Jutamas Ngampasutadol

The Meningococcal Vaccine Candidate GNA1870 Binds the Complement Regulatory Protein Factor H and Enhances Serum Resistance

Neisseria meningitidis binds factor H (fH), a key regulator of the alternative complement pathway. A ∼29 kD fH-binding protein expressed in the meningococcal outer membrane was identified by mass spectrometry as GNA1870, a lipoprotein currently under evaluation as a broad-spectrum meningococcal vaccine candidate. GNA1870 was confirmed as the fH ligand on intact bacteria by 1) abrogation of fH binding upon deleting GNA1870, and 2) blocking fH binding by anti-GNA1870 mAbs. fH bound to whole bacteria and purified rGNA1870 representing each of the three variant GNA1870 families. We showed that the amount of fH binding correlated with the level of bacterial GNA1870 expression. High levels of variant 1 GNA1870 expression (either by allelic replacement of gna1870 or by plasmid-driven high-level expression) in strains that otherwise were low-level GNA1870 expressers (and bound low amounts of fH by flow cytometry) restored high levels of fH binding. Diminished fH binding to the GNA1870 deletion mutants was accompanied by enhanced C3 binding and increased killing of the mutants. Conversely, high levels of GNA1870 expression and fH binding enhanced serum resistance. Our findings support the hypothesis that inhibiting the binding of a complement down-regulator protein to the neisserial surface by specific Ab may enhance intrinsic bactericidal activity of the Ab, resulting in two distinct mechanisms of Ab-mediated vaccine efficacy. These data provide further support for inclusion of this molecule in a meningococcal vaccine. To reflect the critical function of this molecule, we suggest calling it fH-binding protein.

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.

Human Factor H Interacts Selectively with Neisseria gonorrhoeae and Results in Species-Specific Complement Evasion

Complement forms a key arm of innate immune defenses against gonococcal infection. Sialylation of gonococcal lipo-oligosaccharide, or expression of porin 1A (Por1A) protein, enables Neisseria gonorrhoeae to bind the alternative pathway complement inhibitor, factor H (fH), and evade killing by human complement. Using recombinant fH fragment-murine Fc fusion proteins, we localized two N. gonorrhoeae Por1A-binding regions in fH: one in complement control protein domain 6, the other in complement control proteins 18–20. The latter is similar to that reported previously for sialylated Por1B gonococci. Upon incubation with human serum, Por1A and sialylated Por1B strains bound full-length human fH (HufH) and fH-related protein 1. In addition, Por1A strains bound fH-like protein 1 weakly. Only HufH, but not fH from other primates, bound directly to gonococci. Consistent with direct HufH binding, unsialylated Por1A gonococci resisted killing only by human complement, but not complement from other primates, rodents or lagomorphs; adding HufH to these heterologous sera restored serum resistance. Lipo-oligosaccharide sialylation of N. gonorrhoeae resulted in classical pathway regulation as evidenced by decreased C4 binding in human, chimpanzee, and rhesus serum but was accompanied by serum resistance only in human and chimpanzee serum. Direct-binding specificity of HufH only to gonococci that prevents serum killing is restricted to humans and may in part explain species-specific restriction of natural gonococcal infection. Our findings may help to improve animal models for gonorrhea while also having implications in the choice of complement sources to evaluate neisserial vaccine candidates.

Factor H Binding and Function in Sialylated Pathogenic Neisseriae is Influenced by Gonococcal, but Not Meningococcal, Porin

Neisseria gonorrhoeae and Neisseria meningitidis both express the lacto-N-neotetraose (LNT) lipooligosaccharide (LOS) molecule that can be sialylated. Although gonococcal LNT LOS sialylation enhances binding of the alternative pathway complement inhibitor factor H and renders otherwise serum-sensitive bacteria resistant to complement-dependent killing, the role of LOS sialylation in meningococcal serum resistance is less clear. We show that only gonococcal, but not meningococcal, LNT LOS sialylation enhanced factor H binding. Replacing the porin (Por) B molecule of a meningococcal strain (LOS sialylated) that did not bind factor H with gonococcal Por1B augmented factor H binding. Capsule expression did not alter factor H binding to meningococci that express gonococcal Por. Conversely, replacing gonococcal Por1B with meningococcal PorB abrogated factor H binding despite LNT LOS sialylation. Gonococcal Por1B introduced in the background of an unsialylated meningococcus itself bound small amounts of factor H, suggesting a direct factor H-Por1B interaction. Factor H binding to unsialylated meningococci transfected with gonococcal Por1B was similar to the sialylated counterpart only in the presence of higher (20 μg/ml) concentrations of factor H and decreased in a dose-responsive manner by ∼80% at 1.25 μg/ml. Factor H binding to the sialylated strain remained unchanged over this factor H concentration range however, suggesting that LOS sialylation facilitated optimal factor H-Por1B interactions. The functional counterpart of factor H binding showed that sialylated meningococcal mutants that possessed gonococcal Por1B were resistant to complement-mediated killing by normal human serum. Our data highlight the different mechanisms used by these two related species to evade complement.

