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Over the last few decades, several emerging or reemerging viral diseases with no readily available vaccines have ravaged the world. A platform to fastly generate vaccines inducing potent and durable neutralizing antibody and T cell responses is sorely needed. Bioinformatically identified epitope-based vaccines can focus on immunodominant T cell epitopes and induce more potent immune responses than a whole antigen vaccine and may be deployed more rapidly and less costly than whole-gene vaccines. Increasing evidence has shown the importance of the CD4+ T cell response in protection against HIV and other viral infections. The previously described DNA vaccine HIVBr18 encodes 18 conserved, promiscuous epitopes binding to multiple HLA-DR-binding HIV epitopes amply recognized by HIV-1-infected patients. HIVBr18 elicited broad, polyfunctional, and durable CD4 + and CD8+ T cell responses in BALB/c and mice transgenic to HLA class II alleles, showing cross-species promiscuity. To fully delineate the promiscuity of the HLA class II vaccine epitopes, we assessed their binding to 34 human class II (HLA-DR, DQ, and -DP) molecules, and immunized nonhuman primates. Results ascertained redundant 100% coverage of the human population for multiple peptides. We then immunized Rhesus macaques with HIVBr18 under in vivo electroporation. The immunization induced strong, predominantly polyfunctional CD4+ T cell responses in all animals to 13 out of the 18 epitopes; T cells from each animal recognized 7-11 epitopes. Our results provide a preliminary proof of concept that immunization with a vaccine encoding epitopes with high and redundant coverage of the human population can elicit potent T cell responses to multiple epitopes, across species and MHC barriers. This approach may facilitate the rapid deployment of immunogens eliciting cellular immunity against emerging infectious diseases, such as COVID-19., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

During antiretroviral therapy (ART), human immunodeficiency virus type 1 (HIV-1) persists as a latent reservoir in CD4 + T cell subsets in central memory (T CM ), transitional memory (T TM ), and effector memory (T EM ) CD4 + T cells. We have identified differences in mechanisms underlying latency and responses to latency-reversing agents (LRAs) in ex vivo CD4 + memory T cells from virally suppressed HIV-infected individuals and in an in vitro primary cell model of HIV-1 latency. Our ex vivo and in vitro results demonstrate the association of transcriptional pathways of T cell differentiation, acquisition of effector function, and cell cycle entry in response to LRAs. Analyses of memory cell subsets showed that effector memory pathways and cell surface markers of activation and proliferation in the T EM subset are predictive of higher frequencies of cells carrying an inducible reservoir. Transcriptional profiling also demonstrated that the epigenetic machinery (known to control latency and reactivation) in the T EM subset is associated with frequencies of cells with HIV-integrated DNA and inducible HIV multispliced RNA. T CM cells were triggered to differentiate into T EM cells when they were exposed to LRAs, and this increase of T EM subset frequencies upon LRA stimulation was positively associated with higher numbers of p24 + cells. Together, these data highlight differences in underlying biological latency control in different memory CD4 + T cell subsets which harbor latent HIV in vivo and support a role for differentiation into a T EM phenotype in facilitating latency reversal. IMPORTANCE By performing phenotypic analysis of latency reversal in CD4 + T cells from virally suppressed individuals, we identify the T EM subset as the largest contributor to the inducible HIV reservoir. Differential responses of memory CD4 + T cell subsets to latency-reversing agents (LRAs) demonstrate that HIV gene expression is associated with heightened expression of transcriptional pathways associated with differentiation, acquisition of effector function, and cell cycle entry. In vitro modeling of the latent HIV reservoir in memory CD4 + T cell subsets identify LRAs that reverse latency with ranges of efficiency and specificity. We found that therapeutic induction of latency reversal is associated with upregulation of identical sets of T EM -associated genes and cell surface markers shown to be associated with latency reversal in our ex vivo and in vitro models. Together, these data support the idea that the effector memory phenotype supports HIV latency reversal in CD4 + T cells., (Copyright © 2019 Kulpa et al.)

Despite advances in the treatment of HIV infection with ART, elucidating strategies to overcome HIV persistence, including blockade of viral reservoir establishment, maintenance, and expansion, remains a challenge. T cell homeostasis is a major driver of HIV persistence. Cytokines involved in regulating homeostasis of memory T cells, the major hub of the HIV reservoir, trigger the Jak-STAT pathway. We evaluated the ability of tofacitinib and ruxolitinib, two FDA-approved Jak inhibitors, to block seeding and maintenance of the HIV reservoir in vitro. We provide direct demonstration for involvement of the Jak-STAT pathway in HIV persistence in vivo, ex vivo, and in vitro; pSTAT5 strongly correlates with increased levels of integrated viral DNA in vivo, and in vitro Jak inhibitors reduce the frequency of CD4+ T cells harboring integrated HIV DNA. We show that Jak inhibitors block viral production from infected cells, inhibit γ-C receptor cytokine (IL-15)-induced viral reactivation from latent stores thereby preventing transmission of infectious particles to bystander activated T cells. These results show that dysregulation of the Jak-STAT pathway is associated with viral persistence in vivo, and that Jak inhibitors target key events downstream of γ-C cytokine (IL-2, IL-7 and IL-15) ligation to their receptors, impacting the magnitude of the HIV reservoir in all memory CD4 T cell subsets in vitro and ex vivo. Jak inhibitors represent a therapeutic modality to prevent key events of T cell activation that regulate HIV persistence and together, specific, potent blockade of these events may be integrated to future curative strategies.

