Research On The Immunodominant B Cell Epitopes Of Staphylococcus Aureus Enterotoxin B And Epitope Vaccine

Background and aims：Methicillin-resistant Staphylococcus aureus (MRSA) showsmultiple drug-resistant, serious clinical infection and damage, but current clinical antibiotictherapy fail to control MRSA infection, and at present there is no effective MRSA vaccinethat has been successfully developed. The pathogenicity of MRSA depends mainly on itstoxins and invasive enzymes. Staphylococcus aureus enterotoxins (SEs) are the main causeof food poisoning, systemic inflammation and septic shock in MRSA infected patients, ofwhich SEB contains conservative sequences and stable structure among MRSA strains.Besides, SEB has stronger humoral response over other antigens and SEB monoclonalantibody can prevent mouse from the toxic shock syndrome induced by MRSA infection.As SEB itself is a toxin, there was a security for utility, while the recombinant mutant ofSEB (rSEB) without the toxicity, is a good protective antigen of MRSA vaccine. Theimmune protective effect of SEB mainly due to the antibody response, which alwaysfocuses on the immunodominant epitopes. The protective response of an antigen issubstantially induced by the specific protective epitopes. Therefore, the fine-mapping oflinear B-cell epitopes of the SEB antigen in this study will be useful to understand anti-SEBimmunity against MRSA infection and further will be helpful to optimize MRSA vaccinedesigns that are based on the SEB antigen.None of the previous reports was about the immunodominant B cell epitope of SEB orabout the vaccine based on SEB immunodominant epitopes. Therefore, we aimed to screenand identify all of the B cell immunodominant epitopes of SEB, to analyze their biologicalfunction, and to construct and express the multiple-epitope fusion protein and to analyze itsprotective role in MRSA252infection.Methods:I. Identification and functional analysis of the immunodominant B cell epitopes ofSEB 1. As the the toxicity of SEB was closely associated with its superantigen activity, toinvestigate the toxicity of rSEB, splenic mononuclear cells from BALB/c mice werecultured and then various concentrations of SEB or rSEB were added. Cells in mediumalone without treatment were used as controls. We analyzed the ability of rSEB toinduce T cell mitogenesis and IL-2, IFN-γ and TNF-α production in splenic lymphocytes ofBALB/c mice.2. To determine the protective role of rSEB against MRSA infection, BALB/c micewere immunized using rSEB plus AlPO4adjuvant, AlPO4adjuvant alone or PBS alonebefore MRSA252infection. To assess the SEB-specific antibody titer, one week after abooster immunization, the sera from rSEB immunized mice were examined using anELISA.3. To determine the immunodominant peptides of SEB, forty-two18-mer overlappingpeptides that spanned the entire length of the SEB of MRSA252were synthesized, andantiserum samples were collected from BALB/c mice that were immunized with rSEB plusAlPO4adjuvant. Linear B-cell epitope mapping of SEB was determined using an ELISAwith overlapping18-mer peptides. BSA and OVA192-201were the negative control, andnative SEB was the positive control. To statistically analyze the data, we used an unpairedStudent’s t-test in the software SPSS13.0.4. To confirm the reactivity of the immunodominant peptide specific antisera with SEB,BALB/c mice were immunized with KLH-conjugated immunodominant18-mer peptidesand the antisera were acquired after three immunization. Microtiter plates were coated withnative SEB. Then, all of the immunodominant18-mer peptide-specific antisera at thedifferent dilutions were detected using an ELISA.5. To finely map the immunodominant epitopes of SEB, peptides were graduallytruncated from either the carboxy or the amino terminal for each immunodominant peptide.All the truncated peptides were scanned using an ELISA with the diluted antisera from18-mer peptide-immunized mice. Further,18-mer peptide-antisera were diluted at differenttiters, and the truncated peptides with strongest responses to corresponding peptide antiserawere once again scanned using an ELISA, which confirmed the core sequences of theimmunodominant epitopes of SEB.6. To analyze the function of these epitopes, we designed the animal model for immunodominant