Meningococcal Factor H Binding Protein fHbpd184 Polymorphism Influences Clinical Course of Meningococcal Meningitis

<div><p>Factor H Binding protein (fHbp) is an important meningococcal virulence factor, enabling the meningococcus to evade the complement system, and a main target for vaccination. Recently, the structure of fHBP complexed with factor H (fH) was published. Two fHbp glutamic acids, E₂₈₃ and E₃₀₄, form salt bridges with fH, influencing interaction between fHbp and fH. Fifteen amino acids were identified forming hydrogen bonds with fH. We sequenced <em>fHbp</em> of 254 meningococcal isolates from adults with meningococcal meningitis included in a prospective clinical cohort to study the effect of fHbp variants on meningococcal disease severity and outcome. All fHbp of subfamily A had E304 substituted with T304. Of the 15 amino acids in fHbp making hydrogen bonds to fH, 3 were conserved, 11 show a similar distribution between the two fHbp subfamilies as the polymorphism at position 304. The proportion of patients infected with meningococci with fHbp of subfamily A with unfavorable outcome was 2.5-fold lower than that of patients infected with meningococci with fHbp of subfamily B (2 of 40 (5%) vs. 27 of 213 (13%) (<em>P</em> = 0.28). The charge of 2 of 15 amino acids (at position 184 and 306) forming hydrogen bonds was either basic or acidic. The affinity of fHbp_K184 and of fHbp_D184 for recombinant purified human fH was assessed by Surface Plasmon Resonance and showed average K_D of 2.60×10⁻⁸ and 1.74×10⁻⁸, respectively (ns). Patients infected with meningococci with fHbp_D184 were more likely to develop septic shock during admission (11 of 42 [26%] <em>vs.</em> 19 of 211 [9%]; <em>P = </em>0.002) resulting in more frequent unfavorable outcome (9 of 42 [21%] <em>vs.</em> 20 of 211 [10%]; <em>P</em> = 0.026). In conclusion, we dentified fHBP_D184 to be associated with septic shock in patients with meningococcal meningitis.</p> </div

Associations of meningococci containing fHbp_E184 with clinical characteristics^a.

a<p>Data are number/number assessed (%) or median (25th–75th percentile), unless otherwise stated.</p>b<p>Defined as aphasia, monoparesis, or hemiparesis) and cranial nerve palsies.</p><p>Abbreviations: GOS  =  Glasgow Outcome Scale.</p

Distribution of meningococcal fHbp subfamilies and variants among clonal complexes.

a<p>Nomenclature according to Fletcher <i>et al</i>¹⁷.</p>b<p>Nomenclature according to Masignani <i>et al</i>²¹.</p

fHbp protein type and clonal complexes.

a<p>fHbp protein type from the fHbp database at <a href="http://pubmlst.org/neisseria/fHbp/" target="_blank">http://pubmlst.org/neisseria/fHbp/</a>.</p

Severity of disease in patients with HAP or VAP due to PVL positive and PVL negative MRSA.

<p>Severity of disease in patients with HAP or VAP due to PVL positive and PVL negative MRSA.</p

Clinical outcome of patients with HAP or VAP.

<p>Kaplan-Meier survival curves for patients with HAP or VAP due to PVL positive and PVL negative MRSA. Curves were compared using the log-rank test and <i>P</i> values ≤ 0.05 are considered significantly different.</p

Characteristics of patients with HAP or VAP due to PVL positive and PVL negative MRSA.

<p>Characteristics of patients with HAP or VAP due to PVL positive and PVL negative MRSA.</p

Structural basis for the reduced affinity of mfH with fHbp.

<p>(<b>A</b>) Cartoon of hfH₆₇ viewed from through V1 fHbp (solid line) with amino acids changed in hfH with murine residues (outlined by yellow dashes), and those replaced in mfH with human residues (outlined by light blue dashes). (<b>B</b>) SPR analysis of binding of two hfH₆₇ mutants each containing two amino acid changes (shown) with fHbps from each variant family. (<b>C</b>) Far western analysis of V1 fHbp and a control protein, PPX; blots were overlaid with 5 µg/ml of the recombinant proteins mfH, modified mfH (with 14 humanised amino acids) or hfH, or with human serum (1 in 2000 dilution) as indicated; the sizes of the mol. wt. marker are shown. (<b>D</b>) Structure of mfH₆₇ (blue ribbon) superimposed on V1 fHbp (white ribbon) and hfH (green ribbon). While fH₆ from both species are superimposable, the orientation of fH₇ differs significantly between mfH and hfH (indicated in red dashed circle).</p

Non-functional fHbps retain their immunogenicity in transgenic mice.

<p>(<b>A</b>) ELISAs assaying anti-V1 titres elicited in pooled sera following immunisation of transgenic mice with the wild-type protein and non-functional V1 fHbps. (<b>B</b>) SBA titres of sera from individual mice immunisation with fHbps.</p

Effect of mutations at positions equivalent to fHbp V1 residues 283 and 304 (<i>i.e.</i> Thr³⁰⁴ in V2 and V3 fHbp) on the <i>K</i>_D for binding to fH₆₇, shown relative to the wild-type proteins.

<p>DM indicates double Ala substitution; ND, not determined.</p