| Backgroud Streptococcus pneumoniae is a Gram-positive pathogen, which can cause pneumonia, meningitis, septicemia, sinusitis, otitis media and other pneumococcal infectious diseases. Overuse of broad spectrum antibiotics has resulted in increase in resistance to vancomycin and the emergence of multiple drug resistant Streptococcus pneumoniae isolates. Therefore WHO declares S.pn vaccine as one of three most urgent vaccines in the world and recommends that pneumococcal conjugate vaccines should be brought into national immunization programs. Currently capsular transformation, high cost and other disadvantages of 23-valent capsular polysaccharide vaccine and 7-13-valent capsular polysaccharide conjugate vaccines limit their wide use. Therefore, the development of new pneumococcal vaccine is imminent.Attenuated live vaccines are a strong immunogenicity, protective effect and low-cost vaccines. Because it is hard to get the attenuated strain through traditional methods, its development has certain difficulties. Our research group gets a mutant that lost its capsule in the accident. Previous studies have shown its virulence is attenuated even weaker than R6, so it has high security and is expected to develop into an attenuated live vaccine. Therefore, this study will identify its protective effect and preliminary molecular mechanisms based on evaluation the comprehensive security, and provide reliable experimental evidence for the development of live vaccines.Methods The security of D39△CPS-TA was evaluated by capsule detection test, capsule teichoic acid expression level examination, virulence experiments in mice, lung tissue pathological section and bacterial colonization in mice. After mice immunization, the effectiveness of D39△CPS-TA was evaluated by detection of specific antibody titers, colonization protection test, the protective effect of vaccine for pneumonia and sepsis, the protective effect of vaccine against clinical isolates of S. pneumoniae and the long-term protective effect. Finally, the preliminary protection mechanism of D39△CPS-TA was investigated by detection of antibody subtypes, the passive protective effect of antibodies and cytokines secretion examination.Results D39△CPS-TA was a safe attenuated live vaccine. Compared with D39, capsule of D39△CPS-TA became significantly thinner and the expression of capsule teichoic acid was strongly inhibited, as well as with significantly reduced hemolytic activity. At the same time, the virulence of D39△CPS-TA was significantly reduced, only causing minor injury and tolerable inflammation in mice lung tissue. The colonization ability of D39△CPS-TA in mice significantly decreased and bacteria were completely cleared 24 hours post inoculation. The protective effect of attenuated live vaccine D39△CPS-TA was great too. After immunization, D39△CPS-TA could induce high titers of specific antibodies as well as reducing colonization of strains 19F and TIGR4 in mice respectively. It could elicit notable protective effect against pneumonia and sepsis caused by D39. In mucosal immunization model D39△CPS-TA could induce complete protection and the protective effects against clinical strain 6B and 3 infection were both better than the commercial vaccine(p<0.05). Besides long-term protection rates of mucosal immunized and systemic immunized mice were 100% and 75% respectively. Passive protective rate of antibody could reach 100% and the mean antibody subtypes were IgG1, IgG2a, IgG2b. As for the cytokines promoted by D39△CPS-TA, systemic immunity produced high levels of IL-10, and the mucosal immunity produced high levels of IL-10, IL-17A and IFN-γ.Conclusions D39△CPS-TA is relatively safe as an attenuated live vaccine. It induces dramatic protective effect in mice, especially in mucosal immune route, in which protection rate against multi-serotype Streptococcus pneumoniae infection are up to 100%. Systemic immunization mainly induces humoral immunity responses and mucosal immunity induces not only humoral immune responses but also cellular immune responses.
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