Factor H-binding protein (fHbp) is an important antigen of Neisseria meningitidis that is capable of eliciting a robust protective immune response in humans. Previous studies on the interactions of fHbp with antibodies revealed that some anti-fHbp monoclonal antibodies that are unable to trigger complement-mediated bacterial killing in vitro are highly co-operative and become bactericidal if used in combination. Several factors have been shown to influence such co-operativity, including IgG subclass and antigen density. To investigate the structural basis of the anti-fHbp antibody synergy, we determined the crystal structure of the complex between fHbp and the Fab (fragment antigen-binding) fragment of JAR5, a specific anti-fHbp murine monoclonal antibody known to be highly co-operative with other monoclonal antibodies. We show that JAR5 is highly synergic with monoclonal antibody (mAb) 12C1, whose structure in complex with fHbp has been previously solved. Structural analyses of the epitopes recognized by JAR5 and 12C1, and computational modeling of full-length IgG mAbs of JAR5 and 12C1 bound to the same fHbp molecule, provide insights into the spatial orientation of Fc (fragment crystallizable) regions and into the possible implications for the susceptibility of meningococci to complement-mediated killing.

Introduction

Factor H-binding protein (fHbp) is a surface-exposed meningococcal lipoprotein that binds human complement factor H (fH) and is one of the recombinant protein components of Bexsero® and Trumenba®, two recently licensed vaccines against serogroup B meningococcus [1]. Anti-fHbp antibodies can elicit complement-mediated bactericidal activity [2] and inhibit the binding of fH to the bacterial surface. Both mechanisms contribute to the susceptibility of meningococci to complement-mediated killing [3]. While some anti-fHbp monoclonal antibodies (mAbs) fail to promote bacterial killing in the presence of human complement, combinations of two anti-fHbp mAbs frequently are bactericidal [4].

The classical complement pathway is triggered when antigen-bound immunoglobulins bind the complement component 1 (C1) complex formed by the C1q, C1r and C1s subunits [5]. C1q is the recognition subunit, having a bouquet-like structure formed by six heterotrimeric subunits. Each subunit is formed by three helices merging at their C-terminal ends into globular heads [69]. Complement activation is triggered by the initial binding of the globular domains to the Fc (fragment crystallizable) portions of antigen-bearing immunoglobulins. While C1q binds a single Fc with low affinity, the more avid stable binding of two or more of the six globular heads activates the serine protease subunits C1r and C1s, which in turn initiates the downstream reactions of the classical cascade, ultimately resulting in bacteriolysis. In the case of an antigen such as fHbp, which is present on the surface of virtually all pathogenic meningococcal strains [10], efficient C1q engagement by multiple copies of the same mAb can occur under conditions of high expression levels, which ensure a sufficient antigen density on the bacterial surface. Alternatively, the ability of two or more distinct mAbs that bind simultaneously to nonoverlapping epitopes on the same antigen molecule could, in principle, efficiently activate the C1 complex even under conditions of low antigen density [4,11].

Despite the wealth of information on the epitopes recognized by various anti-fHbp mAbs [4,1215], there still is a relatively poor understanding of the structural basis for the synergy of two mAbs that elicit complement-mediated bactericidal activity when binding simultaneously to fHbp. In the present study, we investigated the structural basis underlying the synergy between JAR5 and 12C1, two murine IgG2b mAbs. Although individually these mAbs display bactericidal activity in the presence of rabbit complement, the mAbs were previously reported to have negligible bactericidal activity in the presence of human complement [4,13]. However, JAR5 displayed high bactericidal activity in the presence of human complement when used in combination with other mAbs [4,16].

Here, we report the crystal structure of the complex between the Fab (fragment antigen-binding) fragment of mAb JAR5 and fHbp variant 1.1 (also referred to as peptide ID 1), and present a model of the ternary complex of fHbp bound to mAbs JAR5 and 12C1. We used a serum bactericidal activity assay to assess the possible co-operation of the two mAbs and discuss the spatial orientations of the Fc portions predicted by molecular modeling. Collectively, this work provides a detailed characterization of the fHbp epitope recognized by JAR5, and it helps to elucidate the structural basis for the synergistic complement-mediated bactericidal activity by co-operative mAbs targeting the same antigen molecule.

Materials and methods

Cloning, site-specific mutagenesis and protein purification

A gene fragment (residues 73–320 of the UniProtKB database entry Q9JXV4) encoding fHbp variant 1.1 (variant group 1, peptide ID 1 as designated in the public database at http://pubmlst.org/neisseria/fHbp) was cloned into the pET21b expression plasmid (Novagen) as described previously [2]. Plasmids encoding mutant fHbps were constructed using the Phusion Site-Directed Mutagenesis Kit (Thermo Scientific) or the QuikChange II Site-Directed Mutagenesis Kit (Agilent). The mutant fHbp genes were confirmed by DNA sequencing (Davis Sequencing, Davis, CA, U.S.A.). Escherichia coli strain BL21(DE3) cells were transformed with the plasmids described above to enable IPTG (isopropyl β-d-1-thiogalactopyranoside)-inducible fHbp production at 37°C.

The fHbp mutants were purified by Ni2+ affinity chromatography, using a 5 ml HiTrap Chelating HP column (GE Life Sciences). The binding buffer was 20 mM sodium phosphate, 500 mM NaCl and 20 mM imidazole, pH 7.4, and fHbp was eluted with a linear gradient from 20 to 250 mM imidazole. A second purification step was performed using ion exchange chromatography, using a 5 ml HiTrap SP HP column (GE Life Sciences). The binding buffer was 25 mM MES and 250 mM NaCl, pH 5.5, and fHbp was eluted with a linear gradient from 250 to 750 mM NaCl. Fractions containing purified fHbp were pooled and dialyzed against phosphate-buffered saline (PBS) containing 3% (w/v) sucrose. The size and purity of the proteins was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE; Invitrogen 4–12% NuPAGE, Bis–Tris) and Coomassie blue G-250 staining (Invitrogen Simply Blue SafeStain).

