Effect of complement Factor H on antibody repertoire and protection elicited by meningococcal capsular group B vaccines containing Factor H binding protein

ABSTRACT

Bacteria produce surface ligands for host complement regulators including Factor H (FH), which allows the bacteria to evade immunity. Meningococcal Factor H binding protein (FHbp) is both a virulence factor and a vaccine antigen. Antibodies to FHbp can neutralize its function by inhibiting binding of FH to the bacteria and confer robust complement-mediated protection. However, in the presence of human or primate FH, antibodies to FHbp do not inhibit FH binding and the protective antibody responses are decreased. This immune suppression can be overcome by modification of the FHbp antigen to decrease FH binding, which modulates the antibody repertoire to inhibit FH binding and increase protection. When FHbp is present at sufficient density on the bacterial surface, two or more antibodies can synergize to activate the complement system. Thus, modification of FHbp antigens to decrease FH binding expands the anti-FHbp antibody repertoire and increases the potential for synergistic activity.

KEYWORDS:

• Bactericidal
• capsular group B
• Factor H
• Factor H binding protein
• meningococcal vaccine
• MenB
• mutant
• protective antibody responses
• structure

Introduction

Meningococcal vaccines based on the polysaccharide capsule have been available since the 1970s, but this approach was not possible against capsular group B strains because the group B polysaccharide, polysialic acid, is present in human tissues.¹ Other approaches have focused on sub-capsular protein antigens, such as PorA, PorB, Rmp and Opa, however, these integral membrane proteins are difficult to produce and formulate into vaccines. Moreover, these proteins are antigenically variable, which likely would lead to poor cross-protection against genetically diverse strains of the bacteria. Since the genomic sequence of a meningococcal group B strain became available, new approaches to develop a vaccine against group B were made possible by the identification of protein antigens irrespective of their abundance in the bacteria.^2,3 More than a dozen years after the first genome sequence was determined and initial candidate vaccine antigens were identified and tested in pre-clinical and clinical studies, two protein-based group B vaccines were developed: MenB-4C (Bexsero®; GlaxoSmithKline Biologicals), which was first licensed in the European Union in 2013 and MenB-FHbp (Trumenba®; Pfizer), first licensed in the United States in 2014.^4–7

Both vaccines contain a key protective antigen known as Factor H binding protein (FHbp), which is a lipoprotein present on the meningococcal surface that was previously referred to as GNA1870 or LP2086.^8,9 These vaccines are described in detail elsewhere.^10–15 Among the differences between the two vaccines, in MenB-4C FHbp is present as a soluble fusion to the GNA2091 protein, whereas, in MenB-FHbp the antigen is present as a tri-palmitoylated lipoprotein.^5,8,16 Further, because FHbp is sequence variable and is classified into two sub-families, MenB-FHbp includes two different sequence variants.^6,9 Another difference is that MenB-4C contains three other protective antigens, NadA, NHba, and PorA serosubtype 7–2,4 (the latter included in the outer membrane vesicle (OMV) component). Thus, two different strategies were employed to broaden protection against diverse group B strains; MenB-4C uses a cocktail of antigens and MenB-FHbp uses two complementary FHbp antigens. Each of these strategies has unique advantages and limitations, which are beyond the scope of this review.

Protection against meningococcal infection is measured in a surrogate assay in which serum antibodies are tested for their ability to decrease survival of the bacteria in the presence of human serum complement proteins (“complement-mediated serum bactericidal activity”). The bactericidal titer is reported as the dilution of serum antibody that yields 50% survival, and a titer of greater than or equal to 1:4 has long been considered to be protective.¹⁷ The ability of the group B vaccines to elicit bactericidal antibody responses depends on the degree to which the sequences of the vaccine antigens match those in the meningococcal strains and on antigen expression in the target strain, as previously shown for FHbp.^18,19

Prior to licensure, the protective antibody responses of humans to the two licensed group B vaccines were tested against a limited number of strains. In the case of MenB-4C, as many as seven strains were chosen to evaluate the efficacy of each of the four protective antigens.^20,21 In the case of MenB-FHbp, up to five strains were tested; two strains expressed FHbp sequence variants that were closely matched to those in the vaccine and three strains expressed FHbp variants with lower sequence similarity compared to those in the vaccine.^22–24 Although it would have been valuable to know better the extent of cross-protective antibody responses elicited by the two vaccines prior to licensure, it was not practical to test individual serum samples for bactericidal activity against many strains.

