Molecular Engineering of Ghfp, the Gonococcal Orthologue of Neisseria meningitidis Factor H Binding Protein

Abstract

Knowledge of the sequences and structures of proteins produced by microbial pathogens is continuously increasing. Besides offering the possibility of unraveling the mechanisms of pathogenesis at the molecular level, structural information provides new tools for vaccine development, such as the opportunity to improve viral and bacterial vaccine candidates by rational design. Structure-based rational design of antigens can optimize the epitope repertoire in terms of accessibility, stability, and variability. In the present study, we used epitope mapping information on the well-characterized antigen of Neisseria meningitidis factor H binding protein (fHbp) to engineer its gonococcal homologue, Ghfp. Meningococcal fHbp is typically classified in three distinct antigenic variants. We introduced epitopes of fHbp variant 1 onto the surface of Ghfp, which is naturally able to protect against meningococcal strains expressing fHbp of variants 2 and 3. Heterologous epitopes were successfully transplanted, as engineered Ghfp induced functional antibodies against all three fHbp variants. These results confirm that structural vaccinology represents a successful strategy for modulating immune responses, and it is a powerful tool for investigating the extension and localization of immunodominant epitopes.

FIG 1

(A) NMR mapping of the epitopes recognized by JAR5 and MAb502. The residues involved in the interaction with JAR5 are depicted in green. (B) The epitope of MAb502 is colored in red and is reported according to Scarselli et al. (23). C-term, C terminus; N-term, N terminus.

FIG 2

Classification tree of the different fHbp alleles used in this study. Ranges of amino acid sequence identity of fHbp variants (var.) 1, 2, and 3 to Ghfp are reported in parentheses. Multiple-sequence alignment has been carried out with Clustal W (39), available at the NPS@ server. The dendrogram was obtained at Phylogeny.fr server with TreeDyn (40).

FIG 3

Multiple-sequence alignment of the engineered proteins (NG_5.2, NG_5.6, and NG_5.8) to the wild-type Ghfp and the fHbp subvariant 1.1. The asterisk marks positions 163, 178, and 204, which are critical for MAb502 binding to fHbp subvariant 1.1.

FIG 4

DSC analysis of engineered Ghfp proteins. (A) The overlapping peaks in the melting curve of Ghfp (gray line) have been calculated by applying a non-2-state fitting model according to the Levenberg-Marquardt nonlinear least-squares method using the Origin 7 software. (B) All the mutants generated two very distinct peaks, consistent with two unfolding events. Cp, heat capacity normalized for the concentration.

FIG 5

Interaction of immobilized engineered proteins with factor H (fH) analyzed by SPR. Biacore sensorgrams show the dose-dependent response over time (resonance units [RU]) during the binding of increasing concentrations of factor H (up to 2 μM) on immobilized recombinant fHbp, while no binding is observed with the immobilized Ghfp proteins.

FIG 6

Interaction of engineered Ghfp proteins with JAR5 (A) and MAb502 (B) analyzed by SPR. Representative Biacore sensorgrams show the response over time (resonance units [RU]) during the binding of purified recombinant proteins to immobilized MAbs.

FIG 7

FACS analysis of fHbp surface expression and factor H binding of N. meningitidis strains used in this study. The presence of fHbp on the meningococcal cell surface was detected by binding of mice polyclonal sera elicited by the same fHbp subvariant, when available, or by closely related alleles. In each panel, the amino acid identity between fHbp used to immunize mice and the genetic variant expressed by the strain tested is reported in parentheses. The shaded and white profiles show the reactions with preimmune and immune sera, respectively. Max, maximum; FL1-H, fluorescence intensity.