单纯疱疹病毒2型糖蛋白D和CCL19佐剂融合重组DNA疫苗对小鼠的免疫保护效果  

Protective effects of fusion recombinant DNA vaccine of herpes simplex virus type 2 glycoprotein D and adjuvant CCL19 on mice

作　　者：阎岩 胡勤学[2] Davgadorj Chantsalmaa 王旭 Yan Yan;Hu Qinxue;Davgadorj Chantsalmaa;Wang Xu(Laboratory of Infection and Immunity,the Fifth People′s Hospital of Wuxi,Wuxi 214016,China;State Key Laboratory of Virology,Wuhan Institute of Virology,Chinese Academy of Sciences,Wuhan 430071,China)

机构地区：[1]无锡市第五人民医院感染与免疫实验室,无锡214016 [2]中国科学院武汉病毒研究所病毒学国家重点实验室,武汉430071

出　　处：《中华微生物学和免疫学杂志》2022年第5期333-341,共9页Chinese Journal of Microbiology and Immunology

基　　金：国家病毒学重点实验室开放课题(2019IOV005);无锡市卫健委中青年拔尖人才资助计划(BJ2020094)。

摘　　要：目的通过比较简单混合和融合重组的单纯疱疹病毒2型糖蛋白D(herpes simplex virus type 2 glycoprotein D,HSV-2 gD)联合分子佐剂CCL19的DNA疫苗对免疫小鼠的免疫应答及免疫保护效果的差异,揭示融合重组疫苗的保护作用。方法利用基因重组技术构建HSV-2 gD和CCL19单独表达及融合表达的DNA疫苗,经测序、Western blot和ELISA验证后,肌肉注射免疫BALB/c小鼠两次,免疫后定期收集血清和阴道灌洗液,分离免疫后的脾细胞、肠系膜淋巴结细胞及直肠组织。通过终点法ELISA、体外中和试验、免疫组织化学染色、趋化试验、阴道攻毒试验、荧光定量PCR、称量体重及观察疾病严重程度,比较两种形式的疫苗免疫后小鼠的体液免疫、细胞免疫应答和免疫保护作用的差异。结果融合重组的pgD-IZ-CCL19质粒可在体外正常表达gD蛋白和CCL19蛋白,但pCCL19-IZ-gD质粒的CCL19蛋白表达量比pgD-IZ-CCL19少。免疫pgD-IZ-CCL19的小鼠能产生比pgD+pCCL19组更高水平的血清IgG抗体、阴道灌洗液IgA抗体(P<0.01)和中和能力。与其他组比较,pgD-IZ-CCL19组小鼠免疫后直肠黏膜、脾脏和肠系膜淋巴结均招募了较多的淋巴细胞,攻毒后该组小鼠未出现体重减少和疾病症状,阴道黏膜HSV-2检出率最低,小鼠死亡率也最低,而pCCL19-IZ-gD组的免疫保护无效。结论pgD-IZ-CCL19融合重组的DNA疫苗能诱导小鼠产生较强的免疫应答和免疫保护作用,免疫保护效果优于简单混合DNA疫苗。Objective To compare the immune responses to simply mixed and fused recombinant DNA vaccines of herpes simplex virus type 2 glycoprotein D(HSV-2 gD)and molecular adjuvant CCL19 in mice and to evaluate the protective effects.Methods Gene recombination technology was used to construct recombinant DNA vaccines expressing HSV-2 gD and CCL19 alone or fused together.After verification by sequencing,Western blot and ELISA,BALB/c mice were immunized twice by intramuscular injection.Serum samples and vaginal lavage fluids were collected regularly after immunization.Splenocytes,mesenteric lymph node cells and rectal tissues were collected after immunization.Differences in humoral and cellular immune responses to the two forms of vaccines and their protective effects in mice were analyzed using end-point ELISA,in vitro neutralization assay,immunohistochemical staining,chemotaxis assay,vaginal virus challenge,fluorescence quantitative PCR,weighing and disease severity assessment.Results The fused recombinant pgD-IZ-CCL19 plasmid could express gD protein and CCL19 protein in vitro,but the level of expressed CCL19 protein by pCCL19-IZ-gD plasmid was less than that by pgD-IZ-CCL19.The mice immunized with pgD-IZ-CCL19 showed higher levels of IgG in sera and IgA in vaginal lavage fluids(P<0.01)and stronger neutralization ability than the mice vaccinated with pgD+pCCL19.Compared with other groups,more lymphocytes were recruited in the rectal mucosa,the spleen and mesenteric lymph nodes of mice immunized with pgD-IZ-CCL19.Weight loss or disease symptoms were not observed in the pgD-IZ-CCL19 group after virus challenge.In addition,the positive rate of HSV-2 in vaginal mucosa and the mortality rate in the pgD-IZ-CCL19 group were the lowest.However,pCCL19-IZ-gD turned out ineffective in preventing HSV-2 infection.Conclusions The fused recombinant DNA vaccine pgD-IZ-CCL19 could induce stronger immune responses in mice and provide better protective effects,which was superior to the simply mixed DNA vaccine.

关 键 词：单纯疱疹病毒2型 糖蛋白D CCL19 中和试验 免疫保护 

分 类 号：R392-33[医药卫生—免疫学]