HIV Elite Controllers Research Articles

Expression profile of host restriction factors in HIV-1 elite controllers

BackgroundSeveral host-encoded antiviral factors suppress HIV-1 replication in a cell-autonomous fashion in vitro. The relevance of these defenses to the control of HIV-1 in vivo remains to be elucidated. We hypothesized that cellular restriction of HIV-1 replication plays a significant role in the observed suppression of HIV-1 in "elite controllers", individuals who maintain undetectable levels of viremia in the absence of antiretroviral therapy (ART). We comprehensively compared the expression levels of 34 host restriction factors and cellular activation levels in CD4+ T cells and sorted T cell subsets between elite controllers, HIV-1-infected (untreated) non-controllers, ART-suppressed, and uninfected individuals.ResultsExpression of schlafen 11, a codon usage-based inhibitor of HIV-1 protein synthesis, was significantly elevated in CD4+ T cells from elite controllers as compared to both non-controllers (p = 0.048) and ART-suppressed individuals (p = 0.024), with this effect most apparent in central memory CD4+ T cells. Schlafen 11 expression levels were comparable between controllers and uninfected individuals. Cumulative restriction factor expression was positively correlated with CD4+ T cell activation (r2 = 0.597, p < 0.0001), viral load (r2 = 0.34, p = 0.015), and expression of ISG15 (r2 = 0.73, p < 0.0001), a marker of interferon exposure. APOBEC3C, APOBEC3D, CTR9, TRIM26, and TRIM32 were elevated in elite controllers with respect to ART-suppressed individuals, while levels were comparable to uninfected individuals and non-controllers.ConclusionsHost restriction factor expression typically scales with cellular activation levels. However, the elevated mRNA and protein expression of schlafen 11, despite low activation and viral load, violates the global pattern and may be a signature characteristic of HIV-1 elite control.

High Antibody-Dependent Cellular Cytotoxicity Responses Are Correlated with Strong CD8 T Cell Viral Suppressive Activity but Not with B57 Status in HIV-1 Elite Controllers

The role of Antibody-dependent cellular cytotoxicity (ADCC) responses in HIV-1 controllers is still unclear due to the heterogeneity of these patients. We analyzed 67 HIV-1 controllers and found significantly higher levels of ADCC antibodies in controllers versus viremic subjects (p = 0.017). Moreover, multivariate analysis revealed significantly higher ADCC titers in HLA B57- controllers compared to HLA-B57+ ones (p = 0.0086). These data suggest a role for ADCC in immune control of HIV, especially in HLA B57 negative controllers.

The interplay between the X-DING-CD4, IFN-α and IL-8 gene activity in quiescent and mitogen- or HIV-1-exposed PBMCs from HIV-1 elite controllers, AIDS progressors and HIV-negative controls

X-DING-CD4 blocks HIV-1 long terminal repeat (LTR) and pathogen induced pro-inflammatory response. Increased activity of the X-DING-CD4 gene is associated with cellular resistance to virus; therefore, HIV-1 elite controllers (ECs) should have higher X-DING-CD4 and reduced pro-inflammatory mRNA activity than viremic or uninfected individuals. Also, depending on the cell stimulating factor, expression of X-DING-CD4 mRNA in ECs might be autonomous or contingent on IFN signaling. We compared expression of X-DING-CD4, IFN-α and IL-8 mRNAs in naive, phytohemagglutinin- or HIV-1 exposed PBMCs from ECs, HIV progressors and negative controls; tested correlation between X-DING-CD4 and IFN-α expression; sensitivity of the X-DING-CD4 gene to IFN-α regulation; and evaluated interactions between innate and pro-inflammatory genes. We found that expression of X-DING-CD4 and IFN-α was up-regulated in ECs and correlated in cells stimulated with mitogen, but not HIV-1. The X-DING-CD4 gene was more sensitive to HIV-1 than rIFN-α stimulation. ECs had significantly less IL-8 mRNA when PBMCs were exposed to exogenous HIV-1. Two-way ANOVA showed that control of HIV-1 and virus-induced pro-inflammatory response by ECs stemmed from interactions between expression of innate immunity and pro-inflammatory genes, the state of cell stimulation and the status of virus control. Consequently, interaction of multiple host innate immune responses rather than a single mechanism regulates restriction of HIV-1 in ECs.

