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 BNeisseria meningitidisclinical 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 ofporB3from 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.

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Characterization of Neisseria meningitidis Isolates That Do Not Express the Virulence Factor and Vaccine Antigen Factor H Binding Protein

Clinical and Vaccine Immunology ◽

10.1128/cvi.00055-11 ◽

2011 ◽

Vol 18 (6) ◽

pp. 1002-1014 ◽

Cited By ~ 65

Author(s):

Jay Lucidarme ◽

Lionel Tan ◽

Rachel M. Exley ◽

Jamie Findlow ◽

Ray Borrow ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Binding Protein ◽

Factor H ◽

Negative Regulator ◽

Vaccine Antigen ◽

Natural Immunity ◽

Content Type ◽

Reading Frame ◽

Flanking Sequences

ABSTRACTNeisseria meningitidisremains a leading cause of bacterial sepsis and meningitis. Complement is a key component of natural immunity against this important human pathogen, which has evolved multiple mechanisms to evade complement-mediated lysis. One approach adopted by the meningococcus is to recruit a human negative regulator of the complement system, factor H (fH), to its surface via a lipoprotein, factor H binding protein (fHbp). Additionally, fHbp is a key antigen in vaccines currently being evaluated in clinical trials. Here we characterize strains ofN. meningitidisfrom several distinct clonal complexes which do not express fHbp; all strains were recovered from patients with disseminated meningococcal disease. We demonstrate that these strains have either a frameshift mutation in thefHbpopen reading frame or have entirely lostfHbpand some flanking sequences. No fH binding was detected to other ligands among thefHbp-negative strains. The implications of these findings for meningococcal pathogenesis and prevention are discussed.

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Neisseria cinerea Expresses a Functional Factor H Binding Protein Which Is Recognized by Immune Responses Elicited by Meningococcal Vaccines

Infection and Immunity ◽

10.1128/iai.00305-17 ◽

2017 ◽

Vol 85 (10) ◽

Cited By ~ 3

Author(s):

Hayley Lavender ◽

Katy Poncin ◽

Christoph M. Tang

Keyword(s):

Capsular Polysaccharide ◽

Binding Protein ◽

Alternative Pathway ◽

Factor H ◽

Recent Analysis ◽

Complement Factor ◽

Content Type ◽

Commensal Flora ◽

Commensal Species ◽

Complement Regulator

ABSTRACT Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide. Capsular polysaccharide vaccines are available against meningococcal serogroups A, C, W, and Y. More recently two protein-based vaccines, Bexsero and Trumenba, against meningococcal serogroup B strains have been licensed; both vaccines contain meningococcal factor H binding protein (fHbp). fHbp is a surface-exposed lipoprotein that binds the negative complement regulator complement factor H (CFH), thereby inhibiting the alternative pathway of complement activation. Recent analysis of available genomes has indicated that some commensal Neisseria species also contain genes that potentially encode fHbp, although the functions of these genes and how immunization with fHbp-containing vaccines could affect the commensal flora have yet to be established. Here, we show that the commensal species Neisseria cinerea expresses functional fHbp on its surface and that it is responsible for recruitment of CFH by the bacterium. N. cinerea fHbp binds CFH with affinity similar to that of meningococcal fHbp and promotes survival of N. cinerea in human serum. We examined the potential impact of fHbp-containing vaccines on N. cinerea. We found that immunization with Bexsero elicits serum bactericidal activity against N. cinerea, which is primarily directed against fHbp. The shared function of fHbp in N. cinerea and N. meningitidis and cross-reactive responses elicited by Bexsero suggest that the introduction of fHbp-containing vaccines has the potential to affect carriage of N. cinerea and other commensal species.

