Non-human Primate Models to Investigate Mechanisms of Infection-Associated Fetal and Pediatric Injury, Teratogenesis and Stillbirth.

Preterm labor and preterm birth is strongly associated with intrauterine infection during early gestation ( Kelleher et al., 2020 ). Invasion of the amniotic cavity by Ureaplasma parvum (U. parvum) in particular triggers a strong inflammatory response, and has been implicated in neonatal sepsis, neurodevelopmental abnormalities, and bronchopulmonary dysplasia ( Yoon et al., 1998 ; Novy et al., 2009 ; Waites et al., 2009 ). However, due to the polymicrobial nature of such intrauterine infections, the exact mechanism by which U. parvum induces inflammation has been difficult to elucidate in human clinical studies. The pregnant NHP model thus has been instrumental in elucidating the causal relationship between U. parvum infection, preterm labor, and the associated sequelae. In a chronically catheterized rhesus macaque, intraamniotic inoculation with clinical isolates of U. parvum serovar 1 resulted in upregulation of pro-inflammatory cytokines, prostaglandins and leukocytes in the amniotic fluid, chorioamnionitis, fetal pneumonia and increased uterine activity ( Novy et al., 2009 ). Collectively, these results implicated U. parvum as an important pathogen to fetal lung injury and preterm labor ( Novy et al., 2009 ). Studies of U. parvum infection in pregnant baboon models demonstrated similar findings ( Walsh et al., 1993 ; Yoder et al., 2003 ). U. parvum inoculated into the amniotic fluid was also associated with decreased neonatal brain growth and white matter maturation in a rhesus macaque model ( Kelleher et al., 2017 ). Whether antibiotics to treat a U. parvum infection in utero might prevent preterm birth and fetal injury was unclear from clinical studies ( Grigsby et al., 2012 ). The pregnant NHP model provides the valuable opportunity for researchers to study antenatal antibiotic therapy under controlled experimental conditions. Indeed, maternal administration of azithromycin has been demonstrated to inhibit preterm uterine contractions, minimize fetal lung injury, and improve fetal hemodynamics due to a. parvum intraamniotic infection in the chronically catheterized rhesus macaque ( Grigsby et al., 2012 ; Kelleher et al., 2020 ). Moving forward, NHP studies will further inform current understanding of the pathobiology of U. parvum and other bactrerial intra-amniotic infections.

Group B streptococcus is a gram-positive, chain-forming bacterium that colonizes the rectovaginal tract of about 20% of women worldwide ( Russell et al., 2017 ). Vaginal colonization is generally asymptomatic, but GBS invasion into the placenta, amniotic cavity and fetus can lead to severe complications during pregnancy. Annually, GBS is responsible for at least 409,000 cases of invasive disease in infants, 147,000 cases of stillbirth or infant deaths, and up to 3.5 million cases of preterm labor ( Figure 4 ; Seale et al., 2017 ). Furthermore, invasive GBS infection can cause fetal injury associated with a number of chronic sequelae ( Seale et al., 2017 ). Studying the underlying mechanisms through which GBS exerts these outcomes is challenging. GBS serotype prevalence varies greatly by region, making it difficult to compare cases and outcomes across human cohorts ( Madrid et al., 2017 ; Russell et al., 2017 ). Further, bacterial invasion of the amniotic cavity and fetal compartments occurs silently; investigating the pathogenesis is not possible until there is a fulminant infection of the placenta or amniotic cavity. Studies in pregnant women have thus underscored the inflammatory cascade associated with GBS and other bacterial infections but fell short of elucidating the early events of pathogenesis. And as previously discussed, other mammalian models do not adequately recapitulate important characteristics of human pregnancy, further complicating this effort ( Adams Waldorf et al., 2011b ). Clinical outcomes of invasive GBS infection. In humans, invasive GBS infections can result in a spectrum of obstetrical and neonatal systemic, pulmonary, and neurological complications. The majority of these complications are also observed in the NHP model. To overcome these challenges, a chronically catheterized pregnant NHP model was developed to monitor GBS-induced disease progression, immune response, and pregnancy outcomes following an experimental GBS inoculation ( Gravett et al., 1994 ; Adams Waldorf et al., 2011a ; Boldenow et al., 2016 ; Coleman et al., 2020 , 2021 ). This model is highly translational based on several characteristics: (1) inoculation of GBS in the choriodecidual space where GBS is hypothesized to first invade the placenta, (2) serial sampling of maternal and fetal blood and amniotic fluid, (3) monitoring of uterine contractions and fetal heart rate over time, and (4) administration of antibiotics or immunotherapeutics to prevent preterm birth or fetal injury ( Figure 5 ). This data can be used to delineate the timing of microbial invasion of the amniotic cavity and subsequent fetal infection relative to the progression of adverse outcomes. Host-pathogen responses, innate immunity and –omics approaches to systems biology can also be applied to the study of preterm birth and fetal injury. Chronically catheterized pregnant NHP model of GBS infection and preterm labor. Catheters are also inserted into the dam (maternal vein), amniotic fluid to monitor pressure and enable inoculations and into the fetus (fetal vein). ECG leads may also be placed in the fetal chest wall and arm to monitor the fetal heart rate. The choriodecidual catheter is inserted in between the uterus and the chorioamniotic membranes of the lower uterine segment (within the decidua) of the pigtail macaque ( M. nemestrina ). Inoculation of GBS through the choriodecidual catheter in the lower uterine segment simulates an ascending infection of bacteria from the vagina, which is hypothesized to occur in human cases of preterm labor induced by GBS or other bacteria. Early studies of infection-associated preterm birth were performed in rhesus