaccines | (2024)9:157 6 https://doi.org/10.1038/s41541-024-00927-8 Article Fig. 5 | Histopathologic scoring for neuropathol- ogy in macaque infants. Summary of neuropatho- logic lesions in the CNS (see also Table 1). Scoring system is defined as previously described in ref. 33. PFC prefrontal cortex, FC frontal cortex, BG basal ganglia, TH thalamus, SN substantia nigra, HC hippocampus, OC occipital cortex, PC parietal cortex, TC temporal cortex, CB cerebellum, BS brain stem, CSC cervical spinal cord, TSC thoracic spinal cord, LSC lumbosacral spinal cord, DRG dorsal root ganglia. protects against virus in the placenta and fetal brain during periods of robust were research naive. Upon arrival and standard quarantine procedures, systemic viral replication. In addition, fewer neuropathological findings animals were tested for tuberculosis (TB) at least three times at intervals of were detected in fetuses from non-vaccinated, challenged dams in the two weeks. Animals were also screened for Herpes B, Simian retrovirus current study as compared to our previous report33 highlighting the need for (SRV), Simian Immunodeficiency Virus (SIV), Simian T-cell Leukemia studies with large numbers of animals to capture the full spectrum of fetal Virus (STLV), and Measles. All animals were Herpes B, SRV, SIV, and STLV outcomes. Such large-scale studies are typically not possible due to negative. Study animals were selected based on age. Females were all aged 4- restrictions on the use of NHP for research and limitations in animal 8 years old with similar age and weight distribution per study group. The availability. Likewise, we observed two gross anomalies in the fetal brains of study protocol was reviewed and approved by the Alphagenesis Institutional non-vaccinated, ZIKV challenged animals, but given the limited evaluation Care and Use Committee (IACUC). All experiments conformed to reg- of fetal macaque brains at term in normal colony macaques, we cannot rule ulatory standards outlined by the American Veterinary Medical Association out that the gross lesions observed represent normal variation in macaque (AVMA) and American Association of Laboratory Animal Medi- brains despite their absence in the vaccinated group. Furthermore, questions cine (AALAM). remain regarding the potential role for previous infection with other related viruses such as Dengue fever virus (DENV) in predisposing to ZIKV- Breeding, immunization, and ZIKV challenge induced development of CZS54. Recent studies in both pregnant macaques Seven weeks prior to immunization, females were removed from their and marmosets have shown evidence of enhanced neuropathology and breeding group. For Group 1 (Ad26.M.Env vaccinated, ZIKV-challenge), placental pathology in previously DENV exposed, ZIKV infected nine dams that were not pregnant according to ultrasound were intra- animals55,56. Future studies on ZIKV vaccine efficacy in DENV pre-exposed muscularly immunized with 1×1011 vp Ad26.M.Env. The nine immunized dams may be warranted. Lastly, we were unable to follow a cohort of infants dams were reintroduced to their breeding groups 17 days later. All animals longitudinally in this study to determine whether vaccination of dams were provided enrichment according to recommended guidelines. Dams prevented neurocognitive deficits in offspring57). were monitored for pregnancy every 2 weeks by ultrasound until confirmed All non-vaccinated dams in this study had persistent virus detected in pregnant. After confirmed pregnancy, dams were monitored by ultrasound lymphoid organs at term, supporting that ZIKV infection during pregnancy every 4 weeks. For Group 1, all nine dams had confirmed pregnancy. For can have severe consequences for pregnant women in the absence of clinical Group 2 (non-vaccinated, ZIKV-challenge controls), 7 dams were included signs. Here we show that vaccination with Ad26.M.Env prevents persistent in the study, of which 5 were confirmed pregnant. Pregnant control dams ZIKV replication in lymphoid organs, placenta and fetal tissue including (Group 3, no vaccination, no ZIKV-challenge) were included from the brain in the non-human primate model with a single immunization. Future breeding colony and were of similar age and source as vaccinated and non- studies evaluating Ad26.M.Env protection during the acutely viremic phase vaccinated, challenged animals (Groups 1&2). in pregnant macaques would extend these findings. These