Since licensure of the two MenB vaccines, several studies have focused on the extent of cross-protection against diverse group B strains. In two parallel studies, sera from subjects vaccinated with each of the two licensed vaccines (collected 1 month after the recommended number of vaccine doses) were tested against a panel of reference and outbreak strains. The percentages of subjects with protective titers (≥1:4) were 70–100 for MenB-4C and 90–100 for MenB-FHbp.^25,26 In another study, serum samples from adolescent human subjects vaccinated with an investigational vaccine containing the four protein antigens present in MenB-4C, along with polysaccharide conjugates from non-group B strains, were tested against 110 group B strains. The breadth of coverage was 67% and 71% after two and three doses, respectively.²⁷ The strain coverage observed was similar to that determined from studies in mice (78%) or from a surrogate ELISA-based approach using pooled human serum from adolescents or adults (72% or 85%, respectively).^5,28 In two other studies, sera from humans vaccinated with the recommended three doses of MenB-FHbp were tested against diverse panels of group B and non-group B strains. Against 27 group B strains, the percentages of subjects with serum bactericidal antibody titers of ≥1:8 (samples collected 1 month after dose 3) were 56–100% and against six non-group B strains the protection was between 28% and 100%.^29,30 It is noteworthy that the bactericidal antibody responses against the one group A strain tested were lower than against the group C, W, X and Y strains, which is likely due to low FHbp expression among group A isolates.³¹

The two group B vaccines are increasingly being used in humans, including the routine immunization of infants in the United Kingdom and vaccination of at-risk groups during outbreaks of meningococcal disease.^32,33 The vaccines are widely considered to be safe and effective, however, there are limitations on their immunogenicity in infants, and on the duration and breadth of protection against genetically diverse strains.³⁴ Several studies have shown that binding of the complement protein Factor H (FH) decreases the protective antibody responses to FHbp.^35–37 It had been suggested that modification of a vaccine antigen such as FHbp to decrease binding of a host protein such as FH would be expected to increase immunogenicity.^38,39 While the superior immunogenicity of mutant FHbp antigens with decreased FH binding compared with the respective wild-type (WT) antignes has been demonstrated in animal models, there are not yet any comparable data available from humans.

Effect of binding of FH on antibody responses

In contrast to the human immunogenicity studies, the studies in animal models had differences in the specificity of complement Factor H (FH) for the FHbp vaccine antigen. In particular, FH from humans, chimpanzees and rhesus macaques can bind FHbp, whereas FH from mice, rats and rabbits cannot.^35,40–42 As a result, in mice and rabbits in which FH does not bind FHbp, the isolated FHbp antigen or the licensed MenB-4C vaccine elicited relatively high serum bactericidal antibody titers, generally between 1:1,000 and 1:10,000 when measured with human complement against different strains with FHbp sequence variants that matched the vaccine antigen.^6,9,36 In contrast, the bactericidal titers of macaques elicited by the isolated FHbp antigen or by MenB-4C, or of humans to either of the licensed MenB vaccines, were lower, generally between 1:10 and 1:100.^{22,25,26,37,43} Although MenB-4C contains antigens in addition to FHbp, the other antigens in strain H44/76 used to measure the anti-FHbp antibody responses either were absent (NadA), mismatched in sequence (PorA), or expressed at a relatively low level (NHba).⁴⁴ Thus, the differences in the bactericidal responses among species, at least against this strain, likely resulted from differences in the specificity of FHbp for FH from primates.

