No abstract

We used a novel approach for the direct ex vivo identification and characterization of T cell epitopes based on the screening of peptide spot libraries with freshly isolated splenocytes in a sensitive enzyme-linked immunospot (ELISPOT) assay. This technique was applied for the analysis of splenocytes from Listeria monocytogenes-infected BALB/c and C57BL/6 mice. The screening of peptide spot libraries covering the whole listeriolysin O and p60 of L. monocytogenes confirmed all known CD4 and CD8 T cell epitopes of these proteins and additionally revealed six new H-2d and six new H-2b-restricted T cell epitopes. New epitopes were categorized into CD4 and CD8 T cell epitopes by ex vivo ELISPOT analysis with separated T cell populations. The quantitative analysis of cells reactive with these CD4 and CD8 T cell epitopes revealed the existence of dominant and subdominant CD4 and CD8 T cell populations during L. monocytogenes infection. As a consequence of these data we suggest that ELISPOT-based screening of peptide spot libraries could be a general approach for the rapid identification and characterization of pathogen-specific T cell populations during various infectious diseases.

IntroductionIn the steady state, dendritic cells (DCs) capture self-antigens and maintain low expression of costimulatory molecules but nevertheless migrate to lymph nodes where they tolerize self-reactive T cells. [1][2][3][4][5][6][7] Physiologically arising apoptotic cells (ACs) are one significant source of self-antigens for DCs. 5,7 However, following their encounter with ACs, DCs can be rendered immunologically inert, immunosuppressive, or immunostimulatory. [8][9][10][11][12][13][14][15] These contradictory findings have been difficult to resolve due to the complexity of receptors on DCs and the model systems used (eg, ex vivo versus in vivo or rodent versus human sources of ACs).ACs are recognized and captured by human DCs via an array of receptors, including LOX-1, CD36, ␣v␤3, ␣v␤5, and the complement receptors (CRs) CR3 and CR4. 16 The precise contribution of individual receptors in the binding/uptake of ACs, the initiation of downstream signaling pathways, and cross-presentation of cellassociated antigens remains undefined. Blocking antibodies targeting individual receptors inhibit no more than 50% of the association with ACs, and CD36 Ϫ/Ϫ mice have no obvious defects in phagocytosis or in cross-presentation of antigens encoded within ACs, indicating a redundancy in the system and/or the incomplete characterization of AC receptors on DCs. [17][18][19][20] More recent studies have begun to evaluate the ability of specific AC receptors to modulate DC function. DCs exposed to ACs opsonized with C3bi fragments are inhibited from maturing upon stimulation with LPS or CD40L. This effect is presumably mediated through CR3 and CR4, the receptors for C3bi. 21 Studies of the scavenger receptor CD36 and the ␣v-integrin receptor CD51 have led to similar conclusions regarding their ability to modulate DC function. 12,22 However, it is not known whether such function extends to other members or components of the ␣v-integrin receptor family, particularly the well-established phagocytic receptor ␣v␤5. 17,23 Nor is it clear whether ligation of AC receptors that interfere with DC maturation consequently alters their T-cell-stimulating potential.To gain a better understanding of the phagocytic and immunomodulatory role of the various AC receptors on human DCs, we made use of an AC surrogate system 24 that permitted us to evaluate the function of individual AC receptors. We focused on ␣v␤5 and the CRs, especially CR3. We established that these different AC receptors are not equivalent in function, as might be presumed from published studies, but that there is a distinct division of labor, at least with respect to phagocytosis and tolerance induction potential. While ␣v␤5 mediates efficient phagocytosis, it does not interfere with the DC's capacity to undergo maturation or stimulate T cells. In contrast, engagement of CR3 (or CR4) inhibits the ability of DCs to undergo maturation, produce proinflammatory cytokines or M.Š . conceptualized, designed, and performed experiments; acquired, analyzed, and interpreted data; draft...

bImmunotherapeutic herpes simplex virus 2 (HSV-2) vaccine efficacy depends upon the promotion of antigen-specific immune responses that inhibit reactivation or reactivated virus, thus controlling both recurrent lesions and viral shedding. In the present study, a candidate subunit vaccine, GEN-003/MM-2, was evaluated for its ability to induce a broad-spectrum immune response in mice and therapeutic efficacy in HSV-2-infected guinea pigs. GEN-003 is comprised of HSV-2 glycoprotein D2 (gD2⌬TMR 340-363 ) and a truncated form of infected cell polypeptide 4 (ICP4 383-766 ), formulated with Matrix M-2 (MM-2) adjuvant (GEN-003/MM-2). In addition to eliciting humoral immune responses, CD4؉ and CD8 ؉ T cells characterized by the secretion of multiple cytokines and cytolytic antigen-specific T cell responses that were able to be recalled at least 44 days after the last immunization were induced in immunized mice. Furthermore, vaccination with either GEN-003 or GEN-003/MM-2 led to significant reductions in both the prevalence and severity of lesions in HSV-2-infected guinea pigs compared to those of phosphate-buffered saline (PBS) control-vaccinated animals. While vaccination with MM-2 adjuvant alone decreased recurrent disease symptoms compared to the PBS control group, the difference was not statistically significant. Importantly, the frequency of recurrent viral shedding was considerably reduced in GEN-003/MM-2-vaccinated animals but not in GEN-003-or MM-2-vaccinated animals. These findings suggest a possible role for immunotherapeutic GEN-003/MM-2 vaccination as a viable alternative to chronic antiviral drugs in the treatment and control of genital herpes disease. H erpes simplex virus 2 (HSV-2) is one of the most prevalent sexually transmitted diseases, having infected more than 500 million people worldwide, with an estimated 23 million new infections occurring annually (1). HSV-2 infects epithelial cells of the genital mucosa during primary infection, followed by the establishment of latency in neuronal dorsal root ganglia via retrograde transport along nerve axons. Throughout latency, virus can reactivate, causing genital lesions and/or asymptomatic shedding of virus. Although suppressive antiviral therapy has shown promise in reducing both symptomatic recurrent lesions and overall viral shedding, subclinical HSV reactivation persists, likely contributing significantly to the observed continued transmission (2). The development of an efficacious immunotherapeutic vaccine targeting HSV-2 likely represents the best strategy for preventing both lesion outbreaks and the continued spread of virus.Despite considerable effort, all vaccine candidates to date have failed to meet their defined endpoints in clinical trials. The majority of clinical trials to date have focused on prophylactic subunit vaccines, largely using the HSV-2 surface glycoproteins as immunogens. The viral envelope glycoproteins gD and gB are the dominant targets for neutralizing antibody production (3, 4), making them logical candidates for vaccine devel...