Circulating monocytes are not a major reservoir of HIV-1 in elite suppressors

Circulating HIV-1-infected monocytes have been identified in patients on highly active antiretroviral therapy and may represent an important barrier to viral eradication. The nature of these cells in HIV-1-infected patients who maintain undetectable viral loads and preserved CD4(+) T cell counts without antiretroviral therapy (known as elite controllers or elite suppressors [ES]) is unknown. We describe here infrequent recovery of proviral HIV-1 DNA from circulating monocytes relative to CD4(+) T cells in ES, despite permissiveness of these cells to HIV-1 viral entry ex vivo. Thus, monocytes do not appear to be a major reservoir of HIV-1 in ES.     

Neutralizing antibodies do not mediate suppression of human immunodeficiency virus type 1 in elite suppressors or selection of plasma virus variants in patients on highly active antiretroviral therapy

Neutralizing antibodies (NAb) against autologous virus can reach high titers in human immunodeficiency virus type 1 (HIV-1)-infected patients with progressive disease. Less is known about the role of NAb in HIV-1-infected patients with viral loads of <50 copies/ml of plasma, including patients on effective highly active antiretroviral therapy (HAART) and elite suppressors, who control HIV-1 replication without antiretroviral therapy. In this study, we analyzed full-length env sequences from plasma viruses and proviruses in resting CD4(+) T cells of HAART-treated patients, elite suppressors, and untreated HIV-1-infected patients with progressive disease. For each patient group, we assessed plasma virus neutralization by autologous, contemporaneous plasma. The degree of env diversity, the number of N-linked glycosylation sites, and the lengths of variable loops were all lower in elite suppressors than in HAART-treated and untreated viremic patients. Both elite suppressors and HAART-treated patients had lower titers of NAb against HIV-1 lab strains than those of untreated viremic patients. Surprisingly, titers of NAb against autologous, contemporaneous plasma viruses were similarly low in chronic progressors, elite suppressors, and HAART-treated patients. In elite suppressors and HAART-treated patients, titers of NAb against autologous plasma viruses also did not differ significantly from titers against autologous proviruses from resting CD4(+) T cells. These results suggest that high-titer NAb are not required for maintenance of viral suppression in elite suppressors and that NAb do not select plasma virus variants in most HAART-treated patients. Both drug-mediated and natural suppression of HIV-1 replication to levels below 50 copies/ml may limit the stimulation and maintenance of effective NAb responses.     

Differential Expression of CD96 Surface Molecule Represents CD8+ T Cells with Dissimilar Effector Function during HIV-1 Infection

During HIV-1 infection, immune dysregulation and aberrant lymphocyte functions are well-established characteristics. Cell surface molecules are important for immunological functions and changes in expression can affect lymphocyte effector functions, thereby contributing to pathogenesis and disease progression. In this study we have focused on CD96, a member of the IgG superfamily receptors that have generated increasing recent interest due to their adhesive and co-stimulatory functions in addition to immunoregulatory capacity. CD96 is expressed by both T and NK cells. Although the function of CD96 is not completely elucidated, it has been shown to have adhesive functions and enhance cytotoxicity. Interestingly, CD96 may also have inhibitory functions due to its immunoreceptor tyrosine-based inhibitory motif (ITIM). The clinical significance of CD96 is still comparatively limited although it has been associated with chronic Hepatitis B infection and disease progression. CD96 has not previously been studied in the context of HIV-1 infection, but due to its potential importance in immune regulation and relevance to chronic disease, we examined CD96 expression in relation to HIV-1 pathogenesis. In a cross-sectional analysis, we investigated the CD8⁺ T cell expression of CD96 in cohorts of untreated HIV-1 infected adults with high viral loads (non-controllers) and low viral loads (“elite” controllers). We demonstrated that elite controllers have significantly higher CD96 mean fluorescence intensity on CD8⁺ T cells compared to HIV-1 non-controllers and CD96 expression was positively associated with CD4⁺ T cell counts. Functional assessment showed that CD8⁺ T cells lacking CD96 expression represented a population that produced both perforin and IFN-γ following stimulation. Furthermore, CD96 expression on CD8⁺ T cells was decreased in presence of lipopolysaccharide in vitro. Overall, these findings indicate that down-regulation of CD96 is an important aspect of HIV-1 pathogenesis and differential expression is related to cell effector functions and HIV-1 disease course.     