Generation and purification of antibodies and Fabs

The hybridoma cell line expressing JAR5 was kindly provided by Prof. D.M. Granoff (UCSF Benioff Children's Hospital Oakland). The murine IgG2b subclass mAbs JAR5 and 12C1 and the corresponding Fab fragments were produced and purified by Areta International SrL (Gerenzano, Italy).

mAbs were purified from culture supernatant by Protein G affinity columns (GE Healthcare). For Fab generation, the antibodies were dialyzed against 20 mM sodium phosphate buffer (pH 7); before digestion, 20 mM cysteine–HCl and 10 mM EDTA were added to the dialyzed antibodies and the solution was incubated with agarose immobilized papain (PIERCE) for 3 h at 37°C under stirring. At the end of the incubation, the reaction mixture was applied to spin columns to separate antibodies from immobilized papain by centrifugation. Fc fragments were removed from the supernatant using Protein A Sepharose. The solution was loaded on the matrix and washed with PBS, and the Fab portion was harvested in the flow-through fraction that was then dialyzed against PBS.

Protein crystallization

The complex of fHbp with Fab JAR5 was prepared by co-incubation at 4°C overnight followed by preparative size-exclusion chromatography in 20 mM Tris–HCl, 150 mM NaCl, pH 8.0. Crystallization screening experiments were prepared by mixing equal volumes (200 nl) of the fHbp–Fab JAR5 complex (15 mg/ml) with crystallization reservoir solution, using a Crystal Gryphon liquid handling robot (Art Robbins Instruments). Crystals were grown at 22°C in a sitting-drop vapor-diffusion format using 96-well low-profile Intelliplates (Art Robbins Instruments) and obtained within 48 h using a reservoir consisting of 20% polyethylene glycol (PEG) 6000 and 0.1 M trisodium citrate, pH 5.0.

Structure determination

Before data collection, crystals were washed in a cryoprotectant solution made of 20% PEG 6000, 0.1 M trisodium citrate and 20% ethylene glycol, and were flash-cooled in liquid nitrogen for subsequent data collection. X-ray diffraction data were collected at 100K, at wavelength λ = 1.0 Å, on beamline X06DA at the Swiss Light Source (SLS, Paul Scherrer Institute, Villigen, Switzerland). Data were indexed and integrated using iMOSFLM [17] and were reduced using Scala within the CCP4 program suite [18]. Crystals of the fHbp–Fab JAR5 complex belong to space group C2221, with the asymmetric unit containing one complex and a solvent content of 65.6% (Matthews coefficient of 3.57 Å3/Da). The structure of the fHbp–Fab JAR5 complex was determined at 2.98 Å resolution by molecular replacement with Phaser [19] using three separate search models obtained from the Protein Data Bank (PDB), namely fHbp from PDB entry 2W80 [20], and trimmed Fab coordinates from PDB entries 1NGP and 1MJJ. Rigid body and restrained refinement were carried out with PHENIX [21] and BUSTER [22] with the target-structure restraints option (LSSR) [23] using as fixed co-ordinates those of fHbp from PDB 2YPV [13]. Manual model building was performed in Coot [24]. The final refined model of the complex contains the sequences of the variable regions of the heavy and light chains of Fab JAR5. Since the constant regions of JAR5 are unknown, those from the highest structural homologs to JAR5, PDB entries 1MJJ and 1NGP that were used as templates for molecular replacement, were included in the final model. Structure quality was assessed using Molprobity [25], while protein–protein interface areas were analyzed and calculated using the Protein Interfaces, Surfaces and Assemblies service (PISA) available at the European Bioinformatics Institute (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) [26]. Figures were generated using PyMOL (http://www.pymol.org). Data collection and refinement statistics are provided in Table 1 and the coordinates have been deposited in PDB (ID 5T5F).

Table 1
X-ray data collection and refinement statistics

Statistics for the highest-resolution shell are shown in parentheses.

Rwork = Σ||F(obs)| − |F(calc)||/Σ|F(obs)|.

Rfree = Rwork but calculated for 5% of the total reflections, chosen at random and omitted from refinement.

 Complex fHbp:Fab JAR5 
Data collection 
 Beamline X06DA–PXIII (SLS) 
 Wavelength (Å) 1.0 
 Resolution range (Å) 73.24–2.98 (3.16–2.98) 
 Space group C2221 
 Unit cell (a, b, c) (Å) 65.37, 146.49, 218.22 
 Total reflections 123 052 (17 673) 
 Unique reflections 21 885 (3437) 
 Multiplicity 5.6 (5.1) 
 Completeness (%) 99.9 (99.7) 
 Mean I/σ(I5.5 (1.3) 
 Wilson B-factor (Å266.47 
Rsym 0.21 (1.096) 
Rmeas 0.255 (1.354) 
CC1/2 0.977 (0.429) 
Refinement 
Rfactor 0.223 (0.248) 
Rfree 0.273 (0.289) 
 Number of nonhydrogen atoms 5057 
  Protein 5023 
  Solvent 34 
 Protein residues 654 
 RMS bonds (Å) 0.014 
 RMS angles (°) 1.23 
 Ramachandran favored (%) 94 
 Ramachandran outliers (%) 0.93 
 Clashscore 8.15 
 Average B-factor (Å269.63 
  Macromolecules 69.84 
  Solvent 39.15 
 PDB ID 5T5F 
 Complex fHbp:Fab JAR5 
Data collection 
 Beamline X06DA–PXIII (SLS) 
 Wavelength (Å) 1.0 
 Resolution range (Å) 73.24–2.98 (3.16–2.98) 
 Space group C2221 
 Unit cell (a, b, c) (Å) 65.37, 146.49, 218.22 
 Total reflections 123 052 (17 673) 
 Unique reflections 21 885 (3437) 
 Multiplicity 5.6 (5.1) 
 Completeness (%) 99.9 (99.7) 
 Mean I/σ(I5.5 (1.3) 
 Wilson B-factor (Å266.47 
Rsym 0.21 (1.096) 
Rmeas 0.255 (1.354) 
CC1/2 0.977 (0.429) 
Refinement 
Rfactor 0.223 (0.248) 
Rfree 0.273 (0.289) 
 Number of nonhydrogen atoms 5057 
  Protein 5023 
  Solvent 34 
 Protein residues 654 
 RMS bonds (Å) 0.014 
 RMS angles (°) 1.23 
 Ramachandran favored (%) 94 
 Ramachandran outliers (%) 0.93 
 Clashscore 8.15 
 Average B-factor (Å269.63 
  Macromolecules 69.84 
  Solvent 39.15 
 PDB ID 5T5F 

