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As part of the CHAPS randomized clinical trial, we sequenced a segment of the bacterial 16S rRNA gene from foreskin tissue of 144 adolescents from South Africa and Uganda collected during surgical penile circumcision after receipt of 1 to 2 doses of placebo, emtricitabine with tenofovir disoproxil fumarate, or emtricitabine with tenofovir alafenamide. We found a large proportion ofCorynebacteriumin addition to other anaerobic species.Cutibacterium acneswas more abundant among participants from South Africa than Uganda, though this made no difference in surgical recovery. We did not find a difference in bacterial populations by treatment received nor bacterial taxa that were differentially abundant between participants who received placebo versus active drug. Using RNAseq libraries from foreskin tissue of the same participants, we found negative correlations between the relative abundance of bacterial taxa and the expression of genes downstream of the innate response to bacteria and regulation of the inflammatory response. When participants were divided into clusters based on bacterial community composition, two main clusters emerged which were distinguished by high and low bacterial diversity. Random forest classification showed higher expression ofNFATC3andSELENOSand lower expression ofSTAP1andNLRP6in the higher diversity group compared to the lower. Our results show no difference in the tissue microbiome of the foreskin with short-course PrEP but that bacterial taxa were largely inversely correlated with gene expression, consistent with non-inflammatory colonization.Author SummaryWe investigated the bacterial community of the foreskin of the penis. Previous studies found increased inflammation with certain anaerobic bacteria from swabs taken under the foreskin, but we found that higher relative abundances of the bacteria were correlated with lower expression of inflammatory genes. We did not find different bacteria in participants who received medicine to prevent HIV. Understanding the relationship between bacteria and inflammation in the penis will help us to understand how interventions like penile circumcision reduce the risk of acquiring sexually transmitted infections such as HIV.

Few studies have investigated immune cell ontogeny throughout the neonatal and early paediatric period, where there is often increased vulnerability to infections. Here, we evaluated the dynamics of two critical T cell populations, regulatory (Treg) cells and Th17 cells, over the first 36 weeks of life. Firstly, we observed distinct CD4+ T cells phenotypes between cord blood and peripheral blood, collected within 12 hours of birth, showing that cord blood is not a surrogate for newborn blood. Secondly, both Treg and Th17 cells expanded in a synchronous fashion over 36 weeks of life. However, comparing infants exposed to HIV in utero, but remaining uninfected (iHEU), with HIV-unexposed uninfected control infants (iHUU), there was a lower frequency of peripheral blood Treg cells at birth, resulting in a delayed expansion, and then declining again at 36 weeks. Focusing on birth events, we found that Treg cells co-expressing CCR4 and α4β7 inversely correlated with plasma concentrations of CCL17 (the ligand for CCR4) and intestinal fatty acid binding protein (iFABP), IL-7 and CCL20. This was in contrast to Th17 cells, which showed a positive association with these plasma analytes. Thus, despite the stereotypic expansion of both cell subsets over the first few months of life, there was a disruption in the balance of Th17 to Treg cells at birth likely being a result of gut damage and homing of newborn Treg cells from the blood circulation to the gut.Key pointsPhenotypic differences between cord and birth peripheral blood CD4 cells.Synchronous increase of Th17-Treg cells is disrupted by HIV/ART exposure.Intrauterine HIV exposure was associated with epithelial gut damage.

Determinants of the acquisition and maintenance of maternal microchimerism (MMc) during infancy and the impact of MMc on infant immune responses are unknown. We examined factors which influence MMc detection and level across infancy and the effect of MMc on T cell responses to BCG vaccination in a cohort of HIV exposed, uninfected and HIV unexposed infants in South Africa. MMc was measured in whole blood from 58 infants using a panel of quantitative PCR assays at day one and 7, 15, and 36 weeks of life. Infants received BCG at birth, and selected whole blood samples from infancy were stimulated in vitro with BCG and assessed for polyfunctional CD4+ T cell responses. MMc was present in most infants across infancy with levels ranging from 0-1,193/100,000 genomic equivalents and was positively impacted by absence of maternal HIV, maternal-infant HLA compatibility, infant female sex, and exclusive breastfeeding. Initiation of maternal antiretroviral therapy prior to pregnancy partially restored MMc level in HIV exposed, uninfected infants. Birth MMc was associated with an improved polyfunctional CD4+ T cell response to BCG. These data emphasize that both maternal and infant factors influence the level of MMc, which may subsequently impact infant T cell responses.Abstract Figure

