Preparation Of The Subunit Vaccine Of The Recombinant Staphylococcus Aureus MntC And Functional Study Of Its Immunodominant Epitope

Objectives:Staphylococcus aureus(S. aureus) is an opportunistic bacterial pathogen, the infection of which can lead to a diverse range of human skin infections and lifethreatening diseases, such as bacteraemia, pneumonia, endocarditis, osteomyelitis, sepsis, and wound infections. These diseases are associated with a high rate of morbidity and mortality,which imposes an increasingly high burden on health care resources. However, current clinical efforts fail to control MRSA infection, for example, antibiotic therapy, and there is no effective S. aureus vaccine that has been successfully developed to resist S.aureus infection, up to now.Manganese is an important metal ion for many pathogens. The uptake of manganese by S. aureus is through the manganese transport protein complex, which is mainly a manganese binding surface lipoprotein(MntC). MntC is essentially a metal-binding protein, which has been shown to confer protective immunity in animal model systems of S.aureus infections. In addition, anti-MntC monoclonal antibodies have been identified as binding to S. aureus cells.Therefore, MntC might be a potential therapeutic target for antibiotics research, and also be a potential antigen for S. aureus vaccines.Antibody response is reported as the major specific immunity against MRSA(Methicillin-resistant Staphylococcus aureus) infection. In this study, we found that immunised purified MntC protein is responsible for eliciting anti-MntC IgG immune responses just as an immunotherapeutic agent, and that it effectively increased the survival rates of BALB/c in MRSA systemic infection mouse model, in which MntC probably functioned through its B cell immunodominant epitopes. However, the particular detailed epitope-mapping and protective mechanism of the potential humoral immune response of MntC antigen remain unclear, further the realization of an epitope-vaccine in MRSA infection remains a challenge. Therefore, screening and identification immuno-dominant epitopes of Staphylococcus aureus will be helpful for understanding of MntC in protection of Staphylococcus aureus infection and optimizing the key mechanisms of MntC antigen vaccine. At present, the immunodominant epitopes of MntC in Staphylococcus aureus epitope vaccine studies have never been reported. Therefore, in this study, we analyzed the protective ability of MntC protein in the mouse infection model by clinical MRSA252 strains, and also tested the immune antibody and cytokine expression levels in this model. Further, we screened and identified the immune advantage of linear B-cell epitopes of MntC; we also analyzed the function of MntC-epitope vaccine based on antibody response.Methods:Ⅰ. Function of Staphylococcus aureus protein MntC Construction and expression of protective immunity and vaccine MntC1. To acquire recombinant MntC protein,we empolyed bioinformatics methods and amino acid sequence information to find MntC Staphylococcus aureus protein gene, and then expressed the plasmid containing the whole protein MntC gene sequence based on the use of genetic engineering techniques by pGEX-6p-2 vector system in E. coli BL21(DE3); expressed recombinant MntC protein(MntC) was identified by SDS-PAGE; MntC was purified firstly using MMC ion exchange chromatography, and then by Q HP removal filler endotoxin; the concentration of MntC was determined using the BCA assay.2. To evaluate MntC vaccine protective efficacy against S. aureus infection,the immune protection assay were carried out by MntC vaccine assisted with Freund’s adjuvant(CFA/IFA) and alum adjuvant(Alum) in systemic infection model MRSA252 strain challenge BALB/c mice, and the survival status of different groups of mice the vaccine was observed in 14 days after infection, then the survival rate of immunized mice was calculated,3. To asset the immune protective effect of MntC vaccine against Staphylococcus aureus infection, we selected three kinds of international standard strains and clinical isolates of S. aureus for infection. Alum adjuvant was used in immunized BALB/c mice. We established mouse infection systemic model, conducted various S.aureus strains challenge for 14 days, in which we observed the survival status and the survival rate of different groups of immune mice.4. To investigate the immune protective mechanism of MntC vaccine, after immunization with MntC protein vaccine assisted Freund’s adjuvant(CFA/IFA) and alum(Alum) adjuvant,we collected immunized mice serum, then detected the IgG, IgG1 and IgG2 a antibody expression level by ELISA; and collected the spleen cell suspension by surgery for intraocular spleens of vaccinated mice; after the splenocytes suspends were stimulated by the MntC protein, we detected the MntC specific cytokine secretion of splenocytes after immunization.