Distinct fecal microbial signatures are linked to sex and chronic immune activation in pediatric HIV infection.

Introduction: Our understanding of HIV-associated gut microbial dysbiosis in children perinatally-infected with HIV (CLWH) lags behind that of adults living with HIV. Childhood represents a critical window for the gut microbiota. Any disturbances, including prolonged exposure to HIV, antiretroviral drugs, and antibiotics are likely to have a significant impact on long-term health, resulting in a less resilient gut microbiome. The objective of our study was to characterize the gut microbiota in CLWH, and compare it with HIV-unexposed and -uninfected children. Methods: We enrolled 31 children aged 3 to 15 years; 15 were CLWH and 16 were HUU. We assessed dietary patterns and quality; quantified soluble and cellular markers of HIV disease progression by flow cytometry, enzyme-linked immunosorbent and multiplex-bead assays, and profiled the gut microbiota by 16S rRNA sequencing. We explored relationships between the gut microbiota, antibiotic exposure, dietary habits, soluble and cellular markers and host metadata. Results: Children had a Western-type diet, their median health eating index score was 67.06 (interquartile range 58.76-74.66). We found no discernable impact of HIV on the gut microbiota. Alpha diversity metrics did not differ between CLWH and HUU. Sex impacted the gut microbiota (R-squared= 0.052, PERMANOVA p=0.024). Male children had higher microbial richness compared with female children. Two taxa were found to discriminate female from male children independently from HIV status: Firmicutes for males, and Bacteroides for females. Markers of HIV disease progression were comparable between CLWH and HUU, except for the frequency of exhausted CD4+ T cells (PD-1+) which was increased in CLWH (p=0.0024 after adjusting for confounders). Both the frequency of exhausted CD4+ and activated CD4+ T cells (CD38+ HLADR+) correlated positively with the relative abundance of Proteobacteria (rho=0.568. false discovery rate (FDR)-adjusted p= 0.029, and rho=0.62, FDR-adjusted p=0.0126, respectively). Conclusion: The gut microbiota of CLWH appears similar to that of HUU, and most markers of HIV disease progression are normalized with long-term ART, suggesting a beneficial effect of the latter on the gut microbial ecology. The relationship between exhausted and activated CD4+ T cells and Proteobacteria suggests a connection between the gut microbiome, and premature aging in CLWH.

The vaginal microbiota of women living with HIV on suppressive antiretroviral therapy and its relation to high-risk human papillomavirus infection.

BACKGROUND: Few studies have investigated the vaginal microbiota (VM) in women living with HIV (WLWH) in the context of high-risk human papillomavirus (HR-HPV) infection, even though WLWH are at an increased risk of HPV-related malignancies, including cervical cancer. To explore the impact of HIV and HPV infection on the VM in WLWH, we determined the prevalence of HR-HPV infection and cervical cytologic abnormalities in a cohort of 44 WLWH and 39 seronegative-women (SNW), characterized the vaginal microbiota by 16S sequencing, assessed genital inflammation and systemic immune activation by multiplex bead assay and flow cytometry, respectively. Finally, we explored relationships between bacterial richness and diversity, the top 20 bacterial genera, genital inflammation and systemic immune activation. RESULTS: We found that HR-HPV prevalence was similar between WLWH and SNW. High-grade squamous intraepithelial lesions (HSIL) were only detected in WLWH negative for HR-HPV infection. In regression analyses, no risk factors were identified. Women co-infected with HIV and HR-HPV had the highest level of systemic immune activation, and these levels were significantly different compared with SNW without HR-HPV infection. Lactobacillus iners was the dominant Lactobacillus species in WLWH and SNW alike. CONCLUSION: We found no evidence of differences in vaginal microbial richness and diversity, microbial community structure, and genital inflammation by HIV, HPV, or HIV and HPV status.

Double-Negative T Cell Number and Phenotype Alterations Before and After Effective Antiretroviral Treatment in Persons Living with HIV.

