Expression de protéines virales dans Saccharomyces cerevisiae. Processus d'assemmblage des protéines d'enveloppe du virus de l'hépatite B et des protéines gag du virus d'immunodéficience humaine

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Subcellular localization of recombinant truncated Gag precursor proteins of HIV expressed in Saccharomyces cerevisiae

Objective: To determine signals contained in the HIV-1 Gag precursor implicated in protein transport. Design: To study the localization of truncated Gag proteins expressed in Saccharomyces cerevisiae. Methods: Thin-section immunoelectron microscopy studies were performed on S. cerevisiae cells producing myristoylated or non-myristoylated Pr55gag, the core protein (p24) and several truncated Gag proteins. Results: Pr55gag and the carboxy-terminal truncated Gag proteins were myristoylated and localized at the plasma membrane. p24 was localized in the nucleus or perinuclear membrane. However, addition of a myristoyl group to p24 targeted this molecule to the plasma membrane. Conclusions: The myristoylated amino-terminal 214 amino acids are sufficient to target Pr55gag to the plasma membrane. Subcellular signals implicated in protein transport are present in the core p24 polypeptide which may become dominant or accessible in the absence of the amino-myristoyl group.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Impact of the Conjugation Method on the Immunogenicity of Streptococcus pneumoniae Serotype 19F Polysaccharide in Conjugate Vaccines ▿

7vCRM (Pfizer, Inc.) and PHiD-CV (GlaxoSmithKline Biologicals) are two pneumococcal conjugate vaccines licensed for the prevention of invasive pneumococcal disease and acute otitis media caused by the vaccine serotypes of Streptococcus pneumoniae. Neither vaccine contains serotype 19A, but both contain the closely related serotype 19F. No decrease in the incidence of serotype 19A disease has been observed following the introduction of 7vCRM, suggesting that this serotype 19F-containing vaccine provides limited cross-protection against serotype 19A. To investigate the impact that conjugation methods may have on antipolysaccharide immune responses and to determine whether this limited cross-protection is characteristic of the serotype 19F polysaccharide or rather of the 19F-CRM (cross-reacting material) conjugate, we compared naturally induced antibodies against serotypes 19F and 19A with antibodies induced after vaccination with different pneumococcal conjugate vaccines. We found that conjugation of the serotype 19F polysaccharide using reductive amination (as in 7vCRM) resulted in the formation of at least one additional epitope that is not present in the native form of the 19F polysaccharide or following 19F conjugation using a bifunctional spacer (as in the prototype vaccine 7vOMPC) or cyanylation (as in PHiD-CV). We also found that pneumococcal vaccines conjugated using cyanylation induce more opsonophagocytic antibodies against serotype 19F and a considerably higher level of cross-opsonophagocytic antibodies against serotype 19A than vaccines conjugated using reductive amination. In conclusion, these results suggest that the conjugation method can influence the functionality of the antibodies induced against the homologous serotype 19F and the cross-reactive serotype 19A of S. pneumoniae

Expression of a recombinant Toxoplasma gondii ROP2 fragment as a fusion protein in bacteria circumvents insolubility and proteolytic degradation.

A 268-amino-acid-residue carboxy-terminal antigenic fragment of the Toxoplasma gondii rhoptry protein ROP2 (recROP2(t), residues 196-464) was expressed in Escherichia coli. This recombinant fragment was produced at low concentration and in a highly insoluble form. By contrast, the level of recROP2(t) production was drastically greater when the same coding sequence was fused to the C-terminus of thioredoxin (TRX) or to the maltose-binding protein (MBP) gene. While both fusion proteins were found to be mainly insoluble, solubilization could be achieved without significant degradation. MBP was more efficient than TRX in increasing the recovery of soluble protein with more than 10% of total MBP-recROP2(t) being readily expressed in a soluble form. Moreover, the insoluble form of MBP-recROP2(t) could be correctly refolded with a recovery of more than 80%. Both forms of MBP-recROP2(t) were purified to homogeneity by amylose chromatography. In contrast, the refolding of TRX-recROP2(t) promoted aggregation of the protein, which was prevented by the use of zwitterionic detergent during the one-step purification by gel filtration. Subsequent proteolytic cleavages of purified TRX-recROP2(t) and of MBP-recROP2(t) led respectively to the complete degradation or to the truncation of the recROP2(t) moiety. However, recROP2(t), despite the presence of the fusion partners, adopted a suitable conformation recognized by human serum-derived antibodies from T. gondii-seropositive individuals. Finally, both fusion proteins were able to induce specific humoral and cell-mediated immune response to the ROP2 fragment. Such fusions could represent an alternative to study the immunogenicity of T. gondii proteins which are difficult to produce because of insolubility and degradation.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

