Saekil Yun^a, Jin Woo Jun^b, Sib Sankar Giri^a, Hyoun Joong Kim^a, Cheng Chi^c, Sang Geun Kim^a, Sang Wha Kim^a, Jung Woo Kang^a, Se Jin Han^a, Jun Kwon^a, Woo Taek Oh^a, Se Chang Park<sup>a,*

a</sup> Laboratory of Aquatic Biomedicine, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul, 08826, Republic of Korea
^b Department of Aquaculture, Korea National College of Agriculture and Fisheries, Jeonju, 54874, Republic of Korea
^c Laboratory of Aquatic Nutrition and Ecology, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China

^* Corresponding author: Se Chang Park, DVM, Ph.D.
Mailing address: College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, 81-417, 1 Gwanak-ro, Gwanak-gu, Seoul, Republic of Korea 08826

Abstract
Over the last 50 years, various approaches have been established for the development of antigens for immunostimulation. We used phage lysate (PL), composed of inactivated antigens by the lytic bacteriophage pAh 6-c for Aeromonas hydrophila JUNAH strain to develop a vaccine for the prevention of A. hydrophila infection in Cyprinus carpio (common carp). We also assessed the poly D,L lactide-co-glycolic acid (PLGA) microparticles encapsulation method to increase the efficiency of the vaccine. Six groups of vaccines involving encapsulated by PLGA, formalin killed cells, or phage lysate at low or high concentration were prepared for intraperitoneal injection in C. carpio. Blood specimens and head kidney samples were collected at various time points for bacterial agglutination assay and to assess relative expression of immune-related genes interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), lysozyme C, and serum amyloid A (SAA). The vaccine groups using high dose phage lysate antigen showed significantly higher agglutination titers than all other groups at 4- and 6-weeks post vaccination (wpv), with the titer of the PLGA encapsulated vaccine group being highest from 10 wpv to the end of the experiment. The survival rate of fish immunized with the phage lysate vaccines were higher than that of fish immunized with the formailin killed cells vaccine in the challenge experiment conducted 6 wpv. Additionally, the PLGA-encapsulated high dose phage lysate antigen vaccinated groups showed the best protective efficacy in the challenge experiment 12 wpv. Vaccines using the phage lysate antigen also showed higher IL-1β and lysozyme C gene expression at 7 days post vaccination (dpv) and 2 wpv, and higher TNF-α gene expression was seen at 7 dpv. Higher SAA gene expression was seen in these groups at 1 dpv. These results suggest that phage lysate antigen has the potential to induce robust immune responses than formalin killed cells-based vaccines, and could be more effective as a novel inactivated antigen in preventing A. hydrophila infection in C. carpio.

Keywords : Inactivated antigen; Phage lysate; Aeromonas hydrophila; Poly D,L lactide-co-glycolic acid (PLGA); Cyprinus carpio