Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA–viral peptide interaction as the major factor modulating durable control of HIV infection.
Immune control of viral infections is modulated by diverse T cell receptor (TCR) clonotypes engaging peptide-MHC class I complexes on infected cells, but the relationship between TCR structure and antiviral function is unclear. Here we apply in silico molecular modeling with in vivo mutagenesis studies to investigate TCR-pMHC interactions from multiple CTL clonotypes specific for a well-defined HIV-1 epitope. Our molecular dynamics simulations of viral peptide-HLA-TCR complexes, based on two independent co-crystal structure templates, reveal that effective and ineffective clonotypes bind to the terminal portions of the peptide-MHC through similar salt bridges, but their hydrophobic side-chain packings can be very different, which accounts for the major part of the differences among these clonotypes. Non-specific hydrogen bonding to viral peptide also accommodates greater epitope variants. Furthermore, free energy perturbation calculations for point mutations on the viral peptide KK10 show excellent agreement with in vivo mutagenesis assays, with new predictions confirmed by additional experiments. These findings indicate a direct structural basis for heterogeneous CTL antiviral function.
Early immunological events during acute HIV infection are thought to fundamentally influence long-term disease outcome. Whereas the contribution of HIV-specific CD8 T cell responses to early viral control is well established, the role of HIV-specific CD4 T cell responses in the control of viral replication following acute infection is unknown. A growing body of evidence suggests that CD4 T cells - besides their helper function - have the capacity to directly recognize and kill virally infected cells. In a longitudinal study of a cohort of individuals acutely infected with HIV, we observed that subjects able to spontaneously control HIV replication in the absence of antiretroviral therapy showed a significant expansion of HIV-specific CD4 T cell responses—but not CD8 T cell responses–compared to subjects who progressed to a high viral set point (p=0.038). Strikingly, this expansion occurred prior to differences in viral load or CD4 T cell count and was characterized by robust cytolytic activity and expression of a distinct profile of perforin and granzymes at the earliest time point. Kaplan-Meier analysis revealed that the emergence of Granzyme A+ HIV-specific CD4 T cell responses at baseline was highly predictive of slower disease progression and clinical outcome (average days to CD4 T cell count <350/μl was 575 versus 306, p=0.001). These data demonstrate that HIV-specific CD4 T cell responses can be used during the earliest phase of HIV infection as an immunological predictor of subsequent viral set point and disease outcome. Moreover, these data suggest that expansion of Granzyme A+ HIV-specific cytolytic CD4 T cell responses early during acute HIV infection contributes substantially to the control of viral replication.
Antigen-specific CD8 T cells play a critical role in controlling HIV infection but eventually lose antiviral functions in part because of expression and signaling through the inhibitory PD-1 receptor. To better understand the impact of prolonged TCR ligation on regulation of PD-1 expression in HIV-specific CD8 T cells we investigated the capacity of virus-specific CD8 T cells to modify the PD-1 epigenetic program following reduction in viral load. We observed that the transcriptional regulatory region was unmethylated in the PD-1hi HIV-specific CD8 T cells while it remained methylated in donor matched naïve cells at acute and chronic stages of infection. Surprisingly, the PD-1 promoter remained unmethylated in HIV-specific CD8 T cells from subjects with a viral load controlled by antiviral therapy for greater than 2 years or from elite controllers. Together these data demonstrate that the epigenetic program at the PD-1 locus becomes fixed following prolonged exposure to HIV virus.
Although many new prevention modalities that include the use of antiretroviral drugs (ARVs) show promise, there is no question that a global solution to the HIV epidemic will not be economically or logistically feasible without the development of vaccine that provides durable protection. In the best case scenario, the vaccine has to protect against acquisition of infection, likely mediated by Env-specific B cell responses combined with CD4+ T cell responses to evoke full maturation and maintenance of protective antibodies. But HIV-specific CD8+ T cell responses are also likely to be a key element, particularly for those inevitable situations in which full vaccine-induced protection from acquisition is not achieved, in which case durable control of established infection will be required. Although there is reason to be optimistic that an effective HIV vaccine is possible, one of the major constraints moving forward will likely be constraints on funding to support a diversity of concepts at a time that the correlates of protection from acquisition and disease progression are still unknown. Given the scope of the epidemic and the economic climate, we must strive to do much more with less and seek to access additional resources, both scientific and monetary, from every possible source.
