A Broadly Cross-Reactive Monoclonal Antibody Against an Epitope on the N-terminus of Meningococcal fHbp

Vu, David M.; Pajon, Rolando; Reason, Donald C.; Granoff, Dan M.

doi:10.1038/srep00341

Download PDF

Article
Open access
Published: 28 March 2012

A Broadly Cross-Reactive Monoclonal Antibody Against an Epitope on the N-terminus of Meningococcal fHbp

David M. Vu¹,
Rolando Pajon¹,
Donald C. Reason¹ &
…
Dan M. Granoff¹

Scientific Reports volume 2, Article number: 341 (2012) Cite this article

1870 Accesses
34 Citations
1 Altmetric
Metrics details

Subjects

An Erratum to this article was published on 20 March 2013

This article has been updated

Abstract

Meningococcal factor H binding protein (fHbp) is an important vaccine antigen for prevention of disease caused by capsular group B strains. The protein has been sub-classified into three variant groups. Most anti-fHbp antibodies are variant group-specific and recognize epitopes on the C-terminal domain. We report a murine IgG1 mAb, JAR 41, which broadly cross-reacted with fHbp sequence variants from all variant groups. The mAb bound to the surface of live meningococci with fHbp from each of the three variant groups. In combination with second non-bactericidal anti-fHbp mAbs, JAR 41 elicited complement-mediated bactericidal activity in vitro and augmented passive protection against meningococcal bacteremia in human fH transgenic rats. The epitope was located on a conserved region of the N-terminal portion of the fHbp molecule opposite that of fH contact residues. The data underscore the importance of broadly cross-reactive, surface-exposed epitopes on the N-terminal domain in the design of protective fHbp vaccines.

Structural basis for cooperativity of human monoclonal antibodies to meningococcal factor H-binding protein

Article Open access 26 June 2019

Selection of diverse strains to assess broad coverage of the bivalent FHbp meningococcal B vaccine

Article Open access 29 January 2020

Monoclonal antibodies from humans with Mycobacterium tuberculosis exposure or latent infection recognize distinct arabinomannan epitopes

Article Open access 12 October 2021

Introduction

Factor H binding protein (fHbp) is an important antigen for vaccines being developed for prevention of meningococcal disease caused by capsular group B strains, for which currently there is no broadly protective vaccine (reviewed in^1,2). In mice and humans, vaccines containing recombinant fHbp elicited serum bactericidal antibody activity^3,4,5,6,7, which is a serologic surrogate of protection against developing meningococcal disease^8,9,10. Based on amino acid sequence homology and antigenic cross-reactivity, fHbp has been sub-classified into two sub-families¹¹ or three variant groups¹². fHbp also can be classified into modular groups based on different combinations of five variable segments, each of which is encoded by genes from one of two lineages¹³. In general, bactericidal activity of anti-fHbp antibodies was largely confined to meningococcal strains with fHbp amino acid sequence variants from the same variant group or sub-family as that of the vaccine antigen^7,12,14.

In previous studies, our laboratory has characterized panels of anti-fHbp mAbs prepared in mice immunized with fHbp from variant groups 1, 2 or 3^15,16. While an occasional mAb reacted with fHbp sequence variants from more than one variant group, to date, none of the mAbs reacted with sequence variants from all three variant groups. In this study, we describe a new anti-fHbp mAb, designated JAR 41, which was isolated from a human factor H (fH) transgenic mouse that had been immunized with a recombinant fHbp from variant group 1¹⁷. The new mAb recognized a surface-exposed, broadly cross-reactive epitope present in all fHbp sequence variants tested from variant groups 1, 2 or 3. Thus, JAR 41 may be useful for developing assays to measure strain expression of fHbp, which is an important determinant of susceptibility to anti-fHbp bactericidal activity^18,19,20,21. Information on the JAR 41 epitope also may be important for the design of improved broadly protective fHbp vaccines^14,22.

Results

Anti-fHbp mAb JAR 41 is broadly cross-reactive

By ELISA, JAR 41 had similar concentration-dependent binding to fHbp ID 1 (variant group 1) as that of a control anti-fHbp, JAR 5, which is specific for variant group 1 (Figure 1a). Two other anti-fHbp mAbs, JAR 11 and JAR 35, which bind fHbp from variant groups 2 and 3, but not from group 1, served as negative IgG controls. JAR 41 also showed similar concentration-dependent binding to fHbp ID 77 (variant group 2) and fHbp ID 28 (variant group 3) as that of the respective positive controls, variant group-specific mAbs (Figures 1b and 1c, respectively). There was no reactivity of these two proteins with JAR 5, which was specific for fHbp variant group 1.

As of December 2011, more than 500 unique fHbp amino acid sequence variants had been deposited in the public database, http://pubmlst.org/neisseria/fHbp/. To select sequence variants representative of fHbp diversity for measuring the breadth of JAR 41 reactivity, we performed a phylogenic analysis of 531 sequences in the public database in the form of a network diagram produced using the program SplitsTree 4²³ (Figure 2a). Each unique sequence is represented by a “twig” (node), which are distributed on the network into two sub-families, A and B, as described by Fletcher et al⁷, or three variant groups, 1, 2 or 3, as described by Masignani et al¹². Sub-family A contains the sequences in variant groups 2 and 3 and sub-family B contains the sequences in variant group 1. The proteins also can be classified into ten modular groups based on the respective lineages of five variable segments^13,18.

