1株人3型副流感病毒减毒活疫苗候选毒株在小鼠动物模型上的安全性、免疫原性及保护效力评价

Safety,immunogenicity and protective efficacy of a candidate strain of live attenuated vaccine against human parainfluenza virus type⁃3 in mouse model

目的评价1株人3型副流感病毒(human parainfluenza virus type-3,HPIV-3)减毒活疫苗候选毒株CPW3在小鼠中的安全性、免疫原性及保护效力,以期为HPIV-3减毒活疫苗的研发提供候选毒株。方法从甘肃省妇幼保健医院下呼吸道感染住院患儿下呼吸道分泌物标本中分离培养HPIV-3,采用诱变剂加逐步降温法筛选具有冷适应(coldadaptation,Ca)及温度敏感(temperature sensitivity,Ts)特性的HPIV-3毒株CPW3。用CPW3经鼻腔免疫雌性BALB/c小鼠,免疫30 d内每天测定小鼠体质量和体温;噬斑减少试验测定小鼠血清中和抗体;qRT-PCR法检测小鼠肺部和鼻洗液中HPIV-3病毒载量;ELIspot法测定小鼠肺脏、脾脏和外周血中HPIV-3特异性IgG/IgA抗体分泌淋巴细胞(antibody secreting cells,ASC)应答;流式细胞术测定小鼠肺脏、脾脏和外周血中IFNγ-CD4+T/IFNγ-CD8+T/IL-4-CD4+T细胞应答。HPIV-3野毒株攻毒试验测定CPW3免疫对小鼠肺脏HPIV-3感染的保护效力。结果CPW3可在小鼠上、下呼吸道增殖,但在小鼠肺脏的病毒载量比其亲本株低约100倍;免疫后30 d内,CPW3对小鼠的体质量和体温无影响;体液免疫应答结果显示,CPW3不仅可诱导小鼠产生显著的血清中和抗体应答(t=7.296,P<0.0001,n=12),还可产生显著的小鼠肺部局部定植记忆性IgG(t=3.296,P=0.0076,n=6)和IgA(t=2.884,P=0.0215,n=6)ASC免疫应答;细胞免疫应答结果显示,CPW3可显著诱导小鼠肺部局部定植记忆性IFNγ-CD4+T(t=4.941,P=0.038,n=6)和IFNγ-CD8+T(t=4.005,P=0.0005,n=6)细胞应答。CPW3免疫对小鼠下呼吸道HPIV-3感染具有显著的保护效力(t=2.836,P=0.0071,n=21)。结论在小鼠动物模型中,CPW3具有安全性和免疫原性,对小鼠HPIV-3感染具有显著的保护效力,是1株有前途的HPIV-3减毒活疫苗候选株。

Objective To evaluate the safety,immunogenicity and protective efficacy of CPW3,a candidate strain of live attenuated vaccine against human parainfluenza virus type-3(HPIV-3)in mice,so as to provide a candidate strain for the vaccine development.Methods HPIV-3 was isolated from the samples of lower respiratory tract secretions of hospitalized children with lower respiratory tract infection in Gansu Provincial Maternity and Child-care Hospital and cultured,from which CPW3 strain with characteristics of cold-adaptation(Ca)and temperature sensitivity(Ts)was screened by mutagen plus stepwise cooling method.Female BALB/c mice were immunized i.n.with CPW3 strain and measured for body mass and temperature every day within 30 d after immunization,while for serum neutralizing antibody by plaque reduction test,and for virus load of HPIV-3 in lung and nasal washes by qRT-PCR.Antibody secreting cells(ASC)of specific IgG or IgA antibody against HPIV-3 in lung,spleen and peripheral blood of mice were determined by ELIspot method,while IFNγ-CD4+T/IFNγ-CD8+T/IL-4-CD4+T cells by flow cytometry,and the protective efficacy against HPIV-3 infection in lung by challenge test with wild HPIV-3 strain.Results CPW3 was replicated in upper and lower respiratory tracts of mice,while the virus load in lung tissue was about 100 times lower than that of its parent strain,which showed no effect on body mass and temperature of mice within 30 d after immunization.Results of humoral immune response showed that CPW3 induced not only significant serum neutralizing antibody response(t=7.296,P<0.0001,n=12),but also locally colonized memory IgG(t=3.296,P=0.0076,n=6)and IgA(t=2.884,P=0.0215,n=6)ASC immune responses in mouse lungs.Results of cellular immune response showed that CPW3 significantly induced locally colonized memory IFNγ-CD4+T(t=4.941,P=0.038,n=6)and IFNγ-CD8+T(t=4.005,P=0.0005,n=6)cell immune responses in mouse lungs.CPW3 immunization showed significantly protective efficacy against HPIV-3 infection of lower respiratory tract in mice(t=2.836,P=0.0071,n=21).Conclusion CPW3 showed safety,immunogenicity and significantly protective efficacy against HPIV-3 infection in mice,which is a promising candidate strain for live attenuated HPIV-3 vaccine.