Jason Flamm

Severe CD4+ T-Cell Depletion in Gut Lymphoid Tissue during Primary Human Immunodeficiency Virus Type 1 Infection and Substantial Delay in Restoration following Highly Active Antiretroviral Therapy

ABSTRACT Gut-associated lymphoid tissue (GALT) harbors the majority of T lymphocytes in the body and is an important target for human immunodeficiency virus type 1 (HIV-1). We analyzed longitudinal jejunal biopsy samples from HIV-1-infected patients, during both primary and chronic stages of HIV-1 infection, prior to and following the initiation of highly active antiretroviral therapy (HAART) to determine the onset of CD4+ T-cell depletion and the effect of HAART on the restoration of CD4+ T cells in GALT. Severe depletion of intestinal CD4+ T cells occurred during primary HIV-1 infection. Our results showed that the restoration of intestinal CD4+ T cells following HAART in chronically HIV-1-infected patients was substantially delayed and incomplete. In contrast, initiation of HAART during early stages of infection resulted in near-complete restoration of intestinal CD4+ T cells, despite the delay in comparison to peripheral blood CD4+ T-cell recovery. DNA microarray analysis of gene expression profiles and flow-cytometric analysis of lymphocyte homing and cell proliferation markers demonstrated that cell trafficking to GALT and not local proliferation contributed to CD4+ T-cell restoration. Evaluation of jejunal biopsy samples from long-term HIV-1-infected nonprogressors showed maintenance of normal CD4+ T-cell levels in both GALT and peripheral blood. Our results demonstrate that near-complete restoration of mucosal immune system can be achieved by initiating HAART early in HIV-1 infection. Monitoring of the restoration and/or maintenance of CD4+ T cells in GALT provides a more accurate assessment of the efficacy of antiviral host immune responses as well as HAART.

Viral Suppression and Immune Restoration in the Gastrointestinal Mucosa of Human Immunodeficiency Virus Type 1-Infected Patients Initiating Therapy during Primary or Chronic Infection

ABSTRACT Although the gut-associated lymphoid tissue (GALT) is an important early site for human immunodeficiency virus (HIV) replication and severe CD4+ T-cell depletion, our understanding is limited about the restoration of the gut mucosal immune system during highly active antiretroviral therapy (HAART). We evaluated the kinetics of viral suppression, CD4+ T-cell restoration, gene expression, and HIV-specific CD8+ T-cell responses in longitudinal gastrointestinal biopsy and peripheral blood samples from patients initiating HAART during primary HIV infection (PHI) or chronic HIV infection (CHI) using flow cytometry, real-time PCR, and DNA microarray analysis. Viral suppression was more effective in GALT of PHI patients than CHI patients during HAART. Mucosal CD4+ T-cell restoration was delayed compared to peripheral blood and independent of the time of HAART initiation. Immunophenotypic analysis showed that repopulating mucosal CD4+ T cells were predominantly of a memory phenotype and expressed CD11α, αEβ7, CCR5, and CXCR4. Incomplete suppression of viral replication in GALT during HAART correlated with increased HIV-specific CD8+ T-cell responses. DNA microarray analysis revealed that genes involved in inflammation and cell activation were up regulated in patients who did not replenish mucosal CD4+ T cells efficiently, while expression of genes involved in growth and repair was increased in patients with efficient mucosal CD4+ T-cell restoration. Our findings suggest that the discordance in CD4+ T-cell restoration between GALT and peripheral blood during therapy can be attributed to the incomplete viral suppression and increased immune activation and inflammation that may prevent restoration of CD4+ T cells and the gut microenvironment.

Rapid Onset of Intestinal Epithelial Barrier Dysfunction in Primary Human Immunodeficiency Virus Infection Is Driven by an Imbalance between Immune Response and Mucosal Repair and Regeneration

ABSTRACT Gut-associated lymphoid tissue (GALT) is an early target for human immunodeficiency virus type 1 (HIV-1) infection and is a site for severe CD4+ T-cell depletion. HIV-associated enteropathy is well-documented in chronic HIV-1 infection. However, the initial host responses to HIV infection in GALT and the early molecular correlates of HIV enteropathogenesis have not been characterized during primary HIV infection. In this study, we provide evidence of viral replication in GALT resident CD4+ T cells and macrophages in primary-stage patients and identify early patterns of host mucosal responses and changes in the molecular microenvironment through gene expression profiling. High levels of viral replication in GALT and marked CD4+ T-cell depletion correlated with decreased expression levels of genes regulating epithelial barrier maintenance and digestive/metabolic functions. These changes coincided with a marked increase in the transcription of immune activation-, inflammation-, and apoptosis-associated genes. Our findings indicate that HIV-induced pathogenesis in GALT emerges at both the molecular and cellular levels prior to seroconversion in primary HIV infection, potentially setting the stage for disease progression by impairing the ability to control viral replication and repair and regenerate intestinal mucosal tissues.