We selected 21 amino acid sequence variants representative of the known fHbp amino acid sequence diversity for expression of recombinant proteins for testing JAR 41 reactivity (Table 1). This panel included variants from both sub-families A and B, all three variant groups¹² and all ten modular groups^13,18 (Figure 2b). Note that fHbp ID 283, which is a natural hybrid of variant groups 1 and 3, has A, B and C segments from lineage 2 and D and E segments from lineage 1. This variant was assigned to a new modular group (X).

Table 1 Recombinant fHbp amino acid sequence variants tested for JAR 41 binding

Full size table

JAR 41 showed similar respective concentration-dependent binding with all of the amino acid sequence variants in variant groups 1, 2 and 3 (Figure 3a–c) and with two natural hybrids, ID 207 and ID 283 (Figure 3d). Binding was specific for fHbp in that JAR 41 did not bind to two unrelated recombinant meningococcal surface proteins, NadA and GNA1030 (Figure 3e). Also, with each of the sequence variants tested by ELISA, we included negative control IgG anti-fHbp mAbs that were specific for fHbps from heterologous variant groups, which showed negligible binding to the sequence variant tested for binding with JAR 41 (See for example, Figure 1).

The JAR 41 epitope is exposed on the surface of live meningococci

By flow cytometry, JAR 41 bound to the surface of live bacteria from N. meningitidis strains. Figure 4a compares binding of JAR 41 (25 µg/ml) to wild-type group B strain H44/76 (fHbp ID 1, variant group 1) and an isogenic mutant of strain H44/76 with lower fHbp expression (H44/76-LE). There is ∼4-fold lower binding with the mutant strain. There was no detectable binding to either strain with a control IgG mAb, JAR 11 (specific for fHbps in variant groups 2 or 3). Binding of an anti-PorA mAb was similar with both the wild-type and mutant (Figure 4b), which is evidence that similar numbers of wild-type and mutant cells were tested for JAR 41 binding.

We next tested binding of JAR 41 to strains H44/76 and its isogenic mutant using a range of mAb concentrations (Figures 4c and 4d, respectively). As little as 0.2 µg/ml of JAR 41 showed significant binding to the H44/76-WT or -LE mutants over that of background binding, which was determined by testing 25 µg/ml of JAR 41 with a H44/76 fHbp knockout mutant (light gray shaded area). JAR 41 also bound to the surface of group B strains 8047 (fHbp ID 77, variant group 2) and M1239 (fHbp ID 28, variant group 3) (Figures 4e and 4f, respectively). For each of these strains the respective JAR 41 binding was similar when tested at the two highest concentrations (5 µg/ml or 25 µg/ml for 8047, or 5 and 50 µg/ml for M1239 [grey-shaded area and solid lines, respectively]). Strain 8047 also was tested with lower concentrations of the mAb. Binding above that of the IgG negative control (an IgG anti-fHbp mAb specific for variant group 1) was detected with as little as 0.2 µg/ml of JAR 41.

JAR 41 elicits human complement-mediated bactericidal activity with second anti-fHbp mAbs

JAR 41 was not bactericidal when tested individually at concentrations of up to 100 µg/ml against any of the four test strains (H44/76-WT [Figure 5a], the H44/76 LE mutant [Figure 5b], 8047 [Figure 5c] or M1239 [data not shown]). Activation of the classical complement pathway by IgG requires binding of two IgG mAbs to appropriately spaced epitopes such that the respective IgG Fc regions can engage C1q²⁴. When an individual mAb binds to a sparse antigen, there may be insufficient immune complex formed to activate bacteriolysis, whereas combinations of more than one mAb may be sufficient²⁴. When tested in combination with other non-bactericidal anti-fHbp mAbs specific for fHbp in variant group 1, JAR 41 elicited cooperative bactericidal activity against strains H44/76 (Figure 5a) or the H44/76 LE mutant with lower fHbp expression (Figure 5b). In contrast, JAR 41 was not bactericidal in combination with anti-fHbp mAbs specific for variant groups 2 or 3 when tested against strains 8047 (fHbp variant group 2, Figure 5c) or M1239 (fHbp variant group 3, data not shown). Both of these strains were susceptible to bactericidal activity elicited by anti-fHbp mAb JAR 4 when tested with the anti-fHbp mAbs specific for variant groups 2 or 3 (for example, JAR 4 + JAR 11 or JAR 13 against strain 8047, Figure 4c).

JAR 41 augments passive protective activity of anti-fHbp mAb JAR 5 against experimental meningococcal bacteremia

Binding of human fH to the surface of meningococci down-regulates complement activation, which enables the organism to evade host defenses^25,26,27. Binding is specific for human fH²⁷. We recently developed a human fH transgenic infant rat model to investigate the effect of human fH on meningococcal bacteremia²⁸. After challenge of wild-type rats, strain H44/76 was cleared rapidly from the blood stream whereas the presence of human fH in the transgenic animals enhanced bacteremia. In the present study we used the human fH transgenic rat model to investigate the ability of JAR 41 given individually or in combination with other non-bactericidal anti-fHbp mAbs to confer passive protection against bacteremia caused by challenge with wild-type group B strain H44/76.

