High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2.

Heterologous polyclonal antibodies might represent an alternative to the use of convalescent plasma or monoclonal antibodies (mAbs) in coronavirus disease (COVID-19) by targeting multiple antigen epitopes. However, heterologous antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrates, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the α1,3-galactose, potentially leading to serum sickness or allergy. Here, we immunized cytidine monophosphate-N-acetylneuraminic acid hydroxylase and α1,3-galactosyl-transferase (GGTA1) double KO pigs with the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor binding domain to produce glyco-humanized polyclonal neutralizing antibodies lacking Neu5Gc and α1,3-galactose epitopes. 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 interaction at a concentration <1 µg/mL, and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. We also found that pig GH-pAb Fc domains fail to interact with human Fc receptors, thereby avoiding macrophage-dependent exacerbated inflammatory responses and a possible antibody-dependent enhancement. These data and the accumulating safety advantages of using GH-pAbs in humans warrant clinical assessment of XAV-19 against COVID-19.

Motor neuron degeneration, severe myopathy and TDP-43 increase in a transgenic pig model of SOD1-linked familiar ALS.

Amyotrophic Lateral Sclerosis (ALS) is a neural disorder gradually leading to paralysis of the whole body. Alterations in superoxide dismutase SOD1 gene have been linked with several variants of familial ALS. Here, we investigated a transgenic (Tg) cloned swine model expressing the human pathological hSOD1G93A allele. As in patients, these Tg pigs transmitted the disease to the progeny with an autosomal dominant trait and showed ALS onset from about 27 months of age. Post mortem analysis revealed motor neuron (MN) degeneration, gliosis and hSOD1 protein aggregates in brainstem and spinal cord. Severe skeletal muscle pathology including necrosis and inflammation was observed at the end stage, as well. Remarkably, as in human patients, these Tg pigs showed a quite long presymptomatic phase in which gradually increasing amounts of TDP-43 were detected in peripheral blood mononuclear cells. Thus, this transgenic swine model opens the unique opportunity to investigate ALS biomarkers even before disease onset other than testing novel drugs and possible medical devices.

Generation of cattle knockout for galactose-α1,3-galactose and N-glycolylneuraminic acid antigens.

Two well-characterized carbohydrate epitopes are absent in humans but present in other mammals. These are galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc) which are introduced by the activities of two enzymes including α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Gc hydroxylase (encoded by the CMAH gene) that are inactive in humans but present in cattle. Hence, bovine-derived products are antigenic in humans who receive bioprosthetic heart valves (BHVs) or those that suffer from red meat syndrome. Using programmable nucleases, we disrupted (knockout, KO) GGTA1 and CMAH genes encoding for the enzymes that catalyse the synthesis of αGal and Neu5Gc, respectively, in both male and female bovine fibroblasts. The KO in clonally selected fibroblasts was detected by polymerase chain reaction (PCR) and confirmed by Sanger sequencing. Selected fibroblasts colonies were used for somatic cell nuclear transfer (SCNT) to produce cloned embryos that were implanted in surrogate recipient heifers. Fifty-three embryos were implanted in 33 recipients heifers; 3 pregnancies were carried to term and delivered 3 live calves. Primary cell cultures were established from the 3 calves and following molecular analyses confirmed the genetic deletions. FACS analysis showed the double-KO phenotype for both antigens confirming the mutated genotypes. Availability of such cattle double-KO model lacking both αGal and Neu5Gc offers a unique opportunity to study the functionality of BHV manufactured with tissues of potentially lower immunogenicity, as well as a possible new clinical approaches to help patients with red meat allergy syndrome due to the presence of these xenoantigens in the diet.

hCTLA4-Ig transgene expression in keratocytes modulates rejection of corneal xenografts in a pig to non-human primate anterior lamellar keratoplasty model.

