Michèle Haumont

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.

Protective Immunity against Congenital Toxoplasmosis with Recombinant SAG1 Protein in a Guinea Pig Model

ABSTRACT Primary infection with Toxoplasma gondii during pregnancy can induce fetal pathology and abortion in both humans and animals. The present study describes the development of an experimental model of congenital toxoplasmosis in the guinea pig. In this animal model, we evaluated the protective effect of vaccination with a recombinant form of SAG1 against maternofetal transmission of tachyzoites. The presence of parasites in fetuses was determined by nested PCRs and by an in vivo readout after fetal brain homogenate injections in mice. The absence of parasites was demonstrated in 66 to 86% of fetuses derived from adult guinea pigs immunized with SAG1 and challenged with the mildly virulent T. gondii strain C56. In contrast, more than 80% of fetuses from mock-immunized guinea pigs were infected. The protection was not correlated with titers of antibody to SAG1. Our results indicated that this experimental model constitutes a relevant model for evaluation of vaccine candidates against congenital toxoplasmosis and that SAG1 elicits significant protection against maternofetal transmission.

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.

High-Level Expression in Mammalian Cells of Recombinant House Dust Mite Allergen ProDer p 1 with Optimized Codon Usage

Background: The major house dust mite allergen Der p 1 is associated with allergic diseases such as asthma. Production of recombinant Der p 1 was previously attempted, but with limited success. The present study describes the expression of recombinant (rec) ProDer p 1, a recombinant precursor form of Der p 1, in CHO cells. Methods: As optimization of the codon usage may allow successful overexpression of protein in mammalian cells, a synthetic gene encoding ProDer p 1 was designed on the basis of the codon usage frequently found in highly expressed human genes. Gene synthesis was accomplished from a set of 14 mutually priming overlapping oligonucleotides and after two runs of polymerase chain reaction. Results: COS cells transiently transfected with the synthetic ProDer p 1 gene produced up to 5–10 times as much ProDer p 1 compared with the expression level obtained after transfection with the authentic gene. To stably express the recombinant allergen, CHO-K1 cells were transfected with the ProDer p 1 synthetic gene, and one amplified recombinant clone produced up to 30 mg of recProDer p 1 per liter in the culture medium before purification. recProDer p 1 was secreted as an enzymatically inactive single-chain molecule presenting three glycosylated immunoreactive forms of 41, 38 and 36 kD. When examined with respect to direct binding, recProDer p 1 and natural Der p 1 displayed very similar IgE reactivities. However, IgE inhibition and histamine release assays showed a much higher reactivity to natural Der p 1 compared to recProDer p 1. Conclusions: These data indicated that codon optimization represents an attractive strategy for high-level production of allergen in mammalian cells.

The varicella zoster virus glycoprotein B (gB) plays a role in virus binding to cell surface heparan sulfate proteoglycans

Varicella-zoster virus (VZV) interacts with cell surface heparan sulfate proteoglycans during virus attachment. In the present study, we investigated the potential involvement of two VZV glycoproteins, gB and gE, in the virus adsorption process. We showed that gB, but not gE, binds specifically to cellular heparan sulfate proteoglycans (HSPGs). Indeed, soluble recombinant gB protein (recgB) was found to bind to immobilized heparin and to MRC5 and L cells, a binding which was inhibited by heparin. Furthermore, recgB binding to two heparan sulfate-minus mutant L cell lines, gro2C and sog9 cells, was markedly reduced as compared to the parental L strain. Under the same experimental conditions, soluble recombinant VZV gE protein did not interact with heparin or with cell surfaces. We also demonstrated that the gB-HSPGs interactions were relevant to the VZV attachment to cells. Indeed, although polyclonal antibodies directed to gB did not impair the VZV binding, recgB could delay the virus adsorption. Our results thus strongly suggest that the interactions between gB and heparan sulfate proteoglycans take part in the initial VZV attachment to cell surfaces.

Stable integration and expression of the Plasmodium falciparum circumsporozoite protein coding sequence in mycobacteria.

