Defining the Binding Region in Factor H to Develop a Therapeutic Factor H-Fc Fusion Protein against Non-Typeable Haemophilus influenzae

Non-typeable Haemophilus influenzae (NTHi) cause a range of illnesses including otitis media, sinusitis, and exacerbation of chronic obstructive pulmonary disease, infections that contribute to the problem of antibiotic resistance and are themselves often intractable to standard antibiotic treatment regimens. We investigated a strategy to exploit binding of the complement inhibitor Factor H (FH) to NTHi as a functional target for an immunotherapeutic containing the NTHi binding domain of FH fused to the Fc domain of IgG1. Chimeric proteins containing the regions that most FH-binding bacteria use to engage human FH, domains 6 and 7 (FH6,7/Fc) and/or 18 through 20 (FH18-20/Fc), were evaluated for binding to NTHi. FH6,7/Fc bound strongly to each of seven NTHi clinical isolates tested and efficiently promoted complement-mediated killing by normal human serum. FH18-20/Fc bound weakly to three of the strains but did not promote complement dependent killing. Outer-membrane protein P5 has been implicated in FH binding by NTHi, and FH6,7/Fc binding was greatly diminished in five of seven P5 deficient isogenic mutant strains tested, implicating an alternative FH binding protein in some strains. Binding of FH18-20/Fc was decreased in the P5 mutant of one strain. A murine model was used to evaluate potential therapeutic application of FH6,7/Fc. FH6,7/Fc efficiently promoted binding of C3 to NTHi exposed to mouse serum, and intranasal delivery of FH6,7/Fc resulted in significantly enhanced clearance of NTHi from the lung. Moreover, a P5 deficient mutant was attenuated for survival in the lung model, suggesting that escape mutants lacking P5 would be less likely to replace strains susceptible to FH6,7/Fc. These results provide evidence for the potential utility of FH6,7/Fc as a therapeutic against NTHi lung infection. FH binding is a common property of many respiratory tract pathogens and FH/Fc chimeras may represent promising alternative or adjunctive therapeutics against such infections, which are often polymicrobial

Factor H-Dependent Alternative Pathway Inhibition Mediated by Porin B Contributes to Virulence of Neisseria meningitidis

The identification of factor H binding protein (fHbp)-null invasive meningococcal isolates and the realization that widespread use of fHbp-based vaccines could herald selection of such strains prompted us to characterize novel mechanisms of alternative pathway (AP) inhibition on meningococci. Of seven strains engineered to lack four known AP-inhibiting molecules, capsular polysaccharide, lipooligosaccharide sialic acid, fHbp, and neisserial surface protein A (quadruple mutants), four strains inhibited human AP-mediated C3 deposition. All four expressed the porin B2 (PorB2) molecule, and three strains belonged to the hypervirulent ST-11 lineage. Consistent with reduced C3 deposition, the rate of C3a generation by a PorB2 isolate was lower than that by a PorB3 strain. Allelic replacement of PorB3 with PorB2, in both encapsulated and unencapsulated strains, confirmed the role of PorB2 in AP inhibition. Expression of PorB2 increased resistance to complement-dependent killing relative to that seen in an isogenic PorB3-expressing strain. Adult rabbit and mouse APs were unimpeded on all mutants, and human fH inhibited nonhuman C3 deposition on PorB2-expressing strains, which provided functional evidence for human fH-dependent AP regulation by PorB2. Low-affinity binding of full-length human fH to quadruple mutants expressing PorB2 was demonstrated. fH-like protein 1 (FHL-1; contains fH domains 1 through 7) and fH domains 6 and 7 fused to IgG Fc bound to one PorB2-expressing quadruple mutant, which suggested that fH domains 6 and 7 may interact with PorB2. These results associate PorB2 expression with serum resistance and presage the appearance of fHbp-null and hypervirulent ST-11 isolates that may evade killing by fHbp-based vaccines. IMPORTANCE The widespread use of antimeningococcal vaccines based on factor H (fH) binding protein (fHbp) is imminent. Meningococci that lack fHbp were recently isolated from persons with invasive disease, and these fHbp-null strains could spawn vaccine failure. Our report provides a molecular basis for an explanation of how fHbp-null strains may evade the host immune system. Meningococci possess several mechanisms to subvert killing by the alternative pathway (AP) of complement, including production of the fHbp and NspA fH binding proteins. Here we show that a meningococcal protein called porin B2 (PorB2) contributes to inhibition of the AP on the bacterial surface. A majority of the fHbp-null isolates identified, as well as all members of a hypervirulent lineage (called ST-11), express PorB2. Our findings highlight the potential for the emergence of fHbp-negative strains that are able to regulate the AP and may be associated with fHbp vaccine failure

