Structure-guided T cell vaccine design for SARS-CoV-2 variants and sarbecoviruses.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that escape convalescent and vaccine-induced antibody responses has renewed focus on the development of broadly protective T-cell-based vaccines. Here, we apply structure-based network analysis and assessments of HLA class I peptide stability to define mutationally constrained CD8+ T cell epitopes across the SARS-CoV-2 proteome. Highly networked residues are conserved temporally among circulating variants and sarbecoviruses and disproportionately impair spike pseudotyped lentivirus infectivity when mutated. Evaluation of HLA class I stabilizing activity for 18 globally prevalent alleles identifies CD8+ T cell epitopes within highly networked regions with limited mutational frequencies in circulating SARS-CoV-2 variants and deep-sequenced primary isolates. Moreover, these epitopes elicit demonstrable CD8+ T cell reactivity in convalescent individuals but reduced recognition in recipients of mRNA-based vaccines. These data thereby elucidate key mutationally constrained regions and immunogenic epitopes in the SARS-CoV-2 proteome for a global T-cell-based vaccine against emerging variants and SARS-like coronaviruses.

Loss of Bcl-6-Expressing T Follicular Helper Cells and Germinal Centers in COVID-19.

Humoral responses in coronavirus disease 2019 (COVID-19) are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined post mortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers and a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+ TFH cell differentiation together with an increase in T-bet+ TH1 cells and aberrant extra-follicular TNF-α accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+ TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections, and suggest that achieving herd immunity through natural infection may be difficult.

TCR clonotypes modulate the protective effect of HLA class I molecules in HIV-1 infection.

The human leukocyte antigens HLA-B27 and HLA-B57 are associated with protection against progression of disease that results from infection with human immunodeficiency virus type 1 (HIV-1), yet most people with alleles encoding HLA-B27 and HLA-B57 are unable to control HIV-1. Here we found that HLA-B27-restricted CD8(+) T cells in people able to control infection with HIV-1 (controllers) and those who progress to disease after infection with HIV-1 (progressors) differed in their ability to inhibit viral replication through targeting of the immunodominant epitope of group-associated antigen (Gag) of HIV-1. This was associated with distinct T cell antigen receptor (TCR) clonotypes, characterized by superior control of HIV-1 replication in vitro, greater cross-reactivity to epitope variants and enhanced loading and delivery of perforin. We also observed clonotype-specific differences in antiviral efficacy for an immunodominant HLA-B57-restricted response in controllers and progressors. Thus, the efficacy of such so-called 'protective alleles' is modulated by specific TCR clonotypes selected during natural infection, which provides a functional explanation for divergent HIV-1 outcomes.

Profound Treg perturbations correlate with COVID-19 severity.

The hallmark of severe COVID-19 is an uncontrolled inflammatory response, resulting from poorly understood immunological dysfunction. We hypothesized that perturbations in FoxP3+ T regulatory cells (Treg), key enforcers of immune homeostasis, contribute to COVID-19 pathology. Cytometric and transcriptomic profiling revealed a distinct Treg phenotype in severe COVID-19 patients, with an increase in Treg proportions and intracellular levels of the lineage-defining transcription factor FoxP3, correlating with poor outcomes. These Tregs showed a distinct transcriptional signature, with overexpression of several suppressive effectors, but also proinflammatory molecules like interleukin (IL)-32, and a striking similarity to tumor-infiltrating Tregs that suppress antitumor responses. Most marked during acute severe disease, these traits persisted somewhat in convalescent patients. A screen for candidate agents revealed that IL-6 and IL-18 may individually contribute different facets of these COVID-19-linked perturbations. These results suggest that Tregs may play nefarious roles in COVID-19, by suppressing antiviral T cell responses during the severe phase of the disease, and by a direct proinflammatory role.

Concanamycin A counteracts HIV-1 Nef to enhance immune clearance of infected primary cells by cytotoxic T lymphocytes.

