Immunoproteomic identification of in vivo response toward residual antigenic proteins in xenogeneic heart valve biomaterials

Abstract

Valve replacement is an important therapeutic approach for management of valvular heart disease, accounting for over 200,000 surgeries annually worldwide. Deficiencies of current heart valve prostheses led the NHLBI cardiac surgery working group to highlight the need for improved heart valve leaflet biomaterials. Recently, our group has engineered a novel method to eliminate antigens from bovine pericardium (BP) extracellular matrix (ECM) scaffolds, while retaining biomaterial structure-function properties and regenerative capacity. However, despite reducing BP-ECM scaffold antigen burden by >92%, graft-specific humoral IgG response persisted at ~30% of the level associated with native BP in a rabbit model. In this study, we sought to determine the identities of residual antigens present in antigen removed BP-ECM scaffolds. New Zealand White Rabbits were subpannicularly implanted with either native, glutaraldehyde fixed, or antigen-removed BP-ECM scaffolds (n = 6 per group). Rabbit serum, collected pre- and at 56 d post-implantation, was used to generate IgG affinity chromatography columns. Native BP protein extracts were loaded onto columns, and captured antigens were identified by LC-MS/MS. A total of 259 antigens were identified in native BP rabbits. The glutaraldehyde fixed and antigen-removed rabbits responded to ~4% and ~12% of these same antigens, respectively. A small subset of antigens were uniquely identified in each of the treatment groups, not identified in native BP. These results inform ongoing efforts to eliminate antigens from xenogeneic tissues in development of ECM scaffolds, and may provide insight into the relative immunogenicity of individual xenoantigens in the context of tissue engineering.