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Biomedical subjects

R Rappuoli

Publications and source records attributed to R Rappuoli.

At least 109 records · Page 6Linked to original sources
PubMed1998

Mucosal immunogenicity of genetically detoxified derivatives of heat labile toxin from Escherichia coli.

Using a fixed dose of antigen, the immune response to detoxified mutants of LT-WT following intranasal (i.n.), subcutaneous (s.c.) and oral (i.g.) immunisation has been studied. When given i.n., both LT-WT and mutant toxin, K63, generated significant levels of toxin-specific IgG in the serum, and the levels of IgA in nasal and lung lavages were greater than those induced by rLT-B. In comparison, i.g. immunisation of mice with a similar quantity of either LT-WT or K63 toxin induced barely detectable levels of IgG in the sera. However, if the amount of protein used for i.g. immunisation was increased tenfold, relatively good levels of toxin-specific IgG were induced in the sera by both LT-WT or K63. Low levels of toxin-specific IgA were also observed in intestinal washes from these mice. Western blotting of the sera, using the native toxin as an antigen, demonstrated the presence of both anti-A and anti-B subunit antibodies. Most significantly, toxin-neutralising antibodies were induced in the serum, with the strongest activity being induced by the LT-WT, an intermediate activity induced by mutant K63 and a lower response by rLT-B. Together, these data show that ADP-ribosyltransferase is not necessary for mucosal immunogenicity of these proteins, and that the i.n. route of immunisation is more effective than the i.g. route of immunisation for the generation of both systemic (IgG) and mucosal (IgA) immune responses.

Amino Acid Sequence↗
PubMed1998

Helicobacter pylori: molecular evolution of a bacterial quasi-species.

Helicobacter pylori persists chronically within individuals and as they spread the mutating bacteria migrate with them. The continuous selection and microevolution generates a population of closely related but different bacteria that behave like a quasi-species. Within this heterogeneity, H. pylori strains fall into distinct types, into the virulent (type I) and less virulent (type II) strains, based on the presence of a pathogenicity island (cag) that encodes a specialized secretion machinery. We propose that during chronic infection a dynamic equilibrium between bacteria expressing a disparate degree of virulence is established, and that diverse forms prevail at different times.

Animals↗
PubMed1998

Functional analysis of the Helicobacter pylori principal sigma subunit of RNA polymerase reveals that the spacer region is important for efficient transcription.

We have cloned the rpoD gene encoding the principal sigma (sigma) factor of Helicobacter pylori. The deduced amino acid sequence reveals a predicted polypeptide of 676 residues that has amino acid homology with the principal sigma factors of a number of divergent prokaryotes. We have designated this factor sigma80. Amino acid sequence analysis suggests that region 1.1 is missing in sigma80 and that a region with homology to a regulatory protein from Bacillus subtilis phage SPO1 is present. Genetic studies have indicated that sigma80 is not compatible with the transcriptional machinery of Escherichia coli. However, in vitro sigma80 could be assembled into the E. coli RNA polymerase and could bind to E. coli and H. pylori promoters, suggesting that the sigma80-containing RNA polymerase has the same stoichiometry as the native complex. By exchanging protein domains between E. coli and H. pylori sigma factors, we demonstrate that the sigma80 domain inhibiting transcription from E. coli promoters is confined within the non-conserved spacer region, implying that the spacer region of prokaryotic primary sigma factors plays an important role in the process of transcription. Consistent with its restricted niche and with the availability of a very restricted number of transcriptional regulators, H. pylori may have evolved a spacer region of the sigma factor to modulate total transcription and to quickly respond to microenvironmental changes.

Amino Acid Sequence↗
PubMed1998

Enterotoxic effect of the vacuolating toxin produced by Helicobacter pylori in Caco-2 cells.

Preliminary clinical evidence suggests that Helicobacter pylori may be associated with diarrhea through its vacuolating toxin (VacA). To establish whether VacA induces intestinal secretion, epithelial damage, or both, purified pH-activated VacA was added to Caco-2 cell monolayers mounted in Ussing chambers, and electrical parameters were monitored. Mucosal addition of VacA induced an increase in short circuit current, consistent with enterotoxic effect. The effect was time- and dose-dependent and saturable. It was not found if the toxin was not pH-activated, added to the serosal side, or preheated. In cells preloaded with the Ca2+ buffering compound BAPTA/AM or with the Cl- channel inhibitor 5-nitro-2-3-(3-phenylpropylamino)benzoic acid, short circuit current did not change, indicating that VacA induces activation of Ca2+-dependent Cl- channels. VacA did not show cytopathic effects, as judged by tissue resistance. These results support the hypothesis that H. pylori may be associated with diarrhea through production of VacA.

