Aziz Alami Chentoufi

A genital tract peptide epitope vaccine targeting TLR-2 efficiently induces local and systemic CD8 + T cells and protects against herpes simplex virus type 2 challenge

The next generation of needle-free mucosal vaccines is being rationally designed according to rules that govern the way in which the epitopes are recognized by and stimulate the genital mucosal immune system. We hypothesized that synthetic peptide epitopes extended with an agonist of Toll-like receptor 2 (TLR-2), that are abundantly expressed by dendritic and epithelial cells of the vaginal mucosa, would lead to induction of protective immunity against genital herpes. To test this hypothesis, we intravaginally (IVAG) immunized wild-type B6, TLR-2 (TLR2−/−) or myeloid differentiation factor 88 deficient (MyD88−/−) mice with a herpes simplex virus type 2 (HSV-2) CD8+ T-cell peptide epitope extended by a palmitic acid moiety (a TLR-2 agonist). IVAG delivery of the lipopeptide generated HSV-2-specific memory CD8+ cytotoxic T cells both locally in the genital tract draining lymph nodes and systemically in the spleen. Moreover, lipopeptide-immunized TLR2−/− and MyD88−/− mice developed significantly less HSV-specific CD8+ T-cell response, earlier death, faster disease progression, and higher vaginal HSV-2 titers compared to lipopeptide-immunized wild-type B6 mice. IVAG immunization with self-adjuvanting lipid-tailed peptides appears to be a novel mucosal vaccine approach, which has attractive practical and immunological features.

HLA-A*0201-Restricted CD8+ Cytotoxic T Lymphocyte Epitopes Identified from Herpes Simplex Virus Glycoprotein D

Evidence obtained from both animal models and humans suggests that T cells specific for HSV-1 and HSV-2 glycoprotein D (gD) contribute to protective immunity against herpes infection. However, knowledge of gD-specific human T cell responses is limited to CD4+ T cell epitopes, with no CD8+ T cell epitopes identified to date. In this study, we screened the HSV-1 gD amino acid sequence for HLA-A*0201-restricted epitopes using several predictive computational algorithms and identified 10 high probability CD8+ T cell epitopes. Synthetic peptides corresponding to four of these epitopes, each nine to 10 amino acids in length, exhibited high-affinity binding in vitro to purified human HLA-A*0201 molecules. Three of these four peptide epitopes, gD53–61, gD70–78, and gD278–286, significantly stabilized HLA-A*0201 molecules on T2 cell lines and are highly conserved among and between HSV-1 and HSV-2 strains. Consistent with this, in 33 sequentially studied HLA-A*0201-positive, HSV-1-seropositive, and/or HSV-2-seropositive healthy individuals, the most frequent and robust CD8+ T cell responses, assessed by IFN-γ ELISPOT, CD107a/b cytotoxic degranulation, and tetramer assays, were directed mainly against gD53–61, gD70–78, and gD278–286 epitopes. In addition, CD8+ T cell lines generated by gD53–61, gD70–78, and gD278–286 peptides recognized infected target cells expressing native gD. Lastly, CD8+ T cell responses specific to gD53–61, gD70–78, and gD278–286 epitopes were induced in HLA-A*0201 transgenic mice following ocular or genital infection with either HSV-1 or HSV-2. The functional gD CD8+ T cell epitopes described herein are potentially important components of clinical immunotherapeutic and immunoprophylactic herpes vaccines.

Natural Regulatory T Cells Control Coronary Arteriolar Endothelial Dysfunction in Hypertensive Mice