Western blotting and inhibition enzyme-linked immunosorbent assay

Anti-fHbp mAb binding to fHbp mutants was studied by western blotting and inhibition enzyme-linked immunosorbent assay (ELISA). For western blotting, the purified proteins (0.5 µg) were subjected to SDS–PAGE as above and transferred to polyvinylidene fluoride membrane (Millipore Immobilon-FL). Nonspecific binding to the membrane was blocked with PBS containing 0.1% Tween-20 (Sigma–Aldrich; PBST) and 1% nonfat dry milk (Nestle Carnation). fHbp was detected with anti-fHbp mAb JAR1 (0.5 µg/ml) or JAR5 (0.1 µg/ml) by incubation for 1 h at room temperature (18–26°C). After washing with PBST, rabbit anti-mouse IgG conjugated to IRDye 800 nm (1:20 000 dilution; Rockland Immunochemicals) was added and the membrane was incubated for 1 h at room temperature. After washing again, the bound secondary antibody was visualized using an infrared scanner (Li-Cor Odyssey).

For inhibition ELISA, the wells of microtiter plates were coated with purified wild–type (WT) fHbp variant 1.1 (100 µl of a 2 µg/ml solution) at 4°C overnight. The next day, the wells were washed with PBST, 0.01% NaN3 and blocked with PBST, 0.01% NaN3 and 1% (w/v) BSA (Cohn Fraction IV; Lifeblood). The soluble WT or mutant fHbp inhibitor (serial five-fold dilutions from 50 to 0.02 µg/ml) was added followed by the anti-fHbp mAb (1 µg/ml for JAR1 or 0.2 µg/ml for JAR5). After incubation at 37°C for 1 h, the wells were washed as described above, and bound anti-fHbp mAbs were detected with goat anti-mouse IgG conjugated to alkaline phosphatase (1:5000 dilution; Sigma–Aldrich) for 1 h at room temperature. After washing again, phosphatase substrate para-nitrophenyl phosphate (1 mg/ml; Sigma–Aldrich) was added and the absorbance at 405 nm was read after incubation for 30 min at room temperature.

Differential scanning calorimetry

fHbp proteins were dialyzed against PBS overnight at 4°C (SpectraPor 10 K MWCO). The next day, the absorbance at 280 nm was measured (Nanodrop 1000) and the protein concentration was determined using a molar extinction coefficient of 8940 M−1 cm−1, which was calculated with ProtParam (http://web.expasy.org/protparam/) [27]. The dialyzed protein was diluted to 0.5 mg/ml with PBS, and the thermal stability was determined using a VP-DSC calorimeter (MicroCal) operating at a scan rate of 60°C/h and in the passive feedback mode. Transition midpoint (Tm) values were determined with a non-two-state unfolding model using the Origin 5 software (MicroCal).

Surface plasmon resonance

Surface plasmon resonance (SPR) was used to compare the binding affinity of fHbp mutants with that of the murine mAb JAR5. All SPR experiments were performed using a Biacore T200 instrument at 25°C (GE Healthcare). For the single-cycle kinetics (SCK) experiments, a commercially available Mouse Antibody Capture Kit (GE Healthcare) was used to immobilize anti-mouse IgG antibodies by amine coupling on a carboxymethylated dextran sensor chip (CM-5; GE Healthcare). A density level yielding ∼10 000 response units (RUs) was achieved. The immobilized anti-mouse IgG was used then to capture ∼1000 RU murine mAb JAR5. Experimental SPR running buffer contained 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% (vol/vol) P20 surfactant, pH 7.4 (HBS-EP). For the determination of dissociation constant (KD) and kinetic rate constants, a titration series of five consecutive injections of purified fHbp protein diluted in HBS-EP at increasing concentration (range 1.875–30 nM; flow rate of 40 µl/min) followed by a single final surface regeneration step with buffer containing 10 mM glycine, pH 1.7 (180 s; 10 µl/min), was performed, using the standard SCK method [28] implemented by the Biacore T200 Control Software (GE Healthcare). Anti-mouse IgG-coated surfaces without captured mAb were used as the reference channel. The reference sensorgrams were subtracted from experimental sensorgrams, and a blank injection of buffer only was subtracted from each curve to yield curves representing specific binding. The data shown are representative of two independent experiments. SPR data were analyzed using the Biacore T200 Evaluation software (GE Healthcare). Each sensorgram was fitted with the 1:1 Langmuir binding model, including a term to account for potential mass transfer, to obtain the individual kon and koff kinetic constants; the individual values were then combined to derive the single averaged KD values reported.

For the competition experiments, purified fHbp was covalently immobilized by amine coupling on a CM5 chip to reach a density of ∼500 RU. Then, mAbs at a concentration of 100 nM in HBS-EP were sequentially injected. First, two injections of 180 s at 10 µl/min of one mAb were performed to reach saturation; then, the second mAb was injected and binding levels were compared. Regeneration between injections was achieved by two sequential injections of 10 mM glycine pH 1.7 and 50 mM NaOH (20 s each at 30 µl/min).

Bactericidal activity assay

Bactericidal activity of anti-fHbp mAbs individually or in combination was evaluated against the MC58 strain [B:15:P1.7,16-2: ST-74 (cc32)], which expresses fHbp variant 1.1, as described previously [29] with minor modifications, using human plasma as the source of complement obtained from volunteer donors under informed written consent.