BACKGROUND: Human corneal allografting is an established procedure to cure corneal blindness. However, a shortage of human donor corneas as well as compounding economic, cultural, and organizational reasons in many countries limit its widespread use. Artificial corneas as well as porcine corneal xenografts have been considered as possible alternatives. To date, all preclinical studies using de-cellularized pig corneas have shown encouraging graft survival results; however, relatively few studies have been conducted in pig to non-human primate (NHP) models, and particularly using genetically engineered donors. METHODS: In this study, we assessed the potential benefit of using either hCTLA4-Ig transgenic or α1,3-Galactosyl Transferase (GT) Knock-Out (KO) plus transgenic hCD39/hCD55/hCD59/fucosyl-transferase pig lines in an anterior lamellar keratoplasty pig to NHP model. RESULTS: Corneas from transgenic animals expressing hCTLA4-Ig under the transcriptional control of a neuron-specific enolase promoter showed transgene expression in corneal keratocytes of the stroma and expression was maintained after transplantation. Although a first acute rejection episode occurred in all animals during the second week post-keratoplasty, the median final rejection time was 70 days in the hCTLA4-Ig group vs. 21 days in the wild-type (WT) control group. In contrast, no benefit for corneal xenograft survival from the GTKO/transgenic pig line was found. At rejection, cell infiltration in hCTLA4Ig transgenic grafts was mainly composed of macrophages with fewer CD3+ CD4+ and CD79+ cells than in other types of grafts. Anti-donor xenoantibodies increased dramatically between days 9 and 14 post-surgery in all animals. CONCLUSIONS: Local expression of the hCTLA4-Ig transgene dampens rejection of xenogeneic corneal grafts in this pig-to-NHP lamellar keratoplasty model. The hCTLA4-Ig transgene seems to target T-cell responses without impacting humoral responses, the control of which would presumably require additional peripheral immunosuppression.

Changes in glial cell activation and extracellular vesicles production precede the onset of disease symptoms in transgenic hSOD1G93A pigs.

SOD1 gene is associated with progressive motor neuron degeneration in the familiar forms of amyotrophic lateral sclerosis. Although studies on mutant human SOD1 transgenic rodent models have provided important insights into disease pathogenesis, they have not led to the discovery of early biomarkers or effective therapies in human disease. The recent generation of a transgenic swine model expressing the human pathological hSOD1G93A gene, which recapitulates the course of human disease, represents an interesting tool for the identification of early disease mechanisms and diagnostic biomarkers. Here, we analyze the activation state of CNS cells in transgenic pigs during the disease course and investigate whether changes in neuronal and glial cell activation state can be reflected by the amount of extracellular vesicles they release in biological fluids. To assess the activation state of neural cells, we performed a biochemical characterization of neurons and glial cells in the spinal cords of hSOD1G93A pigs during the disease course. Quantification of EVs of CNS cell origin was performed in cerebrospinal fluid and plasma of transgenic pigs at different disease stages by Western blot and peptide microarray analyses. We report an early activation of oligodendrocytes in hSOD1G93A transgenic tissue followed by astrocyte and microglia activation, especially in animals with motor symptoms. At late asymptomatic stage, EV production from astrocytes and microglia is increased in the cerebrospinal fluid, but not in the plasma, of transgenic pigs reflecting donor cell activation in the spinal cord. Estimation of EV production by biochemical analyses is corroborated by direct quantification of neuron- and microglia-derived EVs in the cerebrospinal fluid by a Membrane Sensing Peptide enabled on-chip analysis that provides fast results and low sample consumption. Collectively, our data indicate that alteration in astrocytic EV production precedes the onset of disease symptoms in the hSODG93A swine model, mirroring donor cell activation in the spinal cord, and suggest that EV measurements from the cells first activated in the ALS pig model, i.e. OPCs, may further improve early disease detection.

LIS1, a glyco-humanized swine polyclonal anti-lymphocyte globulin, as a novel induction treatment in solid organ transplantation.

