Human cytomegalovirus (HCMV) infection is transmitted from the infected mother to the placenta and fetus. Virus replicates in the decidua, invasive cytotrophoblasts that breach the uterine vasculature and villous cytotrophoblasts underlying syncytiotrophoblasts, then reaches blood vessels in the villus core. Virus replication, fibrosis, and edema result in a hypoxic intrauterine environment and release of cytokines that stimulates compensatory development of the placenta. We employed villous explant cultures to study viral effects on differentiation and test novel approaches to rescue the placenta from infection.

Human cytomegalovirus (HCMV) infection is transmitted from the infected mother to the placenta and fetus. Virus replicates in the decidua, invasive cytotrophoblasts that breach the uterine vasculature and villous cytotrophoblasts underlying syncytiotrophoblasts, then reaches blood vessels in the villus core. Virus replication, fibrosis, and edema result in a hypoxic intrauterine environment and release of cytokines that stimulates compensatory development of the placenta. We employed villous explant cultures to study viral effects on differentiation and test novel approaches to rescue the placenta from infection.

Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, yet there are no established treatments for preventing maternal-fetal transmission. During first trimester, HCMV replicates in basal decidua that functions as a reservoir for virus and source of transmission to the attached placenta and fetal hemiallograft but also contains immune cells, including natural killer cells, macrophages, and T cell subsets, that respond to pathogens, protecting the placenta and fetus. However, the specific cellular and cytokine responses to infection are unknown, nor are the immune correlates of protection that guide development of therapeutic strategies. Here we survey immune cell phenotypes in intact explants of basal decidua infected with a clinical pathogenic HCMV strain ex vivo and identify specific changes occurring in response to infection in the tissue environment. Using 4-color immunofluorescence microscopy, we found that at 3 days postinfection, virus replicates in decidual stromal cells and epithelial cells of endometrial glands. Infected cells and effector memory CD8+ T cells (TEM) in contact with them make IFN-γ. CD8+ TEM cells produce granulysin and cluster at sites of infection in decidua and the epithelium of endometrial glands. Quantification indicated expansion of two immune cell subtypes-CD8+ TEM cells and, to a lesser extent, iNKT cells. Approximately 20% of immune cells were found in pairs in both control and infected decidua, suggesting frequent cross-talk in the microenvironment of decidua. Our findings indicate a complex immune microenvironment in basal decidua and suggest CD8+ TEM cells play a role in early responses to decidual infection in seropositive women.

Background

During pregnancy, the Zika flavivirus (ZIKV) infects human placentas, inducing defects in the developing fetus. The flavivirus nonstructural protein 1 (NS1) alters glycosaminoglycans on the endothelium, causing hyperpermeability in vitro and vascular leakage in vivo in a tissue-dependent manner. The contribution of ZIKV NS1 to placental dysfunction during ZIKV infection remains unknown.

Methods

We examined the effect of ZIKV NS1 on expression and release of heparan sulfate (HS), hyaluronic acid (HA), and sialic acid on human trophoblast cell lines and anchoring villous explants from first-trimester placentas infected with ZIKV ex vivo. We measured changes in permeability in trophoblasts and stromal cores using a dextran-based fluorescence assay and changes in HA receptor expression using immunofluorescent microscopy.

Results

ZIKV NS1 in the presence and absence of ZIKV increased the permeability of anchoring villous explants. ZIKV NS1 induced shedding of HA and HS and altered expression of CD44 and lymphatic endothelial cell HA receptor-1, HA receptors on stromal fibroblasts and Hofbauer macrophages in villous cores. Hyaluronidase was also stimulated in NS1-treated trophoblasts.

Conclusions

These findings suggest that ZIKV NS1 contributes to placental dysfunction via modulation of glycosaminoglycans on trophoblasts and chorionic villi, resulting in increased permeability of human placentas.

The emergence of congenital Zika virus (ZIKV) disease, with its devastating effects on the fetus, has prompted development of vaccines and examination of how ZIKV breaches the maternal-fetal barrier. Infection of placental and decidual tissue explants has demonstrated cell types at the uterine-placental interface susceptible to infection and suggests routes for transmission across the placenta and amniochorionic membrane. ZIKV replicates in proliferating Hofbauer cells within chorionic villi in placentas from severe congenital infection. Explants of anchoring villi recapitulate placental architecture and early-stage development and suggest infected Hofbauer cells disseminate virus to fetal blood vessels. ZIKV infection of explants represents a surrogate human model for evaluating protection against transmission by antibodies in vaccine recipients and passive immune formulations and novel therapeutics.

Human cytomegalovirus (HCMV) is the major viral cause of congenital infection and birth defects. Primary maternal infection often results in virus transmission, and symptomatic babies can have permanent neurological deficiencies and deafness. Congenital infection can also lead to intrauterine growth restriction, a defect in placental transport. HCMV replicates in primary cytotrophoblasts (CTBs), the specialized cells of the placenta, and inhibits differentiation/invasion. Human trophoblast progenitor cells (TBPCs) give rise to the mature cell types of the chorionic villi, CTBs and multi-nucleated syncytiotrophoblasts (STBs). Here we report that TBPCs are fully permissive for pathogenic and attenuated HCMV strains. Studies with a mutant virus lacking a functional pentamer complex (gH/gL/pUL128-131A) showed that virion entry into TBPCs is independent of the pentamer. In addition, infection is blocked by a potent human neutralizing monoclonal antibody (mAb), TRL345, reactive with glycoprotein B (gB), but not mAbs to the pentamer proteins pUL130/pUL131A. Functional studies revealed that neutralization of infection preserved the capacity of TBPCs to differentiate and assemble into trophospheres composed of CTBs and STBs in vitro. Our results indicate that mAbs to gB protect trophoblast progenitors of the placenta and could be included in antibody treatments developed to suppress congenital infection and prevent disease.

