Biology subjects

Barry, P. A.

Publications and source records attributed to Barry, P. A..

7 recordsLinked to original sources

bioRxiv2023

The pentameric complex is not required for vertical transmission of cytomegalovirus in seronegative pregnant rhesus macaques

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 SummaryCongenital CMV transmission frequency in seronegative rhesus macaques is not affected by the deletion of the viral pentameric complex.

microbiology↗

bioRxiv2023

Relationship of maternal cytomegalovirus-specific antibody responses and viral load to vertical transmission risk following primary maternal infection in a rhesus macaque model

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. Author summaryCytomegalovirus (CMV) is the most common infectious cause of birth defects globally, but we still do not have licensed medical interventions to prevent vertical transmission of CMV. We utilized a non-human primate model of primary CMV infection during pregnancy to study virological and humoral factors that influence congenital infection. Unexpectedly, we found that the levels virus in maternal plasma were not predictive of virus transmission to the amniotic fluid (AF) in immunocompetent dams. In contrast, CD4+ T cell depleted pregnant rhesus macaques with virus detected in AF had higher plasma viral loads than dams not showing placental transmission. Virus-specific antibody binding, neutralizing, and Fc-mediated antibody effector antibody responses were not different in immunocompetent animals with and without virus detectable in AF, but passively infused neutralizing antibodies and antibodies binding to key glycoproteins were higher in CD4+ T cell-depleted dams who did not transmit the virus compared to those that did. Our data suggests that the natural development of virus-specific antibody responses is too slow to prevent congenital transmission following maternal infection, highlighting the need for the development of vaccines that confer levels of pre-existing immunity to CMV-naive mothers that can prevent congenital transmission to their infants during pregnancy.

immunology↗

bioRxiv2023

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-RhCMV{Delta}Rh13.1/SIVgag, a wild-type-like RhCMV clone with SIVgag 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-RhCMV{Delta}Rh13.1/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-RhCMV{Delta}Rh13.1/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. 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.

immunology↗

bioRxiv2022

FcRγ- NK cell induction by specific CMV and expansion by subclinical viral infections in rhesus macaques

Long-lived memory-like NK cells, characterized by FcR{gamma}-deficiency and enhanced responsiveness to antibody-bound virus-infected cells, have been found in certain human cytomegalovirus (HCMV)-seropositive individuals. Because humans are exposed to numerous microbes and environmental agents, specific relationships between HCMV and FcR{gamma}-deficient NK cells (also known as g-NK cells) have been challenging to define. Here, we show that a subgroup of rhesus cytomegalovirus (RhCMV)-seropositive macaques possesses FcR{gamma}-deficient NK cells that stably persist and display phenotype resembling human FcR{gamma}-deficient NK cells. Moreover, these macaque NK cells resembled human FcR{gamma}-deficient NK cells with respect to functional characteristics, including enhanced responsiveness to RhCMV-infected target in an antibody-dependent manner and hypo-responsiveness to tumor and cytokine stimulation. These cells were not detected in specific-pathogen-free (SPF) macaques free of RhCMV and six other viruses; however, experimental infection of SPF animals with RhCMV strain UCD59, but not RhCMV strain 68-1 or SIV, led to induction of FcR{gamma}-deficient NK cells. In non-SPF macaques, co-infection by RhCMV with other common viruses was associated with higher frequencies of FcR{gamma}-deficient NK cells. These results support a causal role for specific cytomegalovirus strain(s) in the induction of FcR{gamma}-deficient NK cells, and suggest that co-infection by other viruses further expands this memory-like NK cell pool.

