Oral Probiotic 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

J Virol. 2021 May 24;95(12):e00415-21. doi: 10.1128/JVI.00415-21. Print 2021 May 24.


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 a farm level is highly desirable. In this study, we utilized chitosan-alginate capsules as an 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 probiotic 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 probiotic vaccine effectively against extreme digestive environments, and a significant level (P < 0.01) of antigen-specific IgM with KHV-neutralizing activity was detected, which provided a 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.IMPORTANCE An oral probiotic vaccine, pYG-KHV-ORF81/LR CIQ249, encapsulated by chitosan-alginate capsules as an oral delivery system was developed for koi carp against koi herpesvirus (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 a 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 a farm level, suggesting a promising vaccine strategy for fish.

Keywords: ORF81 protein; anti-KHV immune protection; chitosan-alginate capsule; koi herpesvirus (KHV); live probiotic vaccine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Administration, Oral
  • Alginates
  • Animals
  • Antibodies, Neutralizing / blood
  • Antibodies, Viral / blood
  • Antigens, Viral / immunology
  • Capsules
  • Carps*
  • Cell Proliferation
  • Chitosan
  • Fish Diseases / prevention & control*
  • Herpesviridae / immunology*
  • Herpesviridae Infections / prevention & control
  • Herpesviridae Infections / veterinary*
  • Herpesvirus Vaccines / administration & dosage*
  • Herpesvirus Vaccines / immunology
  • Immunogenicity, Vaccine
  • Immunoglobulin M / blood
  • Lacticaseibacillus rhamnosus
  • Lymphocytes / physiology
  • Mass Vaccination / veterinary
  • Probiotics*
  • Recombinant Fusion Proteins
  • Spleen / immunology
  • Spleen / metabolism
  • Vaccines, Synthetic / administration & dosage
  • Viral Proteins / genetics
  • Viral Proteins / immunology*


  • Alginates
  • Antibodies, Neutralizing
  • Antibodies, Viral
  • Antigens, Viral
  • Capsules
  • Herpesvirus Vaccines
  • Immunoglobulin M
  • Recombinant Fusion Proteins
  • Vaccines, Synthetic
  • Viral Proteins
  • Chitosan

Supplementary concepts

  • Cyprinid herpesvirus 3