Cell-intrinsic HIV-1 immune responses in conventional dendritic cells from HIV-1 elite controllers (P6171)

Abstract Introduction: Recent data suggest that in most HIV-1-infected individuals, cell-intrinsic immune responses of conventional dendritic cells (cDC) are blocked by the host proteins Samhd1 and Trex1. Elite controllers (EC) control HIV-1 replication in the absence of treatment, but immune defense mechanisms in these patients are not well understood. Here, we investigated cell-intrinsic immune responses to HIV-1 in these specific patients. Methods: PBMC from EC, untreated chronic progressors (CP), HAART-treated and HIV-1 negative subjects were ex vivo infected with HIV-1. Expression of viral replication products, type I interferons, Samhd1 and Trex1 were analyzed by qPCR. Results: cDC from HIV-1 negative persons were moderately susceptible to HIV-1, while cDC from EC and CP supported HIV-1 replication very weakly. However, in CP, HIV-1 replication was blocked at the level of early reverse transcription, likely as a result of high-level Samdh1 expression, while In EC, reverse transcription was unaltered, and restriction of viral replication mostly occurred at the level of integration. Functionally, these altered patterns of viral restriction in cDC from EC were associated with increased cellular activation, secretion of type I interferons and improved abilities to prime T cell responses. Conclusion: cDC from EC can mount cell-intrinsic immune responses against HIV-1, which may support the generation of highly effective HIV-1-specific T cell responses in these patients.

Attenuation of multiple Nef functions in HIV-1 elite controllers

BackgroundImpaired HIV-1 Gag, Pol, and Env function has been described in elite controllers (EC) who spontaneously suppress plasma viremia to < 50 RNA copies/mL; however, activity of the accessory protein Nef remains incompletely characterized. We examined the ability of 91 Nef clones, isolated from plasma of 45 EC and 46 chronic progressors (CP), to down-regulate HLA class I and CD4, up-regulate HLA class II invariant chain (CD74), enhance viral infectivity, and stimulate viral replication in PBMC.ResultsIn general, EC Nef clones were functional; however, all five activities were significantly lower in EC compared to CP. Nef clones from HLA-B*57-expressing EC exhibited poorer CD4 down-regulation function compared to those from non-B*57 EC, and the number of EC-specific B*57-associated Nef polymorphisms correlated inversely with 4 of 5 Nef functions in these individuals.ConclusionResults indicate that decreased HIV-1 Nef function, due in part to host immune selection pressures, may be a hallmark of the EC phenotype.

Increased coronary atherosclerosis and immune activation in HIV-1 elite controllers

HIV-1 elite controllers spontaneously maintain suppressed levels of viremia, but exhibit significant immune activation. We investigated coronary atherosclerosis by coronary computed tomography angiography (CTA) in elite controllers, nonelite controller, chronically HIV-1 infected, antiretroviral therapy (ART)-treated patients with undetectable viral load ('chronic HIV'), and HIV-negative controls. Prevalence of atherosclerosis (78 vs. 42%, P < 0.05) and markers of immune activation were increased in elite controllers compared with HIV-negative controls. sCD163, a monocyte activation marker, was increased in elite controllers compared with chronic HIV-1 (P < 0.05) and compared with HIV-negative controls (P < 0.05). These data suggest a significant degree of coronary atherosclerosis and monocyte activation among elite controllers.

T cell receptor clonotypes modulate the protective effect of human leukocyte antigen class I alleles in HIV-1 infection

Methods Clonality of tetramer-sorted HIV-1 epitope-specific CD8 T cells from HIV-1 elite controllers and chronic progressors expressing HLA B*27 or B*57 were determined by T cell receptor (TCR) gene sequencing. Polyfunctionality, proliferation, avidity and differentiation phenotypes were analyzed by flow cytometry. Recognition of a GFP reporter cell line or autologous CD4 T cells infected with HIV-1 containing known mutations by HIV-1 epitope-specific bulk CD8 T cells or clonotypic CD8 T cell clones was analyzed by flow cytometry or analysis of p24 production. Immunological synapses between different CD8 T cell clones and infected target cells and lytic granule loading and delivery on a per cell basis were examined using three-dimensional confocal microscopy.