Vaccine; Antiretrovirals; Economy
Regulatory T cells (Tregs) are potent immune modulators, but their precise role in HIV pathogenesis remains incompletely understood. Most studies to date have focused on frequencies or phenotypes of “bulk” Treg populations. However, although antigen-specific Tregs have been reported in other diseases, HIV-1-epitope specific Tregs have not been described to date. We here report the first identification of functional HIV-1-Gag-specific regulatory T cells using human leukocyte antigen class II tetramer staining in HIV-1-infected individuals.
HIV Gag-specific CD4+ and CD8+ T-cell responses are important for HIV immune control. Pulsing overlapping Gag peptides on autologous lymphocytes (OPAL) has proven immunogenic and effective in reducing viral loads in multiple pigtail macaque studies, warranting clinical evaluation.
We performed a phase I, single centre, placebo-controlled, double-blinded and dose-escalating study to evaluate the safety and preliminary immunogenicity of a novel therapeutic vaccine approach ‘OPAL-HIV-Gag(c)’. This vaccine is comprised of 120 15mer peptides, overlapping by 11 amino acids, spanning the HIV Gag C clade sequence proteome, pulsed on white blood cells enriched from whole blood using a closed system, followed by intravenous reinfusion. Patients with undetectable HIV viral loads (<50 copies/ml plasma) on HAART received four administrations at week 0, 4, 8 and 12, and were followed up for 12 weeks post-treatment. Twenty-three people were enrolled in four groups: 12 mg (n = 6), 24 mg (n = 7), 48 mg (n = 2) or matching placebo (n = 8) with 18 immunologically evaluable. T-cell immunogenicity was assessed by IFNγ ELIspot and intracellular cytokine staining (ICS).
The OPAL-HIV-Gag(c) peptides were antigenic in vitro in 17/17 subjects. After vaccination with OPAL-HIV-Gag(c), 1/6 subjects at 12 mg and 1/6 subjects at 24 mg dose groups had a 2- and 3-fold increase in ELIspot magnitudes from baseline, respectively, of Gag-specific CD8+ T-cells at week 14, compared to 0/6 subjects in the placebo group. No Gag-specific CD4+ T-cell responses or overall change in Rev, Nef, Tat and CMV specific responses were detected. Marked, transient and self-limiting lymphopenia was observed immediately post-vaccination (4 hours) in OPAL-HIV-Gag(c) but not in placebo recipients, with median fall from 1.72 to 0.67 million lymphocytes/mL for active groups (P<0.001), compared to post-placebo from 1.70 to 1.56 lymphocytes/ml (P = 0.16).
Despite strong immunogenicity observed in several Macaca nemestrina studies using this approach, OPAL-HIV-Gag(c) was not significantly immunogenic in humans and improved methods of generating high-frequency Gag-specific T-cell responses are required.
Name of Registry
ClinicalTrials.gov, Registry number: NCT01123915, URL trial registry database: http://www.clinicaltrials.gov/ct2/results?term=OPAL-HIV-1001&Search=Search
After antigen encounter, naive lymphocytes differentiate into populations of memory cells that share a common set of functions including faster response to antigen re-exposure and the ability to self-renew. However memory lymphocytes in different lymphocyte lineages are functionally and phenotypically diverse. It is not known whether discrete populations of T and B cells use similar transcriptional programs during differentiation into the memory state. We used cross-species genomic analysis to examine the pattern of genes upregulated during the differentiation of naive lymphocytes into memory cells in multiple populations of human CD4, CD8 and B cell lymphocytes as well as two mouse models of memory development. We identified and validated a signature of genes that was upregulated in memory cells compared to naive cells in both human and mouse CD8 memory differentiation, suggesting marked evolutionary conservation of this transcriptional program. Surprisingly, this conserved CD8 differentiation signature was also upregulated during memory differentiation in CD4 and B cell lineages. To validate the biologic significance of this signature we showed that alterations in this signature of genes could distinguish between functional and exhausted CD8 T cells from a mouse model of chronic viral infection. Finally, we generated genome-wide microarray data from tetramer-sorted human T cells and showed profound differences in this differentiation signature between T cells specific for HIV from those specific for influenza. Thus, our data suggest that in addition to lineage-specific differentiation programs, T and B lymphocytes employ a common transcriptional program during memory development that is disrupted in chronic viral infection.
