Anti-CfaE nanobodies provide broad cross-protection against major pathogenic enterotoxigenic Escherichia coli strains, with implications for vaccine design.

Enterotoxigenic Escherichia coli (ETEC) is estimated to cause approximately 380,000 deaths annually during sporadic or epidemic outbreaks worldwide. Development of vaccines against ETEC is very challenging due to the vast heterogeneity of the ETEC strains. An effective vaccines would have to be multicomponent to provide coverage of over ten ETEC strains with genetic variabilities. There is currently no vaccine licensed to prevent ETEC. Nanobodies are successful new biologics in treating mucosal infectious disease as they recognize conserved epitopes on hypervariable pathogens. Cocktails consisting of multiple nanobodies could provide even broader epitope coverage at a lower cost compared to monoclonal antibodies. Identification of conserved epitopes by nanobodies can also assist reverse engineering of an effective vaccine against ETEC. By screening nanobodies from immunized llamas and a naïve yeast display library against adhesins of colonization factors, we identified single nanobodies that show cross-protective potency against eleven major pathogenic ETEC strains in vitro. Oral administration of nanobodies led to a significant reduction of bacterial colonization in animals. Moreover, nanobody-IgA fusion showed extended inhibitory activity in mouse colonization compared to commercial hyperimmune bovine colostrum product used for prevention of ETEC-induced diarrhea. Structural analysis revealed that nanobodies recognized a highly-conserved epitope within the putative receptor binding region of ETEC adhesins. Our findings support further rational design of a pan-ETEC vaccine to elicit robust immune responses targeting this conserved epitope.

Neisseria meningitidis Urethritis Outbreak Isolates Express a Novel Factor H Binding Protein Variant That Is a Potential Target of Group B-Directed Meningococcal (MenB) Vaccines.

Factor H binding protein (FHbp) is an important Neisseria meningitidis virulence factor that binds a negative regulator of the alternative complement pathway, human factor H (FH). Binding of FH increases meningococcal resistance to complement-mediated killing. FHbp also is reported to prevent interaction of the antimicrobial peptide (AMP) LL-37 with the meningococcal surface and meningococcal killing. FHbp is a target of two licensed group B-directed meningococcal (MenB) vaccines. We found a new FHbp variant, peptide allele identification no. 896 (ID 896), was highly expressed by an emerging meningococcal pathotype, the nonencapsulated urethritis clade (US_NmUC). This clade has been responsible for outbreaks of urethritis in multiple U.S. cities since 2015, other mucosal infections, and cases of invasive meningococcal disease. FHbp ID 896 is a member of the variant group 1 (subfamily B), bound protective anti-FHbp monoclonal antibodies, bound high levels of human FH, and enhanced the resistance of the clade to complement-mediated killing in low levels of human complement likely present at human mucosal surfaces. Interestingly, expression of FHbp ID 896 resulted in augmented killing of the clade by LL-37. FHbp ID 896 of the clade was recognized by antibodies elicited by FHbp in MenB vaccines.

Oral administration of an anti-CfaE secretory IgA antibody protects against Enterotoxigenic Escherichia coli diarrheal disease in a nonhuman primate model.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea-associated illness in developing countries. There is currently no vaccine licensed to prevent ETEC and the development of an efficacious prophylaxis would provide an intervention with significant impact. Recent studies suggested that effective protection could be achieved by inducing immunity to block colonization of ETEC. Here, we evaluated the efficacy of secretory (s) IgA2 and dimeric (d) IgA2 of an anti-colonization factor antigen antibody, 68-61, in the Aotus nancymaae nonhuman primate (NHP) ETEC challenge model via oral and parental delivery. Thirty-nine animals were distributed across 3 groups of 13, and challenged with 5.0x1011 colony forming unit (CFU) of H10407 on Day 0. Group 1 received a dIgA2 68-61 subcutaneously on day 0. Group 2 received a SIgA2 68-61 orally on days -1, 0, and +1, and Group 3 received an irrelevant SIgA2 antibody orally on days -1, 0, and +1. All animals were observed for symptoms of diarrhea, and stools were collected for ETEC colony counts. Anti-CfaE SIgA2 treatment significantly lowered the attack rate, resulting in a protective efficacy of 74.1% (p = 0.025) in Group 2 as compared to Group 3. The anti-CfaE dIgA2 treatment group had reduced diarrheal attack rate, although the reduction did not reach significance (57.1%; p = 0.072) as compared to the irrelevant SIgA2 Group 3. Our results demonstrated the feasibility of oral administration of SIgA as a potential immunoprophylaxis against enteric infections. To our knowledge, this is the first study to demonstrate the efficacy of administrated SIgA in a nonhuman primate model.

