Identification and validation of a linear protective neutralizing epitope in the β-pore domain of alpha toxin.

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

Alpha toxin and protective antigen share structural and functional homology but only limited sequence identity.

<p>Comparison of protein structural models of the heptameric AT (PDB7AHL) (A) and PA (PDB1V36) (B). Sequences in red represent aligned sequence shown in (C). Amino acid alignment of PA and AT in the region of the LND of PA demonstrates 37% sequence identity. Asterisks denote positions of amino acid identity, while periods and colons denote semi-conservative and conservative substitutions, respectively.</p

PND-specific Ab is a minor component of the repertoire of Abs elicited following AT immunization of rabbits and mice.

<p>(A) Two independent lots of commercially available antisera each representing pooled antisera from 2–3 rabbits immunized with full length AT, were evaluated for reactivity with immobilized AT or with peptides by ELISA. The geometric mean titer for antibody reactive with the 1–19 peptide sequence was significantly higher than the titers of Ab reactive with the 119–139 or irrelevant peptide sequences (*<i>p</i> = 0.0002, One way ANOVA; Tukey’s multiple comparison test: *<i>p</i><0.05, 1–19 vs. 119–139 and 1–19 vs Irrelevant). The titers of Ab reactive with the 119–139 and Irrelevant peptide sequences were not statistically different (B) Individual mouse antiserum obtained from mice immunized with H35L were evaluated for reactivity with immobilized AT or with peptides by ELISA. Geometric mean antibody titers specific for the 1–19 peptide sequence was significantly higher than the responses to either the 119–139 or irrelevant peptide sequences (<i>p</i> = 0.0001, One way ANOVA; Tukey’s multiple comparison test: *<i>p</i><0.05, 1–19 vs. 119–139 and 1–19 vs Irrelevant). The titers of Ab reactive with the 119–139 and irrelevant peptide sequences were not statistically different. Each immobilized MAP was validated with a control antiserum (not shown). Bars represent geometric means. The lower limit of quantitation for analysis of rabbit and mouse antibody titers were 16 and 4, respectively.</p

Antibody and TNA responses from rabbits immunized with MAP constructs displaying overlapping residues from AT.

<p>Groups of rabbits were immunized 4 times at two-week intervals with a mixture of two MAPs each containing the respective B cell target sequence (A) linked separately to the T* and P30 helper T cell epitopes. Ten days after the final immunization, rabbits were bled and sera were evaluated by ELISA for reactivity with immobilized full length AT (B) or the in the TNA (C). Bar charts represent arithmetic means. Error bars represent SEM. * <i>p</i> = 0.022, one-way ANOVA; <i>p</i>< 0.05, Tukey’s multiple comparison test: 119–139 vs all other groups.</p

Passive immunization of mice with AT-neutralizing antisera specific for a.a. 122–137 protects mice from dermonecrosis.

<p>Five C57BL/6 mice/group were passive immunized i.p. with 0.5 mls of rabbit serum specific for a.a. 122–137 of AT, or irrelevant control rabbit serum. One day later, all mice were challenged i.d. with 20 x 10⁶ CFUs of <i>S</i>. <i>aureus</i> 8325–4 and were followed for 48 hours. After 48 hours, all mice were euthanized and dermonecrotic lesion areas were photographed (A) and lesion areas were determined as described in <i>Material and Methods</i> and displayed graphically (B). Horizontal bars are geometric means. * <i>p</i> = 0.0037, Mann-Whitney U test, one-tailed.</p

The 122–137 peptide inhibits AT neutralization activity in the sera of rabbits immunized with MAP-119–139.

<p>Antiserum from each of 3 rabbits immunized four times with the MAP-119–139 shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116882#pone.0116882.g005" target="_blank">Fig. 5</a>, was preincubated with 32 μM of the linear peptide, a.a. 122–137 or a control linear peptide for 1 hour at RT prior to assessment in the TNA.</p

Antibody against a.a. 122–137 binds AT by ELISA and protects Jurkat T cells in vitro.

