Fig 1.
A. Amino acid sequence alignment of NMB0345 (NEIS1825, Cell Binding Factor, CBF) from N. meningitidis MC58, N.gonorrhoeae FA1090 and the Campylobacter jejuni PEB3/CBF2 protein. * denotes identical amino acids between all three proteins. Yellow-shaded amino acid sequences denote the putative signal leader peptides. The blue-shaded amino acid sequence denotes the presence of a putative conserved PPIC-type peptidyl prolyl cis/trans isomerase domain (interval 133–259) belonging to the Rotamase_2 superfamily. B) Putative structure of meningococcal CBF based on C.jejuni CBF. The structure of meningococcal CBF was putatively modelled in SWISS-model, using as a template the crystallized structure of 3rfw.1.A protein (PEB4), which is the most closely related structure to C. jejuni PEB3/CBF2 that we have available in Protein Data Bank.
Fig 2.
Purification of recombinant (r)CBF.
The protein was purified to homogeneity using nickel-nitrilotriacetic acid metal affinity chromatography under denaturing conditions. A) SDS-PAGE analysis of purified rCBF. Lane 1, molecular mass markers; lane 2, rCBF (2 μg) showing a single band of ~36 kDa. B) Western blot analysis of purified rCBF reacted with anti-HIS tag antibody. Lane 1, molecular mass markers. Lane 2, protein reactivity with anti-HIS antibody (1/500) showing a single band of ~36 kDa. C) Western blot analysis of purified rCBF reacted with rabbit polyclonal anti-rCBF serum. Lane 1, molecular mass markers. Lane 2, protein reactivity with rabbit anti-rCBF serum (1/400) showing a single band of ~36 kDa.
Fig 3.
ELISA reactivity of antisera raised to different rCBF formulations against pure rCBF protein, outer membranes (OM) and whole meningococcal cells.
Antisera from individual animals immunized with rCBF in various adjuvant and delivery formulations and from animals immunised with OM, were reacted in ELISA against A) purified rCBF protein, B) MC58 OM and C) whole MC58 meningococcal cells. The columns represent the geometric mean reciprocal ELISA titres (n = 4 or 5 animals per group) and the error bars represent the 95% confidence limits. No significant reactivity with rCBF, OM or whole cells in ELISA was observed with sera from sham-immunized animals or with normal mouse serum (absorbance values λ450nm <0.1 for serum dilutions of 1/10). The * denotes that the responses were statistically higher than the corresponding sham-immunized animals (p<0.05), using an independent t-Test to compare differences between the mean values.
Fig 4.
A) Specificity of murine anti-rCBF sera, examined by western immunoblot reactivity with CBF protein present in MC58 lysates. Pooled antisera (1/100 dilution) were reacted against wild-type MC58 lysate (Wt) and MC58 Δnmb0345 knock-out lysate (Δ) in western blot. CBF was recognised as a single protein band of Mr ~29.5 kDa (identified by the arrow) in wild-type MC58 (CBF+) but not in the knock-out strain (CBF-). All sham immunization sera and normal mouse serum were non-reactive against both Wt and knock-out strains, as demonstrated by representative strip blots (control*). B) CBF is expressed on the surface of MC58 meningococci as demonstrated by FACS analysis. On the top panel, the area within the red lines show no reactivity of wild-type MC58 (CBF+) bacteria with murine sham-immunised serum (1/10) and the area within the blue lines shows the significant FACS reactivity of murine antisera (1/10) raised against rCBF in the various formulations. The lower panel shows corresponding reactivity of the same murine sham-immunised sera and antisera against the MC58 Δnmb0345 (CBF-) knock-out strain. Rabbit post-immune serum (1/100) also reacted with the wild-type MC58 (CBF+) (area within the blue line), compared to pre-immune serum (area within the red lines) and no reactivity was observed with the MC58 Δnmb0345 (CBF-) knock-out strain. The numbers within each panel refer to the FITC-mean value. The * denotes the significant (p<0.05) and right-shifted increases in FITC-fluorescence recorded events, using a one sample t-Test to compare mean fluorescence values of test murine antisera against sham-immunised murine sera or rabbit post-immune antiserum against pre-immune serum. Data are representative of n = 2 experiments.
Table 1.
Serum bactericidal activity of murine anti-rCBF sera.
Fig 5.
Production of CBF in different meningococcal strains examined by western blotting.
A) Western blot reactivity of rabbit anti-rCBF sera (1/100 dilution) with CBF in lysates (10 μg of total protein) of meningococcal strains MC58, MC54, MC90, MC161, MC168, MC172, MC173, MC174, MC179 and MC180 (CBF encoded by Allele 1); L2470 (protein encoded by Allele 18) and MC162 and MENC11 (protein encoded by Allele 3). The blot is representative of n = 4 independent western blot experiments. B) For each of the blots, band intensities were quantified by ImageJ and a mean value for each meningococcal strain was calculated with standard deviation (for the bands from the n = 4 independent blots) shown by the error lines, as described previously [32]. For each blot, a ratio of band intensity relative to MC58 was also calculated for each strain and the mean ratio values with standard deviations (n = 4) are tabulated and compared with a student’s t-Test (p values >0.05 for all strains relative to MC58).