Objective and design
The objective of this study was to determine changes in TLR responses of monocytes, myeloid dendritic cells and plasmacytoid dendritic cells during primary and chronic HIV-1 infection. Toll-like receptors (TLRs) serve as important innate receptors to sense pathogens, and have been implicated in mediating immune activation in HIV-1 infection. Studies assessing the consequences of HIV-1 infection on the ability of innate immune cells to respond to TLR stimulation have come to varying conclusions.
Using intracellular flow cytometry, cytokine production by cryopreserved PBMCs from healthy controls and HIV-1 infected individuals were examined after TLR stimulation.
We observed that the effect of HIV-1 infection on TLR responses not only depended on the stage of HIV-1 infection, but was also dependent on the individual receptor and cell type examined. Monocyte and mDC responses to TLR8 stimulation were associated with HIV-1 viral load and CD4+ T cell count, while pDC responses to TLR7 stimulation were not. Responses to TLR2 stimulation were not affected by HIV-1 infection while responses to TLR9 stimulation were universally decreased in all HIV-1 infected individuals examined regardless of treatment or clinical parameters.
Responsiveness to TLR7/8 stimulation, which have been shown to recognize HIV-1 ssRNA, did not decrease in chronic infection, and may represent a contributing factor to ongoing T cell immune activation in the setting of chronic viremic HIV-1 infection.
HIV-1; Toll-like receptor; Innate immunity; Pathogenesis; Dendritic Cells; Monocytes
Host genetic variation is presently estimated to account for about one-fourth of the observed differences in control of HIV across infected individuals. Genome-wide association studies have confirmed that polymorphism within the HLA class I locus is the primary host genetic contributor to determining outcome after infection. Here we progress beyond the genetic associations alone to consider the functional explanations for these correlations. In this process, the complex and multidimensional effects of HLA molecules in viral disease become apparent.
HLA-B; HLA-C 3′ UTR; genome-wide association study; cytotoxic T lymphocytes; microRNA
HIV-1 elite controllers are able to control virus replication to levels below the limits of detection by commercial assays, but the actual level of viremia in these individuals is not well defined. Here we quantify plasma HIV-1 RNA in elite controllers and correlate this with specific immunologic parameters.
Plasma HIV- 1 RNA levels were quantified in 90 elite controllers using a real time RT-PCR assay with a sensitivity of 0.2 copies/ml. HIV-specific immune responses and longitudinal CD4+ T cell counts were examined.
Median plasma HIV-1 RNA was 2 copies/ml (IQR 0.2–14 copies/ml). Longitudinal analysis of 31 elite controllers showed 2–5 fold fluctuations in viremia in the majority of individuals; 6 had persistent levels below 1 copy/ml. Viremia correlated directly with HIV specific neutralizing antibodies and Western Blot reactivity, but not with CD8+ T cell responses. Absolute CD4+ T cell decline was more common among individuals with detectable viremia (p=0.04).
Low-level viremia is present in the majority of elite controllers and is associated with higher HIV specific antibody responses. Absolute CD4+ T cell loss is more common among viremic individuals, suggesting that even very low-level viremia has negative consequences over time.
HIV-1; elite controllers; plasma viral load; CD4+T cells; single copy assay
The balance between proinflammatory mechanisms and dampening of excessive immune activation is critical for successful clearance of a pathogen without harm to the host. In particular, molecules of the B7:CD28 family play a critical role in regulating T cell activation and peripheral tolerance. Chronic pathogens like HIV, which is characterized by ongoing viral replication in spite of detectable virus-specific T cell responses, and cancer cells have exploited these pathways to attenuate antigen-specific T cell immunity. This review summarizes evidence that the molecules of the B7:CD28 family PD-1, CTLA-4 and their ligands play an active and reversible role in virus- specific T cell exhaustion associated with HIV infection in humans and in the SIV model in macaques. We discuss the potential for immunotherapeutic interventions based on manipulation of these inhibitory networks, the promising data obtained with blockade of the PD-1 pathway in animal models, and the challenges to such therapies.
CD4 T cell; CD8 T cell; HIV; SIV; PD-1 (CD279); CTLA-4 (CD152); T cell exhaustion; immune activation
The ability of cytotoxic T lymphocytes (CTL) to clear virus-infected cells is dependent on the presentation of viral peptides processed intracellularly and displayed by MHC-I. Most CTL functional assays utilize exogenously added peptides, which does not account for kinetics and quantity of antigenic peptides produced by infectable cells. Here we examined the relative ability of two major HIV-infectable cell subsets - CD4 T lymphocytes and monocytes- to produce antigenic peptides, using cytosol as a source of peptidases and mass spectrometry to define the degradation products. We show clear subset-specific differences in the kinetics of peptide production and the ability of the peptides produced to sensitize cells for lysis by CTL, with primary CD4 T lymphocytes possessing significantly lower proteolytic activities than monocytes. These differences in epitope processing by cell subsets may impact the efficiency of CTL-mediated clearance of infected subsets and contribute to the establishment of chronic infection.
