No abstract

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.

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.

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.

Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) -specific CD8+ T cells that reside in sensory ganglia, appears to control recurrent herpetic disease by aborting or reducing spontaneous and sporadic reactivations of latent virus. A reliable animal model is the ultimate key factor to test the efficacy of therapeutic vaccines that boost the level and the quality of sensory ganglia-resident CD8+ T cells against spontaneous herpes reactivation from sensory neurons, yet its relevance has been often overlooked. Herpes vaccinologists are hesitant about using mouse as a model in pre-clinical development of therapeutic vaccines because they do not adequately mimic spontaneous viral shedding or recurrent symptomatic diseases, as occurs in human. Alternatives to mouse models are rabbits and guinea pigs in which reactivation arise spontaneously with clinical features relevant to human disease. However, while rabbits and guinea pigs develop spontaneous HSV reactivation and recurrent ocular and genital disease none of them can mount CD8+ T cell responses specific to Human Leukocyte Antigen- (HLA-) restricted epitopes. In this review, we discuss the advantages and limitations of these animal models and describe a novel “humanized” HLA transgenic rabbit, which shows spontaneous HSV-1 reactivation, recurrent ocular disease and mounts CD8+ T cell responses to HLA-restricted epitopes. Adequate investments are needed to develop reliable preclinical animal models, such as HLA class I and class II double transgenic rabbits and guinea pigs to balance the ethical and financial concerns associated with the rising number of unsuccessful clinical trials for therapeutic vaccine formulations tested in unreliable mouse models.