Throughout evolution, the placenta has diversified in structure and composition while maintaining its essential role in supporting fetal development. Trophoblasts, cells responsible for nutrient exchange and immune modulation, are a conserved feature of all placentas. Although primate placentas share morphological similarities, species-specific differences in gene expression remain poorly characterized, largely due to the lack of tractable in vitro models. To address this gap, we developed rhesus macaque placental organoids representing trophoblast and maternal-derived decidual cell types and compared them with human placental organoids. Using integrated single-cell and single-nucleus RNA sequencing, we identified shared and species-specific transcriptional programs across corresponding trophoblast lineages. We further reconstructed lineage trajectories to multinucleated syncytiotrophoblast and extravillous trophoblast populations, revealing conserved differentiation pathways alongside divergent gene expression signatures. This work establishes in vitro models of the nonhuman primate placenta and defines molecular distinctions between human and rhesus trophoblasts, offering insights into evolutionary adaptations underlying placental development.

Human cytomegalovirus (HCMV) encodes four viral Fc-gamma receptors (vFcγRs) that counteract antibody-mediated activation in vitro, but their role in infection and pathogenesis is unknown. To examine their in vivo function in an animal model evolutionarily closely related to humans, we identified and characterized Rh05, Rh152/151 and Rh173 as the complete set of vFcγRs encoded by rhesus CMV (RhCMV). Each one of these proteins displays functional similarities to their prospective HCMV orthologs with respect to antagonizing host FcγR activation in vitro. When RhCMV-naïve male rhesus macaques were infected with vFcγR-deleted RhCMV, peak plasma DNAemia levels and anti-RhCMV antibody responses were comparable to wildtype infections of both male and female animals. However, the duration of plasma DNAemia was significantly shortened in immunocompetent, but not in CD4 + T cell-depleted animals. Since vFcγRs were not required for superinfection of rhesus macaques, we conclude that these proteins can prolong lytic replication during primary infection by evading virus-specific adaptive immune responses, particularly antibodies. The role of viral Fc-gamma receptors in rhesus cytomegalovirus (RhCMV) infection is unclear. Here, the authors characterized RhCMV vFcγRs and report that their deletion did not affect virus replication, tropism or superinfection in rhesus macaques but increased susceptibility of the virus to antibody control.

Congenital cytomegalovirus (cCMV) is the leading infectious cause of birth defects worldwide, yet immune determinants of protection to inform maternal vaccine design remain elusive due to the lack of a translational animal model. Here, we characterized the outcome of primary rhesus CMV (RhCMV) infection in pregnant, immunocompetent, CMV-naïve rhesus macaques. RhCMV DNA was detected in amniotic fluid and/or fetal tissues in six of 12 (50% placental transmission) dams following early second trimester gestation RhCMV inoculation. Widespread tissue dissemination dominated by one of two inoculated RhCMV strains was present in one fetus (8.3% cCMV disease). Placental transmission was associated with elevated fetal and maternal plasma TNF-alpha and reduced maternal brain-derived neurotrophic factor and IL-10 levels. CMV exposure during pregnancy had a broad impact on the placenta and fetus even in the absence of congenital infection, as evidenced by ubiquitous maternal-fetal interface infection, and reduced placental efficiency and small-for-gestation age fetuses compared to control pregnancies. This model recapitulates key aspects of human cCMV and provides new insights into the complexity of CMV vertical transmission.

