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HIV frequently escapes CD8 T cell responses, leading to the accumulation of viral adaptations. We recently demonstrated that during chronic HIV infection, adapted epitopes can promote maturation of dendritic cells (DCs) through direct CD8 T cell interactions and lead to enhanced HIV trans-infection of CD4 T cells. Here, we sought to determine the role of such adaptations following HIV MRKAd5 vaccination. We observed that vaccine-induced adapted epitope-specific CD8 T cells promoted higher levels of DC maturation than nonadapted ones and that these matured DCs significantly enhanced HIV trans-infection. These matured DCs were associated with higher levels of interleukin 5 (IL-5) and IL-13 and a lower level of CXCL5, which have been shown to impact DC maturation, as well as a lower level of CXCL16. Finally, we observed that vaccine recipients with high HLA-I-associated adaptation became HIV infected more quickly. Our results offer another possible mechanism for enhanced infection among MRKAd5 vaccinees. IMPORTANCE Despite the well-established contribution of CD8 T cells in HIV control, prior CD8 T cell-based HIV vaccines have failed to demonstrate any efficacy in preventing viral infection. One such vaccine, known as the MRKAd5 vaccine, showed a potential increased risk of viral infection among vaccine recipients. However, the underlying mechanism(s) remains unclear. In this study, we observed that vaccine recipients with high adaptation to their HLA-I alleles were associated with an increased HIV infection risk in comparison to the others. Similar to what we observed in HIV infection in the prior study, adapted epitope-specific CD8 T cells obtained from vaccine recipients exhibit a greater capacity in facilitating viral infection by promoting dendritic cell maturation. Our findings provide a possible explanation for the enhanced viral acquisition risk among MRKAd5 vaccine recipients and highlight the importance of optimizing vaccine design with consideration of HLA-I-associated HIV adaptation.

Background During early HIV-1 infection, immunodominant T cell responses to highly variable epitopes lead to the establishment of immune escape virus variants. Here we assessed a type 1-polarized monocyte-derived dendritic cell (MDC1)-based approach to selectively elicit cytotoxic T lymphocyte (CTL) responses against highly conserved and topologically important HIV-1 epitopes in HIV-1-infected individuals from the Thailand RV254/SEARCH 010 cohort who initiated antiretroviral therapy (ART) during early infection (Fiebig stages I-IV). Methods Autologous MDC1 were used as antigen presenting cells to induce in vitro CTL responses against HIV-1 Gag, Pol, Env, and Nef as determined by flow cytometry and ELISpot assay. Ultra-conserved or topologically important antigens were respectively identified using the Epigraph tool and a structure-based network analysis approach and compared to overlapping peptides spanning the Gag proteome. Findings MDC1 presenting either the overlapping Gag, Epigraph, or Network 14–21mer peptide pools consistently activated and expanded HIV-1-specific T cells to epitopes identified at the 9–13mer peptide level. Interestingly, some CTL responses occurred outside known or expected HLA associations, providing evidence of new HLA-associated CTL epitopes. Comparative analyses demonstrated more sequence conservation among Epigraph antigens but a higher magnitude of CTL responses to Network and Gag peptide groups. Importantly, CTL responses against topologically constrained Gag epitopes contained in both the Network and Gag peptide pools were selectively enhanced in the Network pool-initiated cultures. Interpretation Our study supports the use of MDC1 as a therapeutic strategy to induce and focus CTL responses toward putative fitness-constrained regions of HIV-1 to prevent immune escape and control HIV-1 infection. Funding A full list of the funding sources is detailed in the Acknowledgment section of the manuscript.

Recent advances in γδ T cell biology have focused on the unique attributes of these cells and their role in regulating innate and adaptive immunity, promoting tissue homeostasis, and providing resistance to various disorders. Numerous bacterial and viral pathogens, including human immunodeficiency virus-1 (HIV), greatly alter the composition of γδ T cells in vivo. Despite the effectiveness of antiretroviral therapy (ART) in controlling HIV and restoring health in those affected, γδ T cells are dramatically impacted during HIV infection and fail to reconstitute to normal levels in HIV-infected individuals during ART for reasons that are not clearly understood. Importantly, their role in controlling HIV infection, and the implications of their failure to rebound during ART are also largely unknown and understudied. Here, we review important aspects of human γδ T cell biology, the effector and immunomodulatory properties of these cells, their prevalence and function in HIV, and their immunotherapeutic potential.

Dendritic cells (DCs) play a fundamental and central role in the immune response, acting as a critical link between the innate and adaptive branches of immunity [...]