Despite lack of bactericidal activity in vitro, pretreatment of human fH transgenic infant rats with 25 µg of JAR 5 conferred partial protection against bacteremia, as evidenced by a lower geometric mean CFU/ml in blood obtained 6 hrs after IP challenge, compared to that of control rats that received PBS (p<0.05, Experiments 1 and 2, Figure 6). In contrast, 25 µg/rat of JAR 41 did not confer significant protection in either experiment (p>0.05). To determine whether JAR 41 could augment JAR 5 protective activity, both antibodies (12.5 µg/rat of each mAb) were given in combination to groups of human fH transgenic infant rats. In both experiments, none of these animals had bacteremia 6 hrs after the IP challenge. In Experiment 2, in which we tested larger numbers of animals, the augmentation of protection by the combination of JAR 41 and JAR 5 was statistically significant (6 of 9 with bacteremia in the JAR 5 or JAR 41 alone groups, vs. 0 of 8 in the JAR 5+JAR 41 combination group, p = 0.009).

JAR 41 recognizes an epitope that overlaps that of anti-fHbp mAb JAR 4

While the combination of JAR 4 with JAR 5, or JAR 4 with mAb502, was bactericidal against group B strain H44/76, JAR 41 was not bactericidal in combination with JAR 4 (Figure 5a). One possible explanation was that JAR 4 and JAR 41 recognized overlapping epitopes. To investigate this question, we measured the ability of JAR 4 to inhibit binding of alkaline phosphatase (AP)-conjugated JAR 41 to fHbp (Supplemental Figure S1a). The positive control, unconjugated JAR 41, gave >95% inhibition of binding while the negative control, unconjugated JAR 5, gave <5% inhibition. At the highest concentration of mAb tested (100 µg/ml), JAR 4 gave ∼70% inhibition of binding of AP-JAR 41 to fHbp. We also measured the ability of unconjugated JAR 41 (IgG1) to inhibit binding of unconjugated JAR 4 (IgG2a) using an AP-conjugated secondary antibody that was specific for IgG2a (Supplemental Figure S1b). JAR 41 gave >95% inhibition of binding of JAR 4 to fHbp. The ability of JAR 41 and JAR 4 to inhibit binding of each other to fHbp implied that the respective epitopes overlapped.

The fHbp epitope recognized by JAR 41 is eliminated by substitution of Ala for Asp at residue 25 in fHbp

We used a mutant fHbp ID 1 library displayed on the surface of yeast to identify amino acid residues that potentially affect JAR 41 binding. The yeast library was sorted to select yeast clones that had lost their ability to bind JAR 41, but which retained their ability to bind JAR 3 (which verified surface-expression of the construct). Individual JAR 41-negative/JAR 3-positive yeast colonies were expanded and the fHbp gene inserts were sequenced. After eliminating sequences containing large deletions or multiple amino acid substitutions, we aligned the predicted amino acid sequences of 13 full-length mutant fHbp genes (Supplemental Figure S2). The mutations that abolished JAR 41 binding in the yeast library included three substitutions for Asp at position 25 (D25G, D25H, D25A), one substitution for Lys at position 27 (K27N) and one substitution for His at position 26 (H26N, which was present in combination with D25A). Substitutions at residue K27 and, to a lesser extent, D25A, corresponded to the region of the N-Terminal domain of fHbp previously shown to be important for binding of JAR 4 to fHbp²⁹.

To confirm the importance of the individual residues on binding of JAR 41, we used site-directed mutagenesis to prepare mutant recombinant proteins with individual alanine substitutions at these positions. While the D25A mutation abolished binding of JAR 41 (Figure 7a, circles with dashed line), there was no significant effect of the K27A or H26A substitutions on JAR 41 binding to the recombinant mutant proteins. It is possible that the lack of an effect of K27A is explained by the different substituted residue (K27N in yeast, vs. K27A in the recombinant protein). The lack of an effect of H26A can be explained by the absence of the D25A substitution, since in yeast the mutant H26A fHbp that did not bind JAR 41 also contained D25A, which individually was sufficient to eliminate JAR 41 binding.

Disruption of JAR 41 binding with the D25A mutant was not a result of disruption of overall protein folding since the recombinant D25A mutant protein retained epitopes needed for binding with two control anti-fHbp mAbs, JAR 1 and JAR 5 (Figures 7c and 7d, respectively). JAR 1 recently was shown to recognize an epitope involving residue 204³⁰ and JAR 5 recognized an epitope involving residues 121 and 122¹⁶ (Figure 7d). Interestingly, the D25A substitution also had minimal effect on binding of JAR 4 (Figure 7b), which has an epitope on the N-terminal domain²⁹ that overlaps that of JAR 41.

Discussion

Binding of most previously described anti-fHbp mAbs was limited to sequence variants belonging to the same variant group or sub-family as that of the vaccine antigen^15,16,31. To our knowledge only one anti-fHbp mAb (MN86-994-11) was reported to cross-react with fHbp variants from all three variant groups³². Insufficient details, however, were provided on this mAb to determine the actual breadth of reactivity, functional activity, or amino acid residues affecting the epitope.

In the present study, the ability of JAR 41 to bind equally well by ELISA to all 21 recombinant fHbp sequence variants tested and to bind by flow cytometry to the surface of live bacteria with fHbp from variant groups 1, 2, or 3 indicated that the epitope was nearly universal and was surface-exposed and accessible to antibody.

The fHbp molecule consists of two domains of antiparallel β-strands connected by a linker^33,34. The epitope recognized by JAR 41 appears to be located on the N-terminal domain since a single amino acid substitution, Ala for Asp at residue 25 of fHbp ID 1 (D25A), eliminated JAR 41 binding. Among the 531 known amino acid sequence variants in the public fHbp peptide database (http://pubmlst.org/neisseria/fHbp/, as of December 2011), Asp at residue 25 was invariant. Further, the adjacent residues in this region of the N-terminal domain also are highly conserved.

