Immunoproteomic Identification of Noncarbohydrate Antigens Eliciting Graft‐Specific Adaptive Immune Responses in Patients with Bovine Pericardial Bioprosthetic Heart Valves

Abstract

Purpose: This case-control retrospective discovery study is to identify antigenic bovine pericardium (BP) proteins that stimulate graft-specific humoral immune response in patients implanted with glutaraldehyde fixed bovine pericardial (GFBP) heart valves. Experimental design: Banked serum is collected from age-and sex-matched patients who received either a GFBP or mechanical heart valve replacement. Serum IgG is isolated and used to generate poly-polyclonal antibody affinity chromatography columns from each patient. Native and deglycosylated BP protein extracts are separately added to individual patient affinity chromatography columns, with unbound proteins washed through the column. Proteins captured in the affinity chromatography columns are submitted for proteomic identification. Differences between GFBP and mechanical heart valve replacement recipients are analyzed with Gaussian linearized modeling. Results: Carbohydrate antigens overwhelm protein capture in the column, requiring BP protein deglycosylation prior to affinity chromatography. Nineteen BP protein antigens, which stimulated graft-specific IgG production, are identified in patients who received GFBP valve replacements. Identified antigens are significantly over-represented for calcium-binding proteins. Conclusions and clinical relevance: Patients implanted with GFBP valves develop a graft-specific humoral immune response toward BP protein antigens, with 19 specific antigens identified in this work. The molecular functions of over-represented antigens, specifically calcium-binding proteins, may aid in understanding the underlying factors that contribute to structural valve deterioration.

Toward acellular xenogeneic heart valve prostheses: Histological and biomechanical characterization of decellularized and enzymatically deglycosylated porcine pulmonary heart valve matrices

“…Nevertheless, it is noteworthy to mention once more that the sole removal of carbohydrates might not be sufficient to prevent chronic graft rejection; indeed, further studies will be necessary to investigate the appropriate removal of SLA class I molecules as well as non‐carbohydrate xenoantigens reported in the literature, including both proteins and lipids 17,21 . Nonetheless, SLA class I molecules and identified non‐carbohydrates xenoantigens are part of the cell membrane or are located in the subcellular space, and since cells and cellular debris are destroyed and washed out by decellularization, these molecules should not play a role anymore in decellularized tissues.…”

“…18 Furthermore, non-self minor histocompatibility antigens, associated with MHC, have been reported to be recognized by T cells, initiating immune rejection. 17,19,20 Gates et al 21 In addition to the above-mentioned protein xenoantigens, carbohydrate antigens seem to play a major role in tissue immunogenicity, due to their large variety within and between species, much greater than that of proteins. 22 Carbohydrates like galactose-alpha-1,3-galactose (α-Gal) and N-glycolylneuraminic acid (Neu5Gc) were identified as responsible structures for hyperacute or acute immune rejection of the transplant by pig-to-human transplantation.…”

“…Gates et al 21 identified several subcellular and a few transmembrane proteins as xenoantigens present in GA‐fixed valves made of bovine pericardium.…”

Small Diameter Xenogeneic Extracellular Matrix Scaffolds for Vascular Applications

“…165,166 Indeed for example, in human patients receiving GTA-fixed heart valves, chronic immune responses toward 19 individual non-HLA antigens were recently reported. 167 Similarly, GTA-fixed heart valves explanted due to development of structural valve deterioration have been shown to be universally positive for non-a-Gal IgG presence. 89,168 GTA fixation is also associated with alterations in ECM scaffold mechanical properties and renders the resultant biomaterial prone to calcification due to free aldehyde residues in the resultant tissue.…”

Mechanisms and Drug Therapies of Bioprosthetic Heart Valve Calcification

“…The additional two immunogenic carbohydrate antigens that have been identified are N-glycolylneuramic acid (Neu5Gc) antigen (Lee et al, 2016;Reuven et al, 2016) and the Sid blood group antigen (Sda) (Li, 2019). Apart from carbohydrate antigens, Katherine et al (Gates et al, 2019) have identified 19 specific protein antigens from GLUT fixed bovine pericardial heart valves that stimulate the graft-specific humoral immune response in patients. Intriguingly, they found calcium-binding proteins were the most highly over-represented biological function of antigens, but such antibody-binding effect of those proteins on BHV calcification is yet to be elucidated.…”