Synthesis of α-gal epitopes on influenza virus vaccines, by recombinant α1,3galactosyltransferase, enables the formation of immune complexes with the natural anti-Gal antibody

Abstract

Synthesis of the carbohydrate structure Galα1-3Galβ1-4GlcNAc-R (termed the α-gal epitope) on viral glycoproteins is of interest because of the large amounts of natural antibody (anti-Gal) produced in humans against this epitope. The presence of α-gal epitopes on inactivated virus or subviral vaccines is likely to enhance vaccine immunogenicity through in vivo complexing with anti-Gal and the subsequent targeting of the vaccine to Fcγ receptors on antigen presenting cells. Our previous studies have demonstrated the increased in vitro immunogenicity of inactivated influenza virus complexed with the anti-Gal antibody. Here we demonstrate a method for engineering the expression of α-gal epitopes on influenza virus hemagglutinin (HA) by recombinant α1,3galactosyltransferase (rα1,3GT). We further demonstrate the formation of immune complexes between this de novo synthesized epitope and anti-Gal. HA has multiple N-acetyllactosamine structures which serve as excellent acceptors for rα1,3GT. The luminal portion of marmoset α1,3GT cDNA was produced in large amounts in the baculo virus system and isolated by affinity chromatography on nickel-Sepharose columns. rα1,3GT effectively transferred galactose from UDP-Gal to the N-acetyllactosamine residues of HA on the intact virion or to isolated HA molecules. At least 3000 α-gal epitopes were de novo synthesized per virion. The natural anti-Gal antibody bound to these epitopes in ELISA, in western blots and in solution, forming distinct immune complexes. These data suggest that in vivo administration of such vaccines will result in their complexing with anti-Gal, and thus may lead to their increased immunogenicity.