Bach, J.-M. | bioRxiv Science

bioRxiv2020

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 1,3-galactose (Gal), 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 1,3-galactosyl-transferase (GGTA1) enzymes to produce glyco-humanized polyclonal antibodies (GH-pAb) lacking Neu5Gc and -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 < 1g/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.

immunology↗

bioRxiv2020

Molecular and functional diversity of distinct subpopulations of extracellular vesicles from stressed pancreatic beta cells: implications for autoimmunity

Beta cell failure and apoptosis following islet inflammation have been associated with autoimmune type 1 diabetes pathogenesis. As conveyors of biological active material, extracellular vesicles (EV) act as mediators in communication with immune effectors fostering the idea that EV from inflamed beta cells may contribute to autoimmunity. Evidence accumulates that beta exosomes promote diabetogenic responses, but relative contributions of larger vesicles as well as variations in the composition of the beta cells vesiculome due to environmental changes have not been explored yet. Here, we made side-by-side comparisons of the phenotype and function of apoptotic bodies (AB), microvesicles (MV) and small EV (sEV) isolated from an equal amount of MIN6 beta cells exposed to inflammatory, hypoxic or genotoxic stressors. Under normal conditions, large vesicles represent 93% of the volume, but only 2% of the number of the vesicles. Our data reveal a consistently higher release of AB and sEV and to a lesser extent of MV, exclusively under inflammatory conditions commensurate with a 4-fold increase in the total volume of the vesiculome and enhanced export of immune-stimulatory material including the autoantigen insulin, microRNA, and cytokines. Whilst inflammation does not change the concentration of insulin inside the EV, specific Toll-like receptor-binding microRNA sequences preferentially partition into sEV. Exposure to inflammatory stress engenders drastic increases in the expression of monocyte chemoattractant protein 1 in all EV and of interleukin-27 solely in AB suggesting selective sorting towards EV subspecies. Functional in vitro assays in mouse dendritic cells and macrophages reveal further differences in the aptitude of EV to modulate expression of cytokines and maturation markers. These findings highlight the different quantitative and qualitative imprints of environmental changes in subpopulations of beta EV that may contribute to the spread of inflammation and sustained immune cell recruitment at the inception of the (auto-) immune response. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/003145v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@1bf7c79org.highwire.dtl.DTLVardef@85098dorg.highwire.dtl.DTLVardef@362e33org.highwire.dtl.DTLVardef@11349ab_HPS_FORMAT_FIGEXP M_FIG C_FIG Inflammation stimulates release of a heterogeneous population of beta EV with differential expression of immunogenic substances involved in immune cell recruitment and activation. HighlightsO_LIStress engenders an up to four-fold increase in the volume of the vesiculome and enhanced auto-antigen release C_LIO_LICytokines are selectively sorted into EV subspecies C_LIO_LITLR-binding microRNAs are enriched in sEV C_LIO_LIEV from stressed beta cells promote dendritic and macrophage cell activation C_LI

immunology↗