肺炎链球菌减毒活菌疫苗D39△CPS-TA的安全性、免疫保护效果及机制研究
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摘要
目的：肺炎链球菌是一种革兰阳性条件致病菌，可引起肺炎、败血症、脑膜炎、中耳炎等多种急性感染性疾病，在全世界范围内特别是发展中国家具有极高的发病率和病死率。疫苗是预防肺炎链球菌感染的有效手段。但目前可用的荚膜多糖疫苗和荚膜多糖蛋白结合疫苗均存在生产成本高、可覆盖血清型有限及血清型置换等问题，极大地限制了这些疫苗在低收入国家的推广使用。因此近年来，新型肺炎链球菌疫苗一直是研究的热点。
减毒活菌疫苗具有免疫原性强、血清型覆盖广、生产成本较低等特点，是一类具有发展潜力的疫苗，但其安全性必须重点考虑。本课题组在前期肺炎链球菌缺陷菌构建实验中得到了一株2型无荚膜的肺炎链球菌突变株D39△CPS-TA。该菌株由于spd1672缺失致细菌磷壁酸表达量显著减少，但其荚膜缺失的原因一直未知。该菌株高度减毒，高剂量感染不致小鼠死亡。我们尝试将其作为减毒活菌疫苗免疫小鼠，初步发现可以产生一定的保护作用，有望开发成商品化的减毒活菌疫苗。本研究拟在探索D39△CPS-TA荚膜缺失原因、明确D39△CPS-TA基因背景的基础上，系统性地评价其安全性和粘膜免疫小鼠的保护效果，并对其免疫保护机制进行深入研究，为该疫苗的进一步开发提供实验依据。
方法：对D39△CPS-TA进行全基因组测序，根据测序结果以相关实验证明其荚膜缺失的原因；通过小鼠毒力实验、感染小鼠肺组织病理评估、小鼠定植实验以及细胞毒性实验来确定D39△CPS-TA的安全性；将D39△CPS-TA粘膜免疫C57BL/6J小鼠，通过评价疫苗对不同血清型的肺炎链球菌定植和致死性感染的保护效果来评价D39△CPS-TA作为减毒活菌疫苗的有效性；最后利用免疫缺陷小鼠模型及相关体外实验，探讨体液免疫反应、T细胞免疫反应及T细胞免疫反应主要免疫亚型（Th1/Th2/Th17/Treg）在疫苗保护性中的作用。
结果：定量PCR结果显示，D39△CPS-TA的荚膜合成相关基因cpsA-D的表达量均较D39野生菌下降约75%。全基因组测序结果分析显示，D39△CPS-TA的cps基因簇上游调控序列中出现点突变T→C（313713），LacZ和GFP报告基因实验显示，突变启动子的报告基因的表达量较野生型分别降低了80%和76%，提示该突变降低了启动子的转录效率。
D39△CPS-TA高度减毒，作为活菌疫苗安全性较高：在动物模型中，D39△CPS-TA鼻腔和腹腔1×10~~8CFU高剂量感染后，小鼠100%存活；HE染色显示，感染D39△CPS-TA小鼠的肺部组织损伤和炎症反应较感染野生菌的小鼠轻，在感染后72小时恢复到正常状态，而感染野生菌小鼠肺组织可见严重的出血、坏死及炎症细胞浸润，且肺组织破坏不可恢复，感染72小时小鼠全部死亡；定植实验结果显示，D39△CPS-TA在小鼠体内定植短暂定植，感染后72小时即可从小鼠体内清除完毕，而D39野生菌则在感染后48小时仍大量存在（1×10~~4CFU）于小鼠小鼠鼻腔灌洗液、肺组织、血液、脑组织中，且感染后48小时小鼠全部死亡。细胞毒性实验显示，在高剂量感染（MOI=100）6小时状态下，D39△CPS-TA对肺上皮细胞和脐静脉内皮细胞的毒性较轻（细胞存活率分别为75%和80%），与R6相似（细胞存活率分别为70%和75%）。
D39△CPS-TA与CT佐剂联用粘膜免疫C57BL/6J小鼠，可显著降低肺炎链球菌19F和TIGR4在小鼠上呼吸道中的定植，免疫小鼠鼻腔灌洗液和肺组织中的细菌载量为对照组小鼠的1/10左右，对D39、14型、3型、6B型感染的生存率保护分别是95%、80%、100%和80%，与阳性对照23价荚膜多糖疫苗的保护效果（70%-75%）相当。
