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Congenital cytomegalovirus (cCMV) is the leading infectious cause of neurologic defects in newborns with particularly severe sequelae in the setting of primary CMV infection in the first trimester of pregnancy. The majority of cCMV cases worldwide occur after non-primary infection in CMV-seropositive women; yet the extent to which pre-existing natural CMV-specific immunity protects against CMV reinfection or reactivation during pregnancy remains ill-defined. We previously reported on a novel nonhuman primate model of cCMV in rhesus macaques where 100% placental transmission and 83% fetal loss were seen in CD4+T lymphocyte-depleted rhesus CMV (RhCMV)-seronegative dams after primary RhCMV infection. To investigate the protective effect of preconception maternal immunity, we performed reinfection studies in CD4+ T lymphocyte-depleted RhCMV-seropositive dams inoculated in late first / early second trimester gestation with RhCMV strains 180.92 (n=2), or RhCMV UCD52 and FL-RhCMVΔRh13.1/SIVgag, a wild-type-like RhCMV clone with SIVgaginserted as an immunological marker (n=3). An early transient increase in circulating monocytes followed by boosting of the pre-existing RhCMV-specific CD8+ T lymphocyte and antibody response was observed in the reinfected dams but not in control CD4+ T lymphocyte-depleted dams. Emergence of SIV Gag-specific CD8+ T lymphocyte responses in macaques inoculated with the FL-RhCMVΔRh13.1/SIVgagvirus confirmed reinfection. Placental transmission was detected in only one of five reinfected dams and there were no adverse fetal sequelae. Viral whole genome, short-read, deep sequencing analysis confirmed transmission of both reinfection RhCMV strains across the placenta with ∼30% corresponding to FL-RhCMVΔRh13.1/SIVgagand ∼70% to RhCMV UCD52, consistent with the mixed human CMV infections reported in infants with cCMV. Our data showing reduced placental transmission and absence of fetal loss after non-primary as opposed to primary infection in CD4+ T lymphocyte-depleted dams indicates that preconception maternal CMV-specific CD8+ T lymphocyte and/or humoral immunity can protect against cCMV infection.Author SummaryGlobally, pregnancies in CMV-seropositive women account for the majority of cases of congenital CMV infection but the immune responses needed for protection against placental transmission in mothers with non-primary infection remains unknown. Recently, we developed a nonhuman primate model of primary rhesus CMV (RhCMV) infection in which placental transmission and fetal loss occurred in RhCMV-seronegative CD4+ T lymphocyte-depleted macaques. By conducting similar studies in RhCMV-seropositive dams, we demonstrated the protective effect of pre-existing natural CMV-specific CD8+ T lymphocytes and humoral immunity against congenital CMV after reinfection. A 5-fold reduction in congenital transmission and complete protection against fetal loss was observed in dams with pre-existing immunity compared to primary CMV in this model. Our study is the first formal demonstration in a relevant model of human congenital CMV that natural pre-existing CMV-specific maternal immunity can limit congenital CMV transmission and its sequelae. The nonhuman primate model of non-primary congenital CMV will be especially relevant to studying immune requirements of a maternal vaccine for women in high CMV seroprevalence areas at risk of repeated CMV reinfections during pregnancy.

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 innate and adaptive immunity, and modulate inflammation. Here, we report the effects of in vivo iNKT activation by a novel 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 of administration. 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 CMKLR1, ARG2 and NLRP12. NKTT320 also 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, which supports its future use as an immunotherapeutic and vaccine adjuvant.SummaryiNKTs are known immunomodulatory cells whose activation is a potential target for immunotherapies and use as an adjuvant. Here we report the potential utility of in vivo iNKT activation using the novel humanized monoclonal antibody NKTT320 for this purpose.

Human cytomegalovirus (HCMV) infection is the leading non-genetic cause of congenital birth defects worldwide. While several studies have investigated the genetic composition of viral populations in newborns diagnosed with HCMV, little is known regarding mother-to-child viral transmission dynamics and how therapeutic interventions may impact within-host viral populations. Here, we investigate how preexisting CMV-specific antibodies shape the maternal viral population and intrauterine virus transmission. Specifically, we characterize the genetic composition of CMV populations in a monkey model of congenital CMV infection to examine the effects of passively-infused hyperimmune globulin (HIG) on viral population genetics in both maternal and fetal compartments. In this study, 11 seronegative, pregnant monkeys were challenged with rhesus CMV (RhCMV), including a group pretreated with a standard potency HIG preparation (n = 3), a group pretreated with a high-neutralizing potency HIG preparation (n = 3), and an untreated control group (n = 5). Targeted amplicon deep sequencing of RhCMV glycoprotein B and L genes revealed that one of the three strains present in the viral inoculum (UCD52) dominated maternal and fetal viral populations. We identified de novo minor haplotypes of this strain and characterized their dynamics. Many of the identified haplotypes were consistently detected at multiple timepoints within sampled maternal tissues, as well as across tissue compartments, indicating haplotype persistence over time and transmission between maternal compartments. However, haplotype numbers and diversity levels were not appreciably different across HIG pretreatment groups. We found that while the presence of maternal antibodies reduced viral load and congenital infection, it has no apparent impact in the intrahost viral genetic diversity at the investigated loci. Interestingly, some haplotypes present in fetal and maternal-fetal interface tissues were also identified in maternal samples of corresponding dams, providing evidence for a wide RhCMV mother-to-fetus transmission bottleneck even in the presence of preexisting antibodies.Author summaryHuman cytomegalovirus (CMV) is the most common infectious cause of birth defects worldwide. Knowledge gaps remain regarding how maternal immunity impacts the genetic composition of CMV populations and the incidence of congenital virus transmission. Addressing these gaps is important to inform vaccine development efforts. Using viral samples collected from a monkey model of congenital CMV infection, we investigated the impact of passively-administered maternal antibodies on the genetic composition of the maternal virus population and that transmitted to the fetus. Our analysis focused on two CMV genes that encode glycoproteins that facilitate viral cellular entry and are known epitope targets of the humoral immune response. By identifying and analyzing variants across sampled maternal tissues, we found no impact in CMV genetic diversity by preexisting CMV-specific antibodies, despite the observation that such antibodies reduce viral load and confer some protection against congenital transmission. We further found that some minor variants identified in fetal and maternal-fetal interface tissues were also present in corresponding maternal tissues, indicating that a large number of viral particles passed from dam to fetus in observed cases of congenital transmission.

Human cytomegalovirus (HCMV) is the most common congenital infection and a known cause of microcephaly, sensorineural hearing loss, and cognitive impairment among newborns worldwide. Natural maternal HCMV immunity reduces the incidence of congenital infection, but does not prevent the disease altogether. We employed a nonhuman primate model of congenital CMV infection to investigate the ability of preexisting antibodies to protect against placental CMV transmission in the setting of primary maternal infection and subsequent viremia, which is required for placental virus exposure. Pregnant, CD4+ T cell-depleted, rhesus CMV-seronegative (RhCMV-seronegative) rhesus monkeys were treated with either standardly produced hyperimmune globulin (HIG) from RhCMV-seropositive macaques or dose-optimized, potently RhCMV-neutralizing HIG prior to intravenous challenge with an RhCMV mixture. HIG passive infusion provided complete protection against fetal loss in both groups. The dose-optimized, RhCMV-neutralizing HIG additionally inhibited placental transmission of RhCMV and reduced viral replication and diversity. Our findings suggest that the presence of durable and potently neutralizing antibodies at the time of primary infection can prevent transmission of systemically replicating maternal RhCMV to the developing fetus, and therefore should be a primary target of vaccines to eliminate this neonatal infection.