Background

Maternal Zika virus (ZIKV) infection with prolonged viremia leads to fetal infection and congenital Zika syndrome. Previously, we reported that ZIKV infects primary cells from human placentas and fetal membranes. Here, we studied viral replication in numerous explants of anchoring villi and basal decidua from first-trimester human placentas and midgestation amniotic epithelial cells (AmEpCs).

Methods

Explants and AmEpCs were infected with American and African ZIKV strains at low multiplicities, and ZIKV proteins were visualized by immunofluorescence. Titers of infectious progeny, cell proliferation, and invasiveness were quantified.

Results

In anchoring villus, ZIKV replicated reproducibly in proliferating cytotrophoblasts in proximal cell columns, dividing Hofbauer cells in villus cores, and invasive cytotrophoblasts, but frequencies differed. Cytotrophoblasts in explants infected by Nicaraguan strains were invasive, whereas those infected by prototype MR766 largely remained in cell columns, and titers varied by donor and strain. In basal decidua, ZIKV replicated in glandular epithelium, decidual cells, and immune cells. ZIKV-infected AmEpCs frequently occurred in pairs and expressed Ki67 and phosphohistone H3, indicating replication in dividing cells.

Conclusions

ZIKV infection in early pregnancy could target proliferating cell column cytotrophoblasts and Hofbauer cells, amplifying infection in basal decidua and chorionic villi and enabling transplacental transmission.

Human cytomegalovirus (HCMV) is the leading viral cause of birth defects, including microcephaly, neurological deficits, hearing impairment, and vision loss. We previously reported that epithelial cells in amniotic membranes of placentas from newborns with intrauterine growth restriction and underlying congenital HCMV infection contain viral proteins in cytoplasmic vesicles. Herein, we immunostained amniotic membranes from 51 placentas from symptomatic and asymptomatic congenital infection with HCMV DNA in amniotic fluid and/or newborn saliva, intrauterine growth restriction, preterm deliveries, and controls. We consistently observed HCMV proteins in amniotic epithelial cells (AmEpCs) from infected placentas, sometimes with aberrant morphology. Primary AmEpCs isolated from mid-gestation placentas infected with pathogenic VR1814 proliferated and released infectious progeny for weeks, producing higher virus titers than late-gestation cells that varied by donor. In contrast to intact virion assembly compartments in differentiated retinal pigment epithelial cells, infected AmEpCs made dispersed multivesicular bodies. Primary AmEpCs and explants of amniochorionic membranes from mid-gestation placentas formed foci of infection, and interferon-β production was prolonged. Infected AmEpCs up-regulated anti-apoptotic proteins survivin and Bcl-x_L by mechanisms dependent and independent of the activated STAT3. Amniotic membranes naturally expressed both survivin and Bcl-x_L, indicating that fetal membranes could foster persistent viral infection. Our results suggest strengthening innate immune responses and reducing viral functions could suppress HCMV infection in the fetal compartment.

Zika virus (ZIKV) infection during pregnancy is linked to severe birth defects, but mother-to-fetus transmission routes are unknown. We infected different primary cell types from mid- and late-gestation placentas and explants from first-trimester chorionic villi with the prototype Ugandan and a recently isolated Nicaraguan ZIKV strain. ZIKV infects primary human placental cells and explants-cytotrophoblasts, endothelial cells, fibroblasts, and Hofbauer cells in chorionic villi and amniotic epithelial cells and trophoblast progenitors in amniochorionic membranes-that express Axl, Tyro3, and/or TIM1 viral entry cofactors. ZIKV produced NS3 and E proteins and generated higher viral titers in amniotic epithelial cells from mid-gestation compared to late-gestation placentas. Duramycin, a peptide that binds phosphatidylethanolamine in enveloped virions and precludes TIM1 binding, reduced ZIKV infection in placental cells and explants. Our results suggest that ZIKV spreads from basal and parietal decidua to chorionic villi and amniochorionic membranes and that targeting TIM1 could suppress infection at the uterine-placental interface.

Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects worldwide, yet the most effective strategies for preventing virus transmission during pregnancy are unknown. We measured the efficacy of human monoclonal antibodies (mAbs) to HCMV attachment/entry factors glycoprotein B (gB) and the pentameric complex, gH/gL-pUL128-131, in preventing infection and spread of a clinical strain in primary placental cells and explants of developing anchoring villi. A total of 109 explants from five first-trimester placentas were cultured, and infection was analyzed in over 400 cell columns containing ~120,000 cytotrophoblasts (CTBs). mAbs to gB and gH/gL, 3-25 and 3-16, respectively, neutralized infection in stromal fibroblasts and trophoblast progenitor cells. mAbs to pUL128-131 of the pentameric complex, 1-103 and 2-18, neutralized infection of amniotic epithelial cells better than mAbs 3-25 and 3-16 and hyperimmune globulin. Select mAbs neutralized infection of cell column CTBs, with mAb 2-18 most effective, followed by mAb 3-25. Treatment of anchoring villi with mAbs postinfection reduced spread in CTBs and impaired formation of virion assembly compartments, with mAb 2-18 achieving better suppression at lower concentrations. These results predict that antibodies generated by HCMV vaccines or used for passive immunization have the potential to reduce transplacental transmission and congenital disease.