■变异型伪狂犬病病毒(PRV)是一种新出现的人畜共患病原体,可导致人类失明。PRV可以利用其大基因组和多个非必需基因构建携带外源基因的重组减毒疫苗。然而,一个主要问题是重组PRV中的外源基因只能整合到基因组中进行独立表达,而不是组装在病毒体的表面。
■我们报道了使用Cre-loxP重组系统和CRISPR-Cas9基因编辑系统,将gE/TK基因缺失,并将猪圆环病毒2型(PCV2)Cap基因插入PRVgE基因胞外域的重组PRV。这种重组PRV(PRV-Cap),带有包膜包埋的Cap蛋白,表现出与其亲本病毒相似的复制能力。
■免疫原性测定显示,PRV-Cap免疫的小鼠对致死性PRV和PCV2攻击具有100%抗性。中和抗体和ELISPOT检测表明,PRV-Cap可以增强针对PRV的中和抗体,并产生对PRV和PCV2均具有特异性的分泌IFN-γ的T细胞。免疫机制研究表明,PRV-Cap的初始免疫显着刺激CD69T细胞的早期激活和扩增,促进CD4Tfh细胞依赖性生发B细胞的激活,并产生有效的特异性效应记忆T和B细胞。用PRV-Cap加强免疫回顾了PRV特异性IFN-γ+IL-2+CD4+T细胞和IFN-γ+TNF-α+CD8+T细胞的激活,以及PCV2特异性IFN-γ+TNF-α+CD8+T细胞。
■集体,我们的数据提示了一种免疫学机制,即带有包膜组装的PCV2Cap蛋白的重组PRV可以作为针对PRV和PCV2的联合免疫的优秀疫苗候选物,并为生产PRV-PCV2疫苗提供了一种经济有效的方法.
UNASSIGNED: Variant pseudorabies virus (PRV) is a newly emerged zoonotic pathogen that can cause human blindness. PRV can take advantage of its large genome and multiple non-essential genes to construct recombinant attenuated vaccines carrying foreign genes. However, a major problem is that the foreign genes in recombinant PRV are only integrated into the genome for independent expression, rather than assembled on the surface of virion.
UNASSIGNED: We reported a recombinant PRV with deleted gE/TK genes and an inserted porcine circovirus virus 2 (PCV2) Cap gene into the extracellular domain of the PRV gE gene using the Cre-loxP recombinant system combined with the CRISPR-Cas9 gene editing system. This recombinant PRV (PRV-Cap), with the envelope-embedded Cap protein, exhibits a similar replication ability to its parental virus.
UNASSIGNED: An immunogenicity assay revealed that PRV-Cap immunized mice have 100% resistance to lethal PRV and PCV2 attacks. Neutralization antibody and ELISPOT detections indicated that PRV-Cap can enhance neutralizing antibodies to PRV and produce IFN-γ secreting T cells specific for both PRV and PCV2. Immunological mechanistic investigation revealed that initial immunization with PRV-Cap stimulates significantly early activation and expansion of CD69+ T cells, promoting the activation of CD4 Tfh cell dependent germinal B cells and producing effectively specific effector memory T and B cells. Booster immunization with PRV-Cap recalled the activation of PRV-specific IFN-γ+IL-2+CD4+ T cells and IFN-γ+TNF-α+CD8+ T cells, as well as PCV2-specific IFN-γ+TNF-α+CD8+ T cells.
UNASSIGNED: Collectively, our data suggested an immunological mechanism in that the recombinant PRV with envelope-assembled PCV2 Cap protein can serve as an excellent vaccine candidate for combined immunity against PRV and PCV2, and provided a cost-effective method for the production of PRV- PCV2 vaccine.