Identification and Characterization of Human Monoclonal Antibodies for Immunoprophylaxis against Enterotoxigenic Escherichia coli Infection.

Enterotoxigenic Escherichia coli (ETEC) causes diarrheal illness in infants in the developing world and travelers to countries where the disease is endemic, including military personnel. ETEC infection of the host involves colonization of the small intestinal epithelium and toxin secretion, leading to watery diarrhea. There is currently no vaccine licensed to prevent ETEC infection. CFA/I is one of the most common colonization factor antigens (CFAs). The CFA/I adhesin subunit, CfaE, is required for ETEC adhesion to host intestinal cells. Human antibodies against CfaE have the potential to block colonization of ETEC and serve as an immunoprophylactic against ETEC-related diarrhea. Mice transgenic for human immunoglobulin genes were immunized with CfaE to generate a panel of human monoclonal IgG1 antibodies (HuMAbs). The most potent IgG1 antibodies identified in the in vitro functional assays were selected and isotype switched to secretory IgA (sIgA) and tested in animal colonization assays via oral administration. Over 300 unique anti-CfaE IgG1 HuMAbs were identified. The lead IgG1 anti-CfaE HuMAbs completely inhibited hemagglutination and blocked adhesion of ETEC to Caco-2 cells. Epitope mapping studies revealed that HuMAbs recognized epitopes in the N-terminal domain of CfaE near the putative receptor binding site. Oral administration of anti-CfaE antibodies in either IgG or sIgA isotypes inhibited intestinal colonization in mice challenged with ETEC. A 2- to 4-log decrease in CFU was observed in comparison to mice challenged with irrelevant isotype controls. We identified fully human monoclonal antibodies against the CfaE adhesion domain that can be potentially employed as an immunoprophylactic to prevent ETEC-related diarrhea.

Breadth and Duration of Meningococcal Serum Bactericidal Activity in Health Care Workers and Microbiologists Immunized with the MenB-FHbp Vaccine.

MenB-FHbp is a meningococcal serogroup B vaccine with two factor H binding protein (FHbp) antigens from subfamilies A and B. For licensure, efficacy was inferred from serum bactericidal antibody (SBA) responses to four reference strains. Only limited information is available on the breadth or duration of protective SBA responses to genetically diverse disease-causing strains. Seventeen health care or laboratory workers were immunized with two (n = 2) or three (n = 15) doses of MenB-FHbp at 0, 2, and 6 months. SBA levels were measured against 14 serogroup B case isolates, including 6 from U.S. college outbreaks and 2 from Quebec during hyperendemic disease. Compared with preimmunization titers, the proportion of subjects with ≥4-fold increases in SBA titer 1 month after 2 doses of vaccine ranged from 35% to 94% for six isolates with FHbp subfamily A and from 24% to 76% for eight isolates with subfamily B FHbp. The respective proportions with ≥4-fold titer increases at 1 month after dose 3 were 73% to 100% and 67% to 100%. At that time point, the proportion of subjects with titers of ≥1:4 (presumed sufficient for short-term protection) ranged from 93% to 100% for all 14 isolates. By 9 to 11 months after dose 3, 50% or fewer of the subjects with follow-up sera had protective titers of ≥1:4 for 4 of 9 isolates tested. Three doses of MenB-FHbp elicited short-term protective SBA responses to diverse disease-causing serogroup B strains. For some strains, serum titers declined to <1:4 by 9 to 11 months, which raises concerns about the duration of broad, long-term protection. (This study has been registered at ClinicalTrials.gov under registration no. NCT02569632.).

