Removal of bowel aerobic gram‐negative bacteria is more effective than immunosuppression with cyclophosphamide and steroids to decrease natural α‐Galactosyl IgG antibodies

Abstract

The use of organs from transgenic pigs for xenotransplantation may be associated with the risk of transmission of microorganisms, especially when the transgenic pigs express human proteins influencing complement activation. The porcine endogenous retroviruses (PERVs) are of particular concern as they can infect human cells in vitro. However, it is unknown whether PERVs can infect transplant recipients in vivo and, if so, whether they are pathogenic. It is therefore essential for experimental and clinical xenotransplantation procedures that specific and sensitive screening methods for PERVs are established. We developed Western blot and enzyme-linked immunosorbant assays (ELISA) based on purified PERVs produced by pig and human cells or recombinant viral protein and synthetic peptides corresponding to PERVs' transmembrane envelope protein, respectively. PERV-specific anti-sera generated against purified virus particles, purified viral proteins and synthetic peptides served as positive controls. Both assays were used for screening the sera of healthy blood donors, pregnant women, patients treated with pig tissues, and butchers with extensive contact to living porcine material to detect antibodies against PERV. None of the individuals showed an antibody pattern characteristic for retroviral infections. Some individuals had antibodies reactive against the major capsid protein p27, against smaller viral proteins of the group specific antigen (Gag) in Western blot assays, or against peptides in the ELISA, probably due to cross-reactivity. Here, we present specific and highly sensitive screening methods applicable for future xenotransplantation procedures, but using these methods we found no evidence of PERV-infection among humans potentially at risk.

Induction of Cytolytic Anti-Gal Antibodies in α-1,3-Galactosyltransferase Gene Knockout Mice by Oral Inoculation withEscherichia coliO86:B7 Bacteria

“…This unanticipated finding may simply be due to differences in environmental exposure to strains of E. coli, Salmonella, Serratia, and Interestingly, the E. coli O86:B7 strain was originally isolated from a human infant (30) and is occasionally detected in contaminated ground meat for human consumption (unpublished data from Richard Wilson, E. coli Reference Center, Pennsylvania State University) and thus likely contributes to the development of anti-Gal antibodies in the human population. Studies in baboons have shown significant decreases in anti-Gal antibody production after elimination of endogenous gram-negative enteric bacteria (20), supporting the premise that anti-Gal antibodies are produced in response to bacteria expressing [Gal ␣-1,3-Gal] antigens.…”

Porcine Endogenous Retroviruses in Xenotransplantation—Molecular Aspects

“…In addition, recombinant transmembrane protein rp15E was developed, which corresponds to the central domain of PERV-A p15E without the hydrophobic N-terminal fusion peptide or the C-terminal transmembrane and cytoplasmic domains (amino acids 488–597). Protein from whole virus was obtained following purification of a virus produced by porcine PK-15 cells by ultracentrifugation and pelleting through a sucrose gradient [137]. Recombinant Gag protein, and more precisely, the C-terminal half, is thought to be more antigenic and has been produced using recombinant DNA technology [138].…”

“…An example is the ELISA test for the detection of anti-PERV antibodies developed by Tacke et al [137]. This test is based on synthetic peptides that correspond to specific regions of p15E, which are highly conserved in different retroviruses—the highly conserved immunosuppressive domain and the adjacent highly immunogenic and immunodominant domain.…”

“…To confirm positive results, a Western blot technique was used with recombinant TM protein (rp15E), purified capsid protein (p27 Gag) and purified whole virus produced by HEK293 cells. A minimum of two reactions against different proteins (Gag and Env), were considered necessary to confirm infection, as is the case with verifying HIV infections [137]. This step is intended to eliminate false positive reactions caused by the presence of cross-reacting antibodies.…”

Boosted Rat Natural Xenoantibodies Cross-React with <b><i>Enterococcus faecalis</i></b> by Targeting Melibiose and <smlcap>L</smlcap>-Rhamnose

“…In humans, xenoantibodies comprise IgM and IgG directed against the galactose-α1,3-galactose (αGal) carbohydrate epitope [5], which is expressed in most mammalian species. Anti-αGal antibodies react with various bacteria, including strains of Escherichia coli, Klebsiella and Salmonella [6,7], and drop after antibiotic treatment that removes Gram-negative enteric flora [8]. These antibodies also bind to senescent human erythrocytes and tumor cells [9].…”