Early-log phase cultures of Neisseria meningitidis were diluted in Dulbecco's PBS (Sigma) containing 1% BSA and 0.1% glucose (assay buffer) at a working dilution of 104–105 colony-forming units per milliliter (CFU/ml). Bacteria were incubated with serial two-fold dilutions of test mAbs (50 µg/ml to 0.1 µg/ml) and 25% of human plasma. The bactericidal activity was defined as the mAb concentration that resulted in at least 50% decrease in CFU/ml after incubation for 1 h in the reaction mixture compared with the CFU/ml in negative control wells at time 0. Control wells contained bacteria incubated with 25% of human plasma without mAbs or bacteria incubated with mAbs in the presence of 25% of human plasma inactivated by heating at 56°C for 30 min.

Molecular modeling

All the computer-generated molecular models were built with the Swiss PDB v. 4.0.4 software [30]. The ternary complex of JAR5 and 12C1 Fabs with fHbp was obtained by superimposing the atomic co-ordinates of fHbp from the Fab JAR5–fHbp complex (the present study) and the Fab 12C1–fHbp complex previously reported (PDB 2YPV) [13].

The ternary complex formed by full-length JAR5 and 12C1 mAbs complexed with fHbp was generated by superimposing the light chain domain and heavy chain domain 1 (CH1) from each Fab onto the corresponding regions of a murine IgG2a structure (PDB 1IGT). Hinge regions were adjusted manually to accommodate the Lys32–His327 insertion in the IgG2b models. The final model was energy-minimized using the GROMOS implementation in Swiss PDB viewer [31].

Results

The crystal structure of the fHbp–JAR5 Fab complex reveals shape complementarity at the epitope–paratope interface

The X-ray crystal structure of the complex between fHbp and the Fab fragment of JAR5 was solved by molecular replacement at 2.98 Å resolution. Continuous electron density allowed modeling of residues 30–255 of fHbp, and residues 1–218 and 1–216 of the heavy and light chains of the Fab JAR5, respectively. The final model was refined to an Rwork/Rfree of 22.3/27.3% (Table 1). The interface between fHbp and JAR5 is formed by 21 fHbp and 30 JAR5 residues, which contribute a total of ∼800 (on fHbp) and ∼700 (on JAR5) Å2 of surface area to the interaction surface. These buried surfaces at the epitope–paratope interface correspond to only ∼7 and ∼4% of the total surface area of fHbp and Fab JAR5, respectively. The epitope of fHbp recognized by JAR5 is composed of two loops comprising residues 84–91 and 115–123, which are localized on the more polar side of the fHbp N-terminal domain (Figure 1A). The interface formed by fHbp and Fab JAR5 reveals notable shape complementarities, with the two epitope loops of fHbp trapping the heavy chain complementarity-determining regions 1, 2 and 3 of JAR5 (Figure 1B,C). Remarkable epitope–paratope complementarity can also be observed in the distribution of electrostatic potentials (Supplementary Figure S1).

Overall fold and epitope–paratope interface of the complex fHbp:Fab JAR5.

Figure 1.
Overall fold and epitope–paratope interface of the complex fHbp:Fab JAR5.

(A) The structure of the complex is depicted as surface representation, with fHbp on top and colored in cyan and green for the C- and N-terminal domains, respectively, while the epitope for JAR5 is colored in red. Heavy and light chains of Fab JAR5 are colored in dark gray and blue, while the paratope is colored in yellow. (B and C) Closer views of the epitope–paratope interface, with the surfaces of the epitope and of the paratope shown in red and yellow. Epitope residues that directly contact JAR5 are labeled white, in (B) and in (C), and are also shown as spheres in (C).

Figure 1.
Overall fold and epitope–paratope interface of the complex fHbp:Fab JAR5.

(A) The structure of the complex is depicted as surface representation, with fHbp on top and colored in cyan and green for the C- and N-terminal domains, respectively, while the epitope for JAR5 is colored in red. Heavy and light chains of Fab JAR5 are colored in dark gray and blue, while the paratope is colored in yellow. (B and C) Closer views of the epitope–paratope interface, with the surfaces of the epitope and of the paratope shown in red and yellow. Epitope residues that directly contact JAR5 are labeled white, in (B) and in (C), and are also shown as spheres in (C).

Of the 21 total fHbp residues that form the interface with the Fab of JAR5, 18 contact the heavy chain of the Fab, burying a surface area of ∼650 Å2, whereas 7 residues interact with the light chain of the Fab, with a buried accessible surface area (ASA) of ∼200 Å2. Epitope residues 119–123 are located near the interface between chains H and L and make interactions with both chains (Figure 2A), while six residues of fHbp make direct polar interactions only with the heavy chain of JAR5 (JAR5-H). The six fHbp residues directly bonding to JAR5-H (Asp85, Gln87, Gln115, Ser117, Gly121 and Lys122) are asymmetrically distributed between two loops consisting of residues 84–91 and 115–123. On fHbp loop 84–91, both side chain oxygen (O) atoms of Asp85 are optimally positioned and oriented to make hydrogen (H) bonds with Thr28 and Tyr32 of JAR5-H (Figure 2A), whereas the side chain N of Gln87 is located at a distance of ∼4 Å from the side chain O of Thr106 of JAR5-H. Although this distance is compatible with the formation of an H-bond, electron densities of the Gln87 side chains starting from the Cγ atom are not fully resolved and suggest it might be flexible. On fHbp loop 115–123, the side chain N and O atoms of Gln115 are both positioned within H-bonding distance from the main chain O atom of Asp103 of JAR5-H, whereas residues Ser117 and Gly121 contact side chain atoms of Trp33 and Arg108 of JAR5-H through their backbone O atoms. Finally, the side chain of fHbp Lys122 makes H-bonds with Tyr99 of JAR5-H. Interestingly, the side chain of Lys122 points toward a proximal region filled with backbone O atoms or side chain OH groups that belong to residues 91, 92, 95 and 96 of JAR5-L, and Lys122 seems to adopt an unusual rotamer conformation in order to interact with Tyr99 of JAR5-H. An alignment of representative fHbp sequences from variant groups 1, 2 and 3 (Figure 2B) reveals that while Asp85 and Gln87 are conserved, other residues (Gln115, Ser117, Gly121 and Lys122) that interact with JAR5 are not conserved in fHbp variant groups 2 and 3. This might explain the variant group 1 specificity of JAR5 [16].