Microbial virulence factors that bind to host proteins represent attractive vaccine candidates since antibodies to the virulence factors could interfere with the interactions with the host proteins.³⁹ However, binding of the host protein to the vaccine antigen might decrease the protective antibody responses. Two human FH transgenic mouse lines were developed, which fortuitously enabled the effect of FH binding on immunogenicity of meningococcal FHbp to be evaluated.^35,45 One of the transgenic mouse lines expressed both mouse and human FH, and its serum concentrations of human FH were similar to those of humans.³⁵ The second line had a chimeric FH comprising mouse FH with domains 6 and 7, which are the domains that bind to FHbp, replaced by the orthologous human sequences, and the FH concentrations appeared to be lower than those of humans.⁴⁵ In a previous review article, we described the effect of binding of human FH on decreasing the immunogenicity of FHbp by comparing the protective antibody responses of WT and human FH transgenic mice to a purified, recombinant FHbp vaccine.⁴⁶ This result was bolstered by a subsequent study that used the licensed MenB-4C vaccine, which also elicited lower protective anti-FHbp antibody responses in human FH transgenic mice than in WT mice.³⁶

A meningococcal native outer membrane vesicle vaccine with over-expressed FHbp was tested in rhesus macaques, however at the time, it was not known whether macaque FH bound to FHbp.⁴⁷ A previous study had shown weak binding of macaque FH to meningococci but it was not investigated whether FHbp was the meningococcal ligand responsible for binding.⁴⁰ Subsequently, we discovered that rhesus macaques have sequence polymorphisms of two amino acid residues of FH domain 6.³⁸ The FH variants resulted in high or low binding to FHbp.^41,42 Approximately 70% of the macaques tested from the California National Primate Research Center (University of California, Davis) had serum FH with low binding to FHbp and 30% had high binding to FHbp. The affinity of the high binding macaque FH for FHbp was similar to that of human FH, with a dissociation constant (K_D) value of approximately 20 nM.⁴² Therefore, given both the genetic and immunologic similarity of rhesus macaques to humans, we proposed that macaques with high binding of FH to FHbp would be a more relevant model for FHbp immunogenicity than the human FH transgenic mouse model. In a pilot study, we tested the immunogenicity of the MenB-4C vaccine in macaques with high or low binding of FH to FHbp.^37,48 Similar to the results of testing MenB-4C in human FH transgenic versus WT mice, the protective antibody responses of macaques with FH binding similar to humans were lower than the responses of macaques with low FH binding.^36,37 Collectively, the results from human FH transgenic mice and rhesus macaques immunized with a licensed group B vaccine indicated that binding of FH significantly decreased the protective antibody responses to FHbp. It was further postulated that modification of a microbial vaccine antigen so as not to bind its cognate host protein would increase immunogenicity, and in particular for FHbp.^38,39

Structures of FHbp variants in the licensed group B vaccines

The crystal structure of a complex between meningococcal FHbp ID1 and a fragment of human FH comprising domains 6 and 7 provided the critical structural information needed to test this hypothesis (Figure 1).³⁸ Because of the large number (>1220) of natural FHbp sequence variants, identification (ID) numbers are assigned in a FHbp peptide database (http://pubmlst.org/neisseria/fHbp). Also, because of the antigenic variability of FHbp, which can be as low as 61% identical, structures of other sequence variants, with or without bound FH, have been determined. There is a published NMR structure of the FHbp sub-family B antigen ID55 (also known as LP2086-B01), which is a component of MenB-FHbp.⁴⁹ There is also a crystal structure of the FHbp sub-family B antigen ID1, which is contained in MenB-4C, without bound FH.⁵⁰ The NMR structure is an ensemble of 17 energy-minimized conformations; therefore, it is not very useful to compare this “structure” with another determined crystallographically. An experimentally determined structure of the second FHbp variant in MenB-FHbp (ID45 or A05) has not been reported. The structures with the highest sequence identity to ID45 are FHbp ID28 bound to FH (93%) and the ID22 stabilized mutant (87%).^51,52 Overall, the FHbp structures are similar and the FH binding site is in the same location; however, FH binding occurs through different specific interactions for FHbp from the two sub-families.^51,53

