Galactosyltransferase Knockout Research Articles

Lung xenogenic injury: does anticoagulation help?

The shortage of human organ donors remains a major limitation to the field of transplantation [1], and can potentially be solved using xenografts. During the past decade, many advances have been made to address the major initial immunological obstacle to “discordant” pig‐to‐human solid organ transplantation: hyperacute rejection (HAR). As organs from unmodified pigs were evaluated in animal models, HAR was found to be primarily a consequence of the recipient's preformed anti‐pig antibodies binding on porcine vascular endothelial cells leading to subsequent complement activation, thrombosis and graft failure [2]. This process occurs within minutes to hours of human blood perfusion in porcine organs. Since the carbohydrate structure Galactose‐α(1,3‐Galactose (Gal) is recognized by over 80% of anti‐pig antibodies found in man, genetically modified galactosyl transferase knock‐out (GalTKO) pig organs have been developed [3,4]. Endothelium and parenchymal cells from GalTKO animals lack the Galα1,3Gal epitope. As predicted, heart and kidney transplant studies in baboons showed that the GalTKO phenotype is associated with decreased antibody binding, reduced activation of the complement cascade and prolonged graft survival [5–8]. However, delayed xenograft rejection, consumptive coagulopathy and microangiopathy limit long‐term outcomes. Xenogenic lungs are even more sensitive to xenogenic injury [9, 10], even with additional expression of complement regulatory protein human CD46 [Burdorf et al]. Immunohistology and biochemical evidence and work by others implicated inflammation and coagulation cascade activation as residual xenogenic injury pathways. To determine the role of these pathways in lung xenogenic injury, our group is evaluating new transgenic pigs [11] and various pharmacologic approaches. New transgenes include human thrombomodulin (hTM), endothelial protein C receptor (EPCR) and ectonucleoside triphosphate diphosphohydrolase‐1 (CD39). Pharmacologic interventions consist in targeting platelet receptors (GPIb, GPIIbIIIa), thrombin (hirulog) and adding exogenous activated protein C to perfused human blood and administering desmopressin to lung donor pigs. The differential effects of these interventions on lung physiological parameters, platelet and coagulation activation will be summarized. Future work will place emphasis on combined targeting of these pathways.Acknowledgments: Funded in part by NIH (IU19A1090959, 1U01AI066335) and by gifts from Revivicor and United Therapeutics.

Trial using pig cells with the H–D antigen knocked down

This report describes an attempt to reduce the expression level of Hanganutziu-Deicher (H-D) antigens by small interfering RNA (siRNA) for pig cytidine monophospho-N-acetylneuraminic acid hydroxylase (pCMAH). A pig endothelial cell (PEC) line, and PEC and fibroblasts from an α1,3galactosyltransferase knockout (GalT-KO) piglet were used. Real-time PCR was used to evaluate the degradation of mRNA by siRNA. The H-D antigen was stained, and then the cells were incubated with human serum for the FACS analysis. The extent of lysis in human serum was next calculated using an LDH assay. Suppression of the mRNA of pCMAH by each siRNA was first determined. The mixture of siRNAs for pCMAH reduced the expressions of the H-D antigen on the PEC and fibroblasts to a considerable extent. The further reduction in the xenoantigenicity for human serum of the GalT-KO cells was then confirmed. In addition, the PEC line showed a significant downregulation in complement-dependent cytotoxicity by the siRNAs, thus indicating that the anti-H-D antigen in human serum is capable of causing lysis of the pig cells. pCMAH silencing by siRNA reduced the expression of the H-D antigen and its antigenicity, thus confirming that the H-D antigen is one of the major non-Gal antigens in this situation.