During early HIV Infection, immunodominant T cell responses to highly variable epitopes lead to the selection and expansion of immune escape variants. As a potential therapeutic strategy, we assessed a specialized type 1-polarized monocyte-derived DC dendritic cell (MDC1)-based approach to selectively elicit functional CD8+ cytotoxic T lymphocyte (CTL) responses against highly conserved and topologically important HIV epitopes. Cells were obtained from 10 HIV-infected individuals in the Thailand RV254/SEARH010 cohort who initiated suppressive anti-retroviral therapy (ART) during Fiebig stages I to IV of early infection. Autologous MDC1 were generated for use as peptide antigen presenting cells to induce ex vivo CTL responses against HIV Gag, Pol, Env and Nef. Ultra-conserved (Epigraph) or topologically important (Network) antigens were respectively identified using the Epigraph tool and a structure-based network analysis approach, and each compared to overlapping peptides spanning the entire Gag proteome. MDC1 loaded with either overlapping Gag, Epigraph, or Network 14-21mer peptide pools were consistently capable of activating and expanding HIV-specific T cells to epitopes identified at the 9-13mer peptide level. Some CTL responses occurred outside of known or expected HLA associations, providing evidence of new HLA-associated CTL epitopes. Comparative analyses of peptide pools demonstrated more sequence conservation among the Epigraph antigens, but statistically higher magnitude of CTL responses to Network and Gag peptide groups. Importantly, when select Gag antigens used to initiate the cultures were part of the Network peptide pool, CTL responses directed against these topologically important epitopes were enhanced as compared to when they were included within the complete pool of overlapping Gag peptides. Our study supports that MDC1 can be used to effectively focus CTL responses toward potentially fitness-constrained regions of HIV as a therapeutic strategy to prevent HIV immune escape and control viral replication.Author summaryA major hurdle in the development of a successful HIV immunotherapy is the capacity of the virus to evade the immune response by efficiently establishing epitope variants in response to selective pressure. While effective at suppressing viremia, current regimens of antiretroviral therapy (ART) are not curative. Therefore, achieving immune control of HIV upon cessation of ART as a functional cure, similar to that observed in ‘elite controllers’ (EC), has been a major therapeutic goal. Such immune control is realized through the actions of antigen-specific cytotoxic T cell lymphocytes (CTL) capable of specifically targeting sequence-conserved epitopes in HIV. In this study, a specialized, antigen presenting, dendritic cell (DC)-based vaccine strategy was used to elicit HIV specific CTL responses in vitro against carefully selected, ultra-conserved and topologically important epitopes. This DC-based approach yielded broad responses against peptide epitopes of both known and unknown HLA-associations, the latter of which implies the uncovering of potentially novel epitopes. Importantly, we demonstrate that CTL responses can be re-directed or focused toward potentially more fitness-constrained regions of the virus, thus highlighting the potential for DC-based therapies to induce immune responses that circumvent the issue of viral escape.

Background: During early HIV Infection, immunodominant T cell responses to highly variable epitopes lead to the establishment of immune escape virus variants. Here we assessed a type 1-polarized monocyte-derived dendritic cell (MDC1)-based approach to selectively elicit cytotoxic T lymphocyte (CTL) responses against highly conserved and topologically important HIV epitopes, using HIV-infected individuals from the Thailand RV254/SEARH010 cohort who initiated antiretroviral therapy (ART) during early infection (Fiebig stages I-IV). Methods: Autologous MDC1 were used as peptide antigen presenting cells to induce ex vivo CTL responses against HIV Gag, Pol, Env and Nef as determined by flow cytometry and ELISPOT assay. Ultra-conserved or topologically important antigens were respectively identified using the Epigraph tool and a structure-based network analysis approach and compared to overlapping peptides spanning the Gag proteome. Findings: MDC1 presenting either the overlapping Gag, Epigraph, or Network 14-21mer peptide pools consistently activated and expanded HIV-specific T cells specific to epitopes identified at the 9-13mer peptide level. Interestingly, some CTL responses occurred outside known or expected HLA associations, providing evidence of new HLA-associated CTL epitopes. Comparative analyses demonstrated more sequence conservation among Epigraph antigens, but higher magnitude of CTL responses to Network and Gag peptide groups. Importantly, CTL responses against topologically important Gag epitopes contained in both the Network and Gag peptide pools were selectively enhanced in the Network pool-initiated cultures. Interpretation: Our study supports the use of MDC1 as a therapeutic strategy to induce and focus CTL responses toward potentially fitness-constrained regions of HIV as a means to prevent immune escape and control HIV-1 infection. Funding Statement: This study was supported by the NIH, National Institute of Allergy and Infectious Diseases grants R21-AI131763, R21-AI138716, UM1-AI126603, and U01-AI35041. Declaration of Interests: The authors have nothing to disclose. Ethics Approval Statement: The Chulalongkorn University Institutional Review Board and the Walter Reed Army Institute of Research, USA, approved this study.

