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Latency reversal agents (LRA) variably induce HIV re-expression in CD4 T cells but reservoirs are not cleared. Whether HIV epitope presentation is similar between latency reversal and initial infection of CD4 T cells is unknown yet crucial to define immune responses able to detect HIV-infected CD4 T cells after latency reversal. HIV peptides displayed by MHC comes from the intracellular degradation of proteins by proteasomes and post-proteasomal peptidases but the impact of LRAs on antigen processing is not known. Here we show that HDAC inhibitors (HDCAi) reduced cytosolic proteolytic activities while PKC agonists (PKCa) increased them to a lesser extent than that induced by TCR activation. During the cytosolic degradation of long HIV peptides in LRA-treated CD4 T cells extracts, HDACi and PKCa modulated degradation patterns of peptides and altered the production of HIV epitopes in often opposite ways. Beyond known HIV epitopes, HDACi narrowed the coverage of HIV antigenic fragments by 8-11aa degradation peptides while PKCa broadened it. LRAs altered HIV infection kinetics and modulated CD8 T cell activation in an epitope- and time-dependent manner. Interestingly the efficiency of endogenous epitope processing and presentation to CD8 T cells was increased by PKCa Ingenol at early time points despite low levels of antigens. LRA-induced modulations of antigen processing should be considered and exploited to enhance and broaden HIV peptide presentation by CD4 T cells and to improve immune recognition after latency reversal. This property of LRAs, if confirmed with other antigens, might be exploited to improve immune detection of diseased cells beyond HIV., Competing Interests: The authors have declared that no competing interests exist.

The presentation of virus-derived peptides by MHC molecules constitutes the earliest signals for immune recognition by T cells. In HIV infection, immune responses elicited during infection do not enable to clear infection and correlates of immune protection are not well defined. Here we review features of antigen processing and presentation specific to HIV, analyze how HIV has adapted to the antigen processing machinery and discuss how advances in biochemical and computational protein degradation analyses and in immunopeptidome definition may help identify targets for efficient immune clearance and vaccine immunogen design., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Multiple genome-wide studies have identified associations between outcome of human immunodeficiency virus (HIV) infection and polymorphisms in and around the gene encoding the HIV co-receptor CCR5, but the functional basis for the strongest of these associations, rs1015164A/G, is unknown. We found that rs1015164 marks variation in an activating transcription factor 1 binding site that controls expression of the antisense long noncoding RNA (lncRNA) CCR5AS. Knockdown or enhancement of CCR5AS expression resulted in a corresponding change in CCR5 expression on CD4 + T cells. CCR5AS interfered with interactions between the RNA-binding protein Raly and the CCR5 3' untranslated region, protecting CCR5 messenger RNA from Raly-mediated degradation. Reduction in CCR5 expression through inhibition of CCR5AS diminished infection of CD4 + T cells with CCR5-tropic HIV in vitro. These data represent a rare determination of the functional importance of a genome-wide disease association where expression of a lncRNA affects HIV infection and disease progression.