We determined that the JAR 41 epitope overlapped that of a previously reported anti-fHbp mAb, JAR 4, which was specific for fHbp in variant groups 1 and 2²⁹. The molecular architecture of fHbp is modular with five variable segments, designated A, B, C, D and E, each of which is derived from one of two lineages¹³. Among the 21 fHbp sequence variants tested (Supplemental Figure S3), JAR 4 bound only to variants containing “A” segments derived from lineage 1 (previously referred to as alpha lineage¹³). In contrast, JAR 41 bound to all variants tested with A segments from lineage 1 or lineage 2 (previously referred to as beta lineage).

The residues of fHbp in contact with fH have been identified in a crystal structure of fHbp in a complex with a fragment of fH³⁵. The fH contact residues were located on a surface opposite that containing Asp25, which affected the JAR 41 epitope and Asp25 and Lys27, which affected the JAR 4 epitope (See cartoon, Supplemental Figure S4). The respective locations were consistent with ELISA data showing that neither JAR 4^16,29 nor JAR 41 mAb inhibited binding of fH to fHbp (data not shown).

JAR 41 individually did not elicit human complement-mediated bactericidal activity, which is typical of most reported murine anti-fHbp mAbs^15,16,24,29. fHbp is relatively sparsely distributed¹⁵ and it is possible that the distance between most of the fHbp molecules on the bacterial surface exceeds that required for optimal engagement of C1q by binding of individual mAbs. In contrast, binding of two IgG antibodies that recognize appropriately spaced non-overlapping epitopes may permit more efficient engagement of C1q and activation of classical complement pathway bacteriolysis^15,16,24,29. Since binding of fH to N. meningitidis increases resistance of the organism to complement-mediated killing^25,26, the ability of at least one of the anti-fHbp mAbs in the pair to inhibit fH can increase mAb bactericidal activity³⁶.

While JAR 41 bound to the surface of strains H44/76, 8047 and M1239 with fHbp in variant groups 1, 2 or 3, respectively (Figure 4), the mAb elicited cooperative complement-mediated bactericidal activity only against strain H44/76 with fHbp in variant group 1 and an isogenic mutant, H44/76-LE with lower fHbp ID 1 expression (Figure 5). The amount of expression of fHbp variant group 1 by the mutant appeared to be similar to that of the two strains with fHbp in variant groups 2 or 3 that were resistant to anti-fHbp JAR 41 cooperative bactericidal activity. Most likely the resistance of these two test strains reflected testing bactericidal activity with different second anti-fHbp mAbs, which were specific for fHbp in variant groups 2 or 3, than those used to test the strains with fHbp variant group 1. Conceivably, different proximity and/or spatial orientations of JAR 41 bound in combination with JAR 5 or mAb502 in strain H44/76, as compared with JAR 11 or 13 in strain 8047, may have permitted more efficient engagement of C1q and activation of the classical complement pathway with strain H44/76.

In the present study, we used a recently described human fH transgenic infant rat model²⁸ to investigate the ability of JAR 41, individually or in combination with JAR 5, to confer passive protection against meningococcal bacteremia caused by group B strain H44/76. We chose to use the transgenic infant rat model because binding of fH to N. meningitidis is specific for human fH^27,28. Thus, measuring the ability of anti-fHbp mAbs to confer passive protection in the transgenic model likely provided information relevant to protection in humans where bound human fH can down-regulate complement activation on N. meningitidis.

Passive administration of JAR 5 conferred partial protection against meningococcal bacteremia in the transgenic rats (Figure 6). This protective activity was of interest since JAR 5 individually did not elicit human complement-mediated bactericidal activity (Figure 5) but in previous studies was shown to block binding of human fH to fHbp¹⁶. In contrast, JAR 41, which was not bactericidal and which did not block fH binding, was not protective against bacteremia in the transgenic rat model. JAR 41, however, augmented protective activity of JAR 5 (sterile blood cultures in all the rats given the combination). This result was consistent with the ability of JAR 41 to elicit cooperative complement-mediated bactericidal activity when tested with JAR 5.

In summary, JAR 41 is a new murine IgG1 mAb that recognizes a broadly cross-reactive, surface-exposed epitope on the N-terminal domain of fHbp from variant groups 1, 2 and 3. The mAb elicits cooperative human complement-mediated bactericidal activity in combination with other non-bactericidal anti-fHbp mAbs and augments passive protective activity against meningococcal bacteremia. The JAR 41 epitope overlapped that of JAR 4, which also had cooperative bactericidal activity but was specific for fHbp from variant groups 1 and 2. Collectively, the data demonstrate the importance of including cross-reactive epitopes in N-terminal domain of fHbp in the design of optimally protective fHbp vaccines.

Methods

Production of anti-fHbp mAb JAR 41

A human fH transgenic BALB/c mouse was immunized with three injections of a recombinant fHbp vaccine as part of a previous study¹⁷. The fHbp sequence variant was from variant group 1 and assigned ID 1 as designated in the public fHbp peptide database at http://pubmlst.org/neisseria/fHbp/. The mouse received three injections of the vaccine, IP, along with aluminum hydroxide adjuvant followed by a fourth injection without adjuvant. Splenocytes were isolated three days later for fusion to the mouse myeloma cell line P3X63-Ag8.653 using established methods¹⁵. Cells were plated into 96-well plates. After two weeks of incubation, cell culture supernatants were tested for reactivity to recombinant fHbp ID 1 by ELISA (see below). Cells from positive wells were expanded, cloned by limiting dilution and the supernatants confirmed for reactivity to fHbp. The cloning was repeated 2 times to ensure monoclonality.