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.

We aimed to assess the status of naturally occurring CD4(+) and CD8(+) T cell responses to a tumour associated antigen, Mesothelin, in patients with pancreatic carcinoma and study the effects of elevated IL-10 on Mesothelin-specific T cell responses. For that sake, short term T cell lines were generated from PBMCs of 16 healthy controls, 15 patients with benign pancreatic diseases and 25 patients with pancreatic carcinoma and Mesothelin-specific CD4(+) and CD8(+) T cell responses were analysed using intracellular cytokine assays for IFN-γ. Plasma levels of IL-10 and Mesothelin were measured using cytometric bead array and ELISA assay, respectively. The blocking assays were performed to assess the effects of IL-10 on Mesothelin-specific T cell responses. Here, we demonstrate that the plasma levels of Mesothelin and IL-10 are significantly increased in patients with pancreatic carcinoma. Additionally, we found that (a) Mesothelin-specific T cell responses are significantly expanded in cancer patients (p = 0.0053), (b) the multifunctional CD4(+) T cell response is directed toward a broad repertoire of epitopes within the Mesothelin protein. (c) Mesothelin-specific CD4+ T cell response is directly inhibited by elevated IL-10 in cancer patients. These data provides evidence for the use of Mesothelin as an immunogen for tumour-specific T cell response.

CD152 (CTLA-4) is a co-stimulatory molecule that is expressed by T cells and negatively regulates immune responses. Here, we report the identification of a novel ligand, GPC(81-95), with the ability to induce both CD152 and LAP (TGF-β1) on human Foxp3(-) CD25(-) CD4(+) T cells. The results demonstrate that GPC(81-95) peptide-induced cell surface CD152 is endocytosed back into the cell during stimulation. The protein export and exocytosis of CD152 is also induced by this ligand. The inhibitory effects of GPC(81-95) on LPS-induced TNF-α production was shown to be closely associated with its ability to induce both LAP (TGF-β1) and CD152. Taken together, we have shown that a novel peptide ligand stimulates LAP (TGF-β1) and CD152 expression on resting CD4 T cells and have demonstrated that GPC(81-95) is a useful tool to study the functional properties of LAP (TGF-β1)(+) CD152(+) CD4(+) T cells., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

T-cell based vaccine approaches have emerged to counteract HIV-1/AIDS. Broad, polyfunctional and cytotoxic CD4(+) T-cell responses have been associated with control of HIV-1 replication, which supports the inclusion of CD4(+) T-cell epitopes in vaccines. A successful HIV-1 vaccine should also be designed to overcome viral genetic diversity and be able to confer immunity in a high proportion of immunized individuals from a diverse HLA-bearing population. In this study, we rationally designed a multiepitopic DNA vaccine in order to elicit broad and cross-clade CD4(+) T-cell responses against highly conserved and promiscuous peptides from the HIV-1 M-group consensus sequence. We identified 27 conserved, multiple HLA-DR-binding peptides in the HIV-1 M-group consensus sequences of Gag, Pol, Nef, Vif, Vpr, Rev and Vpu using the TEPITOPE algorithm. The peptides bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and also to several molecules such as HLA-DP, -DQ and murine IA(b) and IA(d). Sixteen out of the 27 peptides were recognized by PBMC from patients infected with different HIV-1 variants and 72% of such patients recognized at least 1 peptide. Immunization with a DNA vaccine (HIVBr27) encoding the identified peptides elicited IFN-γ secretion against 11 out of the 27 peptides in BALB/c mice; CD4(+) and CD8(+) T-cell proliferation was observed against 8 and 6 peptides, respectively. HIVBr27 immunization elicited cross-clade T-cell responses against several HIV-1 peptide variants. Polyfunctional CD4(+) and CD8(+) T cells, able to simultaneously proliferate and produce IFN-γ and TNF-α, were also observed. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS.