Antigen processing; HIV; MHC-I; epitope; Cytotoxic T Lymphocytes; CD4 T cells monocytes; proteasome; aminopeptidases; immune recognition
During immune responses, antibodies are selected for their ability to bind to foreign antigens with high affinity, in part by their ability to undergo homotypic bivalent binding. However, this type of binding is not always possible. For example, the small number of gp140 glycoprotein spikes displayed on the surface of the human immunodeficiency virus (HIV disfavours homotypic bivalent antibody binding1–3. Here we show that during the human antibody response to HIV, somatic mutations that increase antibody affinity also increase breadth and neutralizing potency. Surprisingly, the responding naive and memory B cells produce polyreactive antibodies, which are capable of bivalent heteroligation between one high-affinity anti-HIV-gp140 combining site and a second low-affinity site on another molecular structure on HIV. Although cross-reactivity to self-antigens or polyreactivity is strongly selected against during B-cell development4, it is a common serologic feature of certain infections in humans, including HIV, Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134 monoclonal anti-HIV-gp140 antibodies cloned from six patients5 with high titres of neutralizing antibodies are polyreactive. Despite the low affinity of the polyreactive combining site, heteroligation demonstrably increases the apparent affinity of polyreactive antibodies to HIV.
Certain immune-driven mutations in HIV-1, such as those arising in p24Gag, decrease viral replicative capacity. However, the intersubtype differences in the replicative consequences of such mutations have not been explored. In HIV-1 subtype B, the p24Gag M250I mutation is a rare variant (0.6%) that is enriched among elite controllers (7.2%) (P = 0.0005) and appears to be a rare escape variant selected by HLA-B58 supertype alleles (P < 0.01). In contrast, in subtype C, it is a relatively common minor polymorphic variant (10 to 15%) whose appearance is not associated with a particular HLA allele. Using site-directed mutant viruses, we demonstrate that M250I reduces in vitro viral replicative capacity in both subtype B and subtype C sequences. However, whereas in subtype C downstream compensatory mutations at p24Gag codons 252 and 260 reduce the adverse effects of M250I, fitness costs in subtype B appear difficult to restore. Indeed, patient-derived subtype B sequences harboring M250I exhibited in vitro replicative defects, while those from subtype C did not. The structural implications of M250I were predicted by protein modeling to be greater in subtype B versus C, providing a potential explanation for its lower frequency and enhanced replicative defects in subtype B. In addition to accounting for genetic differences between HIV-1 subtypes, the design of cytotoxic-T-lymphocyte-based vaccines may need to account for differential effects of host-driven viral evolution on viral fitness.
It is generally believed that CD8+ cytotoxic T lymphocytes (CTLs) play a critical role in limiting the replication of human immunodeficiency virus type 1 (HIV-1) and in determining the outcome of the infection, and this effect may partly depend on which HIV product is preferentially targeted. To address the correlation between HIV-1-specific CTL responses and virus replication in a cohort of former plasma donors (FPDs), 143 antiretroviral therapy naive FPDs infected with HIV-1 clade B' strains were assessed for HIV-1-specific CTL responses with an IFN-γ Elispot assay at single peptide level by using overlapping peptides (OLPs) covering the whole consensus clade B proteome. By using a Spearman's rank correlation analysis, we found that the proportion of Gag-specific CTL responses among the total virus-specific CTL activity was inversely correlated with viral loads while being positively correlated to CD4 counts, as opposed to Pol- and Env-specific responses that were associated with increased viral loads and decreased CD4 counts. In addition, Vpr-specifc CTL responses showed a similar protective effect with Gag responses, but with a much lower frequency of recognition. Significantly, we also observed an association between HLA-A*30/B*13/Cw*06 haplotype and lower viral loads that was probably due to restricted Gag-specific CTL responses. Thus, our data demonstrate the prominent role of Gag-specific CTL responses in disease control. The advantage of HLA-A*30/B*13/Cw*06 haplotype in viral control may be associated with the contribution of Gag-specific CTL responses in the studied individuals.