Production of cloned pigs expressing human thrombomodulin in endothelial cells

For long-term xenograft survival, coagulation control is one of the remaining critical issues. Our attention has been directed toward human thrombomodulin (hTM), because it is expected to exhibit the following beneficial effects on coagulation control and cytoprotection: (i) to solve the problem of molecular incompatibility in protein C activation; (ii) to exert a role as a physiological regulator, only when thrombin is formed; (iii) to suppress direct prothrombinase activity; and (iv) to have anti-inflammatory properties. hTM gene was transfected into pig (Landrace/Yorkshire) fibroblasts using pCAGGS expression vector and pPGK-puro vector. After puromycin selection, only fibroblasts expressing a high level of hTM were collected by cell sorting and then applied to nuclear transfer. Following electroactivation and subsequent culture, a total of 1547 cleaved embryos were transferred to seven surrogate mother pigs. Two healthy cloned piglets expressing hTM were born, successfully grew to maturity and produced normal progeny. Immunohistochemical staining of organs from F1 generation pigs demonstrated hTM expression in endothelial cells as well as parenchymal cells. High expression was observed particularly in endothelial cells of kidney and liver. Aortic endothelial cells from cloned pigs were found to express hTM levels similar to human umbilical vein endothelial cells (HUVEC) and to make it possible to convert protein C into activated protein C. The blockade of human endothelial cell protein C receptor (hEPCR) significantly reduced APC production in HUVEC, but not in hTM-PAEC. Although no bleeding tendency was observed in hTM-cloned pigs, activated partial thromboplastin time (APTT) was slightly prolonged and soluble hTM was detected in pig plasma. hTM was expressed in platelets and mononuclear cells, but not in RBC. Cloned pigs expressing hTM in endothelial cells at a comparable level to HUVEC were produced. As complete suppression of antigen-antibody reaction in the graft is essential for accurate assessment of transgene related to coagulation control, production of genetically engineered pigs expressing hTM and complement regulatory protein based on galactosyltransferase knockout is desired.

Islet Xenotransplantation Using Gal-Deficient Neonatal Donors Improves Engraftment and Function

Significant deficiencies in understanding of xenospecific immunity have impeded the success of preclinical trials in xenoislet transplantation. Although galactose-α1,3-galactose, the gal epitope, has emerged as the principal target of rejection in pig-to-primate models of solid organ transplant, the importance of gal-specific immunity in islet xenotransplant models has yet to be clearly demonstrated. Here, we directly compare the immunogenicity, survival and function of neonatal porcine islets (NPIs) from gal-expressing wild-type (WT) or gal-deficient galactosyl transferase knockout (GTKO) donors. Paired diabetic rhesus macaques were transplanted with either WT (n = 5) or GTKO (n = 5) NPIs. Recipient blood glucose, transaminase and serum xenoantibody levels were used to monitor response to transplant. Four of five GTKO versus one of five WT recipients achieved insulin-independent normoglycemia; transplantation of WT islets resulted in significantly greater transaminitis. The WT NPIs were more susceptible to antibody and complement binding and destruction in vitro. Our results confirm that gal is an important variable in xenoislet transplantation. The GTKO NPI recipients have improved rates of normoglycemia, likely due to decreased susceptibility of xenografts to innate immunity mediated by complement and preformed xenoantibody. Therefore, the use of GTKO donors is an important step toward improved consistency and interpretability of results in future xenoislet studies.

Xenotransplantation of Galactosyl-Transferase Knockout, CD55, CD59, CD39, and Fucosyl-Transferase Transgenic Pig Kidneys Into Baboons

