Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction.

The implementation and access to combined antiretroviral treatment (cART) have dramatically improved the quality of life of people living with HIV (PLWH). However, some comorbidities, such as neurological disorders associated with HIV infection still represent a serious clinical challenge. Soluble factors in plasma that are associated with control of HIV replication and neurological dysfunction could serve as early biomarkers and as new therapeutic targets for this comorbidity. We used a customized antibody array for determination of blood plasma factors in 40 untreated PLWH with different levels of viremia and found sirtuin-2 (SIRT2), an NAD-dependent deacetylase, to be strongly associated with elevated viral loads and HIV provirus levels, as well as with markers of neurological damage (a-synuclein [SNCA], brain-derived neurotrophic factor [BDNF], microtubule-associated protein tau [MAPT], and neurofilament light protein [NFL]). Also, longitudinal analysis in HIV-infected individuals with immediate (n = 9) or delayed initiation (n = 10) of cART revealed that after 1 year on cART, SIRT2 plasma levels differed between both groups and correlated inversely with brain orbitofrontal cortex involution. Furthermore, targeting SIRT2 with specific small-molecule inhibitors in in vitro systems using J-LAT A2 and primary glial cells led to diminished HIV replication and virus reactivation from latency. Our data thus identify SIRT2 as a novel biomarker of uncontrolled HIV infection, with potential impact on neurological dysfunction and offers a new therapeutic target for HIV treatment and cure. IMPORTANCE Neurocognitive disorders are frequently reported in people living with HIV (PLWH) even with the introduction of combined antiretroviral treatment (cART). To identify biomarkers and potential therapeutic tools to target HIV infection in peripheral blood and in the central nervous system (CNS), plasma proteomics were applied in untreated chronic HIV-infected individuals with different levels of virus control. High plasma levels of sirtuin-2 (SIRT2), an NAD+ deacetylase, were detected in uncontrolled HIV infection and were strongly associated with plasma viral load and proviral levels. In parallel, SIRT2 levels in the peripheral blood and CNS were associated with markers of neurological damage and brain involution and were more pronounced in individuals who initiated cART later in infection. In vitro infection experiments using specific SIRT2 inhibitors suggest that specific targeting of SIRT2 could offer new therapeutic treatment options for HIV infections and their associated neurological dysfunction.

Potent Induction of Envelope-Specific Antibody Responses by Virus-Like Particle Immunogens Based on HIV-1 Envelopes from Patients with Early Broadly Neutralizing Responses.

Longitudinal studies in HIV-1-infected individuals have indicated that 2 to 3 years of infection are required to develop broadly neutralizing antibodies. However, we have previously identified individuals with broadly neutralizing activity (bNA) in early HIV-1 infection, indicating that a vaccine may be capable of bNA induction after short periods of antigen exposure. Here, we describe 5 HIV-1 envelope sequences from individuals who have developed bNA within the first 100 days of infection (early neutralizers) and selected two of them to design immunogens based on HIV-1-Gag virus-like particles (VLPs). These VLPs were homogeneous and incorporated the corresponding envelopes (7 to 9 µg of gp120 in 1010 VLPs). Both envelopes (Envs) bound to well-characterized broadly neutralizing antibodies (bNAbs), including trimer-specific antibodies (PGT145, VRC01, and 35022). For immunogenicity testing, we immunized rabbits with the Env-VLPs or with the corresponding stabilized soluble envelope trimers. A short immunization protocol (105 days) was used to recapitulate the early nAb induction observed after HIV-1 infection in these two individuals. All VLP and trimeric envelope immunogens induced a comparably strong anti-gp120 response despite having immunized rabbits with 30 times less gp120 in the case of the Env-VLPs. In addition, animals immunized with VLP-formulated Envs induced antibodies that cross-recognized the corresponding soluble stabilized trimer and vice versa, even though no neutralizing activity was observed. Nevertheless, our data may provide a new platform of immunogens, based on HIV-1 envelopes from patients with early broadly neutralizing responses, with the potential to generate protective immune responses using vaccination protocols similar to those used in classical preventive vaccines. IMPORTANCE It is generally accepted that an effective HIV-1 vaccine should be able to induce broad-spectrum neutralizing antibodies. Since most of these antibodies require long periods of somatic maturation in vivo, several groups are developing immunogens, based on the HIV envelope protein, that require complex and lengthy immunization protocols that would be difficult to implement in the general population. Here, we show that rabbits immunized with new envelopes (VLP formulated) from two individuals who demonstrated broadly neutralizing activity very early after infection induced specific HIV-1 antibodies after a short immunization protocol. This evidence provides the basis for generating protective immune responses with classic vaccination protocols with vaccine prototypes based on HIV envelope sequences from individuals who have developed early broadly neutralizing responses.

