Critical aspects of HIV-1 infection occur in mucosal tissues, particularly in the gut, which contains large numbers of HIV-1 target cells that are depleted early in infection. We used electron tomography (ET) to image HIV-1 in gut-associated lymphoid tissue (GALT) of HIV-1-infected humanized mice, the first three-dimensional ultrastructural examination of HIV-1 infection in vivo. Human immune cells were successfully engrafted in the mice, and following infection with HIV-1, human T cells were reduced in GALT. Virions were found by ET at all stages of egress, including budding immature virions and free mature and immature viruses. Immuno-electron microscopy verified the virions were HIV-1 and showed CD4 sequestration in the endoplasmic reticulum of infected cells. Observation of HIV-1 in infected GALT tissue revealed that most HIV-1-infected cells, identified by immunolabeling and/or the presence of budding virions, were localized to intestinal crypts with pools of free virions concentrated in spaces between cells. Fewer infected cells were found in mucosal regions and the lamina propria. The preservation quality of reconstructed tissue volumes allowed details of budding virions, including structures interpreted as host-encoded scission machinery, to be resolved. Although HIV-1 virions released from infected cultured cells have been described as exclusively mature, we found pools of both immature and mature free virions within infected tissue. The pools could be classified as containing either mostly mature or mostly immature particles, and analyses of their proximities to the cell of origin supported a model of semi-synchronous waves of virion release. In addition to HIV-1 transmission by pools of free virus, we found evidence of transmission via virological synapses. Three-dimensional EM imaging of an active infection within tissue revealed important differences between cultured cell and tissue infection models and furthered the ultrastructural understanding of HIV-1 transmission within lymphoid tissue.

Lung interstitial CD4+ T cells are critical for protection against pulmonary infections, but the fate of this population during HIV-1 infection is not well described. We studied CD4+ T cells in the setting of HIV-1 infection in human lung tissue, humanized mice, and a Mycobacterium tuberculosis (Mtb)/simian immunodeficiency virus (SIV) nonhuman primate co-infection model. Infection with a CCR5-tropic strain of HIV-1 or SIV results in severe and rapid loss of lung interstitial CD4+ T cells but not blood or lung alveolar CD4+ T cells. This is accompanied by high HIV-1 production in these cells in vitro and in vivo. Importantly, during early SIV infection, loss of lung interstitial CD4+ T cells is associated with increased dissemination of pulmonary Mtb infection. We show that lung interstitial CD4+ T cells serve as an efficient target for HIV-1 and SIV infection that leads to their early depletion and an increased risk of disseminated tuberculosis.

It was widely accepted that HIV-1 downregulates HLA-A/B to avoid CTL recognition while leaving HLA-C unaltered in order to prevent NK cell activation by engaging inhibitory NK cell receptors, but it was recently observed that most primary isolates of HIV-1 can mediate HLA-C downmodulation. Now we report that HIV-1-mediated downmodulation of HLA-C was associated with reduced binding to its respective inhibitory receptors. Despite this, HLA-C-licensed NK cells displayed reduced antiviral activity compared to their unlicensed counterparts, potentially due to residual binding to the respective inhibitory receptors. Nevertheless, NK cells were able to sense alterations of HLA-C expression demonstrated by increased antiviral activity when exposed to viral strains with differential abilities to downmodulate HLA-C. These results suggest that the capability of HLA-C-licensed NK cells to control HIV-1 replication is determined by the strength of KIR/HLA-C interactions and is thus dependent on both host genetics and the extent of virus-mediated HLA-C downregulation.

The Duffy Antigen Receptor for Chemokines (DARC)-null trait, common among persons of African descent and associated with lower absolute neutrophil counts (ANCs), may be linked to increased risk to certain infections including HIV-1 but the underlying causes are poorly understood. We hypothesized that DARC-null-linked neutropenia may negatively impact neutrophil immunoregulatory modulation of other immune cells such as natural killer (NK) and CD8+ T cells leading to altered phenotype, functionality and homeostatic activity of these immune cells. HIV-1 uninfected (n = 20) and HIV-1 chronically infected (n = 19) participants were assessed using multi-parametric flow cytometry to determine NK and CD8+ T cell counts, phenotypic profiles, and cytokine production and degranulation. Annexin V and carboxyfluorescein succinimidyl ester (CFSE) staining were used to examine NK cell survival and NK cell and CD8+ T cell proliferation respectively. Participants were genotyped for the DARC-null polymorphism using allelic discrimination assays and ANCs were measured by full blood count. In HIV uninfected individuals, a reduction of total NK cell counts was noted in the absence of DARC and this correlated with lower ANCs. HIV uninfected DARC-null subjects displayed a less mature NK cell phenotype. However, this did not translate to differences in NK cell activation or effector functionality by DARC state. Whilst HIV-1 infected subjects displayed NK cell profiling that is typical of HIV infection, no differences were noted upon DARC stratification. Similarly, CD8+ T cells from HIV infected individuals displayed phenotypic and functional modulation that is characteristic of HIV infection, but profiling was unaffected by the DARC-null variant irrespective of HIV status. Overall, the data suggests that the DARC-null polymorphism and lower ANCs does not impede downstream cytolytic cell priming and functionality.