The role of anti-NHba antibody in bactericidal activity elicited by the meningococcal serogroup B vaccine, MenB-4C.

BACKGROUND: MenB-4C (Bexsero®) is a multicomponent serogroup B meningococcal vaccine. For vaccine licensure, efficacy was inferred from serum bactericidal antibody (SBA) against three antigen-specific indicator strains. The bactericidal role of antibody to the fourth vaccine antigen, Neisserial Heparin binding antigen (NHba), is incompletely understood. METHODS: We identified nine adults immunized with two or three doses of MenB-4C who had sufficient volumes of sera and >3-fold increases in SBA titer against a strain with high NHba expression, which was mismatched with the other three MenB-4C antigens that elicit SBA. Using 1month-post-immunization sera we measured the effect of depletion of anti-NHba and/or anti-Factor H binding protein (FHbp) antibodies on SBA. RESULTS: Against three strains matched with the vaccine only for NHba, depletion of anti-NHba decreased SBA titers by an average of 43-79% compared to mock-adsorbed sera (P<0.05). Despite expression of sub-family A FHbp (mismatched with the sub-family B vaccine antigen), depletion of anti-FHbp antibodies also decreased SBA by 45-64% (P<0.05). Depletion of both antibodies decreased SBA by 84-100%. Against a strain with sub-family B FHbp and expression of NHba with 100% identity to the vaccine antigen, depletion of anti-NHba decreased SBA by an average of 26%, compared to mock-adsorbed sera (P<0.0001), and depletion of anti-FHbp antibody decreased SBA by 92% (P<0.0001). CONCLUSIONS: Anti-NHba antibody can contribute to SBA elicited by MenB-4C, particularly in concert with anti-FHbp antibody. However, some high NHba-expressing strains are resistant, even with an exact match between the amino acid sequence of the vaccine and strain antigens.

Serum Bactericidal Antibody Responses of Adults Immunized with the MenB-4C Vaccine against Genetically Diverse Serogroup B Meningococci.

MenB-4C is a meningococcal vaccine for the prevention of serogroup B disease. The vaccine contains factor H binding protein (FHbp) and three other antigens that can elicit serum bactericidal antibodies (SBA). For vaccine licensure, efficacy was inferred from the SBA responses against three antigen-specific indicator strains. The relation between those results and broad protection against circulating genetically diverse strains is not known. Twenty adults were immunized with two doses of MenB-4C given 1 to 2 months apart. SBA activity against 3 reference strains and 15 serogroup B test strains (6 from college outbreaks) was measured. Compared to the preimmunization titers, 70%, 95%, and 95% of subjects had ≥4-fold increases in the titers of anti-PorA P1.4, anti-NadA, and anti-FHbp antibodies against the reference strains, respectively. In contrast, only 25 to 45% of the subjects had ≥4-fold increases in responses to 10 of the 15 test strains, including 8 that expressed one to three of the antigens in the vaccine. At 1 month, the majority of subjects with <4-fold titer increases had serum titers of ≥1:4, which are considered sufficient for protection. However, the titers against four strains declined to <1:4 by 4 to 6 months in one-third to greater than 50% of the subjects tested. Clinically relevant isolates are often more resistant to SBA than the indicator strains used to measure antigen-specific SBA. A working model is that the percentage of subjects with titers of ≥1:4 at 1 month postimmunization correlates with short-term protection against that strain, whereas the percentage of subjects with ≥4-fold titer increases represents a more robust response. (The protocol used at the Oxford Vaccine Group has been registered at ClinicalTrials.gov under registration no. NCT02398396.).

