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R Rappuoli

Publications and source records attributed to R Rappuoli.

At least 235 records · Page 13Linked to original sources
PubMed1991

Determination of diphtheria toxin neutralizing antibody titers with a cell protein synthesis inhibition assay.

A method which allows an accurate determination of very low titers of antibodies able to neutralize diphtheria toxin has been developed. The assay is based on the incubation of a reference amount of diphtheria toxin with different dilution of the serum and the evaluation of the residual toxicity of diphtheria toxin on Vero cells. The cells are seeded in 96-well plastic plates with the toxin-serum mixtures and the residual toxin activity is measured as the block of cell protein synthesis after incubation with [14C]leucine. Antitoxin titers as low as 0.002 IU can be reproducibly and accurately measured and the method gives a linear response in the range 0.002 to 8.0 IU/ml.

Animals↗
PubMed1991

Towards third-generation whooping cough vaccines.

To date, the most significant use of recombinant-DNA technologies has been to hyperproduce natural molecules that are difficult to obtain in large quantities by conventional methods. However, genetic manipulation can also be an efficient way to modify the properties of natural molecules in order to make them more suitable for human use. In the development of third-generation whooping cough vaccines, recombinant-DNA methods were used to remove the enzymatic activity of pertussis toxin in order to obtain a new molecule which is devoid of toxicity, and can be used for safer vaccination against this disease.

Animals↗
PubMed1991

Phase I clinical trial of an acellular pertussis vaccine composed of genetically detoxified pertussis toxin combined with FHA and 69 kDa.

An acellular pertussis vaccine composed of genetically detoxified pertussis toxin (PT-9K/129G), filamentous haemagglutinin (FHA) and pertactin (69 kDa protein) was evaluated in adult volunteers, in double blind, versus placebo. No fever was reported in either group. Mild local reactions were reported after injection of both vaccine and placebo. After the first dose a marked increase in antibodies to PT, FHA and 69 kDa protein was seen in vaccinated subjects with the exception of one who responded well to PT and FHA but did not show a humoral response to the 69 kDa protein. All vaccinees acquired cellular immunity against the three antigens. No significant variation was observed in the humoral or cellular responses after the second dose.

Adhesins, Bacterial↗
PubMed1991

Computer modelling of the NAD binding site of ADP-ribosylating toxins: active-site structure and mechanism of NAD binding.

Five ADP-ribosylating bacterial toxins, pertussis toxin, cholera toxin, diphtheria toxin, Escherichia LT toxin and Pseudomonas exotoxin A, show significant homology in selected segments of their sequence. Site-directed mutagenesis and chemical modification of residues within these regions cause loss of catalytic activity and of NAD binding. On the basis of these results and of molecular modelling based on the three-dimensional structure of exotoxin A, the geometry of an NAD binding site common to all the toxins is deduced and described in the paper. For diphtheria toxin, sequence similarity with exotoxin A is such that its preliminary structure can be computed by molecular modelling, whereas for the other toxins similarity appears to be restricted to the NAD binding site. Moreover, an analysis of molecular fitting of the NAD molecule into its binding cavity suggests a new model for the conformation of the bound NAD that better accounts for all available experimental information.

ADP Ribose Transferases↗
PubMed1991

Structural and genetic analysis of the bvg locus in Bordetella species.

The bvg locus contains two genes, bvgA and bvgS, which control the expression of the virulence-associated genes in Bordetella species by a system similar to the two-component systems used by a variety of bacterial species to respond to environmental stimuli. We determined the nucleotide sequence of the bvg loci of Bordetella parapertussis and Bordetella bronchiseptica and compared them with the previously determined sequence of Bordetella pertussis. The nucleotide and amino acid sequences of the bvg loci of these species are well conserved in those regions coding for the protein domains which have putative kinase and DNA-binding activities. In marked contrast, the region of BvgS that codes for the protein domain with putative sensor activity shows a high degree of variability. In total, we find 198 base-pair changes in the bvg loci of B. parapertussis and B. bronchiseptica relative to the bvg locus of B. pertussis. One hundred and seventy-three of these base-pair changes are identical in B. parapertussis and B. bronchiseptica. This confirms our previous observation that B. parapertussis and B. bronchiseptica are more related to each other than to B. pertussis. We have mapped the mutations that cause phase changes in B. bronchiseptica and we have found that in three cases these are due to spontaneous deletions in the bvgS gene. The wild-type bvg locus present on a multicopy plasmid cannot complement avirulent derivatives of B. bronchiseptica to wild-type levels, but it can do so when the bvgA gene on the plasmid is inactivated. This suggests that hyperexpression of bvgA down-regulates the bvg system.