Langerhans cells (LCs) reside in the epidermis as a dense network of immune system sentinels, coordinating both immunogenic and tolerogenic immune responses. To determine molecular switches directing induction of LC immune activation, we performed mathematical modelling of gene regulatory networks identified by single cell RNA sequencing of LCs exposed to TNF, a key pro-inflammatory signal produced by the skin. Our approach delineated three programmes of LC phenotypic activation (immunogenic, tolerogenic or ambivalent), and confirmed that TNF enhanced LC immunogenic programming. Through regulon analysis followed by mutual information modelling, we identified IRF1 as the key transcription factor for the regulation of immunogenicity in LCs. Application of a mathematical toggle switch model, coupling IRF1 with tolerance-inducing transcription factors, determined the key set of transcription factors regulating the switch between tolerance and immunogenicity, and correctly predicted LC behaviour in LCs derived from different body sites. Our findings provide a mechanistic explanation of how combinatorial interactions between different transcription factors can coordinate specific transcriptional programmes in human LCs, interpreting the microenvironmental context of the local tissue microenvironments.

BackgroundImplementation of universal antiretroviral therapy (ART) has significantly lowered vertical transmission rates but has also increased numbers of HIV-exposed uninfected children (HEU), who remain vulnerable to morbidities. Here, we investigated whether T cell alterations in the placenta contribute to altered immune status in HEU.MethodsWe analyzed T cells from term placentae decidua and villous tissue and paired cord blood from pregnant women with HIV (PWH) who initiated ART late in pregnancy (n=21) with pregnant women not living with HIV (PWNH) (n=9).ResultsPlacentae from PWH showed inverted CD4:CD8 ratios and higher proportions of tissue resident CD8+ T cells in villous tissue relative to control placentae. CD8+ T cells in the fetal capillaries, which were of fetal origin, positively correlated with maternal plasma viraemia prior to ART initiation, implying that imbalanced T cells persisted throughout pregnancy. Additionally, the expanded memory differentiation of CD8+ T cells was confined to the fetal placental compartment and cord blood but was not observed in the maternal decidua.ConclusionsT cell homeostatic imbalance in the blood circulation of PWH is reflected in the placenta. The placenta may be a causal link between HIV-induced maternal immune changes during gestation and altered immunity in newborn infants in the absence of vertical transmission.Lay SummaryThe effective prevention of HIV transmission during pregnancy with the rollout of antiretroviral therapy (ART) has resulted in increased numbers of HIV-exposed uninfected children (HEU). These children are vulnerable to infections and health problems and have distorted cellular immune systems at birth. We investigated whether these immune alterations originate in the placenta, as this fetal organ maintains life during pregnancy. After collecting placentae at term from pregnant women living with HIV (PWH), who started ART in the third trimester (n=21) and from pregnant women not living with HIV (PWNH) (n=9), we isolated T cells from dissected placental tissue and matching cord blood. Placentae from PWH showed inverted CD4:CD8 ratios in the placenta and cord blood with higher numbers of CD8+ T cells in the fetal part of the placenta. These CD8+ T cells mirrored events in the blood circulation of the mother and the altered balance of T cell immunity in the PWH was reflected in the placenta. Accordingly, the placenta may be a pivotal link between HIV-induced maternal immune changes and altered immunity in newborn infants in the absence of vertical transmission.

Developing an efficacious vaccine to SARS-CoV-2 infection is critical to stem COVID-19 fatalities and providing the global community with immune protection. We have used a bioinformatic approach to aid in the design of an epitope peptide-based vaccine against the spike protein of the virus. Five antigenic B cell epitopes with viable antigenicity and a total of 27 discontinuous B cell epitopes were mapped out structurally in the spike protein for antibody recognition. We identified eight CD8+T cell 9-mers along with 12 CD4+T cell 14-15-mer as promising candidate epitopes putatively restricted by a large number of MHC-I and II alleles respectively. We used this information to construct anin silicochimeric peptide vaccine whose translational rate was highly expressed when cloned in pET28a (+) vector. The vaccine construct was predicted to elicit high antigenicity and cell-mediated immunity when given as a homologous prime-boost, with triggering of toll-like receptor 5 by the adjuvant linker. The vaccine was characterized by an increase in IgM and IgG and an array of Th1 and Th2 cytokines. Uponin silicochallenge with SARS-CoV-2, there was a decrease in antigen levels using our immune simulations. We therefore propose that potential vaccine designs consider this approach.