Heptose I Glycan Substitutions on Neisseria gonorrhoeae Lipooligosaccharide Influence C4b-Binding Protein Binding and Serum Resistance

ABSTRACT Lipooligosaccharide (LOS) heptose (Hep) glycan substitutions influence gonococcal serum resistance. Several gonococcal strains bind the classical complement pathway inhibitor, C4b-binding protein (C4BP), via their porin (Por) molecule to escape complement-dependent killing by normal human serum (NHS). We show that the proximal glucose (Glc) on HepI is required for C4BP binding to Por1B-bearing gonococcal strains MS11 and 1291 but not to FA19 (Por1A). The presence of only the proximal Glc on HepI (lgtE mutant) permitted maximal C4BP binding to MS11 but not to 1291. Replacing 1291 lgtE Por with MS11 Por increased C4BP binding to levels that paralleled MS11 lgtE, suggesting that replacement of the Por1B molecule dictated the effects of HepI glycans on C4BP binding. The remainder of the strain background did not affect C4BP binding; replacing the Por of strain F62 with MS11 Por (F62 PorMS11) and truncating HepI mirrored the findings in the MS11 background. C4BP binding correlated with resistance to killing by NHS in most instances. F62 PorMS11 and its lgtE mutant were sensitive to NHS despite binding C4BP, secondary to kinetically overwhelming classical pathway activation and possibly increased alternative pathway activation (measured by factor Bb binding) by the F62 background. FA19 lgtF (HepI unsubstituted) resisted killing by only 10% NHS, not 50% NHS, despite binding levels of C4BP similar to those of FA19 and FA19 lgtE (both resistant to 50% serum), suggesting a role for the proximal Glc in serum resistance independently of C4BP binding. This study provides mechanistic insights into how HepI LOS substitutions affect the serum resistance of N. gonorrhoeae.

Molecular Characterization of the Interaction between Porins of Neisseria gonorrhoeae and C4b-Binding Protein

Neisseria gonorrhoeae, the causative agent of gonorrhea, is a natural infection only in humans. The resistance of N. gonorrhoeae to normal human serum killing correlates with porin (Por)-mediated binding to the complement inhibitor, C4b-binding protein (C4BP). The entire binding site for both porin molecules resides within complement control protein domain 1 (CCP1) of C4BP. Only human and chimpanzee C4BPs bind to Por1B-bearing gonococci, whereas only human C4BP binds to Por1A strains. We have now used these species-specific differences in C4BP binding to gonococci to map the porin binding sites on CCP1 of C4BP. A comparison between human and chimpanzee or rhesus C4BP CCP1 revealed differences at 4 and 12 amino acid positions, respectively. These amino acids were targeted in the construction of 13 recombinant human mutant C4BPs. Overall, amino acids T43, T45, and K24 individually and A12, M14, R22, and L34 together were important for binding to Por1A strains. Altering D15 (found in man) to N15 (found in rhesus) introduced a glycosylation site that blocked binding to Por1A gonococci. C4BP binding to Por1B strains required K24 and was partially shielded by additional glycosylation in the D15N mutant. Only those recombinant mutant C4BPs that bound to bacteria rescued them from 100% killing by rhesus serum, thereby providing a functional correlate for the binding studies and highlighting C4BP function in gonococcal serum resistance.

Factor H Facilitates Adherence of Neisseria gonorrhoeae to Complement Receptor 3 on Eukaryotic Cells

Neisseria gonorrhoeae can engage human complement receptor 3 (CR3) directly or through surface-bound iC3b. Factor H (fH) that binds to bacteria facilitates conversion of C3b to iC3b. fH also binds directly to CR3 on professional phagocytes. Certain nonprofessional phagocytes, such as primary cervical epithelial cells, also express CR3. We hypothesized that fH could bridge bacteria to CR3 and facilitate gonococcal association with host cells. Specificity of the fH–CR3 interaction was confirmed using human CR3-transfected Chinese hamster ovary (CHO-CR3) cells. Using recombinant proteins that comprised contiguous fH domains (fH contains 20 short consensus repeat [SCR] domains) fused to murine Fc, we observed strong binding through SCRs 18–20, whereas weaker binding occurred through SCRs 6–10. Both regions also bound to unsialylated porin (Por) B.1A-expressing N. gonorrhoeae. Accordingly, fH-related protein 1 (three of its five SCRs are highly homologous to fH SCRs 18–20) bound to CHO-CR3 and to unsialylated PorB.1A gonococci. An alternatively spliced variant of fH called fH-like protein-1 (contains fH SCRs 1–7) bound to gonococci but minimally to CHO-CR3. An fH SCRs 6–20 construct enhanced binding of unsialylated PorB.1A gonococci to CHO-CR3. However, a construct that contained only the apparently relevant SCRs (6, 7, and 18–20) bound to CHO-CR3 and to gonococci separately, but did not enhance bacteria–CR3 interactions, suggesting that the intervening SCRs (8–17) may impart a configurational and spatial requirement for fH to bridge gonococci to CR3. These results indicate adherence between fH-coated gonococci and CR3 and may provide a means for gonococci to gain sanctuary into nonprofessional phagocytes.

Strategies for mimicking Neisserial saccharide epitopes as vaccines.

Monoclonal antibody (mAb) 2C7 recognizes a conserved and widely expressed oligosaccharide (OS) epitope on Neisseria gonorrhoeae. This OS epitope evokes a significant bactericidal and opsonic immune response after natural infection and vaccination. The OS epitope structure represents an excellent target for a potential protective gonococcal vaccine. Because carbohydrate antigens are T-cell independent, inducing weak antibody responses, OS molecules are not useful immunogens. We developed and examined two different strategies to mimic the 2C7 OS epitope: (i) an anti-idiotope (mAb CAI); and (ii) a peptide (PEP-1). These surrogate immunogens elicited antibody responses in mice (CAI and PEP-1) and rabbits (CAI) that were bactericidal in vitro against gonococci. Both CAI and PEP-1 arc true immunologic mimics of OS and may form a basis for the development of vaccine candidates for human immunization against N. gonorrhoeae.