Targeting Lipooligosaccharide (LOS) for a Gonococcal Vaccine

The increasing incidence of gonorrhea worldwide and the global spread of multidrug-resistant strains of Neisseria gonorrhoeae, constitute a public health emergency. With dwindling antibiotic treatment options, there is an urgent need to develop safe and effective vaccines. Gonococcal lipooligosaccharides (LOSs) are potential vaccine candidates because they are densely represented on the bacterial surface and are readily accessible as targets of adaptive immunity. Less well-understood is whether LOSs evoke protective immune responses. Although gonococcal LOS-derived oligosaccharides (OSs) are major immune targets, often they undergo phase variation, a feature that seemingly makes LOS less desirable as a vaccine candidate. However, the identification of a gonococcal LOS-derived OS epitope, called 2C7, that is: (i) a broadly expressed gonococcal antigenic target in human infection; (ii) a virulence determinant, that is maintained by the gonococcus and (iii) a critical requirement for gonococcal colonization in the experimental setting, circumvents its limitation as a potential vaccine candidate imposed by phase variation. Difficulties in purifying structurally intact OSs from LOSs led to “conversion” of the 2C7 epitope into a peptide mimic that elicited cross-reactive IgG anti-OS antibodies that also possess complement-dependent bactericidal activity against gonococci. Mice immunized with the 2C7 peptide mimic clear vaginal colonization more rapidly and reduce gonococcal burdens. 2C7 vaccine satisfies criteria that are desirable in a gonococcal vaccine candidate: broad representation of the antigenic target, service as a virulence determinant that is also critical for organism survival in vivo and elicitation of broadly cross-reactive IgG bactericidal antibodies when used as an immunogen

Complement alone drives efficacy of a chimeric antigonococcal monoclonal antibody

Multidrug-resistant Neisseria gonorrhoeae is a global health problem. Monoclonal antibody (mAb) 2C7 recognizes a gonococcal lipooligosaccharide epitope that is expressed by \u3e 95% of clinical isolates and hastens gonococcal vaginal clearance in mice. Chimeric mAb 2C7 (human immunoglobulin G1 [IgG1]) with an E430G Fc modification that enhances Fc:Fc interactions and hexamerization following surface-target binding and increases complement activation (HexaBody technology) showed significantly greater C1q engagement and C4 and C3 deposition compared to mAb 2C7 with wild-type Fc. Greater complement activation by 2C7-E430G Fc translated to increased bactericidal activity in vitro and, consequently, enhanced efficacy in mice, compared with Fc-unmodified chimeric 2C7. Gonococci bind the complement inhibitors factor H (FH) and C4b-binding protein (C4BP) in a human-specific manner, which dampens antibody (Ab)-mediated complement-dependent killing. The variant 2C7-E430G Fc overcame the barrier posed by these inhibitors in human FH/C4BP transgenic mice, for which a single 1 mug intravenous dose cleared established infection. Chlamydia frequently coexists with and exacerbates gonorrhea; 2C7-E430G Fc also proved effective against gonorrhea in gonorrhea/chlamydia-coinfected mice. Complement activation alone was necessary and sufficient for 2C7 function, evidenced by the fact that (1) complement-inactive Fc modifications that engaged Fc gamma receptor (FcgammaR) rendered 2C7 ineffective, nonetheless; (2) 2C7 was nonfunctional in C1q-/- mice, when C5 function was blocked, or in C9-/- mice; and (3) 2C7 remained effective in neutrophil-depleted mice and in mice treated with PMX205, a C5a receptor (C5aR1) inhibitor. We highlight the importance of complement activation for antigonococcal Ab function in the genital tract. Elucidating the correlates of protection against gonorrhea will inform the development of Ab-based gonococcal vaccines and immunotherapeutics

Development of Complement Factor H-Based Immunotherapeutic Molecules in Tobacco Plants Against Multidrug-Resistant Neisseria gonorrhoeae

