Selective Capture and Determination of Receptor-Binding Hemagglutinin in Influenza Vaccine Preparations Using a Coupled Receptor-Binding/RP-HPLC Assay.

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.

Vaccination with Tumor-Ganglioside Glycomimetics Activates a Selective Immunity that Affords Cancer Therapy.

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.

Glycosphingolipid synthesis employing a combination of recombinant glycosyltransferases and an endoglycoceramidase glycosynthase.

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.

The genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, Campylobacter jejuni. Biosynthesis of sialylated ganglioside mimics in the core oligosaccharide.

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.