Anti-thymocyte or anti-lymphocyte globulins (ATGs/ALGs) are immunosuppressive drugs used in induction therapies to prevent acute rejection in solid organ transplantation. Because animal-derived, ATGs/ALGs contain highly immunogenic carbohydrate xenoantigens eliciting antibodies that are associated with subclinical inflammatory events, possibly impacting long-term graft survival. Their strong and long-lasting lymphodepleting activity also increases the risk for infections. We investigated here the in vitro and in vivo activity of LIS1, a glyco-humanized ALG (GH-ALG) produced in pigs knocked out for the two major xeno-antigens αGal and Neu5Gc. It differs from other ATGs/ALGs by its mechanism of action excluding antibody-dependent cell-mediated cytotoxicity and being restricted to complement-mediated cytotoxicity, phagocyte-mediated cytotoxicity, apoptosis and antigen masking, resulting in profound inhibition of T-cell alloreactivity in mixed leucocyte reactions. Preclinical evaluation in non-human primates showed that GH-ALG dramatically reduced CD4+ (p=0.0005,***), CD8+ effector T cells (p=0.0002,***) or myeloid cells (p=0.0007,***) but not T-reg (p=0.65, ns) or B cells (p=0.65, ns). Compared with rabbit ATG, GH-ALG induced transient depletion (less than one week) of target T cells in the peripheral blood (<100 lymphocytes/L) but was equivalent in preventing allograft rejection in a skin allograft model. The novel therapeutic modality of GH-ALG might present advantages in induction treatment during organ transplantation by shortening the T-cell depletion period while maintaining adequate immunosuppression and reducing immunogenicity.

Unexpected impairment of INa underpins reentrant arrhythmias in a knock-in swine model of Timothy syndrome.

Timothy syndrome 1 (TS1) is a multi-organ form of long QT syndrome associated with life-threatening cardiac arrhythmias, the organ-level dynamics of which remain unclear. In this study, we developed and characterized a novel porcine model of TS1 carrying the causative p.Gly406Arg mutation in CACNA1C, known to impair CaV1.2 channel inactivation. Our model fully recapitulated the human disease with prolonged QT interval and arrhythmic mortality. Electroanatomical mapping revealed the presence of a functional substrate vulnerable to reentry, stemming from an unforeseen constitutional slowing of cardiac activation. This signature substrate of TS1 was reliably identified using the reentry vulnerability index, which, we further demonstrate, can be used as a benchmark for assessing treatment efficacy, as shown by testing of multiple clinical and preclinical anti-arrhythmic compounds. Notably, in vitro experiments showed that TS1 cardiomyocytes display Ca2+ overload and decreased peak INa current, providing a rationale for the arrhythmogenic slowing of impulse propagation in vivo.

Pregnancy: a cloned horse born to its dam twin.

Several animal species, including sheep, mice, cattle, goats, rabbits, cats, pigs and, more recently, mules have been reproduced by somatic cell cloning, with the offspring being a genetic copy of the animal donor of the nuclear material used for transfer into an enucleated oocyte. Here we use this technology to clone an adult horse and show that it is possible to establish a viable, full-term pregnancy in which the surrogate mother is also the nuclear donor. The cloned offspring is therefore genetically identical to the mare who carried it, challenging the idea that maternal immunological recognition of fetal antigens influences the well-being of the fetus and the outcome of the pregnancy.

Laboratory Production of Equine Embryos.

Assisted reproduction technologies (ART) are well developed in humans and cattle and are gaining momentum also in the equine industry because of the fact that the mare does not respond to superovulation but can donate large numbers of oocytes through ovum pick up (OPU). After collection, the oocytes can be fertilized by intracytoplasmic sperm injection (ICSI) using a variety of stallion semen samples, even of poor quality, and the resulting embryos can establish high pregnancy rates after cryopreservation and transfer. The discoveries that equine oocytes can be held at room temperature without loss of viability and that an increase in vitro maturation time can double the number of embryos produced are fueling the uptake of the OPU technique by several clinics that are shipping oocytes of their client's mares to specialized ICSI laboratories for embryo production and freezing. In this article, we present a retrospective analysis of 10 years of work at Avantea with a special focus on the last 3 years. Based on our data, an average production of 1.7 to 2 embryos per OPU-ICSI procedure can be obtained from warmblood donor mares with a pregnancy rate of 70% and a foaling rate in excess of 50%. OPU-ICSI offers the added value of freezing embryos that allows the development of embryo commercialization worldwide to the benefit of top horse breeders who are endorsing this technology as never before.

High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2.

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.