BackgroundAntiretroviral therapy (ART) is universally recommended for all HIV-infected pregnant women to improve survival and to reduce mother-to-child HIV transmission. With widespread use of ART in areas of high HIV prevalence, there is concern that prolonged in-utero ART exposure may result in adverse birth outcomes, such as preterm delivery and low birthweight infants. Underlying drivers of these effects may be found in the placenta.ObjectiveWe examined whole placentas from live births to seek an association between timing of ART initiation and pregnancy outcome.Study DesignThis was a nested sub-study in a larger cohort of HIV-infected women recruited in a large primary care antenatal clinic in Cape Town, South Africa. Whole placentas (n=130) were collected at delivery and examined for histopathology from two ART groups. The stable group (n=53) initiated ART prior to pregnancy and the initiating group (n=77) started ART at a median of 15 weeks gestation. Adverse birth outcomes were defined by preterm delivery (PTD; n=9), small-for-gestational age (SGA; n=12) and low birthweight (LBW; n=11) in the two groups. Wilcoxon rank-sum and t-tests were used to measure differences in placental histopathology by ART group. Binomial and univariate linear regression models, chi-squared or Fisher’s exact test were used to quantify associations between placenta histopathology and maternal factors including ART timing and the association with pregnancy outcome.ResultsWomen in the stable group were significantly older (median [IQR] 33 years [28 - 37] vs 28 [25 - 32]; p=0.003), had higher CD4 cell counts 455 cells/ml [381 - 602] vs 369.5 [251 - 534.5], p=0.04) and were less likely to be prehypertensive or hypertensive at first antenatal care visit (p=0.03) than women in the initiating group. Overall, 119 (91%), women were on a fixed dose regimen of Tenofovir (TDF) + Lamivudine (3TC) + Efavirenz (EFV) and only 3 (2%; all in the stable group) were on a protease inhibitor (PI)-based regimen with no difference between the groups. Overall, nearly a third of placentas were small, although the fetal-placenta weight ratio was in the normal range for most women. Meconium exposure and chorioamnionitis was seen in 19% and 15% of placentas, respectively. Apart from maternal vascular malperfusion (MVM) and decidual arteriopathy, histopathology obervations did not differ significantly between stable and initiating groups. Placentas from the stable group showed increased MVM (39.6% vs 19.4%, p=0.01) and decidual arteriopathy (11.3% vs 1.3%, p=0.02), with a trend of decreased placental weight (392g vs 422g, p=0.09). MVM, in turn, was significantly associated with PTD and LBW (p=0.002 and pConclusionART initiation prior to pregnancy was associated with an increased risk of maternal vascular malperfusion and decidual arteriopathy, suggestive of placental dysfunction. The association between MVM with PTD and LBW suggests that a placenta-mediated mechanism may link the putative association between ART and adverse birth outcomes.

PurposePIMS, is a prospective cohort study in South Africa investigating the association between antiretroviral therapy (ART) use, preterm delivery (PTD)and small-for gestational age (SGA) live births. PIMS main hypotheses are that ART initiation in pregnancy and ART-induced hypertension are associated with PTD and SGA respectively and that reconstitution of cellular immune responses in women on ART from before pregnancy results in increases in PTD of appropriate-for-gestational age (AGA) infants.ParticipantsPregnant women (n=3972) aged ≥18 years regardless of HIV status recruited from 2015 to 2016 into the overall PIMS cohort (2517 HIV-uninfected, 1455 HIV-infected). A nested cohort contained 551 HIV-infected women who were ≤24 weeks’ GA on ultrasound: 261 initiated ART before pregnancy, 290 initiated during pregnancy.Findings to dateWomen in the overall cohort were followed antenatally through to delivery using routine clinical records; further women in the nested cohort were actively followed up until 12 months postpartum, with data were collected on maternal health (HIV care and ART use, clinical care and inter-current clinical history). Other procedures conducted on the nested cohort included physical examinations (anthropometry, blood pressure measurement), assessment of fetal growth (ultrasound), maternal and infant phlebotomy for storage of plasma, RNA and peripheral blood mononuclear cells, collection of delivery specimens (placenta and cord blood), and infant 12 month developmental assessment. Preliminary findings have contributed to our understanding of risk factors for adverse birth outcomes, and the relationship between pregnancy immunology, HIV/ART and adverse birth outcomes.Future plansUsing specimens collected from HIV-infected study participants throughout pregnancy and first year of life, the PIMS provides a valuable platform for answering a variety of research questions focused on temporal changes of immunology markers in women whose immune status is altered by HIV infection, and how ART initiated during pregnancy affects immune responses. The relationship between these immunological changes with adverse birth outcomes as well as possible longer-term impact of exposure to ART in fetal and early life will be explored. Additionally, further active and passive follow-up of mothers and their infants is planned at school-going age and beyond to chart growth, morbidity and development, as well as changes in family circumstances.