Understanding the Molecular Determinants Driving the Immunological Specificity of the Protective Pilus 2a Backbone Protein of Group B Streptococcus

Abstract

The pilus 2a backbone protein (BP-2a) is one of the most structurally and functionally characterized components of a potential vaccine formulation against Group B Streptococcus. It is characterized by six main immunologically distinct allelic variants, each inducing variant-specific protection. To investigate the molecular determinants driving the variant immunogenic specificity of BP-2a, in terms of single residue contributions, we generated six monoclonal antibodies against a specific protein variant based on their capability to recognize the polymerized pili structure on the bacterial surface. Three mAbs were also able to induce complement-dependent opsonophagocytosis killing of live GBS and target the same linear epitope present in the structurally defined and immunodominant domain D3 of the protein. Molecular docking between the modelled scFv antibody sequences and the BP-2a crystal structure revealed the potential role at the binding interface of some non-conserved antigen residues. Mutagenesis analysis confirmed the necessity of a perfect balance between charges, size and polarity at the binding interface to obtain specific binding of mAbs to the protein antigen for a neutralizing response.

Structural Conservation, Variability, and Immunogenicity of the T6 Backbone Pilin of Serotype M6 Streptococcus pyogenes

“…Although GAS BPs show large variations in size and sequence, the modular nature of the pilus proteins revealed by the current T6 structure and that of M1 Spy0128 (6) may aid future vaccine design. Such structure-based antigen design approaches have proved successful for serogroup B meningococcus and group B streptococcus (49,50). Careful examination of GAS BP structures from common serotypes, alongside studies of the human immune response to BP domains, may reduce the number of BP variants needed to provide protection against widely circulating GAS strains.…”

“…The cultures were induced with 0.3 mM IPTG (isopropyl-␤-D-thiogalactopyranoside) at 37°C for 3 h, and the cells were harvested by centrifugation. Cell pellets were resuspended in lysis buffer (50 8.0]-100 mM NaCl at 4°C for 16 h. T6 was separated from the rTEV-His 6 protease and uncleaved protein by immobilized metal affinity chromatography (IMAC). The unbound protein containing T6 was concentrated using a 30-kDa molecular-mass cutoff protein concentrator (VivaScience) and purified by size-exclusion chromatography on a Superdex S200 10/300 column (GE Healthcare) in crystallization buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl).…”

Identification of fimbrial subunits in the genome of Trueperella pyogenes and association between serum antibodies against fimbrial proteins and uterine conditions in dairy cows

“…Because cell surface proteins mediate the interaction between host and pathogens, they provide a key signature for elimination of infection by immune cells. Accordingly, the identification and characterization of antigenic epitopes is critical for the development of vaccines against gram-positive bacteria (Maione et al, 2005;Nuccitelli et al, 2013). Poor fimbriae expression in vitro by wild strains of T. pyogenes is a major impediment for the characterization of its adhesion mechanisms.…”

Peptides for Infectious Diseases: From Probe Design to Diagnostic Microarrays

“…The presence of the antibody bound to its epitope may protect the cleavage site at or near the epitope itself. This technique is generally fast, relatively inexpensive, and has been shown to be able to quickly identify regions of interaction in an antigen [31,42,43]. In some cases, HDX combined with limited proteolysis has been coupled to liquid chromatography (LC) [44,45].…”