Human intestinal beta defensins inhibit viral replication and are diminished in chronic untreated HIV infection

Background One of the hallmarks of HIV infection is an early and dramatic depletion of CD4 T cells in the intestinal lamina propria. This loss of T cells is accompanied by intestinal epithelial cell disruption resulting in the translocation of luminal bacterial products, persistent systemic immune activation and resultant disease progression. Beta defensins are cationic antimicrobial polypeptides produced by intestinal epithelial cells and phagocytes that are a part of the innate immune response. They have ap ivotal role in maintenance of immune homeostasis in the gut. Although beta defensins have been reported to inhibit bacterial and viral replication in vitro, their role in HIV infection has been incompletely characterized. Methods We investigated anti-viral activity and production of defensins in intestinal mucosal biopsies, stool and plasma from individuals with chronic untreated HIV (chronic progressors), immunologically controlled HIV (elite controllers) and HIV uninfected individuals using ELISA, immunohistochemistry, quantitative PCR and viral inhibition assays. Results Plasma levels of beta defensins were increased in the HIV controller group compared to HIV progressors, whereas levels of beta defensins were significantly decreased in biopsies from chronic progressors. Beta defensin release was induced by HIV in ex vivo cultured intestinal cells. Recombinant beta defensins inhibited HIV replication in vitro. Conclusion Beta defensins are upregulated by HIV and inhibit viral replication in vitro. Chronic progressors, however, had diminished levels of beta defensins in gut biopsies and in the plasma. We propose that beta defensins are induced in HIV infection and have a role in inhibiting viral replication and preventing intestinal epithelial cell disruption. In chronic infection, however, their release is diminished, likely due to epithelial damage in the setting of persistent viremia and mucosal inflammation.

Models of protection against HIV/SIV

Models of protection against HIV/SIV infection focuses on three known scenarios that might help to control the AIDS pandemic by applying three so-called natural experiments in which AIDS was effi ciently avoided in people and monkeys. The fi rst section of the book is dedicated to SIV infection in African non-human primates, which are natural hosts of the virus. These species, which are generally frequently infected in the wild, do not normally progress to have AIDS. The mechanisms of control of disease progression do not rely on an advanced control of viral replication but rather on the control of the deleterious eff ects of lentiviral infection, most notably that of excessive immune activation (accepted as the main driver of progression to AIDS). Application of this information to HIV infection could allow design of immune-based treatments that might improve survival of patients infected with HIV. The second part focuses on people who have been exposed to HIV-1 who remain seronegative. Although this outcome might be a result of good fortune, some high-risk cohorts remain uninfected. Clarifi cation of the determinants of protection against HIV infection is a high priority and could lead to the design of eff ective strategies to prevent infection through microbicides or vaccines. Finally, the third section investigates so-called HIV-1 elite controllers. These patients, although infected with HIV, can control viral replication to near undetectable levels and substantially delay disease progression. This pathogenic outcome provides crucial information for present strategies of vaccination and reservoir eradication (ie, for a cure of HIV infection). This simple enumeration shows that these models of protection are important for HIV research. The editors assembled a group of researchers who have made major contributions to the defi nition of these models. The book is not intended to treat the three models separately, but rather to present them as a combination that could identify common traits and interventions. Models of protection against HIV/SIV is aimed at students and young investigators, and to researchers ready to explore new avenues that may lead to the control of HIV.

Metabolic and Cardiovascular Complications in HIV-Infected Patients: New Challenges for a New Age

Metabolic and Cardiovascular Complications in HIV-Infected Patients: New Challenges for a New Age

A high dimensional immune monitoring model of HIV-specific CD8 T cell responses accurately identifies subjects achieving spontaneous control of viral replication

A major challenge in HIV vaccinology is the development of appropriate immune monitoring models to determine vaccine efficacy. Studies of HIV-specific CD8 T cells (CTL) suggest that these responses play an important role in infected individuals capable of spontaneous viral control (HIV elite controllers) and that they will likely play a role in immune interventions. However, no single CTL assay is uniquely associated with the controller phenotype.