Effective CD4+ T-cell restoration in gut-associated lymphoid tissue of HIV-infected patients is associated with enhanced Th17 cells and polyfunctional HIV-specific T-cell responses

Human immunodeficiency virus (HIV) infection leads to severe CD4+ T-cell depletion in gut-associated lymphoid tissue (GALT) that persists despite the initiation of highly active antiretroviral therapy (HAART). It is not known whether restoration of gut mucosal CD4+ T cells and their functions is feasible during therapy and how that relates to immune correlates and viral reservoirs. Intestinal biopsies and peripheral blood samples from HIV-infected patients who were either HAART naive or on long-term HAART were evaluated. Our data demonstrated that gut CD4+ T-cell restoration ranged from modest (<50%) to high (>50%), compared with uninfected controls. Despite persistent CD4+ T-cell proviral burden and residual immune activation in GALT during HAART, effective CD4+ T-cell restoration (>50%) was achieved, which was associated with enhanced Th17 CD4+ T-cell accumulation and polyfunctional anti-HIV cellular responses. Our findings suggest that a threshold of>50% CD4+ T-cell restoration may be sufficient for polyfunctional HIV-specific T cells with implications in the evaluation of vaccines and therapeutics.

HIV-1 Protease and Reverse-Transcriptase Mutations: Correlations with Antiretroviral Therapy in Subtype B Isolates and Implications for Drug-Resistance Surveillance

Background. It is important, for drug-resistance surveillance, to identify human immunodeficiency virus type 1 (HIV-1) strains that have undergone antiretroviral drug selection.Methods. We compared the prevalence of protease and reverse-transcriptase (RT) mutations in HIV-1 sequences from persons with and without previous treatment with protease inhibitors (PIs), nucleoside RT inhibitors (NRTIs), and nonnucleoside RT inhibitors (NNRTIs). Treatment-associated mutations in protease isolates from 5867 persons and RT isolates from 6247 persons were categorized by whether they were polymorphic (prevalence, >0.5%) in untreated individuals and whether they were established drug-resistance mutations. New methods were introduced to minimize misclassification from transmitted resistance, population stratification, sequencing artifacts, and multiple hypothesis testing.Results. Some 36 established and 24 additional nonpolymorphic protease mutations at 34 positions were related to PI treatment, 21 established and 22 additional nonpolymorphic RT mutations at 24 positions with NRTI treatment, and 15 established and 11 additional nonpolymorphic RT mutations at 15 positions with NNRTI treatment. In addition, 11 PI-associated and 1 NRTI-associated established mutations were polymorphic in viruses from untreated persons.Conclusions. Established drug-resistance mutations encompass only a subset of treatment-associated mutations; some of these are polymorphic in untreated persons. In contrast, nonpolymorphic treatment-associated mutations may be more sensitive and specific markers of transmitted HIV-1 drug resistance.

Simplification to rilpivirine/emtricitabine/tenofovir disoproxil fumarate from ritonavir-boosted protease inhibitor antiretroviral therapy in a randomized trial of HIV-1 RNA-suppressed participants.

