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

Adhikari RP, Karauzum H, Sarwar J, Abaandou L, Mahmoudieh M, Boroun AR, Vu H, Nguyen T, Devi VS, Shulenin S, Warfield KL, Aman MJ - PLoS ONE (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Integrated Biotherapeutics Inc., Gaithersburg, Maryland, United States of America.

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.

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Related in: MedlinePlus

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.
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pone-0038567-g002: 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.

Mentions: Based on these predictions, we generated constructs for AT-50aa, AT-62aa, and AT-79aa with a C-terminal 6xHistidine tag. Proteins were expressed in E. coli and purified over a Ni column. Based on the molecular modeling study, we prepared constructs consisting of variable number of glycine amino acids, and determined that extension of the linker to five glycines resulted in the best protein expression, yield and purity for in vivo studies. Therefore, a five-glycine linker AT-79aa was used in the studies described here. Proteins were analyzed by SDS-PAGE and Western Blot (Figure 2) and tested for endotoxin content as described in the Materials and Methods section.


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

Adhikari RP, Karauzum H, Sarwar J, Abaandou L, Mahmoudieh M, Boroun AR, Vu H, Nguyen T, Devi VS, Shulenin S, Warfield KL, Aman MJ - PLoS ONE (2012)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368876&req=5

pone-0038567-g002: 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.
Mentions: Based on these predictions, we generated constructs for AT-50aa, AT-62aa, and AT-79aa with a C-terminal 6xHistidine tag. Proteins were expressed in E. coli and purified over a Ni column. Based on the molecular modeling study, we prepared constructs consisting of variable number of glycine amino acids, and determined that extension of the linker to five glycines resulted in the best protein expression, yield and purity for in vivo studies. Therefore, a five-glycine linker AT-79aa was used in the studies described here. Proteins were analyzed by SDS-PAGE and Western Blot (Figure 2) and tested for endotoxin content as described in the Materials and Methods section.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Integrated Biotherapeutics Inc., Gaithersburg, Maryland, United States of America.

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.

Show MeSH
Related in: MedlinePlus