Galactosyl-transferase knockout (GT-KO) pigs represent the latest major progress to reduce immune reactions in xenotransplantation. However, their organs are still subject to rapid humoral rejection involving complement activation requiring the ongoing development of further genetic modifications in the pig. In a pig-to-baboon renal transplantation setting, we have used donor pigs that are not only GT-KO, but also transgenic for human CD55 (hCD55), hCD59, hCD39, and fucosyl-transferase (hHT). We studied kidney xenograft survival, physiological and immunologic parameters, xenogeneic rejection characteristics, as well as viral transmission aspects among two groups of baboons: control animals ( n = 2), versus those ( n = 4) treated with a cocktail of cyclophosphamide, tacrolimus, mycophenolate mofetil, steroids, and a recombinant human C1 inhibitor. Whereas control animals showed clear acute humoral rejection at around day 4, the treated animals showed moderately improved graft survival with rejection at around 2 weeks posttransplantation. Biopsies showed signs of acute vascular rejection (interstitial hemorrhage, glomerular thrombi, and acute tubular necrosis) as well as immunoglobulin (Ig)M and complement deposition in the glomerular and peritubular capillaries. The low level of preformed non-Gal-α1.3Gal IgM detected prior to transplantation increased at 6 days posttransplantation, whereas induced IgG appeared after day 6. No porcine endogenous retrovirus (PERV) transmission was detected in any transplanted baboon. Thus, surprisingly, organs from the GT-KO, hCD55, hCD59, hCD39, and hHT transgenic donors did not appear to convey significant protection against baboon anti-pig antibodies and complement activation, which obviously continue to be significant factors under a suboptimal immunosuppression regimen. The association, timing, and doses of immunosuppressive drugs remain critical. They will have to be optimized to achieve longer graft survivals.

Development and characterization of an acellular porcine cartilage bone matrix for use in tissue engineering

The aim of this study was to develop a technique to decellularize a porcine cartilage bone construct with view to using this as a biological scaffold for cartilage substitution. The decellularization protocol applied freeze/thaw cycles; this was followed by cyclic incubation in hypotonic tris buffer and 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer plus protease inhibitors. Nucleases (RNase and DNase) were used to digest nucleic acids followed by disinfection using 0.1% (v/v) peracetic acid. Histological analysis confirmed the absence of visible cells within the decellularized tissue. DNA analysis revealed the near-complete removal of genomic DNA from the decellularized tissues. The decellularization process had minimal effect on the collagen content of the cartilage. However, there was a significant reduction in the glycosaminoglycan content in the decellularized tissues. There was no evidence of the expression of the major xenogeneic epitope, galactose-α-1,3-galactose. Biomechanical indentation testing of decellularized tissues showed a significant change in comparison to the fresh cartilage. This was presumed to be caused by the reduction in the glycosaminoglycan content. Biocompatibility of the acellular scaffold was determined using contact cytotoxicity assays and a galactosyltransferase knockout mouse model. Decellularized porcine cartilage tissue was found to exhibit favorable compatibility in both in vitro and in vivo tests. In conclusion, this study has generated data on the production of an acellular cartilage bone matrix scaffold for use in osteochondral defect repair. To our knowledge, this is the first study that has successfully removed whole cells and α-gal from xenogeneic cartilage and bone tissue.

New transgenic pigs for xenotransplantation, part 2: Strategies to overcome cellular rejection

The successful generation of α-1,3 galactosyltransferase knockout (αGal-KO) pigs and pigs expressing human complement regulators was a crucial step forward in xenotransplantation research. These genetic modifications helped to control hyperacute rejection, but vascular and cellular mechanisms still represent a major hurdle for pig-to-primate xenotransplantation. Coagulation incompatibilities between human blood and porcine endothelium lead to thrombotic microangiopathy, which could be observed as a key rejection mechanism in pig-to-primate transplantation using αGal-KO pigs as organ donors. Circulating human thrombin is bound by porcine thrombomodulin, but the resulting heterodimer fails to activate protein C, which inhibits the coagulation cascade. We established a vector for endothelial-specific expression of human thrombomodulin (hTM) in αGal-KOxCD46 pigs. The hTM construct was combined with a floxed blasticidin resistance cassette and transfected mixed cell populations were used as donors for somatic cell nuclear transfer (SCNT). Two hundred and ninety-eight reconstructed embryos were transferred to four recipients, resulting in one pregnancy, which lead to the birth of three living piglets. All of them showed strong endothelial hTM expression. In contrast to vascularised organs, pancreatic islets are not subject to hyperacute rejection, but to cellular rejection, mainly based on T-cell stimulation as islets of mature pigs express αGal-epitopes only at a very low level and are vascularized by recipient capillaries after transplantation. Human CTLA4-Ig is a potent inhibitor of T-cell activation, excelled by its derivate LEA29Y which has higher binding affinity to the costimulatory B7 receptors. We have established the coding sequence of LEA29Y under the transcriptional control of a regulatory fragment of the porcine insulin gene. The construct also contained a floxed neomycin resistance cassette. Porcine fetal fibroblasts were transfected, followed by selection under geneticin for 7 days. Stably transfected cell clones were pooled and used as donor cells for SCNT. Transfer of 216 reconstructed embryos to three recipients resulted in two pregnancies with nine born piglets, including two stillbirths. Seven piglets were shown to be transgenic. Immunohistochemistry of the pancreata of two transgenic founders showed high expression levels of LEA29Y at an age of 3 months.