An HLA-I signature favouring KIR-educated Natural Killer cells mediates immune control of HIV in children and contrasts with the HLA-B-restricted CD8+ T-cell-mediated immune control in adults.

Natural Killer (NK) cells contribute to HIV control in adults, but HLA-B-mediated T-cell activity has a more substantial impact on disease outcome. However, the HLA-B molecules influencing immune control in adults have less impact on paediatric infection. To investigate the contribution NK cells make to immune control, we studied >300 children living with HIV followed over two decades in South Africa. In children, HLA-B alleles associated with adult protection or disease-susceptibility did not have significant effects, whereas Bw4 (p = 0.003) and low HLA-A expression (p = 0.002) alleles were strongly associated with immunological and viral control. In a comparator adult cohort, Bw4 and HLA-A expression contributions to HIV disease outcome were dwarfed by those of protective and disease-susceptible HLA-B molecules. We next investigated the immunophenotype and effector functions of NK cells in a subset of these children using flow cytometry. Slow progression and better plasma viraemic control were also associated with high frequencies of less terminally differentiated NKG2A+NKp46+CD56dim NK cells strongly responsive to cytokine stimulation and linked with the immunogenetic signature identified. Future studies are indicated to determine whether this signature associated with immune control in early life directly facilitates functional cure in children.

A minor population of macrophage-tropic HIV-1 variants is identified in recrudescing viremia following analytic treatment interruption.

HIV-1 persists in cellular reservoirs that can reignite viremia if antiretroviral therapy (ART) is interrupted. Therefore, insight into the nature of those reservoirs may be revealed from the composition of recrudescing viremia following treatment cessation. A minor population of macrophage-tropic (M-tropic) viruses was identified in a library of recombinant viruses constructed with individual envelope genes that were obtained from plasma of six individuals undergoing analytic treatment interruption (ATI). M-tropic viruses could also be enriched from post-ATI plasma using macrophage-specific (CD14) but not CD4+ T cell-specific (CD3) antibodies, suggesting that M-tropic viruses had a macrophage origin. Molecular clock analysis indicated that the establishment of M-tropic HIV-1 variants predated ATI. Collectively, these data suggest that macrophages are a viral reservoir in HIV-1-infected individuals on effective ART and that M-tropic variants can appear in rebounding viremia when treatment is interrupted. These findings have implications for the design of curative strategies for HIV-1.

Limited Humoral and Specific T-Cell Responses After SARS-CoV-2 Vaccination in PWH With Poor Immune Reconstitution.

BACKGROUND: We analyzed humoral and cellular immune responses induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) messenger RNA (mRNA) vaccines in people with human immunodeficiency virus (HIV; PWH) who had CD4+ T-cell counts <200/µL (HIV<200 group). METHODS: This prospective cohort study included 58 PWH in the HIV<200 group, 36 with CD4+ T-cell counts >500/µL (HIV>500 group), and 33 HIV-1-negative controls (control group). Antibodies against the SARS-CoV-2 spike protein (anti-S immunoglobulin [Ig] G) and the receptor-binding domain (anti-RBD IgG) were quantified before and 4 weeks after the first and the second doses of BNT162b2 or mRNA-1273 (at week 8). Viral neutralization activity and T-cell responses were also determined. RESULTS: At week 8, anti-S/anti-RBD IgG responses increased in all groups (P < .001). Median (interquartile range) anti-S and anti-RBD IgG levels at week 8 were 153.6 (26.4-654.9) and 171.9 (61.8-425.8) binding antibody units (BAU)/mL, respectively, in the HIV<200 group, compared with 245.6 (145-824) and 555.8 (166.4-1751) BAU/mL in the HIV>500 group and 274.7 (193.7-680.4) and 281.6 (181-831.8) BAU/mL in controls (P < .05). Neutralizing capacity and specific T-cell immune responses were absent or reduced in 33% of those in the HIV<200 group, compared with 3.7% in the HIV>500 group (P < .01). CONCLUSIONS: One-third of PWH with CD4+ T-cell counts <200/µL show low anti-S/anti-RBD IgG levels, reduced in vitro neutralization activity against SARS-CoV-2, and no vaccine-induced T cells after receiving coronavirus disease 2019 mRNA vaccines.