Elite Controllers with Low to Absent Effector CD8 + T Cell Responses Maintain Highly Functional, Broadly Directed Central Memory Responses

Analyses of the breadth and specificity of virus-specific CD8(+) T cell responses associated with control of HIV have largely relied on measurement of cytokine secretion by effector T cells. These have resulted in the identification of HIV elite controllers with low or absent responses in which non-T-cell mechanisms of control have been suggested. However, successful control of HIV infection may be associated with central memory T cells, which have not been consistently examined in these individuals. Gag-specific T cells were characterized using a peptide-based cultured enzyme-linked immunosorbent spot assay (ELISpot). Peripheral blood mononuclear cells from HIV elite controllers (n = 10), progressors (n = 12), and antiretroviral-treated individuals (n = 9) were cultured with overlapping peptides for 12 days. Specificity was assessed by tetramer staining, functional features of expanded cells were assessed by cytokine secretion, and virus inhibition and phenotypic characteristics were assessed by cell sorting and coculture assays. After peptide stimulation, elite controllers showed a greater number of previously undetectable (new) responses compared to progressors (P = 0.0008). These responses were highly polyfunctional, with 64.5% of responses having 3 to 5 functions. Expandable epitope-specific CD8(+) T cells from elite controllers had strong virus inhibitory capacity and predominantly displayed a central memory phenotype. These data indicate that elite controllers with minimal T cell responses harbor a highly functional, broadly directed central memory T cell population that is capable of suppressing HIV in vitro. Comprehensive examination of this cell population could provide insight into the immune responses associated with successful containment of viremia.

CD4 T-cell regeneration in HIV-1 elite controllers

Elite controllers spontaneously control HIV-1 replication, which in many cases is associated with preservation of normal CD4 T-cell counts. However, a subset of elite controllers has progressive CD4 T-cell losses despite undetectable viral loads, for reasons that remain undefined. Here, we assessed mechanisms of CD4 T-cell homeostasis in elite controllers with progressive vs. nonprogressive HIV-1 disease courses. Flow cytometry assays were used to determine the proliferation, activation and apoptosis levels of naive T cells in elite controllers with high or low CD4 T-cell counts and reference cohorts of HIV-1-negative and HAART-treated persons. Thymic output was measured by single-joint T-cell receptor excision circle (sjTREC)/β T-cell receptor excision circle (βTREC) ratios, and the frequency of circulating recent thymic emigrants was flow cytometrically determined by surface expression of protein tyrosine kinase 7. Proportions of naive T cells in elite controllers were severely reduced and closely resemble those of HIV-1 patients with progressive disease. Despite reductions in naive T cells, most elite controllers were able to maintain normal total CD4 T-cell counts by preservation of uncompromised thymic function in conjunction with extrathymic processes that led to elevated levels of circulating recent thymic emigrants. In contrast, elite controllers with low CD4 T-cell counts had reduced thymic output that mirrored thymic dysfunction during untreated progressive HIV-1 infection. These results indicate that both thymic and extrathymic mechanisms contribute to CD4 T-cell maintenance in elite controllers and support the idea that CD4 T-cell homeostasis and control of viral replication are distinct but frequently coinciding processes.

Dissecting the HIV-specific immune response

Several unique HIV-infected or HIV-resistant cohorts have been studied over the years to try and delineate the correlates of protection. Although several mechanisms have been put forward, studies aiming to integrate the different mechanisms into a comprehensive model are still lacking. Current systems biology approaches emphasize the importance of unifying independent datasets, provide tools that facilitate hypothesis formulation and testing, and direct us toward uncovering novel therapeutic targets by defining molecular networks perturbed during disease. This review will focus on the current findings that utilized systems biology techniques in order to identify correlates of protection from HIV disease progression and resistance to infection in unique cohorts of individuals as well as in nonhuman primate models of SIV infection. Using systems biology technologies and data analysis tools, the studies described herein have found that pathways implicated in survival, cell cycling, inflammation, and oxidative stress work in unison to limit pathology caused by chronic immune activation. This situation favors the survival of effector lymphocytes and limits the dissemination of viral particles in HIV elite controllers, exposed-uninfected individuals, and natural hosts of SIV infection. Systems and computational biology tools have clearly expanded our understanding of HIV pathogenesis by unifying independent observations and by giving us novel molecular targets to pursue. These molecular signatures have the potential to uncover correlates of protection in HIV disease and, in the era of personalized medicine, to determine predictive signatures of treatment efficacy and/or failure.