Double-negative (DN) T cells represent a small and phenotypically heterogeneous population that display regulatory functions. In HIV infection, DN T cells are decreased in peripheral blood and have been negatively associated with T cell activation. This study was aimed at describing the dynamics and phenotypic characteristics of DN T cells in peripheral blood of people living with HIV (PLHIV) before and after antiretroviral therapy (ART) initiation. We included 41 newly diagnosed, ART-naive individuals with advanced HIV infection, who were followed up for 6 months after ART initiation. The control group included 34 people without HIV (PWHIV), on preexposure prophylaxis for HIV infection. DN T cells in peripheral blood were characterized by flow cytometry. The absolute counts of DN T cells were lower in PLHIV than in PWHIV (p = 0.0223), and were particularly low in individuals with advanced HIV disease (p = 0.0311). Activation of DN T cells before ART initiation was directly associated with viral load (VL) (p = 0.0081, r = 0.4083) and inversely associated with CD4+ T cell counts (p = 0.0004, r = -0.4041). Compared with PWHIV, DN T cells of PLHIV expressed higher levels of CD57 (p = 0.0019), Ki67 (p = 0.0065), PD-1 (p = 0.0187), and CD38/HLA-DR (p < 0.0001). After 6 months on ART, expression of Ki67, PD-1, and CD38/HLA-DR on DN T cells returned to similar levels to those observed in PWHIV (p > 0.05 in all cases). However, expression of CD57 decreased only in individuals that start ART with high VL (p = 0.0127). DN T cell counts are decreased in HIV infection. Low DN T cell counts remained despite ART-induced immune reconstitution and viremia control. DN T cell phenotype is altered during chronic untreated infection with a high proportion of proliferating, activated, exhausted, and senescent cells. Most markers return to levels similar to those observed in PWHIV after ART. The impact of altered phenotype of DN T and their regulatory functions warrants further exploration.

Mucosal immune cell populations and the bacteriome of adenoids and tonsils from people living with HIV on suppressive antiretroviral therapy.

Ear, nose, and throat (ENT) conditions are prevalent in people living with HIV (PLWH) and occur at all strata of CD4 counts and despite antiretroviral therapy (ART). ENT conditions are underreported in PLWH. Also, little is known about the adenotonsillar microbiota and its relation to resident adaptive and innate immune cells. To bridge this gap, we characterized immune cell populations and the bacterial microbiota of two anatomical sites (adenoids, tonsils) and the oral cavity. Adenoids and tonsils were obtained from PLWH (n = 23) and HIV-seronegative individuals (SN, n = 16) after nasal surgery and tonsillectomy and processed for flow cytometry. Nasopharyngeal, oropharyngeal swabs, and oral rinses were collected prior to surgery for 16S sequencing. Wilcoxon rank sum test, principal coordinate analysis, permutational multivariate analysis of variance, and linear discriminant analysis (LEfSe) were used to assess differences between PLWH and SN. Spearman's correlations were performed to explore interactions between the bacteriome and mucosal immune cells. Of the 39 individuals included, 30 (77%) were men; the median age was 32 years. All PLWH were on ART, with a median CD4 of 723 cells. ENT conditions were classified as inflammatory or obstructive, with no differences observed between PLWH and SN. PLWH had higher frequencies of activated CD4+ and CD8+ T cells, increased T helper (Th)1 and decreased Th2 cells; no differences were observed for B cells and innate immune cells. Alpha diversity was comparable between PLWH and SN at all 3 anatomical sites (adenoids, tonsils, and oral cavity). The impact of HIV infection on the bacterial community structure at each site, as determined by Permutational multivariate analysis of variance, was minor and not significant. Two discriminant genera were identified in adenoids using LEfSe: Staphylococcus for PLWH and Corynebacterium for SN. No discriminant genera were identified in the oropharynx and oral cavity. Niche-specific differences in microbial diversity and communities were observed. PLWH shared less of a core microbiota than SN. In the oropharynx, correlation analysis revealed that Th17 cells were inversely correlated with bacterial richness and diversity, Filifactor, Actinomyces and Treponema; and positively correlated with Streptococcus. Our study contributes toward understanding the role of the adenotonsillar microbiota in the pathophysiology of ENT conditions.

Altered Vaginal Microbiota Composition Correlates With Human Papillomavirus and Mucosal Immune Responses in Women With Symptomatic Cervical Ectopy.

