Site-specific characterization of envelope protein N-glycosylation on Sanofi Pasteur's tetravalent CYD dengue vaccine.

Recently, several virus studies have shown that protein glycosylation play a fundamental role in the virus-host cell interaction. Glycosylation characterization of the envelope proteins in both insect and mammalian cell-derived dengue virus (DENV) has established that two potential glycosylation residues, the asparagine 67 and 153 can potentially be glycosylated. Moreover, it appears that the glycosylation of these two residues can influence dramatically the virus production and the infection spreading in either mosquito or mammalian cells. The Sanofi Pasteur tetravalent dengue vaccine (CYD) consists of four chimeric viruses produced in mammalian vero cells. As DENV, the CYDs are able to infect human monocyte-derived dendritic cells in vitro via C-type lectins cell-surface molecules. Despite the importance of this interaction, the specific glycosylation pattern of the DENV has not been clearly documented so far. In this paper, we investigated the structure of the N-linked glycans in the four CYD serotypes. Using MALDI-TOF analysis, the N-linked glycans of CYDs were found to be a mix of high-mannose, hybrid and complex glycans. Site-specific N-glycosylation analysis of CYDs using nanoLC-ESI-MS/MS demonstrates that both asparagine residues 67 and 153 are glycosylated. Predominant glycoforms at asparagine 67 are high mannose-type structures while mainly complex- and hybrid-type structures are detected at asparagine 153. In vitro studies have shown that the immunological consequences of infection by the CYD dengue viruses 1-4 versus the wild type parents are comparable in human monocyte-derived dendritic cells. Our E-protein glycan characterizations of CYD are consistent with those observations from the wild type parents and thus support in vitro studies. In addition, these data provide new insights for the role of glycans in the dengue virus-host cell interactions.

Characterization of surfactants in an oil-in-water emulsion-based vaccine adjuvant using MS and HPLC-MS: structural analysis and quantification.

Mass spectrometry (MS) and high performance liquid chromatography coupled to mass spectrometry (HPLC-MS) techniques were developed to characterize two surfactants, cetheareth-12 and sorbitan oleate, used to manufacture AF03, an emulsified oil-in-water (O/W) adjuvant. MS was first used to characterize the chemical structure and determine the composition of the two surfactants. The two surfactants appeared as complex products, in particular with respect to the nature of the fatty alcohols and the distribution of the number of ethylene oxides in cetheareth-12, and with respect to the different sorbitan-bound fatty acids (oleic, linoleic and palmitic acids) in sorbitan oleate. Subsequently, once the ions of interest were determined and selected, HPLC-MS was developed and optimized to quantify and to "quality control" the two surfactants as raw materials and as ingredients in the final O/W emulsion bulk and filled products.

The teichoic acid (C-polysaccharide) synthesized by Streptococcus pneumoniae serotype 5 has a specific structure.

The teichoic acid synthesized by Streptococcus pneumoniae serotype 5, also known as pneumococcal common antigen (C-polysaccharide), was purified. On the basis of compositional analysis, HPAEC-PAD analysis, MALDI-TOF mass spectrometry and NMR spectroscopy, made on the native polysaccharide and on the dephosphorylated repeating unit, the following structure is proposed: [structure: see text]. This C-polysaccharide (C-PS), differs from those previously described by the replacement of Glc by Gal in its repeating unit structure.

HIV-1 gp41 and gp160 are hyperthermostable proteins in a mesophilic environment. Characterization of gp41 mutants.

HIV gp41(24-157) unfolds cooperatively over the pH range of 1.0-4.0 with T(m) values of > 100 degrees C. At pH 2.8, protein unfolding was 80% reversible and the DeltaH(vH)/DeltaH(cal) ratio of 3.7 is indicative of gp41 being trimeric. No evidence for a monomer-trimer equilibrium in the concentration range of 0.3-36 micro m was obtained by DSC and tryptophan fluorescence. Glycosylation of gp41 was found to have only a marginal impact on the thermal stability. Reduction of the disulfide bond or mutation of both cysteine residues had only a marginal impact on protein stability. There was no cooperative unfolding event in the DSC thermogram of gp160 in NaCl/P(i), pH 7.4, over a temperature range of 8-129 degrees C. When the pH was lowered to 5.5-3.4, a single unfolding event at around 120 degrees C was noted, and three unfolding events at 93.3, 106.4 and 111.8 degrees C were observed at pH 2.8. Differences between gp41 and gp160, and hyperthermostable proteins from thermophile organisms are discussed. A series of gp41 mutants containing single, double, triple or quadruple point mutations were analysed by DSC and CD. The impact of mutations on the protein structure, in the context of generating a gp41 based vaccine antigen that resembles a fusion intermediate state, is discussed. A gp41 mutant, in which three hydrophobic amino acids in the gp41 loop were replaced with charged residues, showed an increased solubility at neutral pH.

The use of microcalorimetry to characterize tetanus neurotoxin, pertussis toxin and filamentous haemagglutinin.

Tetanus neurotoxin (TeNT), pertussis toxin (PT) and pertussis filamentous haemagglutinin (FHA) are major virulence factors of Clostridium tetani and Bordetella pertussis, which are the causative agents of tetanus and whooping cough respectively. Inactivated forms of these virulence factors are the protein components of vaccines against these diseases. Here we report microcalorimetric studies to characterize these proteins. The microcalorimetric titration curves of TeNT with micelles of gangliosides GD1b, GT1b and GQ1b were biphasic. For these gangliosides a high-affinity binding site (KD 45-277 nM) can be distinguished from a lower-affinity binding event (KD 666-1190 nM). This is direct evidence for multiple binding sites for gangliosides of the 1b series at TeNT as proposed by Emsley et al. [Emsley, Fotinou, Black, Fairweather, Charles, Watts, Hewitt and Isaacs (2000) J. Biol. Chem. 275, 8889-8894]. In agreement with previous reports, no binding was observed for gangliosides GM1, GM2, GM3 and GD2. The thermal denaturation of TeNT was characterized by two unfolding transitions centred around 57.4 and 62.4 degrees C. The conversion of TeNT into the toxoid form by formaldehyde treatment was accompanied by a large increase in Tm (the midpoint of protein unfolding transition, that is, the temperature at which half the protein is denatured and the other half is still present in its native form). Fetuin and asialofetuin bound to PT with similar affinities (KD 420 and 335 nM respectively). Binding was largely enthalpy-driven and counterbalanced by an unfavourable entropy change, indicating a loss of conformational flexibility. The latter could account for the observed inhibition of ATP binding after binding to fetuin. Furthermore, the molecular limits of mature PT subunit S5 were defined by MS and N-terminal peptide sequencing. The differential-scanning-calorimetry thermogram of FHA shows four well-resolved unfolding transitions, a finding consistent with the sequential denaturation of four structural domains.