data combined Approximately six weeks after calculated date of conception (based on with the Phase I safety and immunogenicity data in people suggests that the ultrasound results), equivalent to human 1st trimester of pregnancy, dams Ad26.M.Env is likely to be efficacious in preventing maternal ZIKV from groups 1 and 2 were challenged with 1 × 106 vp (103 PFU) of ZIKV-BR infection. via the subcutaneous route. During pregnancy, blood and PBMCs were isolated for immunogenicity readouts. Plasma, cerebrospinal fluid (CFS), Methods urine, colorectal biopsies, inguinal/axillary lymph node (LN) biopsies, rectal, Experimental model and subject details vaginal and saliva secretion were taken to monitor ZIKV vRNA. At Outbred, healthy Indian-origin female rhesus monkeys (Macaca mulatta) approximately week 21–23 of pregnancy ( ≈ week 16 following challenge) were housed at Alphagenesis, Yemassee, SC. Animals selected for this study fetuses of all groups were delivered by C- section except for one (OBE) from npj Vaccines | (2024)9:157 7 npj Vaccines | (2024)9:157 Table 1 | Summary of gross and histopathological findings in dam and neonate tissues Animal ID Condition Group Sex Gross Findings Histopathology-Fetus Histopathology-Placenta 78 Control 3 F NSF Hemorrhage in lateral ventricle moderate calcifications 583 Control 3 M NSF Cortical microcalcification NSF https://doi.org/10.1038/s41541-024-00927-8 541 Control 3 M NSF Increased meningeal cellularity NSF 05K ZIKV 2 F Hard, white lesion noted in liver Cortical/neuroprogenitor dysplasia (mild); focal gliosis placental thinning 560 ZIKV 2 F absent occipital gyrus (L); focal proliferation of Cortical/neuroprogenitor dysplasia (mild); Cortical and periventricular NSF neuropil on the right cerebellar lateral microcalcification; multifocal microhemorrhage; mild neuropil vacuolation/ hemisphere rarefication; multifocal meningeal proliferation; increased meningeal cellularity 05L ZIKV 2 M Dilated lateral ventricle upon examination of Cortical microcalcification thrombosis maternal vessel fixed specimens; asymmetry L parietal lobe 02X ZIKV 2 M Absent gyrus in parietal cortex; cloudy CSF Microcalcification within neuroprogenitor clusters, perivascular edema, and NSF necrosis; mild neuropil vacuolation/rarefication; focal gliosis 558 ZIKV 2 M Enlarged ventricle noted upon examination of Cortical/neuroprogenitor dysplasia (mild); cortical microcalcification; Moderate infarction/necrosis chorionic plate; fixed specimens hemorrhage in lateral ventricle; multifocal gliosis; increased meningeal thrombosis maternal vessel cellularity; multifocal spinal cord microhemorrhage 07G Vaccinated, ZIKV 1 M NSF Cortical/neuroprogenitor dysplasia (mild); mild neuropil vacuolation/ thrombosis maternal vessel rarefication; focal gliosis 0BF Vaccinated, ZIKV 1 M NSF NSF Mild abruption; thrombosis maternal vessel 437 Vaccinated, ZIKV 1 M NSF NSF Mild infarction/necrosis chorionic plate; placental thinning; thrombosis maternal vessel 07M Vaccinated, ZIKV 1 F NSF Cortical microcalcification; multifocal microhemorrhage; mild neuropil NSF vacuolation/rarefication OC1 Vaccinated, ZIKV 1 F NSF NSF NSF OBE Vaccinated, ZIKV 1 F NSF NSF Not evaluated; Vaginal delivery infant 79 Vaccinated, ZIKV 1 F NSF NSF NSF 32 Vaccinated, ZIKV 1 F NSF Mild neuropil vacuolation/rarefication Mild infarction/necrosis chorionic plate; thrombosis maternal vessels Article 8 https://doi.org/10.1038/s41541-024-00927-8 Article the vaccine group that was born naturally due to earlier than predicted measured on the NanoDrop (Thermo Scientific, Waltham, MA, USA) or delivery. One pregnancy was lost in the vaccine group due to culture con- as VP per million cells, as shown in Figs. 3 and 4. Assay sensitivity was firmed staphylococcal placentitis (539) and this dam/infant pair was >100 copies/mL, >100 copies per million cells, and >3 copies/mg removed from the study. Reproductive failure or preterm delivery is sig- total RNA. nificant among primates, and the observations in this study were within the historical control range for the testing facility (AGI), and within expected Neutralization Assays. ZIKV-specific neutralizing antibodies were outcomes for pregnancies in rhesus monkeys. measured by fold reduction neutralization (FRNT) and micro- Five live male infants and three live female infants were delivered neutralization (MN) assays, as previously described in refs. 15,16,26,33. among the