Enhanced protective antibody to a mutant meningococcal factor H-binding protein with low-factor H binding.

Meningococcal factor H-binding protein (FHbp) is an antigen in 2 serogroup B meningococcal vaccines. FHbp specifically binds human and some nonhuman primate complement FH. To investigate the effect of binding of FH to FHbp on protective antibody responses, we immunized infant rhesus macaques with either a control recombinant FHbp antigen that bound macaque FH or a mutant antigen with 2 amino acid substitutions and >250-fold lower affinity for FH. The mutant antigen elicited 3-fold higher serum IgG anti-FHbp titers and up to 15-fold higher serum bactericidal titers than the control FHbp vaccine. When comparing sera with similar IgG anti-FHbp titers, the antibodies elicited by the mutant antigen gave greater deposition of complement component C4b on live meningococci (classical complement pathway) and inhibited binding of FH, while the anti-FHbp antibodies elicited by the control vaccine enhanced FH binding. Thus, the mutant FHbp vaccine elicited an anti-FHbp antibody repertoire directed at FHbp epitopes within the FH binding site, which resulted in greater protective activity than the antibodies elicited by the control vaccine, which targeted FHbp epitopes outside of the FH combining site. Binding of a host protein to a vaccine antigen impairs protective antibody responses, which can be overcome with low-binding mutant antigens.

Effect of complement Factor H on anti-FHbp serum bactericidal antibody responses of infant rhesus macaques boosted with a licensed meningococcal serogroup B vaccine.

FHbp is a major serogroup B meningococcal vaccine antigen. Binding of complement Factor H (FH) to FHbp is specific for human and some non-human primate FH. In previous studies, FH binding to FHbp vaccines impaired protective anti-FHbp antibody responses. In this study we investigated anti-FHbp antibody responses to a third dose of a licensed serogroup B vaccine (MenB-4C) in infant macaques vaccinated in a previous study with MenB-4C. Six macaques with high binding of FH to FHbp (FH(high)), and six with FH(low) baseline phenotypes, were immunized three months after dose 2. After dose 2, macaques with the FH(low) baseline phenotype had serum anti-FHbp antibodies that enhanced FH binding to FHbp (functionally converting them to a FH(high) phenotype). In this group, activation of the classical complement pathway (C4b deposition) by serum anti-FHbp antibody, and anti-FHbp serum bactericidal titers were lower after dose 3 than after dose 2 (p<0.02). In macaques with the FH(high) baseline phenotype, the respective anti-FHbp C4b deposition and bactericidal titers were similar after doses 2 and 3. Two macaques developed serum anti-FH autoantibodies after dose 2, which were not detected after dose 3. In conclusion, in macaques with the FH(low) baseline phenotype whose post-dose 2 serum anti-FHbp antibodies had converted them to FH(high), the anti-FHbp antibody repertoire to dose 3 was skewed to less protective epitopes than after dose 2. Mutant FHbp vaccines that eliminate FH binding may avoid eliciting anti-FHbp antibodies that enhance FH binding, and confer greater protection with less risk of inducing anti-FH autoantibodies than FHbp vaccines that bind FH.

Susceptibility of Meningococcal Strains Responsible for Two Serogroup B Outbreaks on U.S. University Campuses to Serum Bactericidal Activity Elicited by the MenB-4C Vaccine.