Methylation regulation of Antiviral host factors, Interferon Stimulated Genes (ISGs) and T-cell responses associated with natural HIV control.

GWAS, immune analyses and biomarker screenings have identified host factors associated with in vivo HIV-1 control. However, there is a gap in the knowledge about the mechanisms that regulate the expression of such host factors. Here, we aimed to assess DNA methylation impact on host genome in natural HIV-1 control. To this end, whole DNA methylome in 70 untreated HIV-1 infected individuals with either high (>50,000 HIV-1-RNA copies/ml, n = 29) or low (<10,000 HIV-1-RNA copies/ml, n = 41) plasma viral load (pVL) levels were compared and identified 2,649 differentially methylated positions (DMPs). Of these, a classification random forest model selected 55 DMPs that correlated with virologic (pVL and proviral levels) and HIV-1 specific adaptive immunity parameters (IFNg-T cell responses and neutralizing antibodies capacity). Then, cluster and functional analyses identified two DMP clusters: cluster 1 contained hypo-methylated genes involved in antiviral and interferon response (e.g. PARP9, MX1, and USP18) in individuals with high viral loads while in cluster 2, genes related to T follicular helper cell (Tfh) commitment (e.g. CXCR5 and TCF7) were hyper-methylated in the same group of individuals with uncontrolled infection. For selected genes, mRNA levels negatively correlated with DNA methylation, confirming an epigenetic regulation of gene expression. Further, these gene expression signatures were also confirmed in early and chronic stages of infection, including untreated, cART treated and elite controllers HIV-1 infected individuals (n = 37). These data provide the first evidence that host genes critically involved in immune control of the virus are under methylation regulation in HIV-1 infection. These insights may offer new opportunities to identify novel mechanisms of in vivo virus control and may prove crucial for the development of future therapeutic interventions aimed at HIV-1 cure.

TL1A-DR3 Plasma Levels Are Predictive of HIV-1 Disease Control, and DR3 Costimulation Boosts HIV-1-Specific T Cell Responses.

Relative control of HIV-1 infection has been linked to genetic and immune host factors. In this study, we analyzed 96 plasma proteome arrays from chronic untreated HIV-1-infected individuals using the classificatory random forest approach to discriminate between uncontrolled disease (plasma viral load [pVL] >50,000 RNA copies/ml; CD4 counts 283 cells/mm3, n = 47) and relatively controlled disease (pVL <10,000 RNA copies/ml; CD4 counts 657 cells/mm3, n = 49). Our analysis highlighted the TNF molecule's relevance, in particular, TL1A (TNFSF15) and its cognate DR3 (TNFSRF25), both of which increased in the relative virus control phenotype. DR3 levels (in plasma and PBMCs) were validated in unrelated cohorts (including long-term nonprogressors), thus confirming their independence from CD4 counts and pVL. Further analysis in combined antiretroviral treatment (cART)-treated individuals with a wide range of CD4 counts (137-1835 cells/mm3) indicated that neither TL1A nor DR3 levels reflected recovery of CD4 counts with cART. Interestingly, in cART-treated individuals, plasma TL1A levels correlated with regulatory T cell frequencies, whereas soluble DR3 was strongly associated with the abundance of effector HLA-DR+CD8+ T cells. A positive correlation was also observed between plasma DR3 levels and the HIV-1-specific T cell responses. In vitro, costimulation of PBMC with DR3-specific mAb increased the magnitude of HIV-1-specific responses. Finally, in splenocytes of DNA.HTI-vaccinated mice, costimulation of HTI peptides and a DR3 agonist (4C12) intensified the magnitude of T cell responses by 27%. These data describe the role of the TL1A-DR3 axis in the natural control of HIV-1 infection and point to the use of DR3 agonists in HIV-1 vaccine regimens.