Control anti-fHbp mAbs

The following murine anti-fHbp mAbs were used as controls in different binding and functional assays: JAR 1 (IgG3), JAR 3 (IgG3), JAR 4 (IgG2a), JAR 5 (IgG2b)^15,16 and mAb502 (IgG2a)^31,37 were from mice immunized with fHbp ID 1 in variant group 1; JAR 11 (IgG2a) and JAR 13 (IgG2a) were from a mouse immunized with fHbp ID 16 in variant group 2¹⁶ and JAR 31 (IgG2b) (Unpublished data of DMG) and JAR 35¹⁶ were from a mouse immunized with fHbp ID 28 in variant group 3.

Purification of recombinant fHbp

We prepared 21 different recombinant fHbp amino acid sequence variants from variant groups 1, 2 or 3 (Table 1) as previously described³⁰. The proteins were expressed from the T7 promotor using the E. coli plasmid pET21b (Novagen, Madison, WI) as described previously¹⁶. The recombinant proteins were purified by immobilized metal ion chromatography using Ni-NTA agarose (Qiagen, Valencia, CA) as described previously¹⁶. Purified fHbps were dialyzed against PBS, sterilized by filtration (Millex 0.22 µm; Millipore, Billerica, MA) and stored frozen at −30°C. The protein concentrations were determined by UV absorbance (Nanodrop 1000, Wilmington, DE) based on the extinction coefficient calculated from the amino acid sequence³⁸.

Anti-fHbp ELISA

Binding of anti-fHbp mAbs to recombinant fHbp (rfHbp) was measured by ELISA, which was performed as described previously³⁶ with minor modifications. In brief, 2 µg/ml of purified recombinant fHbp diluted in PBS was used to sensitize the microtiter plates. After blocking, serial dilutions of the mAbs were added and incubated at 4°C overnight. The wells were washed and bound antibody was detected with goat anti-mouse IgG conjugated with alkaline phosphatase. The ability of JAR 41 to inhibit binding of fH to fHbp was also measured by ELISA as previously described³⁶.

Epitope mapping using yeast display

Randomly mutated sequences of fHbp allele 1 (http://pubmlst.org/neisseria/fHbp/), which encodes fHbp amino acid sequence variant ID 1, were generated by error-prone PCR. In brief, MnCl₂ concentrations were titrated and conditions selected such that the average number of nucleotide substitutions per fHbp gene copy resulted in 1 to 3 amino acid substitutions. The fragments were ligated into pUC18, expanded in E. coli, excised, inserted into the yeast expression vector pYD1 (Invitrogen, Carlsbad, CA) and transfected into the yeast EBY100 strain (Invitrogen) to form the mutated fHbp expression library. The mutated fHbp peptides were displayed on the surface of yeast as Aga2 fusion proteins^39,40. The library was expanded overnight at 30°C, transferred into galactose-containing YNB medium (Yeast Nitrogen Base w/o amino acids [Difco]) to induce recombinant protein expression and incubated for 48 hrs at 20°C. Bulk yeast cultures were simultaneously stained with anti-fHbp mAb, JAR 41, which was detected with FITC-conjugated antibody to mouse IgG and a chimeric human IgG1 mouse anti-fHbp mAb, JAR 3³⁶, which was detected with DyLight 649-conjugated antibody to human kappa light chain. JAR 3 and JAR 41 recognized non-overlapping epitopes as evident by lack of inhibition of binding by ELISA of either mAb to fHbp ID 1 by the other mAb (data not shown). The yeast cultures were sorted by flow cytometry (FACSaria, BD) to select yeast clones that had lost their ability to bind JAR 41, but which retained the ability to bind JAR 3 (which verified surface-expression of the construct). The sorted yeast were plated and incubated at 30°C to isolate single colonies. The individual clones were re-grown, their binding profiles verified by flow cytometry and the sequence of the respective fHbp insert determined to identify amino acid substitutions that had resulted in the loss of the JAR 41 epitope. Approximately 20 JAR 41-negative/JAR 3-positive yeast clones were analyzed and the sequences of their inserts aligned to identify altered residues that led to loss of JAR 41 binding.

Construction of site-specific mutants

Recombinant fHbp mutants with single amino acid substitutions were constructed using the QuikChange II kit (Stratagene, La Jolla, CA) using the manufacturer's protocols as previously described¹⁶. The mutagenesis reactions were transformed into chemically competent E. coli DH5α (Invitrogen, Carlsbad, CA) and independent mutant clones were verified by DNA sequencing (Davis Sequencing, Davis, CA).

Flow cytometry

Binding of the murine mAbs to the surface of live encapsulated bacteria was measured by flow cytometry, which was performed as described previously³⁶. The group B test strains were H44/76 (B:15:P1.7,16; ST-32) with fHbp ID 1; a previously described isogenic mutant, which had been engineered to have decreased expression of fHbp ID 1¹⁹; 8047 (B:2b:P1.5-1, 2-2; ST-8), which expressed fHbp ID 77 in variant group 2; and M1239 (B:14:P1.23,14; ST-41/44), which expressed fHbp ID 28 in variant group 3. The bacterial cells were grown in Mueller Hinton broth culture, harvested by centrifugation, washed and resuspended in buffer as described elsewhere³⁶.