The development of effective yet nontoxic strategies to target the latent human immunodeficiency virus-1 (HIV-1) reservoir in antiretroviral therapy (ART)-suppressed individuals poses a critical barrier to a functional cure. The ‘kick and kill’ approach to HIV eradication entails proviral reactivation during ART, coupled with generation of cytotoxic T lymphocytes (CTLs) or other immune effectors equipped to eliminate exposed infected cells. Pharmacological latency reversal agents (LRAs) that have produced modest reductions in the latent reservoir ex vivo have not impacted levels of proviral DNA in HIV-infected individuals. An optimal cure strategy incorporates methods that facilitate sufficient antigen exposure on reactivated cells following the induction of proviral gene expression, as well as the elimination of infected targets by either polyfunctional HIV-specific CTLs or other immune-based strategies. Although conventional dendritic cells (DCs) have been used extensively for the purpose of inducing antigen-specific CTL responses in HIV-1 clinical trials, their immunotherapeutic potential as cellular LRAs has been largely ignored. In this review, we discuss the challenges associated with current HIV-1 eradication strategies, as well as the unharnessed potential of ex vivo-programmed DCs for both the ‘kick and kill’ of latent HIV-1.

Reducing the risk of human immunodeficiency virus type 1 (HIV-1) transmission is still a public health priority. The development of effective control strategies relies on the quantification of the effects of prophylactic and therapeutic measures in disease incidence. Although several assays can be used to estimate HIV incidence, these estimates are limited by the poor performance of these assays in distinguishing recent from long-standing infections. To address such limitation, we have developed an assay to titrate p24-specific IgG3 antibodies as a marker of recent infection. The assay is based on a recombinant p24 protein capable to detect total IgG antibodies in sera using a liquid micro array and enzyme-linked immunosorbent assay. Subsequently, the assay was optimised to detect and titrate anti-p24 IgG3 responses in a panel of sequential specimens from seroconverters over 24 months. The kinetics of p24-specific IgG3 titres revealed a transient peak in the 4 to 5-month period after seroconversion. It was followed by a sharp decline, allowing infections with less than 6 months to be distinguished from older ones. The developed assay exhibited a mean duration of recent infection of 144 days and a false-recent rate of ca. 14%. Our findings show that HIV-1 p24-specific IgG3 titres can be used as a tool to evaluate HIV incidence in serosurveys and to monitor the efficacy of vaccines and other transmission control strategies.

HIV-1 frequently escapes from CD8 T cell responses via HLA-I restricted adaptation, leading to the accumulation of adapted epitopes (AE). We previously demonstrated that AE compromise CD8 T cell responses during acute infection and are associated with poor clinical outcomes. Here, we examined the impact of AE on CD8 T cell responses and their biological relevance in chronic HIV infection (CHI). In contrast to acute infection, the majority of AE are immunogenic in CHI. Longitudinal analyses from acute to CHI showed an increased frequency and magnitude of AE-specific IFNγ responses compared to NAE-specific ones. These AE-specific CD8 T cells also were more cytotoxic to CD4 T cells. In addition, AE-specific CD8 T cells expressed lower levels of PD1 and CD57, as well as higher levels of CD28, suggesting a more activated and less exhausted phenotype. During CHI, viral sequencing identified AE-encoding strains as the dominant quasispecies. Despite increased CD4 T cell cytotoxicity, CD8 T cells responding to AE promoted dendritic cell (DC) maturation and CD4 T cell trans-infection perhaps explaining why AE are predominant in CHI. Taken together, our data suggests that the emergence of AE-specific CD8 T cell responses in CHI confers a selective advantage to the virus by promoting DC-mediated CD4 T cell trans-infection., Author summary HIV-1 infection remains a critical public health threat across the world. Over the past two decades, CD8 T cells have been clearly shown to exert immune pressure on HIV and drive viral adaptation. Previously, our group reported that such HLA-I associated adaptations can predict clinical outcomes and are beneficial to HIV-1 as CD8 T cells are unable to recognize epitopes with adaptation in acute HIV infection. However, it is still unclear how HIV-1 adaptation impacts CD8 T cells during chronic HIV infection. In this study, we observed an enhancement of CD8 T cell responses targeting adapted epitopes in chronic infection. Although these responses were cytotoxic, they also exhibited a “helper” effect by promoting viral infection of CD4 T cells via interaction with dendritic cells. This phenomenon may contribute to the persistence of adapted viruses. In summary, these findings present a novel mechanism of CD8 T cell driven HIV-1 adaptation.