Background Although ≈70% of the world's population of people living with HIV reside in sub-Saharan Africa, there are minimal prospective data on the contributions of HIV infection to atherosclerosis in the region. Methods and Results We conducted a prospective observational cohort study of people living with HIV on antiretroviral therapy >40 years of age in rural Uganda, along with population-based comparators not infected with HIV. We collected data on cardiovascular disease risk factors and carotid ultrasound measurements annually. We fitted linear mixed effects models, adjusted for cardiovascular disease risk factors, to estimate the association between HIV serostatus and progression of carotid intima media thickness (cIMT). We enrolled 155 people living with HIV and 154 individuals not infected with HIV and collected cIMT images at 1045 visits during a median of 4 annual visits per participant (interquartile range 3-4, range 1-5). Age (median 50.9 years) and sex (49% female) were similar by HIV serostatus. At enrollment, there was no difference in mean cIMT by HIV serostatus (0.665 versus 0.680 mm, P=0.15). In multivariable models, increasing age, blood pressure, and non-high-density lipoprotein cholesterol were associated with greater cIMT (P<0.05), however change in cIMT per year was also no different by HIV serostatus (0.004 mm/year for HIV negative [95% CI, 0.001-0.007 mm], 0.006 mm/year for people living with HIV [95% CI, 0.003-0.008 mm], HIV×time interaction P=0.25). Conclusions In rural Uganda, treated HIV infection was not associated with faster cIMT progression. These results do not support classification of treated HIV infection as a risk factor for subclinical atherosclerosis progression in rural sub-Saharan Africa. Registration URL: https://www.ClinicalTrials.gov; Unique identifier: NCT02445079.

Bacterial vaginosis (BV), a common syndrome characterized by Lactobacillus-deficient vaginal microbiota, is associated with adverse health outcomes. BV often recurs after standard antibiotic therapy in part because antibiotics promote microbiota dominance by Lactobacillus iners instead of Lactobacillus crispatus, which has more beneficial health associations. Strategies to promote L. crispatus and inhibit L. iners are thus needed. We show that oleic acid (OA) and similar long-chain fatty acids simultaneously inhibit L. iners and enhance L. crispatus growth. These phenotypes require OA-inducible genes conserved in L. crispatus and related species, including an oleate hydratase (ohyA) and putative fatty acid efflux pump (farE). FarE mediates OA resistance, while OhyA is robustly active in the human vaginal microbiota and sequesters OA in a derivative form that only ohyA-harboring organisms can exploit. Finally, OA promotes L. crispatus dominance more effectively than antibiotics in an in vitro model of BV, suggesting a novel approach for treatment.

The presence of HIV in the CNS has been related to chronic immune activation and cognitive dysfunction. The aim of this work was to investigate (1) the presence of neuroinflammation in aviremic people with HIV (PWH) on therapy and in nontreated aviremic PWH (elite controllers [ECs]) using a translocator protein 18 kDa radioligand; (2) the relationship between neuroinflammation and cognitive function in aviremic PWH; and (3) the relationship between [11C]-PBR28 signal and quantitative MRI (qMRI) measures of brain tissue integrity such as T1 and T2 relaxation times (rts).

Alveolar macrophages (AMs) are critical for defense against airborne pathogens and AM dysfunction is thought to contribute to the increased burden of pulmonary infections observed in individuals living with HIV-1 (HIV). While HIV nucleic acids have been detected in AMs early in infection, circulating HIV during acute and chronic infection is usually CCR5 T cell-tropic (T-tropic) and enters macrophages inefficiently in vitro. The mechanism by which T-tropic viruses infect AMs remains unknown. We collected AMs by bronchoscopy performed in HIV-infected, antiretroviral therapy (ART)-naive and uninfected subjects. We found that viral constructs made with primary HIV envelope sequences isolated from both AMs and plasma were T-tropic and inefficiently infected macrophages. However, these isolates productively infected macrophages when co-cultured with HIV-infected CD4+ T cells. In addition, we provide evidence that T-tropic HIV is transmitted from infected CD4+ T cells to the AM cytosol. We conclude that AM-derived HIV isolates are T-tropic and can enter macrophages through contact with an infected CD4+ T cell, which results in productive infection of AMs. CD4+ T cell-dependent entry of HIV into AMs helps explain the presence of HIV in AMs despite inefficient cell-free infection, and may contribute to AM dysfunction in people living with HIV.

