Characterization of a Key Neutralizing Epitope on Pertussis Toxin Recognized by Monoclonal Antibody 1B7

Abstract

Despite over five decades of research and vaccination, infection by Bordetella pertussis remains a serious disease with no specific treatments or validated correlates of protective immunity. Of the numerous monoclonal antibodies binding pertussis toxin (PTx) that have been produced and characterized, the murine IgG2a monoclonal antibody 1B7 is uniquely neutralizing in all in vitro assays and in vivo murine models of infection. 1B7 binds an epitope on the enzymatically active S1-subunit of PTx (PTx-S1) with some linear elements but previous work with S1 scanning peptides, phage displayed peptide libraries, and S1 truncation/deletion variants were unable to more precisely define the epitope. Using computational docking algorithms, alanine scanning mutagenesis, and surface plasmon resonance, we characterize the epitope bound by 1B7 on PTx-S1 in molecular detail and define energetically important interactions between residues at the interface. Six residues on PTx-S1 and six residues on 1B7 were identified which, when altered to alanine, resulted in variants with significantly reduced affinity for the native partner. Using this information, a model of the 1B7-S1 interaction was developed, indicating a predominantly conformational epitope located on the base of S1 near S4. The location of this epitope is consistent with previous data and is shown to be conserved across several naturally occurring strain variants including PTx-S1A, B (Tohama-I), D, and E (18-323) in addition to the catalytically inactive 9K/129G variant. This highly neutralizing but poorly immunogenic epitope may represent an important target for next generation vaccine development, identification of immune correlates and passive immunization strategies in pertussis.

Rapid Fine Conformational Epitope Mapping Using Comprehensive Mutagenesis and Deep Sequencing

“…4, b and c). This proposed epitope is consistent with a previous alanine scanning dataset developed by Sutherland and Maynard (24). The two conserved residues outside of this region, Ala-93 and Asn-163, are most likely of structural importance for the conformational epitope as they are buried in the protein core.…”

“…However, our method relies on counting individual sequences after a single cell sort, resulting in a limited dynamic range. To determine whether dissociation constants calculated from deep sequencing results are quantitative, we compared our results with an alanine scanning dataset for in vitro binding of PTx-S1-220 and murine 1B7 (24). Our results are consistent with scanning data for 16 of the 17 mutations (Table 1).…”

“…The plasmid pETCON_PTx-S1-220 2 was created by amplifying PTxS1-220 from pAK400_PTx-S1-200K (24) and inserting into pETCON at NdeI/XhoI sites. To create pETCOn_TROP2Ex, DNA was isolated from HeLa cells with the GenElute Mammalian Genomic DNA Miniprep kit (Sigma).…”

“…Soluble Expression of PTxS1-BL21(DE3) cells containing pAK400_ PTx-S1-220K (24) with an added C-terminal lysine residue were grown in terrific broth at 25°C to an A 600 of 1.5, then induced with 1 mM isopropyl 1-thio-␤-D-galactopyranoside for 5 h. Cell pellets were collected, and the outer membrane was lysed by osmotic shock. Lysate was purified by immobilized metal affinity chromatography followed by size exclusion chromatography in PBS (S75, AKTA FPLC) as previously reported (24).Yields were between 0.2 and 0.6 mg/liter. 3 g of pertussis toxin (PTx; 26.1 KDa) or PTx-S1-220K (25.9 KDa) were run on a 12% polyacrylamide gel at 130 V and stained with GelCode blue.…”

Antibodies Recognizing Protective Pertussis Toxin Epitopes Are Preferentially Elicited by Natural Infection versus Acellular Immunization

“…Genetically detoxified PTx (PTg) was generously donated by Rino Rappuoli, Novartis. The plasmid encoding a soluble, truncated version of the PTx S1 subunit, PTx-S1-220, and the subsequent expression and purification of this protein in Escherichia coli have been described previously (38). Murine hybridoma cells producing the IgG2a antibody 1B7 or the IgG1 antibody 11E6 (31) were grown in Hybridoma serum-free medium (Gibco) at 37°C with 5% CO 2 until cell death (ϳ1 to 2 months).…”