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Factor H binding protein (fHbp)-mediated differential complement resistance of a serogroup C Neisseria meningitidis isolate from cerebrospinal fluid of a patient with invasive meningococcal disease

Access Microbiology ◽

10.1099/acmi.0.000255 ◽

2021 ◽

Vol 3 (9) ◽

Author(s):

Alessandra Facchetti ◽

Jun X. Wheeler ◽

Caroline Vipond ◽

Gail Whiting ◽

Hayley Lavender ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Meningococcal Disease ◽

Close Contact ◽

Binding Protein ◽

Integral Membrane Protein ◽

Factor H ◽

Invasive Meningococcal Disease ◽

Content Type ◽

Type Iv ◽

Complement Resistance

During an outbreak of invasive meningococcal disease (IMD) at the University of Southampton, UK, in 1997, two Neisseria meningitidis serogroup C isolates were retrieved from a student (‘Case’), who died of IMD, and a close contact (‘Carrier’) who, after mouth-to-mouth resuscitation on the deceased, did not contract the disease. Genomic comparison of the isolates demonstrated extensive nucleotide sequence identity, with differences identified in eight genes. Here, comparative proteomics was used to measure differential protein expression between the isolates and investigate whether the differences contributed to the clinical outcomes. A total of six proteins were differentially expressed: four proteins (methylcitrate synthase, PrpC; hypothetical integral membrane protein, Imp; fructose-1,6-bisphosphate aldolase, Fba; aldehyde dehydrogenase A, AldA) were upregulated in the Case isolate, while one protein (Type IV pilus-associated protein, PilC2) was downregulated. Peptides for factor H binding protein (fHbp), a major virulence factor and antigenic protein, were only detected in the Case, with a single base deletion (ΔT366) in the Carrier fHbp causing lack of its expression. Expression of fHbp resulted in an increased resistance of the Case isolate to complement-mediated killing in serum. Complementation of fHbp expression in the Carrier increased its serum resistance by approximately 8-fold. Moreover, a higher serum bactericidal antibody titre was seen for the Case isolate when using sera from mice immunized with Bexsero (GlaxoSmithKline), a vaccine containing fHbp as an antigenic component. This study highlights the role of fHbp in the differential complement resistance of the Case and the Carrier isolates. Expression of fHbp in the Case resulted in its increased survival in serum, possibly leading to active proliferation of the bacteria in blood and death of the student through IMD. Moreover, enhanced killing of the Case isolate by sera raised against an fHbp-containing vaccine, Bexsero, underlines the role and importance of fHbp in infection and immunity.

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Heterogeneity in Rhesus Macaque Complement Factor H Binding to Meningococcal Factor H Binding Protein (FHbp) Informs Selection of Primates To Assess Immunogenicity of FHbp-Based Vaccines

Clinical and Vaccine Immunology ◽

10.1128/cvi.00517-14 ◽

2014 ◽

Vol 21 (11) ◽

pp. 1505-1511 ◽

Cited By ~ 13

Author(s):

Peter T. Beernink ◽

Jutamas Shaughnessy ◽

Heather Stefek ◽

Sanjay Ram ◽

Dan M. Granoff

Keyword(s):

Binding Protein ◽

Rhesus Macaques ◽

Alternative Pathway ◽

Factor H ◽

Complement Factor ◽

Primate Model ◽

Complement Alternative Pathway ◽

Content Type ◽

Bacterial Surface ◽

Short Consensus Repeats

ABSTRACTNeisseria meningitidiscauses disease only in humans. An important mechanism underlying this host specificity is the ability of the organism to resist complement by recruiting the complement downregulator factor H (FH) to the bacterial surface. In previous studies, binding of FH to one of the major meningococcal FH ligands, factor H binding protein (FHbp), was reported to be specific for human FH. Here we report that sera from 23 of 73 rhesus macaques (32%) tested had high FH binding to FHbp. Similar to human FH, binding of macaque FH to the meningococcal cell surface inhibited the complement alternative pathway by decreasing deposition of C3b. FH contains 20 domains (or short consensus repeats), with domains 6 and 7 being responsible for binding of human FH to FHbp. DNA sequence analyses of FH domains 6 and 7 from macaques with high or low FH binding showed a polymorphism at residue 352 in domain 6, with Tyr being associated with high binding and His with low binding. A recombinant macaque FH 6,7/Fc fragment with Tyr352 showed higher binding to FHbp than the corresponding fragment with His352. In previous studies in human FH transgenic mice, binding of FH to FHbp vaccines decreased protective antibody responses, and mutant FHbp vaccines with decreased FH binding elicited serum antibodies with greater protective activity. Thus, macaques with high FH binding to FHbp represent an attractive nonhuman primate model to investigate further the effects of FH binding on the immunogenicity of FHbp vaccines.