The Genome-wide Methylation Profile of CD4+ T Cells From Individuals With Human Immunodeficiency Virus (HIV) Identifies Distinct Patterns Associated With Disease Progression.

BACKGROUND: Human genetic variation-mostly in the human leukocyte antigen (HLA) and C-C chemokine receptor type 5 (CCR5) regions-explains 25% of the variability in progression of human immunodeficiency virus (HIV) infection. However, it is also known that viral infections can modify cellular DNA methylation patterns. Therefore, changes in the methylation of cytosine-guanine (CpG) islands might modulate progression of HIV infection. METHODS: In total, 85 samples were analyzed: 21 elite controllers (EC), 21 subjects with HIV before combination antiretroviral therapy (cART) (viremic, 93 325 human immunodeficiency virus type 1 [HIV-1] RNA copies/mL) and under suppressive cART (cART, median of 17 months, <50 HIV-1 RNA copies/mL), and 22 HIV-negative donors (HIVneg). We analyzed the methylation pattern of 485 577 CpG in DNA from peripheral CD4+ T lymphocytes. We selected the most differentially methylated gene (TNF) and analyzed its specific methylation, messenger RNA (mRNA) expression, and plasma protein levels in 5 individuals before and after initiation of cART. RESULTS: We observed 129 methylated CpG sites (associated with 43 gene promoters) for which statistically significant differences were recorded in viremic versus HIVneg, 162 CpG sites (55 gene promoters) in viremic versus cART, 441 CpG sites (163 gene promoters) in viremic versus EC, but none in EC versus HIVneg. The TNF promoter region was hypermethylated in viremic versus HIVneg, cART, and EC. Moreover, we observed greater plasma levels of TNF in viremic individuals than in EC, cART, and HIVneg. CONCLUSIONS: Our study shows that genome methylation patterns vary depending on HIV infection status and progression profile and that these variations might have an impact on controlling HIV infection in the absence of cART.

Pharmacokinetic/pharmacodynamic analysis of romidepsin used as an HIV latency reversing agent.

OBJECTIVES: To develop a population pharmacokinetic model for romidepsin given as an HIV latency reversing agent (LRA) and to explore the relationship between romidepsin exposure and its in vivo effects on viral gene expression and antiviral immunity. METHODS: A population pharmacokinetic analysis was performed in 15 HIV-1-infected patients who received three weekly infusions of romidepsin (5 mg/m2) within the BCN02 clinical trial. A full pharmacokinetic profile was obtained for each participant at the first dose, and additional samples thereafter. A population pharmacokinetic model was developed. Bayesian estimates of the individual pharmacokinetic parameters of romidepsin were used to simulate individual time-concentration curves on each occasion. The relationship between romidepsin AUC0-∞ and its in vivo effects was assessed. RESULTS: Romidepsin pharmacokinetics were best described by a three-compartment model with linear kinetics. Body weight influenced romidepsin disposition. A significant relationship was observed between romidepsin AUC0-∞ and increases in expression of exhaustion markers by CD4+ and CD8+ T cells and apoptosis markers in CD4+, but not with histone acetylation levels or HIV-1 cell-associated RNA in CD4+ T cells. For each increase of 100 ng·h/mL in romidepsin AUC0-∞, CD4+ counts decreased by a mean (95% CI) of 74 (42-94) cells/mm3 after dosing. CONCLUSIONS: A population model describing the pharmacokinetics of romidepsin as an HIV LRA was developed. Higher exposure to romidepsin resulted in higher expression of apoptosis markers and declines in CD4+ count but did not increase viral reactivation levels. These observations have important implications for the optimization of effective kick-and-kill strategies for an HIV-1 cure.

Zip6 Transporter Is an Essential Component of the Lymphocyte Activation Machinery.