immunology↗

bioRxiv2020

In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome

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 commonly used 68-1 strain of RhCMV has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptation that cause reduced viremia in RhCMV-naive animals and limited shedding compared to low passage isolates. Using sequence information from primary RhCMV isolates we constructed a full-length (FL) RhCMV by repairing all presumed mutations in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naive RM with the reconstituted virus resulted in significant replication in the blood similar to primary isolates of RhCMV and furthermore led to extensive viremia in many tissues at day 14 post infection. In contrast, viral dissemination and viremia 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. Author SummaryHuman cytomegalovirus (HCMV) infections are generally asymptomatic in healthy immunocompetent individuals, but HCMV can cause serious disease after congenital infection and in individuals with immunocompromised immune systems. Since HCMV is highly species specific and cannot productively infect immunocompetent laboratory animals, experimental infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a closely related animal model for HCMV. By employing the unique ability of CMV to elicit robust and lasting cellular immunity, this model has also been instrumental in developing novel CMV-based vaccines against chronic and recurring infections with pathogens such as the human immunodeficiency virus (HIV) and Mycobacterium tuberculosis (Mtb). However, most of this work was conducted with derivatives of the 68-1 strain of RhCMV which has acquired multiple genomic alterations in tissue culture. To model pathogenesis and immunology of clinical HCMV isolates we generated a full-length (FL) RhCMV clone representative of low passage isolates. Infection of RhCMV-naive RM with FL-RhCMV demonstrated viremia and tissue dissemination that was comparable to that of non-clonal low passage isolates. We further demonstrate that FL-RhCMV is strongly attenuated upon deletion of gene regions absent in 68-1 thus demonstrating the usefulness of FL-RhCMV to study RhCMV pathogenesis.

microbiology↗

bioRxiv2020

Bridging the gap: Using reservoir ecology and human serosurveys to estimate Lassa virus incidence in West Africa

Forecasting the risk of pathogen spillover from reservoir populations of wild or domestic animals is essential for the effective deployment of interventions such as wildlife vaccination or culling. Due to the sporadic nature of spillover events and limited availability of data, developing and validating robust, spatially explicit, predictions is challenging. Recent efforts have begun to make progress in this direction by capitalizing on machine learning methodologies. An important weakness of existing approaches, however, is that they generally rely on combining human and reservoir infection data during the training process and thus conflate risk attributable to the prevalence of the pathogen in the reservoir population with the risk attributed to the realized rate of spillover into the human population. Because effective planning of interventions requires that these components of risk be disentangled, we developed a multi-layer machine learning framework that separates these processes. Our approach begins by training models to predict the geographic range of the primary reservoir and the subset of this range in which the pathogen occurs. The spillover risk predicted by the product of these reservoir specific models is then fit to data on realized patterns of historical spillover into the human population. The result is a geographically specific spillover risk forecast that can be easily decomposed and used to guide effective intervention. Applying our method to Lassa virus, a zoonotic pathogen that regularly spills over into the human population across West Africa, results in a model that explains a modest but statistically significant portion of geographic variation in historical patterns of spillover. When combined with a mechanistic mathematical model of infection dynamics, our spillover risk model predicts that 897,700 humans are infected by Lassa virus each year across West Africa, with Nigeria accounting for more than half of these human infections. Author SummaryThe 2019 emergence of SARS-CoV-2 is a grim reminder of the threat animal-borne pathogens pose to human health. Even prior to SARS-CoV-2, the spillover of pathogens from animal reservoirs was a persistent problem, with pathogens such as Ebola, Nipah, and Lassa regularly but unpredictably causing outbreaks. Machine-learning models that anticipate when and where pathogen transmission from animals to humans is likely to occur would help guide surveillance efforts and preemptive countermeasures like information campaigns or vaccination programs. We develop a novel machine learning framework that uses datasets describing the distribution of a virus within its host and the range of its animal host, along with data on spatial patterns of human immunity, to infer rates of animal-to-human transmission across a region. By training the model on data from the animal host alone, our framework allows rigorous validation of spillover predictions using human data. We apply our framework to Lassa fever, a viral disease of West Africa that is spread to humans by rodents, and use the predictions to update estimates of Lassa virus infections in humans. Our results suggest that Nigeria is most at risk for the emergence of Lassa virus, and should be prioritized for outbreak-surveillance.

ecology↗

bioRxiv2019

Impact of preexisting virus-specific maternal antibodies on cytomegalovirus population genetics 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 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.\n\nAuthor 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.

microbiology↗