Objective:To evaluate the efficacy and safety of antiretroviral simplification from a ritonavir-boosted protease inhibitor-based regimen [protease inhibitor+RTV+two nucleos(t)ide reverse transcriptase inhibitors (NRTIs); ≥6 months of exposure prior to study entry with no prior treatment failure] to the single-tablet regimen (STR) rilpivirine/emtricitabine/tenofovir disoproxil fumarate (RPV/FTC/TDF) in virologically suppressed, HIV-1-infected participants. Design:Phase 3b, randomized, open-label, international, 48-week switch study. Methods:Participants were randomized 2 : 1 to switch to RPV/FTC/TDF immediately or stay on their baseline protease inhibitor+RTV+2NRTIs regimen with a delayed switch to RPV/FTC/TDF at week 24. The primary endpoint was noninferiority (12% margin) of RPV/FTC/TDF compared with protease inhibitor+RTV+ two NRTIs in maintaining plasma HIV-1 RNA less than 50 copies/ml at week 24 by Snapshot analysis. Results:A total of 476 participants were randomized and received at least one dose of study drug. Demographics and baseline characteristics were similar between arms. The primary objective of noninferiority at week 24 was met: HIV-1 RNA less than 50 copies/ml by Snapshot analysis, 93.7% of RPV/FTC/TDF versus 89.9% of protease inhibitor+RTV+ two NRTIs (difference 3.8%, 95% confidence interval −1.6 to 9.1%). Through week 48, 89.3% of participants in the immediate switch group maintained virologic suppression. High rates of suppression were maintained with RPV/FTC/TDF regardless of participants pre-antiretroviral HIV-1 RNA level. Overall development of resistance mutations after switching to RPV/FTC/TDF was low. Decreases in total cholesterol, low-density lipoprotein (LDL), and triglycerides were significantly greater among RPV/FTC/TDF recipients than those in the protease inhibitor+RTV+ two NRTIs group. Conclusion:Switching to the STR RPV/FTC/TDF from an RTV-boosted protease inhibitor regimen in virologically suppressed, HIV-1-infected participants maintained virologic suppression with a low risk of virologic failure, while improving total cholesterol, LDL, and triglycerides.

Tenofovir Alafenamide Versus Tenofovir Disoproxil Fumarate in the First Protease Inhibitor-Based Single-Tablet Regimen for Initial HIV-1 Therapy: A Randomized Phase 2 Study.

Objectives:To evaluate the safety and efficacy of the novel tenofovir prodrug, tenofovir alafenamide (TAF), as part of the first protease inhibitor–based single-tablet regimen (STR) for initial treatment of HIV-1 infection. Methods:Antiretroviral therapy (ART)-naive adults with estimated glomerular filtration rate ≥70 mL/min were randomized 2:1 to receive the darunavir/cobicistat/emtricitabine/tenofovir alafenamide (D/C/F/TAF) STR (TAF: N = 103) or darunavir + cobicistat + emtricitabine/tenofovir disoproxil fumarate (TDF: N = 50) once daily with matched placebos for 48 weeks. Results:At week 24, viral suppression (HIV-1 RNA <50 copies/mL) rates were similar (TAF 74.8% vs. TDF 74.0%). At week 48, rates were TAF 76.7% vs. TDF 84.0%; the difference was driven by higher rate of discontinuations in TAF (6.8%) vs. TDF (2%). Among those with virologic failure, none developed resistance. Most adverse events were of mild/moderate severity. The mean change in serum creatinine from baseline at week 48 was 0.06 mg/dL (95% confidence interval: 0.04 to 0.08) for TAF vs. 0.09 mg/dL (95% confidence interval: 0.05 to 0.14) for TDF (P = 0.053). The % change in retinol binding protein/Cr ratio was +9 (TAF) vs. +54 (TDF), P = 0.003; the % change in urine &bgr;-2 microglobulin/Cr ratio was −42.0 (TAF) vs. +2.3 (TDF), P = 0.002. The % change in hip bone mineral density (BMD) was −0.84 (TAF) vs. −3.82 (TDF), P < 0.001 and in spine BMD was −1.57 (TAF) vs. −3.62 (TDF), P = 0.003. There were no fractures in either group. Conclusions:The TAF arm had significantly improved renal and bone safety parameters: less proteinuria and less change in hip and spine BMD, consistent with results from a similarly designed study of the elvitegravir/C/F/TAF STR. This D/C/F/TAF STR offers a promising option for initial HIV treatment, with the high barrier to resistance of darunavir, and the potential for improved long-term renal and bone safety with TAF.