Interactions at the interface between fHbp and JAR5.

Figure 2.
Interactions at the interface between fHbp and JAR5.

(A) Cartoons of fHbp-N, JAR5-L and JAR5-H are shown in green, blue and gray, respectively. Red color on fHbp-N shows all regions interacting with JAR5, while yellow sticks show fHbp residues (labeled in red) involved in direct polar bonds with residues of JAR5-H (shown with gray sticks and gray labels). Direct bonds are indicated with red dashed lines. (B) Sequence alignment of fHbp variants 1.1, 2.16 and 3.28, with the JAR5 epitope annotated with black bars at the bottom and labeled. Residues of fHbp v1.1 involved in direct interactions with JAR5 are marked with black circles on top.

Figure 2.
Interactions at the interface between fHbp and JAR5.

(A) Cartoons of fHbp-N, JAR5-L and JAR5-H are shown in green, blue and gray, respectively. Red color on fHbp-N shows all regions interacting with JAR5, while yellow sticks show fHbp residues (labeled in red) involved in direct polar bonds with residues of JAR5-H (shown with gray sticks and gray labels). Direct bonds are indicated with red dashed lines. (B) Sequence alignment of fHbp variants 1.1, 2.16 and 3.28, with the JAR5 epitope annotated with black bars at the bottom and labeled. Residues of fHbp v1.1 involved in direct interactions with JAR5 are marked with black circles on top.

Structure-based mutagenesis of fHbp reveals key epitope residues

Based on the analysis of residues involved in polar contacts revealed by the co-crystal structure, we generated seven site-specific mutants of fHbp with the aim of elucidating the relative contributions of each residue to the stability of the complex between fHbp and JAR5. Five alanine mutants (Asp85Ala, Gln87Ala, Gln115Ala, Gly121Ala and Lys122Ala) were designed based on the analysis of the epitope–paratope interface in the co-crystal structure, while the two Gly121Arg and Lys122Ser mutants were designed based on sequence polymorphisms among natural fHbp variants, and were previously shown to decrease binding to JAR5 [4]. Because Ser117 mediates H-bonds with JAR5 only through its backbone O atom, we did not mutate this residue. All of the mutant proteins were highly purified as judged by SDS–PAGE (Supplementary Figure S2). As detected by western blotting, JAR5 bound to WT fHbp. JAR5 bound to the Gln87Ala mutant, but not to the Asp85Ala, Gln115Ala, Gly121Ala and Lys122Ala mutants (Figure 3A). This was not surprising, considering the peripheral localization of Gln87 in the epitope. As expected, JAR5 also did not bind to the two mutants, Gly121Arg and Lys122Ser. The control anti-fHbp mAb JAR1, targeting the C-terminal domain [32], bound to the WT and to all seven mutant proteins (Figure 3B). Next, inhibition ELISA was used to test the ability of the solution-phase mutant proteins to inhibit binding of JAR5 to immobilized WT fHbp. Similar to the results from western blotting, only the WT and Gln87Ala mutant gave significant inhibition (Figure 3C). In contrast, the soluble WT and mutant proteins showed similar inhibition of binding of the control mAb JAR1 (Figure 3D).

Binding of mutant fHbps to anti-fHbp mAbs.

Figure 3.
Binding of mutant fHbps to anti-fHbp mAbs.

Western blotting using anti-fHbp mAbs JAR5 (A) and JAR1 (B). M, molecular mass marker; 1, fHbp WT; 2, Asp85Ala mutant; 3, Gln87Ala; 4, Gln115Ala; 5, Gly121Arg; 6, Gly121Ala; 7, Lys122Ser; 8, Lys122Ala. Inhibition of binding by JAR5 (C) and JAR1 (D) to fHbp was determined by ELISA. WT fHbp was immobilized in the wells of the microtiter plate, and serial dilutions of WT or mutant fHbp inhibitor were added to the wells followed by a fixed concentration of mAb. Percent inhibition was calculated from the optical density compared with control wells without mAb. The mean and range of duplicate measurements are shown.

Figure 3.
Binding of mutant fHbps to anti-fHbp mAbs.

Western blotting using anti-fHbp mAbs JAR5 (A) and JAR1 (B). M, molecular mass marker; 1, fHbp WT; 2, Asp85Ala mutant; 3, Gln87Ala; 4, Gln115Ala; 5, Gly121Arg; 6, Gly121Ala; 7, Lys122Ser; 8, Lys122Ala. Inhibition of binding by JAR5 (C) and JAR1 (D) to fHbp was determined by ELISA. WT fHbp was immobilized in the wells of the microtiter plate, and serial dilutions of WT or mutant fHbp inhibitor were added to the wells followed by a fixed concentration of mAb. Percent inhibition was calculated from the optical density compared with control wells without mAb. The mean and range of duplicate measurements are shown.

SPR-binding studies demonstrate decreased binding to JAR5 by fHbp mutants

The ability of the WT and mutant fHbp proteins to bind JAR5 was measured by SPR, by capturing mAb JAR5 as the ligand and injecting fHbp as the analyte. These experiments showed that the Gln87Ala mutant had a binding profile to JAR5 that was most comparable with WT, whereas all of the other mutants displayed considerably faster dissociation than the WT protein, and the Gly121Arg mutant had no detectable binding. To determine binding affinities, single-cycle kinetic experiments were performed, using a range of concentrations of the fHbp mutants (Supplementary Figure S3). The interaction of Fab JAR5 with fHbp WT was of high affinity, demonstrating an equilibrium dissociation constant (KD) of 0.2 nM. Interestingly, Gln87Ala had an SPR-binding profile similar to the WT, but showed a 10-fold weaker interaction (KD = 1.7 nM). Most of the other mutants showed a dramatically reduced (but measurable) affinity for JAR5, with similar KD values that were ∼100-fold lower than WT fHbp (Table 2).

Table 2
Binding of fHbp epitope mutants to mAb JAR5.