Nef is an HIV-encoded accessory protein that enhances pathogenicity by down-regulating major histocompatibility class I (MHC-I) expression to evade killing by cytotoxic T lymphocytes (CTLs). A potent Nef inhibitor that restores MHC-I is needed to promote immune-mediated clearance of HIV-infected cells. We discovered that the plecomacrolide family of natural products restored MHC-I to the surface of Nef-expressing primary cells with variable potency. Concanamycin A (CMA) counteracted Nef at subnanomolar concentrations that did not interfere with lysosomal acidification or degradation and were nontoxic in primary cell cultures. CMA specifically reversed Nef-mediated down-regulation of MHC-I, but not CD4, and cells treated with CMA showed reduced formation of the Nef:MHC-I:AP-1 complex required for MHC-I down-regulation. CMA restored expression of diverse allotypes of MHC-I in Nef-expressing cells and inhibited Nef alleles from divergent clades of HIV and simian immunodeficiency virus, including from primary patient isolates. Lastly, we found that restoration of MHC-I in HIV-infected cells was accompanied by enhanced CTL-mediated clearance of infected cells comparable to genetic deletion of Nef. Thus, we propose CMA as a lead compound for therapeutic inhibition of Nef to enhance immune-mediated clearance of HIV-infected cells.

Temporal changes in T cell subsets and expansion of cytotoxic CD4+ T cells in the lungs in severe COVID-19.

Many studies have been performed in severe COVID-19 on immune cells in the circulation and on cells obtained by bronchoalveolar lavage. Most studies have tended to provide relative information rather than a quantitative view, and it is a combination of approaches by various groups that is helping the field build a picture of the mechanisms that drive severe lung disease. Approaches employed to date have not revealed information on lung parenchymal T cell subsets in severe COVID-19. Therefore, we sought to examine early and late T cell subset alterations in the lungs and draining lymph nodes in severe COVID-19 using a rapid autopsy protocol and quantitative imaging approaches. Here, we have established that cytotoxic CD4+ T cells (CD4 + CTLs) increase in the lungs, draining lymph nodes and blood as COVID-19 progresses. CD4 + CTLs are prominently expanded in the lung parenchyma in severe COVID-19. In contrast CD8+ T cells are not prominent, exhibit increased PD-1 expression, and no obvious increase is seen in the number of Granzyme B+ CD8+ T cells in the lung parenchyma in severe COVID-19. Based on quantitative evidence for re-activation in the lung milieu, CD4 + CTLs may be as likely to drive viral clearance as CD8+ T cells and may also be contributors to lung inflammation and eventually to fibrosis in severe COVID-19.

HLA-B*14:02-Restricted Env-Specific CD8+ T-Cell Activity Has Highly Potent Antiviral Efficacy Associated with Immune Control of HIV Infection.