Coronary artery disease in patients with hypertension is increasing worldwide and leads to severe cardiovascular complications. The cellular and molecular mechanisms that underlie this pathologic condition are not well understood. Experimental and clinical research indicates that immune cells and inflammation play a central role in the pathogenesis of cardiovascular diseases. Recently, it has been reported that CD4(+)CD25(+) regulatory T cells (Tregs) regulate heart fibrosis in hypertension. In this study, we determined the role of Tregs in coronary arteriolar endothelial dysfunction in angiotensin II-dependent hypertensive mice. Mice infused with angiotensin II had significantly increased blood pressure, as determined using telemetry, and apoptotic Treg numbers, as measured using flow cytometry. The mice displayed inflammation, assessed by macrophage activation/infiltration into coronary arterioles and the heart, and increased local tumor necrosis factor-α release, which participates in reduced coronary arteriolar endothelial-dependent relaxation in response to acetylcholine using an arteriograph. Hypertensive mice injected with Tregs isolated from control mice had significantly reduced macrophage activation and infiltration, reduced tumor necrosis factor-α release, and improved coronary arteriolar endothelium-dependent relaxation. Our novel data indicate that Tregs are important in the development of coronary arteriolar endothelial dysfunction in hypertension. These results suggest a new direction in the investigation of vascular disease in hypertension and could lead to a therapeutic strategy that involves immune system modulation using Tregs.

Antitumor activity of a self-adjuvanting glyco-lipopeptide vaccine bearing B cell, CD4+ and CD8+ T cell epitopes

Molecularly defined synthetic vaccines capable of inducing both antibodies and cellular anti-tumor immune responses, in a manner compatible with human delivery, are limited. Few molecules achieve this target without utilizing external immuno-adjuvants. In this study, we explored a self-adjuvanting glyco-lipopeptide (GLP) as a platform for cancer vaccines using as a model MO5, an OVA-expressing mouse B16 melanoma. A prototype B and T cell epitope-based GLP molecule was constructed by synthesizing a chimeric peptide made of a CD8+ T cell epitope, from ovalbumin (OVA257–264) and an universal CD4+ T helper (Th) epitope (PADRE). The resulting CTL–Th peptide backbones was coupled to a carbohydrate B cell epitope based on a regioselectively addressable functionalized templates (RAFT), made of four α-GalNAc molecules at C-terminal. The N terminus of the resulting glycopeptides (GP) was then linked to a palmitic acid moiety (PAM), obviating the need for potentially toxic external immuno-adjuvants. The final prototype OVA-GLP molecule, delivered in adjuvant-free PBS, in mice induced: (1) robust RAFT-specific IgG/IgM that recognized tumor cell lines; (2) local and systemic OVA257–264-specific IFN-γ producing CD8+ T cells; (3) PADRE-specific CD4+ T cells; (4) OVA-GLP vaccination elicited a reduction of tumor size in mice inoculated with syngeneic murine MO5 carcinoma cells and a protection from lethal carcinoma cell challenge; (5) finally, OVA-GLP immunization significantly inhibited the growth of pre-established MO5 tumors. Our results suggest self-adjuvanting glyco-lipopeptide molecules as a platform for B Cell, CD4+, and CD8+ T cell epitopes-based immunotherapeutic cancer vaccines.

A novel HLA (HLA-A*0201) transgenic rabbit model for preclinical evaluation of human CD8+ T cell epitope-based vaccines against ocular herpes.

We introduced a novel humanized HLA-A*0201 transgenic (HLA Tg) rabbit model to assess the protective efficacy of a human CD8+ T cell epitope-based vaccine against primary ocular herpes infection and disease. Each of the three immunodominant human CD8+ T cell peptide epitopes from HSV-1 glycoprotein D (gD53–61, gD70–78, and gD278–286) were joined with a promiscuous human CD4+ T cell peptide epitope (gD49–82) to construct three separate pairs of CD4–CD8 peptides. Each CD4–CD8 peptide pair was then covalently linked to an Nε-palmitoyl–lysine residue via a functional base lysine amino group to construct CD4–CD8 lipopeptides. HLA Tg rabbits were immunized s.c. with a mixture of the three CD4–CD8 HSV-1 gD lipopeptides. The HSV-gD–specific T cell responses induced by the mixture of CD4–CD8 lipopeptide vaccine and the protective efficacy against acute virus replication and ocular disease were determined. Immunization induced HSV-gD49–82–specific CD4+ T cells in draining lymph node (DLN); induced HLA-restricted HSV-gD53–61, gD70–78, and gD278–286–specific CD8+ T cells in DLN, conjunctiva, and trigeminal ganglia and reduced HSV-1 replication in tears and corneal eye disease after ocular HSV-1 challenge. In addition, the HSV-1 epitope-specific CD8+ T cells induced in DLNs, conjunctiva, and the trigeminal ganglia were inversely proportional with corneal disease. The humanized HLA Tg rabbits appeared to be a useful preclinical animal model for investigating the immunogenicity and protective efficacy of human CD8+ T cell epitope-based prophylactic vaccines against ocular herpes. The relevance of HLA Tg rabbits for future investigation of human CD4–CD8 epitope-based therapeutic vaccines against recurrent HSV-1 is discussed.