CD4 T cell activation is critical to the initiation of adaptive immunity. CD4 T cells are also the main targets of HIV infection, and their activation status contributes to the maintenance and outcome of infection. Although the role of activation in the differentiation and proliferation of CD4 T cells is well studied, its impact on the processing and MHC class I (MHC-I) presentation of epitopes and immune recognition by CD8 T cells are not investigated. In this study, we show that the expression and hydrolytic activities of cellular peptidases are increased upon TCR-dependent and MHC-peptide activation of primary CD4 T cells from healthy or HIV-infected persons. Changes in peptidase activities altered the degradation patterns of HIV Ags analyzed by mass spectrometry, modifying the amount of MHC-I epitopes produced, the antigenicity of the degradation products, and the coverage of Ags by degradation peptides presentable by MHC-I. The computational analysis of 2237 degradation peptides generated during the degradation of various HIV-antigenic fragments in CD4 T cells identified cleavage sites that were predictably enhanced, reduced, or unchanged upon cellular activation. Epitope processing and presentation by CD4 T cells may be modulated by the activation state of cells in a sequence-dependent manner. Accordingly, cellular activation modified endogenous Ag processing and presentation and killing of HIV-infected CD4 T cells by CD8 T cells in a way that mirrored differences in in vitro epitope processing. The clearance of HIV-infected cells may rely on different immune responses according to activation state during HIV infection., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Even with sustained antiretroviral therapy, resting CD4 + T cells remain a persistent reservoir of HIV infection, representing a critical barrier to curing HIV. Here, we demonstrate that CD8 + T cells recognize infected, non-activated CD4 + T cells in the absence of de novo protein production, as measured by immune synapse formation, degranulation, cytokine production, and killing of infected cells. Immune recognition is induced by HLA-I presentation of peptides derived from incoming viral particles, and recognition occurred either following cell-free virus infection or following cell-to-cell spread. CD8 + T cells from HIV controllers mediate more effective immune recognition than CD8 + T cells from progressors. These results indicate that non-activated HIV-infected CD4 + T cells can be targeted by CD8 + T cells directly after HIV entry, before reverse transcription, and thus before the establishment of latency, and suggest a mechanism whereby the immune response may reduce the size of the HIV reservoir., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Major histocompatibility class I (MHC-I)-specific inhibitory receptors on natural killer (NK) cells (iNKRs) tolerize mature NK cell responses toward normal cells. NK cells generate cytolytic responses to virus-infected or malignant target cells with altered or decreased MHC-I surface expression due to the loss of tolerizing ligands. The NKG2A/CD94 iNKR suppresses NK cell responses through recognition of the non-classical MHC-I, HLA-E. We used HIV-infected primary T-cells as targets in an in vitro cytolytic assay with autologous NK cells from healthy donors. In these experiments, primary NKG2A/CD94(+) NK cells surprisingly generated the most efficient responses toward HIV-infected T-cells, despite high HLA-E expression on the infected targets. Since certain MHC-I-presented peptides can alter recognition by iNKRs, we hypothesized that HIV-1-derived peptides presented by HLA-E on infected cells may block engagement with NKG2A/CD94, thereby engendering susceptibility to NKG2A/CD94(+) NK cells. We demonstrate that HLA-E is capable of presenting a highly conserved peptide from HIV-1 capsid (AISPRTLNA) that is not recognized by NKG2A/CD94. We further confirmed that HLA-C expressed on HIV-infected cells restricts attack by KIR2DL(+) CD56(dim) NK cells, in contrast to the efficient responses by CD56(bright) NK cells, which express predominantly NKG2A/CD94 and lack KIR2DLs. These findings are important since the use of NK cells was recently proposed to treat latently HIV-1-infected patients in combination with latency reversing agents. Our results provide a mechanistic basis to guide these future clinical studies, suggesting that ex vivo-expanded NKG2A/CD94(+) KIR2DL(-) NK cells may be uniquely beneficial.

Dendritic cells (DCs), macrophages (MPs), and monocytes are permissive to HIV. Whether they similarly process and present HIV epitopes to HIV-specific CD8 T cells is unknown despite the critical role of peptide processing and presentation for recognition and clearance of infected cells. Cytosolic peptidases degrade endogenous proteins originating from self or pathogens, exogenous Ags preprocessed in endolysosomes, thus shaping the peptidome available for endoplasmic reticulum translocation, trimming, and MHC-I presentation. In this study, we compared the capacity of DCs, MPs, and monocyte cytosolic extracts to produce epitope precursors and epitopes. We showed differences in the proteolytic activities and expression levels of cytosolic proteases between monocyte-derived DCs and MPs and upon maturation with LPS, R848, and CL097, with mature MPs having the highest activities. Using cytosol as a source of proteases to degrade epitope-containing HIV peptides, we showed by mass spectrometry that the degradation patterns of long peptides and the kinetics and amount of antigenic peptides produced differed among DCs, MPs, and monocytes. Additionally, variable intracellular stability of HIV peptides prior to loading onto MHC may accentuate the differences in epitope availability for presentation by MHC-I between these subsets. Differences in peptide degradation led to 2- to 25-fold differences in the CTL responses elicited by the degradation peptides generated in DCs, MPs, and monocytes. Differences in Ag-processing activities between these subsets might lead to variations in the timing and efficiency of recognition of HIV-infected cells by CTLs and contribute to the unequal capacity of HIV-specific CTLs to control viral load., (Copyright © 2014 by The American Association of Immunologists, Inc.)

HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24(gag) elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55(gag) increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist.