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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

Infection and Immunity ◽

10.1128/iai.00462-20 ◽

2020 ◽

Vol 88 (12) ◽

Cited By ~ 1

Author(s):

Yih-Ling Tzeng ◽

Serena Giuntini ◽

Zachary Berman ◽

Soma Sannigrahi ◽

Dan M. Granoff ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Binding Protein ◽

Factor H ◽

Negative Regulator ◽

Protein Variant ◽

Content Type ◽

Mucosal Surfaces ◽

Low Levels ◽

Group B ◽

Variant Peptide

ABSTRACT 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.

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Molecular Engineering of Ghfp, the Gonococcal Orthologue of Neisseria meningitidis Factor H Binding Protein

Clinical and Vaccine Immunology ◽

10.1128/cvi.00794-14 ◽

2015 ◽

Vol 22 (7) ◽

pp. 769-777 ◽

Cited By ~ 4

Author(s):

Valentina Rippa ◽

Laura Santini ◽

Paola Lo Surdo ◽

Francesca Cantini ◽

Daniele Veggi ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Rational Design ◽

Vaccine Development ◽

Structural Information ◽

Binding Protein ◽

Molecular Engineering ◽

Factor H ◽

Microbial Pathogens ◽

Content Type ◽

Content Factor

ABSTRACTKnowledge of the sequences and structures of proteins produced by microbial pathogens is continuously increasing. Besides offering the possibility of unraveling the mechanisms of pathogenesis at the molecular level, structural information provides new tools for vaccine development, such as the opportunity to improve viral and bacterial vaccine candidates by rational design. Structure-based rational design of antigens can optimize the epitope repertoire in terms of accessibility, stability, and variability. In the present study, we used epitope mapping information on the well-characterized antigen ofNeisseria meningitidisfactor H binding protein (fHbp) to engineer its gonococcal homologue, Ghfp. Meningococcal fHbp is typically classified in three distinct antigenic variants. We introduced epitopes of fHbp variant 1 onto the surface of Ghfp, which is naturally able to protect against meningococcal strains expressing fHbp of variants 2 and 3. Heterologous epitopes were successfully transplanted, as engineered Ghfp induced functional antibodies against all three fHbp variants. These results confirm that structural vaccinology represents a successful strategy for modulating immune responses, and it is a powerful tool for investigating the extension and localization of immunodominant epitopes.

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Genome-wide association studies reveal the role of polymorphisms affecting factor H binding protein expression in host invasion by Neisseria meningitidis

PLoS Pathogens ◽

10.1371/journal.ppat.1009992 ◽

2021 ◽

Vol 17 (10) ◽

pp. e1009992

Author(s):

Sarah G. Earle ◽

Mariya Lobanovska ◽

Hayley Lavender ◽

Changyan Tang ◽

Rachel M. Exley ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Binding Protein ◽

