Characterization of murine monoclonal antibodies that recognize defined epitopes of pertussis toxin and neutralize its toxic effect on Chinese hamster ovary cells

Walker, Mark J.; Wehland, Jürgen; Timmis, Kenneth N.; Raupach, Bärbel; Schmidt, M. Alexander

doi:10.1128/iai.59.11.4249-4251.1991

Cited by 12 publications

(8 citation statements)

References 23 publications

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“…Antibodies directed against various $2-or S3-specific peptides showed that those reacting with peptides corresponding to the regions located in the vicinity of residue 105 have the highest toxin-neutralizing activities, presumably because of their interaction with receptor binding (40). On the other hand, most toxin-neutralizing mAbs reactive with the B oligomer, including those presented in this study, appear to recognize conformational epitopes (41,42), suggesting that the receptor-binding of PTX depends on the conformation of the B oligomer.…”

Section: Discussionmentioning

confidence: 99%

Site-specific alterations in the B oligomer that affect receptor-binding activities and mitogenicity of pertussis toxin.

Lobet

Feron

Dequesne

et al. 1993

The Journal of Experimental Medicine

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SummaryPertussis toxin plays a major role in the pathogenesis of whooping cough and is considered an important constituent of vaccines against this disease. It is composed of five different subunits associated in a molar ratio 1S1:1S2:1S3:2S4:1S5. The $1 subunit is responsible for the ADPribosyltransferase activity of the toxin. The B moiety, composed of $2 through $5, recognizes and binds to the target cell receptors and has some ADP-ribosyltransferase-independent activities such as mitogenicity. Site-directed mutagenesis of subunits $2 and $3 allowed us to identify amino acid residues involved in receptor binding. Of all the modifications generated, the deletion of Ash 105 in $2 and of Lys 105 in $3 resulted in the more drastic reduction of binding to haptogiobin and CHO cells, respectively. A holotoxin carrying both deletions presented a mitogenicity reduced to an undetectable level. The combination of these B oligomer mutations with two substitutions in the $1 subunit led to the production of a toxin analog with reduced ADPoribosyltransferasedependent and -independent activities including mitogenicity. As shown by immunoprecipitation with various monodonal antibodies, the mutant holotoxin was correctly assembled and antigenically similar to the native toxin. This toxin analog induced toxin-neutralizing antibodies at the same level as the holotoxin carrying only mutations in the $1 subunit, and may therefore be considered a useful candidate for the development of a new generation vaccine against whooping cough.tel/a pertuss/s, the etiologic agent of whooping cough, duces a number of toxins responsible for the local and systemic manifestations of this important childhood disease (1). Among these toxins, pertussis toxin (PTX) 1 plays a major role in the pathogenesis, in that it expresses a wide variety of biological activities, such as histamine sensitization, activation of insulin secretion, promotion of lymphocytoffs, and others (2). In addition, immunization with PTX has been shown to protect mice against subsequent lethal challenge with virulent R pertuss/s (3). It is therefore considered an important constituent in vaccines against pertussis, and is the major component of the new acellnlar vaccines currently being tested or used in several countries (4,5).Paradoxically, this toxin may itself be responsible for the harmful side effects associated with the current vaccines (6). These side effects range in severity from minor local reactions and simple flushing, to permanent neurological damage t Abbre~aons used in this paper: CRAM, cross-reactive mutant; NAD, nicotinamide adenine dinudeotide; FTX, txn'tussis toxin; WT, wild type. and, in some rare cases, death. Before PTX can effectively and safely be used in pertussis vaccines, complete and irreversible detoxification is therefore required.The toxin is composed of five different subunits, named $1 to $5, based on their decreasing molecular weights. The subunits associate in a molar ratio of 1S1:1S2:1S3:2S4:1S5 to form the holotoxin. Functionally, FTX can be...

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Section: Discussionmentioning

confidence: 99%

Site-specific alterations in the B oligomer that affect receptor-binding activities and mitogenicity of pertussis toxin.