Serum bactericidal assay

Human complement-mediated bactericidal activity was measured as previously described¹⁷ using group B strains H44/76, 8047 and M1239 described above. The complement source was serum from a healthy adult who participated in a protocol that was approved by the Children's Hospital Oakland Institutional Review Board (IRB). Written informed consent was obtained from the subject. The serum had normal total hemolytic complement activity and no detectable serum bactericidal antibodies against the test strains. The serum was depleted of IgG using a protein G column (HiTrap Protein G, GE Life Sciences, Piscataway, NJ) as previously described¹⁷ to remove non-bactericidal antibodies that might augment anti-fHbp mAb activity⁴.

The bactericidal activity (BC_50%) was defined by the mAb concentration that resulted in a 50% decrease in CFU/ml after 60-min incubation in the reaction mixture compared with CFU/ml in negative control wells at time zero.

Passive protective activity against bacteremia

Details of the human fH transgenic rat meningococcal bacteremia model have been recently described²⁸. For measurement of passive protective activity, at time 0 human fH transgenic rats, ages 8 to 9 days (experiment 1) or 6 to 7 days (experiment 2), were administered 25 µg IP of anti-fHbp mAb JAR 5 or JAR 41, or a combination of JAR 5 + JAR 41 (12.5 µg of each). Control rats received buffer alone (phosphate buffered saline, PBS) or 12.5 µg of an anti-PorA mAb (P1.7, experiment 1 only). Total volume of mAb or buffer for each animal was 100 µl. At 1.5 hrs, animals were challenged IP with 4900 CFU (experiment 1) or 760 CFU (experiment 2) of group B strain H44/76. At 7.5 hrs, blood samples were obtained by cardiac puncture into syringes containing 25 U heparin. 1, 10 and 200 µl aliquots of each blood sample were spread onto chocolate agar plates, which were then incubated overnight at 37°C in 5% CO₂. Colony counts were obtained the following day to determine CFU/ml. The lower limit of detection was 5 CFU/ml.

Statistical analyses

Statistical analyses were performed using Prism for Mac version 5.0d (GraphPad Software, La Jolla, CA). Comparisons of the proportions of rats that developed bacteremia after pre-treatment with different mAbs or PBS were performed using the Fisher exact test. For the purpose of calculating the geometric mean CFU/ml of blood, sterile blood cultures were assigned a value of half of the lower limit of detection (i.e., <5 CFU/ml was assigned a value of 2.5 CFU/ml). Comparisons of the geometric mean CFU of bacteria per ml of blood between any two groups of rats were performed on log-transformed values using the Student t-test. All probability values reported are two-tailed.

Live vertebrate experiments

All experiments in mice and infant rats were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, U.S.A. The protocols were approved by the Children's Hospital & Research Center at Oakland Institutional Animal Care and Use Committee. Blood collection was performed under anesthesia and all efforts were made to minimize suffering.

Change history

20 March 2013
A correction has been published and is appended to both the HTML and PDF versions of this paper. The error has not been fixed in the paper.