macaques. The first study in the NHP inoculated GBS directly into the amniotic fluid and observed elevated interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and interleukin-8 (IL-8), followed by preterm labor ( Gravett et al., 1994 ). These experiments confirmed that a bacterial infection could cause preterm birth and underscored the importance of cytokines in preterm labor. To determine if cytokines could induce preterm labor, experimental inoculations of IL-1β, TNF-α, IL-6, and IL-8 were performed in the chronically catheterized NHP model. Inoculation of IL-1β or TNF-α induced preterm labor, while IL-6 or IL-8 did not ( Sadowsky et al., 2006 ). The model was later modified to inoculate GBS into the choriodecidual space of the lower uterine segment, a site that was hypothesized to represent the first site that GBS might infect the placenta. Using this new route of inoculation, low-dose GBS (10 2 –10 4 cfu/mL) caused modest TNF-α production with moderate amniotic fluid prostaglandins and mild uterine activity and did not invade the amniotic cavity. In contrast, GBS administered at a higher dose (10 3 –10 6 cfu/mL) induced dramatic elevations in pro-inflammatory cytokines and invaded the amniotic cavity and fetus ( Grigsby et al., 2010 ). Collectively, these studies culminated in the development of an NHP model whereby choriodecidual inoculation of pregnant animals with GBS leads to preterm labor. The next step was to interrogate how choriodecidual and intraamniotic inflammation, as well as GBS bacterial burden, may influence other adverse outcomes, such as placental and fetal injury. During pregnancy, invasive GBS infection can cause significant fetal lung, cardiac, and brain injury, which are each associated with lifelong sequelae ( Seale et al., 2017 ). In addition to defining GBS-induced preterm labor mechanisms, the GBS NHP model is poised to interrogate GBS-induced fetal injury due to the extensive array of fetal samples collected at necropsy, including the lung, brain, and heart. In an initial study of GBS choriodecidual infection in pigtail macaques, preterm labor occurred in 2 of 5 (40%) animals, but histologic evidence of fetal lung injury occurred in 4 of 5 (80%) ( Adams Waldorf et al., 2011a ). Specifically, these fetal lungs demonstrated neutrophil and macrophage infiltrates and thickened alveolar septa by histology. The most severe case of fetal lung injury also had high IL-6 in fetal plasma, which meets the human clinical criteria for fetal systemic inflammatory response syndrome. Surprisingly, GBS was only recovered at the membrane inoculation site of two monkeys (one of which experienced preterm labor) and all animals were culture negative for GBS in the amniotic fluid ( Adams Waldorf et al., 2011a ). In addition, fetal lung injury was observed in the absence of preterm labor or chorioamnionitis. Thus, fetal lung injury may occur even in the absence of GBS invasion into the amniotic fluid suggesting that inflammation is sufficient to induce this outcome. In a microarray study of these fetal lung tissues, key developmental gene sets in the fetal lung were downregulated (angiogenesis, morphogenesis, and cellular differentiation) in tandem with the innate immune response ( McAdams et al., 2012 ). This data was the first to suggest a transcriptional signature of the fetal origins of bronchopulmonary dysplasia, a neonatal condition of prematurity characterized by simplified airways that failed to complete development. A fulminant GBS infection of the chorioamniotic membranes was known to cause chorioamnionitis, an inflammation of the placental membranes associated with preterm labor. However, the effect of an early, cleared GBS infection on placenta integrity and the placental innate immune response was unknown. Microarray analysis of the chorioamniotic membranes obtained from NHPs following GBS clearance revealed that exposure to GBS resulted in downregulated production of multiple cytokeratin and collagen genes, components of amniotic epithelial tensile strength. Transmission electron microscopy demonstrated cytokeratin retraction and loss of intermediate filaments ( Vanderhoeven et al., 2014 ). These early GBS studies in the NHP identified temporal relationships between a transient choriodecidual infection, inflammation in the amniotic fluid, microbial invasion of the amniotic cavity, preterm labor and fetal lung injury. However, these studies utilized the wild-type GBS serotype III ST-17 strain known as COH1; this strain is clinically relevant, but does not display high expression of certain well-described GBS virulence factors such as the hemolytic pigment and the secreted hyaluronidase enzyme (HylB), both of which are linked to GBS-associated preterm labor in mouse models ( Whidbey et al., 2013 ; Vornhagen et al., 2016 ). Therefore, to better appreciate GBS mechanisms driven by these factors and their role in preterm labor, additional GBS strains were used that either overexpressed the hemolytic pigment ( Whidbey et al., 2013 ) or HylB in the NHP model ( Gendrin et al., 2018 ). Group B streptococcus strains that overexpress GBS hemolytic pigment have a mutation in covR , a two-component system response regulator, and have been isolated from women in preterm labor ( Whidbey et al., 2013 ). In a preterm NHP model, choriodecidual inoculation of hyperpigmented GBS COH1Δ covR resulted in more efficient penetration of the placental chorioamniotic membranes and subsequent infection of the fetus. Microbial invasion of the amniotic cavity was associated with chorioamnionitis. However, despite the significant influx of neutrophils to the site of infection, hyperpigmented GBS subverted primary host defense mechanisms by inducing pigment-mediated neutrophil cell death and resisting neutrophil extracellular traps, which ultimately promoted fetal sepsis and preterm labor ( Boldenow et al., 2016 ). Preterm labor was associated with the fetal inflammatory response syndrome, marked by elevated fetal IL-6 in the plasma. Interestingly, GBS-associated fetal inflammatory response syndrome was also associated with elevated IL-6 