Zinc deficiency causes immune dysfunction. In T lymphocytes, hypozincemia promotes thymus atrophy, polarization imbalance, and altered cytokine production. Zinc supplementation is commonly used to boost immune function to prevent infectious diseases in at-risk populations. However, the molecular players involved in zinc homeostasis in lymphocytes are poorly understood. In this paper, we wanted to determine the identity of the transporter responsible for zinc entry into lymphocytes. First, in human Jurkat cells, we characterized the effect of zinc on proliferation and activation and found that zinc supplementation enhances activation when T lymphocytes are stimulated using anti-CD3/anti-CD28 Abs. We show that zinc entry depends on specific pathways to correctly tune the NFAT, NF-κB, and AP-1 activation cascades. Second, we used various human and murine models to characterize the zinc transporter family, Zip, during T cell activation and found that Zip6 was strongly upregulated early during activation. Therefore, we generated a Jurkat Zip6 knockout (KO) line to study how the absence of this transporter affects lymphocyte physiology. We found that although Zip6KO cells showed no altered zinc transport or proliferation under basal conditions, under activation, these KO cells showed deficient zinc transport and a drastically impaired activation program. Our work shows that zinc entry into activated lymphocytes depends on Zip6 and that this transporter is essential for the correct function of the cellular activation machinery.

HIV-1 DNA decay dynamics in early treated individuals: practical considerations for clinical trial design.

BACKGROUND: Initiation of combination antiretroviral therapy (cART) soon after HIV-1 infection limits the establishment of viral reservoirs. Thus, early treated individuals are preferred candidates to evaluate novel viral remission strategies. However, their cART-dependent HIV-1 DNA decay dynamics are still poorly defined. This can hamper the design and interpretation of results from clinical trials intended to further reduce viral reservoirs. OBJECTIVES: To clarify the duration of cART needed for the HIV-1 reservoir to be stabilized in early treated individuals. METHODS: We characterized the longitudinal decline of total HIV-1 DNA levels by droplet digital PCR in 21 individuals initiating cART within 6 months after estimated HIV-1 acquisition. Measurements were taken at cART initiation, after 6 months and annually until Year 4. Correlations between virological and clinical parameters were statistically analysed. Statistical modelling was performed applying a mixed-effects model. RESULTS: Total HIV-1 DNA experienced a median overall decrease of 1.43 log10 units (IQR = 1.17-1.69) throughout the 4 years of follow-up. Baseline levels for total HIV-1 DNA, viral load, absolute CD4+ T cell count and CD4+/CD8+ ratio correlate with final HIV-1 DNA measurements (R2 = 0.68, P < 0.001; R2 = 0.54, P = 0.012; R2 = -0.47, P = 0.031; and R2 = -0.59, P = 0.0046, respectively). Statistical modelling shows that after 2 years on cART the viral reservoir had reached a set point. CONCLUSIONS: A waiting period of 2 years on cART should be considered when designing interventions aiming to impact latent HIV-1 reservoir levels and viral rebound kinetics after cART discontinuation, in order to facilitate interpretation of results and enhance the chance of viral control.

Mechanisms of Abrupt Loss of Virus Control in a Cohort of Previous HIV Controllers.