human immunodeficiency virus type 1; cytotoxic T lymphocytes; human leukocyte antigen class I; Gag
Genetic variation within the HLA-B locus has the strongest impact on HIV disease progression of any polymorphisms within the human genome. However, identifying the exact mechanism involved is complicated by several factors. HLA-Bw4 alleles provide ligands for NK cells and for CD8 T cells, and strong linkage disequilibrium between HLA class I alleles complicates the discrimination of individual HLA allelic effects from those of other HLA and non-HLA alleles on the same haplotype. Here, we exploit an experiment of nature involving two recently diverged HLA alleles, HLA-B*42:01 and HLA-B*42:02, which differ by only a single amino acid. Crucially, they occur primarily on identical HLA class I haplotypes and, as Bw6 alleles, do not act as NK cell ligands and are therefore largely unconfounded by other genetic factors. We show that in an outbred cohort (n = 2,093) of HIV C-clade-infected individuals, a single amino acid change at position 9 of the HLA-B molecule critically affects peptide binding and significantly alters the cytotoxic T lymphocyte (CTL) epitopes targeted, measured directly ex vivo by gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay (P = 2 × 10−10) and functionally through CTL escape mutation (P = 2 × 10−8). HLA-B*42:01, which presents multiple Gag epitopes, is associated with a 0.52 log10 lower viral-load set point than HLA-B*42:02 (P = 0.02), which presents no p24 Gag epitopes. The magnitude of this effect from a single amino acid difference in the HLA-A*30:01/B*42/Cw*17:01 haplotype is equivalent to 75% of that of HLA-B*57:03, the most protective HLA class I allele in this population. This naturally controlled experiment represents perhaps the clearest demonstration of the direct impact of a particular HIV-specific CTL on disease control.
It is unknown whether favorable HLA class II alleles may attenuate HIV-1 through selection pressure in a manner similar to that of protective HLA class I alleles. We investigated the relationship between HLA class II alleles and in vitro replication capacities of recombinant viruses encoding HIV-1 subtype C Gag-protease from chronically infected individuals. No associations were found between individual alleles and lower replication capacity, suggesting no significant HIV-1 attenuation by HLA class II-restricted Gag-specific CD4+ T cell immune pressure.
A new method is used to isolate neutralizing antibodies recognizing a new epitope on the cell surface–expressed, but not soluble, HIV-1 spike.
Two to three years after infection, a fraction of HIV-1–infected individuals develop serologic activity that neutralizes most viral isolates. Broadly neutralizing antibodies that recognize the HIV-1 envelope protein have been isolated from these patients by single-cell sorting and by neutralization screens. Here, we report a new method for anti–HIV-1 antibody isolation based on capturing single B cells that recognize the HIV-1 envelope protein expressed on the surface of transfected cells. Although far less efficient than soluble protein baits, the cell-based capture method identified antibodies that bind to a new broadly neutralizing epitope in the vicinity of the V3 loop and the CD4-induced site (CD4i). The new epitope is expressed on the cell surface form of the HIV-1 spike, but not on soluble forms of the same envelope protein. Moreover, the new antibodies complement the neutralization spectrum of potent broadly neutralizing anti-CD4 binding site (CD4bs) antibodies obtained from the same individual. Thus, combinations of potent broadly neutralizing antibodies with complementary activity can account for the breadth and potency of naturally arising anti–HIV-1 serologic activity. Therefore, vaccines aimed at eliciting anti–HIV-1 serologic breadth and potency should not be limited to single epitopes.
This study addressed two questions: (1) What fraction of individuals maintain a sustained high HIV-1 RNA load after the acute HIV-1C infection peak? and (2) How long is a high HIV-1 RNA load maintained after acute HIV-1C infection in this subpopulation?
Plasma HIV-1 RNA dynamics were studied in 77 subjects with primary HIV-1C infection from African cohorts in Gaborone, Botswana, and Durban, South Africa. HIV-infected individuals who maintained mean viral load of ≥ 100,000 (5.0 log10) copies/ml after 100 days post-seroconversion (p/s) were termed Extended High Viremics. Individuals were followed longitudinally for a median (IQR) of 573 (226;986) days p/s.
The proportion of Extended High Viremics was 34% (95% CI: 23%–44%) during the period 100 to 300 days p/s and 19% (95% CI: 9%–29%) over the period of 200 to 400 days p/s. The median (IQR) duration of HIV-1 RNA load ≥ 100,000 copies/ml among Extended High Viremics was 271 (188;340) days p/s. For the subset with average viral load ≥ 100,000 copies/ml during 200–400 days p/s, the median (IQR) duration was 318 (282;459) days. The Extended High Viremics had a significantly shorter time to CD4 decline to 350 cells/μl (median: 88 vs. 691 days p/s for those not designated as Extended High Viremics; p<0.0001, Gehan-Wilcoxon test).