In 2013 and 2014, two U.S. universities had meningococcal serogroup B outbreaks (a total of 14 cases) caused by strains from two different clonal complexes. To control the outbreaks, students were immunized with a serogroup B meningococcal vaccine (Novartis) that was not yet licensed in the United States. The vaccine (referred to as MenB-4C) contains four components capable of eliciting bactericidal activity. Both outbreak strains had high expression levels of two of the vaccine antigens (subfamily B factor H binding protein [FHbp] and neisserial heparin binding antigen [NHba]); the university B outbreak strain also had moderate expression of a third antigen, NadA. We investigated the bactericidal activity of sera from mice immunized with FHbp, NHba, or NadA and sera from MenB-4C-immunized infant macaques and an adult human. The postimmunization bactericidal activity of the macaque or human serum against isolates from university B with FHbp identification (ID) 1 that exactly matched the vaccine FHbp sequence variant was 8- to 21-fold higher than that against isolates from university A with FHbp ID 276 (96% identity to the vaccine antigen). Based on the bactericidal activity of mouse antisera to FHbp, NadA, or NHba and macaque or human postimmunization serum that had been depleted of anti-FHbp antibody, the bactericidal activity against both outbreak strains largely or entirely resulted from antibodies to FHbp. Thus, despite the high level of strain expression of FHbp from a subfamily that matched the vaccine antigen, there can be large differences in anti-FHbp bactericidal activity induced by MenB-4C vaccination. Further, strains with moderate to high NadA and/or NHba expression can be resistant to anti-NadA or anti-NHba bactericidal activity elicited by MenB-4C vaccination.

Functional Analysis of the Human Antibody Response to Meningococcal Factor H Binding Protein.

UNLABELLED: Two licensed serogroup B meningococcal vaccines contain factor H binding protein (FHbp). The antigen specifically binds human FH, which downregulates complement. In wild-type mice whose mouse FH does not bind to FHbp vaccines, the serum anti-FHbp antibody response inhibited binding of human FH to FHbp. The inhibition was important for eliciting broad anti-FHbp serum bactericidal activity. In human FH transgenic mice and some nonhuman primates, FHbp was able to form a complex with FH and FHbp vaccination elicited anti-FHbp antibodies that did not inhibit FH binding. To investigate the human anti-FHbp repertoire, we cloned immunoglobulin heavy- and light-chain-variable-region genes of individual B cells from three adults immunized with FHbp vaccines and generated 10 sequence-distinct native anti-FHbp antibody fragments (Fabs). All 10 Fabs bound to live meningococci; only 1 slightly inhibited binding of human FH, while 4 enhanced FH binding. Affinity-purified anti-FHbp antibody from serum of a fourth immunized adult also enhanced binding of human FH to live meningococcal bacteria. Despite the bound FH, the affinity-purified serum anti-FHbp antibodies elicited human complement-mediated bactericidal activity that was amplified by the alternative pathway. The lack of FH inhibition by the human anti-FHbp Fabs and serum antibodies suggests that binding of human FH to the vaccine antigen skews the anti-FHbp antibody repertoire to epitopes outside the FH-binding site. Mutant FHbp vaccines with decreased FH binding may represent a means to redirect the human antibody repertoire to epitopes within the FH binding site, which can inhibit FH binding and, potentially, increase safety and protective activity. IMPORTANCE: Two meningococcal vaccines contain factor H binding protein (FHbp). Immunized mice whose mouse factor H (FH) does not bind to FHbp develop serum anti-FHbp antibodies that block binding of human FH to the bacteria. With less bound FH, the bacteria become more susceptible to complement killing. To investigate human responses, we isolated 10 recombinant anti-FHbp antibody fragments (Fabs) from immune cells of three immunized adults. One slightly inhibited binding of FH to the bacteria, and four enhanced FH binding. Purified serum anti-FHbp antibodies from a fourth immunized adult also enhanced FH binding. Although bound FH would be expected to block the alternative pathway, the human anti-FHbp antibodies retained bactericidal activity and the ability to activate the alternative pathway. Mutant FHbp vaccines with decreased binding to human FH may redirect the human antibody repertoire to epitopes within the FH binding site that inhibit FH binding, which are expected to increase protective activity.

Binding of complement factor H to PorB3 and NspA enhances resistance of Neisseria meningitidis to anti-factor H binding protein bactericidal activity.