Ⅱ. Identification B cell immunodominant epitope of S.aureus protein MntC and Function research of immune protection with epitope vaccine1.To immune mice, MntC vaccine with Freund’s adjuvant(CFA/IFA) were injected into BALB/c mice, after three immunizations, the mice immunized antiserum was collected; then the mice were challenged with Staphylococcus aureus strains MRSA252 and the sera were also collected.2. To identify the immunodominant peptides of MntC, we employed 46 synthesized 18-mer overlapping peptides, which spanned the entire length of the MntC, and two group antiserum samples were collected from BALB/c mice, one is that were immunized with MntC plus CFA/IFA adjuvant and the other is that infection with MRSA252 post immunized with MntC plus CFA/IFA adjuvant. With synthesized overlapping 18-mer peptides, Linear B-cell epitope mapping of MntC was screened using an ELISA. BSA and GST141-158 were the negative control, and MntC protein was the positive control. We used an unpaired Student’s t-test in the software SPSS 13.0 to analyze the data statistically,.3. The conservation of the immunodominant epitopes of MntC is important in the study of immunization response of the antigen, To investigate immunodominant epitopes among different S. aureus strains, we used the Uniprot database and retrieved these MntC sequences from different S. aureus strains, for analyzing a sequence alignment of the corresponding regions on MntC, with performing on NCBI internet for alignment. From PubMed protein database, we downloaded the 3D structure of MntC, and mapped the immunodominant epitopes on the 3D structure of MntC by the Py MOL 1.1 program.5. To further preliminarily evaluate the functions and applications of the immunodominant epitopes of MntC in immunise protection, Synthese identified uncoupling protein MntC three immunodominant epitope peptide, three kinds of protein immunodominant epitope in an equal volume of Freund’s adjuvant(CFA/IFA) adjuvant were mixed, with the whole protein and single MntC uncoupling protein species epitope peptide as a control, immunized BALB/c mice at day 14 after immunization were MRSA252 strain lethal dose of poison attack, the establishment of systemic mouse infection model, and within 14 days after infection survival status was observed in mice, calculate the rate of immune protection in mice.6. For the evaluation of the mechanism of the immunodominant epitopes of MntC, we detected the antibody subtype IgG1, IgG2 a, IgG2 b expression level using an ELISA assay. In addition,we investigated the antibody protection against strains of Staphylococcus aureus MRESA252 phagocytic killing capacity in vitro.Results:Ⅰ. Function of Staphylococcus aureus protein MntC Construction and expression of protective immunity and vaccine MntC1. MntC protein was acqiuired with the purity of over 95%. The recombinant protein MntC has good immunogenicity and immunoreactivity. The concentration of the protein was 1.6 mg/mL.2. The survival of the mice immunized by the r Mt C vaccine was higher than that of the mice immunized by Alum adjuvant control.3. The survival rate for GZ02 and CQ19 clinical Staphylococcus aureus isolates units both can reach about 60%, while immune protection rate of WHO2 and BJ03 strains only are 30% and 40%.4. The expression levels of serum IgG and IgG1 antibodies under Freund’s adjuvant(CFA/IFA) vaccine were significantly higher than alum adjuvant assisted MntC vaccine. The antibodies response reflects TH2 humoral immune sources, and the IgG2 a antibody expression level reflects the TH1 cellular immune response. Meanwhile, there is not significantly difference between the two adjuvant vaccine groups.5. After immunized, the expression levels of cytokines IL-5 in two vaccine group is significantly different, but the expression of cytokine IFN-γ and IL-17 A of the mouse spleen cells in Freund’s adjuvant(CFA/IFA) immune vaccine group are higher than that of the Alum adjuvanted vaccine group.