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A Phalipon

Publications and source records attributed to A Phalipon.

26 records · Page 2Linked to original sources
PubMed1992

Identification and characterization of B-cell epitopes of IpaC, an invasion-associated protein of Shigella flexneri.

Invasion plasmid antigen C (IpaC) is a 43-kDa plasmid-encoded protein associated with the ability of shigellae to invade epithelial cells. This protein is consistently strongly recognized by sera from convalescent patients and monkeys experimentally infected with shigellae. The strong immunogenicity of IpaC in the course of natural infection makes it a good candidate as a potentially protective antigen. To map the B-cell epitopes of this protein, the gene encoding IpaC was cloned and expressed at a high level in Escherichia coli. The partially purified recombinant protein was used to raise rabbit polyclonal antisera and murine monoclonal antibodies. A lambda gt11 ipaC gene library was screened with the antisera and antibodies. Recombinant DNA clones producing specific antigenic determinants were isolated, and the sequence of their DNA inserts was determined. The amino acid sequence of each determinant was deduced from the minimal overlap of DNA inserts of multiple antibody-positive DNA clones. Two distinct epitopes, located between amino acid residues 25 and 33 and 90 and 97, were identified. Two additional B-cell epitopes which were located between residues 297 and 349, near the carboxy-terminal end of the protein, were characterized. Each of these epitopes was also recognized by sera from convalescent humans and monkeys. Therefore, it seems likely that these epitopes are relevant to the humoral response against IpaC during natural infection.

Animals↗
PubMed1990

Structure and interaction with model membranes of a CNBR peptide of diphtheria toxin B fragment.

The mechanism by which diphtheria toxin (DT) crosses the endosomal membrane to exert its biological activity in the cell cytoplasm is still poorly understood. By measuring the change in conductance of planar lipid bilayers induced by cyanogen bromide peptides of fragment B of DT, we have identified a domain that could be involved in the pH-dependent membrane interaction of DT. Moreover, infrared spectroscopy has allowed us to demonstrate that, at low pH, in the presence of a lipid bilayer, this domain is mainly helical with the axis of the helices oriented parallel to the lipid acyl chains. On the basis of these results, we have designed mutants of DT which should provide information about the molecular mechanism of the DT membrane translocation process.

Diphtheria Toxin↗
PubMed1989

The E2 antigen, a 32 kd glycoprotein involved in T-cell adhesion processes, is the MIC2 gene product.

E2 is a 32 kd human T-cell surface glycoprotein involved in spontaneous rosette formation with erythrocytes. A 1.11 kb cDNA was isolated from a lambda gt11 expression library by screening with monoclonal antibodies directed against E2. The primary structure of E2, deduced from the nucleotide sequence of its gene, comprises 185 amino acids and is devoid of N-linked glycosylation sites. The E2 protein is rich in proline residues and displays an organization typical of an integral membrane protein. Northern blotting showed a good correlation between mRNA abundance, E2 surface density and the level of T cell differentiation. In fact, nucleotide sequencing revealed that E2 is the MIC2 gene product, previously identified with the 12E7 Mab. Xg(a-) female individuals have no E2 molecule on the surface of their red cells, in contrast with Xg(a+) individuals, but have the molecule in their cytoplasm, in the form of the 28 kd precursor. These findings show that the E2 antigen, a cell surface molecule involved in T cell adhesion processes, is the product of the MIC2 gene, the only pseudoautosomal gene to be described in man.

12E7 Antigen↗
PubMed1989

Expression of a poliovirus type 1 neutralization epitope on a diphtheria toxin fusion protein.

The diphtheria toxin (DT) secreted by Corynebacterium diphtheriae is used after formolization as an efficient vaccine against diphtheria. In an attempt to evaluate its capacity to present heterologous peptide sequence in a recognized form, we created in-phase insertion in the gene encoding the non-toxic mutant protein CRM228 of DT. The sequence chosen for insertion was the synthetic DNA fragment encoding a poliovirus neutralization epitope. Tripartite fusion proteins comprising the mutant DT, the poliovirus peptide and beta-galactosidase were obtained in E. coli and purified by affinity chromatography. These fusion proteins reacted both with antibodies directed against the DT and a poliovirus specific monoclonal antibody. Moreover, these hybrid toxins induced protective antibodies against the lethal effect of DT and neutralizing antibodies against poliovirus. We conclude that the modification of highly immunogenic DT may provide a means for the presentation of foreign peptide sequences to the immune system.

Animals↗
PubMed1989

Evidence for direct regulation of diphtheria toxin gene transcription by an Fe2+-dependent DNA-binding repressor, DtoxR, in Corynebacterium diphtheriae.

Previous studies provided indirect evidence that in Corynebacterium diphtheriae regulation of diphtheria toxin gene (tox) transcription by iron is mediated by a bacterial repressor. By performing in vitro protein-DNA binding experiments, we establish that a corynebacterial Fe2+-sensitive protein, named DtoxR, can bind to a palindromic motif present in the tox promoter region. Binding of this factor prevents the interaction of the transcription initiation machinery with presumptive critical promoter elements, providing evidence that DtoxR is responsible for the repression of toxinogenesis observed in iron-containing growth medium.

Binding, Competitive↗
PubMed1988

Expression of a biologically active diphtheria toxin fragment B in Escherichia coli.

The toxB gene of Corynebacterium diphtheriae bacteriophage beta encoding the B fragment of diphtheria toxin was cloned into an inducible expression vector. When expressed in Escherichia coli, fragment B was not proteolysed and was indistinguishable, by immunological criteria, from wild-type C. diphtheriae-derived fragment B. Soluble fragment B was partially purified from the cytoplasm by saline precipitation steps and was shown to compete with the wild-type diphtheria toxin for binding to receptors of sensitive eukaryotic cells. A complete diphtheria toxin was reconstituted by formation of the disulphide bridge between purified fragment A and recombinant fragment B, which migrates at the expected Mr on Western blots and which was able to block protein synthesis by ADP-ribosylation of elongation factor-2, thereby indicating that the recombinant fragment B had retained its biological activity.

Animals↗
PubMed1987

Genetically engineered diphtheria toxin fusion proteins carrying the hepatitis B surface antigen.

Tripartite fusion proteins comprising the nontoxic mutant protein CRM228 of diphtheria toxin (DT), the hepatitis B virus surface antigen (HBsAg), and beta-galactosidase were obtained by expression of hybrid genes from the pR promoter of bacteriophage lambda and purification by affinity chromatography. The antigenicity and immunogenicity of the individual protein constituents were analyzed. A major neutralizing epitope of DT was inactivated by the HBsAg insertion into the DT B fragment. The fusion proteins elicited antibodies reactive with 22 nm HBsAg particles. This suggests a novel approach towards the use of DT mutants as immunogenic carriers of heterologous antigens.

Animals↗