Among 25 serogroup B Neisseria meningitidis clinical isolates, we identified four (16%) with high factor H binding protein (FHbp) expression that were resistant to complement-mediated bactericidal activity of sera from mice immunized with recombinant FHbp vaccines. Two of the four isolates had evidence of human FH-dependent complement downregulation independent of FHbp. Since alternative complement pathway recruitment is critical for anti-FHbp bactericidal activity, we hypothesized that in these two isolates binding of FH to ligands other than FHbp contributes to anti-FHbp bactericidal resistance. Knocking out NspA, a known meningococcal FH ligand, converted both resistant isolates to anti-FHbp susceptible isolates. The addition of a nonbactericidal anti-NspA monoclonal antibody to the bactericidal reaction also increased anti-FHbp bactericidal activity. To identify a role for FH ligands other than NspA or FHbp in resistance, we created double NspA/FHbp knockout mutants. Mutants from both resistant isolates bound 10-fold more recombinant human FH domains 6 and 7 fused to Fc than double knockout mutants prepared from two sensitive meningococcal isolates. In light of recent studies showing functional FH-PorB2 interactions, we hypothesized that PorB3 from the resistant isolates recruited FH. Allelic exchange of porB3 from a resistant isolate to a sensitive isolate increased resistance of the sensitive isolate to anti-FHbp bactericidal activity (and vice versa). Thus, some PorB3 variants functionally bind human FH, which in the presence of NspA enhances anti-FHbp resistance. Combining anti-NspA antibodies with anti-FHbp antibodies can overcome resistance. Meningococcal vaccines that target both NspA and FHbp are likely to confer greater protection than either antigen alone.

Binding of Complement Factor H (FH) Decreases Protective Anti-FH Binding Protein Antibody Responses of Infant Rhesus Macaques Immunized With a Meningococcal Serogroup B Vaccine.

BACKGROUND: The meningococcal vaccine antigen, factor H (FH)-binding protein (FHbp), binds human complement FH. In human FH transgenic mice, binding decreased protective antibody responses. METHODS: To investigate the effect of primate FH binding, we immunized rhesus macaques with a 4-component serogroup B vaccine (4CMenB). Serum FH in 6 animals bound strongly to FHbp (FHbp-FH(high)) and, in 6 animals, bound weakly to FHbp (FHbp-FH(low)). RESULTS: There were no significant differences between the respective serum bactericidal responses of the 2 groups against meningococcal strains susceptible to antibody to the NadA or PorA vaccine antigens. In contrast, anti-FHbp bactericidal titers were 2-fold lower in FHbp-FH(high) macaques against a strain with an exact FHbp match to the vaccine (P = .08) and were ≥4-fold lower against 4 mutants with other FHbp sequence variants (P ≤ .005, compared with FHbp-FH(low) macaques). Unexpectedly, postimmunization sera from all 12 macaques enhanced FH binding to meningococci. In contrast, serum anti-FHbp antibodies elicited by 4CMenB in mice whose mouse FH did not bind to the vaccine antigen inhibited FH binding. CONCLUSIONS: Binding of FH to FHbp decreases protective anti-FHbp antibody responses of macaques to 4CMenB. Even low levels of FH binding skew the antibody repertoire to FHbp epitopes outside of the FH-binding site, which enhance FH binding.

fH-dependent complement evasion by disease-causing meningococcal strains with absent fHbp genes or frameshift mutations.

