Rational Development of an Attenuated Recombinant Cyprinid Herpesvirus 3 Vaccine Using Prokaryotic Mutagenesis and In Vivo Bioluminescent Imaging

Cyprinid herpesvirus 3 (CyHV-3) is causing severe economic losses worldwide in common and koi carp industries, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open reading frame 134 (ORF134), we unexpectedly obtained a clone with additional deletion of ORF56 and ORF57. This triple deleted recombinant replicated efficiently in vitro and expressed an in vivo safety/efficacy profile compatible with use as an attenuated vaccine. To determine the role of the double ORF56-57 deletion in the phenotype and to improve further the quality of the vaccine candidate, a series of deleted recombinants was produced and tested in vivo. These experiments led to the selection of a double deleted recombinant lacking ORF56 and ORF57 as a vaccine candidate. The safety and efficacy of this strain were studied using an in vivo bioluminescent imaging system (IVIS), qPCR, and histopathological examination, which demonstrated that it enters fish via skin infection similar to the wild type strain. However, compared to the parental wild type strain, the vaccine candidate replicated at lower levels and spread less efficiently to secondary sites of infection. Transmission experiments allowing water contamination with or without additional physical contact between fish demonstrated that the vaccine candidate has a reduced ability to spread from vaccinated fish to naïve sentinel cohabitants. Finally, IVIS analyses demonstrated that the vaccine candidate induces a protective mucosal immune response at the portal of entry. Thus, the present study is the first to report the rational development of a recombinant attenuated vaccine against CyHV-3 for mass vaccination of carp. We also demonstrated the relevance of the CyHV-3 carp model for studying alloherpesvirus transmission and mucosal immunity in teleost skin.

鲤疱疹病毒3型（Cyprinid herpesvirus 3）可在全球范围内给普通鲤及锦鲤养殖业造成严重经济损失，因此亟需一种安全有效、可用于大规模免疫接种的减毒疫苗。本研究通过原核诱变技术制备了单基因缺失重组病毒。在制备缺失开放阅读框134（ORF134）的重组病毒时，我们意外获得了同时缺失ORF56与ORF57的克隆株。该三重缺失重组病毒可在体外高效复制，且其体内安全性与效力特征符合减毒疫苗的应用要求。为明确ORF56-57双缺失对病毒表型的影响，并进一步优化候选疫苗的品质，本研究制备了一系列缺失重组病毒并开展体内试验。通过上述试验，我们筛选出缺失ORF56与ORF57的双缺失重组病毒作为候选疫苗株。本研究通过生物发光成像系统（IVIS）、实时荧光定量聚合酶链反应（qPCR）及组织病理学检查，对该候选疫苗株的安全性与效力进行了评估，结果显示其与野生毒株一样，可通过皮肤感染途径侵入宿主鱼体。但与亲本野生毒株相比，该候选疫苗株的复制水平更低，向继发感染部位的扩散效率也更弱。通过设置仅污染水体、或同时存在鱼类直接接触的传播试验证实，该候选疫苗株从免疫鱼体向未免疫的哨兵同居鱼的传播能力有所下降。最后，IVIS分析结果显示，该候选疫苗株可在感染门户处诱导保护性黏膜免疫应答。综上，本研究首次报道了针对CyHV-3、可用于鲤大规模免疫接种的合理化重组减毒疫苗开发过程。本研究同时证实，CyHV-3-鲤模型可用于研究异疱疹病毒的传播及硬骨鱼皮肤的黏膜免疫机制。