Lorrie Epling

Valganciclovir Reduces T Cell Activation in HIV-Infected Individuals With Incomplete CD4+ T Cell Recovery on Antiretroviral Therapy

BACKGROUND Elevated immune activation persists during treated human immunodeficiency virus (HIV) infection and is associated with blunted CD4 recovery and premature mortality, but its causes remain incompletely characterized. We hypothesized that asymptomatic cytomegalovirus (CMV) replication might contribute to immune activation in this setting. METHODS Thirty antiretroviral therapy-treated HIV-infected CMV-seropositive participants with CD4 counts <350 cells/mm(3) were randomized to receive valganciclovir 900 mg daily or placebo for 8 weeks, followed by an additional 4-week observation period. The primary outcome was the week 8 change in percentage of activated (CD38(+) HLA-DR(+)) CD8(+) T cells. RESULTS Fourteen participants were randomized to valganciclovir and 16 to placebo. Most participants (21 [70%] of 30) had plasma HIV RNA levels <75 copies/mL. The median CD4 count was 190 (IQR: 134-232) cells/mm(3), and 12 (40%) of 30 had detectable CMV DNA in saliva, plasma, or semen at baseline. CMV DNA continued to be detectable at weeks 4-12 in 7 (44%) of 16 placebo-treated participants, but in none of the valganciclovir-treated participants (P = .007). Valganciclovir-treated participants had significantly greater reductions in CD8 activation at weeks 8 (P = .03) and 12 (P = .02) than did placebo-treated participants. These trends were significant even among those with undetectable plasma HIV RNA levels. CONCLUSIONS CMV (and/or other herpesvirus) replication is a significant cause of immune activation in HIV-infected individuals with incomplete antiretroviral therapy-mediated CD4(+) T cell recovery. CLINICAL TRIALS REGISTRATION NCT00264290.

Differences in HIV Burden and Immune Activation within the Gut of HIV-Positive Patients Receiving Suppressive Antiretroviral Therapy

BACKGROUND The gut is a major reservoir for human immunodeficiency virus (HIV) in patients receiving antiretroviral therapy (ART). We hypothesized that distinct immune environments within the gut may support varying levels of HIV. METHODS In 8 HIV-1-positive adults who were receiving ART and had CD4(+) T cell counts of >200 cells/μL and plasma viral loads of <40 copies/mL, levels of HIV and T cell activation were measured in blood samples and endoscopic biopsy specimens from the duodenum, ileum, ascending colon, and rectum. RESULTS HIV DNA and RNA levels per CD4(+) T cell were higher in all 4 gut sites compared with those in the blood. HIV DNA levels increased from the duodenum to the rectum, whereas the median HIV RNA level peaked in the ileum. HIV DNA levels correlated positively with T cell activation markers in peripheral blood mononuclear cells (PBMCs) but negatively with T cell activation markers in the gut. Multiply spliced RNA was infrequently detected in gut, and ratios of unspliced RNA to DNA were lower in the colon and rectum than in PBMCs, which reflects paradoxically low HIV transcription, given the higher level of T cell activation in the gut. CONCLUSIONS HIV DNA and RNA are both concentrated in the gut, but the inverse relationship between HIV DNA levels and T cell activation in the gut and the paradoxically low levels of HIV expression in the large bowel suggest that different processes drive HIV persistence in the blood and gut. TRIAL REGISTRATION ClinicalTrials.gov identifier: NCT00884793 (PLUS1).

Gut Epithelial Barrier Dysfunction and Innate Immune Activation Predict Mortality in Treated HIV Infection