Amino Acid Sequence↗
PubMed1991

The bvg-dependent promoters show similar behaviour in different Bordetella species and share sequence homologies.

The expression of the virulence-associated genes in Bordetella species is co-ordinately regulated by the gene products encoded by the bvg locus. In Bordetella pertussis the expression of this locus is regulated by the P1, P2, P3 and P4 promoters which are located in a 350 bp DNA fragment also containing the PFHA promoter. Here we report the transcriptional regulation of the bvg locus and the fha gene in Bordetella parapertussis and a sequence analysis of the bvg-regulated promoters. The Pp1, Pp2, Pp4 and PpFHA promoters are indistinguishable, both in transcription initiation sites and environmental regulation, from the corresponding promoters of B. pertussis, while the Pp3 promoter is not active. Sequence homologies from nine bvg-regulated promoters show a conserved dinucleotide, 5'-TG-3', at approximately one turn of helix upstream of the -10 5'-A.AaTat-3' region, and a 5'-TTTCC-3' sequence in the -90 region. Since the nucleotide sequence of the inactive Pp3 promoter shows several base substitutions with respect to the found sequence homologies, it is likely that some of these bases play an essential role in promoter activity.

Amino Acid Sequence↗
PubMed1991

Lymphokine secretion and cytotoxic activity of human CD4+ T-cell clones against Bordetella pertussis.

Human CD4+ T-cell clones specific for pertussis toxin and other Bordetella pertussis antigens have been tested for their cytotoxic activity, lymphokine production, and capacity to induce immunoglobulin synthesis. Clones specific for the S1 subunit of pertussis toxin were cytotoxic for autologous Epstein-Barr virus-transformed B cells, which had been pulsed with the native antigen, the recombinant S1 subunit of pertussis toxin, or synthetic peptides derived from the S1 amino acid sequence. The killing of antigen-pulsed target cells was class II restricted. All of the T-cell clones produced mostly interleukin-2 and gamma interferon and assisted allogeneic B cells in the production of immunoglobulins M and G but not immunoglobulin E. The potential in vivo role of the cytotoxic activity of these clones is discussed.

B-Lymphocytes↗
PubMed1991

Properties of the B oligomer of pertussis toxin.

The B oligomer of pertussis toxin was purified from culture supernatants of Bordetella pertussis strains which do not secrete the S1 subunit. The purified B oligomer is devoid of toxicity for CHO cells and other in vivo properties of the toxin, such as leukocytosis and histamine sensitization, but it retains the abilities to agglutinate erythrocytes and to induce the proliferation of T lymphocytes. The B oligomer is also able to induce protective immunity in mice but is less potent than molecules containing the S1 subunit also.

Animals↗
PubMed1991

Properties of pertussis toxin mutant PT-9K/129G after formaldehyde treatment.