CD4(+) T cells expressing the latent form of transforming growth factor-β [latency-associated peptide (LAP) (TGF-β(1))] play an important role in the modulation of immune responses. Here, we identified a novel peptide ligand (GPC(81-95) ) with an intrinsic ability to induce membrane-bound LAP (TGF-β(1)) expression on a subpopulation of human CD4(+) T cells (using flow cytometry; ranging from 0·8% to 2·6%) and stimulate peripheral blood mononuclear cells to release LAP (TGF-β(1) ) (using ELISPOT assay; ranging from 0·03% to 0·16%). In spite of this low percentage of responding cells, GPC(81-95) significantly reduced Toll-like receptor 4 ligand-induced tumour necrosis factor-α production in a TGF-β(1) - and CD4(+) T-cell-dependent manner. The results demonstrate that GPC(81-95) is a useful tool to study the functional properties of a subpopulation of LAP (TGF-β(1))(+) CD4(+) T cells and suggest a pathway that can be exploited to suppress inflammatory response., (© 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.)

T-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4(+) T cells are important for the generation and maintenance of functional CD8(+) cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18), capable of eliciting broad CD4(+) T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4(+)/CD8(+) T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4(+) and CD8(+) T cells that proliferate and produce any two cytokines (IFNγ/TNFα, IFNγ/IL-2 or TNFα/IL-2) simultaneously in response to HIV-1 peptides. For CD4(+) T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFNγ/TNFα/IL-2). The vaccine also generated long-lived central and effector memory CD4(+) T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4(+) T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8(+) T cells and antibody responses- elicited by other HIV immunogens.

Current HIV vaccine approaches are focused on immunogens encoding whole HIV antigenic proteins that mainly elicit cytotoxic CD8+ responses. Mounting evidence points toward a critical role for CD4+ T cells in the control of immunodeficiency virus replication, probably due to cognate help. Vaccine-induced CD4+ T cell responses might, therefore, have a protective effect in HIV replication. In addition, successful vaccines may have to elicit responses to multiple epitopes in a high proportion of vaccinees, to match the highly variable circulating strains of HIV. Using rational vaccine design, we developed a DNA vaccine encoding 18 algorithm-selected conserved, "promiscuous" (multiple HLA-DR-binding) B-subtype HIV CD4 epitopes - previously found to be frequently recognized by HIV-infected patients. We assessed the ability of the vaccine to induce broad T cell responses in the context of multiple HLA class II molecules using different strains of HLA class II- transgenic mice (-DR2, -DR4, -DQ6 and -DQ8). Mice displayed CD4+ and CD8+ T cell responses of significant breadth and magnitude, and 16 out of the 18 encoded epitopes were recognized. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that can be recognized in the context of widely diverse, common HLA class II alleles, this vaccine concept may cope both with HIV genetic variability and increased population coverage. The vaccine may thus be a source of cognate help for HIV-specific CD8+ T cells elicited by conventional immunogens, in a wide proportion of vaccinees.

T cell epitope-driven vaccine design employs bioinformatic algorithms to identify potential targets of vaccines against infectious diseases or cancer. Potential epitopes can be identified with major histocompatibility complex (MHC)-binding algorithms, and the ability to bind to MHC class I or class II indicates a predominantly CD4(+) or CD8(+) T cell response. Furthermore, an epitope-based vaccine can circumvent evolutionary events favoring immune escape present in native proteins from pathogens. It can also focus on only the most relevant epitopes (i.e. conserved and promiscuous) recognized by the majority of the target population. Mounting evidence points to the critical role of CD4(+) T cells in natural antigen encounter and active immunization. In this paper the need for CD4(+) T cell help in vaccine development, the selection of CD4(+) T cell epitopes for an epitope-based vaccine, and how the approach can be used to induce a protective effect are reviewed.

Necrosis of tumor cells can activate both innate and adaptive antitumor immunity. However, there is little information on the effects of necrosis-inducing cancer treatments on tumor-specific T cell immune responses in humans. We studied the effects of a necrosis-inducing treatment (embolization) on anti-alpha-fetoprotein (AFP)-specific CD4(+) T cell responses in hepatocellular carcinoma (HCC) patients and controls using an array of AFP-derived peptides. In this study, we show that AFP-specific CD4(+) T cell responses to three immunodominant epitopes in HCC patients were significantly expanded during (p < 0.0001) and after embolization (p < 0.002). The development of higher frequencies of AFP-specific CD4(+) T cells after treatment were significantly associated with the induction of >50% necrosis of tumor and an improved clinical outcome (p < 0.007). In addition, we identified two novel HLA-DR-restricted AFP-derived CD4(+) T cell epitopes (AFP(137-145) and AFP(249-258)) and showed that the CD4(+) T cells recognizing these epitopes produce Th1 (IFN-gamma and TNF-alpha) but not Th2 (IL-5)-type cytokines. AFP(137-145)-, AFP(249-258)-, and AFP(364-373)-specific CD4(+) T cells were detected in HCC patients but not in patients with chronic liver diseases or healthy donors. In conclusion; our study shows that induction of tumor necrosis by a conventional cancer treatment can unmask tumor rejection Ag cell-mediated immunity and provides a rationale for combining embolization with immunotherapy in HCC patients.