The DNA coding for the circumsporozoite protein of Plasmodium falciparum (CSP; aa 1-412) has been placed under the control of the mycobacterial promoter derived from the gene encoding the 64-kDa antigen of Mycobacterium bovis-BCG. This expression cassette was cloned into pJRD184, an Escherichia coli multicloning site vector, together with the kanamycin resistance gene from Tn903 and the attachment site and integrase gene from the temperate mycobacteriophage FRAT1. One of the resulting plasmids, pNIV2173, introduced by electroporation into both Mycobacterium smegmatis and M. bovis-BCG, integrated at a specific site in the genome of each recipient. Recombinants expressed immunoreactive polypeptides, ranging in size from 62 to 43 kDa, at a level of about 1% of total soluble proteins. Part of this material was present in the culture medium indicating that mycobacterial recombinants were able to secrete the CSP. The M. smegmatis and M. bovis-BCG recombinants, transformed with pNIV2173 and grown in absence of antibiotic, were followed for more than 400 and 50 generations respectively. Over this time span, neither DNA rearrangement nor loss of expression was observed. Inoculation of the recombinant BCG to mice did not induce humoral response to CSP nor proliferative response to CSP Th2R CD4+ T lymphocyte epitope.

Production, quality control, stability and pharmacotoxicity of cGMP-produced Plasmodium falciparum AMA1 FVO strain ectodomain expressed in Pichia pastoris.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, PfAMA1 ectodomain (amino acid 25-545, FVO strain) was produced in Pichia pastoris by 35L scale fed batch fermentation under current Good Manufacturing Practice (cGMP). Fermentation was followed by a three-step chromatographic purification procedure resulting in a yield of 5.8g of purified protein. As judged by size exclusion chromatography, the cGMP-product comprised >95% PfAMA1 monomer, the remainder being predominantly PfAMA1 dimer. In SDS-PAGE two main bands of 68 and 70kDa and some minor bands were evident. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident; less than 15% of the protein had this internal cleavage. By mass-spectrometric analysis, all bands analyzed in overloaded SDS-PAGE gels comprised PfAMA1 derived products. The protein was quantitatively bound by immobilized 4G2, a monoclonal antibody reactive with a reduction sensitive conformational determinant. The lyophilized product was stable for over 1 year. Immunopotency did not diminish, and storage did not lead to alterations in the behaviour of the protein upon formulation with adjuvants selected for Phase I clinical evaluation. These formulations also showed no pharmacotoxicity in rabbits. The final product conformed to preset criteria and was judged suitable for use in human clinical trials.

Purification, characterization and immunogenicity of recombinant varicella-zoster virus glycoprotein gE secreted by Chinese hamster ovary cells.

The gene of the varicella-zoster virus (VZV) glycoprotein gE, engineered to code for a truncated molecule lacking the anchor and carboxy-terminal tail domains, was transfected into Chinese hamster ovary (CHO) cells via the pEE14 mammalian expression vector. One recombinant cell line, CHO-gE-2-9, secreted high levels of truncated gE into the culture medium. The product was purified to near homogeneity by a combination of anion exchange, hydrophobic and metal-chelate chromatographies. Purified recombinant gE showed the expected amino-terminal sequence and its glycosylation pattern proved similar to that of the natural product. When injected into mice, using either Freunds or alum as adjuvant, the native truncated gE induced complement-dependent neutralizing antibodies. In contrast, when the molecule was first denatured, it lost immunogenicity with alum. These data show that the recombinant gE, although truncated, could potentially be included in a subunit vaccine against VZV infection.

Absence of immunoglobulin E synthesis and airway eosinophilia by vaccination with plasmid DNA encoding ProDer p 1

Background Various studies have shown that immunization with naked DNA encoding allergens induces T helper 1(Th1)‐biased non‐allergic responses.

Neutralizing antibody responses induced by varicella-zoster virus gE and gB glycoproteins following infection, reactivation or immunization

The purpose of this study was to compare the antibody responses to varicella‐zoster virus (VZV) gE and gB after natural VZV infection and after vaccination with live attenuated OKA vaccine in order to determine the relative importance of these proteins as components of a subunit vaccine. Anti‐VZV antibody titers determined by IFA were of the same order of magnitude in sera from individuals with a history of varicella and in vaccinated children but higher in individuals given booster vaccination. The titers of anti‐gE and anti‐gB antibodies were measured by ELISA using recombinant gE or gB as capture antigen. From these experiments, it appears that the ratio of anti‐gE to anti‐gB antibody is highly variable from one individual to another but relatively stable over a long period of time for a particular individual, even after a zoster episode. Neutralizing antibodies directed against gE or gB were also measured by subtracting the neutralization titers obtained before and after depletion of the specific antibodies on immobilized recombinant gE, gB, or both. This showed that, with respect to neutralization, anti‐gE and anti‐gB are equally prevalent in vaccinated children and that anti‐gE is generally, but not always, predominant over anti‐gB in VZV‐infected individuals. Finally, antibodies to these two glycoproteins appear to be predominant among the neutralizing antibodies directed to other VZV antigens. J. Med. Virol. 53:63–68, 1997.