Kinetic dissociation constants (KD) derived from SPR experiments measuring binding of fHbp to mAb JAR5 from two replicates/independent experiments. n.d. stands for ‘not determined’ due to insufficient binding (<10 RU). Standard deviations were obtained from fitting procedures in the Biacore T200 Evaluation software.

fHbp KD (M) 
Asp85Ala 2.0 ± 1.3 × 10−8 
Gln87Ala 1.7 ± 1.1 × 10−9 
Gln115Ala 2.3 ± 0.6 × 10−8 
Gly121Ala 2.0 ± 0.6 × 10−8 
Gly121Arg n.d. 
Lys122Ala 1.7 ± 0.3 × 10−8 
Lys122Ser 1.3 ± 0.5 × 10−8 
WT 1.9 ± 0.2 × 10−10 
fHbp KD (M) 
Asp85Ala 2.0 ± 1.3 × 10−8 
Gln87Ala 1.7 ± 1.1 × 10−9 
Gln115Ala 2.3 ± 0.6 × 10−8 
Gly121Ala 2.0 ± 0.6 × 10−8 
Gly121Arg n.d. 
Lys122Ala 1.7 ± 0.3 × 10−8 
Lys122Ser 1.3 ± 0.5 × 10−8 
WT 1.9 ± 0.2 × 10−10 

Differential scanning calorimetry confirms that all mutant fHbp proteins possess thermal stability similar to WT fHbp

To test whether the decreased binding of JAR5 to the mutant proteins might have been due to decreased thermal stability caused by the amino acid substitutions, we performed differential scanning calorimetry. As had been observed previously [13,33], the WT fHbp (variant 1.1) unfolds with two transitions (Supplementary Figure S4), which correspond to unfolding of the N- and C-terminal structural domains. All of the mutant proteins tested here unfolded with similar profiles to the WT (Supplementary Figure S4). Slight reductions in the Tm for the N-terminal domain were observed for the Gly121Ala, Lys122Ala and Lys122Ser mutants; however, these experiments suggested that the reductions in affinity for JAR5 were not due to large decreases in thermal stability.

Molecular basis of the synergy between 12C1 and JAR5

As previously reported, JAR5 does not elicit human complement-mediated bactericidal activity alone, but can elicit bactericidal activity in combination with other murine mAbs [4,16]. We decided therefore to investigate the synergy between JAR5 and 12C1, which is an mAb of the same IgG2b subclass, and whose epitope on fHbp was previously characterized by several methods [13]. As observed for JAR5, 12C1 alone is unable to kill meningococci with human complement, despite its high affinity for fHbp. A model of the ternary complex fHbp:FabJAR5:Fab12C1 was generated by structural superpositions, showing how the physical separation of the JAR5 and 12C1 epitopes allows their simultaneous binding to the same fHbp molecule (Supplementary Figure S5).

To test this hypothesis, the formation of a ternary complex was also investigated by SPR. For this purpose, fHbp was covalently coupled to a CM5 chip and then, the two mAbs, JAR5 and 12C1, were sequentially injected into the detection cell. To ensure complete saturation of the recognized epitope, two injections of the first mAb were performed until no more binding was detected before proceeding with the injection of the other mAb. The experiment was performed in both orders with mAb JAR5 injected until binding was saturated followed by the injection of mAb 12C1 (Figure 4A) and vice versa (Figure 4B). In both cases, both of the mAbs bound the fHbp protein on the chip. As a control experiment, after the two first injections of mAb 12C1, an additional injection of the same mAb was performed and no additional binding was detected, showing that epitope-saturating conditions had been achieved (Figure 4C).

JAR5 and 12C1 are capable of binding the same fHbp molecule.

Figure 4.
JAR5 and 12C1 are capable of binding the same fHbp molecule.

mAbs JAR5 and 12C1 were sequentially injected onto a CM5 chip with covalently immobilized fHbp, in the order JAR5–12C1 (A) and 12C1–JAR5 (B). The first mAb was injected until binding was saturated, and then the second mAb was injected. (C) Control experiment where, after the two first injections of mAb 12C1, an additional injection of the same mAb was performed and no additional binding was detected. RU, response units.

Figure 4.
JAR5 and 12C1 are capable of binding the same fHbp molecule.

mAbs JAR5 and 12C1 were sequentially injected onto a CM5 chip with covalently immobilized fHbp, in the order JAR5–12C1 (A) and 12C1–JAR5 (B). The first mAb was injected until binding was saturated, and then the second mAb was injected. (C) Control experiment where, after the two first injections of mAb 12C1, an additional injection of the same mAb was performed and no additional binding was detected. RU, response units.

To investigate whether the capability of JAR5 and 12C1 to form a stable complex with fHbp could result in synergistic bactericidal activity, both antibodies were tested individually and in combination for the ability to kill meningococcal strain MC58 expressing fHbp variant 1.1. In the presence of human complement, 12C1 had a titer of >50 µg/ml and JAR5 had a titer of ∼20 µg/ml. However, when meningococci were incubated with an equimolar mixture of mAbs JAR5 and 12C1, the titer was of 1.56 µg/ml, indicating significantly higher bactericidal activity (Figure 5).

Bactericidal activity of anti-fHbp mAbs measured against the MC58 strain.

Figure 5.
Bactericidal activity of anti-fHbp mAbs measured against the MC58 strain.

Percent survival of bacteria is measured after incubation for 60 min at 37°C with 12C1, JAR5 or an equimolar mixture and 25% human plasma as a complement source.

Figure 5.
Bactericidal activity of anti-fHbp mAbs measured against the MC58 strain.

Percent survival of bacteria is measured after incubation for 60 min at 37°C with 12C1, JAR5 or an equimolar mixture and 25% human plasma as a complement source.