Association Studies ◽

Bacterial Genome ◽

Factor H ◽

Genome Wide Association ◽

Genome Wide Association Studies ◽

Complement Factor ◽

Genome Wide ◽

Bacterial Genetic

Many invasive bacterial diseases are caused by organisms that are ordinarily harmless components of the human microbiome. Effective interventions against these microbes require an understanding of the processes whereby symbiotic or commensal relationships transition into pathology. Here, we describe bacterial genome-wide association studies (GWAS) of Neisseria meningitidis, a common commensal of the human respiratory tract that is nevertheless a leading cause of meningitis and sepsis. An initial GWAS discovered bacterial genetic variants, including single nucleotide polymorphisms (SNPs), associated with invasive meningococcal disease (IMD) versus carriage in several loci across the meningococcal genome, encoding antigens and other extracellular components, confirming the polygenic nature of the invasive phenotype. In particular, there was a significant peak of association around the fHbp locus, encoding factor H binding protein (fHbp), which promotes bacterial immune evasion of human complement by recruiting complement factor H (CFH) to the meningococcal surface. The association around fHbp with IMD was confirmed by a validation GWAS, and we found that the SNPs identified in the validation affected the 5’ region of fHbp mRNA, altering secondary RNA structures, thereby increasing fHbp expression and enhancing bacterial escape from complement-mediated killing. This finding is consistent with the known link between complement deficiencies and CFH variation with human susceptibility to IMD. These observations demonstrate the importance of human and bacterial genetic variation across the fHbp:CFH interface in determining IMD susceptibility, the transition from carriage to disease.

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Characterization of the Proliferating Cell Nuclear Antigen of Leishmania donovani Clinical Isolates and Its Association with Antimony Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01847-13 ◽

2014 ◽

Vol 58 (6) ◽

pp. 2997-3007 ◽

Cited By ~ 9

Author(s):

Rati Tandon ◽

Sharat Chandra ◽

Rajendra Kumar Baharia ◽

Sanchita Das ◽

Pragya Misra ◽

...

Keyword(s):

Clinical Isolate ◽

Proliferating Cell Nuclear Antigen ◽

Leishmania Donovani ◽

Clinical Isolates ◽

Resistant Isolate ◽

Nuclear Antigen ◽

Wild Type ◽

Content Type ◽

Sensitive Isolate ◽

Proliferating Cell

ABSTRACTPreviously, through a proteomic analysis, proliferating cell nuclear antigen (PCNA) was found to be overexpressed in the sodium antimony gluconate (SAG)-resistant clinical isolate compared to that in the SAG-sensitive clinical isolate ofLeishmania donovani. The present study was designed to explore the potential role of the PCNA protein in SAG resistance inL. donovani. For this purpose, the protein was cloned, overexpressed, purified, and modeled. Western blot (WB) and real-time PCR (RT-PCR) analyses confirmed that PCNA was overexpressed by ≥3-fold in the log phase, stationary phase, and peanut agglutinin isolated procyclic and metacyclic stages of the promastigote form and by ∼5-fold in the amastigote form of the SAG-resistant isolate compared to that in the SAG-sensitive isolate.L. donovaniPCNA (LdPCNA) was overexpressed as a green fluorescent protein (GFP) fusion protein in a SAG-sensitive clinical isolate ofL. donovani, and modulation of the sensitivities of the transfectants to pentavalent antimonial (SbV) and trivalent antimonial (SbIII) drugs was assessedin vitroagainst promastigotes and intracellular (J774A.1 cell line) amastigotes, respectively. Overexpression of LdPCNA in the SAG-sensitive isolate resulted in an increase in the 50% inhibitory concentrations (IC50) of SbV(from 41.2 ± 0.6 μg/ml to 66.5 ± 3.9 μg/ml) and SbIII(from 24.0 ± 0.3 μg/ml to 43.4 ± 1.8 μg/ml). Moreover, PCNA-overexpressing promastigote transfectants exhibited less DNA fragmentation compared to that of wild-type SAG-sensitive parasites upon SbIIItreatment. In addition, SAG-induced nitric oxide (NO) production was found to be significantly inhibited in the macrophages infected with the transfectants compared with that in wild-type SAG-sensitive parasites. Consequently, we infer that LdPCNA has a significant role in SAG resistance inL. donovaniclinical isolates, which warrants detailed investigations regarding its mechanism.

