Fine Antigenic Specificity and Cooperative Bactericidal Activity of Monoclonal Antibodies Directed at the Meningococcal Vaccine Candidate Factor H-Binding Protein

MAb preparation.Anti-fHbp MAbs JAR 3, 4, and 5 (27) and MAb 502 (13) (a gift of Marzia Giuliani, Novartis Vaccines, Siena, Italy) were prepared from mice immunized with fHbp in the v.1 group. Anti-fHbp MAbs JAR 10, 11, and 13 were generated from a mouse immunized with fHbp in the v.2 group (a gene from strain 2996) (3). We generated four new hybridoma cell lines from the spleens of CD-1 mice immunized with rfHbp in the v.3 group (a gene from strain M1239) using methods previously described (29). The MAbs were precipitated from cell culture supernatants with 50% saturated (NH₄)₂SO₄ and dialyzed against phosphate-buffered saline (PBS; Roche).

Bactericidal activity.Complement-mediated bactericidal activity was measured as described elsewhere using washed, log-phase bacteria grown in Mueller-Hinton broth supplemented with 0.25% glucose and 0.02 mM CMP-N-acetylneuramic acid to an optical density at 620 nm of 0.6 (26). The buffer was Dulbecco's PBS (Mediatech, Inc.) containing 0.9 mM CaCl₂, 0.5 mM MgCl₂, 6H₂O, and 1% (wt/vol) bovine serum albumin (BSA; Sigma). The complement source was human serum from a healthy adult with no detectable intrinsic bactericidal activity. The bactericidal activity of the MAb was defined by the concentration that gave a 50% decrease in the number of CFU (BC₅₀) after 60 min of incubation at 37°C compared to the number of CFU at time zero in the negative control reactions.

ELISA.To measure the binding of the MAbs to bacterial cells, N. meningitidis strains were grown for 3 h in Mueller-Hinton broth at 37°C in 5% CO₂. The culture was heated for 1 h at 65°C, and the heat-killed bacteria were washed once in PBS and resuspended in PBS to an optical density at 620 nm of 0.6. The suspension (100 μl) was added to each well of a 96-well microtiter plate (Immulon 2B; Thermo Electron Corp.) and allowed to dry. The plates were blocked with blocking buffer (PBS containing 0.1% Tween-20 [PBST; Sigma] and 1% BSA). All antibodies were diluted in blocking buffer. The primary antibodies were anti-fHbp MAbs (5 μg/ml; incubated for 1 h at room temperature), and the secondary antibody was rabbit anti-mouse immunoglobulin G (IgG)-alkaline phosphatase (1:5,000; incubated for 1 h at room temperature; Zymed). Alkaline phosphatase substrate (Sigma) was added, and the absorbance at 405 nm was measured after 30 min. A strain was considered positive for binding if the absorbance value was >10-fold greater than that for the background. Values for negative reactions generally were <3-fold greater than the value for the background. The binding of the MAbs to rfHbp was measured using an enzyme-linked immunosorbent assay (ELISA) procedure similar to that described above for binding to bacteria, except that the wells of the microtiter plate were coated with 1 μg/ml of rfHbp v.1, v.2, or v.3, purified as described previously (1), and diluted in PBS.

Inhibition of binding of fH.The ability of an anti-fHbp MAb to inhibit the binding of fH to fHbp was measured by ELISA. The wells of a microtiter plate were coated with rfHbp as described above. Dilutions containing 0.016 to 50 μg/ml of the MAb were added to the wells together with 50 μg/ml purified fH (Complement Technology, Inc.). The plates were incubated overnight at 4°C. Bound fH was detected with goat polyclonal anti-fH (Bethyl Laboratories) (1:1,000) followed by mouse anti-goat IgG-alkaline phosphatase conjugate (Santa Cruz Biotech) (1:2,000). Both of the latter steps were performed at room temperature for 2 h. After being washed, substrate was added and developed as described above for the antibody-binding ELISA.

Prediction of residues involved in epitopes.The DNA sequencing of the fHbp genes from a panel of 104 isolates identified 38 unique encoded protein sequences. Subsets of these sequences were aligned with ClustalW (6) to assess whether specific amino acid residues corresponded with the MAb reactivity patterns as measured by ELISA with bacterial cells. For the amino acid alignments, the numbering of the residues is based on that of the mature protein (i.e., lacking the signal sequence) of fHbp from strain MC58. In reality, the lengths of the mature v.2 protein (encoded by a gene from strain 8047) and v.3 fHbp (encoded by a gene from strain M1239) differ by −1 and +7 amino acid residues, respectively, from that of fHbp encoded by the gene from strain MC58. Thus, when we refer to a lysine residue at position 180, the residue is actually at position 179 of the v.2 protein or at position 187 in the v.3 protein.

