INSERM UMR 1064 Center of Research in Transplantation and Immunology
ITUN Institut Transplantation Urologie Néphrologie
Immune phenotype and IgG characteristics of Neu5Gc and alpha-1-3-Gal double knock-out pigs
Apolline Salama1,2, Sophie Conchon1, Andrea Perota3, Bernard Martinet1, Jean-Paul Judor1, Gwénaëlle Evanno1,4, Stéphanie Le-Bas Bernardet1, Ludmilla Le Berre1, Jérémy Hervouet1, David Minault1, Jean-Paul Concordet5, Emilie Dugast1, Bernard Vanhove1, Jérôme Abadie6, Nicolas Gaide6, Irina Lagutina3, Roberto Duchi3, Giovanna Lazzari3, Robert Floch4, Odile Duvaux4, Pierre-Louis Toutain7, Mike Maillasson8, David H Sachs9, Olivier Gauthier10, Sophie Brouard1, Emanuele Cozzi11, Gilles Blancho1, Hélène Perreault12, Jean-Marie Bach13, Cesare Galli3,14, Jean-Paul Soulillou1.
1Institut de Transplantation-Urologie-Néphrologie ITUN, INSERM UMR 1064, Centre Hospitalier Universitaire de Nantes, Université de Nantes, Nantes, France; 2Société d'Accélération du Transfert de Technologies Ouest Valorisation, Rennes, France; 3Avantea, Laboratory of Reproductive Technologies, Cremona, Italy; 4Xenothera, Nantes, France; 5INSERM U565, CNRS UMR7196, MNHN USM503, Paris, France; 6AMaROC, ONIRIS Nantes-Atlantic College of Veterinary Medicine and Food Sciences, Nantes, France; 7UMR 1331 INRA/ENVT; Ecole nationale Vétérinaire de Toulouse, Toulouse, France; 8Plateforme IMPACT Biogenouest, CRCNA UMR 892 INSERM / Université de Nantes, Nantes, France; 9Massachusetts General Hospital, Transplantation Biology Research Center, Center for Transplantation Sciences, Boston, MA, United States; 10Centre de Recherche et d'Investigation Préclinique, ONIRIS, Nantes, Italy; 11Padua General Hospital and Consortium for Research in Organ Transplantation, Padua, Italy; 12Chemistry Department, University of Manitoba, Winnipeg, MB, Canada; 13IECM, EA4644 University/ONIRIS USC1383 INRA, ONIRIS, Nantes, France; 14Department of Veterinary Medical Sciences, University of Bologna, Bologna, Italy
Xenotransplantation offers several potential applications, including solid organs, pancreatic islets, acellular tissues such as heart valves and skin, but also molecular reagents such as hyperimmune polyclonal IgGs. Polyclonal immunoglobulins are widely used in patients for several indications but, as an animal product, they induce a humoral response against IgG xeno-antigens. Particularly, preexisting and elicited antibodies against the galactose-α(1,3)-galactose (α1-3 Gal) and the glycolyl form of neuraminic acid Neu5Gc may induce immune complexes-related diseases.
We have obtained double knock-out pigs (DKOP) for the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) and galactosyltransferase1 (GT1), lacking Neu5Gc and α1-3 Gal (abstract IXA 2013 Conchon S. et al, Generation of CMAH -/- piglets on GAL-/- genetic background, Xenotransplantation 2013; 20: 368–371) and we present here some characteristics of their immune phenotype. Using ELISA, IB4 lectin binding and mass spectrometry, we found that Neu5Gc was present on IgGs from wild-type and GT1 KO lines but absent in DKOPs, whereas α1-3 Gal was not found in any of the pig lines tested. Following immunization with the Jurkat human T-cell line, DKOPs mount a stronger response than wild-type or GT1 KO pigs, possibly due to their ligand-orphan SIGLEC status. Protein-A purified hyperimmune IgGs from immunized DKOPs exhibit stronger in vitro complement-dependent cytotoxicity than IgGs from wild-type and GT1 KO control pigs. This result suggests a possible increase in the titer of specific antibodies or a better access of complement to DKO IgG Fc lacking Neu5Gc. However, surface plasmon resonance (SPR) technology showed no difference of interaction and affinity between human C1q molecules and IgGs of the three pig strains. Finally, the same half-life was observed for DKOP IgGs and rabbit IgGs, after an in vivo protocol in baboons.
In summary, our data show that besides being potentially less immunogenic, IgG antibodies from immunized DKOPs are more abundantly produced and more cytotoxic than control IgGs. These immunoglobulins may offer opportunities as improved polyclonal hyperimmune sera.
Société d’Accélération du Transfert de Technologies Ouest Valorisation. European FP7 ‘Translink’ research program (n°603049).
15:30 - 16:30
|Non-Gal Antibodies||Increased late graft loss in kidney recipients with serum sickness disease following anti-thymocyte globulin induction: relation with an anti-Neu5Gc response||Room 109|
15:30 - 17:00
|Genetic Engineering; Methodology||Immune phenotype and IgG characteristics of Neu5Gc and alpha-1-3-Gal double knock-out pigs||Room 109|