vaccinated, challenged group. Three live male infants and two For FRNT assay Vero cells were seeded at a concentration of 2 × 104 cells/ live female infants were delivered to the non-vaccinated, challenged group. well in 96-well plates 24 h prior to the assay initiation. Heat inactivated Two live males and one live female infant were delivered to the control serum samples were serially diluted prior to being mixed and incubated group (non-vaccinated, non-ZIKV challenged; Table 1). Dams and fetuses with input virus ZIKV-PR (PRVABC59) for 1 hour at 37 °C. Cell-seeded were euthanized for post-mortem gross pathology, histopathology, and 96-well plates were infected with 100 μL of the virus/serum mixtures for virologic assessments. 1 hour before the addition of overlay media. Each serum dilution was tested in triplicate wells. Approximately 24 h after infection, ZIKV foci Method details were detected using an antiflavivirus detection antibody, a horseradish Ad26.M.Env. Complete details about the construction and production of peroxidase (HRP)-conjugated secondary antibody and True-Blue per- the Ad26.M.Env construct are available15,16,22. Briefly, the vaccine was oxidase substrate. ZIKV foci were visualized and counted using an produced on the human PER.C61 cell line and purified and characterized ImmunoSpot analyzer and software. Each assay run included virus input as described previously in ref. 58. Ad26 particle concentrations were and media-only control wells, as well as negative and positive control determined by optical density at 260 nm and viral infectivity by TCID50 serum samples. Neutralizing antibody titers were reported as the inverse assay. All vaccine preparations were tested for bioburden and endotoxin of the serum dilution estimated to reduce the number of input virus by levels (MicroSafe, Millipore, Leiden, The Netherlands) and have passed 50% (FRNT50) as shown in Fig. 1. pre-set release criteria for animal experiments. Microneutralization assays were performed at WRAIR. Serum samples were serially diluted three-fold in 96-well micro-plates, in a total volume of ZIKV challenge stock preparation. ZIKV-BR (Brazil ZKV2015) was 100 uL. 102 PFU ZIKV-PR (PRVABC59) in a total volume of 100uL was propagated in Vero cells (World Health Organization, NICSC- added and incubated at 35 °C for 2 h. Serum/virus mixtures were then 011038011038) that were maintained in EMEM media supplemented transferred to microtiter plates containing confluent Vero cell monolayers with 10%FBS, 6mM L-glutamine and 1x pen/strep. Cells were passaged (World Health Organization, NICSC-011038011038). After incubation for twice a week and incubated at 37 °C, 10% CO2. 4 days, cells were fixed with absolute ethanol: methanol for 1 h at –20 °C and washed three times with PBS. The pan-flavivirus monoclonal antibody 6B6- Ultrasonography. Ultrasounds were performed every 2–4 weeks in C1 conjugated to HRP (6B6-C1 was a gift from JT Roehrig, CDC) was then the ZIKV-infected pregnant rhesus monkeys as well as in 3 unin- added to each well, incubated at 35 °C for 2 h, and washed with PBS. Plates fected pregnant rhesus monkeys in the same breeding facility. Ani- were washed, developed with 3,3’,5,5’–tetramethylbenzidine (TMB) for mals were sedated with Telazol (5 mg/kg), and a GE Logic E with an 50 min at room temperature, stopped with 1:25 phosphoric acid, and 8CRS Micro-convex transducer (FOV 132, 3.6–10 MHz) was used for absorbance was read at 450 nm. For a valid assay, the average absorbance at multiparameter biometric measurements, including biparietal dia- 450 nm of three non-infected control wells had to be % 0.5, and virus-only meter (BPD), occipitofrontal diameter (OFD), head circumference control wells had to be R 0.9. Normalized absorbance values were calculated, (HC), crown-rump length (CRL), abdominal circumference (AC), the MN50 titer was determined by a log mid-point linear regression model. and femur length (FL). The MN50 titer was calculated as the reciprocal of the serum dilution that neutralized R 50% of ZIKV, and seropositivity was defined as a titer R10, Amniocentesis. Animals were sedated with Telazol HCL (4–7 mg/kg with the maximum measurable titer 7290, as shown in Supplementary IM). The area on the abdomen was clipped and sterilely prepped with Fig. 2. triple alternating applications of betadine and alcohol. Using sterile technique, a 22-gauge 3.34-inch needle on a 3-cc syringe was inserted into Tissue Collection and Histopathology. Within 14 days of estimated the ventral abdomen to the amniotic sac with ultrasound guidance. 