人呼吸道合胞病毒G蛋白在大肠杆菌中的表达及联合不同新型佐剂免疫效果的研究  

Study on the Expression of Human Respiratory Syncytial Virus G Protein in Escherichiaa Coli and Its Immunogenicity Combined with Different Novel Adjuvants

作　　者：黄星湖 宋洋[2] 胡宏俏 李海 任虎 宋晶晶 郭宏 赵建楠 江洁 时雨晴 张燕[2] 许文波[1,2] HUANG Xinghu;SONG Yang;HU Hongqiao;LI Hai;REN Hu;SONG Jingjing;GUO Hong;ZHAO Jiannan;JIANG Jie;SHI Yuqing;ZHANG Yan;XU Wenbo(Medical School,Anhui University of Science and Technology,Huainan 232001,China;National Institute for Viral Diseases Control and Prevention,China CDC,NHC Key Laboratory of Medical Virology and Viral Diseases,WHO WPRO Regional Reference Measles/Rubella Laboratory,WHO WPRO Regional Polio Reference Laboratory,Beijing 102206,China;Shandong First Medical University,Taian 271000,China)

机构地区：[1]安徽理工大学医学院,淮南232001 [2]国家卫生健康委员会医学病毒和病毒病重点实验室,中国疾病预防控制中心病毒病预防控制所,世界卫生组织西太平洋地区麻疹/风疹参比实验室,世界卫生组织西太平洋地区脊髓灰质炎参比实验室,北京102206 [3]山东第一医科大学(山东省医学科学院),济南250117

出　　处：《病毒学报》2023年第3期691-701,共11页Chinese Journal of Virology

摘　　要：人呼吸道合胞病毒(Human respiratory syncytial virus, hRSV)是导致5岁以下儿童和老人住院和死亡的主要原因。hRSV囊膜表面的融合蛋白F(Fusion protein, F)和吸附蛋白G(Attachment protein, G)均能诱导产生保护性中和抗体,但目前临床研究主要集中于F蛋白的亚单位疫苗及保护性单抗的研发,对G蛋白的研究较少。虽然G蛋白的高度糖基化导致其免疫原性较差,但使用佐剂能改善G蛋白的免疫效果。本研究利用大肠杆菌成功表达了hRSVA-Long株G蛋白膜外区67~298氨基酸(命名为REG),并探索了REG与四种不同组合形式的新型佐剂联合免疫BALB/c小鼠的免疫效果:BFA03佐剂、B型CpG OND混合磷酸铝佐剂组合、C型CpG OND混合磷酸铝佐剂组合、BALB/c小鼠CD4+T细胞表位肽混合C型CpG OND和磷酸铝佐剂组合。对每只小鼠大腿肌肉多点注射100μL免疫原,初次免疫后第28天进行加强免疫。实验结果表明,接种REG联合新型BFA03佐剂的小鼠体内诱导产生结合抗体的能力最强,且其与接种REG蛋白联合B型CpG OND混合磷酸铝佐剂的小鼠均产生了最高的中和抗体,并且能有效降低小鼠攻毒后的肺病毒载量。然而,REG与4种形式的佐剂组在降低hRSV攻毒后的肺病理损伤方面与对照组相比未见差异。本研究提示,在选用G蛋白研发hRSV疫苗并筛选高效价中和抗体的同时,还应进一步探索降低肺病理损伤的方法。Bronchiolitis and pneumonia caused by human respiratory syncytial virus(hRSV)are the leading causes of hospitalization and death in children under 5 years of age and the elderly.Although the fusion protein(F)and attachment protein(G)on the envelope surface of hRSV can induce the production of protective neutralizing antibodies,the current clinical studies mainly focus on the development of subunit vaccine and protective mAb for F protein,and there are few studies on G protein.Although the high glycosylation of G protein caused its poor immunogenicity,the use of adjuvants improved the immune effect of G protein.In this study,we successfully expressed amino acids 67 to 298(named REG)of G protein of hRSVA-Long strain,and explored the immune effect of REG with four different combination forms:BFAO3 adjuvant,type B CpG OND mixed aluminum phosphate adjuvant,type C CpG OND mixed aluminum phosphate adjuvant,BALB/c mouse CD4+T cell epitope peptide mixed type C CpG OND and aluminum phosphate adjuvant.Each mouse was given 100μL of immunogen from the thigh muscle,and booster immunization was performed on day 28 after primary immunization.The experimental results showed that mice vacinated with REG combined with the new BFAO3 adjuvant had the strongest ability to induce binding antibodies,and both produced the highest neutralizing antibodies with mice vaccinated with type B CpG OND,and could effectively reduce the lung viral load of mice after challenge.However,no difference was seen between REG and the four forms of adjuvant groups in reducing lung pathological damage after hRSV challenge.This study suggests that the methods to reduce lung pathological damage should be further explored while selecting G protein to develop hRSV vaccine and screening high-valence neutralizing antibodies.

关 键 词：人呼吸道合胞病毒 新型佐剂 G蛋白 

分 类 号：R392.9[医药卫生—免疫学] R373.1[医药卫生—基础医学]