anti-Gal Antibody Research Articles

The α-Gal KO Mouse Animal Model is a Reliable and Predictive Tool for the Immune-Mediated Calcification Assessment of Heart Valve Bioprostheses.

Recent studies highlighted the presence of anti-α-Gal antibodies in patients implanted with commercial bioprosthetic heart valves (BHVs). BHVs expose residual α-Gal xenoantigen and their recognition by the circulating anti-Gal antibodies leads to opsonization of the device's tissue component with the consequent triggering of a deterioration pathway that culminates with calcification. Small animal models such as mice and rats have been broadly involved in the in vivo testing of biomaterials by subcutaneous implantation, especially for the effectiveness of BHVs anti-calcific treatments. However, since models employed for this purpose express α-Gal antigen, the implantation of BHVs' leaflets does not elicit a proper immunological response, so the calcification propensity may be dramatically underestimated. An α-Gal knockout (KO) mouse model has been created, using the CRISP/Cas9 approach, and adopted to assess the calcification potential of commercial BHVs leaflets through the surgical implantation in the back subcutis area. Calcium quantification was performed by inductively coupled plasma analysis; immune response against the BHVs leaflets and α-Gal silencing was evaluated through immunological assays. Two months after the implantation of commercial BHV leaflets, the anti-Gal antibody titers in KO mice doubled when compared with those found in wild-type (WT) ones. Leaflets explanted from KO mice, after one month, showed a four-time increased calcium deposition concerning the ones explanted from WT. The degree of silencing of α-Gal varied, depending on the specific organ that was assessed. In any case, the animal model was suitable for evaluating implanted tissue responses. Such mouse model proved to be an accurate tool for the study of the calcific propensity of commercial BHVs leaflets than those hitherto used. Given its reliability, it could also be successfully used to study even other diseases in which the possible involvement of α-Gal has been observed.

Regeneration in Mice of Injured Skin, Heart, and Spinal Cord by α-Gal Nanoparticles Recapitulates Regeneration in Amphibians.

The healing of skin wounds, myocardial, and spinal cord injuries in salamander, newt, and axolotl amphibians, and in mouse neonates, results in scar-free regeneration, whereas injuries in adult mice heal by fibrosis and scar formation. Although both types of healing are mediated by macrophages, regeneration in these amphibians and in mouse neonates also involves innate activation of the complement system. These differences suggest that localized complement activation in adult mouse injuries might induce regeneration instead of the default fibrosis and scar formation. Localized complement activation is feasible by antigen/antibody interaction between biodegradable nanoparticles presenting α-gal epitopes (α-gal nanoparticles) and the natural anti-Gal antibody which is abundant in humans. Administration of α-gal nanoparticles into injuries of anti-Gal-producing adult mice results in localized complement activation which induces rapid and extensive macrophage recruitment. These macrophages bind anti-Gal-coated α-gal nanoparticles and polarize into M2 pro-regenerative macrophages that orchestrate accelerated scar-free regeneration of skin wounds and regeneration of myocardium injured by myocardial infarction (MI). Furthermore, injection of α-gal nanoparticles into spinal cord injuries of anti-Gal-producing adult mice induces recruitment of M2 macrophages, that mediate extensive angiogenesis and axonal sprouting, which reconnects between proximal and distal severed axons. Thus, α-gal nanoparticle treatment in adult mice mimics physiologic regeneration in amphibians. These studies further suggest that α-gal nanoparticles may be of significance in the treatment of human injuries.

Accelerated Burn Healing in a Mouse Experimental Model Using α-Gal Nanoparticles.

Macrophages play a pivotal role in the process of healing burns. One of the major risks in the course of burn healing, in the absence of regenerating epidermis, is infections, which greatly contribute to morbidity and mortality in such patients. Therefore, it is widely agreed that accelerating the recruitment of macrophages into burns may contribute to faster regeneration of the epidermis, thus decreasing the risk of infections. This review describes a unique method for the rapid recruitment of macrophages into burns and the activation of these macrophages to mediate accelerated regrowth of the epidermis and healing of burns. The method is based on the application of bio-degradable "α-gal" nanoparticles to burns. These nanoparticles present multiple α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R), which bind the abundant natural anti-Gal antibody that constitutes ~1% of immunoglobulins in humans. Anti-Gal/α-gal nanoparticle interaction activates the complement system, resulting in localized production of the complement cleavage peptides C5a and C3a, which are highly effective chemotactic factors for monocyte-derived macrophages. The macrophages recruited into the α-gal nanoparticle-treated burns are activated following interaction between the Fc portion of anti-Gal coating the nanoparticles and the multiple Fc receptors on macrophage cell membranes. The activated macrophages secrete a variety of cytokines/growth factors that accelerate the regrowth of the epidermis and regeneration of the injured skin, thereby cutting the healing time by half. Studies on the healing of thermal injuries in the skin of anti-Gal-producing mice demonstrated a much faster recruitment of macrophages into burns treated with α-gal nanoparticles than in control burns treated with saline and healing of the burns within 6 days, whereas healing of control burns took ~12 days. α-Gal nanoparticles are non-toxic and do not cause chronic granulomas. These findings suggest that α-gal nanoparticles treatment may harness anti-Gal for inducing similar accelerated burn healing effects also in humans.