Eliciting highly functional CD8(+) cytotoxic T lymphocyte (CTL) responses against a broad range of epitopes will likely be required for immunotherapeutic control of HIV-1 infection. However, the combination of CTL exhaustion and the ability of HIV-1 to rapidly establish CTL escape variants presents major hurdles toward this goal. Our previous work highlighted the use of monocyte-derived, mature, high-interleukin-12 (IL-12)-producing type 1 polarized dendritic cells (MDC1) to selectively induce more potent effector CTLs derived from naive, rather than memory, CD8(+) T cell precursors isolated from HIV-1-positive participants in the Multicenter AIDS Cohort Study. In this study, we report that these highly stimulatory antigen-presenting cells also express enhanced levels of the coinhibitory molecule programmed cell death ligand 1 (PD-L1), the ligand for PD-1, which is further upregulated upon subsequent stimulation with the CD4(+) T helper cell-derived factor CD40L. Interestingly, blocking the PD-1 signaling pathway during MDC1 induction of HIV-1-specific CTL responses inhibited the priming, activation, and differentiation of naive CD8(+) T cells into effector T cells expressing high levels of T-box transcription factor (T-bet(hi)) and eomesodermin (Eomes(+)). In contrast, PD-1 blockade enhanced the overall magnitude of memory HIV-specific CTL responses and reversed the exhausted memory phenotype from a T-bet(low)/Eomes(+) to a T-bet(hi)/Eomes(+) phenotype. These results indicate that the PD-L1/PD-1 signaling pathway has a previously unappreciated dual role in the induction and regulation of HIV-1-specific CTL immunity, which is greatly determined by the context and differentiation stage of the responsive CD8(+) T cells. IMPORTANCE Targeting the PD-1/PD-L1 immune checkpoint axis with signaling inhibitors has proven to be a powerful immunotherapeutic strategy to enhance the functional quality and survival of existing antigen-specific effector T cells. However, our study demonstrates that the context and timing of PD-1 signaling in T cells greatly impact the outcome of the effector response. In particular, we show that PD-1 activation plays a positive role during the DC-mediated initiation stage of the primary T cell response, while it serves as an inhibitory mechanism during the effector phase of the response. Therefore, caution should be taken in the design of therapies that include targeting of the PD-1/PD-L1 signaling pathway in order to avoid potential negative impacts on the induction of de novo T cell responses.

Combination antiretroviral therapy (cART) has significantly reduced mortality and morbidity of those infected with HIV-1 [1,2]. During cART, the plasma virus levels become undetectable by standard ...

Despite the success of combination antiretroviral therapy (cART), a latent viral reservoir persists in HIV-1-infected persons. Unfortunately, endogenous cytotoxic T lymphocytes (CTLs) are unable to control viral rebound when patients are removed from cART. A "kick and kill" strategy has been proposed to eradicate this reservoir, whereby infected T cells are induced to express viral proteins via latency-inducing drugs followed by their elimination by CTLs. It has yet to be determined if stimulation of existing HIV-1-specific CTL will be sufficient, or if new CTLs should be primed from naïve T cells. In this review, we propose that dendritic cells (DCs), the most potent antigen presenting cells, act as dog trainers and can induce T cells (the dogs) to do magnificent tricks. We propose the hypothesis that an HIV-1 cure will require targeting of naïve T cells and will necessitate "teaching new dogs new tricks" while avoiding activation of potentially dysfunctional endogenous memory CTLs (letting the sleeping dogs lie).

Although antiretroviral therapy can suppress HIV-1 infection to undetectable levels of plasma viremia, integrated latent HIV-1 genomes that encode replication competent virus persist in resting CD4+ T cells. This latent HIV-1 reservoir represents a major barrier to a cure. Currently, there are substantial ongoing efforts to identify therapeutic approaches that will eliminate or reduce the size of this latent HIV-1 reservoir. In this regard, a sensitive assay which can accurately and rapidly quantify inducible replication competent latent HIV-1 from resting CD4+ T cells is essential for HIV-1 eradication studies. Here we describe a reporter cell-based assay to quantify inducible replication competent latent HIV-1. This assay has several advantages over existing technology in that it: (i) is sensitive; (ii) requires only a small blood volume; (iii) is faster, less labor intensive, and less expensive, and (iv) can be readily adapted to a high-throughput format. Using this assay we show that the size of the inducible latent HIV-1 reservoir in aviremic participants on therapy is approximately 70-fold larger than previous estimates.

Recall T cell responses to HIV-1 antigens are used as a surrogate for endogenous cellular immune responses generated during infection. Current methods of identifying antigen-specific T cell reactivity in HIV-1 infection use bulk peripheral blood mononuclear cells (PBMC) yet ignore professional antigen-presenting cells (APC) that could reveal otherwise hidden responses. In the present study, peptides representing autologous variants of major histocompatibility complex (MHC) class I-restricted epitopes from HIV-1 Gag and Env were used as antigens in gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) and polyfunctional cytokine assays. Here we show that dendritic cells (DC) enhanced T cell reactivity at all stages of disease progression but specifically restored T cell reactivity after combination antiretroviral therapy (cART) to early infection levels. Type 1 cytokine secretion was also enhanced by DC and was most apparent late post-cART. We additionally show that DC reveal polyfunctional T cell responses after many years of treatment, when potential immunotherapies would be implemented. These data underscore the potential efficacy of DC immunotherapy that aims to awaken a dormant, autologous, HIV-1-specific CD8 + T cell response. IMPORTANCE Assessment of endogenous HIV-1-specific T cell responses is critical for generating immunotherapies for subjects on cART. Current assays ignore the ability of dendritic cells to reveal these responses and may therefore underestimate the breadth and magnitude of T cell reactivity. As DC do not prime new responses in these assays, it can be assumed that the observed responses are not detected without appropriate stimulation. This is important because dogma states that HIV-1 mutates to evade host recognition and that CD8 + cytotoxic T lymphocyte (CTL) failure is due to the inability of T cells to recognize the autologous virus. The results presented here indicate that responses to autologous virus are generated during infection but may need additional stimulation to be effective. Detecting the breadth and magnitude of HIV-1-specific T cell reactivity generated in vivo is of the utmost importance for generating effective DC immunotherapies.