BACKGROUND While inflammation predicts mortality in treated human immunodeficiency virus (HIV) infection, the prognostic significance of gut barrier dysfunction and phenotypic T-cell markers remains unclear. METHODS We assessed immunologic predictors of mortality in a case-control study within the Longitudinal Study of the Ocular Complications of AIDS (LSOCA), using conditional logistic regression. Sixty-four case patients who died within 12 months of treatment-mediated viral suppression were each matched to 2 control individuals (total number of controls, 128) by duration of antiretroviral therapy-mediated viral suppression, nadir CD4(+) T-cell count, age, sex, and prior cytomegalovirus (CMV) retinitis. A similar secondary analysis was conducted in the SCOPE cohort, which had participants with less advanced immunodeficiency. RESULTS Plasma gut epithelial barrier integrity markers (intestinal fatty acid binding protein and zonulin-1 levels), soluble CD14 level, kynurenine/tryptophan ratio, soluble tumor necrosis factor receptor 1 level, high-sensitivity C-reactive protein level, and D-dimer level all strongly predicted mortality, even after adjustment for proximal CD4(+) T-cell count (all P ≤ .001). A higher percentage of CD38(+)HLA-DR(+) cells in the CD8(+) T-cell population was a predictor of mortality before (P = .031) but not after (P = .10) adjustment for proximal CD4(+) T-cell count. Frequencies of senescent (defined as CD28(-)CD57(+) cells), exhausted (defined as PD1(+) cells), naive, and CMV-specific T cells did not predict mortality. CONCLUSIONS Gut epithelial barrier dysfunction, innate immune activation, inflammation, and coagulation-but not T-cell activation, senescence, and exhaustion-independently predict mortality in individuals with treated HIV infection with a history of AIDS and are viable targets for interventions.

Antiretroviral Therapy Initiated Within 6 Months of HIV Infection Is Associated With Lower T-Cell Activation and Smaller HIV Reservoir Size

Background. CD4(+)/CD8(+) T-cell activation levels often remain elevated in chronic human immunodeficiency virus (HIV) infection despite initiation of antiretroviral therapy (ART). T-cell activation predicts early death and blunted CD4+ T-cell recovery during ART and may affect persistent HIV reservoir size. We investigated whether very early ART initiation is associated with lower on-therapy immune activation and HIV persistence. Methods. From a cohort of patients with early HIV infection (<6 months duration since infection) we identified persons who started ART early (<6 months after infection) or later (≥2 years after infection) and maintained ≥2 years of virologic suppression; at-risk HIV-negative persons were controls. We measured CD4(+)/CD8(+) T-cell activation (percent CD38(+)/HLA-DR(+)) and HIV reservoir size (based on HIV DNA and cell-associated RNA levels). Results. In unadjusted analyses, early ART predicted lower on-therapy CD8(+) T-cell activation (n = 34; mean, 22.1%) than achieved with later ART (n = 32; mean, 28.8%; P = .009), although levels in early ART remained elevated relative to HIV-negative controls (P = .02). Early ART also predicted lower CD4+ T-cell activation than with later ART (5.3% vs 7.5%; P = .06). Early ART predicted 4.8-fold lower DNA levels than achieved with later ART (P = .005), and lower cell-associated RNA levels (difference in signal-to-cutoff ratio (S/Co), 3.2; P = .035). Conclusions. ART initiation <6 months after infection is associated with lower levels of T-cell activation and smaller HIV DNA and RNA reservoir size during long-term therapy.

Growth hormone enhances thymic function in HIV-1–infected adults

Growth hormone (GH) is an underappreciated but important regulator of T cell development that can reverse age-related declines in thymopoiesis in rodents. Here, we report findings of a prospective randomized study examining the effects of GH on the immune system of HIV-1-infected adults. GH treatment was associated with increased thymic mass. In addition, GH treatment enhanced thymic output, as measured by both the frequency of T cell receptor rearrangement excision circles in circulating T cells and the numbers of circulating naive and total CD4(+) T cells. These findings provide compelling evidence that GH induces de novo T cell production and may, accordingly, facilitate CD4(+) T cell recovery in HIV-1-infected adults. Further, these randomized, prospective data have shown that thymic involution can be pharmacologically reversed in humans, suggesting that immune-based therapies could be used to enhance thymopoiesis in immunodeficient individuals.

The HIV-1 reservoir in eight patients on long-term suppressive antiretroviral therapy is stable with few genetic changes over time