Novel therapeutics against the global threat of multidrug-resistant Neisseria gonorrhoeae are urgently needed. Gonococci possess several mechanisms to evade killing by human complement, including binding of factor H (FH), a key inhibitor of the alternative pathway. FH comprises 20 short consensus repeat (SCR) domains organized in a head-to-tail manner as a single chain. N. gonorrhoeae binds two regions in FH; domains 6 and 7 and domains 18 through 20. We designed a novel anti-infective immunotherapeutic molecule that fuses domains 18-20 of FH containing a D-to-G mutation in domain 19 at position 1119 (called FH*) with human IgG1 Fc. FH*/Fc retained binding to gonococci but did not lyse human erythrocytes. Expression of FH*/Fc in tobacco plants was undertaken as an alternative, economical production platform. FH*/Fc was expressed in high yields in tobacco plants (300-600 mg/kg biomass). The activities of plant- and CHO-cell produced FH*/Fc against gonococci were similar in vitro and in the mouse vaginal colonization model of gonorrhea. The addition of flexible linkers [e.g., (GGGGS)2 or (GGGGS)3] between FH* and Fc improved the bactericidal efficacy of FH*/Fc 2.7-fold. The linkers also improved PMN-mediated opsonophagocytosis about 11-fold. FH*/Fc with linker also effectively reduced the duration and burden of colonization of two gonococcal strains tested in mice. FH*/Fc lost efficacy: i) in C6(-/-) mice (no terminal complement) and ii) when Fc was mutated to abrogate complement activation, suggesting that an intact complement was necessary for FH*/Fc function in vivo. In summary, plant-produced FH*/Fc represent promising prophylactic or adjunctive immunotherapeutics against multidrug-resistant gonococci

Candida albicans Factor H Binding Molecule Hgt1p – A Low Glucose-Induced Transmembrane Protein Is Trafficked to the Cell Wall and Impairs Phagocytosis and Killing by Human Neutrophils

Complement is a tightly controlled arm of the innate immune system, facilitating phagocytosis and killing of invading pathogens. Factor H (FH) is the main fluid-phase inhibitor of the alternative pathway. Many pathogens can hijack FH from the host and protect themselves from complement-dependent killing. Candida albicans is a clinically important opportunistic yeast, expressing different FH binding molecules on its cell surface, which allow complement evasion. One such FH binding molecule is the transmembrane protein “High affinity glucose transporter 1” (Hgt1p), involved in glucose metabolism. This study demonstrated that Hgt1p transcription and expression is induced and highest at the low, but physiological glucose concentration of 0.1%. Thus, this concentration was used throughout the study. We also demonstrated the transport of Hgt1p to the fungal cell wall surface by vesicle trafficking and its release by exosomes containing Hgt1p integrated in the vesicular membrane. We corroborated Hgt1p as FH binding molecule. A polyclonal anti-Hgt1p antibody was created which interfered with the binding of FH, present in normal human serum to the fungal cell wall. A chimeric molecule consisting of FH domains 6 and 7 fused to human IgG1 Fc (FH6.7/Fc) even more comprehensively blocked FH binding, likely because FH6.7/Fc diverted FH away from fungal FH ligands other than Hgt1p. Reduced FH binding to the yeast was associated with a concomitant increase in C3b/iC3b deposition and resulted in significantly increased in vitro phagocytosis and killing by human neutrophils. In conclusion, Hgt1p also exhibits non-canonical functions such as binding FH after its export to the cell wall. Blocking Hgt1p-FH interactions may represent a tool to enhance complement activation on the fungal surface to promote phagocytosis and killing of C. albicans

The Effect of Human Factor H on Immunogenicity of Meningococcal Native Outer Membrane Vesicle Vaccines with Over-Expressed Factor H Binding Protein

The binding of human complement inhibitors to vaccine antigens in vivo could diminish their immunogenicity. A meningococcal ligand for the complement down-regulator, factor H (fH), is fH-binding protein (fHbp), which is specific for human fH. Vaccines containing recombinant fHbp or native outer membrane vesicles (NOMV) from mutant strains with over-expressed fHbp are in clinical development. In a previous study in transgenic mice, the presence of human fH impaired the immunogenicity of a recombinant fHbp vaccine. In the present study, we prepared two NOMV vaccines from mutant group B strains with over-expressed wild-type fHbp or an R41S mutant fHbp with no detectable fH binding. In wild-type mice in which mouse fH did not bind to fHbp in either vaccine, the NOMV vaccine with wild-type fHbp elicited 2-fold higher serum IgG anti-fHbp titers (P = 0.001) and 4-fold higher complement-mediated bactericidal titers against a PorA-heterologous strain than the NOMV with the mutant fHbp (P = 0.003). By adsorption, the bactericidal antibodies were shown to be directed at fHbp. In transgenic mice in which human fH bound to the wild-type fHbp but not to the R41S fHbp, the NOMV vaccine with the mutant fHbp elicited 5-fold higher serum IgG anti-fHbp titers (P = 0.002), and 19-fold higher bactericidal titers than the NOMV vaccine with wild-type fHbp (P = 0.001). Thus, in mice that differed only by the presence of human fH, the respective results with the two vaccines were opposite. The enhanced bactericidal activity elicited by the mutant fHbp vaccine in the presence of human fH far outweighed the loss of immunogenicity of the mutant protein in wild-type animals. Engineering fHbp not to bind to its cognate complement inhibitor, therefore, may increase vaccine immunogenicity in humans

