Identification and Validation of a Linear Protective Neutralizing Epitope in the β-Pore Domain of Alpha Toxin

Abstract

The plethora of virulence factors associated with Staphylococcus aureus make this bacterium an attractive candidate for a molecularly-designed epitope-focused vaccine. This approach, which necessitates the identification of neutralizing epitopes for incorporation into a vaccine construct, is being evaluated for pathogens where conventional approaches have failed to elicit protective humoral responses, like HIV-1 and malaria, but may also hold promise for pathogens like S. aureus, where the elicitation of humoral immunity against multiple virulence factors may be required for development of an effective vaccine. Among the virulence factors employed by S. aureus, animal model and epidemiological data suggest that alpha toxin, a multimeric β-pore forming toxin like protective antigen from Bacillus anthracis, is particularly critical, yet no candidate neutralizing epitopes have been delineated in alpha toxin to date. We have previously shown that a linear determinant in the 2β2-2β3 loop of the pore forming domain of B. anthracis protective antigen is a linear neutralizing epitope. Antibody against this site is highly potent for neutralizing anthrax lethal toxin in vitro and for protection of rabbits in vivo from virulent B. anthracis. We hypothesized that sequences in the β-pore of S. aureus alpha toxin that share structural and functional homology to β-pore sequences in protective antigen would contain a similarly critical neutralizing epitope. Using an in vivo mapping strategy employing peptide immunogens, an optimized in vitro toxin neutralization assay, and an in vivo dermonecrosis model, we have now confirmed the presence of this epitope in alpha toxin, termed the pore neutralizing determinant. Antibody specific for this determinant neutralizes alpha toxin in vitro, and is highly effective for mitigating dermonecrosis and bacterial growth in a mouse model of S. aureus USA300 skin infection. The delineation of this linear neutralizing determinant in alpha toxin could facilitate the development of an epitope-focused vaccine against S. aureus.

Virulence gene profiles: alpha-hemolysin and clonal diversity in Staphylococcus aureus isolates from bovine clinical mastitis in China

“…In this study, the correlation between hla gene genotype/ peptides types and geography partly supported the above conclusion. In addition, variations in peptide sequences may influence Hla functionality and may change antigenic epitopes and potentially cause vaccine failure, as reported [ 15 , 16 ].…”

“…Staphylococcus aureus strain WOOD46 (GenBank accession no. X01645) was selected as the reference strain, as reported [ 13 , 16 , 25 – 27 ], and hla gene sequences were aligned using BioEdit v7.0.9 (Micro Focus, Newbury, UK) to designate genotypes. Corresponding peptide sequences were deduced and aligned to determine the presence of amino acid substitutions.…”

“…For example, a H35L substitution had no hemolytic or lethal activity, whereas a C259T substitution resulted in a premature stop codon and a significant reduction in Hla production [ 11 – 13 ]. Additionally, promising results have been obtained using Hla as a candidate for developing a vaccine to prevent S. aureus infections [ 14 – 16 ]. However, variation in Hla peptide sequences could affect vaccine efficacy.…”

The promise and challenge of epitope-focused vaccines

“…Yet immunization with epitope-focused immunogens was capable of eliciting protective levels of antibody against these cryptic epitopes in animal models of anthrax and S. aureus infections. 21,23,24 The identification of such immunorecessive epitopes has a parallel in the findings of the conserved, yet immunologically silent neutralizing epitopes in the stem region of HA, which one day may represent the focus of a new generation of influenza vaccines with the potential to obviate the requirement for annual influenza immunizations. 5,19 Like any new approach, however, many challenges remain in the development and ultimately, the application of epitopefocused vaccines.…”

“…Indeed, we identified one such cryptic epitope in PA in the anthrax model, and a second cryptic epitope in a hemolysin of S. aureus. 21,22 In both cases, immunization with the fulllength protein from which the epitopes were derived fails to elicit appreciable antibody to the neutralizing epitope in animal models, and for the PA epitope appears almost undetectable in human anthrax vaccinees as well. Yet immunization with epitope-focused immunogens was capable of eliciting protective levels of antibody against these cryptic epitopes in animal models of anthrax and S. aureus infections.…”