Prokaryotic Expression,Safety And Immunogenicity Of Cross-protective Vaccine Candidate Of Streptococcus Suis Type 1,2,7 And 9

Streptococcus suis is an important human-animal pathogen,which can be transmitted through respiratory tract and wounds,and pigs show symptoms of meningitis,myocarditis,arthritis and septicemia after the disease.In recent years,the high prevalence of Streptococcus suis types 1,2,7 and 9 in China has caused serious harm to the pig breeding industry.There are35 serotypes of Streptococcus suis,but there are not universal vaccines for different serotypes of Streptococcus suis pathogens to provide cross-protection.Therefore,it is significant to carry out the research on the candidate antigens of the multi-serotype cross-protective vaccine of Streptococcus suis for the scientific prevention and controlling of the disease.In this study,based on three candidate vaccine antigen protein expression vectors constructed by pre-screening Streptococcus suis types 1,2,7,and 9 with reverse vaccinology technology,we carried out expression and purification of the candidate vaccine antigen protein,immunization of New Zealand White rabbits for antibody titer determination（ELISA）,whole blood bactericidal assay,and cell adhesion inhibition assay,which verify the antiserummediated whole blood bactericidal effect and the inhibition of adhesion of the antiserum.After immunization with BALB/c mice,splenocytes were collected for antigen restimulation to analyze the type of cellular immune response induced by the candidate vaccine antigen protein.We also established an animal model of Streptococcus suis type 2 and 7 in New Zealand white rabbits and conducted a protective test to counts the morbidity and mortality of each group.In addition,safety tests of three candidate antigenic proteins on pigs also was conducted.Three candidate vaccine antigenic proteins were successfully expressed,and the serum antibody titers of three proteins showed significant increasing after booster immunization compared with the initial immunization,which indicate all three proteins could induce an effective humoral immune response in the organism.The results of whole blood bactericidal test showed that the antiserum-mediated whole blood bactericidal test after immunization with antigenic proteins of three candidate vaccines had different degrees of killing effect on Streptococcus suis type 2 and 7,which the bactericidal rate against Streptococcus suis type 2was up to 60%,the highest bactericidal rate against Streptococcus suis type 7 was more than95%,and the bactericidal rate of antiserum 9253 and 9342 against Streptococcus suis type 9was more than 50%.In the adhesion inhibition assay,the three candidate vaccine antigen proteins produced different degrees of adhesion inhibition against Streptococcus suis types 2,7and 9 after immunization with antiserum,among which,the adhesion inhibition of Streptococcus suis types 2,7 and 9 was the most obvious with 49%,96% and 71% for antiserum4959,22%,30% and 71% for antiserum 9253 and 38%,93% and 58% for antiserum 9342,respectively.The results of cellular immune response type experiments showed that 4959,9253 and 9342 proteins could increase the level of cytokine IL-17 A in splenocytes more significantly,meanwhile,4959 and 9342 proteins also promoted the level of cytokine IL-10 by 2.5-4 times and the level of IFN-γ by about 2-4 times.The results of the attack protection test showed that the three candidate vaccine antigen proteins could produce cross-protection against Streptococcus suis types 2 and 7 and the protection rate ranged from 50% to 87.5%.In the safety evaluation,the body temperature,diet and mental status of the pigs were normal,and no adverse reactions such as redness,swelling and hard nodules.during the vaccination period,there was no abnormal death of pigs,and the safety was remarkable.This study successfully expressed three candidate vaccine antigen proteins of Streptococcus suis,which could produce effective humoral and cellular immune responses.In addition,the whole blood sterilization test,cell adhesion inhibition test and animal challenge protection test confirmed that the three candidate vaccine antigen proteins have certain cross protection,and they are safe to target animals,Through the above studies,it is proved that the three candidate antigen proteins have the prospective of developing a cross-protective vaccine.