Protective effect of pre-existing natural immunity in a nonhuman primate reinfection model of congenital cytomegalovirus infection.

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 SIVgag inserted as an immunological marker, administered separately (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/SIVgag virus 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/SIVgag and ~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.

Intrahost cytomegalovirus population genetics following antibody pretreatment in a monkey model of congenital transmission.

Human cytomegalovirus (HCMV) infection is the leading non-genetic cause of congenital birth defects worldwide. While several studies have addressed 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 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 between control, standard-potency, and high-potency pretreatment groups. We found that while the presence of maternal antibodies reduced viral load and congenital infection, it had no apparent impact on intrahost viral genetic diversity at the investigated loci. Interestingly, some minor haplotypes present in fetal and maternal-fetal interface tissues were also identified as minor haplotypes in corresponding maternal tissues, providing evidence for a loose RhCMV mother-to-fetus transmission bottleneck even in the presence of preexisting antibodies.

Maternal CD4+ T cells protect against severe congenital cytomegalovirus disease in a novel nonhuman primate model of placental cytomegalovirus transmission.

Elucidation of maternal immune correlates of protection against congenital cytomegalovirus (CMV) is necessary to inform future vaccine design. Here, we present a novel rhesus macaque model of placental rhesus CMV (rhCMV) transmission and use it to dissect determinants of protection against congenital transmission following primary maternal rhCMV infection. In this model, asymptomatic intrauterine infection was observed following i.v. rhCMV inoculation during the early second trimester in two of three rhCMV-seronegative pregnant females. In contrast, fetal loss or infant CMV-associated sequelae occurred in four rhCMV-seronegative pregnant macaques that were CD4(+) T-cell depleted at the time of inoculation. Animals that received the CD4(+) T-cell-depleting antibody also exhibited higher plasma and amniotic fluid viral loads, dampened virus-specific CD8(+) T-cell responses, and delayed production of autologous neutralizing antibodies compared with immunocompetent monkeys. Thus, maternal CD4(+) T-cell immunity during primary rhCMV infection is important for controlling maternal viremia and inducing protective immune responses that prevent severe CMV-associated fetal disease.

Characterization of MHC class I alleles in sooty mangabeys as a tool for evaluating cellular immunity in natural hosts of SIV infection.

Although immune pressure exerted by MHC class I-restricted cytotoxic T lymphocytes (CTL) are an important determinant of outcome in pathogenic HIV and SIV infection, lack of data on MHC class I genes has hampered study of its role in natural hosts with nonpathogenic SIV infection. In this study, we cloned and characterized full-length MHC class I genes derived from the cDNA library of two unrelated naturally infected sooty mangabeys (Cercocebus atys) in whom SIV-specific CTL epitopes were previously mapped. Twenty one full-length MHC class I alleles consisting of five MHC-A (Ceat-A), 13 MHC-B (Ceat-B), and three MHC-E (Ceat-E) alleles were identified. Sequence-specific primers (SSP) for high-throughput screening of genomic DNA by PCR were developed for 16 of the 18 Ceat-A and Ceat-B alleles. Screening of 62 SIV-negative and 123 SIV-infected sooty mangabeys at the Yerkes National Primate Research Center (YNPRC) revealed the presence of up to four MHC-A and eight MHC-B alleles in individual mangabeys, indicating that similar to macaque species, mangabeys have at least two duplications of the MHC-A locus and four duplications of the MHC-B locus in the absence of an MHC-C locus. Using stable transfectants of Ceat MHC Class I alleles in the MHC-null 721.221 cell line, we identified Ceat-B*12:01 as the restricting allele of a previously reported Nef20-28 CTL epitope. Ceat-B*1201/Nef20-28 tetramers identified tetramer-positive CD8+ T lymphocytes in Ceat-B*1201-positive SIV-infected mangabeys. This study has laid the groundwork for comprehensive analysis of CTL and SIV evolution in a natural host of SIV infection.

Protective effect of pre-existing natural immunity in a nonhuman primate reinfection model of congenital cytomegalovirus infection.

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/SIV gag , a wild-type-like RhCMV clone with SIV gag inserted 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/SIV gag virus 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/SIV gag and ∼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 Summary: Globally, 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.

Immunomodulatory potential of in vivo natural killer T (NKT) activation by NKTT320 in Mauritian-origin cynomolgus macaques.

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.

Immune Profile of the Normal Maternal-Fetal Interface in Rhesus Macaques and Its Alteration Following Zika Virus Infection.

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 αß 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.

Impact of CMV Infection on Natural Killer Cell Clonal Repertoire in CMV-Naïve Rhesus Macaques.

Recent functional, gene expression, and epigenetic studies have suggested the presence of a subset of mature natural killer (NK) cells responsible for maintaining NK cell memory. The lack of endogenous clonal markers in NK cells impedes understanding the genesis of these cell populations. In humans, primates, and mice, this phenotype and memory or adaptive functions have been strongly linked to cytomegalovirus or related herpes virus infections. We have used transplantation of lentivirally-barcoded autologous hematopoietic stem and progenitor cells (HSPC) to track clonal hematopoiesis in rhesus macaques and previously reported striking oligoclonal expansions of NK-biased barcoded clones within the CD56-CD16+ NK cell subpopulation, clonally distinct from ongoing output of myeloid, B cell, T cell, and CD56+16- NK cells from HSPC. These CD56-CD16+ NK cell clones segregate by expression of specific KIR surface receptors, suggesting clonal expansion in reaction to specific environmental stimuli. We have now used this model to investigate the impact of rhesus CMV(RhCMV) infection on NK clonal dynamics. Following transplantation, RhCMVneg rhesus macaques display less dominant and oligoclonal CD16+ NK cells biased clones compared to RhCMVpos animals, however these populations of cells are still clearly present. Upon RhCMV infection, CD16+ NK cells proliferate, followed by appearance of new groups of expanded NK clones and disappearance of clones present prior to RhCMV infection. A second superinfection with RhCMV resulted in rapid viral clearance without major change in the mature NK cell clonal landscape. Our findings suggest that RhCMV is not the sole driver of clonal expansion and peripheral maintenance of mature NK cells; however, infection of macaques with this herpesvirus does result in selective expansion and persistence of specific NK cell clones, providing further information relevant to adaptive NK cells and the development of NK cell therapies.

Preexisting antibodies can protect against congenital cytomegalovirus infection in monkeys.

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.