It is unclear if HIV-1 variants lose the ability to prime naive CD8 + cytotoxic T lymphocytes (CTL) during progressive, untreated infection. We conducted a comprehensive longitudinal analysis of viral evolution and its impact on primary and memory CD8 + T cell responses pre-seroconversion (SC), post-SC, and during combination antiretroviral therapy (cART). Memory T cell responses targeting autologous virus variants reached a nadir by 8 years post-SC with development of AIDS, followed by a transient enhancement of anti-HIV-1 CTL responses upon initiation of cART. We show broad and high magnitude primary T cell responses to late variants in pre-SC T cells, comparable to primary anti-HIV-1 responses induced in T cells from uninfected persons. Despite evolutionary changes, CD8 + T cells could still be primed to HIV-1 variants. Hence, vaccination against late, mutated epitopes could be successful in enhancing primary reactivity of T cells for control of the residual reservoir of HIV-1 during cART.

Curing HIV-1 infection will require elimination of persistent cellular reservoirs that harbor latent virus in the face of combination antiretroviral therapy (cART). Proposed immunotherapeutic strategies to cure HIV-1 infection include enhancing lysis of these infected cells by cytotoxic T lymphocytes (CTL). A major challenge in this strategy is overcoming viral immune escape variants that have evaded host immune control. Here we report that naive CD8(+) T cells from chronic HIV-1-infected participants on long-term cART can be primed by dendritic cells (DC). These DC must be mature, produce high levels of interleukin 12p70 (IL-12p70), be responsive to CD40 ligand (CD40L), and be loaded with inactivated, autologous HIV-1. These DC-primed CD8(+) T cell responders produced high levels of gamma interferon (IFN-γ) in response to a broad range of both conserved and variable regions of Gag and effectively killed CD4(+) T cell targets that were either infected with the autologous latent reservoir-associated virus or loaded with autologous Gag peptides. In contrast, HIV-1-specific memory CD8(+) T cells stimulated with autologous HIV-1-loaded DC produced IFN-γ in response to a narrow range of conserved and variable Gag peptides compared to the primed T cells and most notably, displayed significantly lower cytolytic function. Our findings highlight the need to selectively induce new HIV-1-specific CTL from naive precursors while avoiding activation of existing, dysfunctional memory T cells in potential curative immunotherapeutic strategies for HIV-1 infection.Current immunotherapeutic approaches aim to enhance antiviral immunity against the HIV-1 reservoir; however, it has yet to be shown whether T cells from persons on cART can recognize and eliminate virus-infected cells. We show that in persons on cART a personalized medicine approach using their dendritic cells to stimulate their naive T cells induces potent effector CTL in vitro that recognize and eradicate HIV-1-infected CD4(+) T cells. Additionally, we show that the same stimulation of existing memory T cells results in cytokine secretion but limited effector function. Our study demonstrates that the naive T cell repertoire can recognize persistent HIV-1 during cART and supports immunotherapy strategies for an HIV-1 cure that targets naive T cells, rather than existing, dysfunctional, memory T cells.

Curing HIV-1 infection will require elimination of persistent cellular reservoirs that harbor latent virus in the face of combination antiretroviral therapy (cART). Proposed immunotherapeutic strategies to cure HIV-1 infection include enhancing lysis of these infected cells by cytotoxic T lymphocytes (CTL). A major challenge in this strategy is overcoming viral immune escape variants that have evaded host immune control. Here we report that naive CD8+ T cells from chronic HIV-1-infected participants on long-term cART can be primed by dendritic cells (DC). These DC must be mature, produce high levels of interleukin 12p70 (IL-12p70), be responsive to CD40 ligand (CD40L), and be loaded with inactivated, autologous HIV-1. These DC-primed CD8+ T cell responders produced high levels of gamma interferon (IFN-γ) in response to a broad range of both conserved and variable regions of Gag and effectively killed CD4+ T cell targets that were either infected with the autologous latent reservoir-associated virus or loaded with autologous Gag peptides. In contrast, HIV-1-specific memory CD8+ T cells stimulated with autologous HIV-1-loaded DC produced IFN-γ in response to a narrow range of conserved and variable Gag peptides compared to the primed T cells and most notably, displayed significantly lower cytolytic function. Our findings highlight the need to selectively induce new HIV-1-specific CTL from naive precursors while avoiding activation of existing, dysfunctional memory T cells in potential curative immunotherapeutic strategies for HIV-1 infection., IMPORTANCE Current immunotherapeutic approaches aim to enhance antiviral immunity against the HIV-1 reservoir; however, it has yet to be shown whether T cells from persons on cART can recognize and eliminate virus-infected cells. We show that in persons on cART a personalized medicine approach using their dendritic cells to stimulate their naive T cells induces potent effector CTL in vitro that recognize and eradicate HIV-1-infected CD4+ T cells. Additionally, we show that the same stimulation of existing memory T cells results in cytokine secretion but limited effector function. Our study demonstrates that the naive T cell repertoire can recognize persistent HIV-1 during cART and supports immunotherapy strategies for an HIV-1 cure that targets naive T cells, rather than existing, dysfunctional, memory T cells.