<p>Three rabbits were immunized four times with a MAP displaying a.a. 122–137 from AT in an emulsion with CFA for priming immunizations and IFA for booster immunizations. Approximately 10 days after the final immunization, rabbit were bled and sera was evaluated by ELISA for reactivity with full length AT (A) and in the TNA (B).</p

PND-specific Ab does not block binding of AT to RBC ghosts.

<p>RBC ghosts were immobilized on polystyrene 96 well plates and were blocked with 4% BSA in PBS. Soluble AT was incubated at RT with AT alone, or with AT mixed with identical concentrations of either PND-Ig or Control-Ig, or with rabbit anti-AT or sheep anti-AT at concentrations mediating neutralization equal to that of the PND-Ig. Binding of AT was detected using a mouse mAb specific for the N-terminus of AT as described in methods. *<i>p</i>< 0.0001 by ANOVA; <i>p</i>< 0.05, Tukey’s multiple comparisons test: Rabbit anti-AT and Sheep anti-AT vs both Rabbit anti-PND and Rabbit Control. Bars represent arithmetic means from quadruplicate samples and error bars indicate +/- 1 SEM. % Control AT binding = (OD₆₀₀ with Ab/OD₆₀₀ no Ab) x 100. The results are representative of 2 separate assays.</p

Rabbit-IgG specific for the 119–139 sequence from AT protects mice from LAC/USA300-mediated dermonecrosis.

<p>Groups of BALB/c mice (n = 5 except control IgG, n = 7) were passively immunized s.c. with normalized volumes of the respective affinity purified rabbit sera to establish approximately equivalent serum antibody titers in recipient mice. Approximately 48 hours later, all mice were bled and individual mouse sera were evaluated by ELISA for reactivity with immobilized AT (A) and in the TNA (B). All groups were then challenged i.d. with 38 x 10⁶ LAC/USA300. Forty-eight hours later, all mice were euthanized, lesions were photographed (C) and lesion areas determined (D). Mice passively immunized with rabbit IgG specific for the 119–139 sequence had significantly smaller lesions than mice receiving Control IgG (*<i>p</i> = 0.008, Kruskal Wallis; <i>p</i>< 0.05, Dunn’s multiple comparison test: 119–139 vs Control IgG). Lesion areas from groups of mice receiving the 114–131 and 130–147 IgG were not significantly different from the lesion areas of mice receiving the irrelevant Control-IgG, nor were they significantly different from the lesion sizes of mice administered 119–139 IgG, as assessed using the Dunn’s post-hoc analysis. Horizontal lines in (A), (B) and (D) represent geometric means.</p

Dose-Response Titration of PND Ab for protection of mice from LAC/USA300 Dermonecrosis.

<p>Groups of BALB/c mice (n = 4 except neg. controls, n = 3) were passively immunized s.c. with two-fold dilutions of PND Ab starting with a dose of 300 μl (panel A) or 24 μl (Panel B). 48 hours later, mice were challenged i.d. with 32 x 10⁶ (panel A) or 28 x 10⁶ (panel B) CFUs LAC/USA300. Two days later, all mice were euthanized and photographed and lesion areas determined (C). Group-specific lesion area data were plotted against dose and four parameter non-linear regression was used to determine the EC₅₀ of 14 μl (9.66 μg) (R² = 0.55) which represents the dose of PND-Ig which prevented 50% of the maximal lesion area. Antibody and neutralization titers from the sera of mice passively immunized with PND-Ig were determined by ELISA (D) and in the TNA (E) from serum obtained from individual mice on the day of challenge. Each circle represents an individual mouse data point and horizontal lines represent geometric means. Regression lines derived from the serum antibody and TNA data vs. dose were then used to interpolate the PA₅₀ and PN₅₀ which are expressed as reciprocal titers. The regression equation for PA₅₀ determination was log ELISA EC₅₀ = 0.9504 x log Dose + 1.75, and for PN₅₀ determination was log TNA ED₅₀ = 0.858 x log Dose + 0.3589.</p