2 cc of term gestation (26 weeks), dams and fetuses were euthanized with amniotic fluid was collected and frozen immediately. intravenous sodium pentobarbital, and delivery was by caesarian section. Complete necropsies were performed by a veterinarian RT-PCR. RT-PCR assays were utilized to monitor viral loads in plasma, (A.J.M) on fetuses immediately following euthanasia, utilizing stan- CSF, lymph node biopsies, colorectal biopsies, colorectal weck samples, dard necropsy procedures with standard sterile surgical grade and urine longitudinally every 2–4 weeks as indicated in the experimental necropsy instruments and dissection blades. Briefly, peripheral lym- design (see Fig. 1a) and amniotic fluid collected by amniocentesis at day phoid tissues were collected, followed by the gastrointestinal tract 14 post-ZIKV infection, and from tissues collected at necropsy, essen- and abdominal organs. The pleural cavity was opened and the ton- tially as previously described in refs. 15,16,26,33. RNA was extracted with gue, pharynx, trachea, esophagus, heart, and lungs (“pluck”) were a QIAcube HT (Qiagen, Germany). Liquid samples were extracted using removed en masse. Reproductive organs were collected, followed by the Qiacube 96 Cador pathogen HT, and tissue samples were lysed in brain, spinal cord, and eyes. Ruskin-Liston bone cutting forceps were Qiazol, using the Tissuelyser II (Qiagen, Germany), chloroform treated used to expose the spinal cord to the level of the cauda equina. and extracted with the Qiacube 96 RNeasy HT kit. The wildtype ZIKV Limited necropsies were performed on dams for tissues previously BeH815744 Cap gene was utilized as a standard. RNA standards were shown to harbor viral RNA including reproductive organs, lymphoid generated using the AmpliCap-Max T 7 High Yield Message Maker Kit tissues, spleen, and placenta. Fresh tissues were collected utilizing (Cell Script) and purified with RNA clean and concentrator kit (Zymo sterile blades for viral RT-PCR in RNAlater (Ambion). Frozen tissue Research, CA, USA). RNA quality and concentration was assessed by the for histopathology was prepared by trimming tissue, placing tissue BIDMC Molecular Core Facility. Log dilutions of the RNA standard were samples into cryomolds with optimal cutting temperature medium reverse transcribed and included with each RT-PCR assay. Viral loads (OCT, Tissue-Tek), and flash freezing on-site. Additional tissues were were calculated as virus particles (VP) per microgram of total RNA as fixed in 10% neutral buffered formalin (NBF) for histopathology. npj Vaccines | (2024)9:157 9 https://doi.org/10.1038/s41541-024-00927-8 Article Formalin-fixed tissues were trimmed, processed, and embedded in 450 nm/550 nm on a VersaMax microplate reader using Softmax Pro paraffin, sectioned, and stained with hematoxylin and eosin, and 6.0 software (Molecular Devices, CA, USA). ELISA endpoint titers were evaluated independently by two blinded veterinary pathologists defined as the highest reciprocal serum dilution that yielded an absor- (A.J.M., R.B.). Placenta was evalualated by a blinded gynecologic bance > 2-fold over background values and plotted as Log10 pathologist (J.L.H). endpoint titer. Immunohistochemistry and in situ hybridization. Immunohis- Data availability tochemistry and in situ hybridization (RNAscopeTM) were performed All data generated and analyzed in this study are available from the Lead as previously described in ref. 33. Briefly, tissue sections were Contact upon reasonable request. deparaffinized in xylene and rehydrated through graded ethanol solutions to distilled water. Endogenous peroxidase activity was Received: 15 February 2024; Accepted: 23 July 2024; blocked by incubation with 3% hydrogen peroxide followed by heat induced epitope retrieval (HIER) in citrate buffer (Vector Labs) using a slide steamer (IHC World). Tissues were treated for nonspecific References protein binding (Protein Block, DAKO) followed by application of 1. Brasil, P. et al. Zika virus infection in pregnant women in Rio de mouse-anti ZIKV envelope (BioFront Technologies; BF-1176-56, Janeiro. N. Engl. J. Med. 375, 2321–2334 (2016). 1:200) for 30 min at room temperature. A biotin-free polymer-based 2. Driggers, R. W. et al. 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