Elite and viremic HIV controllers are able to control their HIV infection and maintain undetectable or low-level viremia in the absence of antiretroviral treatment. Despite extensive studies, the immune factors responsible for such exclusive control remain poorly defined. We identified a cohort of 14 HIV controllers that suffered an abrupt loss of HIV control (LoC) to investigate possible mechanisms and virological and immunological events related to the sudden loss of control. The in-depth analysis of these subjects involved the study of cell tropism of circulating virus, evidence for HIV superinfection, cellular immune responses to HIV, as well as an examination of viral adaptation to host immunity by Gag sequencing. Our data demonstrate that a poor capacity of T cells to mediate in vitro viral suppression, even in the context of protective HLA alleles, predicts a loss of viral control. In addition, the data suggest that inefficient viral control may be explained by an increase of CD8 T-cell activation and exhaustion before LoC. Furthermore, we detected a switch from C5- to X4-tropic viruses in 4 individuals after loss of control, suggesting that tropism shift might also contribute to disease progression in HIV controllers. The significantly reduced inhibition of in vitro viral replication and increased expression of activation and exhaustion markers preceding the abrupt loss of viral control may help identify untreated HIV controllers that are at risk of losing control and may offer a useful tool for monitoring individuals during treatment interruption phases in therapeutic vaccine trials.IMPORTANCE A few individuals can control HIV infection without the need for antiretroviral treatment and are referred to as HIV controllers. We have studied HIV controllers who suddenly lose this ability and present with high in vivo viral replication and decays in their CD4+ T-cell counts to identify potential immune and virological factors that were responsible for initial virus control. We identify in vitro-determined reductions in the ability of CD8 T cells to suppress viral control and the presence of PD-1-expressing CD8+ T cells with a naive immune phenotype as potential predictors of in vivo loss of virus control. The findings could be important for the clinical management of HIV controller individuals, and it may offer an important tool to anticipate viral rebound in individuals in clinical studies that include combination antiretroviral therapy (cART) treatment interruptions and which, if not treated quickly, could pose a significant risk to the trial participants.

Differential Immunodominance Hierarchy of CD8+ T-Cell Responses in HLA-B*27:05- and -B*27:02-Mediated Control of HIV-1 Infection.

The well-characterized association between HLA-B*27:05 and protection against HIV disease progression has been linked to immunodominant HLA-B*27:05-restricted CD8+ T-cell responses toward the conserved Gag KK10 (residues 263 to 272) and polymerase (Pol) KY9 (residues 901 to 909) epitopes. We studied the impact of the 3 amino acid differences between HLA-B*27:05 and the closely related HLA-B*27:02 on the HIV-specific CD8+ T-cell response hierarchy and on immune control of HIV. Genetic epidemiological data indicate that both HLA-B*27:02 and HLA-B*27:05 are associated with slower disease progression and lower viral loads. The effect of HLA-B*27:02 appeared to be consistently stronger than that of HLA-B*27:05. In contrast to HLA-B*27:05, the immunodominant HIV-specific HLA-B*27:02-restricted CD8+ T-cell response is to a Nef epitope (residues 142 to 150 [VW9]), with Pol KY9 subdominant and Gag KK10 further subdominant. This selection was driven by structural differences in the F pocket, mediated by a polymorphism between these two HLA alleles at position 81. Analysis of autologous virus sequences showed that in HLA-B*27:02-positive subjects, all three of these CD8+ T-cell responses impose selection pressure on the virus, whereas in HLA-B*27:05-positive subjects, there is no Nef VW9-mediated selection pressure. These studies demonstrate that HLA-B*27:02 mediates protection against HIV disease progression that is at least as strong as or stronger than that mediated by HLA-B*27:05. In combination with the protective Gag KK10 and Pol KY9 CD8+ T-cell responses that dominate HIV-specific CD8+ T-cell activity in HLA-B*27:05-positive subjects, a Nef VW9-specific response is additionally present and immunodominant in HLA-B*27:02-positive subjects, mediated through a polymorphism at residue 81 in the F pocket, that contributes to selection pressure against HIV.IMPORTANCE CD8+ T cells play a central role in successful control of HIV infection and have the potential also to mediate the eradication of viral reservoirs of infection. The principal means by which protective HLA class I molecules, such as HLA-B*27:05 and HLA-B*57:01, slow HIV disease progression is believed to be via the particular HIV-specific CD8+ T cell responses restricted by those alleles. We focus here on HLA-B*27:05, one of the best-characterized protective HLA molecules, and the closely related HLA-B*27:02, which differs by only 3 amino acids and which has not been well studied in relation to control of HIV infection. We show that HLA-B*27:02 is also protective against HIV disease progression, but the CD8+ T-cell immunodominance hierarchy of HLA-B*27:02 differs strikingly from that of HLA-B*27:05. These findings indicate that the immunodominant HLA-B*27:02-restricted Nef response adds to protection mediated by the Gag and Pol specificities that dominate anti-HIV CD8+ T-cell activity in HLA-B*27:05-positive subjects.

HIV T-Cell Vaccines.