Figure 1. Crystal structure of the complex between meningococcal FHbp and an FH fragment comprising domains 6 and 7. The two molecules were separated along the Y-axis to show the respective binding surfaces (color). Mutations to decrease FH binding to FHbp target the top, green surface of FHbp. Coordinates are from PDB ID 2w80.³⁸ Figure was constructed with PyMol (The PyMOL Molecular Graphics System, Version 2.3 Schrödinger, LLC)

FHbp mutants with low binding of FH to FHbp

Based on the crystal structure of the complex between FHbp ID1 and an FH fragment, a double mutant FHbp was constructed in which two charged residues, E218 and E239, which are involved in intermolecular salt bridges with FH, were replaced with alanine (the numbering used here to designate the FHbp mutants is based on the mature protein sequence of FHbp ID1). Replacement of these two amino acid residues with alanine decreased binding of human FH to the FHbp mutant >100-fold compared with WT FHbp as measured by surface plasmon resonance.³⁸ Since that pioneering study, our laboratory and others’ have used protein structural information to engineer improved FHbp antigens with decreased binding of FH, including the well-characterized R41S mutant.^35,54,55,56 A candidate mutant FHbp antigen would not only exhibit low binding of human FH compared to WT FHbp but would also display similar binding of control anti-FHbp monoclonal antibodies (MAbs) to confirm the preservation of certain immunogenic epitopes and retain thermal stability. Suitable mutants would also elicit similar immunogenicity in WT mice (in which FH does not bind to FHbp) and higher immunogenicity in human FH transgenic mice (in which human FH does bind to FHbp). To date, several dozen FHbp mutants have been reported and characterized biochemically.^{35,38,51,53,57–59} A subset of the mutants has been tested for immunogenicity in human FH transgenic mice.^35,51,58,59

Site-specific mutagenesis is used often to introduce alanine substitutions to abrogate specific amino acid side-chain interactions and the success of this approach to disrupt protein–protein interactions is not always predictable. Our hypothesis was that new mutants with even lower FH binding than known mutants would be better vaccine candidates. To test this hypothesis, we generated error-prone PCR mutant FHbp libraries and expressed the clones on the surface of E. coli.^60,61 FHbp mutants with low binding of human FH and high binding to control anti-FHbp MAbs were isolated by fluorescence-activated cell sorting. One advantage of the library approach is that different amino acid substitutions at the same position can be identified, and some of these substitutions might be more effective at decreasing FH binding compared with alanine substitutions.⁶⁰ Using this approach, we discovered FHbp mutants in each of the two sub-families that had even lower FH binding than some of the previously identified mutants.^60,61 Further, most of the mutant FHbp antigens elicited similar protective antibody responses as a control WT FHbp antigen in WT mice (in which mouse FH does not bind to either antigen). In human FH transgenic mice with serum human FH concentrations similar to humans, the mutant FHbp antigens elicited 8- to 20-fold higher bactericidal antibody responses than the respective WT FHbp antigens.^35,54,60,61 A summary of the relationship between the decreases observed in binding of human FH and the increases in serum bactericidal antibody responses are shown in Figure 2. The most promising mutants are represented at the top, right of the Figure, and include S223R and H248L in sub-family B, and G220S in sub-family A.^60,61

Figure 2. Relationship between decreases in FH binding of mutant FHbp antigens and serum bactericidal antibody responses, each relative to the respective wild-type FHbp antigen. Magenta circles represent sub-family A (ID22) antigens and blue squares depict sub-family B (ID1) antigens. Fold changes in FH binding were measured by surface plasmon resonance, with the exception of K219N and G220S, which were measured by ELISA. The data from the latter two mutants are from proteins with two additional stabilizing substitutions as previously described.^52,61 It should be noted that the increases in bactericidal activity elicited by mutants as compared to the wild-type antigens depend on the FHbp sequence and expression in the target strain.^43,58.