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Meningococcal Antigen Typing System (MATS)-Based Neisseria meningitidis Serogroup B Coverage Prediction for the MenB-4C Vaccine in the United States

mSphere ◽

10.1128/msphere.00261-17 ◽

2017 ◽

Vol 2 (6) ◽

Cited By ~ 21

Author(s):

Gowrisankar Rajam ◽

Maria Stella ◽

Ellie Kim ◽

Simon Paulos ◽

Giuseppe Boccadifuoco ◽

...

Keyword(s):

United States ◽

Vaccine Coverage ◽

Binding Protein ◽

The United States ◽

Factor H ◽

Enzyme Linked Immunosorbent Assay ◽

Immune Reactivity ◽

Content Type ◽

Efficacy Trials ◽

Typing System

ABSTRACT The meningococcal antigen typing system (MATS) is an enzyme-linked immunosorbent assay (ELISA)-based system that assesses the levels of expression and immune reactivity of the three recombinant MenB-4C antigens and, in conjunction with PorA variable 2 (VR2) sequencing, provides an estimate of the susceptibility of NmB isolates to killing by MenB-4C-induced antibodies. MATS assays or similar antigen phenotype analyses assume importance under conditions in which analyses of vaccine coverage predictions are not feasible with existing strategies, including large efficacy trials or functional antibody screening of an exhaustive strain panel. MATS screening of a panel of NmB U.S. isolates (n = 442) predicts high MenB-4C vaccine coverage in the United States. Neisseria meningitidis is the most common cause of bacterial meningitis in children and young adults worldwide. A 4-component vaccine against N. meningitidis serogroup B (MenB) disease (MenB-4C [Bexsero]; GSK) combining factor H binding protein (fHBP), neisserial heparin binding protein (NHBA), neisserial adhesin A (NadA), and PorA-containing outer membrane vesicles was recently approved for use in the United States and other countries worldwide. Because the public health impact of MenB-4C in the United States is unclear, we used the meningococcal antigen typing system (MATS) to assess the strain coverage in a panel of strains representative of serogroup B (NmB) disease in the United States. MATS data correlate with killing in the human complement serum bactericidal assay (hSBA) and predict the susceptibility of NmB strains to killing in the hSBA, the accepted correlate of protection for MenB-4C vaccine. A panel of 442 NmB United States clinical isolates (collected in 2000 to 2008) whose data were down weighted with respect to the Oregon outbreak was selected from the Active Bacterial Core Surveillance (ABCs; CDC, Atlanta, GA) laboratory. MATS results examined to determine strain coverage were linked to multilocus sequence typing and antigen sequence data. MATS predicted that 91% (95% confidence interval [CI95], 72% to 96%) of the NmB strains causing disease in the United States would be covered by the MenB-4C vaccine, with the estimated coverage ranging from 88% to 97% by year with no detectable temporal trend. More than half of the covered strains could be targeted by two or more antigens. NHBA conferred coverage to 83% (CI95, 45% to 93%) of the strains, followed by factor H-binding protein (fHbp), which conferred coverage to 53% (CI95, 46% to 57%); PorA, which conferred coverage to 5.9%; and NadA, which conferred coverage to 2.5% (CI95, 1.1% to 5.2%). Two major clonal complexes (CC32 and CC41/44) had 99% strain coverage. The most frequent MATS phenotypes (39%) were fHbp and NHBA double positives. MATS predicts over 90% MenB-4C strain coverage in the United States, and the prediction is stable in time and consistent among bacterial genotypes. IMPORTANCE The meningococcal antigen typing system (MATS) is an enzyme-linked immunosorbent assay (ELISA)-based system that assesses the levels of expression and immune reactivity of the three recombinant MenB-4C antigens and, in conjunction with PorA variable 2 (VR2) sequencing, provides an estimate of the susceptibility of NmB isolates to killing by MenB-4C-induced antibodies. MATS assays or similar antigen phenotype analyses assume importance under conditions in which analyses of vaccine coverage predictions are not feasible with existing strategies, including large efficacy trials or functional antibody screening of an exhaustive strain panel. MATS screening of a panel of NmB U.S. isolates (n = 442) predicts high MenB-4C vaccine coverage in the United States.

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