Summary Detailed knowledge of dengue virus (DENV) cell-mediated immunity is limited. In this study we characterize CD8+T lymphocytes recognizing three novel and two known non-structural protein 3 peptide epitopes in DENV-infected dendritic cells. Three epitopes displayed high conservation (75–100%), compared to the others (0–50%). A hierarchy ranking based on magnitude and polyfunctionality of the antigen-specific response showed that dominant epitopes were both highly conserved and cross-reactive against multiple DENV serotypes. These results are relevant to DENV pathogenesis and vaccine design.

Cryptococcus neoformans is one of the most common causes of fungal disease in HIV-infected persons, but not all of those who are infected develop cryptococcal disease (CD). Although CD4+ T cell deficiency is a risk factor for HIV-associated CD, polymorphisms of phagocytic Fc gamma receptors (FCGRs) have been linked to CD risk in HIV-uninfected persons. To investigate associations between FCGR2A 131 H/R and FCGR3A 158 F/V polymorphisms and CD risk in HIV-infected persons, we performed PCR-based genotyping on banked samples from 164 men enrolled in the Multicenter AIDS Cohort Study (MACS): 55 who were HIV infected and developed CD and a matched control group of 54 who were HIV infected and 55 who were HIV uninfected. Using additive and allelic statistical models for analysis, the high-affinity FCGR3A 158V allele was significantly associated with CD status after adjusting for race/ethnicity (odds ratio [OR], 2.1; P = 0.005), as was the FCGR3A 158 VV homozygous genotype after adjusting for race/ethnicity, rate of CD4+ T cell decline, and nadir CD4+ T cell count (OR, 21; P = 0.005). No associations between CD and FCGR2A 131 H/R polymorphism were identified. In binding studies, human IgG (hIgG)-C. neoformans complexes exhibited more binding to CHO-K1 cells expressing FCGR3A 158V than to those expressing FCGR3A 158F, and in cytotoxicity assays, natural killer (NK) cells expressing FCGR3A 158V induced more C. neoformans-infected monocyte cytotoxicity than those expressing FCGR3A 158F. Together, these results show an association between the FCGR3A 158V allele and risk for HIV-associated CD and suggest that this polymorphism could promote C. neoformans pathogenesis via increased binding of C. neoformans immune complexes, resulting in increased phagocyte cargo and/or immune activation., IMPORTANCE HIV-associated CD4+ T cell deficiency is a sine qua non for HIV-associated cryptococcal disease (CD), but not all patients with CD4+ T cell deficiency develop CD despite serological evidence of previous infection. At present, there are no biomarkers that predict HIV-associated CD risk. The goal of our study was to understand whether Fc gamma receptor (FCGR) polymorphisms that have been shown to portend CD risk in HIV-uninfected people are associated with CD risk in HIV-infected people. Such biomarkers could identify those who would benefit most from targeted prophylaxis and/or earlier treatment, particularly in sub-Saharan Africa, where there are nearly a million cases of HIV-associated CD annually. A biomarker of risk could also identify potential candidates for immunization, should there be a vaccine for Cryptococcus neoformans.

