Table 1.
Summary of mAbs binding affinity by SPR analysis.
Data are shown for all twelve cross-reactive mAbs.
Fig 1.
Binding of mAbs to live meningococci as measured by flow cytometry.
In blue the MC58 expressing v1, in red the UK104 expressing v2 and in green the UK320 exposing the v3. Gray-filled area represents negative control bacteria incubated with PBS and secondary antibodies FITC-conjugated antibodies.
Fig 2.
Histogram of bactericidal assay (SBA) titers using rabbit (rSBA) (a) and human (hSBA) (b) serum as source of complement against meningococcal strains expressing v1, v2 and v3 colored in blue, red and green respectively.
Fig 3.
Flow cytometry binding of fH pre-incubation with PBS (red line) and pre-incubation with mAbs (green line). Gray-filled area represents negative control bacteria incubated with PBS and secondary antibodies FITC-conjugated antibodies. mAbs 3G7, 4F9 and 4B3 are able to inhibit fH binding, while mAb 1E6 is an example of those not competing with fH binding.
Table 2.
V-REGION mutations analysis.
Table 3.
X-ray data collection and refinement statistics.
Fig 4.
Crystal structure of Fab 4B3 in complex with the fHbp v1 (wheat).
The heavy chain and the light chain of the Fab 4B3 are depicted as cartoons, colored in raspberry and pale green respectively. The CDRs of both variable light chain (VL) and variable heavy chain (VH) are colored pale yellow.
Fig 5.
CDRs residues which are directly involved in fHbp interactions.
Spheres are the edge of CDRs except for the pale green which represents the first N-term extra-CDR1 residue. In brackets reported the original residues replaced by somatic mutations. Panel (a) shows, P32, F33 and D93 of CDR1 and CDR3 of Variable Light (VL) respectively, engaging constructive interactions. Y91 is the only residue not conserved within all CDRs and has no role in the interaction. In case of Variable Heavy (VH) CDR2 and CDR3 (panel b and c), N52 and R102 contribute respectively to strengthen the network of interactions. these observations suggest an enforcement of the overall interactions with respect to the germline pattern.
Table 4.
Molecular interactions between key residues constituting the Fab 4B3:fHbp v1 epitope.
Fig 6.
Allelic conservation of the key residues of fHbp v1 engaged in the binding with Fab 4B3.
The figure shows the C-terminal β-barrel of fHbp v1 and the surface of the epitope of the Fab 4B3. Each residue is colored based on its conservation among the fHbp gene repertoire which includes 1161 allelic sequences. Where available, the KD value of each single alanine mutant of fHbp has been added.
Table 5.
Impact of fHbp alanine point mutations on binding to mAb 4B3.
Fig 7.
Surface plot of the competition of Fab 4B3 with human factor H for fHbp binding site.
The epitope of the Fab 4B3 is represented in lemon instead the human factor H binding site in blue. In red are highlighted the common residues within the two complexes. The label colors indicate the percentage of conservation as reported in Fig 6.
Fig 8.
Overall representation of possible accommodations between the complex Fab 4B3/fHbp v1and human factor H.
(a) SPR-based factor H competition assay with purified fHbp v1.1 captured on immobilized mAb 4B3, Fab 4B3, and the mAb 1A12. Left panel, start of injection of factor H is indicated by a black arrow, end of injection by a red one. Right panel, buffer control, start of injection of running buffer HBS-EP+ is indicated by a black arrow, end of injection by a red one. (b) On the left superimposition of the crystal structure of Fab 4B3 and the domain 6 and 7 of the human factor H (PDB 2W80) in complex with the fHbp v1. All components are depicted both as cartoons and as solid surface, with the heavy chain (raspberry), the light chain (pale green), the fHbp v1 (wheat) and the human factor H colored in orange. The blue box highlights the region (red) of hfH domain 6 and 4B3 VL overlapping, in the model, suggesting a high likelihood of steric hindrance and clashes within the two proteins in the physiological context. Right, superimposition of the model reported in on the left side and the domain 5, 6 and 7 from X-ray scattering structure of human complement fH (PDB 3GAV) in one of the likely location of domain 5 of fH. In this case we can observe a dramatic collision within domain 5 of fH and human Fab 4B3. The fH domains are depicted as ribbon; while domain 6 and 7 are colored in orange, domain 5 is colored in blue.