Sandrina DaFonseca

Interleukin-7 promotes HIV persistence during antiretroviral therapy

HIV persists in latently infected memory CD4(+) T cells during antiretroviral therapy (ART). When administered to HIV-infected subjects receiving suppressive ART, interleukin-7 (IL-7) increases the number of CD4(+) T cells by promoting their survival and proliferation. However, little is known about the impact of IL-7 on HIV persistence during ART. By isolating large numbers of CD4(+) T cells from HIV-infected subjects, we demonstrate that IL-7 enhances viral production in productively infected cells but does not disrupt viral latency in latently infected cells. When administered to virally suppressed subjects, IL-7 led to the rapid proliferation of memory CD4(+) T cells, which resulted in a 70% increase in the absolute number of circulating CD4(+) T cells harboring integrated HIV DNA 4 weeks after therapy. The genetic diversity of the viral reservoir increased transiently in the majority of the subjects studied before returning to baseline values. Altogether, our results indicate that IL-7 promotes the mechanisms of HIV persistence during ART by enhancing residual levels of viral production and inducing proliferation of latently infected cells, and suggest that IL-7 does not represent a suitable candidate therapeutic strategy for HIV eradication. This trial was registered at www.clinicaltrials.gov as #NCT00099671 (AIDS Clinical Trials Group protocol 5214).

Loss of memory B cells during chronic HIV infection is driven by Foxo3a- and TRAIL-mediated apoptosis

Loss of memory B cells occurs from the onset of HIV-1 infection and persists into the chronic stages of infection. Lack of survival of these cells, even in subjects being treated, could primarily be the consequence of an altered local microenvironment induced by HIV infection. In this study we showed that memory B cell survival was significantly decreased in aviremic successfully treated (ST) subjects compared with subjects who control viral load as a result of natural immunity (elite controller [EC]) or with uninfected control (HIV-) subjects. The lower survival levels observed in memory B cells from ST subjects were the result of disrupted IL-2 signaling that led to increased transcriptional activity of Foxo3a and increased expression of its proapoptotic target TRAIL. Notably, memory B cell survival in ST subjects was significantly enhanced by the addition of exogenous IL-2 in a Foxo3a-dependent manner. We further showed that Foxo3a silencing by siRNA resulted in decreased expression of TRAIL and apoptosis levels in memory B cells from ST subjects. Our results thus establish a direct role for Foxo3a/TRAIL signaling in the persistence of memory B cells and provide a mechanism for the reduced survival of memory B cells during HIV infection. This knowledge could be exploited for the development of therapeutic and preventative HIV vaccines.

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.

Cross-Clade Ultrasensitive PCR-Based Assays To Measure HIV Persistence in Large-Cohort Studies

ABSTRACT A small pool of infected cells persists in HIV-infected individuals receiving antiretroviral therapy (ART). Here, we developed ultrasensitive assays to precisely measure the frequency of cells harboring total HIV DNA, integrated HIV DNA, and two long terminal repeat (2-LTR) circles. These assays are performed on cell lysates, which circumvents the labor-intensive step of DNA extraction, and rely on the coquantification of each HIV molecular form together with CD3 gene sequences to precisely measure cell input. Using primary isolates from HIV subtypes A, B, C, D, and CRF01_A/E, we demonstrate that these assays can efficiently quantify low target copy numbers from diverse HIV subtypes. We further used these assays to measure total HIV DNA, integrated HIV DNA, and 2-LTR circles in CD4+ T cells from HIV-infected subjects infected with subtype B. All samples obtained from ART-naive subjects were positive for the three HIV molecular forms (n = 15). Total HIV DNA, integrated HIV DNA, and 2-LTR circles were detected in, respectively, 100%, 94%, and 77% of the samples from individuals in which HIV was suppressed by ART. Higher levels of total HIV DNA and 2-LTR circles were detected in untreated subjects than individuals on ART (P = 0.0003 and P = 0.0004, respectively), while the frequency of CD4+ T cells harboring integrated HIV DNA did not differ between the two groups. These results demonstrate that these novel assays have the ability to quantify very low levels of HIV DNA of multiple HIV subtypes without the need for nucleic acid extraction, making them well suited for the monitoring of viral persistence in large populations of HIV-infected individuals. IMPORTANCE Since the discovery of viral reservoirs in HIV-infected subjects receiving suppressive ART, measuring the degree of viral persistence has been one of the greatest challenges in the field of HIV research. Here, we report the development and validation of ultrasensitive assays to measure HIV persistence in HIV-infected individuals from multiple geographical regions. These assays are relatively inexpensive, do not require DNA extraction, and can be completed in a single day. Therefore, they are perfectly adapted to monitor HIV persistence in large cohorts of HIV-infected individuals and, given their sensitivity, can be used to monitor the efficacy of therapeutic strategies aimed at interfering with HIV persistence after prolonged ART.

Gold drug auranofin restricts the viral reservoir in the monkey AIDS model and induces containment of viral load following ART suspension.