Formaldehyde treatment is a method routinely used to detoxify diphtheria, tetanus, and pertussis toxins as well as other molecules suitable for vaccine production. To investigate whether chemical detoxification alters the immunological properties of vaccine components, we have treated the pertussis toxin mutant PT-9K/129G with formaldehyde and tested the properties of the resulting molecules. Very low concentrations of formaldehyde stabilize the molecule without affecting the physicochemical and immunological parameters. Increasing doses of formaldehyde abolish the mitogenic and hemagglutinating activities of PT-9K/129G. At the same time, the molecule loses the ability to be recognized by a monoclonal antibody specific for a major protective epitope on the S1 subunit of pertussis toxin and its affinity for anti-pertussis toxin polyclonal antibodies is also reduced. In marked contrast, the ability of PT-9K/129G to be recognized by human T-cell clones is not affected by Formalin treatment. In vivo, the formaldehyde-treated molecules induce amounts of specific antibodies comparable with those of untreated molecules but significantly lower levels of toxin-neutralizing antibodies. Furthermore, the formaldehyde-treated molecules also show a reduced protective activity in the intracerebral challenge assay.

Animals↗
PubMed1991

Further analysis of the sequence of the S1 subunit of pertussis toxin.

The two published sequences of the pertussis toxin operon differ in 3 bp in the S1 subunit gene. In this report, we provide evidence that Bordetella pertussis strains are able to produce active pertussis toxin only when they contain one of the two possible nucleotide sequences.

Amino Acid Sequence↗
PubMed1991

Identification of subregions of Bordetella pertussis filamentous hemagglutinin that stimulate human T-cell responses.

Filamentous hemagglutinin (FHA), a 220-kDa protein that mediates the adhesion of Bordetella pertussis to eukaryotic cells, is a component of acellular vaccines against whooping cough. To identify the subregions of FHA that are immunogenic for T cells, 16 human T-cell clones were raised against purified FHA and tested for the recognition of recombinant and proteolytic fragments. The clones were found to map either in the carboxy-terminal or the amino-terminal part of the FHA molecule, but none of them recognized the central region, which contains a sequence that is homologous to that of the eukaryotic protein fibronectin. These data suggest that subregions of FHA that do not contain sequences that are potentially cross-reactive with self proteins may be sufficient to induce an immune response against the whole protein.

Adhesins, Bacterial↗
PubMed1991

Differential response of the bvg virulence regulon of Bordetella pertussis to MgSO4 modulation.

Magnesium sulfate is known to repress the expression of the virulence factors of Bordetella pertussis that are coordinately regulated by the bvg locus. We have tested the time required by MgSO4 to repress the synthesis of several bvg-regulated mRNA species and found that the promoters of the virulence genes (pertussis toxin, adenylate cyclase, and filamentous hemagglutinin) are repressed in 6 min, while the autogenously regulated promoters of the bvg locus (P1, P3, and P4) are repressed only several hours later. These data show a differential behavior between regulated and autoregulated genes of the bvg regulon.

Adenylate Cyclase Toxin↗
PubMed1991

Human T cell immunity against Bordetella pertussis analyzed at clonal level.

Cloned peripheral blood T lymphocytes from an immune donor were grown in interleukin 2 and tested for proliferation in response to inactivated Bordetella species (B. pertussis, B. parapertussis and B. bronchiseptica) and mutants deficient for the expression of virulence-associated antigens. All the T cell clones obtained were CD4+8- and recognized specifically the Bordetella antigens when presented by autologous B cells. On the basis of the responsiveness to the whole inactivated bacteria, it was possible to cluster the twelve clones obtained into four groups with the following specificity: 1) filamentous hemagglutinin (FHA); 2) B. pertussis specific antigens; 3) virulence-associated Bordetella specific antigens; 4) non-virulence-associated Bordetella specific antigens. Employing two new B. pertussis deletion mutants, clone 6 (representative of cluster 1), was found to recognize the C-terminus of FHA. Furthermore, 3 out of 4 clones of cluster 3 were specifically stimulated by the soluble 69,000 M.W. protein from the outer membrane of B. pertussis. Surprisingly, none of the twelve clones obtained by stimulation in vitro with whole inactivated bacteria recognized PT. Thus, PT does not seem to be the most representative antigen on the whole inactivated bacteria. However, when a new generation of clones was obtained using soluble PT as the in vitro stimulus, it was observed that 11 clones of this group recognized this antigen. Furthermore, the majority of them was against the subunit Sl of PT. Therefore, we can conclude that a T cell memory against PT exists in a donor who has had pertussis several years before. In conclusion, these results provide useful information in the attempt to obtain a simplified acellular vaccine for whooping cough.