L-Rhamnose Antigen: A Promising Alternative to α-Gal for Cancer Immunotherapies

The targeting of autologous vaccines toward antigen presenting cells (APCs) via the in vivo complexation between anti α-Gal (anti-Gal) antibodies and α-Gal antigens presents a promising cancer immunotherapy with enhanced immunogenicity. This strategy takes advantage of the ubiquitous anti-Gal antibody in human serum. In contrast to the α-Gal epitope, the recent identification of high titers of anti-l-rhamnose (anti-Rha) antibodies in humans reveals a new approach toward immunotherapy employing l-rhamnose (Rha) monosaccharides. In order to evaluate this simple antigen in preclinical applications, we have synthesized Rha-conjugated immunogens and successfully induced high titers of anti-Rha antibodies in wildtype mice. Moreover, our studies demonstrate for the first time that wildtype mice could replace α1,3galactosyltransferase knockout (α1,3GT KO) mice in such antigen/antibody-mediated vaccine design when developing cancer immunotherapies.

Investigation of the Regenerative Capacity of an Acellular Porcine Medial Meniscus for Tissue Engineering Applications

Previously, we have described the development of an acellular porcine meniscal scaffold. The aims of this study were to determine the immunocompatibility of the scaffold and capacity for cellular attachment and infiltration to gain insight into its potential for meniscal repair and replacement. Porcine menisci were decellularized by exposing the tissue to freeze-thaw cycles, incubation in hypotonic tris buffer, 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer plus protease inhibitors, nucleases, hypertonic buffer followed by disinfection using 0.1% (v/v) peracetic, and final washing in phosphate-buffered saline. In vivo immunocompatibility was assessed after implantation of the acellular meniscal scaffold subcutaneously into galactosyltransferase knockout mice for 3 months in comparison to fresh and acellular tissue treated with α-galactosidase (negative control). The cellular infiltrates in the explants were assessed by histology and characterized using monoclonal antibodies against: CD3, CD4, CD34, F4/80, and C3c. Static culture was used to assess the potential of acellular porcine meniscal scaffold to support the attachment and infiltration of primary human dermal fibroblasts and primary porcine meniscal cells in vitro. The explants were surrounded by capsules that were more pronounced for the fresh meniscal tissue compared to the acellular tissues. Cellular infiltrates compromised mononuclear phagocytes, CD34-positive cells, and nonlabeled fibroblastic cells. T-lymphocytes were sparse in all explanted tissue types and there was no evidence of C3c deposition. The analysis revealed an absence of a specific immune response to all of the implanted tissues. Acellular porcine meniscus was shown to be capable of supporting the attachment and infiltration of primary human fibroblasts and primary porcine meniscal cells. In conclusion, acellular porcine meniscal tissue exhibits excellent immunocompatibility and potential for cellular regeneration in the longer term.