Differential Immune Response to Bioprosthetic Heart Valve Tissues in the α1,3Galactosyltransferase-Knockout Mouse Model.

Structural valve deterioration (SVD) of bioprosthetic heart valves (BHVs) has great clinical and economic consequences. Notably, immunity against BHVs plays a major role in SVD, especially when implanted in young and middle-aged patients. However, the complex pathogenesis of SVD remains to be fully characterized, and analyses of commercial BHVs in standardized-preclinical settings are needed for further advancement. Here, we studied the immune response to commercial BHV tissue of bovine, porcine, and equine origin after subcutaneous implantation into adult α1,3-galactosyltransferase-knockout (Gal KO) mice. The levels of serum anti-galactose α1,3-galactose (Gal) and -non-Gal IgM and IgG antibodies were determined up to 2 months post-implantation. Based on histological analyses, all BHV tissues studied triggered distinct infiltrating cellular immune responses that related to tissue degeneration. Increased anti-Gal antibody levels were found in serum after ATS 3f and Freedom/Solo implantation but not for Crown or Hancock II grafts. Overall, there were no correlations between cellular-immunity scores and post-implantation antibodies, suggesting these are independent factors differentially affecting the outcome of distinct commercial BHVs. These findings provide further insights into the understanding of SVD immunopathogenesis and highlight the need to evaluate immune responses as a confounding factor.

Antibody production and tolerance to the α-gal epitope as models for understanding and preventing the immune response to incompatible ABO carbohydrate antigens and for α-gal therapies.

This review describes the significance of the α-gal epitope (Galα-3Galβ1-4GlcNAc-R) as the core of human blood-group A and B antigens (A and B antigens), determines in mouse models the principles underlying the immune response to these antigens, and suggests future strategies for the induction of immune tolerance to incompatible A and B antigens in human allografts. Carbohydrate antigens, such as ABO antigens and the α-gal epitope, differ from protein antigens in that they do not interact with T cells, but B cells interacting with them require T-cell help for their activation. The α-gal epitope is the core of both A and B antigens and is the ligand of the natural anti-Gal antibody, which is abundant in all humans. In A and O individuals, anti-Gal clones (called anti-Gal/B) comprise >85% of the so-called anti-B activity and bind to the B antigen in facets that do not include fucose-linked α1-2 to the core α-gal. As many as 1% of B cells are anti-Gal B cells. Activation of quiescent anti-Gal B cells upon exposure to α-gal epitopes on xenografts and some protozoa can increase the titer of anti-Gal by 100-fold. α1,3-Galactosyltransferase knockout (GT-KO) mice lack α-gal epitopes and can produce anti-Gal. These mice simulate human recipients of ABO-incompatible human allografts. Exposure for 2-4weeks of naïve and memory mouse anti-Gal B cells to α-gal epitopes in the heterotopically grafted wild-type (WT) mouse heart results in the elimination of these cells and immune tolerance to this epitope. Shorter exposures of 7days of anti-Gal B cells to α-gal epitopes in the WT heart result in the production of accommodating anti-Gal antibodies that bind to α-gal epitopes but do not lyse cells or reject the graft. Tolerance to α-gal epitopes due to the elimination of naïve and memory anti-Gal B cells can be further induced by 2 weeks in vivo exposure to WT lymphocytes or autologous lymphocytes engineered to present α-gal epitopes by transduction of the α1,3-galactosyltransferase gene. These mouse studies suggest that autologous human lymphocytes similarly engineered to present the A or B antigen may induce corresponding tolerance in recipients of ABO-incompatible allografts. The review further summarizes experimental works demonstrating the efficacy of α-gal therapies in amplifying anti-viral and anti-tumor immune-protection and regeneration of injured tissues.

Correlations between the alpha-Gal antigen, antibody response and calcification of cardiac valve bioprostheses: experimental evidence obtained using an alpha-Gal knockout mouse animal model.