References

Granoff, D. M. Review of meningococcal group B vaccines. Clin. Infect. Dis. 50 Suppl 2, S54–65 (2010).
Article PubMed CAS Google Scholar
Sadarangani, M. & Pollard, A. J. Serogroup B meningococcal vaccines-an unfinished story. Lancet Infect. Dis. 10, 112–124 (2010).
Article CAS PubMed Google Scholar
Giuliani, M. M. et al. A universal vaccine for serogroup B meningococcus. Proc. Natl. Acad. Sci. U. S. A. 103, 10834–10839 (2006).
Article CAS ADS PubMed PubMed Central Google Scholar
Vu, D. M., Wong, T. T. & Granoff, D. M. Cooperative serum bactericidal activity between human antibodies to meningococcal factor H binding protein and Neisserial heparin binding antigen. Vaccine. 29, 1968–1973 (2011).
Article CAS PubMed PubMed Central Google Scholar
Snape, M. D. et al. Immunogenicity of two investigational serogroup B meningococcal vaccines in the first year of life: a randomized comparative trial. Pediatr. Infect. Dis. J. 29, e71–79 (2010).
Article PubMed Google Scholar
Findlow, J. et al. Multicenter, open-label, randomized phase II controlled trial of an investigational recombinant meningococcal serogroup B vaccine with and without outer membrane vesicles, administered in infancy. Clin. Infect. Dis. 51, 1127–1137 (2010).
Article PubMed Google Scholar
Fletcher, L. D. et al. Vaccine potential of the Neisseria meningitidis 2086 lipoprotein. Infect. Immun. 72, 2088–2100 (2004).
Article CAS PubMed PubMed Central Google Scholar
Frasch, C. E., Borrow, R. & Donnelly, J. Bactericidal antibody is the immunologic surrogate of protection against meningococcal disease. Vaccine. 27 Suppl 2, B112–116 (2009).
Article PubMed CAS Google Scholar
Borrow, R., Balmer, P. & Miller, E. Meningococcal surrogates of protection--serum bactericidal antibody activity. Vaccine. 23, 2222–2227 (2005).
Article CAS PubMed Google Scholar
Granoff, D. M. Relative importance of complement-mediated bactericidal and opsonic activity for protection against meningococcal disease. Vaccine. 27 Suppl 2, B117–125 (2009).
Article PubMed CAS Google Scholar
Murphy, E. et al. Sequence diversity of the factor H binding protein vaccine candidate in epidemiologically relevant strains of serogroup B Neisseria meningitidis. J. Infect. Dis. 200, 379–389 (2009).
Article CAS PubMed Google Scholar
Masignani, V. et al. Vaccination against Neisseria meningitidis using three variants of the lipoprotein GNA1870. J. Exp. Med. 197, 789–799 (2003).
Article CAS PubMed PubMed Central Google Scholar
Beernink, P. T. & Granoff, D. M. The modular architecture of meningococcal factor H-binding protein. Microbiology. 155, 2873–2883 (2009).
Article CAS PubMed PubMed Central Google Scholar
Beernink, P. T. & Granoff, D. M. Bactericidal antibody responses induced by meningococcal recombinant chimeric factor H-binding protein vaccines. Infect. Immun. 76, 2568–2575 (2008).
Article CAS PubMed PubMed Central Google Scholar
Welsch, J. A., Rossi, R., Comanducci, M. & Granoff, D. M. Protective activity of monoclonal antibodies to genome-derived neisserial antigen 1870, a Neisseria meningitidis candidate vaccine. J. Immunol. 172, 5606–5615 (2004).
Article CAS PubMed Google Scholar
Beernink, P. T. et al. Fine antigenic specificity and cooperative bactericidal activity of monoclonal antibodies directed at the meningococcal vaccine candidate, factor H-binding protein. Infect. Immun. 76, 4232–4240 (2008).
Article CAS PubMed PubMed Central Google Scholar
Beernink, P. T. et al. A meningococcal factor H binding protein mutant that eliminates factor H binding enhances protective antibody responses to vaccination. J. Immunol. 186, 3606–3614 (2011).
Article CAS PubMed PubMed Central Google Scholar
Pajon, R., Beernink, P. T., Harrison, L. H. & Granoff, D. M. Frequency of factor H-binding protein modular groups and susceptibility to cross-reactive bactericidal activity in invasive meningococcal isolates. Vaccine. 28, 2122–2129 (2010).
Article CAS PubMed Google Scholar
Pajon, R., Fergus, A. M., Koeberling, O., Caugant, D. A. & Granoff, D. M. Meningococcal factor H binding proteins in epidemic strains from Africa: Implications for vaccine development. PLoS Negl. Trop. Dis. 5, e1302 (2011).
Article PubMed PubMed Central Google Scholar
Donnelly, J. et al. Qualitative and quantitative assessment of meningococcal antigens to evaluate the potential strain coverage of protein-based vaccines. Proc. Natl. Acad. Sci. U. S. A. 107, 19490–19495 (2010).
Article CAS ADS PubMed PubMed Central Google Scholar
Jiang, H. Q. et al. Broad vaccine coverage predicted for a bivalent recombinant factor H binding protein based vaccine to prevent serogroup B meningococcal disease. Vaccine. 28, 6086–6093 (2010).
Article CAS PubMed Google Scholar
Scarselli, M. et al. Rational design of a meningococcal antigen inducing broad protective immunity. Sci. Transl. Med. 3, 91ra62 (2011).
Article CAS PubMed Google Scholar
Huson, D. H. & Bryant, D. Application of phylogenetic networks in evolutionary studies. Mol. Biol. Evol. 23, 254–267 (2006).
Article CAS PubMed Google Scholar
Welsch, J. A., Ram, S., Koeberling, O. & Granoff, D. M. Complement-dependent synergistic bactericidal activity of antibodies against factor H-binding protein, a sparsely distributed meningococcal vaccine antigen. J. Infect. Dis. 197, 1053–1061 (2008).
Article CAS PubMed Google Scholar
Madico, G. et al. The meningococcal vaccine candidate GNA1870 binds the complement regulatory protein factor H and enhances serum resistance. J. Immunol. 177, 501–510 (2006).
Article CAS PubMed PubMed Central Google Scholar
Schneider, M. C. et al. Functional significance of factor H binding to Neisseria meningitidis. J. Immunol. 176, 7566–7575 (2006).
Article CAS PubMed Google Scholar
Granoff, D. M., Welsch, J. A. & Ram, S. Binding of complement factor H (fH) to Neisseria meningitidis is specific for human fH and inhibits complement activation by rat and rabbit sera. Infect. Immun. 77, 764–769 (2009).
Article CAS PubMed Google Scholar
Vu, D. M. et al. Enhanced bacteremia in human factor H transgenic rats infected by Neisseria meningitidis. Infect. Immun. 80, 643–650 (2012).
Article CAS PubMed PubMed Central Google Scholar
Beernink, P. T., Lopasso, C., Angiolillo, A., Felici, F. & Granoff, D. A region of the N-terminal domain of meningococcal factor H-binding protein that elicits bactericidal antibody across antigenic variant groups. Mol. Immunol. 46, 1647–1653 (2009).
Article CAS PubMed PubMed Central Google Scholar
Giuntini, S., Beernink, P. T., Reason, D. C. & Granoff, D. M. Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity. PloS One, In press (2012).
Giuliani, M. M. et al. The region comprising amino acids 100 to 255 of Neisseria meningitidis lipoprotein GNA 1870 elicits bactericidal antibodies. Infect. Immun. 73, 1151–1160 (2005).
Article CAS PubMed PubMed Central Google Scholar
McNeil, L. K. et al. Detection of LP2086 on the cell surface of Neisseria meningitidis and its accessibility in the presence of serogroup B capsular polysaccharide. Vaccine. 27, 3417–3421 (2009).
Article CAS PubMed Google Scholar
Cantini, F. et al. Solution structure of the factor H-binding protein, a survival factor and protective antigen of Neisseria meningitidis. J. Biol. Chem. 284, 9022–9026 (2009).
Article CAS PubMed PubMed Central Google Scholar
Mascioni, A. et al. Structural basis for the immunogenic properties of the meningococcal vaccine candidate LP2086. J. Biol. Chem. 284, 8738–8746 (2009).
Article CAS PubMed PubMed Central Google Scholar
Schneider, M. C. et al. Neisseria meningitidis recruits factor H using protein mimicry of host carbohydrates. .Nature 458, 890–893 (2009).
Article CAS ADS PubMed PubMed Central Google Scholar
Giuntini, S., Reason, D. C. & Granoff, D. M. Complement-mediated bactericidal activity of anti-factor H binding protein monoclonal antibodies against the meningococcus relies upon blocking factor H binding. Infect. Immun. 79, 3751–3759 (2011).
Article CAS PubMed PubMed Central Google Scholar
Scarselli, M. et al. Epitope mapping of a bactericidal monoclonal antibody against the factor H binding protein of Neisseria meningitidis. J. Mol. Biol. 386, 97–108 (2009).
Article CAS PubMed Google Scholar
Gasteiger, J. Chemoinformatics: a new field with a long tradition. Anal. Bioanal. Chem. 384, 57–64 (2006).
Article CAS PubMed Google Scholar
Chao, G., Cochran, J. R. & Wittrup, K. D. Fine epitope mapping of anti-epidermal growth factor receptor antibodies through random mutagenesis and yeast surface display. J. Mol. Biol. 342, 539–550 (2004).
Article CAS Google Scholar
Boder, E. T. & Wittrup, K. D. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15, 553–557 (1997).
Article CAS PubMed Google Scholar
Granoff, D. M. et al. Bactericidal monoclonal antibodies that define unique meningococcal B polysaccharide epitopes that do not cross-react with human polysialic acid. J. Immunol. 160, 5028–5036 (1998).
CAS PubMed Google Scholar
Beernink, P. T. et al. Prevalence of factor H-binding protein variants and NadA among meningococcal group B isolates from the United States: implications for the development of a multicomponent group B vaccine. J. Infect. Dis. 195, 1472–1479 (2007).
Article CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

