Avantea

Cancer vaccine is an active immunotherapy approach that aims to stimulate the host's immune responses toward specific targets on cancer cells, to direct their killing. Cancer cells commonly express aberrant cell surface glycosylation that support their immune evasion. Therefore, cancer glycosylation could be used as an efficient target for therapy. Lipid-based glyconanoparticles that express cancer glycosylation could mimic cancer cells and be used as therapeutic cancer vaccines. Here, we describe generation of biomimetic glyconanoparticles cancer vaccine based on porcine red blood cells that express cancer glycosylation containing the dietary nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc).

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.

Background & aims: To be clinically efficient, beta-cell replacement therapies such as pig islet xenotransplantation must ensure sufficient insulin secretion from grafted islets. While protection from host immune reactions is essential for islet engraftment and their subsequent functioning, intrinsic physiological properties of used cells are also a key factor. We have previously shown that islets from transgenic pigs expressing a dipeptidyl peptidase-resistant form of glucagon-like-peptide-1 (GLP-1) and a constitutively activated form of type 3 muscarinic receptor (M3R) in their beta-cells have greatly improved insulin secretory response to glucose stimulation that is otherwise 4-10 times lower than human islets. Our aim in the current study was to use such modified islets to treat insulin-dependent diabetes in a xenotransplantation model. Material & methods: Pancreatic islets were isolated from transgenic and wild-type neonatal piglets and kept in culture in a maturation medium for 8 days. Glucose-stimulated insulin secretion from mature islets was evaluated in vitro during dynamic perifusion experiments. Streptozotocin-induced diabetic immunodeficient NSG mice received 2500 IEQ transgenic or control islets under the left kidney capsule. Blood glucose was monitored weekly and porcine c-peptide was measured monthly to evaluate graft function. Normoglycemic mice had their graft-bearing kidney removed 9 months post-implantation and follow-up continued for hyperglycemia to occur. Sections of graft bearing kidneys and of recipient micee pancreas were stained for insulin and glucagon to evaluate graft maturation and exclude endogenous beta-cell regeneration. Results: Islets isolated from transgenic InsGLP-1M3R piglets secreted 5.5 to 7.5-fold more insulin compared to controls upon stimulation with 15 mM glucose and had G15/G1 stimulation ratio of 3.7 to 7.5 compared to 2.6 for wild type islets. 80% of InsGLP-1M3R islet mice recipients became normoglycemic within 4 weeks vs. 11% of wild-type islet recipients. Porcine c-peptide could be detected in mice blood by the 3rd week post-implantation and gradually increased throughout the 9-month follow-up period with the highest values recorded in recipients of transgenic islets. Graft removal resulted in hyperglycemia in all normoglycemic mice. Immunohistological analysis showed a significant increase of insulin-positive cells within grafted islets whereas no insulin could be detected in the pancreas 9 months following streptozotocin treatment. Conclusion & perspectives: InsGLP-1M3R neonatal pig islets showed significantly improved insulin secretion both in vitro and in vivo. They were found to be more efficient in treating insulin-dependent diabetes in a xenotransplantation immunodeficient model compared to non-modified islets. These islets sourced from a specific pathogen-free herd represent a very promising beta-cell replacement therapy alternative for use in research and in the clinic. Service public de Wallonie, SPW Economie, Emploi, Recherche: Grant ‘‘8318 SPW’’.

Perfusion of convalescent plasma (CP) has demonstrated a potential to improve the pneumonia induced by SARS-CoV-2, but procurement and standardization of CP are barriers to its wide usage. Many monoclonal antibodies (mAbs) have been developed but appear insufficient to neutralize SARS-CoV-2 unless two or three of them are being combined. Therefore, heterologous polyclonal antibodies of animal origin, that have been used for decades to fight against infectious agents might represent a highly efficient alternative to the use of CP or mAbs in COVID-19 by targeting multiple antigen epitopes. However, conventional heterologous polyclonal antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrate epitopes, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the Gal α1,3-galactose (αGal), ultimately forming immune complexes and potentially leading to serum sickness or allergy. To circumvent these drawbacks, we engineered animals lacking the genes coding for the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) and α1,3-galactosyl-transferase (GGTA1) enzymes to produce glyco-humanized polyclonal antibodies (GH-pAb) lacking Neu5Gc and α-Gal epitopes. We found that pig IgG Fc domains fail to interact with human Fc receptors and thereby should confer the safety advantage to avoiding macrophage dependent exacerbated inflammatory responses, a drawback possibly associated with antibody responses against SARS-CoV-2 or to avoiding a possible antibody-dependent enhancement (ADE). Therefore, we immunized CMAH/GGTA1 double knockout (DKO) pigs with the SARS-CoV-2 spike receptor-binding domain (RBD) to elicit neutralizing antibodies. Animals rapidly developed a hyperimmune response with anti-SARS-CoV-2 end-titers binding dilutions over one to a million and end-titers neutralizing dilutions of 1:10,000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV-19, neutralized Spike/angiotensin converting enzyme-2 (ACE-2) interaction at a concentration < 1μg/mL and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. These data and the accumulating safety advantages of using glyco-humanized swine antibodies in humans warranted clinical assessment of XAV-19 to fight against COVID-19.

The northern white rhinoceros (NWR, Ceratotherium simum cottoni) is the most endangered mammal in the world with only two females surviving. Here we adapt existing assisted reproduction techniques (ART) to fertilize Southern White Rhinoceros (SWR) oocytes with NWR spermatozoa. We show that rhinoceros oocytes can be repeatedly recovered from live SWR females by transrectal ovum pick-up, matured, fertilized by intracytoplasmic sperm injection and developed to the blastocyst stage in vitro. Next, we generate hybrid rhinoceros embryos in vitro using gametes of NWR and SWR. We also establish embryonic stem cell lines from the SWR blastocysts. Blastocysts are cryopreserved for later embryo transfer. Our results indicate that ART could be a viable strategy to rescue genes from the iconic, almost extinct, northern white rhinoceros and may also have broader impact if applied with similar success to other endangered large mammalian species.

The purpose of this study was to assess whether perfusion-decellularization technology could be applied to facial grafts. Facial allotransplantation remains an experimental procedure. Regenerative medicine techniques allow fabrication of transplantable organs from an individual's own cells, which are seeded into extracellular matrix (ECM) scaffolds from animal or human organs. Therefore, we hypothesized that ECM scaffolds also can be created from facial subunits. We explored the use of the porcine ear as a clinically relevant face subunit model to develop regenerative medicine-related platforms for facial bioengineering. Porcine ear grafts were decellularized and histologic, immunologic, and cell culture studies done to determine whether scaffolds retained their 3D framework and molecular content; were biocompatible in vitro and in vivo, and triggered an anti-MHC immune response from the host. The cellular compartment of the porcine ear was completely removed except for a few cartilaginous cells, leaving behind an acellular ECM scaffold; this scaffold retained its complex 3D architecture and biochemical components. The framework of the vascular tree was intact at all hierarchical levels and sustained a physiologically relevant blood pressure when implanted in vivo. Scaffolds were biocompatible in vitro and in vivo, and elicited no MHC immune response from the host. Cells from different types remained viable and could even differentiate at the scale of a whole-ear scaffold. Acellular scaffolds were produced from the porcine ear, and may be a valuable platform to treat facial deformities using regenerative medicine approaches.