and IL-8 levels and a greater bacterial burden but minimal neutrophil infiltration in fetal cardiac tissue. In these animals, gene sets involved in fetal heart development, such as cardiac morphogenesis and vasculogenesis gene networks, were downregulated; premature arrest of the fetal cardiac development program may contribute to cardiac dysfunction later in life ( Mitchell et al., 2018 ). Collectively, these findings suggest that inflammation at the maternal-fetal interface is ineffective in curtailing fetal bacteremia, fetal injury and preterm labor in the context of infection with hyperpigmented, hypervirulent GBS. In contrast to hyperpigmented GBS strains, clinical isolates of non-pigmented GBS with increased hyaluronidase activity have also been associated with adverse pregnancy outcomes and neonatal infection ( Vornhagen et al., 2016 ). GBS HylB breaks down hyaluronan (HA) to into HA disaccharides that block recognition and subsequent signaling by toll-like receptor (TLR) 2 and TLR4 ( Kolar et al., 2015 ). NHP infected with GBS that highly express HylB (strain GB37; Gendrin et al., 2018 ) consistently exhibited microbial invasion of the amniotic cavity, fetal sepsis and preterm labor ( Kolar et al., 2015 ; Coleman et al., 2021 ). Notably, infection with GB37 yielded delayed cytokine responses at the choriodecidual membranes, despite rapid invasion of the amniotic cavity and neutrophil and CD8+ T recruitment; this is starkly in contrast to the rapid proinflammatory responses associated with infection by hyperpigmented GBS. Despite significant immune cell infiltrate to the maternal-fetal interface, digital spatial profiling of the uterine mucosa in GB37-infected NHP revealed that even in tissues where GBS had been detected, inflammation-associated markers were dampened relative to baseline. GB37 also blunted ROS production by neutrophils in a TLR2/4 dependent manner, suggesting that GBS uses HylB to evade host immune recognition by blocking TLR2/4 signaling and downstream ROS production in neutrophils ( Coleman et al., 2021 ). In combination with the prior work on hyperpigmented GBS strains ( Boldenow et al., 2016 ), this study highlights that a chronically catheterized NHP model can demonstrate the diverse roles of various GBS virulence factors in disease pathogenesis that would otherwise be difficult to discern in other animal models. As IL-1β and TNF-α are strong inducers of preterm labor ( Sadowsky et al., 2006 ), inhibition of chemokine and cytokine responses is an attractive therapeutic approach for the prevention of GBS-induced preterm labor. In pregnant NHP, pre-treatment with a broad-spectrum chemokine inhibitor dampens pro-inflammatory cytokine levels, such as IL-8 in the maternal plasma and amniotic fluid, and IL-6, IL-1β, and IL-7 in fetal tissues. Although prophylactic administration of a broad spectrum chemokine inhibitor decreased the frequency of GBS-induced uterine contractility and preterm labor, it failed to protect the fetus from adverse sequelae such as microbial invasion of the amniotic cavity and fetal bacteremia. Suppression of pro-inflammatory cytokines led to uncontrolled bacterial replication and dissemination, causing increased neutrophil and macrophage infiltration to chorioamniotic membranes and fetal lungs ( Coleman et al., 2020 ). These findings suggest that a better strategy would likely be to utilize immunomodulators in combination with antibiotics to allow control of both GBS replication and the pro-inflammatory immune response. In the NHP model, the combination of antibiotics and immunomodulators was tested in the context of an amniotic fluid inoculation of GBS. In these experiments, uterine activity was closely monitored after GBS inoculation. Once uterine activity doubled over a 2-h period, a combination of ampicillin with and without dexamethasone and indomethacin was administered. Although ampicillin treatment extended gestation by a few days, ampicillin plus immunomodulators prolonged gestation by a full week in 4 of 5 (80%) monkeys. Both treatments were successful in eradicating, or drastically reducing recovery of, GBS from AF and fetal blood, meninges, and lung. Immunomodulators with ampicillin therapy was also associated with a reduction in IL-1β in the amniotic fluid and fewer neutrophils in the chorioamniotic membranes ( Gravett et al., 2007 ). These studies highlight the translational strength of this model.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

Listeria monocytogenes (Lm) is a facultative intracellular gram-positive food-borne bacterium that is widespread in the environment. Although classically associated with unpasteurized dairy products, soft cheeses and preserved meats and fish, recently outbreaks have been associated with produce such as cantaloupes, lettuce, caramel apples, and others ( Garner and Kathariou, 2016 ). Most individuals infected with Lm have an asymptomatic or mild illness, but listeriosis can cause sepsis and mortality in immunocompromised individuals, the elderly and young children ( Hof, 2003 ). Pregnant women are at particular risk with listeriosis, not manifesting as maternal illness, but instead as stillbirth. Maternal listeriosis results in an increased risk for miscarriage and stillbirth, and even if pregnancy proceeds to delivery of a live infant, neonatal listeriosis causes meningitis, sepsis, and death ( Wadhwa Desai and Smith, 2017 ). The antecedents and sequelae of listeriosis in pregnancy, particularly acute events, are difficult to study in the clinical setting, because infection may be asymptomatic in pregnancy. While rodent models have been valuable for understanding aspects of the biology of listeriosis in pregnancy, those models are complicated by a crucial amino acid difference in mice that results in failure of CDH1 ( E -cadherin) to bind internalin A on the bacterial surface – an interaction critical to the ability of Lm to invade host cells ( Bonazzi et al., 2009 ). The NHP model is an important adjunct with significant histological and functional relevance of the maternal-fetal interface, in particular the immune environment ( Golos et al., 2010 ). Notably, cynomolgus macaques ( Macaca fascicularis ) have a well-characterized maternal-fetal interface making it a valuable Lm model for assessment of fetal impact ( Bondarenko et al., 2009 ; Dambaeva et al., 2009 ). The risk of first trimester listeriosis is particularly difficult to address in human clinical cases, and macaques have been used in a series of studies of the impact of Lm inoculation on pregnancy ( Figure 3 ; Smith et al., 2003 ; Smith et al., 2008 ; Wolfe et al., 2017 ; Wolfe et al., 2019 ). Intragastric inoculation in later gestation has previously been shown to result in an elevated risk for pregnancy loss or stillbirth, however there was no assessment of the bacterial burden or histopathology of the maternal-fetal interface or the fetus itself. Inoculation during the fifth and sixth week of gestation resulted in a very high incidence (∼80%) of fetal demise, as early as 7 days after maternal infection. Nearly all dams had bacteremia and fecal shedding that preceded fetal demise ( Wolfe et al., 2017 ). The macaque model allowed surgical collection of both fetal and maternal tissues upon recognition of fetal demise. Examination of the bacterial burden in maternal tissues revealed, as expected, modest colonization of the usual target tissues in the dam liver, spleen and lymph nodes; remarkably, the bacterial burden in the decidua and the placental bed was 3–4 orders of magnitude higher. At the maternal-fetal interface, suppurative inflammation and necrosis in the decidua and vasculitis of the maternal spiral arteries was consistently noted with microabscesses in the extravillous cytotrophoblastic shell of the placenta, placental villous necrosis and suppurative intervillositis. Fetal membranes also displayed chorioamnionitis and there were gram-positive rods within the umbilical cord. NHP cynomolgus macaque ( M. fascicularis ) model of listeriosis in pregnancy. Intragastric inoculation of L. monocytogenes in the first trimester results in bacteremia that is associated with a modest bacterial burden in classic Listeria target organs (liver, spleen, lymph nodes), but profound infection and histopathology at the maternal-fetal interface, fetal infection, and fetal demise. These studies demonstrated that in early pregnancy, there was significant risk to the fetus of rapid miscarriage. Fetal bacteria was widely disseminated indicating hematogenous spread, as would be inferred by the presence of inflammatory cells in the villous mesenchyme of the placenta ( Wolfe et al., 2017 ); gram-positive tissues included the lung, liver, periosteum, perichondrium, and fetal cranium, as well as ocular muscles, neural tissue, and the developing skull. This profile of bacterial burden suggested that rapidly growing highly vascularized structures had greater risk for colonization ( Wolfe et al., 2019 ). Interestingly, the highly efficient rate of vertical transmission and adverse pregnancy outcomes was not seen when maternal inoculation was done in the third trimester ( Smith et al., 2008 ; Wolfe et al., 2019 ); although if an increased inoculum was used, 3 of 4 subjects inoculated early in the third trimester had fetal demise ( Wolfe et al., 2019 ). The first trimester may thus be more sensitive to maternal listeriosis. In the early pregnancy decidua, there is active neovascularization and macrophage infiltration as decidual spiral arteries are remodeled by invading endovascular trophoblasts ( Bondarenko et al., 2012 ). In this situation, a more robust immune response may be mounted in the decidua, whereby acute inflammation creates greater damage to the maternal fetal interface, allowing bacteria to cross the placental trophoblast barrier ( Bondarenko et al., 2007 ; Bondarenko et al., 2012 ; Carter et al., 2015 ). Histopathology of maternal-fetal interface tissues in third trimester pregnancy was scored as a sum of abnormal histological findings ( Wolfe et al., 2019 ). Of significant interest was the presence of an elevated histopathological placental score, even in pregnancies that progressed to term without fetal loss or evidence of infection. Likewise, PCR analysis of inflammation markers assessed by Luminex assay demonstrated that Lm exposure, regardless of pregnancy loss or survival, clustered apart from control pregnancy. While this was a terminal study for the fetus, the developmental sequelae of infection impact on the placenta and decidua in the offspring is not known from these studies. The macaque model can be used for further studies to address the potential long-term impact on the infant, as is being done with macaque models of maternal Zika virus infection ( Koenig et al., 2020 ). With the expanding occurrence of listeriosis outbreaks due to contamination of the food supply, relevant animal models will be important to develop potential mitigation. While antibiotic therapy is available for pregnant women, the mother is often asymptomatic and thus disease progresses to threaten adverse pregnancy outcomes without the opportunity to intervene. Further research with NHPs may ultimately provide insight into the mechanisms that determine why adverse outcomes are more likely in the first trimester, or which specific factors allow early identification of potentially devastating early pregnancy infection and thus quicker antibiotic intervention. Other important inquiries might be addressed through NHP models as well; it is unclear whether vaccines based on an Lm vector backbone ( Morrow et al., 2019 ) would provide additional protection during pregnancy or if such a vaccine would trigger sensitization and lead to a more profound impact on immune and inflammatory outcomes at the decidual interface. Other approaches, such as developing probiotic treatment ( Corr et al., 2007 ; Ryan et al., 2021 ) may prove productive and should be evaluated in NHP models. It is also unknown whether pre-pregnancy exposure to Lm provides protection from adverse outcomes in pregnancy, or what genetic determinants of pathogenesis might target the maternal-fetal interface; these questions can be answered using the NHP model.