Immune control of human immunodeficiency virus type 1 (HIV) infection is typically associated with effective Gag-specific CD8+ T-cell responses. We here focus on HLA-B*14, which protects against HIV disease progression, but the immunodominant HLA-B*14-restricted anti-HIV response is Env specific (ERYLKDQQL, HLA-B*14-EL9). A subdominant HLA-B*14-restricted response targets Gag (DRYFKTLRA, HLA-B*14-DA9). Using HLA-B*14/peptide-saporin-conjugated tetramers, we show that HLA-B*14-EL9 is substantially more potent at inhibiting viral replication than HLA-B*14-DA9. HLA-B*14-EL9 also has significantly higher functional avidity (P < 0.0001) and drives stronger selection pressure on the virus than HLA-B*14-DA9. However, these differences were HLA-B*14 subtype specific, applying only to HLA-B*14:02 and not to HLA-B*14:01. Furthermore, the HLA-B*14-associated protection against HIV disease progression is significantly greater for HLA-B*14:02 than for HLA-B*14:01, consistent with the superior antiviral efficacy of the HLA-B*14-EL9 response. Thus, although Gag-specific CD8+ T-cell responses may usually have greater anti-HIV efficacy, factors independent of protein specificity, including functional avidity of individual responses, are also critically important to immune control of HIV.IMPORTANCE In HIV infection, although cytotoxic T lymphocytes (CTL) play a potentially critical role in eradication of viral reservoirs, the features that constitute an effective response remain poorly defined. We focus on HLA-B*14, unique among HLAs associated with control of HIV in that the dominant CTL response is Env specific, not Gag specific. We demonstrate that Env-specific HLA-B*14-restricted activity is substantially more efficacious than the subdominant HLA-B*14-restricted Gag response. Env immunodominance over Gag and strong Env-mediated selection pressure on HIV are observed only in subjects expressing HLA-B*14:02, and not HLA-B*14:01. This reflects the increased functional avidity of the Env response over Gag, substantially more marked for HLA-B*14:02. Finally, we show that HLA-B*14:02 is significantly more strongly associated with viremic control than HLA-B*14:01. These findings indicate that, although Gag-specific CTL may usually have greater anti-HIV efficacy than Env responses, factors independent of protein specificity, including functional avidity, may carry greater weight in mediating effective control of HIV.

A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4+ T-Cells to Recognition by Cytotoxic T-Lymphocytes.

Resting CD4+ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8+ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8+ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8+ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8+ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8+ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam3CSK4. In contrast, we did not observe CD8+ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist 'ALT-803', an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8+ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8+ T-cells in HIV eradication strategies.

Anti-APOBEC3G activity of HIV-1 Vif protein is attenuated in elite controllers.

UNLABELLED: HIV-1-infected individuals who control viremia to below the limit of detection without antiviral therapy have been termed elite controllers (EC). Functional attenuation of some HIV-1 proteins has been reported in EC. The HIV-1 accessory protein Vif (virion infectivity factor) enhances viral infectivity through anti-retroviral factor apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G (APOBEC3G) degradation; however, little is known regarding Vif function in EC. Here, the anti-APOBEC3G activities of clonal, plasma HIV RNA-derived Vif sequences from 46 EC, 46 noncontrollers (NC), and 44 individuals with acute infection (AI) were compared. Vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped viruses were generated by cotransfecting 293T cells with expression plasmids encoding patient-derived Vif, human APOBEC3G, VSV-G, and a vif/env-deficient luciferase-reporter HIV-1 proviral DNA clone. Viral stocks were used to infect 293T cells, and Vif anti-APOBEC3G activity was quantified in terms of luciferase signal. On average, the anti-APOBEC3G activities of EC-derived Vif sequences (median log10 relative light units [RLU], 4.54 [interquartile range {IQR}, 4.30 to 4.66]) were significantly lower than those of sequences derived from NC (4.75 [4.60 to 4.92], P < 0.0001) and AI (4.74 [4.62 to 4.94], P < 0.0001). Reduced Vif activities were not associated with particular HLA class I alleles expressed by the host. Vif functional motifs were highly conserved in all patient groups. No single viral polymorphism could explain the reduced anti-APOBEC3G activity of EC-derived Vif, suggesting that various combinations of minor polymorphisms may underlie these effects. These results further support the idea of relative attenuation of viral protein function in EC-derived HIV sequences. IMPORTANCE: HIV-1 elite controllers (EC) are rare individuals who are able to control plasma viremia to undetectable levels without antiretroviral therapy. Understanding the pathogenesis and mechanisms underpinning this rare phenotype may provide important insights for HIV vaccine design. The EC phenotype is associated with beneficial host immunogenetic factors (such as HLA-B*57) as well as with functions of attenuated viral proteins (e.g., Gag, Pol, and Nef). In this study, we demonstrated that HIV-1 Vif sequences isolated from EC display relative impairments in their ability to counteract the APOBEC3G host restriction factor compared to Vif sequences from normal progressors and acutely infected individuals. This result extends the growing body of evidence demonstrating attenuated HIV-1 protein function in EC and, in particular, supports the idea of the relevance of viral factors in contributing to this rare HIV-1 phenotype.