Immune and viral correlates of "secondary viral control" after treatment interruption in chronically HIV-1 infected patients

Upon interruption of antiretroviral therapy, HIV-infected patients usually show viral load rebound to pre-treatment levels. Four patients, hereafter referred to as secondary controllers (SC), were identified who initiated therapy during chronic infection and, after stopping treatment, could control virus replication at undetectable levels for more than six months. In the present study we set out to unravel possible viral and immune parameters or mechanisms of this phenomenon by comparing secondary controllers with elite controllers and non-controllers, including patients under HAART. As candidate correlates of protection, virus growth kinetics, levels of intracellular viral markers, several aspects of HIV-specific CD4+ and CD8+ T cell function and HIV neutralizing antibodies were investigated. As expected all intracellular viral markers were lower in aviremic as compared to viremic subjects, but in addition both elite and secondary controllers had lower levels of viral unspliced RNA in PBMC as compared to patients on HAART. Ex vivo cultivation of the virus from CD4+ T cells of SC consistently failed in one patient and showed delayed kinetics in the three others. Formal in vitro replication studies of these three viruses showed low to absent growth in two cases and a virus with normal fitness in the third case. T cell responses toward HIV peptides, evaluated in IFN-γ ELISPOT, revealed no significant differences in breadth, magnitude or avidity between SC and all other patient groups. Neither was there a difference in polyfunctionality of CD4+ or CD8+ T cells, as evaluated with intracellular cytokine staining. However, secondary and elite controllers showed higher proliferative responses to Gag and Pol peptides. SC also showed the highest level of autologous neutralizing antibodies. These data suggest that higher T cell proliferative responses and lower replication kinetics might be instrumental in secondary viral control in the absence of treatment.     

HLA class I-restricted T-cell responses may contribute to the control of human immunodeficiency virus infection, but such responses are not always necessary for long-term virus control

A rare subset of human immunodeficiency virus (HIV)-infected individuals maintains undetectable HIV RNA levels without therapy ("elite controllers"). To clarify the role of T-cell responses in mediating virus control, we compared HLA class I polymorphisms and HIV-specific T-cell responses among a large cohort of elite controllers (HIV-RNA < 75 copies/ml), "viremic" controllers (low-level viremia without therapy), "noncontrollers" (high-level viremia), and "antiretroviral therapy suppressed" individuals (undetectable HIV-RNA levels on antiretroviral therapy). The proportion of CD4(+) and CD8(+) T cells that produce gamma interferon (IFN-gamma) and interleukin-2 (IL-2) in response to Gag and Pol peptides was highest in the elite and viremic controllers (P < 0.0001). Forty percent of the elite controllers were HLA-B*57 compared to twenty-three percent of viremic controllers and nine percent of noncontrollers (P < 0.001). Other HLA class I alleles more common in elite controllers included HLA-B*13, HLA-B*58, and HLA-B*81 (P < 0.05 for each). Within elite and viremic controller groups, those with protective class I alleles had higher frequencies of Gag-specific CD8(+) T cells than those without these alleles (P = 0.01). Noncontrollers, with or without protective alleles, had low-level CD8(+) responses. Thus, certain HLA class I alleles are enriched in HIV controllers and are associated with strong Gag-specific CD8(+)IFN-gamma(+)IL-2(+) T cells. However, the absence of evidence of T cell-mediated control in many controllers suggests the presence of alternative mechanisms for viral control in these individuals. Defining mechanisms for virus control in "non-T-cell controllers" might lead to insights into preventing HIV transmission or preventing virus replication.     

Autophagy plays an important role in the containment of HIV-1 in nonprogressor-infected patients

Recent in vitro studies have suggested that autophagy may play a role in both HIV-1 replication and disease progression. In this study we investigated whether autophagy protects the small proportion of HIV-1 infected individuals who remain clinically stable for years in the absence of antiretroviral therapy, these named long-term nonprogressors (LTNP) and elite controllers (EC). We found that peripheral blood mononuclear cells (PBMC) of the HIV-1 controllers present a significantly higher amount of autophagic vesicles associated with an increased expression of autophagic markers with respect to normal progressors. Of note, ex vivo treatment of PBMC from the HIV-1 controllers with the MTOR inhibitor rapamycin results in a more efficient autophagic response, leading to a reduced viral production. These data lead us to propose that autophagy contributes to limiting viral pathogenesis in HIV-1 controllers by targeting viral components for degradation.     