The conformation of purified Toxoplasma gondii SAG1 antigen, secreted from engineered Pichia pastoris, is adequate for serorecognition and cell proliferation.

A truncated form of SAG1, the immunodominant surface antigen of Toxoplasma gondii, has been produced in the methylotrophic yeast, Pichia pastoris. By construction, the recombinant protein lacks C-terminal residues 308-336 which, in native SAG1, encompass the glycosylphosphatidylinositol anchorage site. Secretion of anchor-less SAG1 proceeded via the yeast prepro alpha-mating factor signal peptide and yielded two immunoreactive protein species having apparent molecular masses of 31.5 and 34.5 kDa, respectively, and differing only by N-glycosylation of the single Asn-X-Ser site present in the molecule. Purification of the anchor-less SAG1 was achieved by a combination of ion-exchange and size-exclusion chromatographies. N-terminal amino acid sequencing of the products indicated the presence of additional residues glutamic acid--alanine at the N-terminal end of the products. Despite incomplete processing and unnatural glycosylation, anchor-less SAG1 proteins apparently adopted a suitable conformation recognized by monoclonal and human serum-derived antibodies, specific for the native SAG1. In addition, the recombinant anchor-less SAG1 proved competent for inducing proliferation, in vitro, of mononuclear cells from seropositive individuals. Finally, properly adjuvanted anchor-less SAG1 was able to induce protection of mice against a lethal challenge with T. gondii tachyzoites.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

The surface antigen SAG3 mediates the attachment of Toxoplasma gondii to cell-surface proteoglycans.

The attachment of Toxoplasma gondii to target cells is mediated by recognition of cellular heparan sulfate proteoglycans (HSPGs). The present study was performed to determine whether SAG1 and SAG3, two of the parasite surface antigens anchored to the membrane via glycosylphosphatidylinositol groups (GPIs), are involved in the tachyzoite binding to proteoglycans. The use of recombinant soluble forms of these proteins allowed us to demonstrate that SAG3, but not SAG1, interacts specifically with cellular HSPGs. Indeed, soluble recombinant SAG3 protein (recSAG3) was found to bind to immobilized heparin, whereas recSAG1 did not interact with this glycoaminoglycan. The specific adherence of recSAG3 to CHO cells was inhibited by soluble glycoconjugates, of which heparin, fucoidan and dextran sulfate were the most effective. Moreover, binding of recSAG3 to two HSPGs-deficient cell mutants was reduced by up to 80%. Proteoglycan sulfation was critical for SAG3 adherence to HSPGs as incubation of cells in the presence of sodium chlorate drastically reduced the recSAG3 binding. Finally, preincubation of CHO cells with recSAG3 blocked the adsorption of radiolabelled Toxoplasma tachyzoites. Taken together, these results indicate that SAG3 is a first glycoaminoglycan-binding protein associated with Toxoplasma, and SAG3-HSPGs interactions are involved in the parasite attachment to target cells.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

DNA immunisation. New histochemical and morphometric data

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Porcine D-amino acid oxidase: determination of the mRNA nucleotide sequence by the characterization of genomic and cDNA clones

SCOPUS: ar.jinfo:eu-repo/semantics/publishe