Cloning and site-specific mutagenesis.The gene encoding fHbp was amplified from various meningococcal strains by PCR using primers described previously (17). The PCR products were cloned into a T/A plasmid (pGEM-T-Easy; Promega) and subcloned into the NdeI and XhoI sites of pET21b (Novagen) to encode a C-terminal hexahistidine (His₆) tag. The plasmid encoded the full-length fHbp except for the amino-terminal 26 amino acids, which contained the signal sequence. Site-specific mutagenesis was performed using the QuikChange II kit (Stratagene), using 10 ng of plasmid template and the manufacturer's protocols. Wild-type and mutant fHbp expression plasmids were confirmed by DNA sequence determination.

fHbp expression.Expression plasmids were transformed into Escherichia coli BL21(DE3) (Novagen). Cultures (2 ml) were grown in Luria-Bertani medium containing 100 μg/ml ampicillin, and fHbp expression was induced with 1.0 mM isopropyl-β-d-thiogalactopyranoside for 2 to 3 h at 37°C. Cells were collected by centrifugation in an Eppendorf 5415D microcentrifuge at 16,100 × g and resuspended in 1× LDS sample buffer (Invitrogen) containing 25 mM 2-mercaptoethanol (Bio-Rad).

Western blotting.Bacterial lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 4 to 12% NuPAGE polyacrylamide gels and morpholinepropanesulfonic acid sodium dodecyl sulfate-polyacrylamide gel electrophoresis buffer (Invitrogen). The proteins were transferred to polyvinyl difluoride membranes (Immobilon-P; Millipore). The membranes were blocked using PBST containing 2% nonfat dry milk (Carnation; Nestle). The membranes were washed and incubated with the different anti-fHbp MAbs (1 to 5 μg/ml) or, as a control for protein expression by the different clones, 0.02 μg/ml of Penta-His MAb (Qiagen). The membranes were washed in PBST and incubated with a 1:10,000 dilution of a rabbit anti-mouse IgG-horseradish peroxidase conjugate (Zymed) and washed again. The membranes were developed with a chemiluminescent substrate (ECL⁺; GE Healthcare) and visualized on a Storm 840 imager (Molecular Dynamics).

Nucleotide sequence accession numbers.The DNA sequences for fHbp from strains M3153, GB988 (also referred to as M01-0240988), 03S-0408, and RM1090 have been deposited in GenBank (accession numbers EU337062, EU337063, EU828659, and EU310268, respectively).

MAbs prepared against v.2 or v.3 fHbp.We investigated concentration-dependent binding by performing ELISAs of three MAbs from a mouse immunized with recombinant fHbp v.2 and four MAbs from a mouse immunized with fHbp v.3 (Fig. 1). The MAbs prepared against the v.2 protein were designated JAR 10, 11, and 13 and were IgG1, IgG2a, and IgG2a, respectively. The reactivities of these MAbs against different N. meningitidis strains (3) and recombinant chimeric fHbp vaccines (1) were previously described, but the characteristics of their binding to recombinant proteins and functional activity were not characterized. Four newly prepared MAbs against the v.3 protein were designated JAR 32, 33, 35, and 36. Two of these, JAR 32 and 33, were IgG2a, and the other two, JAR 35 and 36, were IgG2b. As determined by ELISA, all seven MAbs showed concentration-dependent binding to the respective recombinant proteins that were used for the immunization of the mice (Fig. 1). In addition, JAR 13 cross-reacted with the v.3 protein (Fig. 1B), and JAR 36 cross-reacted with the v.2 protein (Fig. 1A). By flow cytometry, each of the MAbs showed similar respective patterns of binding and cross-reactivity to live, encapsulated bacteria that expressed fHbp v.2 (8047) or v.3 (M1239) (data not shown).

• Open in new tab
• Download powerpoint

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.

Although JAR 32, 33, and 35, which were from a mouse immunized with fHbp v.3, did not cross-react with the control recombinant v.2 protein used in this study (expressed from the gene from strain 8047), these three MAbs cross-reacted with fHbp expressed by other strains with subvariants of fHbp in the v.2 group (for example, strain RM1090 [2]). These results were consistent with the reported cross-reactivity between polyclonal antisera prepared against v.2 and v.3 proteins (17). JAR 10, which was from a mouse immunized with fHbp in the v.2 group, also cross-reacted with subvariants of fHbp in the v.1 group (for example, strain NZ98/254 [3]).