Increased Immunogenicity of Tumor-Associated Antigen, Mucin 1, Engineered to Express α-Gal Epitopes: A Novel Approach to Immunotherapy in Pancreatic Cancer

Mucin 1 (MUC1), a bound mucin glycoprotein, is overexpressed and aberrantly glycosylated in >80% of human ductal pancreatic carcinoma. Evidence suggests that MUC1 can be used as a tumor marker and is a potential target for immunotherapy of pancreatic cancer. However, vaccination with MUC1 peptides fails to stimulate the immune response against cancer cells because immunity toward tumor-associated antigens (TAA), including MUC1, in cancer patients is relatively weak, and the presentation of these TAAs to the immune system is poor due to their low immunogenicity. We investigated whether vaccination with immunogenetically enhanced MUC1 (by expressing alpha-gal epitopes; Galalpha1-3Galbeta1-4GlcNAc-R) can elicit effective antibody production for MUC1 itself as well as certain TAAs derived from pancreatic cancer cells and induced tumor-specific T-cell responses. We also used alpha1,3galactosyltransferase (alpha1,3GT) knockout mice that were preimmunized with pig kidney and transplanted with B16F10 melanoma cells transfected with MUC1 expression vector. Vaccination of these mice with alpha-gal MUC1 resulted in marked inhibition of tumor growth and significant improvement of overall survival time compared with mice vaccinated with MUC1 alone (P = 0.003). Furthermore, vaccination with pancreatic cancer cells expressing alpha-gal epitopes induced immune responses against not only differentiated cancer cells but also cancer stem cells. The results suggested that vaccination using cells engineered to express alpha-gal epitopes is a novel strategy for treatment of pancreatic cancer.

Increased immunogenicity of HIV-1 p24 and gp120 following immunization with gp120/p24 fusion protein vaccine expressing α-gal epitopes

Developing an effective HIV-1 vaccine will require strategies to enhance antigen presentation to the immune system. In a previous study we demonstrated a marked increase in immunogenicity of the highly glycosylated HIV-1 gp120 protein following enzymatic addition of α-gal epitopes to the carbohydrate chains. In the present study we determined whether gp120 αgal can also serve as an effective platform for targeting other HIV-1 proteins to APC and thus increase immunogenicity of both proteins. For this purpose we produced a recombinant fusion protein between gp120 and the HIV-1 matrix p24 protein (gp120/p24). Multiple α-gal epitopes were synthesized enzymatically on the gp120 portion of the fusion protein to generate a gp120 αgal/p24 vaccine. Immune responses to gp120 αgal/p24 compared to gp120/p24 vaccine lacking α-gal epitopes were evaluated in α1,3galactosyltransferase knockout (KO) mice. These mice lack α-gal epitopes and, therefore, are capable of producing the anti-Gal antibody. T cell responses to p24, as assessed by ELISPOT and by CD8+ T cells intracellular staining assays for IFNγ, was on average 12- and 10-fold higher, respectively, in gp120 αgal/p24 immunized mice than in mice immunized with gp120/p24. In addition, cellular and humoral immune responses against gp120 were higher by 10–30-fold in mice immunized with gp120 αgal/p24 than in gp120/p24 immunized mice. Our data suggest that the α-gal epitopes on the gp120 portion of the fusion protein can significantly augment the immunogenicity of gp120, as well as that of the fused viral protein which lacks α-gal epitopes. This strategy of anti-Gal mediated targeting to APC may be used for production of effective HIV-1 vaccines comprised of various viral proteins fused to gp120.