Significance Identifying the source and dynamics of persistent HIV-1 during combinational antiretroviral therapy (cART) is crucial for understanding the barriers to curing HIV infection. Through genetic characterization of HIV-1 DNA in infected cells from peripheral blood and gut-associated lymphoid tissue from patients after long-term suppressive cART, our study reveals that the primary barrier to a cure is a remarkably stable pool of infected memory CD4+ T cells. Through in-depth phylogenetic analyses, we determined that the HIV-1 reservoir in these cells from eight patients is kept stable during long-term cART and, with little evidence of viral replication, this population could be maintained by homeostatic cell proliferation or other processes. The source and dynamics of persistent HIV-1 during long-term combinational antiretroviral therapy (cART) are critical to understanding the barriers to curing HIV-1 infection. To address this issue, we isolated and genetically characterized HIV-1 DNA from naïve and memory T cells from peripheral blood and gut-associated lymphoid tissue (GALT) from eight patients after 4–12 y of suppressive cART. Our detailed analysis of these eight patients indicates that persistent HIV-1 in peripheral blood and GALT is found primarily in memory CD4+ T cells [CD45RO+/CD27(+/−)]. The HIV-1 infection frequency of CD4+ T cells from peripheral blood and GALT was higher in patients who initiated treatment during chronic compared with acute/early infection, indicating that early initiation of therapy results in lower HIV-1 reservoir size in blood and gut. Phylogenetic analysis revealed an HIV-1 genetic change between RNA sequences isolated before initiation of cART and intracellular HIV-1 sequences from the T-cell subsets after 4–12 y of suppressive cART in four of the eight patients. However, evolutionary rate analyses estimated no greater than three nucleotide substitutions per gene region analyzed during all of the 4–12 y of suppressive therapy. We also identified a clearly replication-incompetent viral sequence in multiple memory T cells in one patient, strongly supporting asynchronous cell replication of a cell containing integrated HIV-1 DNA as the source. This study indicates that persistence of a remarkably stable population of infected memory cells will be the primary barrier to a cure, and, with little evidence of viral replication, this population could be maintained by homeostatic cell proliferation or other processes.

The Distribution of HIV DNA and RNA in Cell Subsets Differs in Gut and Blood of HIV-Positive Patients on ART: Implications for Viral Persistence

Even with optimal antiretroviral therapy, human immunodeficiency virus (HIV) persists in plasma, blood cells, and tissues. To develop new therapies, it is essential to know what cell types harbor residual HIV. We measured levels of HIV DNA, RNA, and RNA/DNA ratios in sorted subsets of CD4+ T cells (CCR7+, transitional memory, and effector memory) and non-CD4+ T leukocytes from blood, ileum, and rectum of 8 ART-suppressed HIV-positive subjects. Levels of HIV DNA/million cells in CCR7+ and effector memory cells were higher in the ileum than blood. When normalized by cell frequencies, most HIV DNA and RNA in the blood were found in CCR7+ cells, whereas in both gut sites, most HIV DNA and RNA were found in effector memory cells. HIV DNA and RNA were observed in non-CD4+ T leukocytes at low levels, particularly in gut tissues. Compared to the blood, the ileum had higher levels of HIV DNA and RNA in both CD4+ T cells and non-CD4+ T leukocytes, whereas the rectum had higher HIV DNA levels in both cell types but lower RNA levels in CD4+ T cells. Future studies should determine whether different mechanisms allow HIV to persist in these distinct reservoirs, and the degree to which different therapies can affect each reservoir.

Hematopoietic Precursor Cells Isolated From Patients on Long-term Suppressive HIV Therapy Did Not Contain HIV-1 DNA

BACKGROUND We address the key emerging question of whether Lin(-)/CD34(+) hematopoietic precursor cells (HPCs) represent an important latent reservoir of human immunodeficiency virus type 1 (HIV-1) during long-term suppressive therapy. METHODS To estimate the frequency of HIV-1 infection in bone marrow, we sorted Lin(-)/CD34(+) HPCs and 3 other cell types (Lin(-)/CD34(-), Lin(-)/CD4(+), and Lin(+)/CD4(+)) from 8 patients who had undetectable viral loads for 3-12 years. Using a single-proviral sequencing method, we extracted, amplified, and sequenced multiple single HIV-1 DNA molecules from these cells and memory CD4(+) T cells from contemporaneous peripheral blood samples. RESULTS We analyzed 100,000-870,000 bone marrow Lin(-)/CD34(+) HPCs from the 8 patients and found no HIV-1 DNA. We did isolate HIV-1 DNA from their bone marrow Lin(+)/CD4(+) cells that was genetically similar to HIV-1 DNA from lymphoid cells located in the peripheral blood, indicating an exchange of infected cells between these compartments. CONCLUSIONS The absence of infected HPCs provides strong evidence that the HIV-1 infection frequency of Lin(-)/CD34(+) HPCs from bone marrow, if it occurred, was <.003% (highest upper 95% confidence interval) in all 8 patients. These results strongly suggest that Lin(-)/CD34(+) HPCs in bone marrow are not a source of persistent HIV-1 in patients on long-term suppressive therapy.