The Breadth of Expandable Memory CD8+ T Cells Inversely Correlates with Residual Viral Loads in HIV Elite Controllers.

UNLABELLED: Previous studies have shown that elite controllers with minimal effector T cell responses harbor a low-frequency, readily expandable, highly functional, and broadly directed memory population. Here, we interrogated the in vivo relevance of this cell population by investigating whether the breadth of expandable memory responses is associated with the magnitude of residual viremia in individuals achieving durable suppression of HIV infection. HIV-specific memory CD8(+) T cells were expanded by using autologous epitopic and variant peptides. Viral load was measured by an ultrasensitive single-copy PCR assay. Following expansion, controllers showed a greater increase in the overall breadth of Gag responses than did untreated progressors (P = 0.01) as well as treated progressors (P = 0.0003). Nef- and Env-specific memory cells expanded poorly for all groups, and their expanded breadths were indistinguishable among groups (P = 0.9 for Nef as determined by a Kruskal-Wallis test; P = 0.6 for Env as determined by a Kruskal-Wallis test). More importantly, we show that the breadth of expandable, previously undetectable Gag-specific responses was inversely correlated with residual viral load (r = -0.6; P = 0.009). Together, these data reveal a direct link between the abundance of Gag-specific expandable memory responses and prolonged maintenance of low-level viremia. Our studies highlight a CD8(+) T cell feature that would be desirable in a vaccine-induced T cell response. IMPORTANCE: Many studies have shown that the rare ability of some individuals to control HIV infection in the absence of antiretroviral therapy appears to be heavily dependent upon special HIV-specific killer T lymphocytes that are able to inhibit viral replication. The identification of key features of these immune cells has the potential to inform rational HIV vaccine design. This study shows that a special subset of killer lymphocytes, known as central memory CD8(+) T lymphocytes, is at least partially involved in the durable control of HIV replication. HIV controllers maintain a large proportion of Gag-specific expandable memory CD8(+) T cells involved in ongoing viral suppression. These data suggest that induction of this cell subset by future HIV vaccines may be important for narrowing possible routes of rapid escape from vaccine-induced CD8(+) T cell responses.

Histone deacetylase inhibitors impair the elimination of HIV-infected cells by cytotoxic T-lymphocytes.

Resting memory CD4+ T-cells harboring latent HIV proviruses represent a critical barrier to viral eradication. Histone deacetylase inhibitors (HDACis), such as suberanilohydroxamic acid (SAHA), romidepsin, and panobinostat have been shown to induce HIV expression in these resting cells. Recently, it has been demonstrated that the low levels of viral gene expression induced by a candidate HDACi may be insufficient to cause the death of infected cells by viral cytopathic effects, necessitating their elimination by immune effectors, such as cytotoxic T-lymphocytes (CTL). Here, we study the impact of three HDACis in clinical development on T-cell effector functions. We report two modes of HDACi-induced functional impairment: i) the rapid suppression of cytokine production from viable T-cells induced by all three HDACis ii) the selective death of activated T-cells occurring at later time-points following transient exposures to romidepsin or, to a lesser extent, panobinostat. As a net result of these factors, HDACis impaired CTL-mediated IFN-γ production, as well as the elimination of HIV-infected or peptide-pulsed target cells, both in liquid culture and in collagen matrices. Romidepsin exerted greater inhibition of antiviral function than SAHA or panobinostat over the dose ranges tested. These data suggest that treatment with HDACis to mobilize the latent reservoir could have unintended negative impacts on the effector functions of CTL. This could influence the effectiveness of HDACi-based eradication strategies, by impairing elimination of infected cells, and is a critical consideration for trials where therapeutic interruptions are being contemplated, given the importance of CTL in containing rebound viremia.

