Dalgliesh, Ailsa J.

American Heart Association estimates the prevalence of valvular heart disease to be 2.5% in the United States. Biological heart valve replacements, such as glutaraldehyde‐fixed bovine pericardium (BP), are considered the current standard of care. The NHLBI xenotransplantation working group identified xenoantigenicity as the major barrier to expanding use of such xenogeneic tissues. Previous research has utilized an immunoproteomic approach based on two‐dimensional western blots to identify a small number of BP xenoantigens, which is time consuming and technically challenging. These limitations have resulted in an ever increasing use of shotgun proteomic approaches due to their superior throughput and reproducibility. However, traditional Liquid Chromatography‐Mass Spectrometry (LC‐MS) fails to separate proteins based on their antigenicity, and therefore it is impossible to determine which identified proteins are antigenic versus not. We hypothesized that an affinity chromatography method utilizing poly‐polyclonal serum would bridge this gap and represent an innovative shotgun proteomic antigen identification approach. Using affinity chromatography rabbit IgG antibodies were isolated from whole serum with Protein A HP SpinTrap (GE Healthcare). IgG capture was confirmed by serum protein electrophoresis and ELISA. Two types of antibody affinity columns were generated, with naive serum (D0) or serum from rabbits immunized with bovine pericardium (D84). Bovine pericardial protein extracts were run through both affinity columns, allowing for the specific capture of antigens, which were later eluted and collected. To confirm specificity of antigen capture, eluants were subjected to western blot, and probed with D84 serum. Affinity enriched antigenic proteins were identified by LC‐MS/MS, with statistical comparisons made between D0 and D84. This approach identified 133 putative antigenic proteins that were enriched on D84 compared to D0 columns. Ten of these antigens corresponded to previously published BP xenoantigens. Of the remaining 123 previously unidentified antigens, 30 have been indicated to be immunogenic in other fields such as cancer and autoimmunity. This method was then applied to three humans who had been implanted with bovine pericardial heart valves. Surprisingly, 300 to 500 proteins were identified from patient columns. Between 57 to 79% of the identified proteins were shared among the three individuals, as well as almost all of the proteins identified in the rabbit model were also present in the human. This work therefore represents the first high throughput method of antigen identification that can now be used to monitor currently implanted patients as well as inform the development of BP‐based biomaterials in the future. Support or Funding Information Research supported by grant number R01HL115205 from the National Heart Lung and Blood Institute (NHLBI) at the National Institutes of Health (NIH) and grant number 5T32OD010931‐10 from the NIH.

Despite a staggering 100,000 heart valve replacements performed in the United States per year, current glutaraldehyde‐fixed xenogeneic bioprosthetic replacements are far from ideal. Although glutaraldehyde‐fixation effectively masks tissue antigenicity in the short term, chronic graft‐specific immune responses still commonly occur, limiting valve life expectancy. Additionally, glutaraldehyde‐fixation crosslinks extracellular matrix proteins preventing remodeling and growth in juvenile patients. A heart valve bioprosthesis formed from an unfixed xenogeneic ECM scaffold, in which antigenic burden has been eliminated or significantly reduced, has potential to overcome deficiencies of current heart valve replacements and improve patient outcomes. Based on protein chemistry principles of sequential, differential solubilization, we hypothesized that amidosulfobetaines would improve lipophilic antigen solubilization and subsequent removal from bovine pericardium following antigen removal (BP‐AR). Antigen removal (AR) was achieved by subjecting native BP samples to hydrophilic AR (48 h), followed by lipophilic AR (hydrophilic solution plus ASB‐14 or ASB‐16 at 0, 1, 2, 5, 8 and 0.1, 2, 3, 4, 5% respectively) (48 h), nuclease digestion (24 h) and washout (48 h). Residual protein was extracted from minced BP‐AR samples using 0.1% sodium dodecyl sulfate (SDS) (hydrophile extract) followed by 1% SDS (lipophile extract). Hydrophile and lipophile extracts ( n = 6 per group) were assessed for residual antigenicity using 1‐DE western blots assessed for IgG positivity using anti‐BP immune serum. Uniaxial tensile testing was performed on BP‐AR scaffolds to determine Young's modulus and Ultimate Tensile Strength. No significant change in scaffold residual hydrophile antigenicity was found between groups, with an average reduction in hydrophile antigenicity of 93.59% compared to untreated control tissue. Both ASB‐14 and ASB‐16 demonstrated statistically significant concentration dependent decreases in residual lipophile antigenicity of BP‐AR, which plateaued at 5% for ASB‐14 and 3% for ASB‐16 (79% and 92% lipophilic antigen removal respectively) ( p < 0.0001). Compared to native BP, there were no significant changes in Young's modulus and Ultimate Tensile Strength between BP‐AR scaffolds generated in the presence of ASB‐14 at 5% or ASB‐16 at 3% ( p < 0.6198) This study demonstrates that increasing amidosulfobetaine concentration increases removal of lipophilic antigens from candidate xenogeneic heart valve biomaterial. The extent to which the achieved reduction in biomaterial antigenicity equates to avoidance of in vivo recipient graft‐specific adaptive immune responses remains to be determined. Additionally, future studies are necessary to quantify ECM protein composition and histology of the resultant xenogeneic BP‐AR scaffolds. However, BP‐AR scaffolds generated using 5% ASB‐14 or 3% ASB‐16 represent promising xenogeneic heart valve prosthesis biomaterials. Support or Funding Information National Institutes of Health (NIH) R01HL115205

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 &gt;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.