A variety of strategies have been attempted in the past to stably transduce natural killer (NK) cells with cytokine or other cellular genes. Here, we demonstrate the successful delivery of the interleukin-2 (IL-2) gene into two human NK cell lines, IL-2–dependent NK-92 and IL-2–independent YT, by retroviral transduction. An MuLV-based retroviral vector expressing human IL-2 andneor markers from a polycistronic message was constructed and transduced into a CRIP packaging cell line. By coincubation of NK cells with monolayers of CRIP cells or by using retrovirus-containing supernatants in a flow-through method, 10% to 20% of NK cells were stably transduced. Upon selection in the presence of increasing G418 concentrations, transduced NK cells were able to proliferate independently of IL-2 for more than 5 months and to secrete up to 5.5 ng/106 cells/24 h of IL-2. IL-2 gene-transduced NK-92 cells had an in vitro cytotoxicity against tumor targets that was significantly higher than that of parental cells and secreted interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) in addition to IL-2. Moreover, the in vivo antitumor activity of IL-2 gene-transduced NK-92 cells against established 3-day liver metastases in mice was greater than that of parental nontransduced NK cells. Stable expression of the IL-2 transgene in NK cells improved their therapeutic potential in tumor-bearing hosts. Thus, transduced NK cells secreted sufficient quantities of bioactive IL-2 to proliferate in vitro and mediated the antitumor effects both in vitro and in vivo in the absence of exogenous IL-2. These results suggest that genetic modification of NK cells ex vivo could be useful for clinical cancer therapy in the future.

For HIV-1 to effectively establish infection and persistence, it must circumvent host immune detection. The capacity of HIV-1 to efficiently mutate and generate multivariant strains in response to host immune selective pressure provides the virus with a particularly effective means of evading cytotoxic T-lymphocyte (CTL) recognition and elimination.1 However, a novel concept has emerged suggesting that HIV-1 can actually benefit from the strategic “baiting” and partial activation of HIV-1-specific CTL responders.2 Driven by immune pressure, CTL epitope divergence can result in the occurrence of surviving epitope variants capable of selectively activating the helper activity of preexisting cross-reactive CTL to create a safe haven for the virus within antigen-presenting dendritic cells (DC). Instead of dampening the immune response through CTL recognition and subsequent killing of these HIV-1 antigen-expressing DC targets, a dysfunctional CTL:DC cross-talk occurs. As a result, these DC become activated and programmed to differentiate into proinflammatory cells displaying unique features that contribute to viral dissemination and persistence.2 We have found that CTL-activated DC are capable of producing high levels of proinflammatory cytokines such as interleukin (IL)-12 and IL-6, but also simultaneously express some immunosuppressive factors such as IL-10 and PD-L1, which may contribute to their resistance to direct cellular attack. They are also programmed to produce a wide range of chemokines, including CCL4, CCL5, CXCL9, and CXCL10, that can preferentially attract activated CD4+ T helper (Th) cells, the main target of HIV-1 infection. Moreover, the production of CCL19 by these DC also promotes their interaction with CCR7+/CD45RA+ naive Th cells as well as CCR7+/CD45RO+ central memory Th cells, the major cellular reservoir of latent HIV-1.3 During this secondary interaction with Th cells, these CTL-programmed DC uniquely undergo fantastic morphological changes, characterized by their rapid development of numerous, far-reaching tunneling nanotube-like membrane protrusions. This intriguing phenomenon ultimately leads to the formation of complex nanotube networks, which facilitate the direct transfer of both cell surface and cytoplasmic components between interconnected neighboring and distant cells. Importantly, these membrane bridges can also serve as highways for HIV-1 to utilize for efficient cell-to-cell transfer (Fig. 1). FIG. 1. Live-cell differential interference contrast image of eukaryotic green fluorescent protein (EGFP)-expressing HIV-1-like particles trafficking via dendritic cell (DC) membrane bridges. Our findings add a new dimension to the proposed concept of “original antigenic sin”4 in which the promiscuous nature of preexisting CTL can be exploited by an evolving virus to modulate the functional and physical characteristics of the DC, and to create an inflammatory environment suitable for viral spread and persistence within the host. These findings also highlight the need to consider the detrimental potential of selectively activating dysfunctional memory CTL responses when implementing anti-HIV-1 vaccine strategies.

Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4. TgM were inoculated with peptides pools and the T-cell immunogenic peptides were identified by ELISPOT. Nine HLA class I and 97 HLA class II novel DENV3 epitopes were identified based on immunogenicity in TgM and their HLA affinity was further confirmed by binding assays analysis. A subset of these epitopes activated memory T-cells from DENV3 immune volunteers and was also capable of priming naïve T-cells, ex vivo, from dengue IgG negative individuals. Analysis of inter- and intra-serotype variation of such an epitope (A02-restricted) allowed us to identify altered peptide ligands not only in DENV3 but also in other DENV serotypes. These studies also characterized the HLA promiscuity of 23 HLA class II epitopes bearing highly conserved sequences, six of which could bind to more than 10 different HLA molecules representing a large percentage of the global population. These epitope data are invaluable to investigate the role of T-cells in dengue immunity/pathogenesis and vaccine design., Author Summary Although there is an increased recognition of the role of T-cells in both dengue pathogenesis and protection, comprehensive analysis of T-cell activation during dengue infection is hampered by the small repertoire of known human dengue T-cell epitopes. Although dengue serotype 3 (DENV3) is responsible for numerous outbreaks worldwide, most of the known epitopes are from studies of dengue 2 serotype (DENV2). In this study, we identified novel DENV3 T-cell epitopes in HLA transgenic mice that were confirmed by HLA binding assays. A subset of these epitopes activated memory T-cells from subjects who were dengue IgG positive and primed naïve T-cells from dengue IgG negative individuals. Notably, some of HLA class II epitopes bearing highly conserved regions common to all four dengue serotypes could bind to multiple HLAs. We postulate that these highly conserved and HLA promiscuous T-helper epitopes can be important components of a dengue tetravalent vaccine.