Preformed antibodies against αGal in the human and the presence of αGal antigens on the tissue constituting the commercial bioprosthetic heart valves (BHVs, mainly bovine or porcine pericardium), lead to opsonization of the implanted BHV, leading to deterioration and calcification. Murine subcutaneous implantation of BHVs leaflets has been widely used for testing the efficacy of anti-calcification treatments. Unfortunately, commercial BHVs leaflets implanted into a murine model will not be able to elicit an αGal immune response because such antigen is expressed in the recipient and therefore immunologically tolerated. This study evaluates the calcium deposition on commercial BHV using a new humanized murine αGal knockout (KO) animal model. Furtherly, the anti-calcification efficacy of a polyphenol-based treatment was deeply investigated. By using CRISPR/Cas9 approach an αGal KO mouse was created and adopted for the evaluation of the calcific propensity of original and polyphenols treated BHV by subcutaneous implantation. The calcium quantification was carried out by plasma analysis; the immune response evaluation was performed by histology and immunological assays. Anti-αGal antibodies level in KO mice increases at least double after 2 months of implantation of original commercial BHV compared to WT mice, conversely, the polyphenols-based treatment seems to effectively mask the antigen to the KO mice's immune system. Commercial leaflets explanted after 1 month from KO mice showed a four-time increased calcium deposition than what was observed on that explanted from WT. Polyphenol treatment prevents calcium deposition by over 99% in both KO and WT animals. The implantation of commercial BHV leaflets significantly stimulates the KO mouse immune system resulting in massive production of anti-Gal antibodies and the exacerbation of the αGal-related calcific effect if compared with the WT mouse. The polyphenol-based treatment applied in this investigation showed an unexpected ability to inhibit the recognition of BHV xenoantigens by circulating antibodies almost completely preventing calcific depositions compared to the untreated counterpart.

Genetically engineered sheep: A new paradigm for future preclinical testing of biological heart valves

BackgroundHeart valve implantation in juvenile sheep to demonstrate biocompatibility and physiologic performance is the accepted model for regulatory approval of new biological heart valves (BHVs). However, this standard model does not detect the immunologic incompatibility between the major xenogeneic antigen, galactose-α-1,3-galactose (Gal), which is present in all current commercial BHVs, and patients who universally produce anti-Gal antibody. This clinical discordance leads to induced anti-Gal antibody in BHV recipients, promoting tissue calcification and premature structural valve degeneration, especially in young patients. The objective of the present study was to develop genetically engineered sheep that, like humans, produce anti-Gal antibody and mirror current clinical immune discordance. MethodsGuide RNA for CRISPR Cas9 nuclease was transfected into sheep fetal fibroblasts, creating a biallelic frame shift mutation in exon 4 of the ovine α-galactosyltransferase gene (GGTA1). Somatic cell nuclear transfer was performed, and cloned embryos were transferred to synchronized recipients. Cloned offspring were analyzed for expression of Gal antigen and spontaneous production of anti-Gal antibody. ResultsTwo of 4 surviving sheep survived long-term. One of the 2 was devoid of the Gal antigen (GalKO) and expressed cytotoxic anti-Gal antibody by age 2 to 3 months, which increased to clinically relevant levels by 6 months. ConclusionsGalKO sheep represent a new, clinically relevant advanced standard for preclinical testing of BHVs (surgical or transcatheter) by accounting for the first time for human immune responses to residual Gal antigen that persists after current BHV tissue processing. This will identify the consequences of immune disparity preclinically and avoid unexpected past clinical sequelae.

Paleo-immunology of human anti-carbohydrate antibodies preventing primate extinctions.

Two human natural anti-carbohydrate antibodies appeared in critical evolutionary events that brought primates and hominins to brink of extinction. The first is the anti-Gal antibody, produced in Old-World monkeys (OWM), apes and humans. It binds the carbohydrate-antigen 'α-gal epitope' (Galα1-3Galβ1-4GlcNAc-R) on carbohydrate-chains (glycans) synthesized by non-primate mammals, lemurs and New-World monkeys (NWM). The second is anti-N-glycolylneuraminic-acid (anti-Neu5Gc) antibody binding Neu5Gc on glycans synthesized by OWM, apes and most non-primate mammals. Ancestral OWM and apes synthesized α-gal epitopes and were eliminated ~20-30 million-years-ago (mya). Only few accidentally mutated offspring lacking α-gal epitopes, produced anti-Gal and survived. Hominin-populations living ~3 mya synthesized Neu5Gc and were eliminated, but few mutated offspring that accidently lost their ability to synthesize Neu5Gc, produced natural anti-Neu5Gc antibody. These hominins survived and ultimately evolved into present-day humans. It is argued that these two near-extinction events were likely to be the result of epidemics caused by highly virulent and lethal enveloped viruses that killed parental-populations. These viruses presented α-gal epitopes or Neu5Gc synthesized in host-cells of the parental-populations. Mutated offspring survived the epidemics because they were protected from the lethal virus by the natural anti-Gal or anti-Neu5Gc antibodies they produced due to loss of immune-tolerance to α-gal epitopes or to Neu5Gc, respectively.

