Identification Of Neutralizing Epitopes With Hla Antigen Using Human Serum Antibodies By Staphylococcus Aureus Vaccination And Preliminary Study On Its Application

Background:Staphylococcus aureus（SA）is an important Gram-positive bacterial pathogen,which can infect the body under certain conditions and cause acute,purulent infection.It can not only cause local purulent infections in skin and soft tissue,endocardium,lung,bone marrow,but also lead to sepsis and even death.The latest national bacterial drug resistance monitoring report in 2019 showed that the isolation rate of nosocomial infection of SA ranked first in Gram-positive bacteria.The antibiotic resistance of SA led to unusual difficulties in the treatment of infectious diseases,especially methicillin-resistant Staphylococcus aureus（MRSA）.The high drug resistance of MRSA made the clinical treatment of conventional antibiotics ineffective and limited.SA is a serious threat to human health.The prevention and treatment of its infection has become a difficult clinical problem.Vaccine immunization is the most economical and effective method to prevent and control SA infection,but there is no successful vaccine for clinical use.Alpha-hemolysin（Hla）is one of the key pathogenic factors of SA and an important candidate antigen for vaccines.The protective effect of the vaccine mainly depends on the immune response induced by protective epitopes.Identifying the protective epitopes of Hla to design the vaccine can remove invalid and pathogenic epitopes in the whole antigen,reduce the risk of vaccine use and improve the effectiveness of the vaccine.Although some studies have reported the protective epitopes of Hla,due to the limitation of conditions,the identification results of these epitopes are mainly derived from animal experiments,which have not been verified in the human body.The protective epitopes identified from animal models provide very limited information for the study of human vaccines,and cannot be directly used in the design of human vaccines against SA.As a self-assembly protein,ferritin has very low toxicity.Coupling epitope peptides to the surface of ferritin to prepare nano-epitope vaccine can enhance the immunogenicity of epitope.Since 2009,our laboratory has developed a new drug class 1.1,recombinant five SA antigen vaccine（r FSAV）.The main antigenic components of the vaccine are Hla,Isd B,m SEB,Sp A5 and Mnt C,and phase II clinical trials have been completed.In this study,the serum of 144 volunteers collected from phase Ib clinical trials was used to identify the B-cell immunodominant epitopes with neutralizing activity against Hla based on Luminex multi-chip detection technology.On this basis,the nano-vaccine based on self-assembly ferritin vector was designed and constructed,which laid the foundation for clarifying the response law of the vaccine and the optimization of the vaccine in the next step.Objective:1.To establish the evaluation method of hemolytic neutralization activity of Hla toxin.2.To identify the immunodominant epitopes with neutralizing activity against Hla,via the serum of 144 volunteers before（D0）and 14 days after（D14）SA vaccine phase Ib was used.3.To prepare self-assembled ferritin nano-vaccine based on neutralizing epitope and to elucidate its protective effect in mouse skin infection model.Methods:1.We expressed and purified Hla toxin protein,preparation of high quality anti-Hla neutralizing antibody,obtaining fresh rabbit red blood cells,and the establishment of Hla toxin hemolytic neutralization activity evaluation method.2.The neutralization activity of 144 serum samples from 144 volunteers before（D0）and14 days after（D14）phase Ib of SA vaccine was detected by the established method.The results were reflected by the antibody dilution（IC50）when inhibiting 50%hemolytic activity,that is,each serum had the corresponding IC50 value.3.Twenty-three Hla epitope peptides were synthesized by step-by-step method.Twenty-four serum samples 14 days after vaccination were randomly selected.The reaction intensity of Hla epitope peptides to these 24 serum samples was detected by Luminex multiple chip detection technology.4.The correlation between the reaction intensity of Hla epitope peptide and serum neutralization activity was analyzed by statistical software SPSS.The polyclonal antibodies against neutralizing activity-related epitopes were prepared,and the neutralizing activity was evaluated to identify the Hla epitopes with neutralizing activity.5.Epitope based nanoparticle（EpNP）was constructed by fusing epitopes with neutralizing activity to the N-terminal of self-assembled ferritin.EpNP was expressed in Escherichia coli,and its physicochemical properties were detected by molecular sieve chromatography,dynamic light scattering and transmission electron microscopy.6.EpNP adsorbed by Al（OH）₃ adjuvant was used to immunize mice on day 0,14 and 21.Serum was collected and anti-Hla antibody titer and in vivo were detected.Results:1.We expressed and purified Hla toxin with a molecular weight of about 33k Da.The hemolytic activity of Hla toxin and its blocking effect by antibody were verified in vitro,and the evaluation method of hemolytic neutralization activity of Hla toxin was successfully established.2.The serum neutralization activity of 288 vaccine clinical trials was determined.3.Twenty-four serum samples of volunteers 14 days after SA vaccination were randomly selected.The immunodominant epitopes with strong reactivity to Hla were detected by Luminex multiple chip detection technology.4.The neutralizing epitope Hla_121-138 of Hla was successfully identified by statistical analysis of the epitope positively correlated with serum neutralization activity.5.The self-assembled ferritin nano-vaccine（EpNP）carrying neutral epitope Hla_121-138was constructed,purified and obtained,with a molecular weight of about 25 k Da.Molecular sieve molecular weight of 562k Da,is 24 times the monomer molecular weight.Dynamic light scattering and electron microscopy results showed that Hla ferritin could self-assemble into nanoparticles with a diameter of 13.2 nm and good uniformity in vitro.6.EpNP immunization could induce mice to produce strong anti-Hla antibody levels,which had obvious neutralizing activity in vitro.EpNP immunization can reduce the degree of skin infection,reduce the colonization of bacteria in the skin,liver,spleen,lung and kidney,and play a good protective role in mouse skin infection model.Conclusions:1.Hla_121-138 is a neutralizing epitope of Hla antigen of human SA vaccine.2.EpNP can self-assemble to form nanoparticles with good uniformity in vitro.3.EpNP immunization can induce the production of neutralizing antibodies and play a protective role in mouse skin infection model.