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INTRODUCTION

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.

METHODS

We reported a recombinant PRV with deleted gE/TK genes and an inserted porcine circovirus virus 2 (PCV2) 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.

RESULTS

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-γIL-2CD4 T cells and IFN-γTNF-αCD8 T cells, as well as PCV2-specific IFN-γTNF-αCD8 T cells.

CONCLUSION

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.

INTRODUCTION

METHODS

RESULTS

CONCLUSION

简介

变异伪狂犬病病毒（PRV）是一种新出现的人畜共患病病原体，可导致人类失明。PRV 可以利用其庞大的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 中的外源基因仅整合到基因组中进行独立表达，而不是组装在病毒粒子的表面。

方法

我们报道了一种使用 Cre-loxP 重组系统结合 CRISPR-Cas9 基因编辑系统构建的缺失 gE/TK 基因和插入猪圆环病毒 2（PCV2）基因到 PRV gE 基因胞外区的重组 PRV。这种带有包膜嵌入的 Cap 蛋白的重组 PRV（PRV-Cap）具有与其亲本病毒相似的复制能力。

结果

免疫原性试验表明，PRV-Cap 免疫的小鼠对致死性 PRV 和 PCV2 攻击具有 100%的抗性。中和抗体和 ELISPOT 检测表明，PRV-Cap 可以增强针对 PRV 的中和抗体，并产生针对 PRV 和 PCV2 的 IFN-γ 分泌 T 细胞。免疫机制研究表明，初始免疫 PRV-Cap 可显著早期激活和扩增 CD69 T 细胞，促进依赖 CD4 Tfh 细胞的生发 B 细胞的激活，并有效地产生特异性效应记忆 T 和 B 细胞。PRV-Cap 的加强免疫可召回 PRV 特异性 IFN-γIL-2CD4 T 细胞和 IFN-γTNF-αCD8 T 细胞以及 PCV2 特异性 IFN-γTNF-αCD8 T 细胞的激活。