α-Gal Nanoparticles Mediated Homing of Endogenous Stem Cells for Repair and Regeneration of External and Internal Injuries by Localized Complement Activation and Macrophage Recruitment.

This review discusses a novel experimental approach for the regeneration of original tissue structure by recruitment of endogenous stem-cells to injured sites following administration of α-gal nanoparticles, which harness the natural anti-Gal antibody. Anti-Gal is produced in large amounts in all humans, and it binds the multiple α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R) presented on α-gal nanoparticles. In situ binding of anti-Gal to α-gal nanoparticles activates the complement system and generates complement cleavage chemotactic-peptides that rapidly recruit macrophages. Macrophages reaching anti-Gal coated α-gal nanoparticles bind them via Fc/Fc receptor interaction and polarize into M2 pro-reparative macrophages. These macrophages secrete various cytokines that orchestrate regeneration of the injured tissue, including VEGF inducing neo-vascularization and cytokines directing homing of stem-cells to injury sites. Homing of stem-cells is also directed by interaction of complement cleavage peptides with their corresponding receptors on the stem-cells. Application of α-gal nanoparticles to skin wounds of anti-Gal producing mice results in decrease in healing time by half. Furthermore, α-gal nanoparticles treated wounds restore the normal structure of the injured skin without fibrosis or scar formation. Similarly, in a mouse model of occlusion/reperfusion myocardial-infarction, near complete regeneration after intramyocardial injection of α-gal nanoparticles was demonstrated, whereas hearts injected with saline display ~20% fibrosis and scar formation of the left ventricular wall. It is suggested that recruitment of stem-cells following anti-Gal/α-gal nanoparticles interaction in injured tissues may result in induction of localized regeneration facilitated by conducive microenvironments generated by pro-reparative macrophage secretions and “cues” provided by the extracellular matrix in the injury site.

α-Gal antigen-deficient rabbits with GGTA1 gene disruption via CRISPR/Cas9

BackgroundPrevious studies have identified the carbohydrate epitope Galα1–3Galβ1–4GlcNAc-R (termed the α-galactosyl epitope), known as the α-Gal antigen as the primary xenoantigen recognized by the human immune system. The α-Gal antigen is regulated by galactosyltransferase (GGTA1), and α-Gal antigen-deficient mice have been widely used in xenoimmunological studies, as well as for the immunogenic risk evaluation of animal-derived medical devices. The objective of this study was to develop α-Gal antigen-deficient rabbits by GGTA1 gene editing with the CRISPR/Cas9 system.ResultsThe mutation efficiency of GGTA1 gene-editing in rabbits was as high as 92.3% in F0 pups. Phenotype analysis showed that the α-Gal antigen expression in the major organs of F0 rabbits was decreased by more than 99.96% compared with that in wild-type (WT) rabbits, and the specific anti-Gal IgG and IgM antibody levels in F1 rabbits increased with increasing age, peaking at approximately 5 or 6 months. Further study showed that GGTA1 gene expression in F2-edited rabbits was dramatically reduced compared to that in WT rabbits.Conclusionsα-Gal antigen-deficient rabbits were successfully generated by GGTA1 gene editing via the CRISPR/Cas9 system in this study. The feasibility of using these α-Gal antigen-deficient rabbits for the in situ implantation and residual immunogenic risk evaluation of animal tissue-derived medical devices was also preliminarily confirmed.

Immunological Risk Assessment of Xenogeneic Dural Patch by Comparing with Raw Material via GTKO Mice.

Objective In this study, α-Gal epitope-deficient (GGTA1 knockout (GTKO)) mice were used to assess the immunological risks of xenogeneic dural patch by comparing with raw material. Methods The xenogeneic dural patch (T2) was prepared from bovine pericardium (T1, raw material) through decellularization and carboxymethyl chitosan (CMCS) coating. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to characterize the collagen fibers and surface microstructural changes in the T1 and T2 samples. The remnant α-Gal epitopes and DNA of implants were detected by standardized method. T1 and T2 were implanted subcutaneously into GTKO mice for 4 and 12 weeks, respectively, and the negative control group (Con) was only performed sham operation. The total serum antibody, anti-Gal antibody, and splenic lymphocyte subtypes were analyzed by ELISA or flow cytometry, and histological analysis of implant-tissue was performed by H&E and Masson stain. Results TEM and Sirius red staining showed that the collagen fibers in the dural patch were closely arranged, and SEM showed that a loose three-dimensional structure was successfully constructed on the surface of the dural patch after CMCS coating. The remnant DNA in T2 was 24.64 ± 8.73 ng/mg (dry weight), and clearance of α-Gal epitope was up to 99.83% compared to T1. The significant increases in serum total IgM, anti-Gal IgG, and anti-Gal IgM at 4 weeks and the significant changes in anti-Gal IgG and spleen lymphocyte at 12 weeks were observed in the T1 group, but no significant change was observed in the T2 group, compared to the control group. Histological semiquantitative analysis showed severe cell and tissue responses at 4 weeks and a moderate response at 12 weeks in the T1 group, while a moderate response at 4 weeks and a slight response at 12 weeks in the T2 group. Conclusions The results demonstrated that the xenogeneic dural patch has a lower and acceptable immunological risk compared to the raw material and control, respectively. On the other hand, it was suggested that GTKO mice are useful experimental model for immunological risk assessment of animal tissue-derived biomaterials.