HIV-1 increases susceptibility to pulmonary infection and disease, suggesting pathogenesis in the lung. However, the lung immune environment during HIV infection remains poorly characterized. This study examined T cell activation and the cytokine milieu in paired bronchoalveolar lavage (BAL) and blood from 36 HIV-uninfected and 32 HIV-infected participants. Concentrations of 27 cytokines were measured by Luminex, and T cells were phenotyped by flow cytometry. Blood and BAL had distinct cytokine profiles (p=0.001). In plasma, concentrations of inflammatory cytokines like IFN-γ (p=0.004) and TNF-α (p=0.004) were elevated during HIV infection, as expected. Conversely, BAL cytokine concentrations were similar in HIV-infected and uninfected individuals, despite high BAL viral loads (VL; median 48,000 copies/ml epithelial lining fluid). HIV-infected individuals had greater numbers of T cells in BAL compared to uninfected individuals (p=0.007); and BAL VL positively associated with CD4+ and CD8+ T cell numbers (p=0.006 and p=0.0002, respectively) and CXCL10 concentrations (p=0.02). BAL T cells were highly activated in HIV-infected individuals, with nearly 2-3 fold greater frequencies of CD4+CD38+ (1.8-fold; p=0.007), CD4+CD38+HLA-DR+ (1.9-fold; p=0.0006), CD8+CD38+ (2.8-fold; p=0.0006), CD8+HLA-DR+ (2-fold; p=0.022) and CD8+CD38+HLA-DR+ (3.6-fold; p<0.0001) cells compared to HIV-uninfected individuals. Overall, this study demonstrates a clear disruption of the pulmonary immune environment during HIV infection, with readily detectable virus and activated T lymphocytes, which may be driven to accumulate by local chemokines.

Diverse and non-Lactobacillus-dominated vaginal microbial communities are associated with adverse health outcomes such as preterm birth and the acquisition of sexually transmitted infections. Despite the importance of recognizing and understanding the key risk-associated features of these communities, their heterogeneous structure and properties remain ill-defined. Clustering approaches are commonly used to characterize vaginal communities, but they lack sensitivity and robustness in resolving substructures and revealing transitions between potential sub-communities. Here, we address this need with an approach based on mixed membership topic models. Using longitudinal data from cohorts of pregnant and non-pregnant study participants, we show that topic models more accurately describe sample composition, longitudinal changes, and better predict the loss of Lactobacillus dominance. We identify several non-Lactobacillus-dominated sub-communities common to both cohorts and independent of reproductive status. In non-pregnant individuals, we find that the menstrual cycle modulates transitions between and within sub-communities, as well as the concentrations of half of the cytokines and 18% of metabolites. Overall, our analyses based on mixed membership models reveal substructures of vaginal ecosystems which may have important clinical and biological associations.

Metagenomic sequencing has the potential to transform microbial detection and characterization, but new tools are needed to improve its sensitivity. Here we present CATCH, a computational method to enhance nucleic acid capture for enrichment of diverse microbial taxa. CATCH designs optimal probe sets, with a specified number of oligonucleotides, that achieve full coverage of, and scale well with, known sequence diversity. We focus on applying CATCH to capture viral genomes in complex metagenomic samples. We design, synthesize, and validate multiple probe sets, including one that targets the whole genomes of the 356 viral species known to infect humans. Capture with these probe sets enriches unique viral content on average 18-fold, allowing us to assemble genomes that could not be recovered without enrichment, and accurately preserves within-sample diversity. We also use these probe sets to recover genomes from the 2018 Lassa fever outbreak in Nigeria and to improve detection of uncharacterized viral infections in human and mosquito samples. The results demonstrate that CATCH enables more sensitive and cost-effective metagenomic sequencing.