Cervical ectopy is a benign condition of the lower genital tract that is frequently detected in women of reproductive age. Although cervical ectopy is regarded as a physiological condition, some women experience symptoms such as leucorrhoea, persistent bleeding and recurrent vaginal infections that require medical intervention. Cervical ectopy has not been linked to cervical cancer, but it is thought to facilitate the acquisition of sexually transmitted diseases (STDs), like Human Papillomavirus (HPV) infection, as it provides a favorable microenvironment for virus infection and dissemination. We and others have described the presence of oncogenic HPV types in women with symptomatic cervical ectopy. The relevance of this finding and the impact of symptomatic cervical ectopy on the cervicovaginal microenvironment (vaginal microbiota, immune and inflammatory responses) are currently unknown. To shed some light into the interplay between HPV, the vaginal microbiota and mucosal immune and inflammatory responses in the context of this condition, we enrolled 156 women with symptomatic cervical ectopy and determined the presence of HPV using a type-specific multiplex genotyping assay. Overall, HPV was detected in 54.48% women, oncogenic HPV types were found in more than 90% of HPV-positive cases. The most prevalent HPV types were HPV16 (29.4%), HPV31 (21.17%) and HPV18 (15.29%). Next, we evaluated the vaginal microbial composition and diversity by 16S rDNA sequencing, and quantified levels of cytokines and chemokines by flow cytometry using bead-based multiplex assays in a sub-cohort of 63 women. IL-21 and CXCL9 were significantly upregulated in HPV-positive women (p=0.0002 and p=0.013, respectively). Women with symptomatic cervical ectopy and HPV infection had increased diversity (p<0.001), and their vaginal microbiota was enriched in bacterial vaginosis-associated anaerobes (Sneathia, Shuttleworthia, Prevotella, and Atopobium) and depleted in Lactobacillus spp. Furthermore, the vaginal microbiota of women with symptomatic cervical ectopy and HPV infection correlated with vaginal inflammation (IL-1ß, rho=0.56, p=0.0004) and increased mucosal homeostatic response (IL-22, rho=0.60, p=0.0001). Taken together, our results suggest that HPV infection and dysbiotic vaginal communities could favor a vaginal microenvironment that might delay the recovery of the cervical epithelium in women with symptomatic cervical ectopy and favor STDs acquisition.

Protease and gag diversity and drug resistance mutations among treatment-naive Mexican people living with HIV.

INTRODUCTION: In Mexico, HIV genotyping is performed in people living with HIV (PLWH) failing their first-line antiretroviral (ARV) regimen; it is not routinely done for all treatment-naive PLWH before ARV initiation. The first nationally representative survey published in 2016 reported that the prevalence of pretreatment drug mutations in treatment-naive Mexican PLWH was 15.5% to any antiretroviral drug and 10.6% to non-nucleoside reverse transcriptase inhibitors (NNRTIs) using conventional Sanger sequencing. Most reports in Mexico focus on HIV pol gene and nucleoside and non-nucleoside reverse transcriptase inhibitor (NRTI and NNRTI) drug resistance mutations (DRMs) prevalence, using Sanger sequencing, next-generation sequencing (NGS) or both. To our knowledge, NGS has not be used to detect pretreatment drug resistance mutations (DRMs) in the HIV protease (PR) gene and its substrate the Gag polyprotein. METHODS: Treatment-naive adult Mexican PLWH were recruited between 2016 and 2019. HIV Gag and protease sequences were obtained by NGS and DRMs were identified using the WHO surveillance drug resistance mutation (SDRM) list. RESULTS: One hundred PLWH attending a public national reference hospital were included. The median age was 28 years-old, and most were male. The median HIV viral load was 4.99 [4.39-5.40] log copies/mL and median CD4 cell count was 150 [68.0-355.78] cells/mm3. As expected, most sequences clustered with HIV-1 subtype B (97.9%). Major PI resistance mutations were detected: 8 (8.3%) of 96 patients at a detection threshold of 1% and 3 (3.1%) at a detection threshold of 20%. A total of 1184 mutations in Gag were detected, of which 51 have been associated with resistance to PI, most of them were detected at a threshold of 20%. Follow-up clinical data was available for 79 PLWH at 6 months post-ART initiation, seven PLWH failed their first ART regimen; however no major PI mutations were identified in these individuals at baseline. CONCLUSIONS: The frequency of DRM in the HIV protease was 7.3% at a detection threshold of 1% and 3.1% at a detection threshold of 20%. NGS-based HIV drug resistance genotyping provide improved detection of DRMs. Viral load was used to monitor ARV response and treatment failure was 8.9%.

Characterization of CD31 expression in CD4+ and CD8+T cell subpopulations in chronic untreated HIV infection.

