The splenic T lymphocytes were enriched with nylon wool columns, and 100 l of 2 106 cells/ml in RPMI-5 culture medium (RPMI 1640 containing 5% fetal bovine serum, 100 nM l-glutamine, 10 nM penicillin-streptomycin, and 5 10?5 M 2-mercaptoethanol) was added to each well in 96-well plates
The splenic T lymphocytes were enriched with nylon wool columns, and 100 l of 2 106 cells/ml in RPMI-5 culture medium (RPMI 1640 containing 5% fetal bovine serum, 100 nM l-glutamine, 10 nM penicillin-streptomycin, and 5 10?5 M 2-mercaptoethanol) was added to each well in 96-well plates. safeguarded from viral challenge, indicating a strong correlation between anti-E antibodies and the protecting capacity. This observation was confirmed by adoptive transfer experiments. Intravenous transfer of E-specific antisera but not crude or T-cell-enriched immune splenocytes to SMYD3-IN-1 sublethally irradiated hosts conferred safety against a lethal JEV challenge. Furthermore, experiments with gene knockout mice showed that DNA vaccination did not induce anti-E titers and protecting immunity SMYD3-IN-1 in Ig?/? and I-A?/? mice, whereas in CD8?/? mice the pE-induced antibody titers and protecting rate were comparable to those produced in the wild-type mice. Taken together, these results demonstrate the anti-E antibody is the most critical protecting component with this JEV challenge model and that production of anti-E antibody by pE DNA vaccine is dependent on the presence of CD4+ T cells but self-employed of CD8+ T cells. (JEV) is definitely a member of the that causes diseases of the human being central nervous system in many areas of the world, especially SMYD3-IN-1 in Southeast Asia. Among those with medical symptoms, the mortality rate can be as high as 10 to 30%, and a majority of individuals who recover suffer severe neurological sequelae (22). Vaccination remains probably one of the most encouraging approaches to reducing JEV infections. Inactivated JEV vaccines prepared from Rabbit Polyclonal to ARG1 infected mouse brains or main hamster kidney cells and a live-attenuated SA14-14-2 vaccine have been used in many parts of Asia with measurable success (31). However, there are several disadvantages to the currently used vaccines. The mouse brain-derived inactivated JEV vaccine is definitely costly to prepare, is unable to induce long-term immunity (26), and most importantly carries the risk of inducing allergic reactions (M. M. Andersen and T. Ronne, Letter, Lancet 337:1044, 1991). The SA14-14-2 attenuated vaccine is definitely efficacious; however, production and regulatory requirements for this vaccine are not established yet. As a result, there has been a significant effort in recent years aimed at utilizing recombinant DNA technology to produce improved JEV vaccines. Successful development of efficacious vaccines will become expedited if the immune reactions that contribute to disease control are recognized. In JEV illness, the immunity against SMYD3-IN-1 membrane (M), envelope (E), and NS1 nonstructural proteins is effective in host defense. The antibody reactions elicited by these viral proteins appear to play the major protecting role. Passive transfer of monoclonal antibodies against E proteins protects mice against JEV encephalitis (10, SMYD3-IN-1 18). Recombinant vaccinia viruses expressing precursor M (pre-M) and E proteins or E protein alone are highly effective at eliciting neutralizing antibodies and protection against JEV challenge in immunized mice (9, 19) and pigs (14). The NS1 protein also evokes a strong antibody response that protects the host against challenge (16). The role of T-cell immunity in JEV protection is less well defined. In JEV-infected patients, the virus-specific CD4+ and CD8+ T lymphocytes have been isolated and found to proliferate in response to JEV stimulation (11). Vaccinees receiving the formalin-inactivated JEV vaccine (1) or the poxvirus-based JEV vaccine (13) have been shown elsewhere to produce CD4+ or CD8+ T cells, respectively, that can mediate JEV-specific cytotoxic activities. In the murine model, JEV-specific cytotoxic T lymphocytes (CTLs) are induced by JEV contamination (24) and by immunization with extracellular particle-based (15) or poxvirus-based (12) JEV vaccines. Whether these specific T-cell responses are protective against JEV contamination is still controversial and remains to be resolved. Adoptive transfer of immune splenocytes or T lymphocytes was reported previously to protect mice from a lethal JEV challenge (20, 25). However, under some circumstances the adoptively transferred T cells were not protective, owing to the different routes of transfer as well as the age and strain of the recipient animals (21, 25). A more comprehensive study using JEV vaccines that can efficiently induce cellular immune responses is required to address this question. DNA vaccines have been demonstrated previously in many animal models to induce a broad range of immune responses, including antibodies, CD8+ CTLs, CD4+ helper T (Th) lymphocytes, and protective immunity against challenge with the pathogen (7, 8). Several recent clinical trials have demonstrated the ability of DNA vaccines to induce antigen-specific CTLs in humans, although their potency is limited (4, 32). The ability of DNA immunization to elicit both antibody and CTL immunity makes it an ideal vaccination approach to evaluate the relative roles of these immune responses in host defense against viral contamination. We previously showed that a plasmid (pE) encoding the JEV E protein produced high titers of E-specific antibodies and provided protection against a lethal JEV challenge (6). Immunization with plasmids encoding other structural (capsid) or nonstructural (NS1-2A, NS3, and NS5) proteins was ineffective..