Influenza A Virus (IAV) is an orthomyxovirus characterized by rapidly acquired mutations in its surface-proteins, hemagglutinin and neuraminidase, that allow for evasion of adaptive human immune responses ( Peteranderl, Herold and Schmoldt, 2016 ). This process of antigenic drift underlies the need for annual revision and alterations of influenza vaccines. Antigenic shift, in which new viral strains emerge through genomic reassortment, has contributed to global influenza pandemics - the most recent of which followed the emergence of the 2009 H1N1 viral strain. Sequelae of the initial respiratory illness of IAV has made it one of the leading causes of death and morbidity among vulnerable populations in the U.S. Despite pregnant women representing a particularly high-risk cohort, they remain a largely understudied population and the impact of IAV on fetal health and development has yet to be fully determined. Vertical transmission of IAV is thought to be rare, but has not been studied comprehensively ( Raj et al., 2014 ). However, IAV has been isolated from both placental tissue and amniotic fluid, having reached the affect cells following infection of the uterine decidua. It has been posited that IAV favors decidual tissue for viral replication before spreading to the fetal chorion and amnion ( Raj et al., 2014 ). This process of viral replication may also have a direct cytopathic effect on chorionic cells, contributing to influenza-associated pregnancy loss. Some studies suggest that fetal death may also be secondary to the maternal inflammatory response following initial infection ( Rasmussen et al., 2008 ). Human epidemiologic studies have raised concern that IAV might act to subtly injure the fetal central nervous system and predispose to the development of neuropsychiatric disorders like schizophrenia decades after birth ( Hare et al., 1973 ; Parker and Neilson, 1976 ; Machon et al., 1983 ; Mednick et al., 1988 ; Barr et al., 1990 ; Brown et al., 2009 ; Al-Haddad et al., 2019 ). In rodents, prenatal exposure to influenza viral infection results in neural abnormalities in the offspring, with reduced cortical thickness and hippocampal volumes ( Fatemi et al., 2002 , 2008 ; Nawa and Takei, 2006 ). Rodents infected with IAV have a different disease course compared to humans, and IAV’s impact on fetal development varies significantly between rodent models and their human counterparts, limiting the impact of this translational research. NHP, however, share a hemochorial placenta with their human counterparts and give birth to only one infant at a time, ensuring advanced brain maturation that mirrors brain development in human children. However, only one NHP study of IAV has been performed to evaluate the impact of infection on fetal development. In an influenza model in pregnant rhesus macaque with the goal of studying the impact of infection on the developing fetal brain, pregnant dams were inoculated with A/Sydney/5/97 (H3N2) in the early third trimester. The offspring, despite no evidence of direct viral exposure, were found to have a significant reduction in bilateral cortical gray matter (cingulate and parietal lobes) at 1 year of age ( Short et al., 2010 ). This translational research underscores an urgency to understand the impact of IAV on fetal development and NHP models will need to continue to be at the center of the assessment of this disease. Understanding the viral-host factors driving enhanced maternal influenza disease and/or fetal brain injury in a NHP model will enable investigating the role of vaccines and therapeutics in preventing maternal and fetal injury.

Animal models using pregnant mice, rats, guinea pigs, hamsters, and rabbits are often used to determine reproductive toxicity in pre-clinical testing and development of new drugs. Yet, these models differ significantly from women in both placentation and hormonal events surrounding parturition, limiting their translational impact – particularly for testing the efficacy of new therapeutics and vaccines ( Table 1 ). While teratogenic phenotypes are observed in many animal models, species-specific differences in critical periods of fetal development also limit the range of investigable research questions. Nevertheless, animal models, especially NHP, provide a critical mechanism for exploring reproductive toxicity and teratogenesis ( Table 2 ). Comparison of animal models for investigation of teratogenesis. Comparison of similarities and differences with humans across common non-human primate models. Placental permeability is determined by the structure and composition of the cell layers that separate maternal and fetal blood and varies among eutherian animals ( Carter, 2020 ). The human placenta is hemochorial, with trophoblasts bathed directly in maternal blood; this thin maternal-fetal barrier makes oxygen and nutrient exchange highly efficient ( Grigsby, 2016 ). Several animal species can model specific aspects of the human placenta. For example, the guinea pig has historically been an excellent animal model of human placental transfer and fetal growth restriction ( Kelleher et al., 2011 ; Dyson et al., 2012 ; Morrison et al., 2018 ); the guinea pig hemomonochorial placenta deeply invades the decidua with proliferating trophoblast cells in a manner most similar to the human placenta ( Kaufmann et al., 2003 ; Carter et al., 2006 ). Similarly, the sheep and its cotyledonary epitheliochorial placenta has a vascular structure similar to that of humans and can tolerate invasive procedures during pregnancy, making it a common model for studying fetal physiology and placenta vascular development ( Grigsby, 2016 ). While these animals, and others, continue to make important contributions to an understanding of pregnancy and placentation, important