Unbiased Identification of Immunogenic Staphylococcus aureus Leukotoxin B-Cell Epitopes

Abstract

Unbiased identification of individual immunogenic B-cell epitopes in major antigens of a pathogen remains a technology challenge for vaccine discovery. We therefore developed a platform for rapid phage display screening of deep recombinant libraries consisting of as few as one major pathogen antigen. Using the bicomponent pore-forming leukocidin (Luk) exotoxins of the major pathogen Staphylococcus aureus as a prototype, we randomly fragmented and separately ligated the hemolysin gamma A (HlgA) and LukS genes into a custom-built phage display system, termed pComb-Opti8. Deep sequence analysis of barcoded amplimers of the HlgA and LukS gene fragment libraries demonstrated that biopannng against a cross-reactive anti-Luk monoclonal antibody (MAb) recovered convergent molecular clones with short overlapping homologous sequences. We thereby identified an 11-amino-acid sequence that is highly conserved in four Luk toxin subunits and is ubiquitous in representation within S. aureus clinical isolates. The isolated 11-amino-acid peptide probe was predicted to retain the native three-dimensional (3D) conformation seen within the Luk holotoxin. Indeed, this peptide was recognized by the selecting anti-Luk MAb, and, using mutated peptides, we showed that a particular amino acid side chain was essential for these interactions. Furthermore, murine immunization with this peptide elicited IgG responses that were highly reactive with both the autologous synthetic peptide and the full-length Luk toxin homologues. Thus, using a gene fragment- and phage display-based pipeline, we have identified and validated immunogenic B-cell epitopes that are cross-reactive between members of the pore-forming leukocidin family. This approach could be harnessed to identify novel epitopes for a much-needed S. aureus-protective subunit vaccine.

Convergent Evolution of Neutralizing Antibodies to Staphylococcus aureus γ-Hemolysin C That Recognize an Immunodominant Primary Sequence-Dependent B-Cell Epitope

“…Generation of murine MAbs reactive with HlgC. Monoclonal antibodies (MAbs) reactive with the HlgC subunit of the Luk family were generated by immunization of BALB/c mice with recombinant HlgC, using a previously reported standard protocol (24).…”

“…Monoclonal antibody gene sequence determinations. For determination of antibody gene rearrangements coding for MAbs, we applied a method incorporating rapid amplification of cDNA ends (RACE) along with constant-region primers for unbiased generation of H and chain amplimers for Sanger sequencing, as previously reported (24,40,41). Alignments to closest germ line genes used the Immunogenetics (IMGT) tool suite (42).…”

“…Using the anti-HIgC2 MAb as bait, we performed four rounds of biopanning, which yielded an increase in the phage-out/phage-in ratio for each of the sequentially selected sublibraries ( Fig. 1A) that suggested the successful selection of specific fragment phage clones (24). Furthermore, the pan4 sublibrary selected by the anti-HlgC2 MAb displayed the highest-level dose-dependent binding interactions with the anti-HlgC2 MAb (Fig.…”

“…This phagemid system enables the generation of large libraries of individual phagemid members, each carrying a gene insertion that is expressed as a fusion protein product with a coat protein on the phage surface that provides a physical linkage with the encoding gene (26). Here, we utilized a proven phage-display library composed of random gene fragments of the highly homologous Luk/PFT family member, LukS (24), that was recognized by all of these MAbs (Fig. S1).…”

“…As the HlgCB complex is a potent and broadly produced virulence factor responsible for S. aureus subversion of host innate immunity, we sought to characterize the molecular features associated with the recognition of the antigenic determinants by HlgCB-neutralizing monoclonal antibodies (MAbs). We utilized a recently developed filamentous phage-display system that enables the identification of the molecular surfaces recognized by an antibody (24), which we probed with a panel of murine MAbs generated by immunization with the HlgC subunit. From these studies, we identified a minimal toxin subregion that contains a stable ␤-hairpin loop previously implicated in the functional properties of the toxin.…”