Multiple B-cell epitope vaccine induces a Staphylococcus enterotoxin B-specific IgG1 protective response against MRSA infection

Abstract

No vaccine against methicillin-resistant Staphylococcus aureus (MRSA) has been currently approved for use in humans. Staphylococcus enterotoxin B (SEB) is one of the most potent MRSA exotoxins. In the present study, we evaluated the efficacy and immunologic mechanisms of an SEB multiple B-cell epitope vaccine against MRSA infection. Synthetic overlapping peptide ELISA identified three novel B-cell immunodominant SEB epitopes (in addition to those previously known): SEB31-48, SEB133-150, and SEB193-210. Six B-cell immunodominant epitopes (amino acid residues 31-48, 97-114, 133-150, 193-210, 205-222, and 247-261) were sufficient to induce robust IgG1/IgG2b-specific protective responses against MRSA infection. Therefore, we constructed a recombinant MRSA SEB-specific multiple B-cell epitope vaccine Polypeptides by combining the six SEB immunodominant epitopes and demonstrated its ability to induce a robust SEB-specific IgG1 response to MRSA, as well as a Th2-directing isotype response. Moreover, Polypeptides-induced antisera stimulated synergetic opsonophagocytosis killing of MRSA. Most importantly, Polypeptides was more effective at clearing the bacteria in MRSA-infected mice than the whole SEB antigen, and was able to successfully protect mice from infection by various clinical MRSA isolates. Altogether, these results support further evaluation of the SEB multiple B-cell epitope-vaccine to address MRSA infection in humans.

Figure 1. Staphylococcus enterotoxin B (SEB) B-cell epitope mapping.

B cell epitope mapping of SEB using an overlapping 18-mer peptide ELISA. To determine the immunodominant peptides of SEB, microtiter plates were coated with synthetic overlapping peptides that spanned the entire length of the SEB of MRSA252 or BSA and OVA_192–201 (negative control peptides). Then, sera samples from BALB/c mice that were directly infected with MRSA252, and that were immunised with rSEB plus AlPO₄ adjuvant before MRSA252 infection were detected (The antisera were diluted to 1:300). The absorbance was read at 450 nm. The raw O.D. values shown were obtained using serum (diluted by a factor of 1:300) from three independent experiments assayed concurrently. Data are represented as the means ± SEM. Probability values of p < 0.05 were considered significant and are denoted by an asterisk (*). **p < 0.01. (A) Antiserum samples from BALB/c mice immunised with rSEB plus AlPO₄ adjuvant following infection with MRSA252 were detected for the immunodominant response. (B) Antiserum samples from non-immunised BALB/c mice infected with MRSA252 were detected for the immunodominant response.

Figure 2. The protective role of the individual immunodominant SEB epitopes against MRSA252 challenge.

(A) Percent survival in mice immunised with individual epitopes plus AlPO₄ or CFA/IFA adjuvant, AlPO₄ or CFA/IFA adjuvant alone, or PBS alone. The significance of protective immunity generated by the individual epitope vaccine was measured with Fisher’s exact test: : for CFA/IFA adjuvanted vaccine, SEB_31–48-KLH (P = 0.011), SEB_97–114-KLH (P = 0.011), SEB_133–150-KLH (P = 0.033), SEB_193–210-KLH (P = 0.003), SEB_205–222-KLH (P = 0.087) SEB_247–261-KLH (P = 0.033); for AlPO₄ adjuvanted vaccine, SEB_31–48-KLH (P = 0.033), SEB_97–114-KLH (P = 0.087), SEB_133–150-KLH (P = 0.033), SEB_193–210-KLH (P = 0.011), SEB_205–222-KLH (P = 0.087), SEB_247–261-KLH (P = 0.087). (B) Antibody production of Polypeptides or the individual immunodominant epitope -vaccinated mice. ELISA detection of the individual immunodominant epitope specific antibody levels in mice immunised by the individual immunodominant epitope. (C) ELISA detection of Polypeptides specific antibody subtype in mice immunised with the individual epitopes. IgG subclass distribution in mice immunised with different formulations. Results are representative of three experiments and data are mean ± SD of samples from ten mice. (D) ELISA detection of SEB specific antibody subtype in mice immunised with the individual epitopes. IgG subclass distribution in mice immunised with different formulations. Results are representative of three experiments and data are mean ± SD of samples from ten mice.

Figure 3. The multiple epitope-vaccine induced protective response against MRSA252 infection in the immunised mice.

(A) Percent survival against MRSA252 infection in the immunised mice. (B) Bacterial burden in the organs of mice after challenge with MRSA 252. **P < 0.01(N = 10; independent experiments).

Figure 4. Histological analysis of MRSA challenged, Polypeptides-immunised mice revealed normal physiological architecture.

Hematoxylin and eosin staining of kidney sections at day 21 after infection. Microscopic images of kidneys, hearts and lungs, 200 × (top row) and 400 × (bottom row). Abscess formation or scattered colonies of bacteria were only found in mock-immunised animals (black arrow).

Figure 5. Polypeptides-specific antibody production after MRSA252 challenge.

(A) Antibody production in Polypeptides or rSEB-vaccinated mice. (B) Polypeptides-specific antibody isotype analysis in Polypeptides or rSEB-vaccinated mice. Results are representative of three experiments and data represent the mean ± SD of samples from ten mice. (C) The individual immunodominant epitope-specific antibody response following Polypeptides or rSEB vaccination.

Figure 6. Opsonophagocytosis stimulated by antisera against SEB_31–48, SEB_97–114, SEB_133–150, SEB_193–210, SEB_205–222, SEB_247–261, and Polypeptides.

Opsonsonophagocytic killing of MRSA252 in the presence of complement was significantly higher for Polypeptides antisera than for individual immunodominant epitope protein antisera (P < 0.05). Percent killing was defined as the reduction in CFU/mL after 2 h compared with that at time zero. Error bars represent the SEM.

Figure 7. Immunisation with Polypeptides generates protective immunity against lethal challenge with six different clinical MRSA isolates.

Survival of Polypeptides-immunised mice following challenge with clinical MRSA isolates (n = 20 for every isolate, of which n = 10 for Polypeptides-immunised mice and n = 10 for PBS-immunised mice). Compared with animals receiving mock-immunization (PBS), the significance of protective immunity generated by the combined vaccine was measured with Fisher’s exact test: (A) CQ19: P <  0.001; (B) SJZ30: P <  0.001; (C) BJ2: P =  0.005; (D) SJZ18: P =  0.003; (E) GZ9: P =  0.001; (F) MRSA252: P <  0.001.