CD4+ T Cells Expressing PD-1, TIGIT and LAG-3 Contribute to HIV Persistence during ART

HIV persists in a small pool of latently infected cells despite antiretroviral therapy (ART). Identifying cellular markers expressed at the surface of these cells may lead to novel therapeutic strategies to reduce the size of the HIV reservoir. We hypothesized that CD4+ T cells expressing immune checkpoint molecules would be enriched in HIV-infected cells in individuals receiving suppressive ART. Expression levels of 7 immune checkpoint molecules (PD-1, CTLA-4, LAG-3, TIGIT, TIM-3, CD160 and 2B4) as well as 4 markers of HIV persistence (integrated and total HIV DNA, 2-LTR circles and cell-associated unspliced HIV RNA) were measured in PBMCs from 48 virally suppressed individuals. Using negative binomial regression models, we identified PD-1, TIGIT and LAG-3 as immune checkpoint molecules positively associated with the frequency of CD4+ T cells harboring integrated HIV DNA. The frequency of CD4+ T cells co-expressing PD-1, TIGIT and LAG-3 independently predicted the frequency of cells harboring integrated HIV DNA. Quantification of HIV genomes in highly purified cell subsets from blood further revealed that expressions of PD-1, TIGIT and LAG-3 were associated with HIV-infected cells in distinct memory CD4+ T cell subsets. CD4+ T cells co-expressing the three markers were highly enriched for integrated viral genomes (median of 8.2 fold compared to total CD4+ T cells). Importantly, most cells carrying inducible HIV genomes expressed at least one of these markers (median contribution of cells expressing LAG-3, PD-1 or TIGIT to the inducible reservoir = 76%). Our data provide evidence that CD4+ T cells expressing PD-1, TIGIT and LAG-3 alone or in combination are enriched for persistent HIV during ART and suggest that immune checkpoint blockers directed against these receptors may represent valuable tools to target latently infected cells in virally suppressed individuals.

Impact of HIV on CD8+ T cell CD57 expression is distinct from that of CMV and aging

Background Chronic antigenic stimulation by cytomegalovirus (CMV) is thought to increase “immunosenesence” of aging, characterized by accumulation of terminally differentiated CD28- CD8+ T cells and increased CD57, a marker of proliferative history. Whether chronic HIV infection causes similar effects is currently unclear. Methods We compared markers of CD8+ T cell differentiation (e.g., CD28, CD27, CCR7, CD45RA) and CD57 expression on CD28- CD8+ T cells in healthy HIV-uninfected adults with and without CMV infection and in both untreated and antiretroviral therapy (ART)-suppressed HIV-infected adults with asymptomatic CMV infection. Results Compared to HIV-uninfected adults without CMV (n = 12), those with asymptomatic CMV infection (n = 31) had a higher proportion of CD28-CD8+ T cells expressing CD57 (P = 0.005). Older age was also associated with greater proportions of CD28-CD8+ T cells expressing CD57 (rho: 0.47, P = 0.007). In contrast, untreated HIV-infected CMV+ participants (n = 55) had much lower proportions of CD28- CD8+ cells expressing CD57 than HIV-uninfected CMV+ participants (P<0.0001) and were enriched for less well-differentiated CD28- transitional memory (TTR) CD8+ T cells (P<0.0001). Chronically HIV-infected adults maintaining ART-mediated viral suppression (n = 96) had higher proportions of CD28-CD8+ T cells expressing CD57 than untreated patients (P<0.0001), but continued to have significantly lower levels than HIV-uninfected controls (P = 0.001). Among 45 HIV-infected individuals initiating their first ART regimen, the proportion of CD28-CD8+ T cells expressing CD57 declined (P<0.0001), which correlated with a decline in percent of transitional memory CD8+ T cells, and appeared to be largely explained by a decline in CD28-CD57- CD8+ T cell counts rather than an expansion of CD28-CD57+ CD8+ T cell counts. Conclusions Unlike CMV and aging, which are associated with terminal differentiation and proliferation of effector memory CD8+ T cells, HIV inhibits this process, expanding less well-differentiated CD28- CD8+ T cells and decreasing the proportion of CD28- CD8+ T cells that express CD57.