Processing and presentation of exogenous HLA class I peptides by dendritic cells from human immunodeficiency virus type 1-infected persons

Dendritic cells (DCs) loaded with viral peptides are a potential form of immunotherapy of human immunodeficiency virus type 1 (HIV-1) infection. We show that DCs derived from blood monocytes of subjects with chronic HIV-1 infection on combination antiretroviral drug therapy have increases in expression of HLA, T-cell coreceptor, and T-cell activation molecules in response to the DC maturation factor CD40L comparable to those from uninfected persons. Mature DCs (mDCs) loaded with HLA A*0201-restricted viral peptides of the optimal length (9-mer) were more efficient at activating antiviral CD8(+) T cells than were immature DCs or peptide alone. Optimal presentation of these exogenous peptides required uptake and vesicular trafficking and was comparable in DCs derived from HIV-1-infected and uninfected persons. Furthermore, DCs from HIV-1-infected and uninfected persons had similar capacities to process viral peptides with C-terminal and N-terminal extensions through their proteasomal and cytosolic pathways, respectively. We conclude that DCs derived from HIV-1-infected persons have similar abilities to process exogenous peptides for presentation to CD8(+) T cells as those from uninfected persons. This conclusion supports the use of DCs loaded with synthetic peptides in immunotherapy of HIV-1 infection.     

A Correlate of HIV-1 Control Consisting of Both Innate and Adaptive Immune Parameters Best Predicts Viral Load by Multivariable Analysis in HIV-1 Infected Viremic Controllers and Chronically-Infected Non-Controllers

HIV-1 infected viremic controllers maintain durable viral suppression below 2000 copies viral RNA/ml without anti-retroviral therapy (ART), and the immunological factor(s) associated with host control in presence of low but detectable viral replication are of considerable interest. Here, we utilized a multivariable analysis to identify which innate and adaptive immune parameters best correlated with viral control utilizing a cohort of viremic controllers (median 704 viral RNA/ml) and non-controllers (median 21,932 viral RNA/ml) that were matched for similar CD4⁺ T cell counts in the absence of ART. We observed that HIV-1 Gag-specific CD8⁺ T cell responses were preferentially targeted over Pol-specific responses in viremic controllers (p = 0.0137), while Pol-specific responses were positively associated with viral load (rho = 0.7753, p = 0.0001, n = 23). Viremic controllers exhibited significantly higher NK and plasmacytoid dendritic cells (pDC) frequency as well as retained expression of the NK CD16 receptor and strong target cell-induced NK cell IFN-gamma production compared to non-controllers (p<0.05). Despite differences in innate and adaptive immune function however, both viremic controllers (p<0.05) and non-controller subjects (p<0.001) exhibited significantly increased CD8⁺ T cell activation and spontaneous NK cell degranulation compared to uninfected donors. Overall, we identified that a combination of innate (pDC frequency) and adaptive (Pol-specific CD8⁺ T cell responses) immune parameters best predicted viral load (R² = 0.5864, p = 0.0021, n = 17) by a multivariable analysis. Together, this data indicates that preferential Gag-specific over Pol-specific CD8⁺ T cell responses along with a retention of functional innate subsets best predict host control over viral replication in HIV-1 infected viremic controllers compared to chronically-infected non-controllers.     

