Vaccine Potential and Diversity of the Putative Cell Binding Factor (CBF, NMB0345/NEIS1825) Protein of Neisseria meningitidis

Abstract

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.

Structure of the Recombinant Neisseria gonorrhoeae Adhesin Complex Protein (rNg-ACP) and Generation of Murine Antibodies with Bactericidal Activity against Gonococci

“…In an attempt to increase the immunogenicity of the rNg-ACP proteins, we also prepared rNg-ACP liposomes and rNg-ACP-ZW 3-14 micelles that contained the immunomodulator monophosphoryl lipid A (MPLA). MPLA is an adjuvant that is increasingly being used with human vaccines ( 32 ), and we have previously shown that it can increase the immunogenicity of different Neisseria recombinant proteins in animal immunization studies ( 23 , 26 , 29 , 30 ). rNg-ACP proteins were also adsorbed to Al(OH) 3 , the standard adjuvant licensed routinely for human vaccines.…”

Immunization with recombinant truncated Neisseria meningitidis-Macrophage Infectivity Potentiator (rT-Nm-MIP) protein induces murine antibodies that are cross-reactive and bactericidal for Neisseria gonorrhoeae

“…The final HC eluate was dialyzed twice against filter-sterilized ice-cold TBS, 0.1 mM EDTA and brought back to 2.5 ml in the same buffer. The bactericidal activity of pooled murine anti-rT-Nm-MIP sera was determined as described previously [29] , using 25% (v/v) HC for meningococcal strains [22] , [30] and 17% (v/v) HS as a source of exogenous complement for gonococcal strains [31] .…”

Neisseria genes required for persistence identified via in vivo screening of a transposon mutant library

“…They function in LOS synthesis (rfaCDEF, kdsA), iron acquisition and detoxification (bfrAB, hemolysin III, bcp), efflux (macA, farA), transcriptional regulation (farR,), and cell wall recycling, synthesis and repair (ltgD/mltB, dacC), among others [55][56][57][58][59][60][61][62][63][64][65]. At least three ORFs, (mip, nspA, cbf), encode homologs of surface expressed immunomodulatory proteins [60,[66][67][68]. A transporter of surface associated proteins (slam1) was also required for persistence [69,70].…”