Some of the strongest immune correlates of controlled HIV infection include markers related to antiviral T-cell responses, especially responses mediated by CD8+ cytotoxic T lymphocytes (CTL). These observations and lessons learned from other viral infections have motivated the development of T-cell vaccine candidates to HIV in the preventive and especially in the therapeutic setting. While none of the T-cell vaccine concepts tested to date have shown sufficient efficacy, the last few years have seen some advances indicating that strategies activating the cellular adaptive immune response to HIV may present a critical component of an effective therapeutic and possibly preventive HIV vaccine. Some of the important components that a successful T-cell vaccine may need to contain and additional considerations for the design and delivery of such vaccines are discussed in this chapter.

Defining the HLA class I-associated viral antigen repertoire from HIV-1-infected human cells.

Recognition and eradication of infected cells by cytotoxic T lymphocytes is a key defense mechanism against intracellular pathogens. High-throughput definition of HLA class I-associated immunopeptidomes by mass spectrometry is an increasingly important analytical tool to advance our understanding of the induction of T-cell responses against pathogens such as HIV-1. We utilized a liquid chromatography tandem mass spectrometry workflow including de novo-assisted database searching to define the HLA class I-associated immunopeptidome of HIV-1-infected human cells. We here report for the first time the identification of 75 HIV-1-derived peptides bound to HLA class I complexes that were purified directly from HIV-1-infected human primary CD4(+) T cells and the C8166 human T-cell line. Importantly, one-third of eluted HIV-1 peptides had not been previously known to be presented by HLA class I. Over 82% of the identified sequences originated from viral protein regions for which T-cell responses have previously been reported but for which the precise HLA class I-binding sequences have not yet been defined. These results validate and expand the current knowledge of virus-specific antigenic peptide presentation during HIV-1 infection and provide novel targets for T-cell vaccine development.

Identification of effective subdominant anti-HIV-1 CD8+ T cells within entire post-infection and post-vaccination immune responses.

Defining the components of an HIV immunogen that could induce effective CD8+ T cell responses is critical to vaccine development. We addressed this question by investigating the viral targets of CD8+ T cells that potently inhibit HIV replication in vitro, as this is highly predictive of virus control in vivo. We observed broad and potent ex vivo CD8+ T cell-mediated viral inhibitory activity against a panel of HIV isolates among viremic controllers (VC, viral loads <5000 copies/ml), in contrast to unselected HIV-infected HIV Vaccine trials Network (HVTN) participants. Viral inhibition of clade-matched HIV isolates was strongly correlated with the frequency of CD8+ T cells targeting vulnerable regions within Gag, Pol, Nef and Vif that had been identified in an independent study of nearly 1000 chronically infected individuals. These vulnerable and so-called "beneficial" regions were of low entropy overall, yet several were not predicted by stringent conservation algorithms. Consistent with this, stronger inhibition of clade-matched than mismatched viruses was observed in the majority of subjects, indicating better targeting of clade-specific than conserved epitopes. The magnitude of CD8+ T cell responses to beneficial regions, together with viral entropy and HLA class I genotype, explained up to 59% of the variation in viral inhibitory activity, with magnitude of the T cell response making the strongest unique contribution. However, beneficial regions were infrequently targeted by CD8+ T cells elicited by vaccines encoding full-length HIV proteins, when the latter were administered to healthy volunteers and HIV-positive ART-treated subjects, suggesting that immunodominance hierarchies undermine effective anti-HIV CD8+ T cell responses. Taken together, our data support HIV immunogen design that is based on systematic selection of empirically defined vulnerable regions within the viral proteome, with exclusion of immunodominant decoy epitopes that are irrelevant for HIV control.

Consequences of HLA-B*13-Associated Escape Mutations on HIV-1 Replication and Nef Function.