None of the MAbs individually was bactericidal with human complement (Table 1). However, each of the MAbs was bactericidal when combined with a second anti-fHbp MAb. The respective minimal bactericidal MAb concentrations (BC₅₀) for each of the pairs are summarized in Table 1, which also shows the pair-wise activity of three additional previously described MAbs, JAR 3, 4, and 5, which were from a mouse immunized with fHbp v.1 (28, 29). For each of the individual MAbs, the minimum antibody concentration when combined with a second MAb was as low as 1 to 5 μg/ml (i.e., 0.5 to 2.5 μg/ml of each MAb). Note that JAR 4, which cross-reacted with fHbp in the v.1 and v.2 groups, had cooperative bactericidal activity with other MAbs that were specific for fHbp in the v.1 or v.2 group (i.e., with JAR 3 or JAR 5 against strain NZ98/254, which expressed fHbp in the v.1 group, or with JAR 11 or JAR 13 against strain 8047, which expressed fHbp in the v.2 group).

Inhibition of binding of fH.We previously reported that anti-fHbp MAbs JAR 3 and 5, but not JAR 4, inhibited the binding of human fH to the surface of live bacterial cells of N. meningitidis as measured by flow cytometry (16). For the present study, we used an ELISA to measure the ability of each of the MAbs to inhibit the binding of fH to recombinant fHbp from the v.1, v.2, or v.3 group. The anti-fHbp MAbs JAR 3 and 5 gave nearly complete inhibition of fH binding to fHbp v.1, whereas JAR 4 gave no inhibition (Fig. 2A). Three of the MAbs isolated from mice immunized with fHbp v.2 or v.3 (JAR 13, 32, and 35), gave >75% inhibition of the binding of fH to rfHbp v.2 or v.3 (Fig. 2B and C, respectively), while four other MAbs gave partial (JAR 11 and 36) or no inhibition (JAR 10 and 33). All of the MAbs that gave >75% inhibition of fH binding by ELISA also gave strong inhibition of the binding of fH to live, encapsulated bacteria as measured by flow cytometry, while the MAbs that gave no inhibition by ELISA also did not inhibit the binding of fH to bacteria (data not shown). MAbs JAR 11 and 36, which showed a partial inhibition of fH binding by ELISA, gave inconsistent inhibition by flow cytometry (data not shown).

• Open in new tab
• Download powerpoint

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.

Mapping amino acid residues affecting epitope expression.From the amino acid sequence alignments of fHbp from informative strains (Table 2), we identified polymorphisms that were consistent with certain residues contributing to MAb reactivity for six of the seven MAbs prepared against v.2 or v.3 protein. For these six MAbs (JAR 10, 11, 13, 32, 33, and 35), the residues predicted to affect epitope expression were between positions 174 and 216 (inclusive). These residues were in the part of the protein previously designated the C domain (13), which formed an eight-stranded beta-barrel in the solution structure of fHbp (4). Predictions of residues involved in the epitopes recognized by these six MAbs were tested by the site-specific mutagenesis of plasmid-encoded fHbp expressed in E. coli. A residue from a reactive protein was changed to another residue naturally present in a nonreactive protein (knockout [KO]). A converse change at the same position in the sequence from a nonreactive protein to the corresponding residue of a reactive protein also was performed to test whether the epitope could be introduced (knock-in [KI]).

JAR 10 and 33.The epitope for MAb JAR 10, which was raised against a v.2 fHbp, was predicted from sequence alignments to involve both a lysine (K) at position 180 and a glutamate (E) at 192 (Table 2). The respective residues in the JAR 10-negative strains were R180 (strains RM1090, M1239, and GB988) or D192 (strains MC58 and GB988). The immunoblotting of recombinant fHbps containing single-amino-acid substitutions showed that JAR 10 binding was greatly decreased in K180R or E192D mutants of v.2 fHbp from strain 8047 (Fig. 3A, lanes 4 and 5). Moreover, the epitope could be introduced by a converse single-amino-acid substitution, R180K, in the nonreactive v.3 fHbp from strain M1239 (Fig. 3A, lane 7), which naturally contained E192 at the second position of the pair (Table 2), or by D192E in the v.1 fHbp from strain MC58 (Fig. 3A, lane 9), which naturally contained K180 at the first position (Table 2). A control blot probed with a Penta-His MAb specific for the C-terminal His₆ tag showed that similar amounts of the protein were expressed from wild-type and mutant clones (Fig. 3B), which indicated that the mutant proteins were not destabilized globally.

• Open in new tab
• Download powerpoint

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.