免疫缺陷小鼠的研究显示：粘膜免疫D39△CPS-TA对体液免疫缺陷小鼠和T细胞免疫缺陷小鼠的定植和生存率均无保护作用，免疫小鼠体内的19F载量与对照小鼠相当，D39攻毒的免疫小鼠生存率为0%和20%。D39△CPS-TA特异的抗血清和脾细胞培养上清对肺炎链球菌TIGR4的杀伤率均为70%左右。将IFN-γ、IL-4和IL-17缺陷的C57BL/6J小鼠D39△CPS-TA分别鼻腔免疫，进行定植实验和生存率实验，定植实验中，IFN-γ、IL-4缺陷小鼠对照组上呼吸道中的19F载量为免疫组的100倍左右，而免疫IL-17缺陷小鼠的19F定植量与对照小鼠无差别，说明Th17型免疫反应参与对细菌定植的保护；D39生存率实验中，免疫IFN-γ、IL-17缺陷小鼠的生存率分别为60%和80%，而免疫IL-4缺陷小鼠全部死亡，说明Th4型免疫反应参与对小鼠生存率的保护。D39△CPS-TA免疫的C57BL/6J小鼠体内存在大量Treg细胞，多肽P17抑制Treg后，免疫小鼠的细菌定植量恢复到对照小鼠水平，小鼠生存率由70%降至15%，说明D39△CPS-TA特异的Treg免疫反应参与对细菌定植和小鼠生存率的保护。
结论：D39△CPS-TA cps调控序列的点突变导致细菌荚膜缺失，高度减毒，作为减毒活菌疫苗安全性较高。D39△CPS-TA粘膜免疫C57BL/6J小鼠，可抵抗多种不同血清型肺炎链球菌感染，具有良好的非血清型依赖的保护性。体液免疫反应和T细胞免疫反应均参与疫苗对于定植和致死性感染的保护，在参与保护作用的T细胞免疫反应中，Th2免疫途径提供对肺炎链球菌致死性感染的保护，Th17免疫途径及其介导的中性粒细胞、巨噬细胞等炎细胞的招募作用提供对肺炎链球菌定植的保护；D39△CPS-TA诱导的调节性T细胞（Treg）反应在D39△CPS-TA对于定植和致死性感染的保护中均发挥了重要作用。
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.
减毒活菌疫苗具有免疫原性强、血清型覆盖广、生产成本较低等特点，是一类具有发展潜力的疫苗，但其安全性必须重点考虑。本课题组在前期肺炎链球菌缺陷菌构建实验中得到了一株2型无荚膜的肺炎链球菌突变株D39△CPS-TA。该菌株由于spd1672缺失致细菌磷壁酸表达量显著减少，但其荚膜缺失的原因一直未知。该菌株高度减毒，高剂量感染不致小鼠死亡。我们尝试将其作为减毒活菌疫苗免疫小鼠，初步发现可以产生一定的保护作用，有望开发成商品化的减毒活菌疫苗。本研究拟在探索D39△CPS-TA荚膜缺失原因、明确D39△CPS-TA基因背景的基础上，系统性地评价其安全性和粘膜免疫小鼠的保护效果，并对其免疫保护机制进行深入研究，为该疫苗的进一步开发提供实验依据。
方法：对D39△CPS-TA进行全基因组测序，根据测序结果以相关实验证明其荚膜缺失的原因；通过小鼠毒力实验、感染小鼠肺组织病理评估、小鼠定植实验以及细胞毒性实验来确定D39△CPS-TA的安全性；将D39△CPS-TA粘膜免疫C57BL/6J小鼠，通过评价疫苗对不同血清型的肺炎链球菌定植和致死性感染的保护效果来评价D39△CPS-TA作为减毒活菌疫苗的有效性；最后利用免疫缺陷小鼠模型及相关体外实验，探讨体液免疫反应、T细胞免疫反应及T细胞免疫反应主要免疫亚型（Th1/Th2/Th17/Treg）在疫苗保护性中的作用。
结果：定量PCR结果显示，D39△CPS-TA的荚膜合成相关基因cpsA-D的表达量均较D39野生菌下降约75%。全基因组测序结果分析显示，D39△CPS-TA的cps基因簇上游调控序列中出现点突变T→C（313713），LacZ和GFP报告基因实验显示，突变启动子的报告基因的表达量较野生型分别降低了80%和76%，提示该突变降低了启动子的转录效率。