Meningococci bind human fH to down-regulate complement, which enhances survival of the bacteria in serum. A major fH ligand is the vaccine candidate, factor H-binding protein (fHbp). Although fHbp has been considered an essential meningococcal virulence factor, rarely, invasive isolates with absent fHbp genes or frameshift mutations have been identified. In previous studies fH binding to these isolates was not detected. The aim of the present study was to investigate fH binding and complement evasion by invasive meningococcal serogroup B clinical isolates with absent fHbp genes or frameshift mutations. Four of the seven isolates tested bound human fH by flow cytometry and survived in IgG-depleted human serum. In all four, fH binding was decreased after inactivating the gene encoding NspA. Binding of fH to fHbp and NspA is specific for human fH. To investigate fH-dependent evasion of host defenses, human fH transgenic infant rats, or control littermates negative for human fH, were challenged IP with 10(3)-10(4)CFU of two of the isolates with no detectable fH binding by flow cytometry. At 6h, bacteremia caused by both strains was higher in human fH transgenic rats than in control rats (P<0.002). In conclusion, six of the seven isolates had evidence of fH binding and/or human fH-dependent complement evasion in transgenic rats. In four, NspA was as an alternative fH ligand. fHbp vaccination may select for mutants that do not require fHbp for complement evasion. Inclusion of additional target antigens in vaccines containing fHbp may delay emergence of these mutants.

Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity.

BACKGROUND: Meningococcal factor H binding protein (fHbp) is a promising vaccine candidate. Anti-fHbp antibodies can bind to meningococci and elicit complement-mediated bactericidal activity directly. The antibodies also can block binding of the human complement down-regulator, factor H (fH). Without bound fH, the organism would be expected to have increased susceptibility to bacteriolysis. Here we describe bactericidal activity of two anti-fHbp mAbs with overlapping epitopes in relation to their different effects on fH binding and bactericidal activity. METHODS AND PRINCIPAL FINDINGS: Both mAbs recognized prevalent fHbp sequence variants in variant group 1. Using yeast display and site-specific mutagenesis, binding of one of the mAbs (JAR 1, IgG3) to fHbp was eliminated by a single amino acid substitution, R204A, and was decreased by K143A but not by R204H or D142A. The JAR 1 epitope overlapped that of previously described mAb (mAb502, IgG2a) whose binding to fHbp was eliminated by R204A or R204H substitutions, and was decreased by D142A but not by K143A. Although JAR 1 and mAb502 appeared to have overlapping epitopes, only JAR 1 inhibited binding of fH to fHbp and had human complement-mediated bactericidal activity. mAb502 enhanced fH binding and lacked human complement-mediated bactericidal activity. To control for confounding effects of different mouse IgG subclasses on complement activation, we created chimeric mAbs in which the mouse mAb502 or JAR 1 paratopes were paired with human IgG1 constant regions. While both chimeric mAbs showed similar binding to fHbp, only JAR 1, which inhibited fH binding, had human complement-mediated bactericidal activity. CONCLUSIONS: The lack of human complement-mediated bactericidal activity by anti-fHbp mAb502 appeared to result from an inability to inhibit binding of fH. These results underscore the importance of inhibition of fH binding for anti-fHbp mAb bactericidal activity.

Combined roles of human IgG subclass, alternative complement pathway activation, and epitope density in the bactericidal activity of antibodies to meningococcal factor h binding protein.

Meningococcal vaccines containing factor H binding protein (fHbp) are in clinical development. fHbp binds human fH, which enables the meningococcus to resist complement-mediated bacteriolysis. Previously, we found that chimeric human IgG1 mouse anti-fHbp monoclonal antibodies (MAbs) had human complement-mediated bactericidal activity only if the MAb inhibited fH binding. Since IgG subclasses differ in their ability to activate complement, we investigated the role of human IgG subclasses on antibody functional activity. We constructed chimeric MAbs in which three different murine fHbp-specific binding domains were each paired with human IgG1, IgG2, or IgG3. Against a wild-type group B isolate, all three IgG3 MAbs, irrespective of their ability to inhibit fH binding, had bactericidal activity that was >5-fold higher than the respective IgG1 MAbs, while the IgG2 MAbs had the least activity. Against a mutant with increased fHbp expression, the anti-fHbp MAbs elicited greater C4b deposition (classical pathway) and greater bactericidal activity than against the wild-type strain, and the IgG1 MAbs had similar or greater activity than the respective IgG3 MAbs. The bactericidal activity against both wild-type and mutant strains also was dependent, in part, on activation of the alternative complement pathway. Thus, at lower epitope density in the wild-type strain, the IgG3 anti-fHbp MAbs had the greatest bactericidal activity. At a higher epitope density in the mutant, the IgG1 MAbs had similar or greater bactericidal activity than the IgG3 MAbs, and the activity was less dependent on the inhibition of fH binding than at a lower epitope density.

