重组病毒BoHV-1 gG-/tk-/gD+和BoHV-1 gG-/tk-/gD5+的兔体内安全性和免疫原性

摘要

[Objective] Infectious bovine rhinotracheitis (IBR) is caused by BoHV-1 and there is no commercialized marker vaccine against IBR in China.Based on the previously constructed BoHV-1 gG-/tk-gene-deleted vaccine in this lab,the glycoprotein gD extracellular region sequence which served as the best immunogenicity in BoHV-1 and BoHV-5 was further inserted into the site where tk is located and recombinant virus strains BoHV-1 gG-/tk-/gD+ and BoHV-1 gG-/tk-/gD5+ were successfully constrcuted.This paper aimed to evaluate the immunogenicity and safety of recombinant virus strains in rabbits and protection against both BoHV-1 and BoHV-5 for development of more effective vaccines against BoHV-1 and BoHV-5 infection.[Methods] The 30 rabbits were divided into 6 groups and vaccinated and challenged by nasal inoculation.After strains were vaccinated,their safety was evaluated by clinical symptoms,temperature,virus shedding.Then the rabbits were challenged with either wt BoHV-1 or wt BoHV-5 at the 28th day after vaccination.The protection was evaluated by clinical signs,temperature,virus shedding,histopathology,isolation and identification of virus.Besides,the immunogenicity was studied by neutralization test,indirect-ELISA and specific PBMC proliferative response.[Results] The rabbits vaccinated with BoHV-1 gG-/tk-/gD+ and BoHV-1 gG-/tk/gD5+ didn't show clinical signs,nasal virus shedding and viral survival in lung tissues.One rabbit vaccinated with BoHV-1 gG-/tk-had viral survival in lung tissues.After challenge,both strains BoHV-1 gG-/tk-/gD+ and BoHV-1 gG/tk-/gD5+ could diminish the clinical signs and nasal virus shedding and maintain the good structure of lung tissues indicating the protection against BoHV-1 and cross-protection against BoHV-5 were improved.Besides,BoHV-1 gG-/tk-/gD+ and BoHV-1 gG-/tk-/gD5+ induced stronger neutralization and ELISA antibodies and PBMC proliferation compared with BoHV-1 gG-/tk-.[Conclusion] BoHV-1 gG-/tk/gD+ and BoHV-1 gG-/tk-/gD5+ are safe and could induce higher levels of immune response than BoHV-1 gG-/tk-strain.%[目的]牛传染性鼻气管炎由BoHV-1感染引起,我国对该病的防控尚缺乏商品化标记疫苗,本实验室前期成功研制了BoHV-1 gG-/tk-基因缺失疫苗,并通过将BoHV-1和BoHV-5的免疫原性最好的糖蛋白gD的胞外区序列分别插入BoHV-1 gG-/tk-的tk位置,构建了重组病毒BoHV-1 gG-/tk-/gD+和BoHV-1 gG/tk-/gD5+.本研究的目的在于评价该重组病毒在兔体内的安全性和免疫原性及对BoHV-1保护力和BoHV-5的交叉保护力,以期开发出更为有效的牛传染性鼻气管炎标记疫苗.[方法]选用30只日本大耳白兔并随机分成6组,鼻腔接种和攻毒.通过临床症状观察、体温测量和鼻腔排毒检测进行安全性评估.接种28 d后分别使用wt BoHV-1和wt BoHV-5对兔体进行攻击,并通过临床症状观察、体温测量、鼻腔排毒、病理组织学和组织病毒的分离鉴定进行保护力研究.使用间接ELISA、中和实验和外周血单核细胞的增殖水平对重组病毒免疫原性进行评估.[结果]接种BoHV-1 gG-/tk-/gD+和BoHV-1 gG-/tk-/gD5+后兔体均未出现显著临床症状,无鼻腔排毒现象,并且肺组织未分离到病毒;BoHV-1 gG-/tk-接种组有一只兔肺组织分离到病毒.攻毒后,BoHV-1 gG-/tk-/gD+和BoHV-1 gG-/tk-/gD5+可以减少临床症状、鼻腔排毒并维持正常肺组织形态,表现为对BoHV-1的保护力以及BoHV-5的交叉保护力提高.与BoHV-1 gG-/tk-相比,BoHV-1 gG-/tk-/gD+和BoHV-1 gG-/tk-/gD5+免疫诱导更高水平的中和抗体、ELISA抗体以及PBMC增殖. [结论]BoHV-1 gG-/tk-/gD+和BoHV-1gG-/tk-/gD5+安全性良好,同时,与BoHV-1 gG-/tk-相比,免疫原性得到提高.