Characteristics of Plasmacytoid Dendritic Cell and CD4+ T Cell in HIV Elite Controllers

Despite variability, the majority of HIV-1-infected individuals progress to AIDS characterized by high viral load and massive CD4+ T-cell depletion. However, there is a subset of HIV-1-positive individuals that does not progress and spontaneously maintains an undetectable viral load. This infrequent patient population is defined as HIV-1 controllers (HIV controllers), and represents less than 1% of HIV-1-infected patients. HIV-1-specific CD4+ T cells and the pool of central memory CD4+ T cells are also preserved despite immune activation due to HIV-1 infection. The majority of HIV controllers are also defined by the absence of massive CD4+ T-cell depletion, even after 10 years of infection. However, the mechanisms involved in protection against HIV-1 disease progression have not been elucidated yet. Controllers represent a heterogeneous population; we describe in this paper some common characteristics concerning innate immune response and CD4+ T cells of HIV controllers.

Impairment of viral replication capacity by nef alleles from HIV elite controllers

Background Elite controllers (EC) are HIV-1 infected persons who can spontaneously control viremia to extremely low levels. Because it is known that nef is required for HIV1 replication and pathogenesis in vivo, we wish to clarify how HIV-1 nef plays a role in acquiring EC phenotype. Materials and methods Viral RNAs were extracted from plasma samples of 45 ECs and 48 chronic progressors (CP) and used for amplification of nef by RT-PCR. The amplified products were cloned into a plasmid and subsequently sequenced. A representative nef clone was selected for each patient and cloned into an NL43 proviral plasmid backbone. Infectious recombinant viruses were prepared by transfecting 293T cells with these resultant proviral clones and tested for their viral replication capacity in PBMCs obtained from multiple healthy donors. Viral replication was monitored by measuring the concentration of p24 Gag in the culture supernatant by p24 ELISA every 3 days. The same virus stocks were further tested for their infectivity using TZM-bl cells. Results All nef alleles isolated from EC and CP had intact ORFs. Phylogenetic analysis revealed no specific lineage or clustering, suggesting the absence of common genetic defects in EC-derived nef alleles. All viruses expressing patient-derived nef showed a wide range of viral replication in PBMC, confirming the importance of nef alleles in the enhancement of viral replication capacity. The viruses harboring EC-derived nef replicated and peaked on Day 12 after infection or even later; whereas the viruses harboring CP-derived nef peaked on Day 9. Also, the peak level of p24 values was significantly lower in the EC-nef viruses than in the CP-nef viruses. Because Nef activity is highly dependent on PBMC donors, we tested 4 different donors’ PBMC and compared the initial burst of their viral replication at Day 9. In all donors’PBMC, viruses carrying EC-nef displayed lower p24 values than those carrying CP-nef, suggesting that the viral replication capacity was much impaired by nef alleles in EC. Corroboratively, in viral infectivity assay in TZM-bl cells, the EC-nef viruses showed significantly lower infectivity than the CP-nef viruses. Conclusion

IL28B Genotype Does Not Correlate with HIV Control in African Americans

HIV-1 natural viral suppressors (NVS) are individuals that control HIV replication without antiretrovirals (also know as HIV elite controllers). We have recently shown that these individuals have an elevated rate of hepatitis C virus (HCV) clearance. Given the association of IL28B genotype, specifically the rs12979860 single nucleotide polymorphism (SNP) based CC genotype, with HCV clearance, we studied its association with HIV control in 172 African American HIV subjects and 173 race-matched controls. The frequency of the CC genotype was 12.5% in the NVS, 14.7% in the LVL ("low viral load" cohort with 400-20,000 HIV-1 RNA copies/mL), 17.8% in the MHVL ("medium/high viral load" cohort with >20,000 HIV-1 RNA copies/mL), and 11.6% in an HIV-negative cohort. There was no statistical significance in the CC genotype distribution between these cohorts (p= 0.48 between the NVS and non-NVS HIV positive controls, p= 0.85 between NVS and HIV-negatives). We also did not observe any association between CC genotype distribution and HIV RNA viral load, as a continuous measure. The IL28B CC genotype does not account for the noted HIV control in our specific NVS cohort. Further studies will be needed to determine if a common genetic factor can primarily account for any joint clearance of HCV and control of HIV.