D39△CPS-TA高度减毒，作为活菌疫苗安全性较高：在动物模型中，D39△CPS-TA鼻腔和腹腔1×10~~8CFU高剂量感染后，小鼠100%存活；HE染色显示，感染D39△CPS-TA小鼠的肺部组织损伤和炎症反应较感染野生菌的小鼠轻，在感染后72小时恢复到正常状态，而感染野生菌小鼠肺组织可见严重的出血、坏死及炎症细胞浸润，且肺组织破坏不可恢复，感染72小时小鼠全部死亡；定植实验结果显示，D39△CPS-TA在小鼠体内定植短暂定植，感染后72小时即可从小鼠体内清除完毕，而D39野生菌则在感染后48小时仍大量存在（1×10~~4CFU）于小鼠小鼠鼻腔灌洗液、肺组织、血液、脑组织中，且感染后48小时小鼠全部死亡。细胞毒性实验显示，在高剂量感染（MOI=100）6小时状态下，D39△CPS-TA对肺上皮细胞和脐静脉内皮细胞的毒性较轻（细胞存活率分别为75%和80%），与R6相似（细胞存活率分别为70%和75%）。
D39△CPS-TA与CT佐剂联用粘膜免疫C57BL/6J小鼠，可显著降低肺炎链球菌19F和TIGR4在小鼠上呼吸道中的定植，免疫小鼠鼻腔灌洗液和肺组织中的细菌载量为对照组小鼠的1/10左右，对D39、14型、3型、6B型感染的生存率保护分别是95%、80%、100%和80%，与阳性对照23价荚膜多糖疫苗的保护效果（70%-75%）相当。
免疫缺陷小鼠的研究显示：粘膜免疫D39△CPS-TA对体液免疫缺陷小鼠和T细胞免疫缺陷小鼠的定植和生存率均无保护作用，免疫小鼠体内的19F载量与对照小鼠相当，D39攻毒的免疫小鼠生存率为0%和20%。D39△CPS-TA特异的抗血清和脾细胞培养上清对肺炎链球菌TIGR4的杀伤率均为70%左右。将IFN-γ、IL-4和IL-17缺陷的C57BL/6J小鼠D39△CPS-TA分别鼻腔免疫，进行定植实验和生存率实验，定植实验中，IFN-γ、IL-4缺陷小鼠对照组上呼吸道中的19F载量为免疫组的100倍左右，而免疫IL-17缺陷小鼠的19F定植量与对照小鼠无差别，说明Th17型免疫反应参与对细菌定植的保护；D39生存率实验中，免疫IFN-γ、IL-17缺陷小鼠的生存率分别为60%和80%，而免疫IL-4缺陷小鼠全部死亡，说明Th4型免疫反应参与对小鼠生存率的保护。D39△CPS-TA免疫的C57BL/6J小鼠体内存在大量Treg细胞，多肽P17抑制Treg后，免疫小鼠的细菌定植量恢复到对照小鼠水平，小鼠生存率由70%降至15%，说明D39△CPS-TA特异的Treg免疫反应参与对细菌定植和小鼠生存率的保护。
结论：D39△CPS-TA cps调控序列的点突变导致细菌荚膜缺失，高度减毒，作为减毒活菌疫苗安全性较高。D39△CPS-TA粘膜免疫C57BL/6J小鼠，可抵抗多种不同血清型肺炎链球菌感染，具有良好的非血清型依赖的保护性。体液免疫反应和T细胞免疫反应均参与疫苗对于定植和致死性感染的保护，在参与保护作用的T细胞免疫反应中，Th2免疫途径提供对肺炎链球菌致死性感染的保护，Th17免疫途径及其介导的中性粒细胞、巨噬细胞等炎细胞的招募作用提供对肺炎链球菌定植的保护；D39△CPS-TA诱导的调节性T细胞（Treg）反应在D39△CPS-TA对于定植和致死性感染的保护中均发挥了重要作用。
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.
引文
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