Oral probiotics vaccine expressing koi herpesvirus (KHV) ORF81 protein delivered by chitosan-alginate capsules is a promising strategy for mass oral vaccination of carps against KHV infection.

Koi herpesvirus (KHV) is highly contagious and lethal to cyprinid fish, causing significant economic losses to the carp aquaculture industry, particularly to koi carp breeders. Vaccines delivered through intramuscular needle injection or gene gun are not suitable for mass vaccination of carp. So, the development of cost-effective, oral vaccines that are easily applicable at farm level is highly desirable. In this study, we utilized chitosan-alginate capsules as oral delivery system for a live probiotic (Lactobacillus rhamnosus) vaccine pYG-KHV-ORF81/LR CIQ249 expressing KHV ORF81 protein. The tolerance of the encapsulated recombinant Lactobacillus to various digestive environments and the ability of the probiotics strain to colonize the intestine of carp was tested. The immunogenicity and the protective efficacy of the encapsulated probiotic vaccine was evaluated by determining IgM levels, lymphocyte proliferation, expression of immune-related genes, and viral challenge to vaccinated fish. It was clear that the chitosan-alginate capsules protected the probiotics vaccine effectively against extreme digestive environments, and significant level (p < 0.01) of antigen-specific IgM with KHV-neutralizing activity was detected, which provided protection rate of ca. 85% for koi carp against KHV challenge. The strategy of using chitosan-alginate capsules to deliver probiotic vaccines is easily applicable for mass oral vaccination of fish.ImportanceAn oral probiotic vaccine pYG-KHV-ORF81/LR CIQ249 encapsulated by chitosan-alginate capsules as oral delivery system was developed for koi carp against KHV infection. This encapsulated probiotic vaccine can be protected from various digestive environments and maintain effectively high viability, showing a good tolerance to digestive environments. This encapsulated probiotic vaccine has a good immunogenicity in koi carp via oral vaccination, and the significant level of antigen-specific IgM was effectively induced after oral vaccination, displaying effective KHV-neutralizing activity. This encapsulated probiotic vaccine can provide effective protection for koi carp against KHV challenge, which is handling stress-free for the fish, cost-effective, and suitable for the mass oral vaccination of koi carp at farm level, suggesting a promising vaccine strategy for fish.