We subsequently tested the immunogenicity of a mutant FHbp antigen with two amino acid substitutions, R41S and H248L, which decreased FH binding more than 250-fold, in infant rhesus macaques with high binding of FH to FHbp.⁴³ Groups of 11 macaques were vaccinated with the double mutant or a control WT FHbp vaccine and a negative control group of four animals was vaccinated with the adjuvant alone. Against a strain with high expression of a matched FHbp sequence variant, the mutant FHbp antigen gave three-fold higher bactericidal titers than the WT antigen. Against strains with mismatched FHbp sequence variants, the bactericidal titers were up to 15-fold higher for the mutant FHbp than for the WT FHbp.^43,56 Moreover, a strain with a lower level of FHbp expression also showed greater susceptibility to antibodies to the FHbp mutant.⁴³ However, since protection elicited by FHbp antigens is sub-family specific, strains with FHbp in a different sub-family from the vaccine antigen would not be expected to be susceptible even to an improved mutant FHbp antigen.^6,8,9 In summary, the results of testing the immunogenicity of an FHbp mutant with low FH binding in a non-human primate model relevant to humans were similar to those previously observed in a human FH transgenic mouse model.

As noted above, the contribution of anti-FHbp antibodies toward bactericidal activity against different strains depends on the presence, sequence and expression of each of the vaccine antigens in the strains. For MenB-FHbp, all of the bactericidal activity is attributable to anti-FHbp antibodies. For MenB-4C, strains have been identified against which each of the four principal antigens are protective.⁴⁴ Against other strains, two or more antigens are likely protective.²⁸ FHbp is arguably the most important antigen in MenB-4C, and approximately 60–70% of invasive group B isolates in the U.S. and E.U. have FHbp in sub-family B, which is the same sub-family as the FHbp antigen in MenB-4C.^62,63

Effect of human or non-human primate FH on antibody repertoire

Antibodies to a microbial virulence factor have the potential to neutralize the function of the virulence factor. For example, anti-FHbp MAbs can inhibit FH binding to purified FHbp as measured in an ELISA or to FHbp on the bacterial surface as detected by flow cytometry.^64,65 Anti-FHbp MAbs from WT mice whose FH did not bind FHbp generally inhibited FH binding.^64,66,67 Inhibition of FH binding to the bacterial surface increases the susceptibility of the bacteria to host immune responses because FH negatively regulates the amplification loop of the complement alternative pathway.^68–70 Further, inhibition of FH binding by anti-FHbp MAbs was important for bactericidal activity.^71,72 Although most murine anti-FHbp MAbs inhibited FH binding whereas some other MAbs did not, it was noteworthy that one MAb enhanced binding of FH to FHbp.^64,66,68

We hypothesized that binding of human FH to the FHbp vaccine antigen redirects the antibody repertoire to epitopes outside of the FH binding site and, therefore, that antibodies produced in the absence of FH that bound FHbp would better inhibit FH binding than antibodies produced in the presence of FH that bound FHbp. In one study, anti-FHbp antibodies from WT mice immunized with MenB-4C inhibited binding of FH to FHbp, whereas antibodies from human FH transgenic mice enhanced FH binding.³⁶ The enhancement of FH binding can be explained by the possibility that, following vaccination with an FHbp antigen that is bound by FH, antibodies could recognize and stabilize the FH-FHbp complex. As expected, antibodies from human FH transgenic mice immunized with a recombinant FHbp R41S mutant also inhibited binding of FH to FHbp.³⁶ Thus, the antibody repertoire to a mutant FHbp with low FH binding in the presence of human FH could mimic the antibody repertoire of the WT antigen in the absence of human FH.