High-dimensional immunomonitoring models of HIV-1-specific CD8 T-cell responses accurately identify subjects achieving spontaneous viral control.

UNLABELLED: The development of immunomonitoring models to determine HIV-1 vaccine efficacy is a major challenge. Studies suggest that HIV-1­specific CD8 T cells play a critical role in subjects achieving spontaneous viral control (HIV-1 controllers) and that they will be important in immune interventions. However, no single CD8 T-cell function is uniquely associated with controller status and the heterogeneity of responses targeting different epitopes further complicates the discovery of determinants of protective immunity. In the present study, we describe immunomonitoring models integrating multiple functions of epitope-specific CD8 T cells that distinguish controllers from subjects with treated or untreated progressive infection. Models integrating higher numbers of variables and trained with the least absolute shrinkage and selection operator (LASSO) variant of logistic regression and 10-fold cross-validation produce "diagnostic tests" that display an excellent capacity to delineate subject categories. The test accuracy reaches 75% area under the receiving operating characteristic curve in cohorts matched for prevalence of protective alleles. Linear mixed-effects model analyses show that the proliferative capacity, cytokine production, and kinetics of cytokine secretion are associated with HIV-1 control. Although proliferative capacity is the strongest single discriminant, integrated modeling of different dimensions of data leverages individual associations. This strategy may have important applications in predictive model development and immune monitoring of HIV-1 vaccine trials. KEY POINTS: Immune monitoring models integrating multiple functions of HIV-1-specific CD8 T cells distinguish controllers from subjects with progressive HIV-1 infection. This strategy may have important applications in predictive model development and immune monitoring of HIV-1 vaccine trials.

HLA-Cw*0102-restricted HIV-1 p24 epitope variants can modulate the binding of the inhibitory KIR2DL2 receptor and primary NK cell function.

Accumulating evidence suggests an important role for Natural Killer (NK) cells in the control of HIV-1 infection. Recently, it was shown that NK cell-mediated immune pressure can result in the selection of HIV-1 escape mutations. A potential mechanism for this NK cell escape is the selection of HLA class I-presented HIV-1 epitopes that allow for the engagement of inhibitory killer cell immunoglobulin-like receptors (KIRs), notably KIR2DL2. We therefore investigated the consequences of sequence variations within HLA-Cw*0102-restricted epitopes on the interaction of HLA-Cw*0102 with KIR2DL2 using a large panel of overlapping HIV-1 p24 Gag peptides. 217 decameric peptides spanning the HIV-1 p24 Gag consensus sequence were screened for HLA-Cw*0102 stabilization by co-incubation with Cw*0102⁺/TAP-deficient T2 cells using a flow cytometry-based assay. KIR2DL2 binding was assessed using a KIR2DL2-IgG fusion construct. Function of KIR2DL2⁺ NK cells was flow cytometrically analyzed by measuring degranulation of primary NK cells after co-incubation with peptide-pulsed T2 cells. We identified 11 peptides stabilizing HLA-Cw*0102 on the surface of T2 cells. However, only one peptide (p24 Gag209₋218 AAEWDRLHPV) allowed for binding of KIR2DL2. Notably, functional analysis showed a significant inhibition of KIR2DL2⁺ NK cells in the presence of p24 Gag209₋218-pulsed T2 cells, while degranulation of KIR2DL2⁻ NK cells was not affected. Moreover, we demonstrated that sequence variations in position 7 of this epitope observed frequently in naturally occurring HIV-1 sequences can modulate binding to KIR2DL2. Our results show that the majority of HIV-1 p24 Gag peptides stabilizing HLA-Cw*0102 do not allow for binding of KIR2DL2, but identified one HLA-Cw*0102-presented peptide (p24 Gag209₋218) that was recognized by the inhibitory NK cell receptor KIR2DL2 leading to functional inhibition of KIR2DL2-expressing NK cells. Engagement of KIR2DL2 might protect virus-infected cells from NK cell-mediated lysis and selections of sequence polymorphisms that increase avidity to KIR2DL2 might provide a mechanism for HIV-1 to escape NK cell-mediated immune pressure.