Cytomegalovirus (CMV) is the most common congenital infection and cause of birth defects worldwide. Primary CMV infection during pregnancy leads to a higher frequency of congenital CMV (cCMV) than maternal re-infection, suggesting that maternal immunity confers partial protection. However, poorly understood immune correlates of protection against placental transmission contributes to the current lack of an approved vaccine to prevent cCMV. In this study, we characterized the kinetics of maternal plasma rhesus CMV (RhCMV) viral load (VL) and RhCMV-specific antibody binding and functional responses in a group of 12 immunocompetent dams with acute, primary RhCMV infection. We defined cCMV transmission as RhCMV detection in amniotic fluid (AF) by qPCR. We then leveraged a large group of past and current primary RhCMV infection studies in late-first/early-second trimester RhCMV-seronegative rhesus macaque dams, including immunocompetent (n = 15), CD4+ T cell-depleted with (n = 6) and without (n = 6) RhCMV-specific polyclonal IgG infusion before infection to evaluate differences between RhCMV AF-positive and AF-negative dams. During the first 3 weeks after infection, the magnitude of RhCMV VL in maternal plasma was higher in AF-positive dams in the combined cohort, while RhCMV glycoprotein B (gB)- and pentamer-specific binding IgG responses were lower magnitude compared to AF-negative dams. However, these observed differences were driven by the CD4+ T cell-depleted dams, as there were no differences in plasma VL or antibody responses between immunocompetent AF-positive vs AF-negative dams. Overall, these results suggest that levels of neither maternal plasma viremia nor humoral responses are associated with cCMV following primary maternal infection in healthy individuals. We speculate that other factors related to innate immunity are more important in this context as antibody responses to acute infection likely develop too late to influence vertical transmission. Yet, pre-existing CMV glycoprotein-specific and neutralizing IgG may provide protection against cCMV following primary maternal CMV infection even in high-risk, immunocompromised settings.

Cytomegalovirus (CMV) is a top priority in vaccine development due to the high burden of disease in immunocompromised populations and congenitally infected infants. Evidence suggests that CMV-specific Fc-mediated antibody effector responses, such as antibody dependent cellular phagocytosis and cytotoxicity, are key for prevention of multiple CMV disease outcomes. However, human CMV expresses three Fcγ receptors that have been shown in vitro to synergistically interfere with anti-CMV IgG engagement of host Fcγ receptors (FcγRs). We recently identified three functionally homologous viral Fcγ receptors (vFcγRs) in rhesus macaque (RM) CMV (RhCMV), which allow us to validate the function of vFcγRs in vivo. Using a full-length (FL) RhCMV backbone (Taher, et al. 2020), we produced a mutant RhCMV lacking all identified vFcγRs (ΔvFcγR) and compared infection of CMV-seronegative male RMs with ΔvFcγR RhCMV (n=4) to FL RhCMV (n=3). RMs infected with ΔvFcγR and FL RhCMV experienced similar peak DNAemia levels 1 week post infection but ΔvFcγR RhCMV DNAemia was contained by week 3 while FL RhCMV remained detectable in plasma through week 9. The observed early control of infection is concurrent with the development of RhCMV-specific IgG responses which peak 3–4 weeks post infection in both groups. The kinetics of RhCMV-specific binding and neutralizing antibody responses were similar between the two groups of RMs, suggesting that anti-RhCMV Fc-mediated antibody effector responses may have been more effective against the RhCMV deleted of vFcγRs. Thus, vFcγRs are promising candidates for vaccine immunogens to improve Fc mediated effector responses and efficacy to reduce incidence of CMV-associated birth defects and transplant complications. Supported by grants from NIH P01-AI129859 and P01-AI129859_S1, NIH T32-CA009111, National CMV Foundation ECRA

Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neonatal neurological impairment but essential virological determinants of transplacental CMV transmission remain unclear. The pentameric complex (PC), composed of five subunits, glycoproteins H (gH), gL, UL128, UL130, and UL131A, is essential for efficient entry into non-fibroblast cells in vitro. Based on this role in cell tropism, the PC is considered a possible target for CMV vaccines and immunotherapies to prevent cCMV. To determine the role of the PC in transplacental CMV transmission in a non-human primate model of cCMV, we constructed a PC-deficient rhesus CMV (RhCMV) by deleting the homologues of the HCMV PC subunits UL128 and UL130 and compared congenital transmission to PC-intact RhCMV in CD4+ T cell-depleted or immunocompetent RhCMV-seronegative, pregnant rhesus macaques (RM). Surprisingly, we found that the transplacental transmission rate was similar for PC-intact and PC-deleted RhCMV based on viral genomic DNA detection in amniotic fluid. Moreover, PC-deleted and PC-intact RhCMV acute infection led to similar peak maternal plasma viremia. However, there was less viral shedding in maternal urine and saliva and less viral dissemination in fetal tissues in the PC-deleted group. As expected, dams inoculated with PC-deleted RhCMV demonstrated lower plasma IgG binding to PC-intact RhCMV virions and soluble PC, as well as reduced neutralization of PC-dependent entry of the PC-intact RhCMV isolate UCD52 into epithelial cells. In contrast, binding to gH expressed on the cell surface and neutralization of entry into fibroblasts by the PC-intact RhCMV was higher for dams infected with PC-deleted RhCMV compared to those infected with PC-intact RhCMV. Our data demonstrates that the PC is dispensable for transplacental CMV infection in our non-human primate model. One Sentence Summary Congenital CMV transmission frequency in seronegative rhesus macaques is not affected by the deletion of the viral pentameric complex.

Congenital cytomegalovirus (cCMV) is the most common in utero infection and causes major neurodevelopmental deficits, but there remains no licensed vaccine to prevent cCMV. Little is known about maternal immune responses that can prevent placental CMV transmission, which could guide rational design of an effective vaccine. Using the rhesus macaque (RM) model of primary RM CMV (RhCMV) infection during pregnancy, we established that pre-existing RhCMV-neutralizing IgG protected against cCMV, even in the setting of CD4+ T cell depletion, where vertical transmission occurs consistently in RhCMV-seronegative RMs. However, the antibody (Ab) functions mediating this protection have not been fully defined. To identify humoral immune correlates of containment of viremia and protection against cCMV we used samples from previous studies in this model to measure Ab binding to whole RhCMV virus and key glycoproteins, neutralization, Ab dependent cellular phagocytosis (ADCP), and cytotoxicity (ADCC). Immunocompetent RMs developed both RhCMV-specific neutralizing and Fc-mediated effector functions (ADCP, ADCC) 2–4 weeks post infection. However, Ab responses were not statistically distinct between transmitters and non-transmitters during this period. Maternal viremia outperformed maternal Ab responses as a predictor of placental transmission. Therefore, we correlated each Ab response with maternal plasma viral load at day 21 post infection. Interestingly, IgG binding to gB and the pentamer, immunodominant viral glycoproteins involved in viral entry, displayed significant inverse relationships with maternal viremia (n=15, Spearman r=−0.71, p=0.003 for both), highlighting these as critical targets for vaccine design. Supported by grants from NIH (NIAID P01-AI129859, NCI T32-CA009111)

Invariant natural killer T-lymphocytes (iNKT) are unique immunomodulatory innate T cells with an invariant TCRα recognizing glycolipids presented on MHC class-I-like CD1d molecules. Activated iNKT rapidly secrete pro-and anti-inflammatory cytokines, potentiate immunity, and modulate inflammation. Here, we report the effects of in vivo iNKT activation in Mauritian-origin cynomolgus macaques by a humanized monoclonal antibody, NKTT320, that binds to the invariant region of the iNKT TCR. NKTT320 led to rapid iNKT activation, increased polyfunctionality, and elevation of multiple plasma analytes within 24 hours. Flow cytometry and RNA-Seq confirmed downstream activation of multiple immune subsets, enrichment of JAK/STAT and PI3K/AKT pathway genes, and upregulation of inflammation-modulating genes. NKTT320 also increased iNKT frequency in adipose tissue and did not cause iNKT anergy. Our data indicate that NKTT320 has a sustained effect on in vivo iNKT activation, potentiation of innate and adaptive immunity, and resolution of inflammation, which supports its future use as an immunotherapeutic.

