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.

In this report, rat hypothalamic nitric oxide synthase (NOS) activity is shown to be partially inhibited by physiological concentrations of the pineal hormone melatonin. In vitro studies demonstrate that 1 nM melatonin, which approximates the physiological concentration of the hormone at night, significantly inhibited NOS activity. In vivo studies show that administering melatonin or collecting the hypothalamus from animals at night, when endogenous melatonin levels are elevated, results in a significant decrease of NOS activity. Results also show that calmodulin may be involved in this process since its presence in the incubation medium prevents the inhibitory effect of melatonin on NOS activity.

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The muscarinic-gated atrial potassium (I KACh ) channel contributes to the heart rate decrease triggered by the parasympathetic nervous system. I KACh is a heteromultimeric complex formed by Kir3.1 and Kir3.4 subunits, although Kir3.4 homomultimers have also been proposed to contribute to this conductance. While Kir3.4 homomultimers evince many properties of I KACh , the contribution of Kir3.1 to I KACh is less well understood. Here, we explored the significance of Kir3.1 using knock-out mice. Kir3.1 knock-out mice were viable and appeared normal. The loss of Kir3.1 did not affect the level of atrial Kir3.4 protein but was correlated with a loss of carbachol-induced current in atrial myocytes. Low level channel activity resembling recombinant Kir3.4 homomultimers was observed in 40% of the cellattached patches from Kir3.1 knock-out myocytes. Channel activity typically ran down quickly, however, and was not recovered in the inside-out configuration despite the addition of GTP and ATP to the bath. Both Kir3.1 knock-out and Kir3.4 knock-out mice exhibited mild resting tachycardias and blunted responses to pharmacological manipulation intended to activate I KACh . We conclude that Kir3.1 confers properties to I KACh that enhance channel activity and that Kir3.4 homomultimers do not contribute significantly to the muscarinic-gated potassium current.

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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-DRgenotyped and clinically defined symptomatic (n ‫؍‬ 10) and asymptomatic (n ‫؍‬ 10) HSV-1-seropositive healthy individuals. Peptides gB [161][162][163][164][165][166][167][168][169][170][171][172][173][174][175] ؉ T cells from symptomatic patients preferentially recognized gB 661-675 (P < 0.0001). Thus, we identified three previously unrecognized CD4؉ CTL peptide epitopes in HSV-1 gB. Among these, gB 166-180 and gB 666-680 appear to be "asymptomatic" peptide epitopes and therefore should be considered in the design of future herpes vaccines.Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are ubiquitous viruses that infect a majority of people worldwide (3,22,68). Shedding of reactivated HSV is estimated to occur at rates of 3 to 28% in adults who harbor latent HSV in their sensory neurons (32,(66)(67)(68). However, the vast majority of these individuals do not experience recurrent herpetic disease and are designated "asymptomatic patients" (22,40,68). In contrast, in some individuals (symptomatic patients), reactivation of latent virus leads to induction of "pathogenic" HSVspecific CD4 ϩ and CD8 ϩ T cells (22, 68) and recurrent disease, ranging from rare episodes occurring every 5 to 10 years to outbreaks occurring monthly or even more frequently among a small proportion of subjects (22,26,60). Interestingly, for genital herpes, symptomatic and asymptomatic patients have similar virus shedding rates (68). It is likely that the same is true for ocular and oro-facial herpes, since shedding rates in tears and saliva of asymptomatic individuals have been reported to be as high as 33.5% (22,29,40,42). Latently infected patients are at risk of developing severe immunopathology, such as blinding herpetic stromal keratitis (HSK) (primarily due to HSV-1), painful genital ulcerations (primarily due to HSV-2), and in rare cases, fatal HSV encephalitis (reviewed in reference 72).Considering the wealth of data addressing the role of T cells in animal models, it is surprising how few reports explore the immunologic basis of symptomatic and asymptomatic HSV infections in humans. The HSV-1-specific CD4 ϩ T-cell responses in the cornea are much more likely to cause pathology than those in the genital tract or anus/buttocks region. Indeed, the involvement of CD4 ϩ T cells that produce Th1 cyt...

Targeting the mucosal immune system of the genital tract (GT) with subunit vaccines failed to induce potent and durable local CD8+ T cell immunity, crucial for protection against many sexually transmitted viral (STV) pathogens, including herpes simplex virus type 2 (HSV-2) that causes genital herpes. In this study, we aimed to investigate the potential of a novel lipopeptide/adenovirus type 5 (Lipo/rAdv5) prime/boost mucosal vaccine for induction of CD8+ T cell immunity to protect the female genital tract from herpes. The lipopeptide and the rAdv5 vaccine express the immunodominant HSV-2 CD8+ T cell epitope (gB498-505) and both were delivered intravaginally (IVAG) in the progesterone-induced B6 mouse model of genital herpes. Compared to its homologous lipopeptide/lipopeptide (Lipo/Lipo); the Lipo/rAdv5 prime/boost immunized mice: (i) developed potent and sustained HSV-specific CD8+ T cells, detected in both the GT draining nodes (GT-DLN) and in the vaginal mucosa (VM); (ii) had significantly lower virus titers; (iii) had decreased overt signs of genital herpes disease; and (iv) did not succumb to lethal infection (p < 0.005), following intravaginal HSV-2 challenge. Polyfunctional CD8+ T cells, producing IFN-γ, TNF-α and IL-2 and exhibiting cytotoxic activity, were associated with protection (p < 0.005). The protective CD8+ T cell response was significantly compromised in the absence of the adaptor myeloid differentiation factor 88 (MyD88) (p = 0.0001). Taken together, these findings indicate that targeting the VM with a Lipo/rAdv5 prime/boost vaccine elicits a potent, MyD88-dependent, and long-lasting mucosal CD8+ T cell protective immunity against sexually transmitted herpes infection and disease.

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 (OVA(257-264)) 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 alpha-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 OVA(257-264)-specific IFN-gamma 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.