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Effect of factor H-binding protein sequence variation on factor H binding and survival of Neisseria meningitidis in human blood.

ABSTRACT: Binding of the complement inhibitor factor H (fH) to the surface of Neisseria meningitidis is critical for evasion of innate host defenses. The meningococcal vaccine candidate factor H-binding protein (fHbp) serves as an fH ligand. We prepared 16 recombinant fHbp natural sequence variants. By enzyme-linked immunosorbent assay (ELISA), the variants from a New Zealand epidemic strain (fHbp ID 14) and from an endemic United Kingdom strain (ID 15) showed 10-fold lower fH binding than a reference fHbp from an epidemic Norwegian strain (ID 1). By surface plasmon resonance, association rate constants (k(a)) for fHbp ID 14 and 15 were similar to those for ID 1, but dissociation rate constants (k(d)) were 4- to 10-fold higher than those for ID 1. To determine the effect of fH affinity on fHbp fitness, we prepared isogenic mutants of strain H44/76 that expressed fHbp ID 1, 14, or 15. By flow cytometry, mutants expressing fHbp ID 14 or 15 had lower fH binding than ID 1. When incubated in plasma or blood of nonimmune donors, all three mutants showed similar increases in CFU/ml. In contrast, an isogenic fHbp knockout mutant, which grew well in broth, was rapidly killed in plasma or blood. Thus, although fHbp expression was required for survival of strain H44/76 in blood or plasma, expression of two natural fHbp sequence variants with lower fH affinity had minimal or no effect on nonimmune clearance. One reason may be the high fH concentrations in normal serum, which favor saturation of fH binding to fHbp, even when dissociation rates varied over 10-fold.

SUBMITTER: Dunphy KY

PROVIDER: S-EPMC3019890 | biostudies-other | 2011 Jan

REPOSITORIES: biostudies-other

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Effect of factor H-binding protein sequence variation on factor H binding and survival of Neisseria meningitidis in human blood.

Dunphy Kathleen Y KY Beernink Peter T PT Brogioni Barbara B Granoff Dan M DM

Infection and immunity 20101101 1

Binding of the complement inhibitor factor H (fH) to the surface of Neisseria meningitidis is critical for evasion of innate host defenses. The meningococcal vaccine candidate factor H-binding protein (fHbp) serves as an fH ligand. We prepared 16 recombinant fHbp natural sequence variants. By enzyme-linked immunosorbent assay (ELISA), the variants from a New Zealand epidemic strain (fHbp ID 14) and from an endemic United Kingdom strain (ID 15) showed 10-fold lower fH binding than a reference fHb ...[more]

PMID: 21041484

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Project description:BackgroundNeisseria meningitidis can cause severe infection in humans. Polymorphism of Complement Factor H (CFH) is associated with altered risk of invasive meningococcal disease (IMD). We aimed to find whether polymorphism of other complement genes altered risk and whether variation of N. meningitidis factor H binding protein (fHBP) affected the risk association.MethodsWe undertook a case-control study with 309 European cases and 5,200 1958 Birth Cohort and National Blood Service cohort controls. We used additive model logistic regression, accepting P<0.05 as significant after correction for multiple testing. The effects of fHBP subfamily on the age at infection and severity of disease was tested using the independent samples median test and Student's T test. The effect of CFH polymorphism on the N. meningitidis fHBP subfamily was investigated by logistic regression and Chi squared test.ResultsRs12085435 A in C8B was associated with odds ratio (OR) of IMD (0.35 [95% CI 0.19-0.67]; P = 0.03 after correction). A CFH haplotype tagged by rs3753396 G was associated with IMD (OR 0.56 [95% CI 0.42-0.76], P = 1.6x10⁻⁴). There was no bacterial load (CtrA cycle threshold) difference associated with carriage of this haplotype. Host CFH haplotype and meningococcal fHBP subfamily were not associated. Individuals infected with meningococci expressing subfamily A fHBP were younger than those with subfamily B fHBP meningococci (median 1 vs 2 years; P = 0.025).DiscussionThe protective CFH haplotype alters odds of IMD without affecting bacterial load for affected heterozygotes. CFH haplotype did not affect the likelihood of infecting meningococci having either fHBP subfamily. The association between C8B rs12085435 and IMD requires independent replication. The CFH association is of interest because it is independent of known functional polymorphisms in CFH. As fHBP-containing vaccines are now in use, relationships between CFH polymorphism and vaccine effectiveness and side-effects may become important.

Project description:The cbf gene from Neisseria meningitidis strain MC58 encoding the putative Cell Binding Factor (CBF, NMB0345/NEIS1825) protein was cloned into the pRSETA system and a ~36-kDa recombinant (r)CBF protein expressed in Escherichia coli and purified by metal affinity chromatography. High titres of rCBF antibodies were induced in mice following immunization with rCBF-saline, rCBF-Al(OH)3, rCBF-Liposomes or rCBF-Zwittergent (Zw) 3-14 micelles, both with and without incorporated monophosphoryl lipid A (MPLA) adjuvant. Anti-rCBF sera reacted in western blots of meningococcal lysates with a single protein band of molecular mass ~29.5 kDa, indicative of mature CBF protein, but did not react with a lysate of a Δnmb0345 mutant (CBF-), demonstrating specificity of the murine immune responses. CBF protein was produced by all strains of meningococci studied thus far and the protein was present on the surface of MC58 (CBF+) bacteria, but absent on Δnmb0345 mutant (CBF-) bacteria, as judged by FACS reactivity of anti-rCBF sera. Analysis of the NEIS1825 amino acid sequences from 6644 N. meningitidis isolates with defined Alleles in the pubmlst.org/Neisseria database showed that there were 141 ST types represented and there were 136 different allelic loci encoding 49 non-redundant protein sequences. Only 6/6644 (<0.1%) of N. meningitidis isolates lacked the nmb0345 gene. Amongst serogroup B isolates worldwide, ~68% and ~20% expressed CBF encoded by Allele 1 and 18 respectively, with the proteins sharing >99% amino acid identity. Murine antisera to rCBF in Zw 3-14 micelles + MPLA induced significant serum bactericidal activity (SBA) against homologous Allele 1 and heterologous Allele 18 strains, using both baby rabbit serum complement and human serum complement (h)SBA assays, but did not kill strains expressing heterologous protein encoded by Alelle 2 or 3. Furthermore, variable bactericidal activity was induced by murine antisera against different meningococcal strains in the hSBA assay, which may correlate with variable surface exposure of CBF. Regardless, the attributes of amino acid sequence conservation and protein expression amongst different strains and the ability to induce cross-strain bactericidal antibodies indicates that rCBF could be a potential meningococcal vaccine antigen and merits further testing.

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Effect of factor H-binding protein sequence variation on factor H binding and survival of Neisseria meningitidis in human blood.

Publications

Effect of factor H-binding protein sequence variation on factor H binding and survival of Neisseria meningitidis in human blood.

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