Screening Of Antigenic Epitopes Of Tilapia Lake Virus By Phage Display And Evaluation Of Its Immune Efficacy

Tilapia lake virus（Ti LV）is a newly emerged negative sense single-stranded RNA virus,which causes tilapia lake virus disease（Ti LVD）and poses a great threat to the global tilapia culture industry.Vaccination is an effective measure to prevent Ti LVD,and the study of antigenic epitopes is an important basis for the development of safe and efficient vaccines.At present,the function of Ti LV protein is still unclear,which makes the identification of antigenic epitopes relatively difficult.The aim of this study was to screen the mimic epitopes of Ti LV by phage display,and to evaluate their immunogenicity and protective efficacy by immunization.On this basis,the natural epitopes of Ti LV were analyzed and identified by bioinformatics,and epitope vaccine was prepared to evaluate the immune protection effect of Ti LV.The results were as follows:1.Three rounds of biopanning of a random dodecapeptide library by phage display technology,22 randomly selected phage clones were sequenced and analyzed after biopanning,and affinity identification of phage clones was performed in combination with enzyme-linked immunosorbent assay.The results showed that the yield of the first round of biopanning was4.47×10^-6,the yield of the second round of biopanning was 2.12×10^-4,and the yield of the third round of biopanning was 3.87×10^-3.The phage recovery rate increased nearly 866 times after three rounds of biopanning.Sequencing results showed that the 22 phage clones contained a total of 9 different sequences.Phage ELISA results showed that phage clones with6 sequences（named Pep1,Pep2,Pep3,Pep4,Pep5 and Pep6）had good reactivity with anti-Ti LV serum.2.Nile tilapia（0.98±0.20 g）was immunized intraperitoneally by solid-phase synthetic polypeptides（Pep1,Pep2,Pep3,Pep4,Pep5 and Pep6）,emulsified by Freund’s adjuvant,and then immunized by primary-booster strategy at an immunization dose of 20μg.Serum and tissue samples were collected weekly after the initial immunization,and the immune activation effect was evaluated by enzyme-linked immunosorbent assay,enzyme activity assay and fluorescence quantitative PCR.The protective effect of the polypeptide vaccine on tilapia was evaluated 21 days after booster immunization combined with virus challenge test.The results showed that compared with adjuvant group and PBS control group,the levels of serum antibody,immune-related physiological indexes and gene expression in immune group were significantly increased（P<0.05）.The mortality was 93.9%in PBS group and 90.9%in adjuvant group,and the survival rates were 24.1%,21.2%,57.6%,18.2%,21.2%,and 36.4%in Pep1,Pep2,Pep3,Pep4,Pep5 and Pep6 immunization group,respectively.3.The natural epitope of Tilv associated with Pep3 was predicted by sequence bioinformatics,protein structure prediction and analysis,and identified by enzyme-linked immunosorbent assay（ELISA）.The results showed that Pep3 had a high sequence similarity with Tilv Segment 1 at position 399-410（S1399-410）,with a similarity of 58.3%.The structure prediction and analysis of the S1 protein showed that the S1399-410 region formed a long loop,and the S1399-410 region was completely exposed to the solvent accessible surface.Furthermore,the structure of S1399-410 was better than that of S1,and its RMSD was 4.382.The ELISA activity of S1399-410 synthetic peptide was higher than that of PEP3,while the mutant peptides S1399-410Y400A,S1399-410R403A and S1399-410Y400A/R403A were not detected.4.The epitope vaccine（KLH-S1399-410）was prepared by coupling S1399-410 with keyhole limpet hemocyanin（KLH）and immunized Nile tilapia（0.93±0.24 g）by intraperitoneal injection in a primary-booster immunization strategy at a dose of 20μg.Serum samples were collected weekly after the initial immunization,and the immune response capacity of immunized tilapia was assessed using enzyme-linked immunosorbent assay and virus neutralization test.After 21 days of booster immunization,the immune protective effect was evaluated in combination with virus challenge test,virus load determination test and histopathological observation.The results showed that the serum antibody titers of the KLH-S1399-410 immunized group were significantly higher than those of the control group and the s1 recombinant protein immunized group at the 4th week（P<0.05）and lasted until the 6th week（P<0.05）.The results of the virus neutralization test showed that the half-inhibition dilution factor（IC50）of sera from the KLH-S1399-410 immunization group at week 5 was 434.1,which was significantly higher than the IC50 of sera from the S1 immunization group at the same time（124.8）.The mortality rates in the control and KLH groups were 90.9%and 87.9%,respectively,while the survival rates in the KLH-S1399-410 and S1 immunization groups were81.8%and 60.6%,respectively,and the protection rate in the KLH-S1399-410 immunization group was 21.2%higher than that in the S1 immunization group at the same dose.In conclusion,the Ti LV antigen epitope S1399-410 coupled with the KLH carrier protein in tilapia could trigger a high level of neutralizing antibody response,and the serum antibody level could be maintained at a high level for a long period of time,thus achieving an efficient preventive effect against Ti LV.The results of this study can provide theoretical reference for epitope-based Ti LV vaccine design,and have important significance for the healthy development of tilapia industry.