Screening And Activity Evaluation Of Staphylococcus Aureus Enterotoxin B Nanobody

Staphylococcus aureus is a zoonotic pathogen that causes worldwide epidemics of infections,including toxic shock syndrome and bacteremia.Previously,treatment for Staphylococcus aureus infection focused on antibiotics,but with antibiotic abuse,a more pathogenic methicillin-resistant Staphylococcus aureus(MRSA)was produced.Enterotoxin B(SEB),as a superantigen,plays a vital role in the infection of MRSA,which mainly binds to MHC-Ⅱ molecules and Vβregion on TCR,resulting in the uncontrolled release of various inflammatory cytokines such as IL1βand TNF-αand producing inflammatory response.Therefore,to inhibit the function of SEB is very important.At present,antibodies and vaccines against SEB are under the research stage,and most of them focus on the use of monoclonal antibodies to inhibit the binding of SEB to its receptors.However,the monoclonal antibody itself has a large molecular weight,resulting in limited penetration ability,which is difficult to meet the needs of clinical applications.In addition,the nanobody VHH evolved from the variable region of HcAb has stronger and faster tissue penetration,can reach the dense tissues such as solid tumors to function,and at the same time,under the filtering of the kidney,the half-life in the blood is relatively short,and can avoid the accumulation of toxins,which has great advantages in the development of antibody drugs.In this study,eight nanobodies that could specifically bind Staphylococcus aureus SEB were isolated from a nanobody library with a size of 5×10~8,combined with yeast surface display and flow cytometry screening technology.Subsequently,different concentrations of enterotoxin B protein were used to detect the binding ability of these 8 nanobodies,and it was found that their Kd value was 20-150 nM.In addition,SEB S12-20,SEB S10-11,SEB S12-2,SEB S10-7and SEB S12-10 among the 8 nanobodies could be efficiently expressed in E.coli,and the concentration of purified nanobody SEB S12-20 could reach 1.5 mg/mL.Finally,the neutralizing activity of SEB S12-20 and SEB S10-11 were verified by cell experiments,and it was found that only the nanobody SEB S12-20 could significantly reduce the secretion and expression of IL1β,TNF-αin mouse spleen cells at a concentration of 5μg/mL.Compared with the stimulation of enterotoxin B protein alone,the mean transcription levels of IL1β,TNF-αwere reduced by 7.6,8.55 times after combined with nanobody SEB S12-20,respectively,indicating that the nanobody SEB S12-20 selected in this experiment could effectively attenuate the inflammatory caused by SEB.Finally,the structures of the nanobodies were simulated by AlphaFold2,followed by docking with SEB through the ZDOCK and RDOCK in DS software.It was found that the nanobody SEB S12-20 competitively bonded with TCR and MHC-Ⅱ,thus inhibiting the function of SEB.But SEB S10-11 bonded to the other domain in the SEB molecule.In summary,the nanobody SEB S12-20 isolated in this study would be useful in the diagnosis or treatment of related diseases caused by SEB.