To explore the structural basis for synergistic activity, we also modeled the ternary complex formed by fHbp bound to the full-length 12C1 and JAR5 mAbs. The model suggested that when both antibodies were bound to fHbp, four binding sites were potentially available at the Fab–Fc interfaces for the interaction with C1q (Figure 6). Residues Glu318, Lys320 and Lys322, on the Fc region, typically define the C1q-binding site in murine IgG2b [34]. In the model, pairs of Glu318 belonging to different antibodies were separated by distances ranging from 115 to 130 Å. A comparable distance separates Glu318 residues belonging to antibodies located at the opposite sides of the hexameric array of human IgG1 bound to C1q, as recently revealed by TEM [35], suggesting that this grouping of accessible Fc regions may be well suited for co-operative C1q engagement.

Model of the ternary complex fHbp:mAb12C1:mAbJAR5.

Figure 6.
Model of the ternary complex fHbp:mAb12C1:mAbJAR5.

fHbp is shown at the bottom with the N- and C-domains depicted with green and cyan surfaces, respectively. A computer model of the JAR5 mAb is depicted with light-gray and blue surfaces for the H and L chains, whereas the model of the 12C1 mAb is shown with dark gray and violet surfaces for the H and L chains. The Fc regions of the two mAbs are labeled in black, while the distances between residues critical for binding to C1q are highlighted and labeled in red.

Figure 6.
Model of the ternary complex fHbp:mAb12C1:mAbJAR5.

fHbp is shown at the bottom with the N- and C-domains depicted with green and cyan surfaces, respectively. A computer model of the JAR5 mAb is depicted with light-gray and blue surfaces for the H and L chains, whereas the model of the 12C1 mAb is shown with dark gray and violet surfaces for the H and L chains. The Fc regions of the two mAbs are labeled in black, while the distances between residues critical for binding to C1q are highlighted and labeled in red.

Discussion

Antibody co-operativity in engaging C1q is an important phenomenon that potentiates the immune response against pathogenic microorganisms. Despite recent advances in the characterization of the molecules involved in the classical complement activation cascade, there are still few data available on the structural basis of the interaction between C1q and pairs of co-operative antibodies. To investigate the molecular mechanisms that regulate this interaction, we selected as a model a pair of murine mAbs raised against meningococcal fHbp. The reasons for this choice rely principally on the large amount of experimental data available on structure, function and immunological properties of this meningococcal antigen [1]. fHbp is a lipoprotein anchored to the bacterial outer membrane through a lipidated N-terminal cysteine. X-ray and NMR structures reveal that fHbp possesses a solvent-ASA of ∼8300 Å2 [36]. Even if a part of this area is shielded in vivo by the bacterial outer membrane, fHbp appears large enough to simultaneously accomodate on its surface more than one functional antibody, given that typical antibody footprints on an antigen vary in surface area from 200 to 1500 Å2 [3740]. However, to our knowledge, the demonstration of simultaneous binding of mAbs to fHbp has not previously been reported.

Previous epitope-mapping studies revealed that murine mAbs targeting well-spaced residues on the fHbp surface, although unable to induce complement-mediated killing when used alone, efficiently trigger the complement cascade when used in combination [4,12,14,16]. Such co-operative bactericidal activity can be explained by two mAbs binding to the same fHbp molecule with appropriate spatial orientations that render their Fc regions available to engage C1q.

This observed antibody synergy may be influenced by several factors in addition to epitope localization, including antigen density and IgG subclass [11]. It has been reported that while murine JAR5 (IgG2b) lacks bactericidal activity against meningococcal strain H44/76, a chimeric construct in which the human IgG1 constant (Fc) region fused to the murine JAR5 antigen-binding (Fab) domains is bactericidal with human complement [41]. These previous studies highlight the importance of combining antibodies of the same subclass to evaluate the contribution to synergy provided by spatial orientation of the Fc regions of pairs of co-operative mAbs. JAR5 and 12C1 are both IgG2b murine mAbs with little or no bactericidal activity in the presence of human complement, despite their ability to inhibit fHbp binding to fH [13,16]. Therefore, we reasoned that the bactericidal activity that we observed with a mixture of 12C1 and JAR5 in the presence of human complement could provide a convincing indication of their synergy. This assumption was supported by the observation that, in the case of JAR5, the ability to co-operate with other mAbs already had been demonstrated [4,16].

To elucidate the molecular bases for the binding of mAb JAR5 to fHbp, we solved the crystal structure of the Fab–fHbp complex. We thus identified the location of the antigenic residues critical for binding to JAR5 on the fHbp N-terminal loops 84–91 and 115–123 (Figure 2A), and studied the binding and stability of selected mutants of these residues to confirm their role in the interface. While only two residues on loop 84–91 (Asp85 and Gln87) make direct polar interactions with JAR5, at least four residues on loop 115–123 (Gln115, Ser117, Gly121 and Lys 122) directly contact the Fab through both side chain and main chain atoms. In addition, while loop 84–91 is located on the periphery of the epitope–paratope interface, loop 115–123 is more centrally located within the interface, and it reaches into a groove of the Fab JAR5 to become partially sandwiched between heavy and light chains (Figure 1C). In agreement with these structural observations, the SPR-binding data confirm that the contribution of loop 84–91 to binding is smaller than that of loop 115–123; the fHbp–JAR5 interaction seems mainly driven by residues on loop 115–123 since the mutants, Gln115Ala, Gly121Ala, Gly121Arg, Lys122Ala and Lys122Ser, all strongly reduce or abolish binding (Supplementary Figure S3). The Gln87Ala mutation had only a moderate impact, probably due to its peripheral location in the binding interface, and relatively large distance (4 Å) from its binding partner.

The fHbp region containing the epitope recognized by JAR5 already has been reported as the target of murine mAbs, elicited either by variant 1.1 (B24 according to a different classification scheme) or variant 1.55 (B01) [4,33]. Structural superposition of fHbp from the 12C1 and JAR5 complexes showed how the location of the epitopes of the two Fabs is probably compatible with their simultaneous binding to the same fHbp molecule. Accordingly, SPR measurements showed that the two mAbs were indeed capable of binding simultaneously to fHbp. Moreover, JAR5 and 12C1 were highly co-operative in the bactericidal assay.