The Herpes Simplex Virus Type 1 Latency-Associated Transcript Can Protect Neuron-Derived C1300 and Neuro2A Cells from Granzyme B-Induced Apoptosis and CD8 T-Cell Killing

ABSTRACT The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is the only HSV-1 gene transcript abundantly expressed throughout latency. LAT null mutants have a significantly reduced reactivation phenotype. LATs antiapoptosis activity is the major LAT factor involved in supporting the wild-type reactivation phenotype. During HSV-1 latency, some ganglionic neurons are surrounded by CD8 T cells, and it has been proposed that these CD8 T cells help maintain HSV-1 latency by suppressing viral reactivations. Surprisingly, despite injection of cytotoxic lytic granules by these CD8 T cells into latently infected neurons, neither apoptosis nor neuronal cell death appears to occur. We hypothesized that protection of latently infected neurons against cytotoxic CD8 T-cell killing is due to LATs antiapoptosis activity. Since CD8 T-cell cytotoxic lytic granule-mediated apoptosis is critically dependent on granzyme B (GrB), we examined LATs ability to block GrB-induced apoptosis. We report here that (i) LAT can interfere with GrB-induced apoptosis in cell cultures, (ii) LAT can block GrB-induced cleavage (activation) of caspase-3 both in cell culture and in a cell-free in vitro cell extract assay, and (iii) LAT can protect C1300 and Neuro2A cells from cytotoxic CD8 T-cell killing in vitro. These findings support the hypothesis that LATs antiapoptosis activity can protect latently infected neurons from being killed by CD8 T-cell lytic granules in vivo.

New concepts in herpes simplex virus vaccine development: notes from the battlefield

The recent discovery that T cells recognize different sets of herpes simplex virus type 1 and type 2 epitopes from seropositive symptomatic and asymptomatic individuals might lead to a fundamental immunologic advance in vaccine development against herpes infection and diseases. The newly introduced needle-free mucosal (i.e., topical ocular and intravaginal) lipopeptide vaccines provide a novel strategy that might target ocular and genital herpes and possibly provide ‘heterologous protection’ from HIV-1. Indeed, mucosal self-adjuvanting lipopeptide vaccines are easy to manufacture, simple to characterize, extremely pure, cost-effective, highly immunogenic and safe. In this review, we bring together recent published and unpublished data that illuminates the status of epitope-based herpes vaccine development and present an overview of our recent approach to an ‘asymptomatic epitope’-based lipopeptide vaccine.

Asymptomatic Human CD4+ Cytotoxic T-Cell Epitopes Identified from Herpes Simplex Virus Glycoprotein B

ABSTRACT The identification of “asymptomatic” (i.e., protective) epitopes recognized by T cells from herpes simplex virus (HSV)-seropositive healthy individuals is a prerequisite for an effective vaccine. Using the PepScan epitope mapping strategy, a library of 179 potential peptide epitopes (15-mers overlapping by 10 amino acids) was identified from HSV type 1 (HSV-1) glycoprotein B (gB), an antigen that induces protective immunity in both animal models and humans. Eighteen groups (G1 to G18) of 10 adjacent peptides each were first screened for T-cell antigenicity in 38 HSV-1-seropositive but HSV-2-seronegative individuals. Individual peptides within the two immunodominant groups (i.e., G4 and G14) were further screened with T cells from HLA-DR-genotyped and clinically defined symptomatic (n = 10) and asymptomatic (n = 10) HSV-1-seropositive healthy individuals. Peptides gB161-175 and gB166-180 within G4 and gB661-675 within G14 recalled the strongest HLA-DR-dependent CD4+ T-cell proliferation and gamma interferon production. gB166-180, gB661-675, and gB666-680 elicited ex vivo CD4+ cytotoxic T cells (CTLs) that lysed autologous HSV-1- and vaccinia virus (expressing gB)-infected lymphoblastoid cell lines. Interestingly, gB166-180 and gB666-680 peptide epitopes were strongly recognized by CD4+ T cells from 10 of 10 asymptomatic patients but not by CD4+ T cells from 10 of 10 symptomatic patients (P < 0.0001; analysis of variance posttest). Inversely, CD4+ T cells from symptomatic patients preferentially recognized gB661-675 (P < 0.0001). Thus, we identified three previously unrecognized CD4+ CTL peptide epitopes in HSV-1 gB. Among these, gB166-180 and gB666-680 appear to be “asymptomatic” peptide epitopes and therefore should be considered in the design of future herpes vaccines.