Bacterial Proteins↗
PubMed1998

Pertussis toxin potentiates Th1 and Th2 responses to co-injected antigen: adjuvant action is associated with enhanced regulatory cytokine production and expression of the co-stimulatory molecules B7-1, B7-2 and CD28.

Pertussis toxin (PT) is a major virulence factor of Bordetella pertussis which exerts a range of effects on the immune system, including the enhancement of IgE, IgA and IgG production, delayed-type hypersensitivity reactions, and the induction of experimental autoimmune diseases. However, the mechanism by which PT mediates adjuvanticity remains to be defined. In this investigation we have shown that PT can potentiate antigen-specific T cell proliferation and the secretion of IFN-gamma, IL-2, IL-4 and IL-5 when injected with foreign antigens. A chemically detoxified PT and a genetic mutant with substitutions/deletions in the S-1 and B oligomer components that abrogate enzymatic and binding activity displayed no adjuvant properties. In contrast, a non-toxic S-1 mutant devoid of enzymatic activity but still capable of receptor binding retained its adjuvanticity, augmenting the activation of both Th1 and Th2 subpopulations of T cells. In an attempt to address the mechanism of T cell activation, we found that PT stimulated the production of IFN-gamma and IL-2 by naive T cells and IL-1 by macrophages. Therefore potentiation of distinct T cell subpopulations may have resulted in part from the positive influence of IFN-gamma on the development of Th1 cells and the co-stimulatory role of IL-1 for Th2 cells. Furthermore, PT augmented expression of the co-stimulatory molecules B7-1 and B7-2 on macrophages and B cells, and CD28 on T cells, suggesting that the adjuvant effect may also be associated with facilitation of the second signal required for maximal T cell activation. This study demonstrates that the immunopotentiating properties of PT are largely independent of ADP-ribosyltransferase activity, but are dependent on receptor binding activity and appear to involve enhanced activation of T cells.

Adjuvants, Immunologic↗
PubMed1998

Levels of expression and immunogenicity of attenuated Salmonella enterica serovar typhimurium strains expressing Escherichia coli mutant heat-labile enterotoxin.

The effects of heterologous gene dosage as well as Salmonella typhimurium strain variability on immune response toward both the heterologous antigen, the nontoxic mutant of the Escherichia coli heat-labile enterotoxin LTK63, and the carrier Salmonella strain have been analyzed. Effects of a single integration into the host DNA and different-copy-number episomal vectors were compared in S. typhimurium delta cya delta crp delta asd strains of two different serotypes, UK-1 and SR-11. Expression of the enterotoxin in the different Salmonella isolates in vitro was found to vary considerably and, for the episomal vectors, to correlate with the plasmid copy number. LTK63-specific serum immunoglobulin G (IgG) and mucosal immunoglobulin A (IgA) antibodies were highest in mice immunized with the high-level-expression strain. High anti-LTK63 IgG and IgA titers were found to correspond to higher anti-Salmonella immunity, suggesting that LTK63 exerts an adjuvant effect on response to the carrier. Statistically significant differences in anti-LTK63 immune response were observed between groups of mice immunized with the attenuated delta cya delta crp UK-1 and SR-11 derivatives producing the antigen at the same rate. These data indicate that the same attenuation in S. typhimurium strains of different genetic backgrounds can influence significantly the immune response toward the heterologous antigen. Moreover, delivery of the LTK63 enterotoxin to the immune system by attenuated S. typhimurium strains is effective only when synthesis of the antigen is very high during the initial phase of invasion, while persistence of the S. typhimurium strain in deep tissues has only marginal influence.

Adjuvants, Immunologic↗
PubMed1998

Deletion of the major proteolytic site of the Helicobacter pylori cytotoxin does not influence toxin activity but favors assembly of the toxin into hexameric structures.

The Helicobacter pylori cytotoxin is proteolytically cleaved at a flexible hydrophilic loop into two subunits. Deletion of the loop sequences had no effect on biological activity of the toxin in the HeLa cell vacuolation assay but favored the organization of the protein into hexameric rather than heptameric structures.

Bacterial Proteins↗
PubMed1998

Identification of the Helicobacter pylori VacA toxin domain active in the cell cytosol.

