Methylated CpG-containing plasmid activates the immune system.

Bacterial DNA differs from mammalian DNA by the presence of unmethylated cytosine-phosphate-guanosine (CpG) motifs. The immunostimulatory properties of a DNA vaccine have been suspected to be associated with these motifs. The aim of this study was to assess the inactivation of the immunostimulatory potential of a plasmid after methylation of its CpG motifs. We constructed two identical non-coding plasmids, and one of these was de novo methylated on its CG sequences. A single administration of recombinant antigen with methylated or unmethylated CpG-containing plasmid was performed in mice. As expected, only unmethylated CpG-containing plasmid enhanced the specific immune response. However, a study of in vivo activation of Langerhans' cells and analysis of mRNA synthesis indicated that both the plasmids promoted cell emigration and cytokine induction. These data highlight that a methylated CpG-containing plasmid is not inert and carries immunomodulatory properties. The results further emphasize the necessity to definitively identify the mode of action of plasmids used for DNA vaccination.

Adjuvant activity of free Bordetella pertussis filamentous haemagglutinin delivered by mucosal routes.

The development of safe and potent mucosal adjuvants remains a major objective in vaccinology. The potential usefulness of filamentous haemagglutinin (FHA) of Bordetella pertussis as an adjuvant was assessed in a mouse model. The glutathione-S-transferase of Schistosoma mansoni (Sm28GST) was used for intranasal administration, while the gut-resistant keyhole limpet haemocyanin (KLH) was administrated by the oral route. For both antigens, coadministration with FHA increased antigen-specific immunoglobulin titres. This adjuvant effect did not require chemical cross-linking or direct interaction between FHA and the antigen tested. FHA also behaved as an adjuvant by the subcutaneous route, indicating that its adjuvanticity is not restricted to binding to mucosal surfaces. The FHA-induced adjuvanticity was also observed in mice with high anti-FHA antibody titres as a result of antipertussis vaccination, indicating that pre-existing anti-FHA antibodies do not impair FHA adjuvanticity. No mRNA coding for proinflammatory cytokines was induced in the lungs after intranasal FHA administration. However, an increase in the levels of mRNAs coding for B7-1, transforming growth factor (TGF)-beta and major histocompatibility complex (MHC)-II was detected in the lungs after FHA administration. Although the molecular mechanisms of the FHA-induced adjuvanticity remain to be elucidated, the data presented here indicate that this adhesin, already assessed for human use as a pertussis vaccine constituent, represents a promising adjuvant to improve the humoral immune response when given by mucosal routes.

Vaccine properties of antigens entrapped in microparticles produced by spray-drying technique and using various polyester polymers.

The present study investigated the suitability of various microparticles produced by spray-drying technique to entrap and preserve the physiochemical and biological properties of an antigen. These microparticles were constituted either by poly(lactide) polymers characterized by various molecular weight or poly(lactide-co-glycolide) polymers. The recombinant 28 kDa glutathione S-transferase of Schistosoma mansoni (rSm28GST) characterized by major epitopes involved in the active site of this enzyme was selected as model antigen. The microparticles were characterized by a mean size

Single-dose mucosal immunization with biodegradable microparticles containing a Schistosoma mansoni antigen.

The purpose of this work was to assess the immunogenicity of a single nasal or oral administration of recombinant 28-kDa glutathione S-transferase of Schistosoma mansoni (rSm28GST) entrapped by poly(lactide-co-glycolide) (PLG)- or polycaprolactone (PCL)-biodegradable microparticles. Whatever the polymer and the route of administration used, the equivalent of 100 microg of entrapped rSm28GST induced a long-lasting and stable antigen-specific serum antibody response, with a peak at 9 to 10 weeks following immunization. Isotype profiles were comparable, with immunoglobulin G1 being the predominant isotype produced. The abilities of specific antisera to neutralize the rSm28GST enzymatic activity have been used as criteria of immune response quality. Pooled 10-week sera from mice receiving PLG microparticles by the nasal or oral route neutralized the rSm28GST enzymatic activity, whereas sera of mice receiving either PCL microparticles, free rSm28GST, or empty microparticles inefficiently neutralized this enzymatic activity. Finally, this study shows that a single administration of these microparticles could provide distinct and timely release pulses of microencapsulated antigen, which might greatly facilitate future vaccine development.

Bordetella pertussis filamentous hemagglutinin enhances the immunogenicity of liposome-delivered antigen administered intranasally.