The maternal decidua is an immunologically complex environment that balances maintenance of immune tolerance to fetal paternal antigens with protection of the fetus against vertical transmission of maternal pathogens. To better understand host immune determinants of congenital infection at the maternal-fetal tissue interface, we performed a comparative analysis of innate and adaptive immune cell subsets in the peripheral blood and decidua of healthy rhesus macaque pregnancies across all trimesters of gestation and determined changes after Zika virus (ZIKV) infection. Using one 28-color and one 18-color polychromatic flow cytometry panel we simultaneously analyzed the frequency, phenotype, activation status and trafficking properties of αβ T, γδ T, iNKT, regulatory T (Treg), NK cells, B lymphocytes, monocytes, macrophages, and dendritic cells (DC). Decidual leukocytes showed a striking enrichment of activated effector memory and tissue-resident memory CD4+ and CD8+ T lymphocytes, CD4+ Tregs, CD56+ NK cells, CD14+CD16+ monocytes, CD206+ tissue-resident macrophages, and a paucity of B lymphocytes when compared to peripheral blood. t-distributed stochastic neighbor embedding (tSNE) revealed unique populations of decidual NK, T, DC and monocyte/macrophage subsets. Principal component analysis showed distinct spatial localization of decidual and circulating leukocytes contributed by NK and CD8+ T lymphocytes, and separation of decidua based on gestational age contributed by memory CD4+ and CD8+ T lymphocytes. Decidua from 10 ZIKV-infected dams obtained 16-56 days post infection at third (n=9) or second (n=1) trimester showed a significant reduction in frequency of activated, CXCR3+, and/or Granzyme B+ memory CD4+ and CD8+ T lymphocytes and γδ T compared to normal decidua. These data suggest that ZIKV induces local immunosuppression with reduced immune recruitment and impaired cytotoxicity. Our study adds to the immune characterization of the maternal-fetal interface in a translational nonhuman primate model of congenital infection and provides novel insight in to putative mechanisms of vertical transmission.

Astrocytes are an early and important target of Zika virus (ZIKV) infection in the developing brain, but the impacts of infection on astrocyte function remain controversial. Given that nonhuman primate (NHP) models of ZIKV infection replicate aspects of neurologic disease seen in human infections, we cultured primary astrocytes from the brain tissue of infant rhesus macaques and then infected the cells with Asian or African lineage ZIKV to identify transcriptional patterns associated with infection in these cells. The African lineage virus appeared to have greater infectivity and promote stronger antiviral signaling, but infection by either strain ultimately produced typical virus response patterns. Both viruses induced hypoxic stress, but the Asian lineage strain additionally had an effect on metabolic and lipid biosynthesis pathways. Together, these findings describe an NHP astrocyte model that may be used to assess transcriptional signatures following ZIKV infection.

Invariant natural killer T-lymphocytes (iNKT) are a unique subset of immunomodulatory innate T-cells with an invariant TCRα chain recognizing glycolipids presented on the MHC class-I-like CD1d molecule. Activated iNKT rapidly secrete pro-and anti-inflammatory cytokines, potentiate innate and adaptive immunity, and modulate inflammation. While iNKT activation by glycolipid agonists are being explored as an adjuvant, their use depends on CD1d-restricted antigen presentation. Here, we report the effects of iNKT activation by a novel humanized monoclonal antibody, NKTT320, that binds to the invariant region of the iNKT TCR. A single dose of NKTT320 led to rapid iNKT activation, increased polyfunctionality, and elevation of multiple pro-inflammatory and chemotactic plasma analytes within 24 hours in cynomolgus macaques. Flow cytometry and RNA-Seq confirmed downstream effects of NKTT320 on multiple immune cell subsets. Inflammatory response, JAK/STAT and PI3K/AKT pathway genes were enriched along with upregulation of the inflammation-modulating genes CMKLR1, ARG2 and NLRP12. Finally, NKTT320 induced iNKT trafficking to adipose tissue and did not cause iNKT anergy. Our data indicate that NKTT320 has a sustained effect on in vivo iNKT activation, potentiation of innate and adaptive immunity, and resolution of inflammation, properties that support its future application as an immunotherapeutic and vaccine adjuvant.