Polymorphic residues in HLA-B that mediate HIV control distinctly modulate peptide interactions with both TCR and KIR molecules.

Immunogenetic studies have shown that specific HLA-B residues (67, 70, 97, and 156) mediate the impact of HLA class I on HIV infection, but the molecular basis is not well understood. Here we evaluate the function of these residues within the protective HLA-B∗5701 allele. While mutation of Met67, Ser70, and Leu156 disrupt CD8+ T cell recognition, substitution of Val97 had no significant impact. Thermal denaturation of HLA-B∗5701-peptide complexes revealed that Met67 and Leu156 maintain HLA-peptide stability, while Ser70 and Leu156 facilitate T cell receptor (TCR) interactions. Analyses of existing structures and structural models suggested that Val97 mediates HLA-peptide binding to inhibitory KIR3DL1 molecules, which was confirmed by experimental assays. These data thereby demonstrate that the genetic basis by which host immunity impacts HIV outcomes occurs by modulating HLA-B-peptide stability and conformation for interaction with TCR and killer immunoglobulin receptor (KIR) molecules. Moreover, they indicate a key role for epitope specificity and HLA-KIR interactions to HIV control.

Dysregulated Tim-3 expression on natural killer cells is associated with increased Galectin-9 levels in HIV-1 infection.

BACKGROUND: Natural killer (NK) cells constitutively express high levels of Tim-3, an immunoregulatory molecule recently proposed to be a marker for mature and functional NK cells. Whether HIV-1 infection modulates the expression of Tim-3 on NK cells, or the levels of its ligand Galectin-9 (Gal-9), and how signaling through these molecules affects the NK cell response to HIV-1 remains inadequately understood. RESULTS: We analyzed Tim-3 and Gal-9 expression in a cohort of 85 individuals with early and chronic HIV-1 infection, and in 13 HIV-1 seronegative control subjects. HIV-1 infection was associated with reduced expression of Tim-3 on NK cells, which was normalized by HAART. Plasma concentrations of Gal-9 were higher in HIV-1-infected individuals than in healthy individuals. Interestingly, Gal-9 expression in immune cells was significantly elevated in early infection, with monocytes and dendritic cells displaying the highest expression levels, which correlated with HIV-1 viral loads. In vitro, Gal-9 triggered Tim-3 downregulation on NK cells as well as NK cell activation. CONCLUSIONS: Our data suggest that high expression levels of Gal-9 during early HIV-1 infection can lead to enhanced NK cell activity, possibly allowing for improved early control of HIV-1. In contrast, persistent Gal-9 production might impair Tim-3 activity and contribute to NK cell dysfunction in chronic HIV-1 infection.

Elite controllers with low to absent effector CD8+ T cell responses maintain highly functional, broadly directed central memory responses.

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

Epithelial adhesion molecules can inhibit HIV-1-specific CD8⁺ T-cell functions.

Under persistent antigenic stimulation, virus-specific CD8⁺ T cells become increasingly dysfunctional and up-regulate several inhibitory molecules such as killer lectin-like receptor G1 (KLRG1). Here, we demonstrate that HIV-1 antigen-specific T cells from subjects with chronic-progressive HIV-1 infection have significantly elevated KLRG1 expression (P < .001); show abnormal distribution of E-cadherin, the natural ligand of KLRG1, in the intestinal mucosa; and have elevated levels of systemic soluble E-cadherin (sE-cadherin) that significantly correlate with HIV-1 viral load (R = 0.7, P = .004). We furthermore demonstrate that in the presence of sE-cadherin, KLRG1(hi) HIV-1-specific CD8⁺ T cells are impaired in their ability to respond by cytokine secretion on antigenic stimulation (P = .002) and to inhibit viral replication (P = .03) in vitro. Thus, these data suggest a critical mechanism by which the disruption of the intestinal epithelium associated with HIV-1 leads to increased systemic levels of sE-cadherin, which inhibits the effector functions of KLRG1(hi)-expressing HIV-1-specific CD8⁺ T cells systemically.