In a subsequent study in infant rhesus macaques immunized with MenB-4C, serum antibodies from animals with low FH binding to FHbp had greater inhibition than antibodies from animals with high binding.³⁷ Moreover, in rhesus macaques with high binding of FH to FHbp, antibodies elicited by MenB-4C also enhanced FH binding to live meningococci. In a recent study, serum antibodies from macaques immunized with a native OMV vaccine with over-expressed mutant FHbp inhibited FH binding whereas antibodies to a control licensed group B vaccine enhanced FH binding (Figure 3).⁵⁶ In another study, using Fab fragments cloned from B cells from human subjects vaccinated with MenB-4C, none of the 10 Fabs characterized strongly inhibited binding of FH to FHbp.⁶⁵ Finally, serum antibodies isolated from an immunized human subject also enhanced binding of FH to FHbp.⁶⁵ While the effects of serum antibodies from a larger number of vaccinated human subjects on FH binding have not been reported, it is expected that most humans vaccinated with FHbp that binds FH would also develop antibodies that enhance binding of FH.

Figure 3. FH inhibition by macaque serum antibodies elicited by a native OMV vaccine with over-expressed mutant FHbp. After two vaccine doses, antibodies from all 13 macaques immunized with the native OMV vaccine inhibited FH binding to purified, recombinant FHbp, whereas antibodies from animals to a licensed control vaccine did not inhibit and some enhanced FH binding. The p-value for a paired t-test is shown. Macaques immunized with aluminum hydroxide (Al(OH)₃) as a negative control did not show significant inhibition or enhancement. Figure adapted from ref.⁵⁶

Structural analysis of anti-FHbp antibody repertoire

The three-dimensional structures of several complexes between murine anti-FHbp antibody (Fab) fragments have been determined by X-ray crystallography. The first complex to be elucidated was between a murine Fab designated 12C1 and the nominal FHbp antigen, ID1, which is the FHbp sequence variant in the MenB-4C vaccine. This Fab was specific for FHbp in sub-family B (variant group 1) and inhibited FH binding.⁷³ The crystal structure revealed that Fab 12C1 bound to an epitope that significantly overlapped the FH binding site as observed in the structure of the complex between FHbp and FH domains 6 and 7.^38,73 The structure of a second murine Fab designated JAR 5 was also determined in a complex with FHbp. Similar to 12C1, JAR 5 is specific for most FHbp sequence variants in sub-family B (variant group 1) and inhibits binding of FH to FHbp.^68,74 The structure of the complex between Fab JAR 5 and FHbp showed that this Fab bound to several surface-exposed loops and partially overlapped the FH binding site.⁷⁵

The epitopes of other murine anti-FHbp MAbs have been mapped by a variety of methods, including NMR chemical shift analysis, hydrogen-deuterium exchange mass spectrometry (HDX-MS), peptide phage display, an FHbp mutant library displayed on yeast and sequence alignments combined with site-specific mutagenesis.^{64,66,73,76–78} In another study, an epitope was defined using a synthetic peptide derived from the sequence of FHbp that specifically inhibited the binding of a broadly reactive MAb that had been raised against an FHbp antigen from sub-family B.⁴⁹ However, these methods have limitations either in being indirect or not gaining a complete picture of all of the residues in the interaction.

One recent study highlighted the structure of a complex between a human Fab designated 1A12 and FHbp, which showed that the Fab bound to a conserved region that was present in all known FHbp sequences.⁷⁹ The epitope was located opposite from the FH binding site, thus explaining why the Fab lacked FH inhibition.⁶⁵ The epitopes of several other human anti-FHbp antibodies have been mapped by HDX-MS.^80,81 One of these human MAbs, designated 1G3, also did not inhibit human FH and bound to a region previously reported to interact with murine MAbs JAR 4 and JAR 41, neither of which inhibited FH binding.^65,77,78 The three MAbs bound to the same region of the amino-terminal domain of FHbp and cross-reacted with FHbp from different sub-families (or variant groups). Interestingly, the ability of FHbp to elicit cross-reactive antibodies in mice and humans indicates that there is a potential to confer cross-protection against strains expressing many different FHbp sequence variants.

