4CMenB vaccine induces elite cross-protective human antibodies that compete with human factor H for binding to meningococcal fHbp

Abstract

Neisseria meningitidis serogroup B (MenB) is the leading cause of meningococcal meningitis and sepsis in industrialized countries, with the highest incidence in infants and adolescents. Two recombinant protein vaccines that protect against MenB are now available (i.e. 4CMenB and MenB-fHbp). Both vaccines contain the Factor H Binding Protein (fHbp) antigen, which can bind the Human Factor H (fH), the main negative regulator of the alternative complement pathway, thus enabling bacterial survival in the blood. fHbp is present in meningococcal strains as three main variants which are immunologically distinct. Here we sought to obtain detailed information about the epitopes targeted by anti-fHbp antibodies induced by immunization with the 4CMenB multicomponent vaccine. Thirteen anti-fHbp human monoclonal antibodies (mAbs) were identified in a library of over 100 antibody fragments (Fabs) obtained from three healthy adult volunteers immunized with 4CMenB. Herein, the key cross-reactive mAbs were further characterized for antigen binding affinity, complement-mediated serum bactericidal activity (SBA) and the ability to inhibit binding of fH to live bacteria. For the first time, we identified a subset of anti-fHbp mAbs able to elicit human SBA against strains with all three variants and able to compete with human fH for fHbp binding. We present the crystal structure of fHbp v1.1 complexed with human antibody 4B3. The structure, combined with mutagenesis and binding studies, revealed the critical cross-reactive epitope. The structure also provided the molecular basis of competition for fH binding. These data suggest that the fH binding site on fHbp v1.1 can be accessible to the human immune system upon immunization, enabling elicitation of human mAbs broadly protective against MenB. The novel structural, biochemical and functional data are of great significance because the human vaccine-elicited mAbs are the first reported to inhibit the binding of fH to fHbp, and are bactericidal with human complement. Our studies provide molecular insights into the human immune response to the 4CMenB meningococcal vaccine and fuel the rationale for combined structural, immunological and functional studies when seeking deeper understanding of the mechanisms of action of human vaccines.

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.

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.

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 K_D value of each single alanine mutant of fHbp has been added.

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.