Antibody Interactions with M and M-like Proteins

About This Grant

Streptococcus pyogenes (Strep A) is a major global pathogen responsible for significant morbidity and mortality. Efforts to develop a broadly protective immunization strategy against Strep A have been hindered by the extensive sequence variability of its primary virulence factor, the M protein, with over 220 known M types. The immune response to M proteins is typically type-specific, limiting broad protection. This project builds on recent structural and functional discoveries to design M protein-based immunogens that elicit cross-reactive antibodies against diverse Strep A strains. We hypothesize that conserved 3D structural patterns in M proteins can drive broad immune recognition and be leveraged to enhance immunogen design. A promising M protein-based immunogen that completed Phase I trials, StreptAnova, generates cross-reactive antibodies, yet the basis for this cross-reactivity is unknown. Our work suggests that the M type cross-reactivity may be due to conserved three-dimensional (3D) structural patterns within M proteins that mediate binding to human C4b-binding protein (C4BP), an essential virulence factor. Notably, half of the M proteins in StreptAnova contain a C4BP-binding pattern as do about half of the cross-reactive M types. In our own experiments, we have demonstrated that an M2 protein-derived immunogen, containing the C4BP-binding 3D pattern, elicits cross-reactive and opsonophagocytic antibodies against several M types that share this structural pattern. However, the breadth of this response was limited, revealing opportunities for improvement. Our recent findings identified a C4BP-binding pattern in M22 protein that is the most prevalent across Strep A strains. We hypothesize that an M22-based immunogen, particularly with structural modifications, will significantly expand M-type cross-reactivity. Additionally, our collaborator Pontus Nordenfelt (Lund University, Sweden) has identified human monoclonal antibodies that are cross-reactive against M proteins of multiple types, many of which do not bind C4BP. The basis for their M type cross-reactivity is not known and will be investigated here. Our specific aims are the following. (1) Develop an M protein-based immunogen with broad M type cross-reactivity. We will investigate whether an M22-based immunogen, incorporating natural or consensus sequences fused to immunogenic stabilizing sequences, can enhance protection across multiple Strep A strains. (2) Determine the structures of M-type cross-reactive antibodies bound to M proteins. We will use X-ray crystallography and site-directed mutagenesis to investigate antibody interactions with M proteins, testing whether cross-reactivity is driven by recognition of the C4BP-binding 3D pattern. We will also structurally characterize cross-reactive human antibodies isolated from convalescent patients to identify novel mechanisms of broad recognition. This project integrates structural biology, immunology, and rational immunogen design to address a long-standing challenge in Strep A research. Results will provide fundamental insights into M protein cross-reactivity and could serve as the foundation for a broadly protective strategy.