Synergistic bactericidal activity of anti-FHbp mabs

To activate the complement classical pathway that leads to bactericidal activity, multiple antibodies are needed to bind C1q.⁸² When the antigen density is sufficiently high, a single MAb can elicit complement-mediated bactericidal activity.⁷⁴ However, FHbp is sparsely expressed on the surface of many meningococcal strains and, therefore, most individual anti-FHbp MAbs did not elicit human complement-mediated bactericidal activity.^8,74,83 In contrast, certain pairs of anti-FHbp MAbs did elicit bactericidal activity, which was referred to as “synergistic” or “cooperative” bactericidal activity (Figure 4).^64,84 The most likely mechanism for the synergism is that two MAbs that recognize different epitopes on the same FHbp molecule could be oriented such that their Fc regions are in sufficient proximity to bind to the same molecule of C1q. The crystal structures of two Fab fragments, each in a complex with FHbp, allowed modeling of a ternary complex of the two antibodies, which indicated that the respective Fc regions would be ~130 Å apart and be close enough to allow engagement of C1q.^73,75 There was no synergistic bactericidal activity when pairs of MAbs recognized overlapping epitopes because they would not be able to bind simultaneously to a single molecule of FHbp.^64,66,78

Figure 4. Molecular model of two antibody Fab fragments bound to FHbp. The figure was generated by superimposing the FHbp molecules from two experimentally determined structures of FHbp-Fab complexes (Fab 12C1; PDB ID 2YPV and Fab JAR 5; 5T5F).^73,75 FHbp is shown in gray with surface rendering and the heavy and light chains of each Fab are shown in blue and green, respectively. JAR 5 binds to a region of the amino-terminal domain of FHbp (top left) and 12C1 binds to a surface that overlaps the FH binding site (top). The Fabs bind to non-overlapping epitopes and the respective MAbs elicit cooperative bactericidal activity.⁷⁵ Figure was constructed with PyMol (The PyMOL Molecular Graphics System, Version 2.3 Schrödinger, LLC)

From one of our earlier studies of synergistic bactericidal activity of anti-FHbp MAbs, we suggested that inhibition of FH binding by at least one of the MAbs was important for bactericidal activity.⁶⁴ At the time, we did not have a sufficient number of MAbs that recognized the same FHbp sequence variant and lacked FH inhibition to test this hypothesis. Subsequently, our colleagues identified several pairs of murine anti-FHbp MAbs that lacked FH inhibition but elicited synergistic bactericidal activity with human complement.⁷⁸ Pairs of intact IgG molecules constructed from the human Fabs described above elicited bactericidal activity when one MAb was directed to the amino-terminal domain and the second was to the carboxyl-terminal domain.⁸⁰ Further, six different pairs of human anti-FHbp MAbs elicited synergistic bactericidal activity in the absence of FH inhibition, which provided further evidence that FH inhibition by anti-FHbp MAbs is not essential for bactericidal activity.

Other mechanisms of protection by anti-FHbp mabs

In humans, anti-FHbp antibodies exert their functional activity through a variety of mechanisms. Of these, complement-mediated serum bactericidal antibody activity leading to bacterial lysis is likely to be the most important mechanism. One seminal study that underscored the importance of complement-mediated bactericidal activity, which is the result of the complement classical pathway, is from military recruits in whom serum bactericidal antibody titers (measured in vitro) of ≥1:4 were protective and a titer of <1:4 was not.¹⁷ Similar to the synergy of anti-FHbp MAbs described above, it is likely that polyclonal serum antibodies recognizing non-overlapping epitopes confer protection by the same mechanism. It is also possible for anti-FHbp antibodies to cooperate with antibodies to other antigens, such as Neisserial Heparin binding antigen (NHba), which is another component of the MenB-4C vaccine, or potentially other antigens.⁸⁵ A second mechanism of protection, as described above, is the ability of some anti-FHbp antibodies to inhibit binding of FH to FHbp. This inhibition interferes with the ability of the bacteria to recruit FH to their surfaces to down-regulate amplification of the alternative pathway. This mechanism is described in greater detail in a comprehensive review article on FH and Neisserial pathogenesis.⁷⁰

