Mucosal immunization with a Staphylococcus aureus IsdA-cholera toxin A2/B chimera induces antigen-specific Th2-type responses in mice

Abstract

Staphylococcus aureus is a leading cause of opportunistic infection worldwide and a significant public health threat. The iron-regulated surface determinant A (IsdA) adhesin is essential for S. aureus colonization on human nasal epithelial cells and plays an important role in iron acquisition and resistance to human skin defenses. Here we investigated the murine immune response to intranasal administration of a cholera toxin A(2)/B (CTA(2)/B) chimera containing IsdA. Plasmids were constructed to express the IsdA-CTA(2)/B chimera and control proteins in Escherichia coli. Proper construction of the chimera was verified by SDS-PAGE, Western blotting, GM1 enzyme-linked immunosorbent assay (ELISA), and confocal microscopy. Groups of female BALB/c mice were mock immunized or immunized with IsdA-CTA(2)/B, IsdA mixed with CTA(2)/B, or IsdA alone, followed by one booster immunization at 10 days postpriming. Analysis of serum IgG and nasal, intestinal, and vaginal IgA suggested that mucosal immunization with IsdA-CTA(2)/B induces significant IsdA-specific humoral immunity. Functional in vitro assays revealed that immune serum significantly blocks the adherence of S. aureus to human epithelial cells. Splenocytes from mice immunized with IsdA-CTA(2)/B showed specific cellular proliferation and production of interleukin-4 (IL-4) after in vitro stimulation. Immunization with IsdA-CTA(2)/B drove isotype switching to IgG1, indicative of a Th2-type response. Our results suggest that the immunogenicity of the S. aureus IsdA-CTA(2)/B chimera merits further investigation as a potential mucosal vaccine candidate.

Fig. 1.

Expression and purification of the IsdA-CTA₂/B chimera and control proteins. (A) Structure and operon organization of pBA001 for IsdA-CTA₂/B expression. isdA was amplified from MRSA252 and cloned into pARLDR19, which directs the IsdA-CTA₂ and CTB peptides to the periplasm of E. coli for holotoxin assembly. (B) SDS-PAGE analysis of flowthrough (FT), washes (W1 and W2), and elution (E) of IsdA-CTA₂/B from d-galactose affinity purification and anti-CTA/B Western blot of purified IsdA-CTA₂/B (∼38 and 11 kDa). (C) SDS-PAGE analysis of proteins used in vaccination studies: IsdA-CTA₂/B chimera (G1), IsdA plus CTA₂/B mixed (G2), IsdA (G3), and anti-His₆ Western blot of purified IsdA used in G2 and G3 (∼37 kDa). α, anti.

Fig. 2.

Characterization of IsdA-CTA₂/B in vitro. (A) Ganglioside GM1 ELISA using anti-CTA and anti-CTB comparing the receptor binding of IsdA-CTA₂/B chimera and native CT. Error bars are based on triplicates, and results are representative of three independent assays. (B) Confocal microscopy of IsdA-CTA₂/B binding and transport into Vero and DC2.4 cells using polyclonal anti-CT and anti-rabbit-FITC with DAPI. Cells were incubated with IsdA-CTA₂/B for 45 min at 4°C to inhibit, or at 37°C to promote, cellular uptake. FITC/DAPI channel overlay is shown, and results are representative of three independent experiments.

Fig. 3.

Systemic antibody response to IsdA-CTA₂/B in vivo. IsdA-specific IgG ELISA endpoint titers from days 10, 14, and 45 sera pooled by immunization group (n = 6). Significance (P < 0.05) between mice immunized with IsdA-CTA₂/B versus controls (*) is shown, and error bars are based on assays performed in triplicate.

Fig. 4.

Mucosal antibody response to IsdA-CTA₂/B in vivo. Percent IsdA-specific IgA out of total IgA in day 45 nasal (1:2), intestinal (1:8), and vaginal (1:16) wash pooled by immunization group (n = 6). Significance (P < 0.05) between mice immunized with IsdA-CTA₂/B versus controls (*) is shown, and error bars are based on assays performed in triplicate.

Fig. 5.

Analysis of IsdA-specific cellular proliferation in mock-immunized mice or mice immunized with IsdA-CTA₂/B chimera, IsdA plus CTA₂/B mixed, or IsdA. (A) CFSE-labeled splenocytes were cultured in vitro for 84 h with IsdA, stained with anti-CD3-PE-Cy5, and analyzed by flow cytometry. The plots show a representative experiment; CFSE gates were set at the undivided peak of nonstimulated cells to determine proliferation of stimulated CD3⁺ cells. (B) Percent proliferation of IsdA-specific CD3⁺ T lymphocytes from individual mice on day 45 as determined by flow cytometry. Significance is based on n = 6. (C) Resazurin assay of splenocytes from days 14 and 45 cultured in vitro for 84 h with IsdA. The stimulation index equals the ratio of fluorescence of stimulated to nonstimulated cells. Error bars are based on n = 2 (day 14) or n = 6 (day 45). Significance (P < 0.05) between mice immunized with IsdA-CTA₂/B versus controls (*) is shown.

Fig. 6.

Cytokine and IgG subclass profiles of mock-immunized mice or mice immunized with IsdA-CTA₂/B chimera, IsdA plus CTA₂/B mixed, or IsdA. (A) IL-4 and IFN-γ levels in culture supernatants from splenocytes, pooled by immunization group (n = 6), stimulated in vitro for 84 h with IsdA were measured by ELISA. Significance (P < 0.05) between mice immunized with IsdA-CTA₂/B chimera versus controls (*) is shown. Error bars are based on assays performed in triplicate, and results are representative of two independent assays. (B) IsdA-specific IgG1 and IgG2a ELISA titrations from day 45 sera pooled by immunization group (n = 6). Significance (P < 0.05) between mice immunized with IsdA-CTA₂/B chimera versus IsdA (+) and mock (#) is shown, and error bars are based on assays performed in triplicate.

Fig. 7.

Effect of immune serum on S. aureus adhesion to human epithelial cells in vitro. Sera (1:100; day 45) were pooled by immunization group and incubated with MRSA252 (5 × 10⁷ CFU) (A) or MRSA USA300 (5 × 10⁹ CFU) (B) for 1 h at 37°C and then added to confluent HeLa cells. After washing and lysis, the number of internalized and cell-bound bacteria was enumerated. Error bars are based on three (A) or two (B) independent experiments. Significance (P < 0.05) between mice immunized with IsdA-CTA₂/B chimera versus controls (*) is shown.