In an attempt to increase the immunogenicity of mucosally delivered antigens, we incorporated the Bordetella pertussis filamentous hemagglutinin (FHA) adhesin into liposomes containing the glutathione S-transferase of Schistosoma mansoni (Sm28GST) as a model antigen. Outbred mice immunized twice intranasally with liposomes containing a constant suboptimal dose of Sm28GST and increasing doses of FHA produced anti-Sm28GST antibodies in a FHA dose-dependent manner. The addition of 3 microg of FHA to the liposomes induced more than 10-fold-higher anti-Sm28GST antibody titers, compared to those induced by liposomes without FHA. The presence of FHA did not alter the nature of the humoral immune response, and the sera contained anti-Sm28GST immunoglobulin G1 (IgG1), IgG2a, and IgG2b. However, anti-Sm28GST IgA was only detected when at least 3 microg of FHA was added to the preparation. These results show a promising potential for FHA to enhance the immunogenicity of mucosally administered antigens incorporated into liposomes.

Glutathione S-transferases of 28kDa as major vaccine candidates against schistosomiasis.

For the development of vaccine strategies to generate efficient protection against chronic infections such as parasitic diseases, and more precisely schistosomiasis, controlling pathology could be more relevant than controlling the infection itself. Such strategies, motivated by the need for a cost-effective complement to existing control measures, should focus on parasite molecules involved in fecundity, because in metazoan parasite infections pathology is usually linked to the output of viable eggs. In numerous animal models, vaccination with glutathione S-transferases of 28kDa has been shown to generate an immune response strongly limiting the worm fecundity, in addition to the reduction of the parasite burden. Recent data on acquired immunity directed to 28GST in infected human populations, and new development to draw adapted vaccine formulations, are presented.

Role of adhesion molecules of the selectin-carbohydrate families in antibody-dependent cell-mediated cytoxicity to schistosome targets.

The role of adhesion molecules in antibody-dependent cell-mediated cytotoxicity (ADCC) of macrophages toward the extracellular parasite Schistosoma mansoni was investigated by using 1) a panel of blocking mAbs directed against adhesion molecules and 2) different soluble ligands as candidate inhibitors of ADCC. The results show that the beta2-integrin Mac-1 (CD11b/CD18), L-selectin (CD62-L), and the carbohydrate determinant sialyl Lewis(x) (sLe(x); sCD15) are required for macrophage effector function toward schistosomula targets. On the other hand, the parasite counter-receptors involved in ADCC were found to share common motifs with the mammalian selectin-carbohydrate families. One family of parasite receptor(s) involved in ADCC carries the Lewis(x) (Le(x); CD15) carbohydrate structure, whereas a second family of receptor(s) appears to display selectin-like properties with affinity for the sLe(x) tetrasaccharide. Immunostaining experiments confirmed that schistosomula express on their surface hostlike molecules recognized by anti-Le(x) (CD15) and by anti-human E-selectin (CD62-E) mAbs. The double receptor-ligand interaction between macrophages and parasite targets provides new insights into the biologic roles of selectins and Le(x)-related structures in immunity against helminthic parasites.

Local transient induction of inflammatory cytokines after intranasal administration of recombinant Bordetella pertussis.

Inflammatory cytokines have been described to play a critical role in the orientation and amplification of the IgA immune response. In this study, we show that the intranasal administration of a Bordetella pertussis strain expressing the protective antigen glutathione-S-transferase of Schistosoma mansoni (Sm28GST) induced an inflammatory response in the lungs of mice, characterized by the production of inflammatory cytokines, such as Tumor Necrosis Factor alpha, Interleukin-6 and Transforming-Growth Factor beta. The production and the secretion of these cytokines in lung tissues were early and transient. Their presence was observed only during the first week after administration despite the persistence of the bacteria for 1 month. Two weeks after inoculation, Interleukin-10 secretion was detected in the lungs, which could explain the decrease in the production of inflammatory cytokines. These inflammation-regulating cytokines, induced in the lungs by the presence of the bacterial vector, could be part of the process generating the local immune response, in particular the anti-Sm28GST IgA response.

Intradermal immunization of rats with plasmid DNA encoding Schistosoma mansoni 28 kDa glutathione S-transferase.

Direct administration of plasmid DNA encoding an antigen represents an attractive approach to vaccination against infectious diseases, particularly in developing countries where easy-to-handle and cost-effective vaccines are needed. We have investigated the potential of DNA immunization to induce a specific antibody response against Schistosoma mansoni, using plasmid-DNA encoding the protective antigen, S. mansoni 28 kDa glutathione S-transferase (Sm28GST). Since S. mansoni parasite penetrates into its host through the skin, this tissue was chosen for plasmid DNA delivery. Following plasmid DNA administration into the skin of rats, the parasite antigen was detected in skin cells by immunohistochemistry. Three administrations of 200 micrograms plasmid at 14 day intervals led to the induction of a long-lasting specific IgG antibody response in the sera of immunized rats, with a predominance of IgG2a and IgG2b subclasses. Sera of immunized animals were able to mediate antibody-dependent cellular cytotoxicity in vitro, leading to the specific killing of parasite larvae. A parasite challenge performed on plasmid DNA-immunized animals induced a strong and rapid boosting effect on the specific IgG antibody response. These results demonstrate the potential of genetic immunization via the skin with plasmid DNA encoding Sm28GST for inducing immune responses with protective patterns against an S. mansoni infection.