Accelerated healing of skin burns by anti-Gal/α-gal liposomes interaction

Topical application of α-gal liposomes on burns results in rapid local recruitment of neutrophils and macrophages. Recruited macrophages are pivotal for healing of burns because they secrete cytokines/growth factors that induce epidermis regeneration and tissue repair. α-Gal liposomes have glycolipids with α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R) which bind anti-Gal, the most abundant natural antibody in humans constituting ∼1% of immunoglobulins. Interaction of α-gal liposomes with anti-Gal within the fluid film formed on burns, activates complement and generates chemotactic complement cleavage peptides which effectively recruit neutrophils and macrophages. Anti-Gal IgG coating α-gal liposomes further binds to Fcγ receptors on macrophages and activates them to secrete cytokines/growth factors. Efficacy of α-gal liposomes treatment in accelerating burn healing is demonstrated in the experimental model of α1,3galactosyltransferase knockout mice. These mice are the only available nonprimate mammals that can produce anti-Gal in titers similar to those in humans. Pairs of burns in mice were covered either with a spot bandage coated with 10 mg α-gal liposomes, or with a control spot bandage coated with saline. On Day 3 post-treatment, the α-gal liposomes treated burns contained ∼5-fold as many neutrophils as control burns, whereas macrophages were found only in α-gal liposomes treated burns. On Day 6, 50–100% of the surface area of α-gal liposomes treated burns were covered with regenerating epidermis (re-epithelialization), whereas almost no epidermis was found in control burns. The extensive recruitment of macrophages by anti-Gal/α-gal liposomes interaction was further demonstrated in vivo with polyvinyl alcohol (PVA) sponge discs containing α-gal liposomes, implanted subcutaneously. Since anti-Gal is abundant in all humans, it is suggested that treatment with α-gal liposomes will be effective also in patients with burns and other skin wounds.

Immune responses to α1,3 galactosyltransferase knockout pigs

To summarize the current knowledge of the immune response generated against xenografts stemming from alpha1,3-galactosyltransferase knockout (GalT-KO) pigs. In particular, we will address the nature of potentially remaining Gal epitopes, the role of non-Gal xenoantigens, and the cellular response directed against GalT-KO tissues. New findings support the view that porcine cells do not express isoglobotrihexosylceramide 3, and GalT-KO pigs, if at all, express negligible levels of Gal. The anti-non-Gal antibody response to GalT-KO cells allowed the identification of several potentially relevant porcine xenoantigens, mainly carbohydrates. Coculture of wildtype pig aortic endothelial cells but not of GalT-KO pig aortic endothelial cells with whole human blood induces the secretion of porcine and human cytokines and the upregulation of E-selectin; in contrast, the transmigration of human leukocytes across porcine endothelium is not regulated by Gal. New immunological problems are arising after the elimination of Gal by the generation of GalT-KO pigs; these include non-Gal antibodies and the identification of their elusive antigens, as well as cellular components of the immune system, including neutrophils, macrophages, natural killer cells, and T cells.

Mechanism for increased immunogenicity of vaccines that form in vivo immune complexes with the natural anti-Gal antibody

Anti-Gal constitutes ∼1% of circulating IgG in humans and interacts specifically with α-gal epitopes. We reported previously that expression of α-gal epitopes on HIV gp120 and influenza virus vaccines increases immunogenicity by ∼100-fold. We hypothesize that immunogenicity of any microbial vaccine can be markedly increased by linked α-gal epitopes due to in vivo formation of immune complexes with anti-Gal and the effective internalization of such immune complexes by APC, via Fc/FcγR interaction. The increased transport to lymph nodes and processing of anti-Gal complexed vaccines internalized by APC, results in effective activation of vaccine specific CD4 + and CD8 + T cells, and high cellular and humoral immune response. This universal mechanism for anti-Gal mediated increased immunogenicity is demonstrated in α1,3galactosyltransferase knockout mice with ovalbumin as a model vaccine.

