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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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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.
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pone-0038567-g009: 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.

Mentions: Formation of Hla heptamers upon binding to cell surface receptor is a key event in pore formation and subsequent lysis of target cells [26]. Given the role of the N-terminal domain, we hypothesized that antibodies to AT-62aa interfere with the formation of Hla heptamers and consequently inhibit target cell lysis. To examine this hypothesis, we tested the effect of AT62-IgG on heptameric Hla (Hla7) formation in a Western blot assay. Hla was incubated with or without increasing concentrations of the pAbs before incubating the mixture with RBCs. The cells were pelleted, washed, lysed, and subjected to Western blotting without prior boiling. AT62-IgG effectively prevented the formation of the heptameric Hla structure in a dose dependent manner (Figure 9). Taken together, these data suggest that antibodies to AT-62aa neutralize the activity of Hla by binding to the N-terminal domain by preventing oligomerization and the subsequent pore formation.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038567-g009: 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.
Mentions: Formation of Hla heptamers upon binding to cell surface receptor is a key event in pore formation and subsequent lysis of target cells [26]. Given the role of the N-terminal domain, we hypothesized that antibodies to AT-62aa interfere with the formation of Hla heptamers and consequently inhibit target cell lysis. To examine this hypothesis, we tested the effect of AT62-IgG on heptameric Hla (Hla7) formation in a Western blot assay. Hla was incubated with or without increasing concentrations of the pAbs before incubating the mixture with RBCs. The cells were pelleted, washed, lysed, and subjected to Western blotting without prior boiling. AT62-IgG effectively prevented the formation of the heptameric Hla structure in a dose dependent manner (Figure 9). Taken together, these data suggest that antibodies to AT-62aa neutralize the activity of Hla by binding to the N-terminal domain by preventing oligomerization and the subsequent pore formation.

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