Study on immunological status of Chinese HIV-infected individuals

HIV-1 infection is characterized by a gradual decline of immune function, and the immune dysfunction is widely regarded as one of the most important determinants of disease progression. The present study was performed to analyze in more detail the immunological status of HIV-infected people in China. T cell counts, activation of T cells, HIV-1 specific CTL and plasma levels of cytokines were determined with flow cytometry, IFN-gamma Elispot or ELISA techniques. The HIV viral load was negatively correlated with CD4(+), CD8(+) T cell counts (r=-0.654, P<0.001; r=-0.228, P<0.05); the breadth and magnitude of HIV-1 specific CTL responses against HIV-1 Gag peptides was related to disease progression; the activation of CD8(+) T cells was significantly higher than that in HIV-negative controls; the level of plasma IL-12 was much lower and the plasma IFN-gamma, IL-10 and IL-6 were much higher in HIV-infected persons than in HIV-negative controls (P<0.05). Study on immunological status in HIV-infected Chinese is very important in predicting the disease progression and providing information for HAART therapy in China.     

T cell responses to human endogenous retroviruses in HIV-1 infection

Human endogenous retroviruses (HERVs) are remnants of ancient infectious agents that have integrated into the human genome. Under normal circumstances, HERVs are functionally defective or controlled by host factors. In HIV-1-infected individuals, intracellular defense mechanisms are compromised. We hypothesized that HIV-1 infection would remove or alter controls on HERV activity. Expression of HERV could potentially stimulate a T cell response to HERV antigens, and in regions of HIV-1/HERV similarity, these T cells could be cross-reactive. We determined that the levels of HERV production in HIV-1-positive individuals exceed those of HIV-1-negative controls. To investigate the impact of HERV activity on specific immunity, we examined T cell responses to HERV peptides in 29 HIV-1-positive and 13 HIV-1-negative study participants. We report T cell responses to peptides derived from regions of HERV detected by ELISPOT analysis in the HIV-1-positive study participants. We show an inverse correlation between anti-HERV T cell responses and HIV-1 plasma viral load. In HIV-1-positive individuals, we demonstrate that HERV-specific T cells are capable of killing cells presenting their cognate peptide. These data indicate that HIV-1 infection leads to HERV expression and stimulation of a HERV-specific CD8+ T cell response. HERV-specific CD8+ T cells have characteristics consistent with an important role in the response to HIV-1 infection: a phenotype similar to that of T cells responding to an effectively controlled virus (cytomegalovirus), an inverse correlation with HIV-1 plasma viral load, and the ability to lyse cells presenting their target peptide. These characteristics suggest that elicitation of anti-HERV-specific immune responses is a novel approach to immunotherapeutic vaccination. As endogenous retroviral sequences are fixed in the human genome, they provide a stable target, and HERV-specific T cells could recognize a cell infected by any HIV-1 viral variant. HERV-specific immunity is an important new avenue for investigation in HIV-1 pathogenesis and vaccine design.     

Interleukin-21-producing HIV-1-specific CD8 T cells are preferentially seen in elite controllers

A hallmark of human immunodeficiency virus type 1 (HIV-1) pathogenesis is the rapid loss of CD4 T cells leading to generalized immune dysfunction, including an exhausted CD8 T cell phenotype. Understanding the necessary factors that govern the functional quality and protective potential of antiviral T cell responses would facilitate rational vaccine design and improve therapeutic strategies to combat persistent infections. Mouse models of chronic viral infection demonstrate that interleukin-21 (IL-21), produced primarily by CD4 T cells, is required for the generation and maintenance of functionally competent CD8 T cells and viral containment. We reasoned that preserved IL-21 production during HIV-1 infection would be associated with enhanced CD8 T cell function, allowing improved viral control. Here we analyzed the ability of CD4 and CD8 T cells to produce several cytokines in addition to IL-21 ex vivo following stimulation with overlapping HIV-1 peptides. Both CD4 and CD8 T cells were able to produce IL-21 in response to HIV-1 infection, with the latter cell type more closely associated with viral control. Furthermore, IL-21-producing HIV-1-specific CD4 T cells (compared to those producing other cytokines) were the best indicator of functional CD8 T cells. Our results demonstrate that HIV-1-specific IL-21-producing CD8 T cells are induced following primary infection and enriched in elite controllers, suggesting a critical role for these cells in the maintenance of viremia control.     

