Philippe Talaga

Development of a high-performance anion-exchange chromatography with pulsed-amperometric detection based quantification assay for pneumococcal polysaccharides and conjugates

A method, using high-performance anion-exchange chromatography with pulsed-amperometric detection (HPAEC-PAD), has been developed to determine the concentrations of Streptococcus pneumoniae capsular polysaccharides and polysaccharide conjugates used in formulating a conjugate vaccine for the prevention of pneumococcal infections. In an effort to determine optimum hydrolysis conditions for the analysis, pneumococcal polysaccharides were subjected to three different hydrolysis methods: trifluoroacetic acid (TFA) hydrolysis, methanolysis followed by TFA hydrolysis, or hydrofluoric acid (HF) hydrolysis followed by TFA hydrolysis. For quantification purposes, best results were obtained by methanolysis followed by TFA hydrolysis for uronic acid-containing polysaccharides, and by TFA hydrolysis for all the others. For the quantification of all the polysaccharides (from native to conjugated forms), a monosaccharide reference mixture (Rha, Gal and GlcA) hydrolyzed along with the samples can be used as standards for routine analysis. This is much more convenient than to hydrolyze a well-characterized reference polysaccharide (necessary standard only for type 1 capsular polysaccharide). This method is rapid, very sensitive (less than 10 microg of polysaccharide is required), and may replace advantageously the currently used colorimetric assays used to determine polysaccharides content. Moreover, it can be readily adapted for use with other bacterial polysaccharide preparations as well.

Quantitative determination of C-polysaccharide in Streptococcus pneumoniae capsular polysaccharides by use of high-performance anion-exchange chromatography with pulsed amperometric detection

Capsular polysaccharides of Streptococcus pneumoniae are used to formulate polyvalent pneumococcal vaccines. A sensitive method, using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), has been developed to quantify the contamination of pneumococcal capsular polysaccharides (PnPs) with the C-polysaccharide (C-Ps). As this polysaccharide is highly immunogenic, and since anti C-Ps antibodies are not protective, the need to monitor and reduce its level is of uppermost importance. The method is based on the quantification by HPAEC-PAD of ribitol, which is released by a two-step hydrolysis of the PnPs using aqueous hydrofluoric acid (HF) followed by trifluoroacetic acid hydrolysis (TFA). This simple method has been shown to provide both qualitative and quantitative information about the purity of polysaccharide preparations.

Characterization and immunogenicity in mice of recombinant influenza haemagglutinins produced in Leishmania tarentolae

The membrane displayed antigen haemagglutinin (HA) from several influenza strains were expressed in the Leishmania tarentolae system. This non-conventional expression system based on a parasite of lizards, can be readily propagated to high cell density (>10(8)cells/mL) in a simple incubator at 26°C. The genes encoding HA proteins were cloned from six influenza strains, among these being a 2009 A/H1N1 pandemic strain from swine origin, namely A/California/07/09(H1N1). Soluble HA proteins were secreted into the cell culture medium and were easily and successfully purified via a His-Tag domain fused to the proteins. The overall process could be conducted in less than 3 months and resulted in a yield of approximately 1.5-5mg of HA per liter of biofermenter culture after purification. The recombinant HA proteins expressed by L. tarentolae were characterized by dynamic light scattering and were observed to be mostly monomeric. The L. tarentolae recombinant HA proteins were immunogenic in mice at a dose of 10μg when administered twice with an oil-in-water emulsion-based adjuvant. These results suggest that the L. tarentolae expression system may be an alternative to the current egg-based vaccine production.

Genetic and structural characterization of L11 lipooligosaccharide from Neisseria meningitidis serogroup A strains.

The lipooligosaccharide (LOS) of immunotype L11 is unique within serogroup A meningococci. In order to resolve its molecular structure, we conducted LOS genotyping by PCR analysis of genes responsible for α-chain sugar addition (lgtA, -B, -C, -E, -H, and -F) and inner core substituents (lgtG, lpt-3, and lpt-6). For this study, we selected seven strains belonging to subgroup III, a major clonal complex responsible for meningococcal meningitis epidemics in Africa. In addition, we sequenced the homopolymeric tract regions of three phase-variable genes (lgtA, lgtG, and lot-3) to predict gene functionality. The fine structure of the L11 LOS of each strain was determined using composition and glycosyl linkage analyses, NMR, and mass spectrometry. The masses of the dephosphorylated oligosaccharides were consistent with an oligosaccharide composed of two hexoses, one N-acetyl-hexosamine, two heptoses, and one KDO, as proposed previously. The molar composition of LOS showed two glucose residues to be present, in agreement with lgtH sequence prediction. Despite phosphoethanolaminetransferase genes lpt-3 and lpt-6 being present in all seven Neisseria meningitidis strains, phosphoethanolamine (PEtn) was found at both O-3 and O-6 of HepII among the three ST-5 strains, whereas among the four ST-7 strains, only one PEtn was found and located at O-3 of the HepII. The L11 LOS was found to be O-acetylated, as was indicated by the presence of the lot-3 gene being in-frame in all of the seven N. meningitidis strains. To our knowledge, these studies represent the first full genetic and structural characterization of the L11 LOS of N. meningitidis. These investigations also suggest the presence of further regulatory mechanisms affecting LOS structure microheterogeneity in N. meningitidis related to PEtn decoration of the inner core.

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.