The Gut Mucosal Viral Reservoir in HIV-Infected Patients Is Not the Major Source of Rebound Plasma Viremia following Interruption of Highly Active Antiretroviral Therapy

ABSTRACT Interruption of suppressive highly active antiretroviral therapy (HAART) in HIV-infected patients leads to increased HIV replication and viral rebound in peripheral blood. Effects of therapy interruption on gut-associated lymphoid tissue (GALT) have not been well investigated. We evaluated longitudinal changes in viral replication and emergence of viral variants in the context of T cell homeostasis and gene expression in GALT of three HIV-positive patients who initiated HAART during primary HIV infection but opted to interrupt therapy thereafter. Longitudinal viral sequence analysis revealed that a stable proviral reservoir was established in GALT during primary HIV infection that persisted through early HAART and post-therapy interruption. Proviral variants in GALT and peripheral blood mononuclear cells (PBMCs) displayed low levels of genomic diversity at all times. A rapid increase in viral loads with a modest decline of CD4+ T cells in peripheral blood was observed, while gut mucosal CD4+ T cell loss was severe following HAART interruption. This was accompanied by increased mucosal gene expression regulating interferon (IFN)-mediated antiviral responses and immune activation, a profile similar to those found in HAART-naive HIV-infected patients. Sequence analysis of rebound virus suggested that GALT was not the major contributor to the postinterruption plasma viremia nor were GALT HIV reservoirs rapidly replaced by HIV rebound variants. Our data suggest an early establishment and persistence of viral reservoirs in GALT with minimal diversity. Early detection of and therapy for HIV infection may be beneficial in controlling viral evolution and limiting establishment of diverse viral reservoirs in the mucosal compartment.

Sex differences matter in the gut: effect on mucosal immune activation and inflammation

BackgroundWomen and men have diverse responses to many infectious diseases. These differences are amplified following menopause. However, despite extensive information regarding the effects of sex hormones on immune cells, our knowledge is limited regarding the effects of sex and gender on the function of the mucosal immune system. Sex differences also manifest in the prevalence of gut associated inflammatory and autoimmune disorders, including Crohn’s disease, ulcerative colitis and Celiac disease. It is thus hypothesized that a baseline sex-associated difference in immune activation may predispose women to inflammation-associated disease.MethodsPeripheral blood samples and small intestinal biopsies were obtained from 34 healthy men and women. Immunophenotypic analysis of isolated lymphocytes was performed by flow cytometry. Oligonucleotide analysis was used to study the transcriptional profile in the gut mucosal microenvironment while real-time PCR analysis was utilized to identify differential gene expression in isolated CD4+ T cells. Transcriptional analysis was confirmed by protein expression levels for genes of interest using fluorescent immunohistochemistry. Data was analyzed using the GraphPad software package.ResultsWomen had higher levels of immune activation and inflammation-associated gene expression in gut mucosal samples. CD4+ and CD8+ T cells had a significantly higher level of immune activation-associated phenotype in peripheral blood as well as in gut associated lymphoid tissue along with higher levels of proliferating T cells. CD4+ T cells that showed upregulation of IL1β as well as the TH17 pathway-associated genes contributed a large part of the inflammatory profile.ConclusionIn this study, we demonstrated an upregulation in gene expression related to immune function in the gut microenvironment of women compared to men, in the absence of disease or pathology. Upon closer investigation, CD4+ T cell activation levels were higher in the LPLs in women than in men. Sex differences in the mucosal immune system may predispose women to inflammation-associated diseases that are exacerbated following menopause. Our study highlights the need for more detailed analysis of the effects of sex differences in immune responses at mucosal effector sites.

A randomized trial of the efficacy of group therapy in changing viral load and CD4 counts in individuals living with HIV infection

Objective: This randomized pilot study evaluates whether seropositive patients who are randomly assigned to receive a supportive-expressive group therapy plus education intervention show greater improvements in increased immune function and decreased viral load compared to those randomly assigned to an education-only intervention. Method: Fifty-nine individuals who had been HIV-seropositive for at least 6 months prior to inclusion in the study and had been receiving standard pharmacologic treatment were entered in a prospective randomized trial of the effects of weekly supportive-expressive group therapy on changes in immune status. Participants were matched for AIDS status and sex and randomized to receive weekly sessions of group psychotherapy plus educational materials on HIV/AIDS, or to receive the educational materials alone. Participants were assessed before treatment and then 12 weeks later. Results: Individuals who were randomized to group therapy showed a statistically significant increase in CD4 count and decrease in HIV viral load. Among individuals randomized to the education only condition, no significant change occurred in CD4 count or viral load. Conclusions: These results provide preliminary data suggesting that HIV-seropositive individuals who receive supportive-expressive group psychotherapy may experience concomitant improvements in CD4 cell count and viral load. Further research with a larger sample should examine the possible underlying mechanisms of such benefits.