Cells exposed to Helicobacter pylori toxin VacA develop large vacuoles which originate from massive swelling of membranous compartments at late stages of the endocytic pathway. When expressed in the cytosol, VacA induces vacuolization as it does when added from outside. This and other evidence indicate that VacA is a toxin capable of entering the cell cytosol, where it displays its activity. In this study, we have used cytosolic expression to identify the portion of the toxin molecule responsible for the vacuolating activity. VacA mutants with deletions at the C and N termini were generated, and their activity was analyzed upon expression in HeLa cells. We found that the vacuolating activity of VacA resides in the amino-terminal region, the whole of which is required for its intracellular activity.

Bacterial Proteins↗
PubMed1998

A recombinant live attenuated strain of Vibrio cholerae induces immunity against tetanus toxin and Bordetella pertussis tracheal colonization factor.

An attenuated strain of Vibrio cholerae was used as a carrier for the expression of heterologous antigens such as fragment C from tetanus toxin (TetC) and tracheal colonization factor from Bordetella pertussis (Tcf). In vitro, high levels of protein were obtained when the Escherichia coli nirB promoter was used and the bacteria were grown with low aeration. Intranasal immunization of mice with IEM101 expressing TetC elicited serum vibriocidal activity and induced antibodies against tetanus toxin which were protective against lethal challenge with 10 times the 50% lethal dose of tetanus toxin. Bacterial viability was essential for the induction of anti-TetC antibodies. Intranasal administration of IEM101 expressing Tcf induced a significant reduction in bacterial colonization of the tracheas of mice challenged with wild-type B. pertussis. These data are in agreement with the putative role of Tcf in Bordetella tracheal colonization. In conclusion, we have demonstrated that V. cholerae may be used as a live vector to deliver heterologous antigens in vivo and that protection to both systemic and local challenge may be achieved.

Administration, Intranasal↗
PubMed1998

Binding of the Helicobacter pylori vacuolating cytotoxin to target cells.

The vacuolating cytotoxin of Helicobacter pylori, VacA, enters the cytoplasm of target cells and causes vacuolar degeneration by interfering with late stages of endocytosis. By using indirect immunofluorescence and flow cytometry, we have demonstrated that VacA binds to specific high-affinity cell surface receptors and that this interaction is necessary for cell intoxication.

3T3 Cells↗
PubMed1998

Experimental model of Helicobacter pylori infection.

Critical issues in the development of a vaccine against Helicobacter pylori are represented by the definition of molecules important in the pathogenesis of the infection, by the availability of an animal model reproducing several aspects of the human infection, and lastly by the availability of powerful adjuvants allowing strong protection after mucosal delivery of the antigens. A mouse model of Helicobacter pylori infection was established in our laboratories. Vaccination of these animals with Helicobacter pylori antigens, such as VacA, CagA, etc., induced protection, both prophylactic and therapeutic, when antigens were administered orally together with fully non toxic mutants of Escherichia coli heat-labile enterotoxin, as mucosal adjuvants. This experimental mouse model allows the study of the pathogenesis of Helicobacter pylori infection and the development of vaccines.

Animals↗
PubMed1998

Adjuvant effect of non-toxic mutants of E. coli heat-labile enterotoxin following intranasal, oral and intravaginal immunization.

Cholera toxin and Escherichia coli heat-labile enterotoxin (LT) are known to be very effective mucosal adjuvants, but their toxicity limits their use in humans. We genetically detoxified LT by substituting single residues in the active site of the enzymatic A subunit and obtained mutant molecules that retain mucosal adjuvant activity but are devoid of toxicity. These mutant LT molecules induce mucosal and systemic responses to antigens delivered intranasally, orally and intravaginally in mice. Furthermore, mucosal immunization with these molecules confers protection against systemic challenge with tetanus toxin (TT) and mucosal challenge with Helicobacter pylori.

Adjuvants, Immunologic↗
PubMed1997

Effect of helicobacter pylori vacuolating toxin on maturation and extracellular release of procathepsin D and on epidermal growth factor degradation.

The effect of vacuolating toxin (VacA) from Helicobacter pylori on endosomal and lysosomal functions was studied by following procathepsin D maturation and epidermal growth factor (EGF) degradation in HeLa cells exposed to the toxin. VacA inhibited the conversion of procathepsin D (53 kDa) into both the intermediate (47 kDa) and the mature (31 kDa) form. Nonprocessed cathepsin D was partly retained inside cells and partly secreted in the extracellular medium via the constitutive secretion pathway. Intracellular degradation of EGF was also inhibited by VacA with a similar dose-response curve. VacA did not alter endocytosis, cell surface recycling, and retrograde transport from plasma membrane to trans-Golgi network and endoplasmic reticulum, as estimated by using transferrin, diphtheria toxin, and ricin as tracers. Subcellular fractionation of intoxicated cells showed that procathepsin D and nondegraded EGF accumulate in lysosomes. Measurements of intracellular acidification with fluorescein isothiocyanate-dextran revealed a partial neutralization of the lumen of endosomes and lysosomes, sufficient to account for both mistargeting of procathepsin D outside the cell and the decreased activity of lysosomal proteases.