Objectives:A small pool of long-lived memory CD4+ T cells harboring the retroviral genome is one main obstacle to HIV eradication. We tested the impact of the gold compound, auranofin, on phenotype and viability of CD4+ T cells in vitro, and on persistence of lentiviral reservoir cells in vivo. Design:In-vitro and in-vivo study. The pro-differentiating effect of auranofin was investigated in human primary CD4+ T cells, and its capacity to deplete the viral DNA (vDNA) reservoir was tested in a pilot study involving six SIVmac251-infected macaques with viral loads stably suppressed by antiretroviral therapy (ART) (tenofovir/emtricitabine/raltegravir). The study was then amplified by intensifying ART using darunavir/r and including controls under intensified ART alone. All therapies were eventually suspended and viro-immunological parameters were monitored over time. Methods:Cell subpopulations were quantitated by flow cytometry following proper hematological analyses. Viral load and cell-associated vDNA were quantitated by Taqman real-time PCR. Results:In naïve, central memory and transitional memory CD4+ T cells, auranofin induced both phenotype changes and cell death which were more pronounced in the memory compartment. In the pilot study in vivo, auranofin transiently decreased the cell-associated vDNA reservoir in peripheral blood. When ART was intensified, a sustained decrease in vDNA was observed only in auranofin-treated monkeys but not in controls treated with intensified ART alone. After therapy suspension, only monkeys that had received auranofin showed a deferred and subsequently blunted viral load rebound. Conclusion:These findings represent a first step towards a remission of primate lentiviral infections.

Maintenance of CD4+ T-cell memory and HIV persistence: keeping memory, keeping HIV.

Purpose of reviewThe present review summarizes the current challenges for the design of new therapeutic strategies toward HIV eradication in individuals receiving suppressive highly active antiretroviral therapy (HAART). We will focus on the experimental evidence suggesting that immunological mechanisms involved in the generation and maintenance of memory CD4+ T cells are also responsible for the establishment and persistence of a stable reservoir for HIV. Recent findingsRecent studies performed on clinical samples obtained from virally suppressed HIV-infected individuals indicate that T-cell survival and homeostatic proliferation, two major mechanisms involved in the maintenance of immunological memory, contribute to the persistence of latently infected memory CD4+ T cells. Thus, the long lifespan characteristic of the HIV reservoir is likely a consequence of the capacity of the immune system to generate and maintain memory CD4+ T cells for a long period. SummaryThese findings suggest that strategies aimed at reducing the pool of latently infected cells should interfere with the survival pathways responsible for the long-term maintenance of memory CD4+ T cells. Because memory CD4+ T cells are critical for appropriate immune defense, targeted approaches are needed to interfere only with the long-term survival of discrete fractions of memory T cells carrying proviral DNA.

Foxo3a: An integrator of immune dysfunction during HIV infection

Chronic HIV infection, which is primarily characterized by the progressive depletion of total CD4(+) T cells, also causes persistent inflammation and immune activation. This is followed by profound changes in cellular and tissue microenvironments that often lead to prolonged immune dysfunction. The global nature of this immune dysfunction suggests that factors that are involved in immune cell survival, proliferation, differentiation and maturation are all affected. Of particular interest is the transcriptional factor Foxo3a that regulates a number of genes that are critical in the development and the maintenance of T and B cells, dendritic cells (DCs) and macrophages. Alterations in the microenvironment mediated by HIV infection cause significant increase in the transcriptional activity of Foxo3a; this has major impact on T cell and B cell immunity. In fact, recent findings from HIV infected individuals highlight three important points: (1) the alteration of Foxo3a signaling during HIV infection deregulates innate and adaptive immune responses; (2) Foxo3a-mediated effects are reversible and could be restored by interfering with the Foxo3a pathway; and (3) down-regulation of Foxo3a transcriptional activity in elite controllers (ECs) represents a molecular signature, or a correlate of immunity, associated with natural protection and lack of disease progression. In this review, we will discuss how HIV-infection altered microenvironments could result in impaired immune responses via the Foxo3a signaling pathway. Defining precisely the molecular mechanisms of how persistent inflammation and immune activation are able to influence the Foxo3a pathway could ultimately help in the development of novel approaches to improve immune responses in HIV infected subjects.

Nef promotes evasion of human immunodeficiency virus type 1-infected cells from the CTLA-4-mediated inhibition of T-cell activation

CTLA-4 is a negative regulator of T-cell receptor-mediated CD4(+) T-cell activation and function. Upregulation of CTLA-4 during human immunodeficiency virus type 1 (HIV-1) infection on activated T cells, particularly on HIV-specific CD4(+) T cells, correlates with immune dysfunction and disease progression. As HIV-1 infects and replicates in activated CD4(+) T cells, we investigated mechanisms by which HIV-1 modulates CTLA-4 expression to establish productive viral infection in these cells. Here, we demonstrate that HIV-1 infection in activated CD4(+) T cells was followed by Nef-mediated downregulation of CTLA-4. This was associated with a decreased T-cell activation threshold and significant resistance to CTLA-4 triggering. In line with these in vitro results, quantification of pro-viral HIV DNA from treatment-naive HIV-infected subjects demonstrated a preferential infection of memory CD4(+)CTLA-4(+) T cells, thus identifying CTLA-4 as a biomarker for HIV-infected cells in vivo. As transcriptionally active HIV-1 and Nef expression in vivo were previously shown to take place mainly in the CD3(+)CD4(-)CD8(-) [double-negative (DN)] cells, we further quantified HIV DNA in the CTLA-4(+) and CTLA-4(-) subpopulations of these cells. Our results showed that DN T cells lacking CTLA-4 expression were enriched in HIV DNA compared with DN CTLA-4(+) cells. Together, these results suggested that HIV-1 preferential infection of CD4(+)CTLA-4(+) T cells in vivo was followed by Nef-mediated concomitant downregulation of both CD4 and CTLA-4 upon transition to productive infection. This also highlights the propensity of HIV-1 to evade restriction of the key negative immune regulator CTLA-4 on cell activation and viral replication, and therefore contributes to the overall HIV-1 pathogenesis.