The 12C1 and JAR5 epitopes have 15 and 5 residues, respectively, in common with the fH-binding site [20]. The capability of this mAb pair to shield almost completely the fHbp surface interacting with human fH, in principle, could be the main reason for their synergic activity. However, Vu et al. reported that synergic bactericidal activity also can be observed when combining nonbactericidal murine mAbs that target regions outside the fH binding site [14]. The work of Vu and colleagues suggests that mAb binding to the fH-binding site is not a prerequisite to observe synergic bactericidal activity. We hypothesized therefore that mAbs binding to nonoverlapping sites on the same molecule are the main requirement that renders two antibodies capable of co-operating in inducing complement-mediated bacterial killing

To gain insights into the possible spatial orientation adopted by Fc regions when JAR5 and 12C1 bound fHbp, we modeled a ternary complex of fHbp with both the full-length antibodies. The JAR5 and 12C1 mAbs were modeled using the co-ordinates of a murine IgG2a antibody as a template for the hinge and CH2 and CH3 domains. Although sequence similarity suggested that the known IgG2a structure was an appropriate template to model the IgG2b constant regions with a high level of confidence, it should be noted that there is a large variation in the orientation of the Fab regions in the three crystal structures of intact IgG currently available, where observed Fab–Fab angles range from 115° to 172° and Fab–Fc angles vary from 66° to 123° [4244]. Such values are consistent with structural investigations performed in solution by cryo-electron microscopy and cryo-electron tomography on murine IgG2a, which revealed that the Fab–Fab angle can range from 88° to 140° and Fab–Fc from 15° to 156° [45,46]. Collectively, the body of experimental data suggests that a considerable degree of flexibility can exist within the murine IgG2. Thus, the model that we obtained represents that of a single possible conformation and does not describe the dynamic flexibility that probably exists in solution. Despite these caveats, the predicted conformation of the ternary model fHbp–12C1–JAR5 suggests that each mAb presents two binding sites well accessible to C1q.

Although the relative orientation of the JAR5 and 12C1 Fc regions is remarkably different from that observed in the hexameric array recently described by Diebolder et al. [35], the Fc residues responsible for the binding to C1q in the model of the JAR5–fHbp complex are separated by 115–130 Å, which is a comparable distance with that observed by Diebolder. This observation suggests that simultaneous binding to fHbp by two different antibodies can promote a multivalent high-avidity binding to C1q, thus enabling synergistic activity. Whether such a capability is exclusively due to the reciprocal orientation of the Fc portions or whether steric hindrance deriving from simultaneous binding also contributes to prevent more efficiently the interaction with human fH remains to be elucidated. However, the present work has important implications, since it has been demonstrated that susceptibility of meningococci to complement-mediated killing triggered by anti-fHbp polyclonal sera is strongly dependent on the antigen density on the cell surface and that a critical amount of antigen is required to ensure a stable C1q engagement necessary for the efficient activation of the complement cascade [47]. While antigen density certainly plays a role, here we have demonstrated that co-operativity of antibodies recognizing nonoverlapping epitopes on the same antigen molecule can also make an important contribution to efficient C1q activation, particularly in the case of relative antigen scarcity.

Abbreviations

     
  • ASA

    accessible surface area

  •  
  • BSA

    bovine serum albumin

  •  
  • C1

    component 1

  •  
  • CFU

    colony-forming units

  •  
  • CH1

    chain heavy domain 1

  •  
  • ELISA

    enzyme-linked immunosorbent assay

  •  
  • Fab

    fragment antigen-binding

  •  
  • Fc

    fragment crystallizable

  •  
  • fH

    factor H

  •  
  • fHbp

    factor H binding protein

  •  
  • IPTG

    isopropyl β-d-1-thiogalactopyranoside

  •  
  • mAbs

    monoclonal antibodies

  •  
  • MES

    2-(N-morpholino)ethanesulfonic acid

  •  
  • PBS

    phosphate-buffered saline

  •  
  • PBST

    phosphate-buffered saline with Tween® 20

  •  
  • PDB

    Protein Data Bank

  •  
  • PEG

    polyethylene glycol

  •  
  • RU

    response units

  •  
  • SCK

    single-cycle kinetics

  •  
  • SDS–PAGE

    sodium dodecyl sulfate polyacrylamide gel electrophoresis

  •  
  • SPR

    surface plasmon resonance

  •  
  • TEM

    transmission electron microscopy

  •  
  • WT

    wild type.

Author Contribution

M.S. and P.T.B. conceived the work and designed the experiments. E.M. determined and analyzed the crystal structure. P.L.S. peformed DSC and SPR analyses. D.V. generated reagents and carried out crystallizations. L.S. and E.L. performed the serological studies, H.S. generated and characterized the recombinant mutant proteins, and B.B. advised on serum bactericidal activity analysis. M.J.B. co-ordinated the project. M.S. performed computer modeling. E.M., P.L.S., M.J.B., P.T.B. and M.S. wrote the manuscript.

Funding

The present study was sponsored by Novartis Vaccines, which became GSK Vaccines srl (Italy), now part of the GSK group of companies, which was involved in all stages of the study conduct and analysis. The work at UCSF Benioff Children's Hospital Oakland was supported in part by a grant from the National Institute for Allergy and Infectious Diseases, National Institutes of Health [R01 AI099125] to P.T.B.

Acknowledgments

We thank Joachim Diez and Santina Russo (Expose GmbH, Villigen) for assistance with diffraction data collection at the SLS, Prof. E. Richard Moxon (University of Oxford, Oxford, U.K.) for providing N. meningitidis strain MC58 and Prof. Dan M. Granoff for mAb JAR5.

Competing Interests

All authors have declared the following interests: E.M., P.L.S., D.V., L.S., B.B., E.L., M.J.B. and M.S. were employees of Novartis Vaccines at the time of the study. Following the acquisition of Novartis Vaccines by the GSK group of companies in March 2015, they are now employees of the GSK group of companies. M.J.B. reports ownership of GSK shares and/or restricted GSK shares. P.T.B. is listed as an inventor on patents owned by the GSK group of companies. H.S. reports no financial conflict of interest.

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Supplementary data