表达猪瘟病毒E0-E2蛋白的重组腺病毒疫苗免疫效力研究

Immunoefficacy of recombinant adenovirus vaccine expressing E0-E2 protein of Classical swine fever virus

为研究重组猪瘟腺病毒活载体疫苗(rAd-E0-E2株)对猪的免疫保护效力,并确定其最小免疫剂量及免疫期,本研究将40头健康仔猪随机分为8组,每组5头,1~5组为最小免疫剂量测试组,分别接种rAd-E0-E2株活疫苗1/20头份、1/10头份、1/2头份、1头份(1.58×107.0IFU),另设生理盐水对照组。于免疫后7 d、14 d收集血清,采用ELISA方法检测猪瘟病毒抗体,免疫后14 d攻毒;6~8组为免疫期组,分别接种1头份的rAd-E0-E2株活疫苗和1头份的猪瘟活疫苗(脾淋源,C株),于免疫后7 d、14 d、21 d、30 d、60 d、90 d、120 d、150 d、180 d、210 d收集血清,采用ELISA方法检测猪瘟病毒抗体,免疫后7个月攻毒。所有试验组猪攻毒后监测体温变化,观察猪瘟临床症状和剖检病变。最小免疫剂量检测结果显示,1/20头份剂量组攻毒后1/5保护,4/5出现高热稽留、典型CSF症状和剖检病变;1/10头份、1/2头份和1头份剂量组攻毒后均5/5保护,体温无明显变化,无猪瘟临床症状和剖检病变。免疫期结果显示,免疫组攻毒后均为5/5保护,且r Ad-E0-E2株免疫后7个月抗体水平高于C株,阴性对照组猪攻毒后均发病。综上所述,疫苗的最小免疫剂量为1/10头份(1.58×106.0IFU),考虑到临床实际应用影响因素较多,为确保免疫效果,将疫苗的使用剂量定为最小免疫剂量的10倍,即1头份。r Ad-E0-E2株活疫苗免疫猪1头份后免疫期至少为7个月。本研究为r Ad-E0-E2株后续应用提供了参考依据。

In this study,the immunoefficacy of recombinant adenovirus vaccine expressing E0-E2 protein of classical swine fever virus was evaluated,and the minimum immunization dose and immune duration were determined.Forty healthy piglets were randomly divided into 8 groups with 5 pigs in each group.Groups 1 to 5 were used to determine the minimum immunization dose.They were inoculated with live vector vaccine(r Ad-E0-E2 strain)at 1/20 dose,1/10 dose,1/2 dose,1 dose(1.58×107.0 IFU)and saline negative control,respectively.Serum was collected from each individual animal at 7 d and 14 dafter immunization,and CSFV specific antibodies were then detected by ELISA.These animals were challenged at 14 d after vaccination.Groups 6-8 were immunized with one dose of thelive vector vaccine(r Ad-E0-E2 strain),the live swine fever vaccine(splenic lymphogenic source,strain C)and saline control,respectively.Serum was collected at 7 d,14 d,21 d,30 d,60 d,90 d,120 d,150 d,180 d,and 210 d after immunization,and CSFV antibodies were detected by ELISA.These animals were challenged at 7 months after immunization.All experimental groups were monitored for the changes of body temperature after challenge,and the clinical symptoms and post-mortem lesions of CSFV were observed.The results of the minimum immunization dose groups showed that the protection efficacy of 1/20 dose group was 1/5 after challenge,and 4/5 showed high fever retention,typical CSFV symptoms and post-mortem lesions;animals from groups with 1/10 dose,1/2 dose and 1 dose were protected by 5/5 after challenge,and there was no significant change of body temperature,no clinical symptoms and no necropsy.The results of the immunization duration group showed that the protection efficacy of the pigs in the immunized group were up to 5/5 after challenge,but the antibody level of live vector vaccine(r Ad-E0-E2 strain)was higher than that of C strain at 7 months after immunization.All the pigs in the negative control group developed disease after challenge.In summary,the minimum immunization dose of the vaccine(r Ad-E0-E2 strain)is 1/10 dose(1.58×106.0 IFU).Considering that there are many influencing factors in clinical application,in order to ensure the immune effect,the vaccine dose is set to 10 times the minimum immunization dose,1 dose.The immunization period of pigs immunized with 1 does live vector vaccine last for at least 7 months.This study provides a reference for the subsequent application of the vaccine(r Ad-E0-E2 strain).