This work was supported in part by Public Health Service grants R01 AI 046464 and AI 082263 from the National Institute of Allergy and Infectious Diseases, NIH. D.M.G. also is supported in part by an endowment established by the Clorox Company. The laboratory work at Children's Hospital Oakland Research Institute was performed in a facility funded by Research Facilities Improvement Program grant C06 RR 016226 from the National Center for Research Resources, NIH. We are grateful to Denise Playdle Green for expert technical assistance.

Author information

Authors and Affiliations

Children's Hospital Oakland Research Institute, Oakland, CA 94609, U.S.A.
David M. Vu, Rolando Pajon, Donald C. Reason & Dan M. Granoff

Authors

David M. Vu
View author publications
You can also search for this author in PubMed Google Scholar
Rolando Pajon
View author publications
You can also search for this author in PubMed Google Scholar
Donald C. Reason
View author publications
You can also search for this author in PubMed Google Scholar
Dan M. Granoff
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

R.P. performed the phylogenic analyses of fHbp sequence variants and prepared Figure 2 and Supplementary Figure 4. D.C.R. sorted the fHbp ID 1 yeast display mutants and assisted with mapping the JAR 41 epitope. D.M.V. and D.M.G. designed the study, wrote the main manuscript text and prepared the remaining figures and table 1. All authors reviewed and contributed to writing of the manuscript.

Ethics declarations

Competing interests

D.M.G. holds a paid consultancy with Novartis Vaccines and Diagnostics. D.M.G. R.P. and D.M.V. are inventors on patents or patent applications in the area of meningococcal B vaccines. D.C.R. report no conflicts of interest.

Electronic supplementary material

Supplementary Information

Supplementary Figures S1 to S4

Rights and permissions

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareALike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/

Reprints and permissions

About this article

Cite this article

Vu, D., Pajon, R., Reason, D. et al. A Broadly Cross-Reactive Monoclonal Antibody Against an Epitope on the N-terminus of Meningococcal fHbp. Sci Rep 2, 341 (2012). https://doi.org/10.1038/srep00341

Download citation

Received: 11 January 2012
Accepted: 05 March 2012
Published: 28 March 2012
DOI: https://doi.org/10.1038/srep00341

This article is cited by

A homopolymeric adenosine tract in the promoter region of nspA influences factor H-mediated serum resistance in Neisseria meningitidis
- Heike Claus
- Kerstin Hubert
- Kay Johswich
Scientific Reports (2019)
Structural basis for cooperativity of human monoclonal antibodies to meningococcal factor H-binding protein
- Ilaria Peschiera
- Maria Giuliani
- Ilaria Ferlenghi
Communications Biology (2019)
Crystal structure reveals vaccine elicited bactericidal human antibody targeting a conserved epitope on meningococcal fHbp
- Jacinto López-Sagaseta
- Peter T. Beernink
- Matthew J. Bottomley
Nature Communications (2018)
Human protective response induced by meningococcus B vaccine is mediated by the synergy of multiple bactericidal epitopes
- M. Giuliani
- E. Bartolini
- V. Masignani
Scientific Reports (2018)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.