epitope core sequence antisera for the in vitro SEB neutralizationexperiment.The anti-immunodominant epitope sera were produced in BALB/c mice bysubcutaneous injection of KLH-conjugated core sequence of the epitope in Freund’sadjuvant. The immune-serum was collected seven days after the final boost injection. Tnvitro, the ability of the core sequence antisera to inhibit SEB-induced T-cell proliferationand IL-2, IFN-γ and TNF-α production was examined.7. To investigate the conservation of the immunodominant epitopes of SEB amongdifferent S. aureus strains, SEB sequences from different S. aureus strains were retrievedfrom the Uniprot database, a sequence alignment of the corresponding regions on SEB wasperformed for alignment on NCBI internet. The3D structure of SEB was downloaded fromPubMed protein database. Immunodominant epitopes were mapped on the3D structure ofSEB using the PyMOL1.1program.8. To preliminarily evaluate the applications of the immunodominant epitopes of SEBin human body, the cross reactivities of these epitopes with the clinical MRSA (+) humansera were determined by an ELISA. Microtiter plates were coated with theimmunodominant epitopes of SEB. SEB antiserum was the positive control, and the clinicalserum without SEB specific antibody was the negative control.II. Preparation and protective function analysis of multiple-epitope vaccine ofSEB1. We constructed the prokaryotic expression vector which contained three dominantand three subdominant B cell epitopes of SEB by genetic engineering technology. Then therecombinant multiple-epitope fusion protein were expressed in Escherichia coli BL21(DE3).The expression form of the recombinant protein was determined by SDS-PAGE. Theimmunogenicity and immune reactivity of the recombinant protein was analyzed byWestern Blot. The protein was purified by Q Sepharose High Performance. The N terminalamino acids of the protein were sequenced. The concentration of the protein wasdetermined by BCA assay.2. To determine the survival rates of the immunized mice after S. aureus infection, weutilized the SEB multiple-epitope fusion protein or rSEB and an AlPO4adjuvant or Freundadjuvant to immunize BALB/c mice. Two weeks after the last immunization, BALB/c micewere intravenously injected with MRSA252. The survival rates of the infected mice were monitored for14days after infection.3. We utilized an ELISA to assess the titer of the immune sera. To evaluate the role ofthe SEB multiple-epitope fusion protein specific antisera in SEB neutralization, theanti-SEB IgG was analyzed by ELISA. To assess the specific antibody response to thesingle18-peptide, microtiter plates were coated with the single18-peptide, respectively,then the antisera were detected by ELISA.4. For the evaluation of the bacterial colonization in organs of mice after MRSAinfection, the heart, liver, lung and kidney of the mice were aseptically collected. Bacteriasuspensions of these organs was diluted with physiological saline at different dilutions, thenwere coated on the culture plates. For calculating the bacteria of each organs, colonycounting, Gram staining and PCR method were utilized.Results:I. Identification and functional analysis of the immunodominant B cell epitopes ofSEB1. The SEB mutant (rSEB) did not possess SEB-induced superantigen activity inBALB/c mice lymphocyte cultures. rSEB lost the ability of SEB-induced T cell mitogenesisand IL-2, IFN-γ and TNF-α production in splenic lymphocytes of BALB/c mice.2.80%of mice that were immunized with rSEB plus AlPO4adjuvant survived withoutclinical signs, and the survival rate of these mice was higher than that in the AlPO4adjuvantalone group (20%) and higher than that in the PBS group (none survived).The vaccinationof rSEB plus AlPO4adjuvant induced a significant antibody response that was specific tothe native SEB in mice. The mean antibody titer in the mice immunized by rSEB plusAlPO4adjuvant was up to1:64000higher than that in the AlPO4adjuvant or than that in thePBS alone group.3. The strongest IgG antibody reactivity was concentrated on three majorimmunodominant peptides: SEB97-114(NKNIDLFGTNYYYQCYFS), SEB205-222(YETGYIKFIEGNGHSFWY) and SEB247-261(VESKSINVEVHLTKK) of the SEB antigen(Figure2A). Because the regions SEB97-114and SEB247-261of SEB have been