A novel Staphylococcus aureus vaccine: iron surface determinant B induces rapid antibody responses in rhesus macaques and specific increased survival in a murine S. aureus sepsis model

Abstract

Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans.

FIG. 1.

Improved survival of BALB/c and ICR mice immunized with IsdB and challenged with S. aureus Becker. (A, B, and C) Survival curves for three independent experiments performed with 20 to 30 BALB/c mice per group. (D, E, and F) Survival curves for three independent experiments performed with 10 to 20 ICR mice. •, survival of mice immunized with IsdB-APA; ○, survival of mice immunized with AAHSA.

FIG. 2.

Correlation of the survival of S. aureus lethal challenge to IsdB serum titer in IsdB- and sham-immunized mice (n = 160). The data are presented as a reverse cumulative distribution-frequency plot. IsdB serum titers from 160 mice were used for this plot; 120 of the serum samples were from mice immunized with IsdB (circles), and 40 were from naïve adjuvant-immunized mice (triangles and horizontal lines). All serum samples were diluted 1:4,000 and analyzed by ELISA. The x axis indicates the optical density measured at A₄₅₀, and the y axis indicates the percent probability of survival of mice with a specific optical density value. Open circles indicate immunized mice that survived the challenge (n = 65), and filled circles indicate immunized mice that succumbed to infection (n = 55). Likewise, mice that survived in the sham-immunized group are plotted as triangles (n = 13), and mice that died are plotted as horizontal lines (n = 27).

FIG. 3.

Improved survival of mice immunized with three doses of IsdB and challenged with diverse clinical isolates of S. aureus. (A) S. aureus ME60 (median lethal challenge dose, 3.1 × 10⁸ CFU); (B) S. aureus MCL8538 (median lethal challenge dose, 2.9 × 10⁸ CFU); (C) S. aureus ME27 (median lethal challenge dose, 1.0 × 10⁸ CFU; (D) S. aureus ME31 (median lethal challenge dose, 1.6 × 10⁸ CFU); (E) S. aureus ME11 (median lethal challenge dose, 2.1 × 10⁸ CFU). •, survival of mice immunized with IsdB-AAHSA; ○, survival of mice immunized with AAHSA.

FIG. 4.

Surface expression of IsdB by S. aureus Becker grown under different conditions. The histograms indicate the IsdB-specific fluorescence intensities (solid line) and the baseline signals obtained with the second antibody only (dotted line). IsdB expression was measured using murine IsdB immune serum (A) and an IsdB-specific monoclonal antibody (B to F). Cultures were grown overnight in vitro in RPMI 1640 (RPMI) (A and B) or RPMI 1640 supplemented with 20 μM FeSO₄ (Fe⁺⁺) (C), 10 μM heme (Heme) (D), or 20 μM FeCl₃ (Fe⁺⁺⁺) (E). In vivo cultures were generated from dialysis tubes inoculated with S. aureus cells and implanted into rats (F).

FIG. 5.

Lack of IsdB surface expression on S. aureus Becker isdB harA as measured by flow cytometry. (A) IsdB-specific monoclonal antibody staining of S. aureus Becker isdB harA. (B) IsdB-specific monoclonal antibody staining of S. aureus Becker wild type. (C) Convalescent S. aureus mouse serum used for staining S. aureus Becker isdB harA. (D) Convalescent S. aureus mouse serum used for staining wild-type S. aureus Becker. The thick solid line indicates the results for IsdB-specific antibodies, the dotted line shows the results for naïve serum, and the thin solid line shows the results for the second-stage control (without primary antibody). Ab, antibody.

FIG. 6.

Protection is specific for an immune response against surface-expressed IsdB: survival of mice immunized three times with 20 μg of IsdB formulated with AAHSA after challenge with S. aureus Becker isdB harA (n = 20 for the IsdB- and AAHSA-immunized groups; n = 10 for the naïve group). Panels A and B show the results for two independent experiments. •, IsdB-AAHSA-immunized mice; □, naïve mice; ○, AAHSA-immunized mice.

FIG. 7.

Anamnestic and long-lasting response in rhesus macaques immunized with IsdB. (A) Geometric mean titer (GMT) obtained for the rhesus macaque immunization group (n = 3). •, IsdB-AAHSA-immunized rhesus macaques; ○, AAHSA-immunized rhesus macaques. The immunization schedule is indicated by open triangles. (B) End point titer (EPT) range obtained for each group at each time. *, vaccine administration.