Bacterial Proteins↗
PubMed1997

Vacuoles induced by Helicobacter pylori toxin contain both late endosomal and lysosomal markers.

Intoxication of mammalian cells with the vacuolating toxin (VacA) released by Helicobacter pylori causes the formation of large acidic vacuoles containing the vacuolar ATPase proton pump and Rab7, a late endosome marker. Here, we describe a novel subcellular fractionation procedure, and we show that nanomolar concentrations of VacA induce a clear redistribution of lysosomal membrane glycoproteins among endocytic compartments. This redistribution is an early event in the process of cellular intoxication by VacA and precedes the formation of macroscopic vacuoles. The absence of the cation independent mannose 6-P receptor and the presence of Rab7 and of lysosomal membrane proteins in the newly formed compartment suggest that the vacuolating toxin induces the accumulation of a post-endosomal hybrid compartment presenting both late endosomal and lysosomal features.

Animals↗
PubMed1997

The small GTP binding protein rab7 is essential for cellular vacuolation induced by Helicobacter pylori cytotoxin.

The VacA cytotoxin, produced by toxigenic strains of Helicobacter pylori, induces the formation of large vacuoles highly enriched in the small GTPase rab7. To probe the role of rab7 in vacuolization, HeLa cells were transfected with a series of rab mutants and exposed to VacA. Dominant-negative mutants of rab7 effectively prevented vacuolization, whereas homologous rab5 and rab9 mutants were only partially inhibitory or ineffective, respectively. Expression of wild-type or GTPase-deficient rab mutants synergized with VacA in inducing vacuolization. In vitro fusion of late endosomes was enhanced by active rab7 and inhibited by inactive rab7, consistent with vacuole formation by merging of late endosomes in a process that requires functional rab7. Taken together, the effects of overexpressed rab proteins described here indicate that continuous membrane flow along the endocytic pathway is necessary for vacuole growth.

Bacterial Proteins↗
PubMed1997

Crystal structure of a non-toxic mutant of heat-labile enterotoxin, which is a potent mucosal adjuvant.

Two closely related bacterial toxins, heat-labile enterotoxin (LT-I) and cholera toxin (CT), not only invoke a toxic activity that affects many victims worldwide but also contain a beneficial mucosal adjuvant activity that significantly enhances the potency of vaccines in general. For the purpose of vaccine design it is most interesting that the undesirable toxic activity of these toxins can be eliminated by the single-site mutation Ser63Lys in the A subunit while the mucosal adjuvant activity is still present. The crystal structure of the Ser63Lys mutant of LT-I is determined at 2.0 A resolution. Its structure appears to be essentially the same as the wild-type LT-I structure. The substitution Ser63Lys was designed, based on the wild-type LT-I crystal structure, to decrease toxicity by interfering with NAD binding and/or catalysis. In the mutant crystal structure, the newly introduced lysine side chain is indeed positioned such that it could potentially obstruct the productive binding mode of the substrate NAD while at the same time its positive charge could possibly interfere with the critical function of nearby charged groups in the active site of LT-I. The fact that the Ser63Lys mutant of LT-I does not disrupt the wild-type LT-I structure makes the non-toxic mutant potentially suitable, from a structural point of view, to be used as a vaccine to prevent enterotoxigenic E. coli infections. The structural similarity of mutant and wild-type toxin might also be the reason why the inactive Ser63Lys variant retains its adjuvant activity.

Adjuvants, Immunologic↗
PubMed1997

Current developments in new vaccines for adolescents.

Routine immunization in early childhood is well accepted and healthcare policies are designed to achieve optimal immunization coverage in this age group. This machinery is so well designed that when new vaccines become available, they are included in infant immunization schedules, even if not necessarily needed at this age, just because we have no mechanism to get in touch, in a systematic way, with adolescents, adults, and the elderly. However, it is well recognized that adolescence is the age with the greatest risk for exposure to sexually transmitted infectious diseases. As several vaccines targeted at this age group are likely to become available in the near future, healthcare policies and vaccine combinations should be designed to provide an optimal vaccine coverage at this age. Implementing this vaccination policy would not only provide the most effective means of preventing infectious diseases, but it would also anchor adolescents into a chartered channel of comprehensive preventive health care.

Adolescent↗