The Meningococcal Vaccine Candidate Neisserial Surface Protein A (NspA) Binds to Factor H and Enhances Meningococcal Resistance to Complement

Complement forms an important arm of innate immunity against invasive meningococcal infections. Binding of the alternative complement pathway inhibitor factor H (fH) to fH-binding protein (fHbp) is one mechanism meningococci employ to limit complement activation on the bacterial surface. fHbp is a leading vaccine candidate against group B Neisseria meningitidis. Novel mechanisms that meningococci employ to bind fH could undermine the efficacy of fHbp-based vaccines. We observed that fHbp deletion mutants of some meningococcal strains showed residual fH binding suggesting the presence of a second receptor for fH. Ligand overlay immunoblotting using membrane fractions from one such strain showed that fH bound to a ∼17 kD protein, identified by MALDI-TOF analysis as Neisserial surface protein A (NspA), a meningococcal vaccine candidate whose function has not been defined. Deleting nspA, in the background of fHbp deletion mutants, abrogated fH binding and mAbs against NspA blocked fH binding, confirming NspA as a fH binding molecule on intact bacteria. NspA expression levels vary among strains and expression correlated with the level of fH binding; over-expressing NspA enhanced fH binding to bacteria. Progressive truncation of the heptose (Hep) I chain of lipooligosaccharide (LOS), or sialylation of lacto-N-neotetraose LOS both increased fH binding to NspA-expressing meningococci, while expression of capsule reduced fH binding to the strains tested. Similar to fHbp, binding of NspA to fH was human-specific and occurred through fH domains 6–7. Consistent with its ability to bind fH, deleting NspA increased C3 deposition and resulted in increased complement-dependent killing. Collectively, these data identify a key complement evasion mechanism with important implications for ongoing efforts to develop meningococcal vaccines that employ fHbp as one of its components

Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being unable to limit the infection. Herein we describe the course of GAS infection in three human complement inhibitor transgenic (tg) mouse models that examined each inhibitor (human C4BP or FH) alone, or the two inhibitors together (C4BPxFH or 'double' tg). GAS infection with strains that bound C4BP and FH resulted in enhanced mortality in each of the three transgenic mouse models compared to infection in wild type mice. In addition, GAS manifested increased virulence in C4BPxFH mice: higher organism burdens and greater elevations of pro-inflammatory cytokines and they died earlier than single transgenic or wt controls. The effects of hu-C4BP and hu-FH were specific for GAS strains that bound these inhibitors because strains that did not bind the inhibitors showed reduced virulence in the 'double' tg mice compared to strains that did bind; mortality was also similar in wild-type and C4BPxFH mice infected by non-binding GAS. Our findings emphasize the importance of binding of complement inhibitors to GAS that results in impaired opsonization and phagocytic killing, which translates to enhanced virulence in a humanized whole animal model. This novel hu-C4BPxFH tg model may prove invaluable in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy

Functional comparison of the binding of factor H short consensus repeat 6 (SCR 6) to factor H binding protein from Neisseria meningitidis and the binding of factor H SCR 18 to 20 to Neisseria gonorrhoeae porin

Both Neisseria meningitidis and Neisseria gonorrhoeae recruit the alternative pathway complement inhibitory protein factor H (fH) to their surfaces to evade complement-dependent killing. Meningococci bind fH via fH binding protein (fHbp), a surface-exposed lipoprotein that is subdivided into three variant families based on one classification scheme. Chimeric proteins that comprise contiguous domains of fH fused to murine Fc were used to localize the binding site for all three fHbp variants on fH to short consensus repeat 6 (SCR 6). As expected, fH-like protein 1 (FHL-1), which contains fH SCR 6, also bound to fHbp-expressing meningococci. Using site-directed mutagenesis, we identified histidine 337 and histidine 371 in SCR 6 as important for binding to fHbp. These findings may provide the molecular basis for recent observations that demonstrated human-specific fH binding to meningococci. Differences in the interactions of fHbp variants with SCR 6 were evident. Gonococci bind fH via their porin (Por) molecules (PorB.1A or PorB.1B); sialylation of lipooligosaccharide enhances fH binding. Both sialylated PorB.1B- and (unsialylated) PorB.1A-bearing gonococci bind fH through SCR 18 to 20; PorB.1A can also bind SCR 6, but only weakly, as evidenced by a low level of binding of FHL-1 relative to that of fH. Using isogenic strains expressing either meningococcal fHbp or gonococcal PorB.1B, we discovered that strains expressing gonococcal PorB.1B in the presence of sialylated lipooligosaccharide bound more fH, more effectively limited C3 deposition, and were more serum resistant than their isogenic counterparts expressing fHbp. Differences in fH binding to these two related pathogens may be important for modulating their individual responses to host immune attack