Biological Equivalence of GGTA-1 Glycosyltransferase Knockout and Standard Porcine Pericardial Tissue Using 90-Day Mitral Valve Implantation in Adolescent Sheep

ObjectiveThere is growing interest in the application of genetically engineered reduced antigenicity animal tissue for manufacture of bioprosthetic heart valves (BHVs) to reduce antibody induced tissue calcification and accelerated structural valve degeneration (SVD). This study tested biological equivalence of valves made from Gal-knockout (GalKO) and standard porcine pericardium after 90-day mitral valve implantation in sheep.MethodsGalKO (n = 5) and standard (n = 5) porcine pericardial BHVs were implanted in a randomized and blind fashion into sheep for 90-days. Valve haemodynamic function was measured at 30-day intervals. After explantation, valves were examined for pannus, vegetation, inflammation, thrombus, and tissue calcification.ResultsNine of 10 recipients completed the study. There was no difference between study groups for haemodynamic performance and no adverse valve-related events. Explanted BHVs showed mild pannus integration and minimal thrombus, with no difference between the groups. Limited focal mineral deposits were detected by x-ray. Atomic spectroscopy analysis detected tissue calcium levels of 1.0 µg/mg ± 0.2 for GalKO BHVs and 1.9 µg/mg ± 0.9 for standard tissue BHVs (p = 0.4), considered to be both low and equivalent.ConclusionsThis is the first demonstration of biological equivalence between GalKO and standard pig pericardium. The GalKO mutation causes neither intrinsic detrimental biological nor functional impact on BHV performance. Commercial adaptation of GalKO tissue for surgical or transcatheter BHVs would remove the clinical disparity between patients producing anti-Gal antibody and BHVs containing the Gal antigen. GalKO BHVs may reduce accelerated tissue calcification and SVD, enhancing patient choices, especially for younger patients.Graphical

Biosynthesis of α-Gal Epitopes (Galα1-3Galβ1-4GlcNAc-R) and Their Unique Potential in Future α-Gal Therapies.

The α-gal epitope is a carbohydrate antigen which appeared early in mammalian evolution and is synthesized in large amounts by the glycosylation enzyme α1,3galactosyltransferase (α1,3GT) in non-primate mammals, lemurs, and New-World monkeys. Ancestral Old-World monkeys and apes synthesizing α-gal epitopes underwent complete extinction 20–30 million years ago, and their mutated progeny lacking α-gal epitopes survived. Humans, apes, and Old-World monkeys which evolved from the surviving progeny lack α-gal epitopes and produce the natural anti-Gal antibody which binds specifically to α-gal epitopes. Because of this reciprocal distribution of the α-gal epitope and anti-Gal in mammals, transplantation of organs from non-primate mammals (e.g., pig xenografts) into Old-World monkeys or humans results in hyperacute rejection following anti-Gal binding to α-gal epitopes on xenograft cells. The in vivo immunocomplexing between anti-Gal and α-gal epitopes on molecules, pathogens, cells, or nanoparticles may be harnessed for development of novel immunotherapies (referred to as “α-gal therapies”) in various clinical settings because such immune complexes induce several beneficial immune processes. These immune processes include localized activation of the complement system which can destroy pathogens and generate chemotactic peptides that recruit antigen-presenting cells (APCs) such as macrophages and dendritic cells, targeting of antigens presenting α-gal epitopes for extensive uptake by APCs, and activation of recruited macrophages into pro-reparative macrophages. Some of the suggested α-gal therapies associated with these immune processes are as follows: 1. Increasing efficacy of enveloped-virus vaccines by synthesizing α-gal epitopes on vaccinating inactivated viruses, thereby targeting them for extensive uptake by APCs. 2. Conversion of autologous tumors into antitumor vaccines by expression of α-gal epitopes on tumor cell membranes. 3. Accelerating healing of external and internal injuries by α-gal nanoparticles which decrease the healing time and diminish scar formation. 4. Increasing anti-Gal–mediated protection against zoonotic viruses presenting α-gal epitopes and against protozoa, such as Trypanosoma, Leishmania, and Plasmodium, by vaccination for elevating production of the anti-Gal antibody. The efficacy and safety of these therapies were demonstrated in transgenic mice and pigs lacking α-gal epitopes and producing anti-Gal, raising the possibility that these α-gal therapies may be considered for further evaluation in clinical trials.