Antigen-Presenting Cells↗
PubMed1990

The subunit S1 is important for pertussis toxin secretion.

Pertussis toxin is a protein containing five noncovalently linked subunits which are assembled into the monomer A (containing the subunit S1) and the oligomer B (containing subunits S2, S3, S4, and S5 in a 1:1:2:1 ratio). Each of the five subunits is synthesized as a precursor containing a secretory leader peptide and is secreted into the periplasm of Bordetella pertussis where the five subunits are assembled into the oligomeric structure and then released into the culture medium. In the absence of subunit S3 the remaining subunits are not secreted into the medium, thus suggesting that the assembled structure is necessary for the release of the toxin into the supernatant. In this study we describe four B. pertussis mutants which secrete into the medium low amounts of the B oligomer of pertussis toxin. These mutants have single or multiple changes in the gene encoding the S1 subunit and synthesize S1 proteins with altered conformation which are not assembled into the holotoxin and are apparently degraded in the periplasm. These data indicate that while the B oligomer alone has the structural information necessary for the extracellular export of pertussis toxin, the S1 subunit is required for its efficient release into the medium.

Base Sequence↗
PubMed1990

DNA sequence homology between attB-related sites of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, and the attP site of gamma-corynephage.

Chromosomal restriction fragments of Corynebacterium ulcerans and C. diphtheriae, containing an integration site for corynephages of the beta family, show homology on Southern blots. Homologous DNA in also found in the soil isolate C. glutamicum, although this strain is not susceptible to beta-corynephages. Three of these DNA fragments, one for each bacterial strain, and a fragment of gamma-corynephage DNA previously shown to contain the phage integration site, were cloned and sequenced. Alignment of the 3 bacterial sequences shows a very high degree of homology in a stretch of ca 120 nucleotides, whereas the rest of the sequences is generally non-homologous. Within this common bacterial portion, a segment of ca. 96 nucleotides (core sequence) is also highly homologous to the phage sequence. The first half (ca. 50 bp) of the core sequence is identical in all aligned sequences whereas the second half, which is largely occupied by a stem-and-loop structure, contains point mutations peculiar to each clone. The described sequences are likely to be involved in phage integration/excision processes.

Attachment Sites, Microbiological↗
PubMed1990

Histidine-21 is involved in diphtheria toxin NAD+ binding.

Histidine-21 is the sole histidine present in the A chain of diphtheria toxin and recent evidence suggests that it is involved in NAD+ binding. Fluorimetric assays of NAD+ binding and diethylpyrocarbonate modification performed at different pH values provide further insights on the role of this residue and indicate that its pKa value is 6.3. Conformational changes of subunit A of diphtheria toxin have been detected by analysis of tryptophan fluorescence in the pH 2.5-4 and pH 9-10.5 ranges. This indicates that histidine-21 is unlikely to be involved in the low pH-driven conformational change of diphtheria toxin.

Binding Sites↗
PubMed1990

Positive transcriptional feedback at the bvg locus controls expression of virulence factors in Bordetella pertussis.

Regulation of the genes coding for virulence factors in Bordetella pertussis is controlled by the bvg locus, which encodes one putative sensory protein (BvgS) and one positive regulator of transcription (BvgA). We have studied the transcription of the bvg locus and found that this is controlled by a 350-base-pair DNA fragment, which contains five promoters, three of which transcribe the bvg locus, one transcribes an antisense RNA, and one transcribes a virulence-associated gene. Under noninducing conditions, only the promoter P2 is active and this is responsible for the production of low amounts of regulatory proteins. Upon induction, the other four promoters become active and, by a mechanism that may involve transcriptional and translational regulation, cause a 50-fold increase of the transcriptional activator BvgA. A model of the autoregulation of the bvg locus is presented.

Bacterial Proteins↗