Complement-mediated bactericidal activity of anti-factor H binding protein monoclonal antibodies against the meningococcus relies upon blocking factor H binding.

Binding of the complement-downregulating protein factor H (fH) to the surface of the meningococcus is important for survival of the organism in human serum. The meningococcal vaccine candidate factor H binding protein (fHbp) is an important ligand for human fH. While some fHbp-specific monoclonal antibodies (MAbs) block binding of fH to fHbp, the stoichiometry of blocking in the presence of high serum concentrations of fH and its effect on complement-mediated bactericidal activity are unknown. To investigate this question, we constructed chimeric antibodies in which the human IgG1 constant region was paired with three murine fHbp-specific binding domains designated JAR 3, JAR 5, and MAb502. By surface plasmon resonance, the association rates for binding of all three MAbs to immobilized fHbp were >50-fold higher than that for binding of fH to fHbp, and the MAb dissociation rates were >500-fold lower than that for fH. While all three MAbs elicited similar C1q-dependent C4b deposition on live bacteria (classical complement pathway), only those antibodies that inhibited binding of fH to fHbp (JAR 3 and JAR 5) had bactericidal activity with human complement. MAb502, which did not inhibit fH binding, had complement-mediated bactericidal activity only when tested with fH-depleted human complement. When an IgG1 anti-fHbp MAb binds to sparsely exposed fHbp on the bacterial surface, there appears to be insufficient complement activation for bacteriolysis unless fH binding also is inhibited. The ability of fHbp vaccines to elicit protective antibodies, therefore, is likely to be enhanced if the antibody repertoire is of high avidity and includes fH-blocking activity.

Meningococcal outer membrane vesicle vaccines derived from mutant strains engineered to express factor H binding proteins from antigenic variant groups 1 and 2.

Meningococcal outer membrane vesicle (OMV) vaccines, which are treated with detergents to decrease endotoxin activity, are safe and effective in humans. However, the vaccines elicit serum bactericidal antibody responses largely directed against PorA, which is antigenically variable. We previously prepared a native (non-detergent-treated) OMV vaccine from a mutant of group B strain H44/76 in which the lpxL1 gene was inactivated, which resulted in penta-acylated lipid A with attenuated endotoxin activity. To enhance protection, we overexpressed factor H binding protein (fHbp) from the antigenic variant 1 group. The vaccine elicited broad serum bactericidal antibody responses in mice against strains with fHbp variant 1 (approximately 70% of group B isolates) but not against strains with variant 2 or 3. In the present study, we constructed a mutant of group B strain NZ98/254 with attenuated endotoxin that expressed both endogenous variant 1 and heterologous fHbp variant 2. A mixture of the two native OMV vaccines from the H44/76 and NZ98/254 mutants stimulated proinflammatory cytokine responses by human peripheral blood mononuclear cells similar to those stimulated by control, detergent-treated OMV vaccines from the wild-type strains. In mice, the mixture of the two native OMV vaccines elicited broad serum bactericidal antibody responses against strains with heterologous PorA and fHbp in the variant 1, 2, or 3 group. By adsorption studies, the principal bactericidal antibody target was determined to be fHbp. Thus, native OMV vaccines from mutants expressing fHbp variants have the potential to be safe for humans and to confer broad protection against meningococcal disease from strains expressing fHbp from each of the antigenic variant groups.