Near Complete Repair After Myocardial Infarction in Adult Mice by Altering the Inflammatory Response With Intramyocardial Injection of α-Gal Nanoparticles

Background: Neonatal mice, but not older mice, can regenerate their hearts after myocardial-infarction (MI), a process mediated by pro-reparative macrophages. α-Gal nanoparticles applied to skin wounds in adult-mice bind the anti-Gal antibody, activate the complement cascade and generate complement chemotactic peptides that recruit pro-reparative macrophages which are further activated by these nanoparticles. The recruited macrophages decrease wound healing time by ~50%, restore the normal skin structure and prevent fibrosis and scar formation in mice.Objectives: The objective of this study is to determine if α-gal nanoparticles injected into the reperfused myocardium after MI in adult-mice can induce myocardial repair that restores normal structure, similar to that observed in skin injuries.Methods and Results: MI was induced by occluding the mid-portion of the left anterior descending (LAD) coronary artery for 30 min. Immediately following reperfusion, each mouse received two 10 μl injections of 100 μg α-gal nanoparticles in saline into the LAD territory (n = 20), or saline for controls (n = 10). Myocardial infarct size was measured by planimetry following Trichrome staining and macrophage recruitment by hematoxylin-eosin staining. Left ventricular (LV) function was measured by echocardiography. Control mice displayed peak macrophage infiltration at 4-days, whereas treated mice had a delayed peak macrophage infiltration at 7-days. At 28-days, control mice demonstrated large transmural infarcts with extensive scar formation and poor contractile function. In contrast, mice treated with α-gal nanoparticles demonstrated after 28-days a marked reduction in infarct size (~10-fold smaller), restoration of normal myocardium structure and contractile function.Conclusions: Intramyocardial injection of α-gal nanoparticles post-MI in anti-Gal producing adult-mice results in near complete repair of the infarcted territory, with restoration of normal LV structure and contractile function. The mechanism responsible for this benefit likely involves alteration of the usual inflammatory response post-MI, as previously observed with regeneration of injured hearts in adult zebrafish, salamanders and neonatal mice.

Altered Blood Levels of Anti-Gal Antibodies in Alzheimer's Disease: A New Clue to Pathogenesis?

Alzheimer’s disease is a neurodegenerative disorder whose pathological mechanisms, despite recent advances, are not fully understood. However, the deposition of beta amyloid -peptide and neuroinflammation, which is probably aggravated by dysbiotic microbiota, seem to play a key role. Anti-Gal are the most abundant xenoreactive natural antibodies. They are supposed to stem from immunization against the gut microbiota and have been implicated in the pathogenesis of several diseases, including multiple sclerosis. These antibodies target the alpha-Gal epitope, expressed on the terminal sugar units of glycoprotein or glycolipid of all mammals except apes, Old World monkeys and humans. The alpha-Gal is constitutively expressed in several bacteria constituting the brain microbiota, and alpha-Gal-like epitopes have been detected in gray matter, amyloid plaque, neurofibrillary tangles and corpora amylacea of the human brain, suggesting a potential link between anti-Gal and Alzheimer’s disease etiopathogenesis. For the first time, our study searched for possible alterations of anti-Gal immunoglobulin levels in Alzheimer’s disease patients. IgG and IgM blood levels were significantly lower, and IgA significantly higher in patients than in healthy subjects. These results suggest that such immunoglobulins might be implicated in Alzheimer’s disease pathogenesis and open new scenarios in the research for new biomarkers and therapeutic strategies.

In Situ "Humanization" of Porcine Bioprostheses: Demonstration of Tendon Bioprostheses Conversion into Human ACL and Possible Implications for Heart Valve Bioprostheses.