Ⅱ. Identification of MntC of S.aureus protein B cell immunodominant epitope and Function of immune protection with epitope vaccine1. We screened and identified MntC113-136, MntC209-232 and MntC263-286 immunodominant linear B-cell epitope of S. aureus MntC antigens.2. The post-immunization antisera and infection antisera after immunization to further detected integration titer with various peptides, the results showed that IgG antibody titers have been identified in three immunodominant epitope MntC113-136, MntC209-232 and MntC263-286 may up to 1: 600 and 1: 1200, while other epitope peptide antibody response levels were 1: 100 or less.3. The amino acid sequences of three immunodominant epitopes MntC113-136, MntC209-232 and MntC263-286 was 100% conserved in S. aureus strains including MRSA252 and others 17 different strains. In the three-dimensional protein crystals structure of MntC, three immunodominant epitopes located in the surface of antigens structure, and antigens are immunogenic stimulation dominant area.4. The immune protection rate between epitope vaccine and whole MntC protein vaccines were 80% and 70% espectively, both significantly higher than the single epitope vaccine and adjuvant and the buffer control group.5. Immunodominant epitopes conjugate vaccine group and whole MntC protein vaccine group had higher Ig G, Ig G1 and Ig G2 b antibody expression levels, which seemed that TH2 stimulate humoral immune response.The titer of the rerum was up to 1: 1024000. In MntC protein vaccine group, the TH1 cellular immune response is greater than the IgG2 a antibody epitope vaccine group.6. The opsonophagocytic killing rate can reach up to 85% or more after MntC vaccination and during the antiserum of diluted at 1: 3, it’s the ability of opsonophagocytic of Staphylococcus aureus in vitro conditioning can still be maintained above 50%, much higher than that of the single epitope vaccine and adjuvant and the buffer control group.Conclusions:To elaborate further the humoral immune response of MntC antibody and characterize detailed Linear B cell antibody epitopes, the overlapping synthetic peptides were used to detect the MntC-specific antibodies in immunised MntC vaccinations with mice serum and MRSAinfected post MntC immunised mice serum, respectively. The linear B-cell epitopes of MntC were completely mapped, and the vaccine basis of immunodominant epitopes of MntC was evaluated. The conservation of all three immunodominant epitopes was then confirmed and located in a 3-D structural model of MntC. Furthermore, we evaluated the efficacy of the immune protection conferred by the immunodominant-epitope vaccine of MntC by using survival rates, antibody response, and opsonophagocytic activity of immunodominant-epitope peptides-specific antibody in vitro. Our findings authenticated MntC113-136, MntC209-232 and MntC263-286 as three immunodominant epitopes on the MntC of MRSA and confirmed that the vaccine with three epitope-peptides presented better protective efficacy in mice. Moreover, opsonophagocytic assays indicated that the epitope-vaccine specific Ig G was able to kill the S. aureus bacteria in vitro. These studies of MntC epitope will be helpful for understanding the humoral immunity response and epitope-vaccine will be alternative and promising in developing an MRSA vaccine.In this study, we use recombinant MntC protein preparation MntC vaccine in mice, MntC vaccine in mice infected with S. aureus MRSA252 systemic model has protective immunity; humoral immune response played a leading role in immune protection. Screened and identified MntC113-136, MntC209-232 and MntC263-286 the immunodominant epitope antigen Staphylococcus aureus MntC overlapping peptides by ELISA method, these epitopes are highly conserved in different strains of Staphylococcus aureus, and located on the surface of MntC, which is immunogenic antigen stimulation dominant area on the crystal structure of the protein. Synthesis of three epitopes of the protein and KLH conjugate prepared epitope vaccine, three immunodomiant epitopes vaccine was assesed with good immune protection rate in s.aureus MRSA252 systemic infection model mice. Epitope vaccine induced the expression of antibodies such as Ig G; specific antibodies also have a direct opsonophagocytic function in vitro to resist staphylococcal MRSA252 infection. Immunodominant epitope of MntC vaccine play an important role in the immune protection of S. aureus infection immunity, the present study provides a theoretical basis for the prevention and treatment of S. aureus infections, provide new ideas and design strategies for the development of staphylococcal vaccines.