40 years of heart transplantation and the DFG‐Transregio Research Group Xenotransplantation

40 years of heart transplantation and the DFG‐Transregio Research Group Xenotransplantation

Application of cyclophosphamide-induced tolerance in α1,3-galactosyltransferase knockout mice presensitized with Galα1-3Galβ-4-GlcNAc antigens

Hyperacute rejection (HAR) mediated by the natural antibody (nAb) against Gal alpha 1-3Gal beta-4-GlcNAc (alpha Gal) is the major obstacle in xenogeneic organ transplantation. Previously, we reported the acceptance of donor heart grafts in anti-alpha Gal nAb-producing galactosyltransferase knockout (GalT KO) mice after cyclophosphamide (CP)-induced tolerance conditioning. In the present study, we applied our tolerance induction conditioning in presensitized recipient mice. GalT KO (alpha Gal(-/-), H-2(b/d)) recipient mice were presensitized with alpha Gal(+) rabbit red blood cells (RRBCs). Presensitized or nonsensitized recipient mice were treated with CP-induced tolerance conditioning, consisting of AKR (alpha Gal(+/+), H-2(k)) spleen cells (SC), CP, busulfan (BU), and AKR bone marrow cells (BMC). We assessed the survival of donor hearts and skin grafts and analyzed the production of anti-alpha Gal Abs by flow cytometry. Donor mixed chimerism was achieved in the presensitized GalT KO mice treated with CP-induced tolerance conditioning. In parallel with the disappearance of anti-alpha Gal Abs, permanent acceptance of donor heart grafts and skin grafts was observed in presensitized and GalT KO mice treated with CP-induced tolerance conditioning. Both B-cell and T-cell tolerance was achieved in the presence of a higher titer of anti-alpha Gal Abs after treatment with CP-induced tolerance conditioning.

Allogeneic Melanoma Vaccine Expressing αGal Epitopes Induces Antitumor Immunity to Autologous Antigens in Mice Without Signs of Toxicity

Owing to the absence of alphaGal epitopes in human cells and constant stimulation of the immune system by the symbiotic bacterial flora, humans develop high titers of natural antibodies against these epitopes. It has been demonstrated that syngeneic whole cell vaccines modified to express alphaGal epitopes could be used to generate a potent anticancer vaccine. In this study, we tested whether allogeneic whole cell cancer vaccines modified to express alphaGal epitopes would be effective for the treatment of murine melanoma. The alpha(1,3)galactosyltransferase (alphaGT) knockout mice (H-2) with preexisting subcutaneous and pulmonary tumors [alphaGal B16, H-2] received therapeutic vaccinations with S91M3alphaGal (H-2) whole cell allogeneic vaccines. These mice had better survival and reduced pulmonary metastasis burden compared with control mice treated with S91M3 vaccine cells. Vaccination with S91M3alphaGal-induced cytotoxic CD8 T cells recognizing the syngeneic alphaGal B16 tumors measured by adoptive transfer to recipients bearing pulmonary metastases. The presence of allo-antigens did not dominate the induction of immunity to "cryptic" tumor antigens and had helped in the generation of a more efficient vaccine to treat preexisting tumors when compared with classic autologous vaccines. Vaccination with allogeneic alphaGal vaccines did not induce signs of toxicity including changes in weight, hematology, chemistry, and histopathology of major perfused organs or autoimmunity in long-term murine models for breast, lung, and melanoma. This study established the safety and efficacy data of allogeneic alphaGal whole cell vaccines and constituted the basis for the initiation of human clinical trials to treat human malignancies.

QS2. Administration of Clodronate Confers Added Protection Against HALR

Introduction: Performance of galactosyl-transferase knockout (GalTKO) pig lungs perfused with human blood is inconsistent and inferior to that of other GalTKO organs tested in other experimental systems. The purpose of this study was to determine whether resident pulmonary intravascular macrophages (PIMs), which have been previously shown to contribute importantly to hyperacute lung rejection of wild-type pigs, are the primary cause of GalTKO lung xenograft dysfunction. Methods: Heparinized fresh human blood was used to perfuse GalTKO pig lungs that had been previously treated with clodronate to deplete PIMs (N=3).