Some closely related human leukocyte antigen (HLA) alleles are associated with variable clinical outcomes following HIV-1 infection despite presenting the same viral epitopes. Mechanisms underlying these differences remain unclear but may be due to intrinsic characteristics of the HLA alleles or responding T cell repertoires. Here we examine CD8+ T cell responses against the immunodominant HIV-1 Gag epitope TL9 (TPQDLNTML180-188) in the context of the protective allele B*81:01 and the less protective allele B*42:01. We observe a population of dual-reactive T cells that recognize TL9 presented by both B*81:01 and B*42:01 in individuals lacking one allele. The presence of dual-reactive T cells is associated with lower plasma viremia, suggesting a clinical benefit. In B*42:01 expressing individuals, the dual-reactive phenotype defines public T cell receptor (TCR) clones that recognize a wider range of TL9 escape variants, consistent with enhanced control of viral infection through containment of HIV-1 sequence adaptation.

While modulation of regulatory T cell (Treg) function and adoptive Treg transfer are being explored as therapeutic modalities in the context of autoimmune diseases, transplantation and cancer, their role in HIV-1 pathogenesis remains less well defined. Controversy persists regarding their beneficial or detrimental effects in HIV-1 disease, which warrants further detailed exploration. Our objectives were to investigate if functional CD4(+) Tregs can be isolated and expanded from HIV-1-infected individuals for experimental or potential future therapeutic use and to determine phenotype and suppressive capacity of expanded Tregs from HIV-1 positive blood and tissue. Tregs and conventional T cell controls were isolated from blood and gut-associated lymphoid tissue of individuals with HIV-1 infection and healthy donors using flow-based cell-sorting. The phenotype of expanded Tregs was assessed by flow-cytometry and quantitative PCR. T-cell receptor ß-chain (TCR-β) repertoire diversity was investigated by deep sequencing. Flow-based T-cell proliferation and chromium release cytotoxicity assays were used to determine Treg suppressive function. Tregs from HIV-1 positive individuals, including infants, were successfully expanded from PBMC and GALT. Expanded Tregs expressed high levels of FOXP3, CTLA4, CD39 and HELIOS and exhibited a highly demethylated TSDR (Treg-specific demethylated region), characteristic of Treg lineage. The TCRß repertoire was maintained following Treg expansion and expanded Tregs remained highly suppressive in vitro. Our data demonstrate that Tregs can be expanded from blood and tissue compartments of HIV-1+ donors with preservation of Treg phenotype, function and TCR repertoire. These results are highly relevant for the investigation of potential future therapeutic use, as currently investigated for other disease states and hold great promise for detailed studies on the role of Tregs in HIV-1 infection.

Colonization by Lactobacillus in the female genital tract is thought to be critical for maintaining genital health. However, little is known about how genital microbiota influence host immune function and modulate disease susceptibility. We studied a cohort of asymptomatic young South African women and found that the majority of participants had genital communities with low Lactobacillus abundance and high ecological diversity. High-diversity communities strongly correlated with genital pro-inflammatory cytokine concentrations in both cross-sectional and longitudinal analyses. Transcriptional profiling suggested that genital antigen-presenting cells sense gram-negative bacterial products in situ via Toll-like receptor 4 signaling, contributing to genital inflammation through activation of the NF-κB signaling pathway and recruitment of lymphocytes by chemokine production. Our study proposes a mechanism by which cervicovaginal microbiota impact genital inflammation and thereby might affect a woman's reproductive health, including her risk of acquiring HIV.

Immunological damage in acute HIV infection (AHI) may predispose to detrimental clinical sequela. However, studies on the earliest HIV-induced immunological changes are limited, particularly in sub-Saharan Africa. We assessed the plasma cytokines kinetics, and their associations with virological and immunological parameters, in a well-characterized AHI cohort where participants were diagnosed before peak viremia.

