Infection can protect against subsequent disease by induction of both humoral and cellular immunity, but inert protein-based vaccines are not as effective. In this study, we present a new vaccine design, with Ag covalently conjugated to solid core nano-beads of narrowly defined size (0.04–0.05 μm) that localize to dendritic cells (DEC205+ CD40+, CD86+) in draining lymph nodes, inducing high levels of IFN-γ production (CD8 T cells: precursor frequencies 1/5000 to 1/1000) and high Ab titers in mice. Conjugation of Ag to these nano-beads induced responses that were significantly higher (2- to 10-fold) than those elicited by other bead sizes, and higher than a range of currently used adjuvants (alum, QuilA, monophosphoryl lipid A). Responses were comparable to CFA/IFA immunization for Abs and ex vivo peptide-pulsed dendritic cell immunization for CD8 T cells. A single dose of Ag-conjugated beads protected mice from tumors in two different model challenges and caused rapid clearance of established tumors in mice. Thus, a range of Ags conjugated to nano-beads was effective as immunogens in both therapeutic and prophylactic scenarios.

A major problem with pig-to-human-tissue xenograft studies is that humans have natural antibodies to pig cells; these antibodies would cause hyperacute rejection if pig tissues were xenografted to humans. Here we show that most of human IgM antibodies present in the serum of healthy donors and reactive with pig cells react with galactose in an (al-3) linkage with galactose-i.e., Gal(al-3)Gal. Absorption studies demonstrated that the antibodies detected the same or similar epitopes on the surface of pig erythrocytes, blood and splenic lymphocytes, and aortic endothelial cells (EC). The antibodies were sensitive to 2-mercaptoethanol (2ME) treatment, did not bind to protein A or G, and were present in the high molecular weight fraction of serum; they are clearly IgM antibodies. Further, the antibodies did not react with human ABO blood group substances and are not related to human blood group A or B, which carry a terminal galactose. The reaction of human serum with pig erythrocytes was specifically inhibited by mono-and disaccharides: D-galactose, melibiose, stachyose, methyl-a-D-galactopyranoside, and D-galactosamine but not by D-glucose or methyl-(3D-galactopyranoside; demonstrating that the reaction is with galactose in an a and not a 3 linkage. A cDNA clone encoding the murine a-1,3-galactosyltransferase (which transfers a terminal galactose residue with an (al-3) linkage to a subterminal galactose) was isolated by polymerase chain reaction (PCR), cloned, and trnsfected into COS cells, which are of Old World monkey origin and, like humans, do not express Gal(al-3)Gal. After transfection, COS cells became strongly reactive with human serum and with IB4 lectin [which reacts only with Gal(al-3)Gal]; this reactivity could be removed by absorption with pig erythrocytes. As most ofthe antibody reacting with pig cells can be removed by absorption with either melibiose or Gal(al-3)Gal+ COS cells, most of these react with Gal(al-3)Gal. These findings provide the basis for genetic manipulation of the pig a-1,3-galactosyltransferase for future transplantation studies.

No abstract

The transplantation of pig organs to humans (xenotransplantation) is now receiving serious consideration because of the shortage of human donors for organ transplants of kidney, liver and heart, and of islet cell transplantation for diabetes. The problem with such xenografts would be hyperacute rejection--mediated by natural antibodies in humans to pig antigens, complement fixation to endothelial cells, and the rapid onset of intravascular coagulation. It is now clear that the major target of the natural IgM and IgG antibodies is the terminal carbohydrate epitope Gal alpha(1,3)Gal, formed by the alpha 1,3galactosyl transferase, which places a terminal galactose residue in an alpha-linkage to another galactose. The alpha 1,3galactosyl transferase in the pig gives rise to very high endothelial cell expression of Gal alpha(1,3)Gal, a ready explanation for the hyperacute rejection of vascularized organs. In addition the parenchuma of liver and kidneys have high levels of Gal alpha-(1,3)Gal. These tissues will all fail in a pig-to-human transplant in what can now be precisely defined in terms of antigen and antibody. We have already made some suggestions for removal of anti-Gal alpha(1,3)Gal antibodies and if the procedure were technically feasible xenotransplantation could be attempted now, especially in patients doomed to a certain death because of the absence of a donor (especially for liver where ex vivo perfusion could be performed). However, the immune system is far from simple, as is shown by the healthy status of mice lacking MHC Class I, Class II or both Class I & II molecules. Perhaps the curtain is about to go up to reveal a new scene! Islets differ from the other tissues and may well not undergo acute antibody-mediated hyperacute rejection--it will be of interest to see how these fare in xenotransplantation models or even in patients. Again, normal individuals do not have anti-islet antibodies; but a proportion of diabetic patients do have such antibodies--whether these will cause hyperacute or acute rejection or are markers of immunity of T-cell type, remains to be seen. Whatever, the area is exciting, is progressing rapidly and, as indicated elsewhere, within a few years we should know whether modified pig tissue can be grafted to some patients. The isolation of the cDNA clone encoding the pig alpha 1,3 galactosyl transferase is an essential first step in the production of a transgenic pig lacking the alpha 1,3Galactosyltransferase and therefore the Gal alpha(1,3)Gal epitope, and such animals could serve as donor for human transplantation.

Mucins are a family of high molecular weight, highly glycosylated glycoproteins found in the apical cell membrane of human epithelial cells from the mammary gland, salivary gland, digestive tract, respiratory tract, kidney, bladder, prostate, uterus and rete testis. Increased synthesis of the core protein and alterations in the carbohydrates attached to these glycoproteins are believed to play important roles in the function and proliferation of tumour cells. Aberrant glycosylation leads not only to the production of novel carbohydrate structures, but also to the exposure of the core peptide. These novel epitopes may be candidates for diagnosis or therapy, by using either synthetic mucin fragments as vaccines, or monoclonal antibody-based reagents which detect these structures.