Unique features of memory T cells in HIV elite controllers: a systems biology perspective

Elite controllers constitute a rare group of HIV-infected individuals who control HIV replication and maintain normal CD4 cell counts without antiretroviral therapy (ART). The mechanisms involved in the control of infection are poorly understood. This review will focus on the identification of signaling pathways upregulated or downregulated in different memory T-cell subsets in elite controllers by using systems biology approaches. Features of memory T cells in simian immunodeficiency virus (SIV) natural hosts will be also highlighted. Finally, we will discuss how these approaches will guide the development of new vaccines and therapeutic interventions. Studies by our group identified the FOXO3a, STAT5, and Wnt/beta-catenin pathways as unique molecular signatures associated with survival of memory T cells in elite controllers. These discoveries open the path for the design of new strategies to prevent T-cell depletion in HIV-infected individuals. The use of systems biology to identify molecular pathways involved in the survival of memory T cells is a powerful tool toward the understanding of mechanisms of HIV control in elite controllers. This will help to identify correlates of immune protection leading to the design of effective HIV vaccines and new targeted therapeutic interventions.

Increased HIV-specific CD8+ T-cell cytotoxic potential in HIV elite controllers is associated with T-bet expression

AbstractRecent data suggest that CD8+ T-cell effector activity is an important component in the control of HIV replication in elite controllers (ECs). One critical element of CD8+ T-cell effector function and differentiation is the T-box transcription factor T-bet. In the present study, we assessed T-bet expression, together with the effector proteins perforin, granzyme A (Grz A), granzyme B (Grz B), and granulysin, in HIV-specific CD8+ T cells from ECs (n = 20), chronically infected progressors (CPs; n = 18), and highly active antiretroviral therapy (HAART)–suppressed individuals (n = 19). Compared with the other cohort groups, HIV-specific CD8+ T cells among ECs demonstrated a superior ability to express perforin and Grz B, but with no detectable difference in the levels of Grz A or granulysin. We also observed higher levels of T-bet in HIV-specific CD8+ T cells from ECs, with an ensuing positive correlation between T-bet and levels of both perforin and Grz B. Moreover, HIV-specific CD8+ T cells in ECs up-regulated T-bet to a greater extent than CPs after in vitro expansion, with concomitant up-regulation of perforin and Grz B. These results suggest that T-bet may play an important role in driving effector function, and its modulation may lead to enhanced effector activity against HIV.

217 HIV Infection Risk Association with FCG Receptor Polymorphism

Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism for HIV-specific antibodies to target infected cells for destruction by cytotoxic effectors. The Fc portion of IgG antibodies bind cytotoxic effector cells through a family of Fc receptors that include FCGR3A (CD16). Alternately, antibody binding to Fc receptor delivers an activating signal to the effector cells. Thus there is a balance between effector cell stimulation and target cell killing during ADCC. Single nucleotide polymorphisms (SNP) in the Fc receptor gene FCGR3A alter the amino acid sequence and encode high or low functioning proteins. We predicted that elite controller groups (HIV+ individuals with durable viurus suppression without taking antiretroviral drugs) would have increased frequencies of the high functioning allelels, compared to HIV progressors or uninfected controls. Genomic DNA was analyzed from individuals with no HIV infection, Elite controllers and HIV progressors on HAART for a known polymorphism in the FCGR3A gene that has functional relevance to ADCC function. The rs396991 polymorphism results in a T (F amino acid) or a G (V amino acid) allele at the SNP position. PCR was used to amplify a region of FCGR3A and an analysis of rs396691 was done using the ABISnapshot multiplex kit. An allele frequency of T (0.82) and G (0.18) for normal donors, T (0.75) and G (0.25) for elite controllers and T (0.58) and G (0.42) for HIV progressors. These results of higher frequencies of high functioning alleles in HIV progressors suggest that this allele may be responsible for chronic lymphocyte activation that is a feature of progressing HIV disease.