A high proportion of Extended High Viremics – individuals maintaining high plasma HIV-1 RNA load after acute infection – has been identified during primary HIV-1 subtype C infection. These Extended High Viremics likely contribute disproportionately to HIV-1 incidence.
HIV-1 subtype C; primary infection; viral HIV-1 RNA load; Southern Africa; HIV-1 transmission
Accumulating data generated from persons who naturally control HIV without the need for antiretroviral treatment has led to significant insights into the possible mechanisms of durable control of AIDS virus infection. At the center of this control is the HIV-specific CD8 T cell response, and the basis for this CD8-mediated control is gradually being revealed. Genome wide association studies coupled with HLA sequence data implicate the nature of the HLA-viral peptide interaction as the major genetic factor modulating durable control of HIV, but host genetic factors account for only around 20% of the variability in control. Other factors including specific functional characteristics of the TCR clonotypes generated in vivo, targeting of vulnerable regions of the virus that lead to fitness impairing mutations, immune exhaustion, and host restriction factors that limit HIV replication all have been shown to additionally contribute to control. Moreover, emerging data indicate that the CD8+ T cell response may be critical for attempts to purge virus infected cells following activation of the latent reservoir, and thus lessons learned from elite controllers (ECs) are likely to impact the eradication agenda. On-going efforts are also needed to understand and address the role of immune activation in disease progression, as it becomes increasingly clear that durable immune control in ECs comes at a cost. Taken together, the research achievements in the attempt to unlock the mechanisms behind natural control of HIV will continue to be an important source of insights and ideas in the continuous search after an effective HIV vaccine, and for the attempts to achieve a sterilizing or functional cure in HIV positive patients with progressive infection.
HIV; elite controllers; CD8+ T cells; immune activation; HIV vaccine
HIV-1 depends on many host factors for propagation. Other host factors, however, antagonize HIV-1 and may have profound effects on viral activation. Curing HIV-1 requires the reduction of latent viral reservoirs that remain in the face of antiretroviral therapy (ART). Using orthologous genetic screens, we identified bromodomain containing 4 (BRD4) as a negative regulator of HIV-1 replication. Antagonism of BRD4, via RNA interference or with a small molecule inhibitor, JQ1, both increased proviral transcriptional elongation and alleviated HIV-1 latency in cell line models. In multiple instances, JQ1 when used in combination with the NF-κB activators, Prostratin or PHA, enhanced the in vitro reactivation of latent HIV-1 in primary human T cells. These data are consistent with a model wherein BRD4 competes with the virus for HIV-1 dependency factors (HDFs) and suggests that combinatorial therapies that activate HDFs and antagonize HIV-1 competitive factors may be useful for curing HIV-1 infection.
Viruses evade immune detection partly through immune-associated mutations. Analyses of HIV sequences derived from infected persons have identified numerous examples of HLA-associated mutations within or adjacent T cell epitopes, but the potential impact of most mutations on epitope production and presentation remains unclear. The multistep breakdown of proteins into epitopes includes trimming of N-extended peptides into epitopes by aminopeptidases before loading onto MHC-I molecules. Defining sequence signatures that modulate epitope production would lead to a better understanding of factors driving viral evolution and immune escape at the population level. Here we identified cytosolic aminopeptidases cleavage preferences in primary cells, its impact on HIV antigen degradation into epitopes in primary human cell extracts by mass spectrometry, and on epitope presentation to CTL. We observed a hierarchy of preferred amino acid cleavage by cytosolic aminopeptidases. We demonstrated that flanking mutations producing more or less cleavable motifs can increase or decrease epitope production and presentation by up to 14-fold. We found that the efficiency of epitope production correlates with cleavability of flanking residues. These in vitro findings were supported by in vivo population-level analyses of clinically-derived viral sequences from 1134 antiretroviral-naïve HIV-infected persons: HLA-associated mutations immune pressures drove the selection of residues that are less cleavable by aminopeptidases predominantly at N-flanking sites, leading to reduced epitope production and immune recognition. These results underscore an important and widespread role of antigen processing mutations in HIV immune escape and identify molecular mechanisms underlying impaired epitope presentation.
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.