UNLABELLED: HLA-B*13 is associated with superior in vivo HIV-1 viremia control. Protection is thought to be mediated by sustained targeting of key cytotoxic T lymphocyte (CTL) epitopes and viral fitness costs of CTL escape in Gag although additional factors may contribute. We assessed the impact of 10 published B*13-associated polymorphisms in Gag, Pol, and Nef, in 23 biologically relevant combinations, on HIV-1 replication capacity and Nef-mediated reduction of cell surface CD4 and HLA class I expression. Mutations were engineered into HIV-1NL4.3, and replication capacity was measured using a green fluorescent protein (GFP) reporter T cell line. Nef-mediated CD4 and HLA-A*02 downregulation was assessed by flow cytometry, and T cell recognition of infected target cells was measured via coculture with an HIV-specific luciferase reporter cell line. When tested individually, only Gag-I147L and Gag-I437L incurred replicative costs (5% and 17%, respectively), consistent with prior reports. The Gag-I437L-mediated replication defect was rescued to wild-type levels by the adjacent K436R mutation. A novel B*13 epitope, comprising 8 residues and terminating at Gag147, was identified in p24(Gag) (GQMVHQAIGag140-147). No other single or combination Gag, Pol, or Nef mutant impaired viral replication. Single Nef mutations did not affect CD4 or HLA downregulation; however, the Nef double mutant E24Q-Q107R showed 40% impairment in HLA downregulation with no evidence of Nef stability defects. Moreover, target cells infected with HIV-1-NefE24Q-Q107R were recognized better by HIV-specific T cells than those infected with HIV-1NL4.3 or single Nef mutants. Our results indicate that CTL escape in Gag and Nef can be functionally costly and suggest that these effects may contribute to long-term HIV-1 control by HLA-B*13. IMPORTANCE: Protective effects of HLA-B*13 on HIV-1 disease progression are mediated in part by fitness costs of CTL escape mutations in conserved Gag epitopes, but other mechanisms remain incompletely known. We extend our knowledge of the impact of B*13-driven escape on HIV-1 replication by identifying Gag-K436R as a compensatory mutation for the fitness-costly Gag-I437L. We also identify Gag-I147L, the most rapidly and commonly selected B*13-driven substitution in HIV-1, as a putative C-terminal anchor residue mutation in a novel B*13 epitope. Most notably, we identify a novel escape-driven fitness defect: B*13-driven substitutions E24Q and Q107R in Nef, when present together, substantially impair this protein's ability to downregulate HLA class I. This, in turn, increases the visibility of infected cells to HIV-specific T cells. Our results suggest that B*13-associated escape mutations impair HIV-1 replication by two distinct mechanisms, that is, by reducing Gag fitness and dampening Nef immune evasion function.

Immune screening identifies novel T cell targets encoded by antisense reading frames of HIV-1.

Cytotoxic-T lymphocyte (CTL) responses to epitopes in alternative HIV reading frames have been reported. However, the extent of CTL responses to putative proteins encoded in antisense reading frames is unknown. Using sequence alignments and computational approaches, we here predict five potential antisense HIV proteins and characterize common CTL responses against them. Results suggest that antisense-derived sequences are commonly transcribed and translated and could encode functional proteins that contain important targets of anti-HIV cellular immunity.

Cell cycle control and HIV-1 susceptibility are linked by CDK6-dependent CDK2 phosphorylation of SAMHD1 in myeloid and lymphoid cells.

Proliferating cells are preferentially susceptible to infection by retroviruses. Sterile α motif and HD domain-containing protein-1 (SAMHD1) is a recently described deoxynucleotide phosphohydrolase controlling the size of the intracellular deoxynucleotide triphosphate (dNTP) pool, a limiting factor for retroviral reverse transcription in noncycling cells. Proliferating (Ki67(+)) primary CD4(+) T cells or macrophages express a phosphorylated form of SAMHD1 that corresponds with susceptibility to infection in cell culture. We identified cyclin-dependent kinase (CDK) 6 as an upstream regulator of CDK2 controlling SAMHD1 phosphorylation in primary T cells and macrophages susceptible to infection by HIV-1. In turn, CDK2 was strongly linked to cell cycle progression and coordinated SAMHD1 phosphorylation and inactivation. CDK inhibitors specifically blocked HIV-1 infection at the reverse transcription step in a SAMHD1-dependent manner, reducing the intracellular dNTP pool. Our findings identify a direct relationship between control of the cell cycle by CDK6 and SAMHD1 activity, which is important for replication of lentiviruses, as well as other viruses whose replication may be regulated by intracellular dNTP availability.