The ability of cytotoxic T lymphocytes (CTL) to clear virus-infected cells requires the presentation of viral peptides intracellularly processed and displayed by major histocompatibility complex class I. Assays to measure CTL-mediated killing often use peptides exogenously added onto target cells--which does not account for epitope processing--or follow killing of infected cells at a single time point. In this study we established a real-time fluorogenic cytotoxic assay that measures the release of the Glucose-6-phosphate-dehydrogenase by dying target cells every 5 min after addition of CTL. It has comparable sensitivity to (51)chromium-based killing assay with the additional advantage of incorporating the kinetics of epitope presentation. We showed that HIV infection of immortalized or primary CD4 T cells leads to asynchronous killing by two CTL clones specific for epitopes located in different proteins. Real-time monitoring of killing of virus-infected cells will enable identification of immune responses efficiently preventing virus dissemination., (© 2013.)

Viruses evade immune detection partly through immune-associated mutations. Analyses of HIV sequences derived from infected individuals have identified numerous examples of HLA-associated mutations within or adjacent to 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 class I molecules. Definition of sequence signatures that modulate epitope production would lead to a better understanding of factors driving viral evolution and immune escape at the population level. In this study, we identified cytosolic aminopeptidases cleavage preferences in primary cells and its impact on HIV Ag 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-naive HIV-infected individuals: 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 Ag processing mutations in HIV immune escape and identify molecular mechanisms underlying impaired epitope presentation.

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.

Murine models indicate that interleukin-10 (IL-10) can suppress viral clearance, and interventional blockade of IL-10 activity has been proposed to enhance immunity in chronic viral infections. Increased IL-10 levels have been observed during HIV infection and IL-10 blockade has been shown to enhance T-cell function in some HIV-infected subjects. However, the categories of individuals in whom the IL-10 pathway is up-regulated are poorly defined, and the cellular sources of IL-10 in these subjects remain to be determined. Here we report that blockade of the IL-10 pathway augmented in vitro proliferative capacity of HIV-specific CD4 and CD8 T cells in individuals with ongoing viral replication. IL-10 blockade also increased cytokine secretion by HIV-specific CD4 T cells. Spontaneous IL-10 expression, measured as either plasma IL-10 protein or IL-10 mRNA in peripheral blood mononuclear cells (PBMCs), correlated positively with viral load and diminished after successful antiretroviral therapy. IL-10 mRNA levels were up-regulated in multiple PBMC subsets in HIV-infected subjects compared with HIV-negative controls, particularly in T, B, and natural killer (NK) cells, whereas monocytes were a major source of IL-10 mRNA in HIV-infected and -uninfected individuals. These data indicate that multiple cell types contribute to IL-10-mediated immune suppression in the presence of uncontrolled HIV viremia.

Human leukocyte antigen (HLA)-B27-positive subjects are uncommon in their ability to control infection with human immunodeficiency virus type 1 (HIV-1). However, late viral escape from a narrowly directed immunodominant Gag-specific CD8(+) T-lymphocyte (CTL) response has been linked to AIDS progression in these individuals. Identifying the mechanism of the immune-mediated control may provide critical insights into HIV-1 vaccine development. Here, we illustrate that the CTL escape mutation R(264)K in the HLA-B27-restricted KK10 epitope in the capsid resulted in a significant defect in viral replication in vitro. The R(264)K variant was impaired in generating late reverse transcription products, indicating that replication was blocked at a postentry step. Notably, the R(264)K mutation was associated in vivo with the development of a rare secondary mutation, S(173)A, which restored viral replication in vitro. Furthermore, infectivity of the R(264)K variant was rescued by the addition of cyclosporine A or infection of a cyclophilin A-deficient cell line. These data demonstrate a severe functional defect imposed by the R(264)K mutation during an early step in viral replication that is likely due to the inability of this variant to replicate efficiently in the presence of normal levels of cyclophilin A. We conclude that the impact of the R(264)K substitution on capsid structure constrains viral escape and enables long-term maintenance of the dominant CTL response against B27-KK10, providing an explanation for the protective effect of HLA-B27 during HIV infection.

In progressive viral infection, antiviral T cell function is impaired by poorly understood mechanisms. Here we report that the inhibitory immunoregulatory receptor CTLA-4 was selectively upregulated in human immunodeficiency virus (HIV)-specific CD4(+) T cells but not CD8(+) T cells in all categories of HIV-infected subjects evaluated, with the exception of rare people able to control viremia in the absence of antiretroviral therapy. CTLA-4 expression correlated positively with disease progression and negatively with the capacity of CD4(+) T cells to produce interleukin 2 in response to viral antigen. Most HIV-specific CD4(+) T cells coexpressed CTLA-4 and another inhibitory immunoregulatory receptor, PD-1. In vitro blockade of CTLA-4 augmented HIV-specific CD4(+) T cell function. These data, indicating a reversible immunoregulatory pathway selectively associated with CD4(+) T cell dysfunction, provide a potential target for immunotherapy in HIV-infected patients.

