Two human antibodies to a meningococcal serogroup B vaccine antigen enhance binding of complement Factor H by stabilizing the Factor H binding site

Microbial pathogens bind host complement regulatory proteins to evade the immune system. The bacterial pathogen Neisseria meningitidis, or meningococcus, binds several complement regulators, including human Factor H (FH). FH binding protein (FHbp) is a component of two licensed meningococcal vaccines and in mice FHbp elicits antibodies that inhibit binding of FH to FHbp, which defeat the bacterial evasion mechanism. However, humans vaccinated with FHbp develop antibodies that enhance binding of FH to the bacteria, which could limit the effectiveness of the vaccines. In the present study, we show that two vaccine-elicited antibody fragments (Fabs) isolated from different human subjects increase binding of complement FH to meningococcal FHbp by ELISA. The two Fabs have different effects on the kinetics of FH binding to immobilized FHbp as measured by surface plasmon resonance. The 1.7- and 2.0-Å resolution X-ray crystal structures of the Fabs in complexes with FHbp illustrate that the two Fabs bind to similar epitopes on the amino-terminal domain of FHbp, adjacent to the FH binding site. Superposition models of ternary complexes of each Fab with FHbp and FH show that there is likely minimal contact between the Fabs and FH. Collectively, the structures reveal that the Fabs enhance binding of FH to FHbp by altering the conformations and mobilities of two loops adjacent to the FH binding site of FHbp. In addition, the 1.5 Å-resolution structure of one of the isolated Fabs defines the structural rearrangements associated with binding to FHbp. The FH-enhancing human Fabs, which are mirrored in the human polyclonal antibody responses, have important implications for tuning the effectiveness of FHbp-based vaccines.

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Impaired Immunogenicity of a Meningococcal Factor H-Binding Protein Vaccine Engineered To Eliminate Factor H Binding

Clinical and Vaccine Immunology ◽

10.1128/cvi.00103-10 ◽

2010 ◽

Vol 17 (7) ◽

pp. 1074-1078 ◽

Cited By ~ 19

Author(s):

Peter T. Beernink ◽

Jutamas Shaughnessy ◽

Sanjay Ram ◽

Dan M. Granoff

Keyword(s):

Binding Protein ◽

Factor H ◽

Vaccine Antigen ◽

Mouse Strains ◽

Enzyme Linked Immunosorbent Assay ◽

Complement Factor ◽

Protein Vaccine ◽

Wild Type ◽

Bacterial Surface ◽

Cast Doubt

ABSTRACT Meningococcal factor H-binding protein (fHbp) is a promising antigen that is part of two vaccines in clinical development. The protein specifically binds human complement factor H (fH), which downregulates complement activation on the bacterial surface and enables the organism to evade host defenses. In humans, the vaccine antigen forms a complex with fH, which may affect anti-fHbp antibody repertoire and decrease serum bactericidal activity by covering important fHbp epitopes. In a recent study, fHbp residues in contact with fH were identified from a crystal structure. Two fHbp glutamate residues that mediated ion-pair interactions with fH were replaced with alanine, and the resulting E218A/E239A mutant no longer bound the fH fragment. In the present study, we generated the E218A/E239A mutant recombinant protein and confirmed the lack of fH binding. By enzyme-linked immunosorbent assay (ELISA), the mutant fHbp showed similar respective concentration-dependent inhibition of binding of four bactericidal anti-fHbp monoclonal antibodies (MAbs) to fHbp, compared with inhibition by the soluble wild-type protein. In two mouse strains, the mutant fHbp elicited up to 4-fold-lower IgG anti-fHbp antibody titers and up to 20-fold-lower serum bactericidal titers than those elicited by the wild-type fHbp vaccine. Thus, although introduction of the two alanine substitutions to eliminate fH binding did not appear to destabilize the molecule globally, the mutations resulted in decreased immunogenicity in mouse models in which neither the mutant nor the wild-type control vaccine bound fH. These results cast doubt on the vaccine potential in humans of this mutant fHbp.

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Identification and characterization of a new cell surface protein possessing factor H-binding activity in the swine pathogen and zoonotic agent Streptococcus suis

Journal of Medical Microbiology ◽

10.1099/jmm.0.057877-0 ◽

2013 ◽

Vol 62 (7) ◽

pp. 1073-1080 ◽

Cited By ~ 13

Author(s):

Katy Vaillancourt ◽

Laetitia Bonifait ◽

Louis Grignon ◽

Michel Frenette ◽

Marcelo Gottschalk ◽

...

