Immunisation With Immunodominant Linear B Cell Epitopes Vaccine of Manganese Transport Protein C Confers Protection against Staphylococcus aureus Infection

Abstract

Vaccination strategies for Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA) infections have attracted much research attention. Recent efforts have been made to select manganese transport protein C, or manganese binding surface lipoprotein C (MntC), which is a metal ion associated with pathogen nutrition uptake, as potential candidates for an S. aureus vaccine. Although protective humoral immune responses to MntC are well-characterised, much less is known about detailed MntC-specific B cell epitope mapping and particularly epitope vaccines, which are less-time consuming and more convenient. In this study, we generated a recombinant protein rMntC which induced strong antibody response when used for immunisation with CFA/IFA adjuvant. On the basis of the results, linear B cell epitopes within MntC were finely mapped using a series of overlapping synthetic peptides. Further studies indicate that MntC113-136, MntC209-232, and MntC263-286 might be the original linear B-cell immune dominant epitope of MntC, furthermore, three-dimensional (3-d) crystal structure results indicate that the three immunodominant epitopes were displayed on the surface of the MntC antigen. On the basis of immunodominant MntC113-136, MntC209-232, and MntC263-286 peptides, the epitope vaccine for S. aureus induces a high antibody level which is biased to TH2 and provides effective immune protection and strong opsonophagocytic killing activity in vitro against MRSA infection. In summary, the study provides strong proof of the optimisation of MRSA B cell epitope vaccine designs and their use, which was based on the MntC antigen in the development of an MRSA vaccine.

Fig 1. The survival rates and antibody titres of rMntC vaccine.

The survival rates of rMntC vaccine (with CFA/IFA as adjuvant), CFA/IFA adjuvant alone, and histidine buffer as control, challenged for BALB/c mice (n = 10) by S. aureus strains MRSA252 (A). The titres were determined by ELISA for IgG (B). The survival analyses and comparison of rMntC vaccine and adjuvant control were calculated separately by a log rank test. The asterisks represent a statistically significant difference (**P < 0.01, ***P < 0.001).

Fig 2. ELISA detection of B cell epitope mapping of MntC with antiserum of immunised and infected BALB/c mice.

To determine the immunodominant peptides of MntC, microtitre plates were coated with synthetic overlapping peptides that spanned the entire length of the MntC of MRSA252 or GST141-158 (negative control peptides) or MntC protein and BSA. B cell epitope mapping of MntC using an overlapping 18-mer peptide ELISA with antiserum samples from BALB/c mice that were immunised with rMntC plus CFA adjuvant (A) and antiserum samples from BALB/c mice that were infected after immunisation with rMntC plus CFA adjuvant (B) were detected. The absorbances read at 450 nm for peptides MntC113-136, MntC209-232, and MntC263-286 were significantly higher than BSA (P < 0.05) and higher than GST141-158 (P < 0.01).

Fig 3. Sequence alignment and localisation of immunodominant epitopes on MntC.

Sequence alignment of immunodominant peptides of MntC demonstrated that the amino acids of MntC113-136, MntC209-232, and MntC263-286 of MntC were wholly conserved in all S. aureus strains (A). Localisation of immunodominant peptides MntC113-136, MntC209-232, and MntC263-286 on the 3-d crystal structure of MntC. MntC113-136, MntC209-232, and MntC263-286 are shown in red, magenta, and orange respectively (B).

Fig 4. The survival rates of rMntC and peptides vaccine.

The survival rates of rMntC and conjunct peptides with KLH vaccine (with CFA/IFA as adjuvant), and CFA/IFA adjuvant alone as control, challenged for BALB/c mice (n = 10) by S. aureus strains MRSA252. The immunized dose of peptides vaccine was 40μl(100μg peptides-KLH) with 40μl CFA/IFA adjuvant and the mixture polypeptides vaccine was prepared with equal distribution of all three immunodominant epitopes (33.3μg/peptide-KLH) with 40μl CFA/IFA adjuvant. The survival rate comparison of rMntC and polypeptide vaccine were insignificant, but survival analyses and comparison of polypeptide vaccine and adjuvant control were significant when assessed by log rank test (P < 0.01).

Fig 5. Antibody titres of immunisation with rMntC, peptide vaccine, and controls.

The titres were determined by ELISA for IgG (A), IgG1 (B), IgG2a (C), and IgG2b (D). The results represent the means and standard error for a group of mice and the significance was measured by a log rank test (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig 6. OPKA of antisera to peptides (Anti-peptides) or rMntC (anti-rMntC) against MRSA252.

2 × 10⁶ CFU S. aureus strains were incubated in the presence of 2 × 10⁶ HL60 cells and mouse antisera against rMntC and peptide vaccine in the presence of infant rabbit complement. A percentage of MRSA252 was killed in the opsonophagocytic assay. Bars represent percentage killing compared with NMS represented as a mean of quadruplicate samples with SEM. NMS: no mouse antisera. No complement: no infant rabbit complement.