Staphylococcus aureus의 Hla, SEA 및 SEB 재조합 단백질 기반 백신 후보 물질 개발

Abstract

Development of vaccine candidates based on Staphylococcus aureus Hla, SEA, and SEB recombinant proteins Da Hae Kwak Adviser: Prof. Kim Dong-Min., M.D., Ph.D Department of Biomedical Science Graduate School of Chosun University Background: Staphylococcus aureus is the most common gram-positive bacterium causing infections and various diseases. Antibiotics are used in the treatment of S. aureus, but the emergence of antibiotic-resistant strains like methicillin-resistant S. aureus(MRSA) and vancomycin-resistant S. aureus(VRSA) has elevated its global prevalence as a significant infectious agent. Additionally, S. aureus is a complex pathogen known for causing a range of diseases. The virulence of S. aureus can be attributed to the number of exotoxins the strain possesses. Due to the numerous variable infection-related factors, a multivalent vaccine composed of antigens associated with different stages of infection is necessary. Among the toxins of S. aureus, α-hemolysin(Hla) is a pore-forming exotoxin secreted by most S. aureus strains, contributing to various diseases and playing an essential role in biofilm formation. Enterotoxin A(SEA) and enterotoxin B(SEB) act as enterotoxins, commonly causing food poisoning when ingested or inhaled. Especially, SEB is a potent superantigen inducing massive T cell activation, leading to a cytokine storm that threatens life. While several of these virulence factors of S. aureus have been evaluated as vaccine candidates, targeting only one antigen, vaccine development, as known, has failed. Hla, also known as α-hemolysin, is a member of the pore-forming β-barrel toxin family expressed by most S. aureus strains, targeting various cell types and causing several diseases such as skin and soft tissue infections, and pneumonia. Hla forms a complex with ADAM10, the cell receptor for α -hemolysin, interfering with cell adhesion, promoting S. aureus invasion, and inducing cell death, ultimately compromising cell membrane integrity. Mutating the Hla amino acid sequence from 1 to 62 and replacing histidine at the 35th amino acid position with leucine effectively protects mice from S. aureus. S. aureus Enterotoxin A and B are the most common toxins in S. aureus-related food poisoning. They directly bind to the MHC II molecules on antigen-presenting cell surfaces and subsequently attach to the variable beta chain of T cell receptors(TCR), triggering the release of various cytokines that can induce toxic shock syndrome. Mutations in critical residues related to the binding of TCR and MHC II receptors by SEB have become targets for new vaccine development and have been evaluated as effective vaccine candidates through the appropriate combination of these residues. Method: Initially, a literature review was conducted to identify recombinant proteins. Subsequently, five recombinant proteins were expressed and purified: Hla, SEA, SEB, Hla(27-88/H61L), SEB(N50A/Y117A). Protein quantification, SDS-PAGE analysis, and western blot were performed following the expression and purification of the recombinant proteins. Animal experiments were carried out to evaluate the immune response to com binations of the recombinant proteins, establish the lethal dose of S. aureus, a nd conduct challenge studies to assess efficacy as a vaccine candidate. Blood samples obtained from the animal experiments were utilized for hemolysin ass ays to determine the relative hemolytic activity of Hla and Hla(27-88/H61L). An indirect ELISA (RocheⓇ) was conducted to confirm the production of anti bodies against the S. aureus recombinant proteins. The antibody titers of the antisera against the recombinant protein combinations were also verified throu gh an indirect ELISA (RocheⓇ). Result: Through a data review for the discovery of recombinant proteins, three proteins Hla, SEA, SEB were identified. Subsequently, a non-toxic recombinant protein was expressed and purified by mutating the amino acid sequences of the wild-type Hla and SEB. The hemolysin assay confirmed the hemolytic abilities of Hla and Hla(27-88/H61L), revealing lower relative hemolytic activity in Hla(27-88/H61L) compared to Hla. Indirect ELISA (RocheⓇ) using antisera from mice immunized with recombinant proteins coated with the recombinant proteins as antigens showed reactions from the respective antisera. SEB IgG antibody response from mouse anti-Hla, SEB(N50A/Y117A) IgG antibody response from mouse anti-Hla(27-88/H61L), and Hla(27-88/H61L) IgM, IgG antibody response from mouse anti-SEB(N50A/Y117A) were confirmed. The antisera from mice immunized with various recombinant protein combinations for indirect ELISA showed reactions from the corresponding antisera. SEA IgG antibody response from anti-Hla(27-88/H61L), SEB IgG antibody response from anti-Hla(27-88/H61L) + SEA, and SEB IgG antibody response from anti-Hla(27-88/H61L) + SEA were confirmed. Upon checking the antibody titers for Hla IgG using the antisera from mice immunized with various recombinant protein combinations, the group immunized with Hla + alum exhibited the highest antibody titers. For SEB IgG, the group immunized with Hla(27-88/H61L) + SEA + SEB(N50A/Y117A) + ASO3 showed the highest antibody titers. The results from conducting lethal dose experiments with S. aureus 29213 indicated an lethal dose at 1×108 cfu/mL. However, after attacking mice immunized with various recombinant protein combinations with S. aureus 29213 at 1×108 cfu/mL, the following day, all mice were observed to have died. Conclusion: In this study, various recombinant proteins of S. aureus were expressed. The mutated Hla(27-88/H61L) recombinant protein, which lacked the pore-forming ability characteristic of Hla, inhibited mouse RBC hemolysis and exhibited relatively hemolytic activity compared to the Hla recombinant protein. Analysis of antibody responses in immunized mice revealed a significant increase in antibody response when using the ASO3 adjuvant. However, when mice were immunized with a combination of several recombinant proteins, they were not effectively protected in challenge study with S. aureus strain. Because S. aureus harbors numerous toxin and enterotoxins apart from SEA and SEB, it may be difficult to observe vaccine effectiveness by blocking only a few toxins. Therefore, multivalent antigen vaccine targetting both toxins and bacterial structural components would be more effective.