The reactivity of MAb JAR 33, which was raised against a v.3 fHbp, also was associated with residues at positions 180 and 192, which corresponded to those of JAR 10 reactivity with the v.2 protein (Table 2). JAR 33 reactivity could be knocked out by an R180K mutation in fHbp v.3 of strain M1239 (Fig. 3C, lane 4) and introduced in the fHbp of a nonreactive v.2 strain, 8047, by the reciprocal K180R mutation (Fig. 3C, lane 6). A control blot probed with a Penta-His MAb showed that similar amounts of the proteins were expressed from the respective wild-type and mutant clones (Fig. 3B).

JAR 11, 32, and 35.The reactivity of JAR 32 and JAR 35 was associated with the presence of a lysine residue at position 174 (K174) of strains RM1090 and M1239 (Table 2). The immunoblotting of wild-type and KO mutant clones indicated that both JAR 32 and JAR 35 reactivity was abolished in a mutant of M1239 fHbp containing the K174A substitution (Fig. 4A and B, lanes 5). Reactivity could be introduced with the converse substitution, A174K, in the nonreactive fHbp from strain 8047 (Fig. 4A and B, lanes 7). JAR 32 and 35 had different IgG subclasses (IgG2a and IgG2b, respectively). By ELISA, using secondary antibodies specific for each of these subclasses, the addition of JAR 32 gave the concentration-dependent inhibition of the binding of JAR 35 to fHbp v.3 (>90%; data not shown), which was consistent with the two epitopes being overlapping or identical. However, JAR 35 gave only a partial inhibition of the binding of JAR 32 (24%).

• Open in new tab
• Download powerpoint

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.

A replicate blot of the mutant proteins shown (Fig. 4A and B) was probed with JAR 11 and showed that the epitope recognized by this MAb was eliminated by the A174K substitution in fHbp from strain 8047 (Fig. 4C, lane 7). However, JAR 11 reactivity could not be introduced into the nonreactive v.3 protein of M1239 with the converse substitution (K174A) (Fig. 4C, lane 5). This result suggested that additional residues were important for the JAR 11 epitope. The Penta-His control immunoblot showed that the proteins were expressed at similar levels from wild-type and mutant clones (Fig. 4D).

JAR 13.The JAR 13 epitope was associated with the presence of a serine residue at position 216 (Table 2). The substitution of S216G in the reactive fHbp v.3 protein of strain M1239 eliminated the epitope, while the G216S substitution in the nonreactive fHbp from strain RM1090 introduced the epitope (data not shown).

JAR 3 and 5.As described above, we previously characterized a panel of anti-fHbp MAbs prepared from a mouse immunized with recombinant fHbp in the v.1 group (encoded by a gene from strain MC58) (29). Two of the MAbs, designated JAR 3 and JAR 5, reacted broadly with nearly all subvariants of fHbp v.1. Each MAb inhibited the binding of fH to the surface of encapsulated N. meningitidis strains (16), and each was bactericidal against strain NZ98/254 (fHbp v.1) when tested in combination with a second MAb, JAR 4 (Table 1), but not when tested with each other (Table 1).

Based on our previous data, JAR 3 and JAR 5 bound to strains expressing fHbp in the v.1 group, including MC58, 4243, M1390, and NZ98/254, which had a glycine at position 121, but not to strain M6190, which had an arginine at position 121 (29). Another strain, 03S-0408, also did not react with JAR 3 and JAR 5; this strain had residue G121 but contained serine at position 122 rather than lysine, which was present in all of the reactive strains. The data from these natural polymorphisms suggested that this portion of the molecule is important for the expression of the JAR 3 and JAR 5 epitopes. We therefore used site-specific mutagenesis to change the glycine residue at position 121 in the fHbp sequence of strain MC58 to arginine. In a second mutant, we changed the lysine at position 122 to serine. Both substitutions resulted in the loss of JAR 3 and JAR 5 reactivity. The data for the G121R substitution are shown in Fig. 5A and B, lanes 4. A converse change in fHbp from strain M6190, R121G, introduced the JAR 5 epitope (Fig. 5A, lane 6) and, to a lesser extent, the JAR 3 epitope (Fig. 5B, lane 6). The weaker signals for the M6190 mutant R121G protein relative to that of the wild-type protein indicated that additional residues were important for the expression of these epitopes. The Penta-His control MAb showed that the wild-type and mutant proteins were produced in similar quantities (Fig. 5C). A converse change in fHbp from strain 03S-0408, S122K, was not done.

• Open in new tab
• Download powerpoint

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.

Additional evidence that JAR 3 and JAR 5 recognized overlapping epitopes was derived from competitive inhibition experiments. As reported previously, by ELISA JAR 5 inhibited the binding of JAR 3 to fHbp by >90%, and JAR 3 inhibited the binding of JAR 5 by >90% (28). Thus, JAR 3 and JAR 5 recognized overlapping epitopes, since each of these MAbs inhibited the binding of the other to fHbp.