Although the Zika virus (ZIKV) epidemic is subsiding, immune responses that are important for controlling acute infection have not been definitively characterized. Nonhuman primate (NHP) models were rapidly developed to understand the disease and to test vaccines, and these models have since provided an understanding of the immune responses that correlate with protection during natural infection and vaccination. Here, we infected a small group of male rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques with a minimally passaged Brazilian ZIKV isolate and used multicolor flow cytometry and transcriptional profiling to describe early immune patterns following infection. We found evidence of strong innate antiviral responses together with induction of neutralizing antibodies and T cell responses. We also assessed the relative importance of CD8 T cells in controlling infection by carrying out CD8 T cell depletion in an additional two animals of each species. CD8 depletion appeared to dysregulate early antiviral responses and possibly increase viral persistence, but the absence of CD8 T cells ultimately did not impair control of the virus. Together, these data describe immunological trends in two NHP species during acute ZIKV infection, providing an account of early responses that may be important in controlling infection.

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68-1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68-1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques.

CD8+ lymphocytes are critically important in the control of viral infections, but their roles in acute Zika virus (ZIKV) infection remain incompletely explored in a model sufficiently similar to humans immunologically. Here, we use CD8+ lymphocyte depletion to dissect acute immune responses in adult male rhesus and cynomolgus macaques infected with ZIKV. CD8 depletion delayed serum viremia and dysregulated patterns of innate immune cell homing and monocyte-driven transcriptional responses in the blood. CD8-depleted macaques also showed evidence of compensatory adaptive immune responses, with elevated Th1 activity and persistence of neutralizing antibodies beyond the clearance of serum viremia. The absence of CD8+ lymphocytes increased viral burdens in lymphatic tissues, semen, and cerebrospinal fluid, and neural lesions were also evident in both CD8-depleted rhesus macaques. Together, these data support a role for CD8+ lymphocytes in the control of ZIKV dissemination and in maintaining immune regulation during acute infection of nonhuman primates.

The critical importance of CD8+ lymphocytes during chronic viral infection is well established, but their roles in acute ZIKV infection remain incompletely explored. Importantly, antiviral CD8 responses could modulate neurological manifestations that have accompanied recent ZIKV outbreaks. The rhesus macaque model of ZIKV is a particularly valuable tool to understand immune mechanisms of ZIKV control due to similarities in immune function to humans and due to their susceptibility to infection with primary human isolates of the virus. In the present study, we infected four adult male rhesus macaques with ZIKV, two of which had been depleted of CD8+ lymphocytes prior to infection. CD8 depletion resulted in delayed viremia and a near absence of innate immune responses in the blood, demonstrated by a complete lack of neutrophil recruitment to the blood and a striking absence of transcriptional changes in type I interferon response and other key immune genes relative to non-depleted controls during acute infection. Depletion also resulted in differential patterns of monocyte expansion and reduced monocyte activation measured by CD169 expression. Notably, CD8-depleted macaques showed possible evidence of compensatory CD4 T cell responses and persistence of neutralizing antibodies at later timepoints, despite clearance of virus from serum. Neural lesions were also evident in both CD8-depleted animals. One of the depleted animals recovered CD8+ lymphocytes by 21 days post-infection and mounted a high magnitude CD8 T cell response against the virus. The other CD8-depleted animal did not recover CD8+ lymphocytes over the course of the study, and post-mortem histology revealed severe brainstem encephalomalacia as well as enhanced viral dissemination in the semen and seminal vesicle. Together, these data support a potential role for CD8+ lymphocytes in control of ZIKV dissemination and in maintaining immune regulation during acute infection of rhesus macaques.