Other mechanisms for antibody-mediated protection against meningococci have been shown to be important. In one study, whole blood samples from non-immunized human donors were able to kill the bacteria even though the respective sera did not kill, which indicated that cell-mediated immunity played a role in bacterial clearance.⁸⁶ In two related studies, serum antibodies from human subjects vaccinated with investigational vaccines containing FHbp were able to kill the bacteria in the presence of complement component 6- (C6)-depleted serum and polymorphonuclear leukocytes, which provided more direct evidence for opsonophagocytosis as an independent mechanism for protection.^87,88 Although this mechanism has not been demonstrated to be protective specifically by antibodies to FHbp, it has been shown for OMV and/or recombinant protein vaccines including FHbp as one of several antigens. Finally, group B vaccines can theoretically elicit antibody protection by preventing acquisition of colonizing bacteria in the mucous membranes of the nasopharynx. One study assessed the effect of group B vaccination on nasopharyngeal colonization; however, this study was not able to demonstrate a significant impact of vaccination on colonization.⁸⁹ Overall, group B vaccines containing FHbp can elicit antibody protection via multiple mechanisms and the most important of these is complement-mediated bactericidal activity.

Concluding remarks

As previously noted, FHbp is a virulence factor used by the bacteria to evade the host immune system and, therefore, this protein has both advantages and disadvantages as a vaccine antigen. One obvious advantage is that antibodies to the vaccine antigen can interfere with its function as a virulence factor. Since binding of the host protein might render the vaccine antigen less immunogenic or mask important epitopes, a key problem is to develop modified antigens that do not bind to the host protein. With the combination of key protein structural information, two different lines of human FH transgenic mice, and rhesus macaques with high binding of FH to FHbp, we and others have shown that it is possible to modify the FHbp vaccine antigen to increase immunogenicity in relevant animal models. These findings, along with the ability of antibodies to inhibit binding of FH to augment bactericidal activity, suggest that the FHbp antigens in the currently licensed vaccines could be replaced with mutant antigens to provide increased vaccine efficacy. A current direction in the field includes the addition of the MenB-4C protein antigens to the A, C, W and Y polysaccharide conjugates to create a broadly protective pentavalent vaccine.^27,90 Other current advances include the development of an investigational native OMV vaccine with over-expressed mutant FHbp, which induced higher antibody titers than recombinant FHbp or the MenB-4C vaccine.^55,56 In mice and in a subset of rhesus macaques, this native OMV vaccine also elicited bactericidal antibodies against Neisseria gonorrhoeae, suggesting the prospect of a pan-Neisserial vaccine, especially if the OMV can be supplemented with gonococcal antigens.

Although it must be noted that the currently licensed vaccines do elicit protective antibody responses against meningococci, a future direction is to increase the immunogenicity in infants, to increase the breadth of protection against diverse strains, and to extend the duration of protection by achieving higher antibody titers. Since meningococcal infections can progress more rapidly than memory B cell proliferation, future studies are needed to determine whether higher peak anti-FHbp antibody titers will translate into a longer duration of circulating bactericidal antibodies. Collectively, our studies have shown promise for several mutants in each FHbp sub-family; these mutants have the additional benefit of eliciting antibodies that inhibit binding of FH, which defeats one of the bacterium’s key evasion mechanisms.^60,61 Based on a wealth of pre-clinical data from studies in transgenic mice and rhesus macaques, we anticipate such mutant FHbp antigens will soon be tested in humans.

Disclosure of Potential Conflicts of Interest

The author is named as an inventor on patents and patent applications relating to meningococcal capsular group B vaccines, which include Factor H binding proteins with decreased binding of Factor H.

Acknowledgments

We thank Kelsey Sharkey (UCSF Benioff Children’s Hospital Oakland) for critical review of the manuscript.

Additional information

Funding

This work was supported by research grants [R01 AI114701 and R01 AI134868] from the National Institute of Allergy and Infectious Diseases, NIH.