Study On Safety, Protective Effect And Immune Mechanisms Of Streptococcus Pneumoniae Live Attenuated Vaccine D39△CPS-TA

Objective Streptococcus pneumoniae is a Gram-positive conditionedpathogen, causing series of actue infective disease such as pneumonia,sepsis, meningitis, otitis media, and so on. Vaccination is an efficient andcost-effective strategy to decrease the morbidity and mortality ofinfectious pneumococcal diseases worldwide, partically in developingcountries. While the currently licensed pneumococcal polysaccharidevaccine and polysaccharide conjugate vaccine are highly effective againstthe serotypes included, their high cost and limited serotype coverage aswell as serotype replacement limit their use in low-income areas wheremorbidity and mortality from pneumococcal disease are highest. Therefore,an affordable vaccine that confers broad, preferably serotype-independentprotection from pneumococcal disease remains a major global healthpriority.Attenuated live vaccine is of great value, possessing advantages ofstrong immunogenicity, broad serotype coverage and relative low manufacturing complexity and costs. And the primary issue that should betaken into consideration when utilizing a live attenuated vaccine is itssafety. In previous study we got an unencapsulated mutant of serotype2pneumococcal strain D39△CPS-TA. Due to deletion of spd1672, adecreased expression of teichoic acid was found in D39△CPS△TA,however, the reason for loss of capsule was still unknown. D39△CPS△TA was highly attenuated, causing no mice dead when infected with highdose of this strain. We have found that as attenuated live vaccine, D39△CPS△TA could afford some protection against pneumococcal infection inBALB/c mice and was expected to be developed into a commercialattenuated live vaccine. Therefore, in this study, on the base ofinvestigation of the reason for capsule loss, we will further identify thesafety, protection efficacy and immune mechanism of D39△CPS-TA inC57BL/6J mice model, providing experimental evidence for the furtherdevelopment of D39△CPS-TA.Methods Genome sequencing of D39△CPS-TA was done andrelevant experiments were utilized to explore the reason for capsule loss.The safety of D39△CPS-TA was evaluated through virulence experimentin mice, pathological evaluation of infected mice lungs, colonizationexperiment in mice and cytotoxicity assay. C57BL/6J mice wereintranasally immunized with D39△CPS-TA and protections againstcolonization and lethal infection of pneumococcal strains with various serotypes were observed to assess the effectiveness of D39△CPS-TA.Finally, the roles of humoral immune response, T cellular immuneresponse as well as Th1/Th2/Th17/Treg immune subsets inD39△CPS-TA-induced protection were assessed in immune deficientmice model.Results The expression of CPS biosynthesis gene cps inD39△CPS-TA was decreased by75%compared with that in D39. Therewas a single base mutation in the upstream regulatory region ofpneumococcal CPS biosynthesis (cps) locus of D39△CPS-TA. The LacZreport gene and GFP report gene experiments showed that the expressionsof LacZ and GFP regulated by mutant promoter were reduced by80%and70%compared with those regulated by wild promoter, respectively,indicating this single base mutation suppressed the regulatory efficiency ofcps promoter.D39△CPS-TA was highly attenuated as a live vaccine. In micemodel, D39△CPS-TA was completely avirulent independent of infectionroutes. Infection with D39△CPS-TA caused mild destroy to alveoli andpartial inflammatory cell infiltration in lungs, and the mice performedapparent inflammatory response resolution at72hours post infection,whereas the lungs of D39-infected mice showed severe destroy to alveoli,strong inflammatory cell infiltration and necrosis, and these changes couldnot be recovered. D39△CPS-TA was absolutely cleared at72h post infectin, however, at48h post infection there was still numerous of D39detected in D39infected mice. The cytotoxicity assay also showed highlyattenuation of D39△CPS-TA. When infected with D39△CPS-TA(MOI=100) about6hours, MLE-12cells and HUVEC cells showedaround75%and80%viability, respectively.Intranasal immunization with D39△CPS-TA plus CT inducesremarkable protection against pneumococcal infection in aserotype-independent manner. Vaccination could significantly reducecolonization of pneumococcal strains19F and TIGR4in C57BL/6J mice.Approximately10-fold reduction of bacterial loads was observed in nasalwashes and lung homogenates of immunized mice compared with thecontrol group. The survival rates of immune mice infected withpneumococcal strains D39, serotype14,3and6B were95%、80%、100%and80%, respectively, which were comparable with that provided by23-valent polysaccharide pneumococcal vaccine (70%-75%).There was no protection against pneumococcal colonization andlethanl challenge in D39△CPS-TA immunized humoral immune deficientmice and T cellular immune deficient mice. The bacterial loads of19F inimmune mice were comparable with that in control mice, and the survivalrates of D39challenged immune mice were0%and20%, respectively.The killing of TIGR4by D39△CPS-TA specific humoral and cellularimmune response was about70%. The bacterial loads in control IFN-γor IL-4deficient mice were almost100times of those in immune one, andthe immune IL-17deficient mice had comparable19F loads with thecontrol mice, indicating that Th17immune response participatedprotection against colonization. When challenged with D39, the survivalrates of immune IFN-γor IL-17deficient mice were60%and80%,respectively, however, all the immue IL-4deficient mice dead,implyingprotection against lethal pneumococcal challenge was mediated by Th4immune response. There was srong Treg cell response in D39△CPS-TAimmunized C57BL/6J mice, and when the Treg function was suppressedby peptide P17, the bacterial loads in immune mice was recoverd to that incontrol mice whereas the survival rate was declined from70%to15%,suggesting that D39△CPS-TA specific Treg immune response participatedin the protection against colonization as well as lethal challenge.Conclusion The single base mutation in the upstream regulatoryregion of pneumococcal CPS biosynthesis (cps) locus leads to the capsuleloss of D39△CPS-TA, which is completely attenuated in-vivo and in-vitroas a live attenuated vaccine. Remarkable protection against pneumococcalcolonization and lethal challenge was acquired by intranasal immunizationwith D39△CPS-TA. Both humoral and cellular immunity aredemonstrated to be required for full protection against pneumococcalcolonization and lethal infection. Moreover, Th17and Th2immunesubsets are responsible for the mucosal and systemic protection, respectively. In particular, D39△CPS△TA-elicited regulatory T cellsparticipate in the protection against pneumococcal colonization and lethalpneumococcal infection.