Novel structurally designed vaccine for S. aureus α-hemolysin: protection against bacteremia and pneumonia

Abstract

Staphylococcus aureus (S. aureus) is a human pathogen associated with skin and soft tissue infections (SSTI) and life threatening sepsis and pneumonia. Efforts to develop effective vaccines against S. aureus have been largely unsuccessful, in part due to the variety of virulence factors produced by this organism. S. aureus alpha-hemolysin (Hla) is a pore-forming toxin expressed by most S. aureus strains and reported to play a key role in the pathogenesis of SSTI and pneumonia. Here we report a novel recombinant subunit vaccine candidate for Hla, rationally designed based on the heptameric crystal structure. This vaccine candidate, denoted AT-62aa, was tested in pneumonia and bacteremia infection models using S. aureus strain Newman and the pandemic strain USA300 (LAC). Significant protection from lethal bacteremia/sepsis and pneumonia was observed upon vaccination with AT-62aa along with a Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE) that is currently in clinical trials. Passive transfer of rabbit immunoglobulin against AT-62aa (AT62-IgG) protected mice against intraperitoneal and intranasal challenge with USA300 and produced significant reduction in bacterial burden in blood, spleen, kidney, and lungs. Our Hla-based vaccine is the first to be reported to reduce bacterial dissemination and to provide protection in a sepsis model of S. aureus infection. AT62-IgG and sera from vaccinated mice effectively neutralized the toxin in vitro and AT62-IgG inhibited the formation of Hla heptamers, suggesting antibody-mediated neutralization as the primary mechanism of action. This remarkable efficacy makes this Hla-based vaccine a prime candidate for inclusion in future multivalent S. aureus vaccine. Furthermore, identification of protective epitopes within AT-62aa could lead to novel immunotherapy for S. aureus infection.

Figure 1. Structural analysis of Hla.

(A) The relative topology of 1–62 and 1–62(GGG)–(223–236) AT constructs on the protein surface of a subunit from the 7AHL heptameric hemolysin crystal structure. The protein surface for the 1–62 segment is colored green, the 223–236 sequence colored dark green, and the remaining structure colored purple. (B) Topology of the secondary structural elements in α-hemolysin for peptide segments examined in this study.

Figure 2. SDS-PAGE and Western blot analysis of purified Hla constructs.

(A) SDS-PAGE. Lane 1: Biorad pre-stained protein standard; Lane 2: AT-50aa; Lane 3: AT-62aa; Lane 4: AT-79aa. All proteins were loaded at 1 µg/ lane and stained with Coomassie blue. (B) Western Blot. Lane 1: Biorad pre-stained protein standard; Lane 2: AT-50aa; Lane 3: AT-62aa; Lane 4: AT-79aa; Lane 5: Full length alpha toxin. All proteins were detected by anti-alpha toxin mAb (6C12) generated against AT-79aa construct.

Figure 3. Comparative antibody response to vaccine candidates.

(A) Individual serum antibody titers towards wild-type Hla determined after three immunizations (B) Neutralization titers of pooled sera towards wild-type Hla after three immunizations.

Figure 4. Comparative efficacy study of vaccine candidates in S. aureus bacteremia and pneumonia infection models.

Survival of mice vaccinated with the three vaccine candidates and control mice after IP challenge with 5×10⁴ CFU of USA300 along with Hog Mucin (A) or IN challenge with 6×10⁷ CFU of S. aureus strain Newman (B). Survival of mice vaccinated with AT-62aa and challenged IN with 1.5×10⁸ CFU of S. aureus USA300 (C). Symbol key: AT-50aa (open circle), AT-62aa (black square), AT-79aa (open triangles) and mock-immunized mice (grey diamond). Statistical analysis was performed using Log-Rank (Mantel-Cox) test.

Figure 5. Passive protection with rabbit polyclonal AT-62aa (AT62-IgG) in bacteremia model.

Protection from lethal challenge with S. aureus USA300 (A) or USA400 (B) after passive immunization with polyclonal rabbit antibodies AT62-IgG (black square) compared to mock-treated mice (grey diamond). Statistical analysis was performed using Log-Rank (Mantel-Cox) test, P<0.0001.

Figure 6. Passive protection against bacterial dissemination in bacteremia model.

Bacterial burden in organs and blood was determined after passive immunization with polyclonal AT62-IgG or naïve IgG followed by IP infection with S. aureus USA300 in 3% Hog mucin. Statistical analysis was performed with Mann-Whitney Test with two-tailed P value (P<0.0001 for all tested CFU).

Figure 7. Passive protection against bacterial dissemination in pneumonia model.

Bacterial burden in organs and blood was determined after passive immunization with polyclonal AT62-IgG or naïve IgG followed by IN infection with S. aureus USA300. Mann-Whitney Test with two-tailed P value (P values as indicated in figures).

Figure 8. Neutralization of Hla with AT62-IgG.

100 ng of purified alpha toxin was pre-incubated at RT with different concentration of polyclonal antibody ranging from 500 µg/ml to 0.5 µg/ml and then incubated with 2% rabbit blood at 37°C for 30 min. The suspension was centrifuged and hemolysis was measured at OD_{416 nm}.

Figure 9. Inhibition of toxin oligomerization with AT62-IgG.

Rabbit RBCs were incubated with Hla alone or Hla pre-incubated with pAb. The mixtures were incubated with 10% rabbit RBC for 45 min at 37°C, cells were pelleted, washed, lysed, and loaded in SDS-PAGE without heating. Lane 1: boiled; lane 2 at 4°C, lane 3: Hla control without RBC; lanes 4–10: 15 µg/ml of Hla neutralized with decreasing concentration of anti AT-62aa pAb (two fold diluted from 400 to 6.25 ug/ml). Western blot was developed with sheep anti-Hla polyclonal antibody.