The Herpes Simplex Virus 1 Latency-Associated Transcript Promotes Functional Exhaustion of Virus-Specific CD8+ T Cells in Latently Infected Trigeminal Ganglia: a Novel Immune Evasion Mechanism

ABSTRACT Following ocular herpes simplex virus 1 (HSV-1) infection of C57BL/6 mice, HSV-specific (HSV-gB498–505 tetramer+) CD8+ T cells are induced, selectively retained in latently infected trigeminal ganglia (TG), and appear to decrease HSV-1 reactivation. The HSV-1 latency-associated transcript (LAT) gene, the only viral gene that is abundantly transcribed during latency, increases reactivation. Previously we found that during latency with HSV-1 strain McKrae-derived viruses, more of the total TG resident CD8 T cells expressed markers of exhaustion with LAT+ virus compared to LAT− virus. Here we extend these findings to HSV-1 strain 17syn+-derived LAT+ and LAT− viruses and to a virus expressing just the first 20% of LAT. Thus, the previous findings were not an artifact of HSV-1 strain McKrae, and the LAT function involved mapped to the first 1.5 kb of LAT. Importantly, to our knowledge, we show here for the first time that during LAT+ virus latency, most of the HSV-1-specific TG resident CD8 T cells were functionally exhausted, as judged by low cytotoxic function and decreased gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) production. This resulted in LAT− TG having more functional HSV-gB498–505 tetramer+ CD8+ T cells compared to LAT+ TG. In addition, LAT expression, in the absence of other HSV-1 gene products, appeared to be able to directly or indirectly upregulate both PD-L1 and major histocompatibility complex class I (MHC-I) on mouse neuroblastoma cells (Neuro2A). These findings may constitute a novel immune evasion mechanism whereby the HSV-1 LAT directly or indirectly promotes functional exhaustion (i.e., dysfunction) of HSV-specific CD8+ T cells in latently infected TG, resulting in increased virus reactivation.

Towards a Rational Design of an Asymptomatic Clinical Herpes Vaccine: The Old, the New, and the Unknown

The best hope of controlling the herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) pandemic is the development of an effective vaccine. However, in spite of several clinical trials, starting as early as 1920s, no vaccine has been proven sufficiently safe and efficient to warrant commercial development. In recent years, great strides in cellular and molecular immunology have stimulated creative efforts in controlling herpes infection and disease. However, before moving towards new vaccine strategy, it is necessary to answer two fundamental questions: (i) why past herpes vaccines have failed? (ii) Why the majority of HSV seropositive individuals (i.e., asymptomatic individuals) are naturally “protected” exhibiting few or no recurrent clinical disease, while other HSV seropositive individuals (i.e., symptomatic individuals) have frequent ocular, orofacial, and/or genital herpes clinical episodes? We recently discovered several discrete sets of HSV-1 symptomatic and asymptomatic epitopes recognized by CD4+ and CD8+ T cells from seropositive symptomatic versus asymptomatic individuals. These asymptomatic epitopes will provide a solid foundation for the development of novel herpes epitope-based vaccine strategy. Here we provide a brief overview of past clinical vaccine trials, outline current progress towards developing a new generation “asymptomatic” clinical herpes vaccines, and discuss future mucosal “asymptomatic” prime-boost vaccines that could optimize local protective immunity.