differences in placental morphology (e.g., maternal-fetal interface, histological structure; Furukawa et al., 2014 ) have notable implications for studying teratogenesis. Non-human primates have been used to model human implantation, placentation, parturition, and endometriosis ( Grigsby, 2016 ). While placentation in NHP is characterized by generally superficial implantation and a less developed decidua lobe ( Roberts et al., 2012 ), placental transfer in species like the rhesus macaque ( Macaca mulatta ) are analogous to the human placenta, making the pregnant NHP an ideal model for studying placenta permeability, pharmacodynamics, and toxicant transfer ( Grigsby, 2016 ). Of note, complications in human pregnancy characterized by improper trophoblast invasion of the endometrium (e.g., preeclampsia, fetal growth restriction) currently lack a fully suited animal model ( Carter and Pijnenborg, 2011 ; Carter, 2020 ). Common laboratory animal models have contributed significantly to an understanding of fetal development. Researchers have used the chick embryo, frog, and zebrafish embryos and eggs to study the effect of teratogens and pathogens like ZIKV on the early precursors of the peripheral nervous system by loss of function analysis ( Barriga et al., 2015 ; Narasimhan et al., 2020 ). Rabbit models have also been used to study the impact of factors like maternal dietary restriction and environmental pollutants on early embryonic and fetal-placental development ( Fischer et al., 2012 ; Lopez-Tello et al., 2019 ; Carter, 2020 ). However, these animals and rodent models are altricial ( Tran et al., 2000 ; Cronise et al., 2001 ), meaning that significant organ development occurs postnatally; this limits the translational ability of these models to study the temporal impact of infectious diseases on organogenesis and brain analogous to mid− to late human gestation. A precocial animal, born in a more advanced state of development (e.g., sheep, NHP; Grigsby, 2016 ; Carter, 2020 ), is better suited to evaluating the teratogenic potential of a pathogen on human fetal brain growth and differentiation, which largely occur in the third trimester of gestation ( Dobbing and Sands, 1979 ). NHP models also offer the additional opportunity for researchers to assess deviations in neonatal behavior after birth ( Nelson and Winslow, 2009 ; Bauman et al., 2014 ; Machado et al., 2015 ; Grant et al., 2019 ). Non-human primates most closely emulate the human immune system, an important consideration when analyzing the maternal-fetal-placental immune response to a pathogen ( Safronetz et al., 2013 ). However, for many practical and scientific reasons, rodents and other animals are often used as the primary disease models ( Table 1 ; Safronetz et al., 2013 ). For example, the pregnant mouse model is useful for investigating how the host immune system balances the need to maintain fetal tolerance with pathogen defense; the murine immune system is well characterized and research tools are commercially available ( Lowe et al., 2018 ). Additionally, some animal models are natural hosts for the disease of interest, like adenoviruses for rodents and guinea pigs ( Safronetz et al., 2013 ), and ZIKV for NHP ( Narasimhan et al., 2020 ). However, many human pathogens need to be adapted to a specific animal model that may not adequately manifest human disease and pathology, like the mouse-adapted or guinea-pig-adapted Ebola virus ( Safronetz et al., 2013 ). Similarly, L. monocytogenes , does not naturally infect the mouse gut and experimental modifications to change the method of inoculation or adapting humanized mice models sacrifice the integrity of placental infection ( Lowe et al., 2018 ). Even among NHP, different species are not equally susceptible to all pathogens ( Safronetz et al., 2013 ). The pigtail macaque ( Macaca nemestrina ) is known to be especially susceptible to multiple flaviviruses including dengue virus, Japanese encephalitis virus, chikungunya virus, hepatitis C, and other human pathogens (malaria, tuberculosis, chlamydia, Kaposi’s sarcoma) ( Putaporntip et al., 2010 ; Bruce et al., 2013 ; Sourisseau et al., 2013 ; Nakgoi et al., 2014 ). Overall, the NHP represents an excellent model of human infectious disease. Although NHP are often susceptible to human infectious diseases, the incidence of a teratogenic phenotype is often low in both humans and NHP models and may be highly dependent upon the gestational age at inoculation. For example, several species of NHP including African Green (or vervet) monkeys ( Chlorocebus aethiops ) ( Sigurdardottir et al., 1963 ), patas monkeys (Erythrocebus patas) ( Draper and Laurence, 1969 ), baboons ( Horstmann, 1969 ), chimpanzees ( Horstmann, 1969 ), and rhesus macaques ( Parkman et al., 1965a ) are susceptible to rubella virus, but rarely manifest clinical illness. In two studies challenging pregnant rhesus macaques with rubella virus in the first ( Parkman et al., 1965b ; Sever et al., 1966 ) and third trimesters ( Parkman et al., 1965b ), the classical findings of the congenital rubella syndrome were not observed; however, these studies were limited by small numbers [ N = 4 ( Sever et al., 1966 ), N = 6 ( Parkman et al., 1965b )] and may not have captured infrequent events. In a third study that challenged rhesus macaques ( N = 14) with rubella virus in the early first trimester, spontaneous abortion occurred in 9 of 14 (64%) pregnancies and congenital cataracts were observed in 2 of the 5 (40%) viable fetuses ( Delahunt and Rieser, 1967 ). These studies demonstrate the challenge of studying teratogenesis in any animal model, which involves consideration of pathogen sensitivity, temporal susceptibility across gestation and the infrequent nature of some teratogenic phenotypes.