Mosaic HIV-1 Gag antigens can be processed and presented to human HIV-specific CD8+ T cells.

Polyvalent mosaic HIV immunogens offer a potential solution for generating vaccines that can elicit immune responses against genetically diverse viruses. However, it is unclear whether key T cell epitopes can be processed and presented from these synthetic Ags and recognized by epitope-specific human T cells. In this study, we tested the ability of mosaic HIV immunogens expressed by recombinant, replication-incompetent adenovirus serotype 26 vectors to process and present major HIV clade B and clade C CD8 T cell epitopes in human cells. A bivalent mosaic vaccine expressing HIV Gag sequences was used to transduce PBMCs from 12 HIV-1-infected individuals from the United States and 10 HIV-1-infected individuals from South Africa; intracellular cytokine staining, together with tetramer staining, was used to assess the ability of mosaic Gag Ags to stimulate pre-existing memory responses compared with natural clade B and C vectors. Mosaic Gag Ags expressed all eight clade B epitopes tested in 12 United States subjects and all 5 clade C epitopes tested in 10 South African subjects. Overall, the magnitude of cytokine production induced by stimulation with mosaic Ags was comparable to clade B and clade C Ags tested, but the mosaic Ags elicited greater cross-clade recognition. Additionally, mosaic Ags induced HIV-specific CD4 T cell responses. Our studies demonstrate that mosaic Ags express major clade B and clade C viral T cell epitopes in human cells, as well as support the evaluation of mosaic HIV-1 vaccines in humans.

Preserved function of regulatory T cells in chronic HIV-1 infection despite decreased numbers in blood and tissue.

Regulatory T cells (Tregs) are potent immune modulators, but their role in human immunodeficiency virus type 1 (HIV-1) pathogenesis remains poorly understood. We performed a detailed analysis of the frequency and function of Tregs in a large cohort of HIV-1-infected individuals and HIV-1 negative controls. While HIV "elite controllers" and uninfected individuals had similar Treg numbers and frequencies, the absolute numbers of Tregs declined in blood and gut-associated lymphoid tissue in patients with chronic progressive HIV-1 infection. Despite quantitative changes in Tregs, HIV-1 infection was not associated with an impairment of ex vivo suppressive function of flow-sorted Tregs in both HIV controllers and untreated chronic progressors.

Molecular basis of differential HLA class I-restricted T cell recognition of a highly networked HIV peptide.

Cytotoxic-T-lymphocyte (CTL) mediated control of HIV-1 is enhanced by targeting highly networked epitopes in complex with human-leukocyte-antigen-class-I (HLA-I). However, the extent to which the presenting HLA allele contributes to this process is unknown. Here we examine the CTL response to QW9, a highly networked epitope presented by the disease-protective HLA-B57 and disease-neutral HLA-B53. Despite robust targeting of QW9 in persons expressing either allele, T cell receptor (TCR) cross-recognition of the naturally occurring variant QW9_S3T is consistently reduced when presented by HLA-B53 but not by HLA-B57. Crystal structures show substantial conformational changes from QW9-HLA to QW9_S3T-HLA by both alleles. The TCR-QW9-B53 ternary complex structure manifests how the QW9-B53 can elicit effective CTLs and suggests sterically hindered cross-recognition by QW9_S3T-B53. We observe populations of cross-reactive TCRs for B57, but not B53 and also find greater peptide-HLA stability for B57 in comparison to B53. These data demonstrate differential impacts of HLAs on TCR cross-recognition and antigen presentation of a naturally arising variant, with important implications for vaccine design.