Fine antigenic specificity and cooperative bactericidal activity of monoclonal antibodies directed at the meningococcal vaccine candidate factor h-binding protein

Abstract

No broadly protective vaccine is available for the prevention of group B meningococcal disease. One promising candidate is factor H-binding protein (fHbp), which is present in all strains but often sparsely expressed. We prepared seven murine immunoglobulin G monoclonal antibodies (MAbs) against fHbp from antigenic variant group 2 (v.2) or v.3 ( approximately 40% of group B strains). Although none of the MAbs individually elicited bactericidal activity with human complement, all had activity in different combinations. We used MAb reactivity with strains expressing fHbp polymorphisms and site-specific mutagenesis to identify residues that are important for epitopes recognized by six of the v.2 or v.3 MAbs and by two v.1 MAbs that were previously characterized. Residues affecting v.2 or v.3 epitopes resided between amino acids 174 and 216, which formed an eight-stranded beta-barrel in the C domain, while residues affecting the v.1 epitopes included amino acids 121 and 122 of the B domain. Pairs of MAbs were bactericidal when their respective epitopes involved residues separated by 16 to 20 A and when at least one of the MAbs inhibited the binding of fH, a downregulatory complement protein. In contrast, there was no cooperative bactericidal activity when the distance between residues was >or=27 A or <or=14 A, which correlated with the inhibition of the binding of one MAb by the other MAb. Thus, a model for anti-fH MAb bactericidal activity against strains expressing low levels of fHbp requires the binding of two MAbs directed at nonoverlapping epitopes, which activates the classical complement pathway as well as inhibits fH binding. The latter increases the susceptibility of the organism to complement-mediated bacteriolysis.

FIG. 1.

Concentration-dependent binding of anti-fHbp MAbs to recombinant fHbp v.2 or v.3 as measured by ELISA. JAR 10, 11, and 13 (solid lines) were from a mouse immunized with a recombinant v.2 protein. JAR 32, 33, 35, and 36 (dashed lines) were from a mouse immunized with a recombinant v.3 protein. (A) Binding to fHbp v.2 (encoded by a gene from strain 8047). (B) Binding to fHbp v.3 (encoded by a gene from strain M1239). OD, optical density.

FIG. 2.

Inhibition of the binding of human fH to recombinant fHbp by anti-fHbp MAbs as measured by ELISA. (A) Inhibition of binding to fHbp v.1. (B) Inhibition of binding to fHbp v.2. (C) Inhibition of binding of fH to fHbp v.3. The recombinant v.1 protein is from the gene of strain MC58. Respective v.2 and v.3 recombinant proteins are those used in experiments shown in Fig. 1.

FIG. 3.

Western blot showing residues that are important for the binding of anti-fHbp MAbs JAR 10 and JAR 33. Residues lysine (K) 180 and glutamate (E) 192 were involved in the JAR 10 epitope, and arginine (R) 180 was essential for the JAR 33 epitope. The numbering was based on the amino acid sequence of MC58 v.1 fHbp lacking the signal sequence (17) (see Materials and Methods). wt, wild type. (A) JAR 10. (B) Penta-His MAb from the same samples as those used for panel A. (C) JAR 33.

FIG. 4.

Western blot showing residues that are important for the binding of anti-fHbp MAbs JAR 11, JAR 32, and JAR 35. Residue lysine (K) 174 was essential for the JAR 32 and JAR 35 epitopes, and an alanine (A) residue at position 174 was involved in the JAR 11 epitope. The numbering is described in the legend to Fig. 3. wt, wild type. (A) JAR 32. (B) JAR 35. (C) JAR 11. (D) Penta-His MAb.

FIG. 5.

Western blot showing a residue that is important for the binding of anti-fHbp MAbs JAR 3 and JAR 5. The epitopes for JAR 3 and JAR 5 were eliminated by the replacement of glycine (G) by arginine (R) at position 121 (G121R) in fHbp from strain MC58 and were introduced with substitution R121G in fHbp from strain M6190. The numbering is described in the legend to Fig. 3. (A) JAR 5. (B) JAR 3. (C) Penta-His MAb.

FIG. 6.

Model of the locations of amino acid residues affecting the expression of fHbp epitopes. Coordinates were from the solution structure of fHbp v.1 of strain MC58 (4). The B and C domains are depicted in dark and light gray, respectively. The positions of the N and C termini are indicated. (Left) Locations of amino acid residues involved in the MAb epitopes are shown in black, with their respective residue numbers shown in parentheses. (Right) Molecule rotated 120° relative to the image in the left panel. The location of the residue previously reported to be involved in the epitope of MAb 502 is shown (13). Note that not all epitopes are present on fHbp v.1 from strain MC58, but the corresponding amino acid residues involved in the epitopes expressed by fHbp v.2 and v.3 proteins are indicated. The numbering is described in the legend to Fig. 3. The figure was generated using PyMol (http://www.pymol.org).