Human immunodeficiency virus (HIV)-induced changes in immune cells during the acute phase of infection can cause irreversible immunological damage and predict the rate of disease progression. Antiretroviral therapy (ART) remains the most effective strategy for successful immune restoration in immunocompromised people living with HIV and the earlier ART is initiated after infection, the better the long-term clinical outcomes. Here we explored the effect of ART on peripheral antigen presenting cell (APC) phenotype and function in women with HIV-1 subtype C infection who initiated ART in the hyperacute phase (before peak viremia) or during chronic infection. Peripheral blood mononuclear cells obtained longitudinally from study participants were used for immunophenotyping and functional analysis of monocytes and dendritic cells (DCs) using multiparametric flow cytometry and matched plasma was used for measurement of inflammatory markers IL-6 and soluble CD14 (sCD14) by enzyme-linked immunosorbent assay. HIV infection was associated with expansion of monocyte and plasmacytoid DC (pDC) frequencies and perturbation of monocyte subsets compared to uninfected persons despite antiretroviral treatment during hyperacute infection. Expression of activation marker CD69 on monocytes and pDCs in early treated HIV was similar to uninfected individuals. However, despite early ART, HIV infection was associated with elevation of plasma IL-6 and sCD14 levels which correlated with monocyte activation. Furthermore, HIV infection with or without early ART was associated with downmodulation of the co-stimulatory molecule CD86. Notably, early ART was associated with preserved toll-like receptor (TLR)-induced IFN-α responses of pDCs. Overall, this data provides evidence of the beneficial impact of ART initiated in hyperacute infection in preservation of APC functional cytokine production activity; but also highlights persistent inflammation facilitated by monocyte activation even after prolonged viral suppression and suggests the need for therapeutic interventions that target residual immune activation.

Mucosa-associated invariant T (MAIT) cells are innate-like T cells with specialized antimicrobial functions. Circulating MAIT cells are depleted in chronic human immunodeficiency virus (HIV) infection, but studies examining this effect in peripheral tissues, such as the female genital tract, are lacking.

Heterosexual transmission of human immunodeficiency virus type 1 (HIV-1) is associated with a significant bottleneck in the viral quasispecies population, yet the timing of that bottleneck is poorly understood. We characterized HIV-1 diversity in the blood and female genital tract (FGT) within 2 weeks after detection of infection in three women enrolled in a unique prospective cohort in South Africa. We assembled full-length HIV-1 genomes from matched cervicovaginal lavage (CVL) samples and plasma. Deep sequencing allowed us to identify intrahost single-nucleotide variants (iSNVs) and to characterize within-sample HIV-1 diversity. Our results demonstrated very little HIV-1 diversity in the FGT and plasma by the time viremia was detectable. Within each subject, the consensus HIV-1 sequences were identical in plasma and CVL fluid. No iSNV was present at >6% frequency. One subject had 77 low-frequency iSNVs across both CVL fluid and plasma, another subject had 14 iSNVs in only CVL fluid from the earliest time point, and the third subject had no iSNVs in CVL fluid or plasma. Overall, the small amount of diversity that we detected was greater in the FGT than in plasma and declined over the first 2 weeks after viremia was detectable, compatible with a very early HIV-1 transmission bottleneck. To our knowledge, our study represents the earliest genomic analysis of HIV-1 in the FGT after transmission. Further, the use of metagenomic sequencing allowed us to characterize other organisms in the FGT, including commensal bacteria and sexually transmitted infections, highlighting the utility of the method to sequence both HIV-1 and its metagenomic environment.IMPORTANCE Due to error-prone replication, HIV-1 generates a diverse population of viruses within a chronically infected individual. When HIV-1 is transmitted to a new individual, one or a few viruses establish the new infection, leading to a genetic bottleneck in the virus population. Understanding the timing and nature of this bottleneck may provide insight into HIV-1 vaccine design and other preventative strategies. We examined the HIV-1 population in three women enrolled in a unique prospective cohort in South Africa who were followed closely during the earliest stages of HIV-1 infection. We found very little HIV-1 diversity in the blood and female genital tract during the first 2 weeks after virus was detected in the bloodstream. These results are compatible with a very early HIV-1 population bottleneck, suggesting the need to study the HIV-1 population in the female genital tract before virus is detectable in the bloodstream.

γδ T cells predominate in the intestinal mucosa and help maintain gut homeostasis and mucosal immunity. Although HIV infection significantly alters these cells, what drives these perturbations is unclear. Growing evidence suggests that impaired intestinal immune function in HIV leads to chronic immune activation and disease progression. This occurs even in HIV controllers - individuals with undetectable HIV viremia without antiretroviral therapy (ART). We show that Vδ1+ cells, a subset of γδ T cells described as being important in intestinal barrier function, increase in frequency in HIV-infected individuals, including HIV controllers. These cells resemble terminally differentiated effector memory cells, producing the pro-inflammatory cytokines IFNγ, TNFα, and MIP-1β upon stimulation. Importantly, pro-inflammatory Vδ1+ cell frequency correlates with levels of HIV RNA in intestinal tissue but not in plasma. This study supports a model in which local viral replication in the gut in HIV controllers disrupts the phenotype and function of Vδ1+ cells, a cell type involved in the maintenance of epithelial barrier integrity, and may thereby contribute to systemic immune activation and HIV disease progression.

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.