Design and evaluation of meningococcal vaccines through structure-based modification of host and pathogen molecules

Abstract

Neisseria meningitis remains a leading cause of sepsis and meningitis, and vaccines are required to prevent infections by this important human pathogen. Factor H binding protein (fHbp) is a key antigen that elicits protective immunity against the meningococcus and recruits the host complement regulator, fH. As the high affinity interaction between fHbp and fH could impair immune responses, we sought to identify non-functional fHbps that could act as effective immunogens. This was achieved by alanine substitution of fHbps from all three variant groups (V1, V2 and V3 fHbp) of the protein; while some residues affected fH binding in each variant group, the distribution of key amino underlying the interaction with fH differed between the V1, V2 and V3 proteins. The atomic structure of V3 fHbp in complex with fH and of the C-terminal barrel of V2 fHbp provide explanations to the differences in the precise nature of their interactions with fH, and the instability of the V2 protein. To develop transgenic models to assess the efficacy of non-functional fHbps, we determined the structural basis of the low level of interaction between fHbp and murine fH; in addition to changes in amino acids in the fHbp binding site, murine fH has a distinct conformation compared with the human protein that would sterically inhibit binding to fHbp. Non-functional V1 fHbps were further characterised by binding and structural studies, and shown in non-transgenic and transgenic mice (expressing chimeric fH that binds fHbp and precisely regulates complement system) to retain their immunogenicity. Our findings provide a catalogue of non-functional fHbps from all variant groups that can be included in new generation meningococcal vaccines, and establish proof-in-principle for clinical studies to compare their efficacy with wild-type fHbps.

Figure 1. Structure and immunogenicity of V1 fHbps with impaired fH binding.

(A) Structures of V1 fHbp mutants with reduced binding. Top left shows overlay of cartoon representation of fHbp structures from V1 fHbp (grey), fHbp^DM (gold), and fHbp^R106A (teal). Zoom boxes show close-ups of modified residues with different densities (F_O-F_C) contoured at 4 sigma. Inset panels show typical equilibrium fits for binding to fH₆₇, and average K _D and quality of fit indicators based on four repeats. Binding of V1 fHbp and modified fHbps to fH₆₇ by SPR (B), and by Coomassie straining (blue bands) or far Western analysis with fH (black bands) to these proteins and lysates from N. meningitidis strain MC58 and MC58ΔfHbp, the fHbp mutant (C). (D) Antibody titres against V1 fHbps elicited by immunisation with fHbps.

Figure 2. Key residues in fHbp necessary for high affinity interactions with fH.

Amino acids of V1, V2 and V3 fHbps (A, B and C respectively, fill representations) superimposed on the structure of V1 fHbp with fH (black sticks) demonstrating the impact of residues on the K _D as a percentage of results with the corresponding wild-type protein. Substitution of residues coloured yellow increases binding by > fivefold, while amino acids labelled in orange have a significant effect on binding proteins from all three variant groups.

Figure 3. Structures and stability of V2 and V3 fHbps.

(A) Overlay of V1 and V3 fHbp:fH₆₇ complexes shown in a cartoon representation with the V1 complex shown in light grey, and V3 fHbp (rainbow coloured blue at the N-terminus to red at the C-terminus) with fH₆₇ from the V3 complex shown in black. (B) Overlay of V3 fHbp (stick representation) in the region of residue 106 (magenta) and V3 fHbp^P106A (carbon-green, oxygen-red, nitrogen-blue). (C) Overlay of V3 fHbp (rainbow coloured blue to red, N- to C-terminus) with the C terminal barrel of V2 fHbp (grey); pictures drawn by PyMol. (D) DSC of V1 (red line), V2 (blue line) and V3 (green line) fHbp showing unfolding of the C-terminal barrel at around 80°C for all variants, and the N-terminal barrel at lower temperatures.

Figure 4. Structural basis for the reduced affinity of mfH with fHbp.

(A) 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) SPR analysis of binding of two hfH₆₇ mutants each containing two amino acid changes (shown) with fHbps from each variant family. (C) 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. (D) 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).

Figure 5. Non-functional fHbps retain their immunogenicity in transgenic mice.

(A) 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) SBA titres of sera from individual mice immunisation with fHbps.