This review describes the first studies on successful conversion of porcine soft-tissue bioprostheses into viable permanently functional tissue in humans. This process includes gradual degradation of the porcine tissue, with concomitant neo-vascularization and reconstruction of the implanted bioprosthesis with human cells and extracellular matrix. Such a reconstruction process is referred to in this review as “humanization”. Humanization was achieved with porcine bone-patellar-tendon-bone (BTB), replacing torn anterior-cruciate-ligament (ACL) in patients. In addition to its possible use in orthopedic surgery, it is suggested that this humanization method should be studied as a possible mechanism for converting implanted porcine bioprosthetic heart-valves (BHV) into viable tissue valves in young patients. Presently, these patients are only implanted with mechanical heart-valves, which require constant anticoagulation therapy. The processing of porcine bioprostheses, which enables humanization, includes elimination of α-gal epitopes and partial (incomplete) crosslinking with glutaraldehyde. Studies on implantation of porcine BTB bioprostheses indicated that enzymatic elimination of α-gal epitopes prevents subsequent accelerated destruction of implanted tissues by the natural anti-Gal antibody, whereas the partial crosslinking by glutaraldehyde molecules results in their function as “speed bumps” that slow the infiltration of macrophages. Anti-non gal antibodies produced against porcine antigens in implanted bioprostheses recruit macrophages, which infiltrate at a pace that enables slow degradation of the porcine tissue, neo-vascularization, and infiltration of fibroblasts. These fibroblasts align with the porcine collagen-fibers scaffold, secrete their collagen-fibers and other extracellular-matrix (ECM) components, and gradually replace porcine tissues degraded by macrophages with autologous functional viable tissue. Porcine BTB implanted in patients completes humanization into autologous ACL within ~2 years. The similarities in cells and ECM comprising heart-valves and tendons, raises the possibility that porcine BHV undergoing a similar processing, may also undergo humanization, resulting in formation of an autologous, viable, permanently functional, non-calcifying heart-valves.

Increasing Efficacy of Enveloped Whole-Virus Vaccines by In situ Immune-Complexing with the Natural Anti-Gal Antibody.

The appearance of variants of mutated virus in course of the Covid-19 pandemic raises concerns regarding the risk of possible formation of variants that can evade the protective immune response elicited by the single antigen S-protein gene-based vaccines. This risk may be avoided by inclusion of several antigens in vaccines, so that a variant that evades the immune response to the S-protein of SARS-CoV-2 virus will be destroyed by the protective immune response against other viral antigens. A simple way for preparing multi-antigenic enveloped-virus vaccines is using the inactivated whole-virus as vaccine. However, immunogenicity of such vaccines may be suboptimal because of poor uptake of the vaccine by antigen-presenting-cells (APC) due to electrostatic repulsion by the negative charges of sialic-acid on both the glycan-shield of the vaccinating virus and on the carbohydrate-chains (glycans) of APC. In addition, glycan-shield can mask many antigenic peptides. These effects of the glycan-shield can be reduced and immunogenicity of the vaccinating virus markedly increased by glycoengineering viral glycans for replacing sialic-acid units on glycans with α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R). Vaccination of humans with inactivated whole-virus presenting α-gal epitopes (virusα-gal) results in formation of immune-complexes with the abundant natural anti-Gal antibody that binds to viral α-gal epitopes at the vaccination site. These immune-complexes are targeted to APC for rigorous uptake due to binding of the Fc portion of immunecomplexed anti-Gal to Fcγ receptors on APC. The APC further transport the large amounts of internalized vaccinating virus to regional lymph nodes, process and present the virus antigenic peptides for the activation of many clones of virus specific helper and cytotoxic T-cells. This elicits a protective cellular and humoral immune response against multiple viral antigens and an effective immunological memory. The immune response to virusα-gal vaccine was studied in mice producing anti-Gal and immunized with inactivated influenza-virusα-gal. These mice demonstrated 100-fold increase in titer of the antibodies produced, a marked increase in T-cell response, and a near complete protection against challenge with a lethal dose of live influenza-virus, in comparison to a similar vaccine lacking α-gal epitopes. This glycoengineering can be achieved in vitro by enzymatic reaction with neuraminidase removing sialic-acid and with recombinant α1,3galactosyltransferase (α1,3GT) synthesizing α-gal epitopes, by engineering host-cells to contain several copies of the α1,3GT gene (GGTA1), or by transduction of this gene in a replication-defective adenovirus vector into host-cells. Theoretically, these methods for increased immunogenicity may be applicable to all enveloped viruses with N-glycans on their envelope.