Immunogenicity of Influenza Virus Vaccine Is Increased by Anti-Gal-Mediated Targeting to Antigen-Presenting Cells

This study describes a method for increasing the immunogenicity of influenza virus vaccines by exploiting the natural anti-Gal antibody to effectively target vaccines to antigen-presenting cells (APC). This method is based on enzymatic engineering of carbohydrate chains on virus envelope hemagglutinin to carry the alpha-Gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R). This epitope interacts with anti-Gal, the most abundant antibody in humans (1% of immunoglobulins). Influenza virus vaccine expressing alpha-Gal epitopes is opsonized in situ by anti-Gal immunoglobulin G. The Fc portion of opsonizing anti-Gal interacts with Fc gamma receptors on APC and induces effective uptake of the vaccine virus by APC. APC internalizes the opsonized virus to transport it to draining lymph nodes for stimulation of influenza virus-specific T cells, thereby eliciting a protective immune response. The efficacy of such an influenza vaccine was demonstrated in alpha 1,3galactosyltransferase (alpha 1,3GT) knockout mice, which produce anti-Gal, using the influenza virus strain A/Puerto Rico/8/34-H1N1 (PR8). Synthesis of alpha-Gal epitopes on carbohydrate chains of PR8 virus (PR8(alpha gal)) was catalyzed by recombinant alpha1,3GT, the glycosylation enzyme that synthesizes alpha-Gal epitopes in cells of nonprimate mammals. Mice immunized with PR8(alpha gal) displayed much higher numbers of PR8-specific CD8(+) and CD4(+) T cells (determined by intracellular cytokine staining and enzyme-linked immunospot assay) and produced anti-PR8 antibodies with much higher titers than mice immunized with PR8 lacking alpha-Gal epitopes. Mice immunized with PR8(alpha gal) also displayed a much higher level of protection than PR8 immunized mice after being challenged with lethal doses of live PR8 virus. We suggest that a similar method for increasing immunogenicity may be applicable to avian influenza vaccines.

Xenograft-like destruction of tumors and their conversion into vaccines by intratumoral injection of α-gal glycolipids

3074 Background: We describe a novel immunotherapy exploiting the natural anti-Gal antibody to destroy tumors and convert them into endogenous vaccines. Anti-Gal constitutes 1% of immunoglobulins in humans and interacts specifically with a-gal epitopes (Gala1- 3Galβ1–4GlcNAc-R) on glycolipids and glycoproteins. a-Gal epitopes are abundant in nonprimate mammals. Binding of anti-Gal to a-gal epitopes on pig cells mediates xenograft rejection. We inject glycolipids carrying a-gal epitopes (a-gal glycolipids) into solid tumors. Methods: a-Gal glycolipids extracted from rabbit RBC have carbohydrate chains capped with a-gal epitopes. Efficacy of treatment was studied in a1,3galactosyltransferase knockout mice bearing B16 melanoma. These mice are unique since they lack a-gal epitopes and can produce anti- Gal, like humans. Also B16 cells lack a-gal epitopes. B16 lesions (∼5 mm) were injected with 1mg a-gal glycolipids. Results: Intratumoral injection of a-gal glycolipids results in local inflammation mediated by anti-Gal binding to the ∼2x1016 a-gal epitopes on these glycolipids, activation of complement and generation of chemotactic factors. a-Gal glycolipids insert spontaneously into tumor cell membranes. Binding of anti-Gal to a-gal epitopes on such tumor cells induces lesion destruction, similar to xenograft rejection. Anti-Gal further opsonizes tumor cells for effective uptake by inflammation recruited dendritic cells (DC), via Fcγ receptors of these APC. The DC transport internalized tumor Ags to draining lymph nodes and present tumor Ag peptides, thus activating tumor specific T cells and eliciting an immune response against micrometastases. Conclusions: Injected a-gal glycolipids effectively destroy lesions and convert them into vaccines. This treatment may be even more effective in humans, since complement activity is much higher than in mice. This treatment may also be considered as neo-adjuvant immunotherapy for converting primary tumor into autologous tumor vaccine that elicits a protective immune response against micrometastases, during the period preceding resection of the tumor. No significant financial relationships to disclose.