Lobet

Feron

Dequesne

et al. 1993

The Journal of Experimental Medicine

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“…Samples were heated at 95ЊC for 5 min, and after centrifugation (1 min at 17000 ϫ g), 10 l of each supernatant was spotted onto nitrocellulose membranes (Bio-Rad Laboratories, Munich, Germany). The membranes were blocked for 1 h with 10% low-fat milk (0.3%) in PBS and further incubated for 2 h with a cocktail of appropriately diluted monoclonal antibodies E19, E205, and E251 (38), which are reactive with PT subunits. The proteins were detected by using antibody peroxidase-conjugated goat anti-mouse immunoglobulin G as a secondary antibody (Bio-Rad Laboratories).…”

Section: Methodsmentioning

confidence: 99%

Stable expression of pertussis toxin in Bordetella bronchiseptica under the control of a tightly regulated promoter

Suárez

Staendner

Rohde

et al. 1997

Appl Environ Microbiol

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Pertussis toxin (PT) is an essential component of acellular vaccines against whooping cough. However, the industrial production of PT from Bordetella pertussis is impaired by slow growth and poor yields. To overcome these problems, we have constructed a minitransposon containing the tox operon under the control of a tightly regulated promoter responsive to an aromatic inducer. The expression cassettes have been integrated into the chromosome of Bordetella bronchiseptica 5376 and ATCC 10580 bvg. Five recombinant clones containing the tox operon under the control of the Psal promoter, which is activated by the product of nahR, were further characterized. The recombinant clones expressed PT after only 3 h of induction with sodium salicylate at levels similar to those of B. pertussis grown for 24 h. The stability of the engineered phenotype was 100% after 72 h of growth without selective pressure. The growth pattern was not modified either under noninducing conditions or in the presence of the inducer at low concentrations, suggesting that strain performance would not be affected in bioreactors when uncoupled from gene expression. Recombinant PT, which was localized mainly in the periplasm, was purified by affinity chromatography. The recombinant protein was immunologically indistinguishable from wild-type PT and retained its biological activity as determined by the CHO cell-clustering test. These recombinant clones appear to be useful tools for the cost-effective production of PT under conditions of improved biosafety, as demonstrated by the inducible expression of PT uncoupled from the bacterial biomass in a nonvirulent and fast-growing B. bronchiseptica background.

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“…For immunoelectron microscopy, uncoated or antigencoated liposomes were absorbed onto freshly prepared collodium-covered 300-mesh nickel grids and carefully washed with distilled water. After being air dried at room temperature, the grids were treated with protein A-purified anti-FHA or anti-PT polyclonal (21,50) or monoclonal (14,51) antibodies (125 jig of IgG protein ml-') for 60 min at room temperature. Unbound antibodies were removed by a mild spray of PBS (pH 6.9) from a plastic bottle.…”

Section: Methodsmentioning

confidence: 99%

Antibody responses in the serum and respiratory tract of mice following oral vaccination with liposomes coated with filamentous hemagglutinin and pertussis toxoid

et al. 1993

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Mice were orally vaccinated with liposomes coated with filamentous hemagglutinin (FHA) and detoxified pertussis toxin (PT) of BordeteUla pertussis. FHA-and PT-specific immunoglobulin G (IgG) was detected in serum, and both IgG and IgA were detected in lung washes following the immunization. Antibody responses in mice immunized with liposomes coated with FHA and PT were significantly higher than those in mice immunized with free FHA and PT, which demonstrated the adjuvanticity of the liposome carrier. The results indicate the potential usefulness of this approach for eliciting immune responses against FHA and PT (and perhaps other pertussis antigens) in humans and its possible utility in large-scale vaccination to protect against both B. pertussis infection and disease.

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Characterization of murine monoclonal antibodies that recognize defined epitopes of pertussis toxin and neutralize its toxic effect on Chinese hamster ovary cells

Cited by 12 publications

References 23 publications

Site-specific alterations in the B oligomer that affect receptor-binding activities and mitogenicity of pertussis toxin.

Site-specific alterations in the B oligomer that affect receptor-binding activities and mitogenicity of pertussis toxin.

Stable expression of pertussis toxin in Bordetella bronchiseptica under the control of a tightly regulated promoter

Antibody responses in the serum and respiratory tract of mice following oral vaccination with liposomes coated with filamentous hemagglutinin and pertussis toxoid

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