Discordance between frequency of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferon-producing CD4(+) T cells and HIV-1-specific lymphoproliferation in HIV-1-infected subjects with active viral replication

One hallmark of uncontrolled, chronic human immunodeficiency virus type 1 (HIV-1) infection is the absence of strong HIV-1-specific, CD4(+) T-cell-proliferative responses, yet the mechanism underlying this T helper (Th)-cell defect remains controversial. To better understand the impact of HIV-1 replication on Th-cell function, we compared the frequency of CD4(+) Th-cell responses based on production of gamma interferon to lymphoproliferative responses directed against HIV-1 proteins in HIV-1-infected subjects with active in vivo viral replication versus those on suppressed highly active antiretroviral therapy (HAART). No statistically significant differences in the frequencies of cytokine-secreting, HIV-1-specific CD4(+) T cells between the donor groups were found, despite differences in viral load and treatment status. However, HIV-1-specific lymphoproliferative responses were significantly greater in the subjects with HAART suppression than in subjects with active viral replication. Similar levels of HIV-1 RNA were measured in T-cell cultures stimulated with HIV-1 antigens regardless of donor in vivo viral loads, but only HIV-1-specific CD4(+) T cells from subjects with HAART suppression proliferated in vitro, suggesting that HIV-1 replication in vitro does not preclude HIV-1-specific lymphoproliferation. This study demonstrates a discordance between the frequency and proliferative capacity of HIV-1-specific CD4(+) T cells in subjects with ongoing in vivo viral replication and suggests that in vivo HIV-1 replication contributes to the observed defect in HIV-1-specific CD4(+) T-cell proliferation.     

HIV-1 continues to replicate and evolve in patients with natural control of HIV infection

Elucidating mechanisms leading to the natural control of HIV-1 infection is of great importance for vaccine design and for understanding viral pathogenesis. Rare HIV-1-infected individuals, termed HIV-1 controllers, have plasma HIV-1 RNA levels below the limit of detection by standard clinical assays (<50 to 75 copies/ml) without antiretroviral therapy. Although several recent studies have documented persistent low-grade viremia in HIV-1 controllers at a level not significantly different from that in HIV-1-infected individuals undergoing treatment with combination antiretroviral therapy (cART), it is unclear if plasma viruses are undergoing full cycles of replication in vivo or if the infection of new cells is completely blocked by host immune mechanisms. We studied a cohort of 21 HIV-1 controllers with a median level of viremia below 1 copy/ml, followed for a median of 11 years. Less than half of the cohort carried known protective HLA types (B*57/27). By isolating HIV-1 RNA from large volumes of plasma, we amplified single genome sequences of both pro-rt and env longitudinally. This study is the first to document that HIV-1 pro-rt and env evolve in this patient group, albeit at rates somewhat lower than in HIV-1 noncontrollers, in HLA B*57/27-positive, as well as HLA B*57/27-negative, individuals. Viral diversity and adaptive events associated with immune escape were found to be restricted in HIV-1 controllers, suggesting that replication occurs in the face of less overall immune selection.     

HIV-1-specific interleukin-21+ CD4+ T cell responses contribute to durable viral control through the modulation of HIV-specific CD8+ T cell function

Functional defects in cytotoxic CD8(+) T cell responses arise in chronic human viral infections, but the mechanisms involved are not well understood. In mice, CD4 cell-mediated interleukin-21 (IL-21) production is necessary for the maintenance of CD8(+) T cell function and control of persistent viral infections. To investigate the potential role of IL-21 in a chronic human viral infection, we studied the rare subset of HIV-1 controllers, who are able to spontaneously control HIV-1 replication without treatment. HIV-specific triggering of IL-21 by CD4(+) T cells was significantly enriched in these persons (P = 0.0007), while isolated loss of IL-21-secreting CD4(+) T cells was characteristic for subjects with persistent viremia and progressive disease. IL-21 responses were mediated by recognition of discrete epitopes largely in the Gag protein, and expansion of IL-21(+) CD4(+) T cells in acute infection resulted in lower viral set points (P = 0.002). Moreover, IL-21 production by CD4(+) T cells of HIV controllers enhanced perforin production by HIV-1-specific CD8(+) T cells from chronic progressors even in late stages of disease, and HIV-1-specific effector CD8(+) T cells showed an enhanced ability to efficiently inhibit viral replication in vitro after IL-21 binding. These data suggest that HIV-1-specific IL-21(+) CD4(+) T cell responses might contribute to the control of viral replication in humans and are likely to be of great importance for vaccine design.