Crystal structures of human Fabs targeting the Bexsero meningococcal vaccine antigen NHBA

Martina Maritan, Roberta Cozzi, Paola Lo Surdo, Daniele Veggi, Matthew James Bottomley, Enrico Malito, Martina Maritan, Roberta Cozzi, Paola Lo Surdo, Daniele Veggi, Matthew James Bottomley, Enrico Malito

Abstract

Neisserial heparin-binding antigen (NHBA) is a surface-exposed lipoprotein from Neisseria meningitidis and is a component of the meningococcus B vaccine Bexsero. As part of a study to characterize the three-dimensional structure of NHBA and the molecular basis of the human immune response to Bexsero, the crystal structures of two fragment antigen-binding domains (Fabs) isolated from human monoclonal antibodies targeting NHBA were determined. Through a high-resolution analysis of the organization and the amino-acid composition of the CDRs, these structures provide broad insights into the NHBA epitopes recognized by the human immune system. As expected, these Fabs also show remarkable structural conservation, as shown by a structural comparison of 15 structures of apo Fab 10C3 which were obtained from crystals grown in different crystallization conditions and were solved while searching for a complex with a bound NHBA fragment or epitope peptide. This study also provides indirect evidence for the intrinsically disordered nature of two N-terminal regions of NHBA.

Trial registration: ClinicalTrials.gov NCT02305446.

Keywords: Bexsero; Fabs; NHBA; Neisseria; fragment antigen binding; mAbs; monoclonal antibodies; vaccine.

Figures

Figure 1
Figure 1
Formation and characterization of Fab–NHBA complexes. (a) SDS–PAGE analysis under reducing (left) and nonreducing (right) conditions of purified NHBAp2 (lane 1), Fab 10C3 (lane 2), the 10C3–NHBA complex (lane 3), Fab 12E1 (lane 4) and the 12E1–NHBA complex (lane 5). (b) Size-exclusion chromatography elution profiles of NHBAp2 (grey), Fab 10C3 (green), the 10C3–NHBA complex (magenta), Fab 12E1 (cyan) and the 12E1–NHBA complex (blue). Each chromatogram refers to an independent run.
Figure 2
Figure 2
Overall fold of the apo Fab 12E1 and Fab 10C3 structures. The structures of Fab 12E1 (a) and Fab 10C3 (b) are depicted as cartoons, with the heavy (H) and light (L) chains coloured dark and light grey, respectively. CDR-L1 and CDR-H1 are coloured yellow, CDR-L2 and CDR-H2 green, and CDR-L3 and CDR-H3 cyan. Disulfide bonds are depicted by red sticks in each structure.
Figure 3
Figure 3
Structural comparisons of apo 10C3 structures. (a) All 15 10C3 structures solved in this work are shown as ribbons after superposition, and are coloured black and white for the heavy (H) and light (L) chains, respectively. (b) The two most divergent apo 10C3 structures are depicted superposed as ribbons (structures 6 and 15; see Supplementary Table S1) and coloured as in (a). The regions of maximum divergence between Cα atoms of the two structures are shown as magenta sticks.
Figure 4
Figure 4
Sequences and structural annotations of the Fab 12E1 and Fab 10C3 CDRs. The sequences of Fab 12E1 (top) and Fab 10C3 (bottom) are shown with secondary-structure annotation at the top. CDR residues are highlighted in yellow (CDR-H1 and CDR-L1), green (CDR-H2 and CDR-L2), and cyan (CDR-H3 and CDR-L3). CDR conformations and secondary-structure elements are shown below and above the sequence, respectively. Regions of the Ramachandran plot that define CDR clusters by conformation are annotated as follows: B for β-sheet region, P for polyproline II, A for α-helix, D for δ region (near α-helix but with more negative values of φ), L for left-handed helix and G for γ region (φ > 0° excluding the L and B regions). Lower-case letters in the loop conformations indicate cis residues.
Figure 5
Figure 5
Fab 12E1 and Fab 10C3 CDRs. Top views of the Fab 12E1 (a) and Fab 10C3 (b) CDR regions, coloured according either to the most represented residues (left) or to the electrostatic potential distribution (right). Orange patches indicate aromatic residues (Trp and Tyr), blue patches indicate positively charged residues (Lys and Arg) and green patches indicate polar uncharged residues (Ser, Thr and Asn). Dotted black lines separate the Fab heavy and light chains. The electrostatic potential distribution was calculated with APBS (Lerner & Carlson, 2006 ▸), where red and blue surfaces show negative and positive charges contoured in the range from −3kBT e−1 (red) to +3kBT e−1 (blue), while white surfaces indicate neutral potential.

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