Immune targeting of (glyco)protein tumor markers has been useful to develop cancer and virus vaccines. However, the ganglioside family of tumor-associated glycolipids remains intractable to vaccine approaches. Here we show that synthetic antigens mimicking the carbohydrate moiety of GD2 or GD3 gangliosides can be used as vaccines to activate a selective humoral and cellular immunity that is therapeutic against several cancers expressing GD2 or GD3. Adoptive transfer of T cells generated after vaccination elicits tumor-infiltrating lymphocytes of the γδ T cell receptor and CD8+ phenotypes; and affords a high therapeutic index. The glycomimetic vaccine principles can be expanded to target the family of tumor gangliosides and other carbohydrates expressed primarily in pathological states.

Influenza vaccine potency is determined by the quantification of immunologically active hemagglutinin capable of eliciting neutralizing antibodies upon immunization. Currently, the single radial immunodiffusion (SRID) method is the standard in vitro potency assay used for lot release of seasonal inactivated influenza vaccines. Despite the proven usage of SRID, significant limitations such as the time-consuming preparation of reagents and limited dynamic range warrant the need for the development of alternative potency assays. Such alternative approaches need to discriminate and quantify relevant hemagglutinin material, provide strain identity, and be independent of strain-specific and seasonal reagents. Herein, we present a proof of concept method that combines the capture of conformationally well-folded hemagglutinin via a sialic acid binding step with the resolving power of reversed-phase high-performance liquid chromatography for strain identity and determination. Details of the protocol for the selective capture of receptor-binding hemagglutinin, its release from the receptor, and its relative determination are presented. This approach was found to provide flexibility for the reagents to be used and was adaptable to varying strain compositions of influenza vaccines. This proof of concept approach was developed as an antibody-independent methodology.

Glycosynthase mutants of Rhodococcus sp. endo-glycoceramidase II efficiently synthesize complex glycosphingolipids. Glycosyl fluoride donors may be assembled via sequential glycosyltransferase-catalysed glycosylation of lactosyl fluoride. Alternatively, lactosyl fluoride may be coupled to sphingosine prior to subsequent glycosylation steps.

Glycobiology has rapidly developed into an important area of research devoted to the understanding and analysis of the interactions that carbohydrates have with other biomolecules. To accompany the rapid development of this field, better techniques for the analysis of the biosynthesis of complex carbohydrates are required. To investigate interactions between proteins and carbohydrates, an adequate supply of carbohydrate ligand must be obtained. Isolation of biologically active carbohydrates is possible, but only in small amounts. Recent efforts in the cloning of glycosyltransferase genes from a variety of sources has opened the possibility of enzymatic synthesis or chemienzymatic synthesis of a larger number of relevant oligosaccharides (1–4, ).

We have compared the lipo-oligosaccharide (LOS) biosynthesis loci from 11 Campylobacter jejuni strains expressing a total of 8 different ganglioside mimics in their LOS outer cores. Based on the organization of the genes, the 11 corresponding loci could be classified into three classes, with one of them being clearly an intermediate evolutionary step between the other two. Comparative genomics and expression of specific glycosyltransferases combined with in vitro activity assays allowed us to identify at least five distinct mechanisms that allow C. jejuni to vary the structure of the LOS outer core as follows: 1) different gene complements; 2) phase variation because of homopolymeric tracts; 3) gene inactivation by the deletion or insertion of a single base (without phase variation); 4) single mutation leading to the inactivation of a glycosyltransferase; and 5) single or multiple mutations leading to "allelic" glycosyltransferases with different acceptor specificities. The differences in the LOS outer core structures expressed by the 11 C. jejuni strains examined can be explained by one or more of the five mechanisms described in this work.

We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen, Campylobacter jejuni OH4384, which has been associated with Guillain-Barre syndrome. We first cloned a gene encoding an alpha-2,3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C.jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C, jejuni OH4384, The LOS biosynthesis locus from C, jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C,jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as accepters. We identified genes encoding a beta-1,4-N-acetylgalactosaminyl-transferase (cgtA), a beta-1,3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to 0-3 of galactose and to 0-8 of a sialic acid that is linked alpha-2,3 to a galactose, The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of (3)J(C,H) correlations across the glycosidic bond. The role of cgtA and csf-II in the synthesis of the GT1a mimic in C,jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two related C,jejuni strains that express shorter ganglioside mimics in their LOS.

We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen,Campylobacter jejuni OH4384, which has been associated with Guillain-Barré syndrome. We first cloned a gene encoding an α-2,3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C. jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C. jejuni OH4384. The LOS biosynthesis locus from C. jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C. jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as acceptors. We identified genes encoding a β-1,4-N-acetylgalactosaminyl-transferase (cgtA), a β-1,3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to O-3 of galactose and to O-8 of a sialic acid that is linked α-2,3- to a galactose. The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of3J(C,H) correlations across the glycosidic bond. The role of cgtA and cst-II in the synthesis of the GT1a mimic in C. jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two relatedC. jejuni strains that express shorter ganglioside mimics in their LOS.

Large-scale enzymatic synthesis of oligosaccharides, which contain terminal N-acetyl-neuraminic acid residues requires large amounts of the sialyltransferase and the corresponding sugar-nucleotide synthetase, which is required for the synthesis of the sugar-nucleotide donor, CMP-Neu5Ac. Using genes cloned from Neisseria meningitides, we constructed a fusion protein that has both CMP-Neu5Ac synthetase and α-2,3-sialyltransferase activities. The fusion protein was produced in high yields (over 1200 U/L, measured using an α-2,3-sialyltransferase assay) in Escherichia coli and functionally pure enzyme could be obtained using a simple protocol. In small-scale enzymatic syntheses, the fusion protein could sialylate various oligosaccharide acceptors (branched and linear) with N-acetyl-neuraminic acid as well as N-glycolyl- and N-propionyl-neuraminic acid in high conversion yield. The fusion protein was also used to produce α-2,3-sialyllactose at the 100 g scale using a sugar nucleotide cycle reaction, starting from lactose, sialic acid, phosphoenolpyruvate, and catalytic amounts of ATP and CMP.

The structure and specificity of the recombinant alpha-2,3-sialyltransferase from Neisseria meninigitidis are reported. This enzyme showed an unusual acceptor specificity in that it could use alpha-terminal and beta-terminal Gal residues as acceptors. In addition (beta1-->4)-linked and (beta1-->3)-linked terminal Gal served as acceptors. These properties distinguish the bacterial enzyme from the more widely investigated mammalian equivalents. The protein was expressed as a membrane-associated protein in Escherichia coli at a level of 750 U/l (approximately 250 mg/l). The protein could be extracted with buffers containing 0.2% Triton X-100 and purified to homogeneity using immobilized-metal-affinity chromatography. Electrospray-ionization mass spectrometry of peptides obtained by cleavage with cyanogen bromide and trypsin confirmed over 95% of the deduced amino acid sequence. When used for enzymatic synthesis in coupled reactions with recombinant CMP-Neu5Ac synthetase, the alpha-2,3-sialyltransferase could sialylate fluorescent derivatives of N-acetyllactosamine with N-acetylneuraminic acid, N-propionylneuraminic acid and N-glycoloylneuraminic acid.