Keyword(s):

Cell Surface ◽

Surface Protein ◽

Streptococcus Suis ◽

Binding Activity ◽

Factor H ◽

Complement Factor ◽

Cell Surface Protein ◽

Amino Terminal ◽

Swine Pathogen ◽

Complement Regulator

Streptococcus suis is a major swine pathogen and an emerging zoonotic agent. The ability of pathogenic bacteria to bind the complement regulator factor H on their cell surface may allow them to avoid complement attack and phagocytosis. The aim of this study was to characterize a new cell surface protein possessing factor H-binding activity in S. suis serotype 2. The capacity of S. suis to bind the complement regulator factor H on its surface was demonstrated by ELISA. Using a factor I–cofactor assay, it was found that the functional activity of factor H bound to S. suis was kept. Since the product of gene SSU0186 in S. suis P1/7 shared similarity with a Streptococcus pneumoniae protein (named PspC) possessing factor H-binding activity, it was proposed as a putative factor H receptor in S. suis. SSU0186 has a 1686 bp open reading frame encoding a 561 amino acid protein containing the Gram-positive cell wall anchoring motif (LPXTG) at the carboxy-terminal, an amino-terminal signal sequence, an α-helix domain, a proline-rich region and a G5 domain. The SSU0186 gene was cloned in Escherichia coli and the purified recombinant factor H-binding protein showed a molecular mass of 95 kDa, as determined by SDS-PAGE. The protein possessed the functional property of binding factor H. Sera from S. suis-infected pigs reacted with the recombinant factor H receptor, suggesting that it is produced during the course of infections. In conclusion, we identified a novel S. suis cell surface protein that binds the complement factor H. This cell surface protein may help S. suis to resist complement attack and phagocytosis and contribute to pathogenesis.

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Molecular crypsis by pathogenic fungi using human factor H. A numerical model

10.1101/250662 ◽

2018 ◽

Author(s):

Stefan Lang ◽

Sebastian Germerodt ◽

Christina Glock ◽

Christine Skerka ◽

Peter F. Zipfel ◽

...

Keyword(s):

Immune System ◽

Molecular Mimicry ◽

Pathogenic Fungi ◽

Blood Stream ◽

Factor H ◽

Host Cells ◽

Microbial Pathogens ◽

Complement Factor ◽

Interaction Modelling ◽

Predominant Factor

AbstractMolecular mimicry is the formation of specific molecules by microbial pathogens to avoid recognition and attack by the immune system of the host. Several pathogenic Ascomycota and Zygomycota show such a behaviour by utilizing human complement factor H to hide in the blood stream. We call this type of mimicry molecular crypsis. Such a crypsis can reach a point where the immune system can no longer clearly distinguish between self and non-self cells. Thus, a trade-off between attacking disguised pathogens and erroneously attacking host cells has to be made, which can lead to autoreactivity. Based on signalling theory and protein-interaction modelling, we here present a mathematical model of molecular crypsis of pathogenic fungi using the example of Candida albicans. We tackle the question whether perfect crypsis is feasible, which would imply that protection of human cells by complement factors would be useless. The model identifies pathogen abundance relative to host cell abundance as the predominant factor influencing successful or unsuccessful molecular crypsis. If pathogen cells gain a (locally) quantitative advantage over host cells, even autoreactivity may occur. Our new model enables insights into the mechanisms of candidiasis-induced sepsis and complement associated autoimmune diseases.

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M Protein of the Group A StreptococcusBinds to the Seventh Short Consensus Repeat of Human Complement Factor H

Infection and Immunity ◽

10.1128/iai.66.4.1427-1431.1998 ◽

1998 ◽

Vol 66 (4) ◽

pp. 1427-1431 ◽

Cited By ~ 69

Author(s):

Timothy K. Blackmore ◽

Vincent A. Fischetti ◽

Tania A. Sadlon ◽

Helena M. Ward ◽

David L. Gordon

Keyword(s):

Protein Binding ◽

Binding Site ◽

Regulatory Protein ◽

Factor H ◽

M Protein ◽

Protein Binding Site ◽

Enzyme Linked Immunosorbent Assay ◽

Complement Factor ◽

Heparin Binding ◽

Protein Factor

ABSTRACT Streptococcus pyogenes evades complement by binding the complement-regulatory protein factor H (fH) via the central conserved C-repeat region of M protein. However, the corresponding binding region within fH has not previously been precisely localized. fH is composed of 20 conserved modules called short consensus repeats (SCRs), each of which contains approximately 60 amino acids. A series of fH truncated and deletion mutants were prepared, and their interaction with M6 protein was examined. The M protein binding site was initially localized to SCRs 6 to 15 as demonstrated by ligand dot blotting, chemical cross-linking, and enzyme-linked immunosorbent assay. SCR 7 was then shown to contain the M protein binding site, as a construct consisting of the first seven SCRs bound M protein but a construct containing the first six SCRs did not bind. In addition, deletion of SCR 7 from full-length fH abolished binding to M protein. SCR 7 is known to contain a heparin binding domain, and binding of fH to M6 protein was almost totally inhibited in the presence of 400 U of heparin per ml. These results localize the M6 protein binding site of fH to SCR 7 and indicate that it is in close proximity to the heparin binding site.

