Antigenicity of Bovine Pericardium Determined by a Novel Immunoproteomic Approach

Abstract

Despite bovine pericardium (BP) being the primary biomaterial used in heart valve bioprostheses, recipient graft-specific immune responses remain a significant cause of graft failure. Consequently, tissue antigenicity remains the principal barrier for expanding use of such biomaterials in clinical practice. We hypothesize that our understanding of BP antigenicity can be improved by application of a combined affinity chromatography shotgun immunoproteomic approach to identify antigens that have previously been overlooked. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) analysis of affinity chromatography purified antigens resulted in identification of 133 antigens. Most importantly, antigens were identified from all subcellular locations, including 18 integral membrane protein antigens. Critically, isoforms of several protein families were found to be antigenic suggesting the possibility that shared epitope domains may exist. Furthermore, proteins associated with immune, coagulation, and inflammatory pathways were over-represented, suggesting that these biological processes play a key role in antigenicity. This study brings to light important determinants of antigenicity in a clinically relevant xenogeneic biomaterial (i.e. BP) and further validates a rapid, high-throughput method for immunoproteomic antigen identification.

Graft-specific immune tolerance is determined by residual antigenicity of xenogeneic extracellular matrix scaffolds

“…Recent in vivo studies indicate that lipophilic antigens may engender a greater graft-specific adaptive immune response than hydrophilic antigens [3,13,29]. This dichotomy may result from the fact that majority of lipophilic antigens are membrane associated and thus more readily available for adaptive immune surveillance than are hydrophilic cellular antigens [14,30]. Unfortunately, this concern is compounded by the fact that lipophilic antigens have inherently low solubility in aqueous solutions and are therefore challenging to remove from candidate tissues [23,24,31].…”

“…Xenogeneic ECM scaffolds in which native tissue properties are maintained therefore have potential for in vivo self-recognition allowing for integration, remodeling, and growth [11–13]. However, tissue antigenicity represents the principal barrier toward use of unfixed xenogeneic ECM biomaterials in clinical practice, due to its ability to stimulate recipient graft-specific adaptive immune responses [5,14,15]. Therefore, the primary goal of antigen removal and decellularization protocols is to remove antigenic components and cellular debris from tissue in a manner that preserves the native ECM microenvironment.…”

“…Even though the field of antigen identification and characterization is still evolving, prior studies have shown that antigens can be broadly categorized based on their solubility [14,20,21]. Such classification schemes may also inform removal strategies, since hydrophilic antigens are more readily solubilized and removed from candidate tissues in aqueous extraction solutions than are lipophilic antigens [22,23].…”

Cardiac Non-Human Leukocyte Antigen Identification: Techniques and Troubles

“…Much of the research presented here relied on a comparison of small cohorts with single pre/posttransplant time points ( 29 , 52 ) or matched cases ( 28 ). Our lab has recently validated a novel immunoproteomic method for antigen discovery which is fast, simple and inexpensive compared to the currently available technology ( 69 ). It has been applied to an animal model of xenotransplantation and is currently being applied to cardiac transplant patients ( 69 ).…”

“…Our lab has recently validated a novel immunoproteomic method for antigen discovery which is fast, simple and inexpensive compared to the currently available technology ( 69 ). It has been applied to an animal model of xenotransplantation and is currently being applied to cardiac transplant patients ( 69 ). Although further clinical validation is necessary, this affinity chromatography antigen identification approach has potential to further enhance understanding of recipient non-HLA graft-specific immune responses.…”