previouslyreported to contain the B cell epitopes SEB252-261(INVEVHLTKK) and SEB96-103(KNKNIDLF), respectively, SEB205-222might contain a novel linear B-cell epitope of SEB.4. Antibodies against three SEB immunodominant peptides showed stronger reactivity with SEB than with normal sera at different dilutions (P＜0.05), particularly at the1:1000and1:2000dilutions (P＜0.01).5. By truncated-peptide ELISA, among the truncated peptides, SEB97–112had thestrongest response to the SEB97–114antiserum, SEB205–222had the strongest responseto the SEB205–222antiserum, and SEB247–261had the strongest response to theSEB247–261antiserum. Antisera titration assay further confirmed that the core sequence ofSEB97–114is SEB97-112, the core sequence of SEB207-222is SEB205-222, and the core sequenceof SEB247-257is SEB247-261.6. We acquired all of the immunodominant epitope-specific antisera (anti-SEB97-112,anti-SEB207-222and anti-SEB247-257) in BALB/c mice model. All of the immunodominantepitope-specific antisera had the ability to inhibit SEB-induced T-cell proliferation andcytokine production from splenocytes of BALB/c mice.7. The homology analysis indicated that SEB97–112and SEB207-222were well-conservedamong different S. aureus strains, whereas SEB247-257was not, which indicated thatSEB97–112and SEB207-222might be good candidate molecules for vaccines that are based onSEB. SEB97–112was in the loop region of SEB whereas SEB207-222and SEB247-257were inthe β-slice region of SEB in the3D crystal structure.8. SEB97-112had the cross reactivity with14of14clinical MRSA (+) human sera,SEB207-222had the cross reactivity with13of14clinical MRSA (+) human sera, andSEB247-257had the cross reactivity with14of14clinical MRSA (+) human sera.II. Preparation and protective function analysis of multiple-epitope vaccine ofSEB1. We constructed the prokaryotic expression vector which contained three dominantand three subdominant B cell epitopes of SEB. The expression form of the recombinantmultiple-epitope fusion protein was inclusion body. After purification, the purity of themultiple-epitope fusion protein was more than95%. The multiple-epitope fusion proteinhad good immunogenicity and immune reactivity. The N terminal amino acids of theprotein were the same as those of the target protein. The concentration of the protein was2.924mg/mL.2. We utilized the multiple-epitope fusion protein and an AlPO4adjuvant or Freundadjuvant to immunize BALB/c mice, the mice obtained the protection against infection ability of MRSA252after three times of immunization. The survival of the mice immunizedby the multiple-epitope fusion protein vaccine was higher than that of the mice immunizedby rSEB vaccine, and particularly in the mice immunized by the multiple-epitope fusionprotein and AlPO4adjuvant, the survival was100%.3. The vaccination of SEB multiple-epitope fusion protein induced a significantantibody response, particularly when with AlPO4adjuvant assited, the titer was up to1:736000. The antisera of the SEB multiple-epitope fusion protein had strongly response tonative SEB. The level of the anti-SEB IgG was different when the fusion protein wasassisted with different adjuvants. Meantime, the antisera of the SEB multiple-epitope fusionprotein had response to the single18-mer peptides at different level. It is the synergisticresponse of all of the immunodominant epitopes that enhanced the protective immuneeffect.4. After the immunization and MRSA infection, MRSA252strains mainly colonized inkidney of the mice rather than the heart, liver, and lung. By statistical analysis, the survivalof all of the mice had negative correlation with the amount of MRSA252colonization in theorgans.Conclusion:We screened and identified three immunodominant B cell epitopes of SEB byoverlapping peptide ELISA, toxin activity neutralization test, bioinformatics analysis andclinical serological analysis. Of these three immunodominant B cell epitopes, SEB207-222isa novel epitope, SEB97-112and SEB247-257are the core sequence of the reported epitopes.Further, we constructed and acquired the SEB multi-epitope vaccine by genetic engineeringtechnology, then confirmed its protective role against MRSA infection and bacteriaclearance in mice model, which would provide new ideas and methods for the research ofMRSA vaccine.