Koi Herpes Virus (KHV or Cyprinid Herpesvirus 3, CyHV-3) is among the most threatening pathogens affecting common carp production as well as the highly valuable ornamental koi carp. To date, no effective commercial vaccine is available for worldwide use. A previous study reported that three intramuscular injections with an ORF25-based DNA vaccine, led to the generation of neutralizing antibodies and conferred significant protection against an intraperitoneal challenge with KHV. In the present study, we set out to optimize an ORF25-based DNA vaccination protocol that required fewer injections and would confer protection upon a challenge that better resembled the natural route of infection. To this end, ORF25 was cloned in pcDNA3 either as a soluble protein or as a full-length transmembrane GFP-fusion protein. We tested our ORF25-based DNA vaccines in multiple vaccination trials using different doses, vaccination routes (i.m. injection and oral gavage) and challenge methods (bath and cohabitation). Furthermore, we analysed local and systemic responses to the i.m. injected DNA vaccine through histological and RT-qPCR analysis. We observed a strong protection when fish received three injections of either of the two DNA vaccines. However, this protection was observed only after bath challenge and not after cohabitation challenge. Furthermore, protection was insufficient when fish received one injection only, or received the plasmid orally. The importance of choosing a challenge model that best reflects the natural route of infection and the possibility to include additional antigens in future DNA vaccination strategies against KHV will be discussed.

锦鲤疱疹病毒（KHV或Cyrpinid疱疹病毒3，CyHV-3）是影响鲤鱼生产以及极有价值的观赏锦鲤的最具威胁性的病原体之一。迄今为止，尚无有效的商业疫苗可供全球使用。先前的一项研究报告说，三次肌肉注射基于ORF25的DNA疫苗可导致中和抗体的产生，并赋予针对KHV腹膜内攻击的显着保护作用。在本研究中，我们着手优化基于ORF25的DNA疫苗接种方案，该方案所需的注射次数更少，并且可以在更像自然感染途径的挑战中提供保护。为此，将ORF25作为可溶性蛋白或全长跨膜GFP融合蛋白克隆到pcDNA3中。我们在使用不同剂量，疫苗接种途径（即时注射和口服管饲）和攻击方法（洗澡和同居）的多次疫苗接种试验中测试了基于ORF25的DNA疫苗。此外，我们通过组织学和RT-qPCR分析了对注射的DNA疫苗的局部和全身反应。当鱼类接受两种DNA疫苗中的任一种的三剂注射时，我们观察到了强有力的保护。但是，只有在洗澡后才观察到这种保护作用，而在同居后才观察到这种保护作用。此外，当鱼类仅接受一次注射或口服接受质粒时，保护作用不足。将讨论选择最能反映自然感染途径的挑战模型的重要性，以及在未来针对KHV的DNA疫苗接种策略中包括其他抗原的可能性。