Optimization of recombinant vaccinia-based ELISPOT assay.

The ELISPOT assay has been considered as one of the most sensitive assays to measure antigen-specific CD8 T cells in vitro. Recently, recombinant vaccinia was successfully used to express internally processed target antigens in host cells in direct ex-vivo ELISPOT assays. However, the background in these assays was relatively elevated, and the risk of killing effector T cells was high. Therefore, we examined in this study an alternative approach where the replication of recombinant vaccinia virus was inhibited by the usage of Cidofovir in vitro. Our data indicate that recombinant vaccinia-infected target cells treated with Cidofovir retained their functional activity and present internally processed antigens more efficiently to T cells than non-treated ones. We also identify the optimum doses of Cidofovir to be in the range of 0.75-0.075 microg/ml. Thus, Cidofovir treatment of the target cells prior to antigen stimulation could be a useful methodology to increase the sensitivity of the ELISPOT assay.

DNA immune responses induced by codelivery of IL-12 expression vectors with hepatitis C structural antigens.

OBJECTIVE: To demonstrate the utility of DNA vaccines for the tailored methods, the efficacy of enhanced immune responses, and the types of increased immune responses. METHODS: Four recombinant plasmids constructed included the coding regions for the core protein (pC) and for the core, E1 and E2 together (pCE1E2), IL-12 p35 and p40. These plasmids were transfected into mammalian cells to test their protein expression and were injected into the quadriceps muscles of BALB/C mice for measurement of specific antibodies and cytotoxic T-lymphocyte (CTL) responses. RESULTS: All the recombinant plasmids were shown to express specific antigens stably in mammalian cells. Codelivery of pIL-12 expression cassettes with pC and pCE1E2 in mice resulted in the enhancement of Ag-dependent CTL responses and the reduction of specific Ab response. The CTL activity was: pC=18.65%+/-5.71%, pCE1E2=20.07%+/-11.11%, pC+pIL-12=60.11%+/-17.37%, pCE1E2+pIL-12=67.48%+/-15.57%, respectively. The average A values of anti-HCV were pC=0.415+/-0.127, pCE1E2=0.358+/-0.096, pC+pIL-12=0.210+/-0.086, pCE1E2+pIL-12=0.258+/-0.125. CONCLUSION: Codelivery of pIL-12 with plasmid DNA can enhance the efficacy of immune responses and shift the type of immune responses.

gC1qR expression in chimpanzees with resolved and chronic infection: potential role of HCV core/gC1qR-mediated T cell suppression in the outcome of HCV infection.

Chimpanzee is a unique animal model for HCV infection, in which about 50% of infections resolve spontaneously. It has been reported that the magnitude of T cell responses to HCV core in recovered chimpanzees is greater than that in chronically infected ones. However, the mechanism(s) by which the chimpanzees with resolved infection overcome core-mediated immunosuppression remains unknown. In this study, we examined the effect of HCV core on T cell responsiveness in chimpanzees with resolved and chronic HCV infection. We found that core protein strongly inhibited T cell activation and proliferation in chimpanzees with chronic infection, while this inhibition was limited in chimpanzees with resolved infection. Notably, the level of gC1qR, as well as the binding of core protein, on the surface of T cells was lower in recovered chimpanzees when compared to chimpanzees with chronic HCV infection. Intriguingly, the observed differences in gC1qR expression levels and susceptibility to core-induced suppression amongst HCV-chronically infected and recovered chimpanzees were observed prior to HCV challenge, suggesting a possible genetic determination of the outcome of infection. These findings suggest that gC1qR expression on the surface of T cells is crucial for HCV core-mediated T cell suppression and viral clearance, and that represents a novel mechanism by which a virus usurps host machinery for persistence.