Development and validation of high-performance size exclusion chromatography methods to determine molecular size parameters of Haemophilus influenzae type b polysaccharides and conjugates.

Current vaccines against Haemophilus influenzae type b (Hib) consist of the polyribosyl ribitol phosphate (PRP) capsular polysaccharide chemically conjugated to a carrier protein. Among the various biological and physical analyses to be performed on these vaccines, the determination of the molecular size of the polysaccharide preparations throughout the conjugation process is particularly relevant. Comparison of results from high-performance size exclusion chromatography (HPSEC) with those routinely obtained using conventional gel permeation chromatography (CGPC) methods highlights the correlation between the two methods for determining the values of the chromatographic distribution coefficient (KD) of native and activated polysaccharides. The resulting data showed that the KD value is sufficient to characterize these polysaccharides using an HPSEC method. However, additional molecular size parameters (i.e., molar mass and hydrodynamic radius) are necessary for a reliable characterization of the tetanus conjugate (PRP-T), certainly due to the lattice-like structure of the conjugate. In practice, an absolute detection system in HPSEC composed of a low-angle light scattering detector, a viscometer, and a refractive index (RI) detector was used. As demonstrated, these HPSEC methods are rapid, accurate, and reproducible for the polysaccharides and their glycoconjugates and provide a relevant and more informative alternative to the current CGPC methods.

The combined use of analytical tools for exploring tetanus toxin and tetanus toxoid structures

Aldehyde detoxification is a process used to convert toxin into toxoid for vaccine applications. In the case of tetanus toxin (TT), formaldehyde is used to obtain the tetanus toxoid (TTd), which is used either for the tetanus vaccine or as carrier protein in conjugate vaccines. Several studies have already been conducted to better understand the exact mechanism of this detoxification. Those studies led to the identification of a number of formaldehyde-induced modifications on lab scale TTd samples. To obtain greater insights of the changes induced by formaldehyde, we used three industrial TTd batches to identify repeatable modifications in the detoxification process. Our strategy was to combine seven analytical tools to map these changes. Mass spectrometry (MS), colorimetric test and amino acid analysis (AAA) were used to study modifications on amino acids. SDS-PAGE, asymmetric flow field flow fractionation (AF4), fluorescence spectroscopy and circular dichroism (CD) were used to study formaldehyde modifications on the whole protein structure. We identified 41 formaldehyde-induced modifications across the 1315 amino acid primary sequence of TT. Of these, five modifications on lysine residues were repeatable across TTd batches. Changes in protein conformation were also observed using SDS-PAGE, AF4 and CD techniques. Each analytical tool brought a piece of information regarding formaldehyde induced-modifications, and all together, these methods provided a comprehensive overview of the structural changes that occurred with detoxification. These results could be the first step leading to site-directed TT mutagenesis studies that may enable the production of a non-toxic equivalent protein without using formaldehyde.

Determination of molecular size parameters and quantification of polyacrylic acid by high performance size-exclusion chromatography with triple detection

Synthetic polyelectrolytes are a broad class of vaccine adjuvants. Among them, polyacrylic acid (PAA), a polyanionic polymer, is currently evaluated by Sanofi Pasteur. As chain length is considered to be a critical quality attribute for adjuvant properties of PAA, measurement of precise and accurate molecular size parameters is important for these polymers. In the field of synthetic polymer chemistry, methods for determination of molecular size parameters are well defined. Specifically, high performance size-exclusion chromatography (HPSEC) with multi-detection system is a method of choice. This paper describes the development of HPSEC method to well characterize and precisely quantify PAA in different adjuvant formulations. A first set of characterizations were made, with determination of dn/dc coefficient, which enabled the determination of weight- and number-average molecular weight, viscosimetric radius, and intrinsic viscosity. In-depth characterization was also made with branching study through the use of Mark-Houwink parameter determination. The quantification method was also evaluated according to validation method-like criteria: limit of detection and limit of quantification, repeatability, accuracy, and specificity with recombinant surface glycoprotein gB from human cytomegalovirus (CMV-gB) as model antigen.

Bacterial Polysaccharide Vaccines: Analytical Perspectives

Many bacterial pathogens express cell surface polysaccharides which protect the bacterium during the initial stages of infection and hide the cell surface from innate host immune defense mechanisms. In some cases, pre-existing antibodies directed against these cell surface polysaccharides protect against infection: in these cases the purified polysaccharides can be used as prophylactic vaccines. Purified polysaccharide vaccines prepared from capsular polysaccharides (CPSs) present several unique features. The polysaccharides are relatively simple, and, in the absence of accessible animal models, quality control is largely based on physicochemical and immunochemical methods, and dosage is in SI units. As protection is serotype- (or serogroup-) specific, polysaccharide vaccines are often blends of multiple polysaccharides derived from different strains, e.g., 23 in the case of pneumococcal vaccines. Critical quality attributes for these vaccines are polysaccharide identity, polysaccharide molecular size, and safety factors (such as pyrogenicity). As T cell-independent Type II immunogens, these vaccines fail to invoke immunological memory, so that repeat immunization is required. They are poorly immunogenic in infants, the principal risk group for many of these infections, and technologies such as saccharide conjugation to carrier proteins have been developed to protect this group, while polysaccharide vaccines continue to be used in adolescents and adults. In this chapter, we present background on the physical chemical nature of bacterial polysaccharides, their Critical Quality Attributes (CQA’s), and the analytical methodologies applied to their structural and physical characterization as well as to measure their CQA’s.