BACKGROUND: The platelet endothelial cell adhesion molecule-1 (PECAM-1) or CD31 has been involved in regulation of T-cell tolerance, activation, survival and homing in mice cells. However, there is limited knowledge about the expression pattern and role of this molecule in human T cells, particularly in conditions of chronic immune activation. OBJECTIVES: We explored CD31 expression in T cell differentiation subsets of individuals with untreated HIV infection and in non-HIV-infected controls. We also assessed phenotypic differences between CD31+ and CD31- subsets in memory and terminally differentiated (TEMRA) CD4+ and CD8 + T cells. METHODS: Forty-one individuals with untreated HIV infection and 34 non-HIV-infected controls were included in the study. We compared the expression of CD31 in CD4+ and CD8 + T cells across stages of differentiation in the two study groups by flow cytometry. We also analyzed the expression of CD57 (a marker of senescence), Ki67 (a marker of cycling cells), PD-1 (a marker of exhaustion), and CD38/HLA-DR (a marker of immune activation) on memory and TEMRA CD31+ and CD31- T cells. RESULTS: CD31 expression was significantly higher in CD8 + T cells than in CD4 + T cells, measured as frequency, absolute numbers and median fluorescence intensity (MFI), in both study groups (p < 0.0001 in all cases). Intermediate differentiation subsets of CD4+ and CD8 + T cells expressed higher levels of CD31 in the context of HIV infection (p < 0.001 in all cases). CD31 expression frequency decreased with cellular differentiation of CD4+ and CD8 + T cells in both groups, but this decrease was steeper in individuals without HIV infection (CD4+: p < 0.001 and CD8+: p < 0.0001). As expected, memory and TEMRA CD4+ and CD8 + T cells expressed significantly higher levels of CD57, PD-1, Ki67 and CD38/HLA-DR in HIV-infected compared to non-HIV-infected individuals (p < 0.01 in all cases). CD31 expression was associated with lower activation of memory (but not TEMRA) CD4 + T cells in non-HIV-infected persons, an effect not observed in the HIV-infected group. CD31 expression on memory CD8 + T cells of HIV-infected individuals was associated higher levels of PD-1 (p = 0.0019) and CD38/HLADR (p = 0.0345), and higher PD-1 expression on CD8 + TEMRA (p = 0.0024), an effect not observed in non-HIV-infected individuals. CONCLUSION: In the context of HIV-associated chronic immune activation, specifically on memory CD8 + T cells, CD31 expression was associated with higher PD-1 and CD38/HLA-DR co-expression, suggesting that CD31 expression may result from an insufficient attempt to contain T cell exhaustion and activation. CD31-targeted therapies may contribute to modulate these cellular responses.

Therapeutic avenues for restoring the gut microbiome in HIV infection.

The interplay between the gut microbiota, the intestinal barrier and the mucosal immune system is profoundly altered in Human Immunodeficiency Virus (HIV) infection. An HIV-associated microbial dysbiotic signature has been difficult to define due to the strong impact of confounders that are intimately linked with HIV infection, namely HIV risk behaviors. When controlling for sexual preference and gender, HIV-associated microbial dysbiotic signatures are characterized by an increase in deleterious taxa and a decrease in beneficial bacteria and their respective metabolic end-products. First attempts to restore the gut microbiota of HIV subjects on Antiretroviral Therapy using Fecal Microbiota Transplantation proved to be safe and reported mild transient engraftment of donor microbiota and no effect on markers of HIV disease progression. This review focuses on the current evidence supporting a role for microbial dysbiosis in HIV pathogenesis, and reviews current microbiome-based therapeutics for restoring the gut microbiota in HIV infection.

Associations between recent thymic emigrants and CD4+ T-cell recovery after short-term antiretroviral therapy initiation.

OBJECTIVE: Around 20-30% of HIV-infected individuals (HIV+) on successful antiretroviral therapy (ART) fail to normalize their CD4 T-cell counts. Various factors could contribute to the lack of immune reconstitution, one of them being thymic insufficiency. We aimed to explore associations between recent thymic emigrants (RTEs) and CD4 T-cell recovery. DESIGN: ART-naive HIV+ individuals who started ART with advanced AIDS were selected. Good versus poor immune reconstitution was defined by CD4 gains above or below 100 CD4 T cells/µl. The follow-up period was 6 months. METHODS: Peripheral blood mononuclear cells were isolated and flow cytometry was used to characterize RTEs as the fraction of naive CD4 T cells expressing CD31, the platelet endothelial cell adhesion molecule. Markers of cellular activation, senescence, exhaustion and cycling were also assessed. RESULTS: After 6 months on ART, HIV+ individuals with good immune reconstitution had higher absolute numbers of RTEs, compared with those with poor immune reconstitution, and these strongly correlated with CD4 gains in those individuals with good immune reconstitution but not with poor immune reconstitution. We also found that CD8 T-cell immune activation decreased as early as 2 months post-ART initiation in individuals with good immune reconstitution, but only at month 6 post-ART in individuals with poor immune reconstitution. Levels of immune activation were inversely correlated with the absolute numbers of RTEs in both groups, but more strongly so in individuals with poor immune reconstitution. CONCLUSION: We show that RTEs are linked to CD4 T-cell recovery and that the degree of immune reconstitution is not directly linked to persistent immune activation.