Suggest new approaches focus on engineer antigenic structures capable of exposing a stable conformation of these epitopes and thus inducing B-cell secretion of HIV-1 pnAbs. We postulated that conformationally stable structures are better immunogens and can elicit the production of better antibodies and results show that MD simulations can help engineer vaccine antigens capable of enhancing protective antibody responses.

Background The ability of HIV-1 to rapidly accumulate mutations provides the virus with an effective means of escaping CD8+ cytotoxic T lymphocyte (CTL) responses. Here we describe how subtle alterations in CTL epitopes expressed by naturally occurring HIV-1 variants can result in an incomplete escape from pre-existing CTL recognition to create a pro-inflammatory environment, providing the virus with a selective advantage.

Background: HIV-1 remains sequestered during antiretroviral therapy (ART) and can resume high-level replication upon cessation of ART or development of drug resistance. Reactivity of memory CD8 + T lymphocytes to HIV-1 could potentially inhibit this residual viral replication, but is largely muted by ART in relation to suppression of viral antigen burden. Dendritic cells (DC) are important for MHC class I processing and presentation of peptide epitopes to memory CD8 + T cells, and could potentially be targeted to activate memory CD8 + T cells to a broad array of HIV-1 epitopes during ART. Principal Findings: We show for the first time that HIV-1 peptide-loaded, CD40L-matured DC from HIV-1 infected persons on ART induce IFN gamma production by CD8 + T cells specific for a much broader range and magnitude of Gag and Nef epitopes than do peptides without DC. The DC also reveal novel, MHC class I restricted, Gag and Nef epitopes that are able to induce polyfunctional T cells producing various combinations of IFN gamma, interleukin 2, tumor necrosis factor alpha, macrophage inhibitory protein 1 beta and the cytotoxic de-granulation molecule CD107a. Significance: There is an underlying, broad antigenic spectrum of anti-HIV-1, memory CD8 + T cell reactivity in persons on ART that is revealed by DC. This supports the use of DC-based immunotherapy for HIV-1 infection.

Antiretroviral therapy (ART) decreases HIV-1 viremia and AIDS-associated mortality. Despite this, HIV infected patients are unable to clear virus during treatment interruption due to insufficient cytotoxic T cell (CTL) activity against the autologous reservoir. It is unclear if naive T cells from patients on ART can respond to immunotherapies that induce CTL specific for their own, unique virus. Unfortunately, late-evolving virus and the ART reservoir contain escape epitope variants that confer a lack of CTL control. We hypothesize that a dendritic cell (DC)-based immunotherapy during ART can induce CTL capable of eliminating the autologous reservoir, despite their failure to do so during natural infection.

BackgroundHIV-specific cellular immune responses are associated with control of viremia and delayed disease progression. An effective therapeutic vaccine could mimic these effects and reduce the need for continued antiretroviral therapy. DermaVir, a topically administered plasmid DNA-nanomedicine expressing HIV (CladeB) virus-like particles consisting of 15 antigens, induces predominantly central memory T-cell responses.MethodsTreated HIV-infected adults (HIV RNA 350) were randomized to placebo or escalating DermaVir doses (0.1 or 0.4 mg of plasmid DNA at weeks 1, 7, and 13 in the low- and intermediate-dose groups and 0.8 mg at weeks 0, 1, 6, 7, 12, and 13 in the high-dose group), n = 5-6 evaluable subjects per group. Immunogenicity was assessed by a 12-day cultured interferon-γ enzyme-linked immunosorbent spot assay at baseline and at weeks 9, 17, and 37 using 1 Tat/Rev and 3 overlapping Gag peptide pools (p17, p24, and p15).ResultsGroups were comparable at baseline. The study intervention was well tolerated, without dose-limiting toxicities. Most responses were highest at week 17 (4 weeks after last vaccination) when Gag p24 responses were significantly greater among intermediate-dose group compared with control subjects [median (IQR): 67,600 (5633-74,368) versus 1194 (9-1667)] net spot-forming units per million cells, P = 0.032. In the intermediate-dose group, there was also a marginal Gag p15 response increase from baseline to week 17 [2859 (1867-56,933), P = 0.06], and this change was significantly greater than in the placebo group [0 (-713 to 297), P = 0.016].ConclusionsDermaVir administration was associated with a trend toward greater HIV-specific, predominantly central memory T-cell responses. The intermediate DermaVir dose tended to show the greatest immunogenicity, consistent with previous studies in different HIV-infected patient populations.