First experience of α1,3-galactosyltransferase gene-knockout (GTKO) transgenic pig to nonhuman primate lamellar corneal xenotransplantation

Background: Corneal allotransplantation is a well-known technique to treat corneal blindness. However, the problem of organ scarcity in transplantology has become profound. One promising alternative could be xenograft using pig as an organ donor. Full thickness corneal xenotransplantation needs total immunosuppression. This study is an investigation of the efficacy of α1,3-ga lactosyltransferase gene-knockout (GTKO) transgenic pig-to-nonhuman primate lamellar corneal transplantation with minimal immunosuppression. Methods: We conducted 10 lamellae corneal xenotransplantation between 2016 and 2019. Clinically acceptable graft size (diam eter 7.5 mm, thickness 500 um). The dexamethasone subconjunctival injection (1.5 mg/0.3 mL) was administered for minimal immunosuppression immediately after surgery and eye drops of 0.5% levofloxacin and 1% prednisolone acetate were applied four times a day for 1 week, gradually tapered. No eye drops were added after two months. Three cases are alive without graft rejection. We examined the remaining seven cases for the histopathological features and Immunologic profiles. Results: As a result, three of the ten xenografts survival is significantly longer 1,239, 589, and 316 days. Corneal opacity result ed in graft failure, and terminated in seven cases. Rejected grafts showed extensive polymorphic cellular infiltration, different degrees of epithelial layer irregular attenuation, stromal neovascularization, and inflammatory cell infiltrations including lym phocytes, plasma cells, eosinophils, neutrophils. Stromal irregularity, fibrosis and edema are observed in two of seven cases resulting in a single case of sub epithelial detachment. Immunologic profiles of the recipients with rejected grafts shows minimal increase in anti-Gal antibody IgG and IgM but increase in anti-Gal IgG is seen in one case. Four cases have different systemic in flammatory conditions with regards to plasma C3a and D-dimer levels. The anterior stromal surface of the graft showed epitheli al nests and fibrous proliferation. Conclusions: The GTKO transgenic pig to NHP lamella xeno corneal transplantation could be a promising substitute for human corneal allograft. Lamella xeno corneal transplantation may be a feasible option with minimal immunosuppression.

SARS-CoV-2 replicating in nonprimate mammalian cells probably have critical advantages for COVID-19 vaccines due to anti-Gal antibodies: A minireview and proposals.

Glycoproteins of enveloped viruses replicating in nonprimate mammalian cells carry α‐1,3‐galactose (α‐Gal) glycans, and can bind to anti‐Gal antibodies which are abundant in humans. The antibodies have protected humans and their ancestors for millions of years, because they inhibit replication of many kinds of microbes carrying αGal glycans and aid complements and macrophages to destroy them. Therefore, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) replicating in nonprimate mammalian cells (eg, PK‐15 cells) carry αGal glycans and could be employed as a live vaccine for corona virus 2019 (COVID‐19). The live vaccine safety could be further enhanced through intramuscular inoculation to bypass the fragile lungs, like the live unattenuated adenovirus vaccine safely used in US recruits for decades. Moreover, the immune complexes of SARS‐CoV‐2 and anti‐Gal antibodies could enhance the efficacy of COVID‐19 vaccines, live or inactivated, carrying α‐Gal glycans. Experiments are imperatively desired to examine these novel vaccine strategies which probably have the critical advantages for defeating the pandemic of COVID‐19 and preventing other viral infectious diseases.

Immune risk assessment of residual αGal in xenogeneic decellularized cornea using GTKO mice.

The xenogeneic decellularized corneal matrix (DCM) was expected to be used in lamellar keratoplasty in clinic as the substitute of allogeneic cornea. After decellularization treatment, the remaining risk of xenograft rejection needed to be assessed. The galactose-α1,3-galactose, as the most abundant and closely rejection-related xenogeneic antigen, should be one of the important factors concerned in immunological evaluation. In this study, residual αGal in the DCM was first determined by an enzyme-linked immunosorbent assay method with qualified accuracy and specificity. Then the DCM was implanted subcutaneously into the α1,3-galactosyltransferase gene-knockout (GTKO) mice, accompanied by the implantation in the wild-type C57BL/6 mice as a comparison. The total serum antibody levels, anti-Gal antibody levels, inflammatory cytokines and ratios of splenic lymphocyte subtypes were detected and the histopathological analysis of implants were performed to systematically evaluate the immune responses. The experimental result showed the fresh porcine corneal matrix samples had (9.90 ± 1.54) × 1012 αGal epitope per mg while the content of residual αGal in the DCM was (7.90 ± 2.00) × 1012 epitope per mg. The GTKO mice had similar potential of reaction to immune stimulation to that of wild-type C57BL/6 mice. At 4 weeks after implantation of DCM, in WT mice and GTKO mice there were both innate immunity response to the DCM characterized by macrophage infiltration. But the elevations of anti-Gal IgG level and the percentage of splenic natural killer cells were only detected in GTKO mice. These changes were thought to be pertinent to the residual αGal antigen, which could not be detected in WT mice. No further αGal antibody-mediated cellular immunity and significant changes of serum cytokine contents were found in GTKO mice, which perhaps suggested that the immune reactions to the DCM after 4 weeks of implantation were moderate and had minor effect on the survival of the corneal graft.