Molecular characterization of a synthetic neutralizing antibody targeting p67 of Theileria parva

Abstract

The Theileria parva sporozoite surface antigen p67 is a target of the bovine humoral immune response that generates antibodies capable of providing protection against subsequent infection. As a result, p67 has been the subject of efforts aimed at the development of an anti-sporozoite subunit vaccine. Previous studies have identified neutralizing epitopes in the N- and C-terminal regions of the full-length protein and shown that immunization with a C-terminal fragment of p67 (p67C) alone is capable of eliciting protection. To identify additional neutralizing epitopes in p67C, selections were conducted against it using a phage-displayed synthetic antibody library. An antibody that neutralized the sporozoite in vitro was identified, and the crystal structure of a Fab:peptide complex was elucidated. Mutagenesis studies aimed at validating and further characterizing the Fab:peptide interaction identified critical residues involved in binding and neutralization. This study also validates distinct epitopes for previously reported neutralizing antibodies.

Keywords: Theileria parva; crystal structure; neutralizing; p67; parasite; synthetic antibody.

FIGURE 1

Characterization of Fab B11. (a) Sequences of Fab B11 CDRs. Only positions that were diversified in the phage‐displayed library are shown and are numbered according to the IMGT nomenclature (Lefranc et al., 2003). (b) Biolayer interferometry (BLI) association and dissociation curves for indicated concentrations of Fab B11 (black) binding to immobilized p67. Binding signals were globally fit (gray) and the calculated K _D value is shown. (c) ELISA for binding (y‐axis) of serial dilutions of Fab B11 (x‐axis) to His‐p67C (black squares) and the negative control proteins ubiquitin (white circles) and BSA (white triangles). (d) ELISA for binding (y‐axis) of 50 nM Fab B11 (black), IgG AR21.4 (gray), and IgG AR22.7 (white) to the following antigens (x‐axis): Full‐length p67, p67N (residues 21–225), and p67C (residues 572–651). (e) ELISA for binding (y‐axis) of 50 nM Fab B11 (black) and IgG AR21.4 (gray) to the following antigens (x‐axis): p67C, p67¹, and p67². (f) Neutralization activity in an in vitro sporozoite neutralization assay with bovine PBMCs. Five‐fold serial dilutions of antibody from 250 nM were evaluated in the sporozoite neutralization assay relative to a non‐binding IgG isotype control. Error bars were derived from the standard deviation of technical replicates run in triplicate and results shown are representative of experiments run in quadruplicate.

FIGURE 2

Crystal structure of Fab B11 in complex with p67C¹. (a) Fab B11 is shown as a ribbon with the light chain (LC) and heavy chain (HC) colored light or dark gray, respectively, except for the CDRs (as defined by the IMGT nomenclature (Lefranc et al., 2003)), which are colored as follows: L1, (blue), L2 (magenta), L3 (cyan), H1 (yellow), H2 (orange), H3 (red). p67C^1a (the 20‐residue region of p67C (Nene & Morrison, 2016) that was resolved in the electron density map) is shown as a green ribbon with the N‐terminus rendered as a sphere. The side and top‐down views of Fab B11 are shown at left and right, respectively.

FIGURE 3

Antibodies binding to minimal p67 epitopes. (a) Fab B11 binding to p67 residues 574–598 (biotin‐GGGSLRGLDLSEEEVKKILDEIVKD). (b) IgG AR21.4 binding to p67 residues 612–628 (PSGRSSERQPSLGPSLVGGGSK‐biotin). (c) IgG AR22.7 binding to p67 residues 204–220 (ELKKTLQPGKTSTGETTGGGSK‐biotin). BLI association and dissociation curves (black) are shown for indicated concentrations (black) of antibody binding to immobilized biotinylated peptide. Binding signals were globally fit (gray) and the calculated K _D values are shown.

FIGURE 4

The functional epitope of p67C^1a for binding to Fab B11. (a) Open book view of the p67C¹:Fab‐B11 complex. The 20‐residue p67C^1a peptide (left) and Fab B11 (right) are shown as transparent surfaces. p67C^1a residues are colored red or green if alanine substitutions did or did not reduce binding significantly (Ala/WT >6), respectively, and residues that do not contact Fab B11 are colored gray. Side chains are shown for residues that were hits in the alanine scan. Fab B11 residues are colored red or green if they contact p67C^1a residues that were or were not hits in the alanine scan, respectively, and other residues in the light and heavy chains are colored light or dark gray, respectively. Side chains are shown for residues that contact p67C^1a residues that were hits in the alanine scan. (b) Details of the molecular interactions between Fab B11 and p67C^1a residues (i) G⁵⁸⁰ and Leu⁵⁸¹, (ii) Val⁵⁸⁸ and Leu⁵⁹², and (iii) Asp⁵⁹³ and K⁵⁹⁷. p67C^1a residues are colored green. Fab B11 residues are colored as follows: CDR‐L2, (magenta), CDR‐L3 (cyan), CDR‐H1 (yellow), CDR‐H2 (orange), CDR‐H3 (red), framework (gray). Fab B11 side chains that interact with the relevant p67C^1a residues are shown, and hydrogen bonds are shown as dashed lines.