Immunoglobulin A response in murine schistosomiasis: stimulatory role of egg antigens.

The immunoglobulin G (IgG) and IgA antibody responses to different Schistosoma mansoni antigens have been determined in chronically infected mice as well as in unisexually infected animals. With a panel of enzyme-linked immunosorbent assays (ELISAs), soluble antigens from furcocercariae, adult worms, and eggs were probed with sera collected at 3-week intervals. Bisexually infected animals developed significant IgG and IgA antibody responses to the antigens tested, which increased after egg deposition. In unisexual infections no significant differences were recorded in the IgG antibody profile for furocercaria and adult worm antigens, whereas the IgA antibody response was impaired. Both the IgA and IgG antibody responses toward egg antigens were reduced compared with those in a bisexual infection. Furthermore, a specific mucosal IgA antibody response was observed only in the bisexually infected animals. Histological analysis performed on bisexually infected mice led to the observation of eggs and granulomatous lesions within the Peyer's patch follicles, which are essential sites for the induction of mucosal immunity in the intestine. These data suggest a relationship between egg deposition and the induction of the IgA antibody response toward schistosomes.

Green fluorescent protein as a new expression marker in mycobacteria.

This study describes the use and the advantages of the green fluorescent protein (GFP) as a reporter molecule for mycobacteria. The gfp gene from Aequorea victoria was placed under the control of the hsp60 promoter in the shuttle vector pGFM-11. The gfp expression in the recombinant Mycobacterium smegmatis and BCG was readily detected on agar plates by the development of an intense green fluorescence upon irradiation with long-wave u.v. light. In mycobacteria containing a pGFM-11 derivative that lacks the hsp60 promoter, no fluorescence was observed. However, this plasmid was successfully used as a promoter-probe vector to identify BCG promoters. The fluorescence emission of GFP in mycobacteria harbouring pGFM-11 and grown in liquid media could be quantified by spectrofluorimetry. This allowed for easy assessment of drug susceptibility. As GFP does not require the addition of substrates or co-factors, the green fluorescent bacilli could be directly observed within infected macrophages using fluorescence and laser confocal microscopy, or in tissue sections of infected mice. Finally, infected cells or free-living recombinant mycobacteria could also be analysed by flow cytometry. The GFP thus appears to be a convenient reporter for mycobacteria, allowing tracing of recombinant mycobacteria, isolation of promoters with interesting properties, in vivo drug testing and the development of new diagnostic tools.

Transcytosis of the polymeric Ig receptor requires phosphorylation of serine 664 in the absence but not the presence of dimeric IgA.

MDCK cells expressing the polymeric immunoglobulin (poly-Ig) receptor, cocultured with IgA-producing hybridoma cells, transported dimeric IgA (dIgA) from the basolateral into the lumenal compartment, where it was recovered as secretory component-dIgA complexes. The tail of the receptor was phosphorylated on serines 664 and 726. Each serine was mutated to alanine. Appearance of A726 receptor at the basolateral surface was reduced approximately 5-fold. This was accompanied by a approximately 5-fold reduction in dIgA transcytosis. Basolateral delivery of receptor was not affected by mutation A664, and in the absence of dIgA, the receptor accumulated in recycling basolateral endosomes. In coculture, however, dIgA transcytosis by A664 receptor was normal. Thus, entry of receptor into the transcytotic pathway requires Ser-664 phosphorylation only in the absence of dIgA.

Highly inducible synthesis of heterologous proteins in epithelial cells carrying a glucocorticoid-responsive vector.

A glucocorticoid-responsive vector is described which allows for the highly inducible expression of complementary DNAs (cDNAs) in stably transfected mammalian cell lines. This vector, pLK-neo, composed of a variant mouse mammary tumor virus long terminal repeat promoter, containing a hormone regulatory element, a Geneticin resistance-encoding gene in a simian virus 40 transcription unit, and a polylinker insertion site for heterologous cDNAs, was used to express the polymeric immunoglobulin (poly-Ig) receptor and the thymocyte marker, Thy-1, in Madin-Darby canine kidney (MDCK) cells and in murine fibroblast L cells. A high level of poly-Ig receptor or Thy-1 mRNA accumulation was observed in MDCK cells in response to dexamethasone with a parallel ten- to 200-fold increase in protein synthesis depending on the recombinant protein and the transfected cell clone.