We remain unprepared to protect pregnancies from many teratogenic viral threats, as well as from other emerging pathogens with teratogenic potential. The expanding landscape of emerging pathogens with an unknown potential to induce teratogenesis or fetal injury necessitates a dedicated scientific effort to understand the mechanisms of disease pathogenesis and host response in pregnant and neonatal animal models. Many animal models have provided a critical foundation on which to assess disease pathogenesis, host pathogen response, and teratogenic fetal impact. However, the limited translational capacity of most mammalian models to human pregnancy has brought the NHP model into particular focus. This review demonstrates the critical utility of the NHP model in the study of a select few teratogenic pathogens: ZIKV, HCMV, HIV-1, IAV, Lm, and GBS. The notable similarities between humans and NHPs including the maternal-fetal interface, fetal development and pathogen sensitivity make the NHP ideal for the study of infectious disease and therapeutics in pregnancy. NHP models are essential to determining the potential for fetal injury and teratogenesis due to new pathogens and the efficacy of therapeutics to prevent fetal damage.

The teratogenic potential of pathogens was first realized in 1941, when Australian ophthalmologist Sir Norman McAlister Gregg reported the triad of congenital malformations (cataracts, heart disease, and hearing loss) of children born to mothers with a rubella virus infection in early pregnancy ( Gregg, 1991 ). Vertical transmission from a maternal rubella virus infection is now known to also cause fetal glaucoma, microphthalmia, and developmental delay; the constellation of these symptoms is known as congenital rubella syndrome ( Miller et al., 1982 ; Claus et al., 2020 ). By the late 20th century, additional pathogens like Toxoplasma gondii , human cytomegalovirus (HCMV), parvovirus B19, syphilis, herpes simplex virus (HSV), and varicella-zoster virus (VZV) were also identified as infectious teratogens transmittable to a fetus either trans -placentally or during delivery ( Dudgeon, 1976 ). This list was recently expanded to include Zika virus (ZIKV), when an outbreak in northeastern Brazil in 2015–2016 was shown to be linked to a cluster of cases of neonatal microcephaly ( Duffy et al., 2009 ; Cao-Lormeau et al., 2014 ; Cauchemez et al., 2016 ). As the ZIKV outbreak transformed into a global pandemic, the scientific literature linking maternal infection with fetal teratogenesis and stillbirth was strengthened by key studies in mice and non-human primates (NHP) ( Adams Waldorf et al., 2016 ; Dudley et al., 2018 ; Waldorf et al., 2018 ). Several animal models can be used to study infectious teratogenesis and fetal injury. When developing an animal model with an infectious disease, one must take into consideration microbial infection dynamics, species susceptibility, dose, route of inoculation, methods for assessing injury and translational relevance to humans. Animal models are necessary to perform critical studies of pathogenesis, clinical outcomes, and therapeutics, which cannot be performed in human pregnancies or infants. The NHP model is the closest animal model to human pregnancy and shares many similarities including placentation, pregnancy physiology, maternal-fetal interface, and timeline of fetal development ( Furukawa et al., 2014 ; Grigsby, 2016 ; Stouffer and Woodruff, 2017 ). NHP models are also ideal for pre-clinical investigation of novel therapeutics and vaccines to prevent infectious disease. This review will focus on NHP models of teratogenesis, fetal and pediatric brain injury, preterm birth, stillbirth, and spontaneous abortion. We will also compare the advantages and disadvantages of NHP models to other animal models for the investigation of congenital and pediatric infectious injury. Although there are many infectious teratogens and pathogens with the potential to induce organ injury to fetuses and neonates, we will focus on several that have been studied in the NHP model including ZIKV, HCMV, human immunodeficiency virus 1 (HIV-1), influenza A virus (IAV), Listeria monocytogenes , and group B streptococcus (GBS). Finally, we feature novel advancements in testing vaccines and therapies in NHP models to prevent teratogenesis and injury, highlighting the translational potential to human pregnancy and neonatal care.

