The Experimental Research On Protective Effect And Mechanisms Of Mucosal Immunization With ClpX Against Pneumococcal Infection In Mice

ObjectiveStreptococcus pneumoniae (S. pn) localizes on the mucosal surfaces ofthe host upper respiratory tract. It can be readily extended from carrier stateto diverse life-threatening invasive infection such as pneumonia, meningitisand bacteremia, causing great morbidity and mortality worldwide. As theantibiotic resistance of S. pn develops, the need of a promising preventivestrategy becomes a global problem of great urgency.Pneumococcal ClpX (caseinolytic protease X) protein is a member ofHSP100family, which functions as a molecular chaperone. ClpX is knownas an ATPase subunit, along with proteolytic subunit ClpP, forming afunctional ClpXP complex. It has been revealed that ClpX subunit wasresponsible for heat shock resistance and substrate identification and wasrelevant to the survival and virulence of S. pn, indicating ClpX as apromising vaccine candidate protein. This study was carried out toinvestigate the protective effect and mechanisms of mucosal immunization with ClpX against pneumococcal infection, and to evaluate the possibilityof ClpX as a alternative vaccine candidate protein.MethodsThe full-length S. pn ClpX protein was expressed in Escherichia coli(E. coli) by prokaryotic expression technology, and purified recombinantClpX (rClpX) protein was introduced to BALB/c mice for the preparationof specific polyclonal anti-sera. The surface expression of ClpX waspredicted by bioinformatics analysis, and was verified by flow cytometryand Western blot. The conservation of ClpX in9different serotypes of S.pn was analysed by PCR and Western blot. The anti-ClpX IgG titers in seraof pneumococcal infection children, healthy children and healthy adultswere evaluated by ELISA. rClpX protein was introduced to BALB/c micethrough a mucosal route, with Cholera Toxin (CT) as the adjuvant. Theantibody titers and isotypes and splenocytes-secreted cytokines induced byimmunization were evaluated by ELISA. Finally, mice were challengedwith S. pn CMCC31693(serotype19F), and the bacterial loads in nasalwashes were estimated5days post infection. S. pn D39(serotype2) andCMCC31614(serotype14) were introduced to mice respectively, and thesurvival rate was monitored.ResultsThe recombinant plasmid pET-28(a)-clpX was successfullyconstructed, and the rClpX protein with＞90%purity was successfully prepared. ClpX was shown to be a non-transmembrane protein bybioinformatics analysis. And the secretory-expression of ClpX, instead ofsurface-exposure, was further confirmed by flow cytometry and Westernblot. Furthermore, ClpX was highly conserved in9different serotypes of S.pn by PCR and Western blot. In addition, the anti-ClpX IgG antibody levelsin sera were much higher in healthy children, compared with pediatricpatients, and displayed an age-related increase. Furthermore, the IgG andIgA antibody levels in both sera and saliva were effectively elicited byClpX-immunization (P＜0.01), and IgG1and IgG2b were the majorantibody isotypes in serum samples. Besides, high levels of IL-17A andIL-4secreted by splenocyts were also elicited by ClpX-immunization(P＜0.05). Finally, the protection against serotype19F S. pn nasopharyngealcolonization (P＜0.001) and serotype2S. pn (P＜0.05) and serotype14S.pn (P＜0.01) lethal infection was successfully elicited.ConclusionThe secretory expression, the conservation in various pneumococcalstrains and the immunogenicity in human features of S. pn ClpX proteinmake it meet the demand of S. pn vaccine candidate protein. Mucosalimmunization with rClpX protein could induce Th2-and Th17-mediatedimmune responses, and provide good protective effect againstnasopharyngeal colonization and lethal challenge by various S. pn strains.Our findings suggest that ClpX is a potential candidate antigen that could be incorporated in pneumococcal protein vaccines.