Transfection with synthetic mRNA is a safe and efficient method of delivering antigens to dendritic cells for immunotherapy. Targeting antigens to the lysosome can sometimes enhance the CD4+ T-cell response. We transfected antigen-presenting cells (APCs) with mRNA encoding Gag-p24 and cytoplasmic, lysosomal, and secreted forms of Nef. Antigen-specific cytotoxic T cells were able to lyse the majority of transfected targets, indicating that transfection was efficient. Transfection of APCs with a Nef construct bearing lysosomal targeting signals produced rapid and prolonged antigen presentation to CD4+ and CD8+ T cells. Polyclonal CD4+ and CD8+ T-cell lines recognizing multiple distinct epitopes were expanded by coculture of transfected dendritic cells with peripheral blood mononuclear cells from viremic and aviremic HIV-infected subjects. Importantly, lysosome-targeted antigen drove a significantly greater expansion of Nef-specific CD4+ T cells than cytoplasmic antigen. The frequency of recognition of CD8 but not CD4 epitopes by mRNA-expanded T cells was inversely proportional to sequence entropy and was similar to ex vivo responses from a large chronic cohort. Thus human dendritic cells transfected with mRNA encoding lysosome-targeted HIV antigen can expand a broad, polyclonal repertoire of antiviral T cells, offering a promising approach to HIV immunotherapy.

Human immunodeficiency virus (HIV)-1 amino acid sequence polymorphisms associated with expression of specific human histocompatibility leukocyte antigen (HLA) class I alleles suggest sites of cytotoxic T lymphocyte (CTL)-mediated selection pressure and immune escape. The associations most frequently observed are between expression of an HLA class I molecule and variation from the consensus sequence. However, a substantial number of sites have been identified in which particular HLA class I allele expression is associated with preservation of the consensus sequence. The mechanism behind this is so far unexplained. The current studies, focusing on two examples of "negatively associated" or apparently preserved epitopes, suggest an explanation for this phenomenon: negative associations can arise as a result of positive selection of an escape mutation, which is stable on transmission and therefore accumulates in the population to the point at which it defines the consensus sequence. Such negative associations may only be in evidence transiently, because the statistical power to detect them diminishes as the mutations accumulate. If an escape variant reaches fixation in the population, the epitope will be lost as a potential target to the immune system. These data help to explain how HIV is evolving at a population level. Understanding the direction of HIV evolution has important implications for vaccine development.

The development of HIV-1-specific CD8(+) T-cell responses during acute HIV-1 infection is associated with a dramatic decline in HIV-1 replication and the resolution of the acute retroviral syndrome. These HIV-1-specific CD8(+) T cells typically target a small number of viral epitopes in a distinct hierarchical order, and high-level viremia in chronic progressive infection leads to broadly diversified HIV-1-specific CD8(+) T-cell responses with a less clear immunodominance pattern. It is argued here that the specific hierarchical pattern of immune responses in acute HIV-1 infection is the result of a tightly regulated process that, among other factors, is critically impacted by the kinetics of viral protein expression, the HLA class I background of the infected individual and the autologous sequence of the infecting virus.

Mutations within cytotoxic T lymphocyte (CTL) epitopes impair T cell recognition, but escape mutations arising in flanking regions that alter antigen processing have not been defined in natural human infections. In human histocompatibility leukocyte antigen (HLA)-B57+ HIV-infected persons, immune selection pressure leads to a mutation from alanine to proline at Gag residue 146 immediately preceding the NH2 terminus of a dominant HLA-B57-restricted epitope, ISPRTLNAW. Although N-extended wild-type or mutant peptides remained well-recognized, mutant virus-infected CD4 T cells failed to be recognized by the same CTL clones. The A146P mutation prevented NH2-terminal trimming of the optimal epitope by the endoplasmic reticulum aminopeptidase I. These results demonstrate that allele-associated sequence variation within the flanking region of CTL epitopes can alter antigen processing. Identifying such mutations is of major relevance in the construction of vaccine sequences.