SP is a consultant for Merck, Moderna, Pfizer, and Dynavax vaccines, and collaborates with Merck and Moderna on sponsored research programs around CMV vaccines. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Human cytomegalovirus (HCMV) is a common β-herpesvirus that spreads through bodily fluids such as blood, saliva, and urine as well as through organ transplantation, breast milk and through vertical transmission to a fetus. Though often asymptomatic after the initial 2-to-3-week illness, CMV infections can persist as a latent or chronic infection late into adulthood. As the most common congenital infection globally (1 in ∼200 children), congenital HCMV (cCMV) infection is a frequent cause of infant neurological sequelae, including sensorineural hearing loss (SNHL) and cognitive or motor deficits ( Yow and Demmler, 1992 ; Dollard et al., 2007 ; Kenneson and Cannon, 2007 ; Manicklal et al., 2013 ). Yet, despite the significant global impact of cCMV, there is not currently an effective vaccine to prevent HCMV acquisition or in utero infection. Several animal models have been applied to investigate CMV pathogenesis and transmission, including rodents [e.g., mice ( Rawlinson et al., 1996 ), rats ( Vink et al., 2000 ), and guinea pigs ( McGregor et al., 2004 )] and NHP [e.g., chimpanzees ( Davison et al., 2003 ), rhesus macaques ( Hansen et al., 2003 ), and cynomolgus macaques ( Ambagala et al., 2011 ; Marsh et al., 2011 )]. However, since CMV variants are highly species-specific, these models rely on CMV strains distinct from HCMV. Nevertheless, NHP CMV genomes most closely match that of HCMV. The similarity between HCMV and rhesus macaque cytomegalovirus (RhCMV) at the genome and amino acid sequence level makes rhesus macaques an ideal NHP model to investigate CMV infections. The size of the HCMV genome (229,354 bp) and RhCMV genome (strain 68-1; 221,459 bp) is comparable in length and the two genomes share 97% similarity at the nucleotide level. With regard to open reading frames (ORFs), both HCMV and RhCMV genomes are colinear and have approximately 250–260 potential ORFs. Eighty percent of the RhCMV ORFs are homologous to HCMV ORFs, and more than 90% of the RhCMV ORFs has an ortholog in HCMV genome at the protein family level ( Hansen et al., 2003 ; Murphy et al., 2003 ; Rivailler et al., 2006 ; Oxford et al., 2008 ; Malouli et al., 2012 ; Stern-Ginossar et al., 2012 ). Rhesus macaques are also an ideal model to study cCMV transmission, because the pathogenesis of CMV infection of human and rhesus macaques is remarkably similar ( Bialas et al., 2015 ; Itell et al., 2017 ; Roark et al., 2020 ). Resembling the high seroprevalence of HCMV infection globally (83%) ( Zuhair et al., 2019 ), the seroprevalence of RhCMV infection is approximately 95–100% among animals studied in primate research centers ( Swack and Hsiung, 1982 ; Jones-Engel et al., 2006 ). Moreover, the fetal sequelae of HCMV and RhCMV infection are similar, sharing common manifestations such as hearing impairment, microcephaly, and fetal loss ( London et al., 1986 ; Tarantal et al., 1998 ; Chang et al., 2002 ; Barry et al., 2006 ; Cheeran et al., 2009 ; Bialas et al., 2015 ; Nelson et al., 2017 ). Both antepartum, intrapartum and postpartum transmission (via breast) milk are commonly observed in HCMV infection ( Diosi, 1997 ; Hamprecht et al., 2001 ; Fowler et al., 2003 ; Kenneson and Cannon, 2007 ; Hamprecht and Goelz, 2017 ). In the rhesus macaque model, placental transmission was possible in immunocompetent dams, though it occurred consistently and was more severe when maternal CD4+ T cell were depleted. As shown in Figure 2 , the cCMV transmission rate of rhesus macaques with CD4+ T cell depletion is 6 of 6 (100%) with 80% fetal loss, while that of the immunocompetent animals is 2 of 3 (66%) with no fetal loss ( Bialas et al., 2015 ). Notably, this high rate of transmission and fetal loss in CD4+ T cell-depleted dams was shown to be prevented by administration of passive RhCMV-specific IgG infusion prior to inoculation ( Nelson et al., 2017 ). These findings suggest that rhesus macaques are an ideal NHP model to investigate cCMV transmission, establishing the importance of both maternal antibodies and CD4+ T cells in transmission risk. cCMV transmission in a CD4+ T cell-depleted and immunocompetent rhesus macaque ( M. mulatta ) NHP model. (A) Seronegative dams were CD4+ T cell-depleted at gestational week 7 and inoculated with RhCMV 1 week after CD4+ T cell depletion. All (6/6; 100%) dams transmitted RhCMV vertically and a spontaneous abortion occurred in 5 of 6 (83%) dams. (B) Immunocompetent seronegative dams were inoculated with RhCMV at week 7 of pregnancy. Vertical transmission occurred in two of three (66%) pregnancies with no spontaneous abortions ( Bialas et al., 2015 ; Nelson et al., 2017 ). The genomic similarities between HCMV and RhCMV as well as the multiple shared characteristics between human and rhesus macaque cCMV models make rhesus macaques an ideal preclinical NHP model for vaccine development. Two glycoprotein complexes, glycoprotein B (gB) and pentameric complex gH/gL/Ul128/UL130/UL131 (PC), on the HCMV virion surface have been proposed as vaccine targets due to their ability to elicit neutralizing activity ( Anderholm et al., 2016 ; Gardner and Tortorella, 2016 ). There is a 60% shared amino acid identity between HCMV and RhCMV gB, and RhCMV gB shares a similar role and structure as HCMV gB ( Kravitz et al., 1997 ). HCMV gB/MF59 adjuvanted vaccine provided partial efficacy in phase II clinical trials ( Zhang and Pass, 2004 ; Bernstein et al., 2016 ), and several current HCMV vaccine platforms have added the PC ( Gerna et al., 2017 ). RhCMV gB has been expressed in modified vaccinia Ankara virus (MVA) vectors, DNA expression plasmids, as well as a soluble protein subunits and result in elicitation of neutralizing antibodies ( Abel et al., 2011 ; Valencia et al., 2019 ). Subunits of PC are also conserved between HCMV and RhCMV ( Hansen et al., 2003 ; Rivailler et al., 2006 ; Wussow et al., 2013 ), and potent neutralizing antibodies can be induced in rhesus macaques with RhCMV gB and PC-expressing MVA vectors ( Wussow et al., 2013 ). In conclusion, the rhesus macaque is an excellent preclinical model to examine CMV pathogenesis, immunity, and vaccine efficacy due to the genetic similarity of RhCMV genome and the comparable pathogenesis of RhCMV infection.