口蹄疫重组鸡痘病毒在豚鼠体内的毒性、分布、抗体消长规律的研究

以病毒为重组疫苗的载体构建的活载体基因重组疫苗免疫哺乳动物,抵抗传染病的研究已获得成功,其中鸡痘病毒存在着很大的潜在优势。然而,作为非复制的载体其安全性迫切需要检验。选择重组鸡痘病毒vUTAL 3CP1分别以正常剂量、超大剂量,肌肉注射、静脉注射接种豚鼠,第一次免疫后间隔14 d分别进行二免、三免;对妊娠豚鼠的不同阶段进行免疫,通过PCR、RT-PCR、EL ISA、中和抗体检测和免疫组化等检测方法,对疫苗在豚鼠体内的基因和蛋白分布、抗体消长规律以及毒性,和对妊娠豚鼠和子代体内的基因和蛋白分布、抗体消长规律以及毒性进行研究。实验结果表明:肌肉接种FMD重组疫苗株后,临床观察、病理组织学和检测表明在整个试验期间,试验组免疫动物精神状态良好、饮水、采食等临床表现一切正常;病毒培养表明只在接种的部位免疫3 h可以培养出病毒,其他组织和其他时间点均未能培养出病毒,证明重组鸡痘病毒vUTAL 3CP1在体内是一过性感染,免疫3 h的病毒是未完全吸附的病毒,而不是复制的病毒;通过PCR,RT-PCR检测,可在心脏、肝脏、脾脏、肺脏、肾脏、肌肉、脑、肠系膜淋巴结内检测到了FPV 4b DNA和FMDV VP1 DNA,且在大部分组织能存留5 d左右;增加免疫剂量和进行多次免疫,其在体内存留时间仍然很短,重组疫苗可诱导豚鼠产生较高水平的抗FMDV特异性抗体和中和抗体;静脉注射也在体内检测到病毒的DNA,但其在体内的分布和存留时间短,诱导豚鼠产生的抗FMDV特异性抗体和中和抗体水平比肌肉注射低、且持续时间短,病毒培养表明只在任何组织和任何时间点均未能培养出病毒;对妊娠豚鼠的不同阶段均无毒性,未造成流产、早产、死胎等不良反应,子代未检测到病毒的DNA。以上均证明重组鸡痘病毒vUTAL 3CP1在豚鼠体内存留时间短,对妊娠豚鼠、子代无毒性,且能产生良好的免疫反应,且对环境无污染,为后期其他哺乳动物实验提供必要的基础数据,从而更进一步验证所构建的重组鸡痘活载体疫苗的生物安全性及免疫原性,对免疫动物无安全威胁。

Distribution virulence and antibody variation of the recombinant fowlpox virus vUTAL3CP in cavia cobaya

The possible development and use of vaccinia virus as recombinant vaccines for protective immunization against infectious diseases have stimulated research into the development of other poxviruses as potential recombinant vectors. However,safety shall be paramount importance to construct effective,nonreplicating vaccine vectors.Fowlpoxviruses,which should meet these stringent safety criteria,need to be tested properly for ability to establish productive infection in mammalian origin.A series of trials were conducted to assess the recombinant fowlpox virus encoding the FMDV 3CP1 protein in various prime-boost combinations,doses,and application routes.Clinical,histological,viral culture,PCR,RT-PCR,immunochemistry neutralizing antibody were used to assess the bio-safety and immunogenicity of the recombinant fowlpox virus.During the experiment,the mental status,hydroposia and foraging behaviors of the mice were normal.No detectable pathological change was found in the organs of inoculated cavia cobaya.Virus was successfully isolated from only tissue specimens collected from inoculation spots at 3 hours post inoculation(HPI) by viral culture.Using PCR,viral nucleic acids could be detected as early as 3 HPI in the spleens and inoculated muscles;from 3 days post inoculation(DPI),viral nucleic acids could be found in all organs;none viral nucleic acids could be detected from any organs at 10 DPI.Virus was not isolated from all tissues specimens at hours post inoculation(HPI)with intravenous injection(iv).Using PCR,viral nucleic acids could be detected as early as 3 HPI in the spleens;from 3 days post inoculation(DPI),viral nucleic acids could be found in all organs;none viral nucleic acids could be detected from any organs at 7 DPI.In addition,no pollute environment had also been ensured by detection dejecture and urine after the bio-safety of this vaccine was studied.