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Binding of Complement Factor H to Loop 5 of Porin Protein 1A: A Molecular Mechanism of Serum Resistance of Nonsialylated Neisseria gonorrhoeae

Journal of Experimental Medicine ◽

10.1084/jem.188.4.671 ◽

1998 ◽

Vol 188 (4) ◽

pp. 671-680 ◽

Cited By ~ 191

Author(s):

Sanjay Ram ◽

Daniel P. McQuillen ◽

Sunita Gulati ◽

Christopher Elkins ◽

Michael K. Pangburn ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Binding Site ◽

Molecular Basis ◽

Factor H ◽

Serum Resistance ◽

Complement Factor ◽

Bacterial Surface ◽

Exposed Region ◽

Porin Protein ◽

Serum Bactericidal Assay

Neisseria gonorrhoeae isolated from patients with disseminated infection are often of the porin (Por1A) serotype and resist killing by nonimmune normal human serum. The molecular basis of this resistance (termed stable serum resistance) in these strains has not been fully defined but is not related to sialylation of lipooligosaccharide. Here we demonstrate that Por1A bearing gonococcal strains bind more factor H, a critical downregulator of the alternative complement pathway, than their Por1B counterparts. This results in a sevenfold reduction in C3b, which is >75% converted to iC3b. Factor H binding to isogenic gonococcal strains that differed only in their porin serotype, confirmed that Por1A was the acceptor molecule for factor H. We identified a surface exposed region on the Por1A molecule that served as the binding site for factor H. We used gonococcal strains with hybrid Por1A/B molecules that differed in their surface exposed domains to localize the factor H binding site to loop 5 of Por1A. This was confirmed by inhibition of factor H binding using synthetic peptides corresponding to the putative exposed regions of the porin loops. The addition of Por1A loop 5 peptide in a serum bactericidal assay, which inhibited binding of factor H to the bacterial surface, permitted 50% killing of an otherwise completely serum resistant gonococcal strain. Collectively, these data provide a molecular basis to explain serum resistance of Por1A strains of N. gonorrhoeae.

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Localization of the heparin-binding site on complement factor H.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)55379-5 ◽

1991 ◽

Vol 266 (25) ◽

pp. 16847-16853 ◽

Cited By ~ 1

Author(s):

M.K. Pangburn ◽

M.A. Atkinson ◽

S. Meri

Keyword(s):

Binding Site ◽

Factor H ◽

Complement Factor H ◽

Complement Factor ◽

Heparin Binding ◽

Heparin Binding Site

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Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase

Nature ◽

10.1038/18050 ◽

1999 ◽

Vol 398 (6722) ◽

pp. 84-90 ◽

Cited By ~ 215

Author(s):

Wuyi Meng ◽

Sansana Sawasdikosol ◽

Steven J. Burakoff ◽

Michael J. Eck

Keyword(s):

Binding Site ◽

Amino Terminal ◽

Terminal Domain ◽

Amino Terminal Domain

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The importance of the location of antibody binding on the M6 protein for opsonization and phagocytosis of group A M6 streptococci.

Journal of Experimental Medicine ◽

10.1084/jem.167.3.1114 ◽

1988 ◽

Vol 167 (3) ◽

pp. 1114-1123 ◽

Cited By ~ 75

Author(s):

K F Jones ◽

V A Fischetti

Keyword(s):

Complement Fixation ◽

Hypervariable Region ◽

Antibody Binding ◽

Factor H ◽

Complement Factor ◽

Amino Terminal ◽

Group A ◽

Native Group ◽

Binding Titer

One of 19 mAbs against the native group A streptococcal M6 protein proved opsonic for type 6 organisms in a bactericidal assay. The opsonic and three nonopsonic antibodies were selected for isotype and complement fixation studies based on previous knowledge of their epitope site on the M6 molecule. While mAb 3B8 (IgG3), whose epitope is in the NH2-terminal hypervariable region of the molecule (distal from the cell), and mAbs 10B6 (IgG2a) and 10F5 (IgG2b), both located in the conserved central region of the molecule, all fix complement, 10A11 (IgG1) did not. Only mAb 3B8 was opsonic despite the fact that mAbs 10B6 and 10F5 both exhibited similar complement-fixing capacity, binding titer, and surface exposure of epitopes. Analysis of antibodies raised against synthetic peptides representing various regions of the M6 protein showed that only the amino-terminal peptide (residues 1-21) was capable of eliciting opsonic antibodies, despite the fact that peptides from other areas produced antibodies with high-binding titers to the native M6 protein and also with the ability to bind to intact streptococcal cells. These results not only support the observed type specificity of opsonic antibodies, but also clearly point to the importance of the location of antibody binding on the M molecule relative to the actual functional capacity of the antibody with respect to the opsonization and phagocytosis of M6 streptococci. These results may underscore the recently observed role of complement Factor H in the antiphagocytic activity of the M protein.

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