Pertussis holotoxoid formed in vitro with a genetically deactivated S1 subunit.

Abstract

The cytotoxicity of pertussis toxin, a multisubunit exotoxin produced byBordeteUapertussis, is believed to be due to the ADP-ribosyltransferase activity of the Si subunit. We have previously described the recombinant expression of each of the five individual pertussis toxin subunits in Escherichia coli and the production of an enzymaticaily deficient form of the Si subunit by site-directed mutagenesis. We now report the in vitro assembly of holotoxin from native pertussis toxin B oligomer and recombinant Si subunits, the latter purified and refolded from insoluble inclusion bodies. Holotoxin assembled with recombinant S1 of authentic amino acid sequence was indistinguishable from native pertussis toxin in its electrophoretic migration and ability to elicit a cytopathic response in cultured Chinese hamster ovary cells; in contrast, holotoxin assembled with the genetically deactivated analog of recombinant Si displayed greatly diminished cytopathicity.These results verify that the in vitro cytopathic effects of pertussis toxin are the result of the enzymatic activity of the S1 subunit and illustrate the potential for constructing complex quaternary protein structures in vitro from insoluble, unfolded polypeptides derived from expression in recombinant systems.In an effort to create a fully efficacious yet less reactogenic pertussis vaccine, we have focused on the recombinant production of PTX subunit proteins, detoxification of the Si moiety by site-specific mutagenic inactivation of enzyme activity, and the in vitro assembly of a genetic "holotoxoid." For these purposes, the individual subunit polypeptides were expressed at high levels in Escherichia coli (36), and a region of the recombinant Si subunit (Val8-Pro'5) was identified as both necessary for enzyme activity and critical for the formation of its neutralizing, protective epitope (37). Selective site-directed mutagenesis of the subcloned S1 cistronic element permitted us to derive and analog polypeptide (Arg9 -k Lys) retaining the protective epitope, but with strikingly reduced ADP-ribosyltransferase activity (38). We wished to assess the biological activity of this genetically deactivated S1 mutant subunit and evaluate its potential to participate in the production of a recombinant holotoxoid. To this end, we have assembled holotoxin molecules in vitro that are composed of native PTX B oligomer and recombinant SI subunits of either native or mutant sequence and evaluated their relative abilities to elicit cytotoxic responses in cultured cells.Pertussis toxin (PTX) is both a major virulence factor of Bordetella pertussis (1), the etiologic agent of whooping cough (2), and an important antigenic element in vaccines for immunoprotection against disease (3-7). Certain clinical manifestations of infection (1,8,9) and minor reactions to vaccination (10,11) are attributable to the effects of this complex exotoxin. The origin of the extremely rare adverse neurologic events temporally related to pertussis immunization remains controversial (11-15). Chemic...

Contribution of the B oligomer to the protective activity of genetically attenuated pertussis toxin

“…Protein preparations. The recombinant analog form of the Si subunit, with the Arg-9-to-Lys substitution (rS1/1-4), was purified as previously described (3). B oligomer was prepared as previously described (2) and was determined by the Chinese hamster ovary (CHO) cell assay (12) to be >99.8% free of PT.…”

“…Reassociation. The in vitro assembly of the genetically attenuated holotoxin was performed essentially as described by Bartley et al (3). Briefly, 3 ,ug of rSl/1-4 was mixed with 4.2 ,ug of B oligomer in a total volume of 150 ,ul of 50 mM potassium phosphate buffer, pH 7.5, containing 0.3 M NaCl and 2 M urea.…”

“…We have assembled a holotoxin similar to the one previously described (3); the S1 subunit of this genetically attenuated holotoxin has a lysine residue in place of Arg-9, which results in the loss of 99.9% of the ADP-ribosyltransferase activity of the native Si subunit (7). While its enzymatic activity is dramatically attenuated, the structure of the molecule is believed to be similar to that of the native protein, since this S1 mutant is capable of combining with the B oligomer to form a holotoxinlike molecule (3). In this study, we compared the ability of this genetically attenuated holotoxin with that of the B oligomer to protect mice from lethal aerosol challenge with B. pertussis.…”

Novel Strategies in the Design and Production of Vaccines

“…Ofthe original constructs examined, only the analog with the substitution at arginine-9 had lost essentially all of its catalytic potential without modification to its reactivity with a neutralizing monoclonal antibody; successive site-specific substitution experiments also demonstrated that substitutions at glutamate-I 29 had a comparable effect on 8 I . When reassembled in vitro with native B oligomer and analog 8 I subunits containing such substitutions, the resulting holotoxin molecules were impotent for intoxicating cultured cells (Bartley et al, 1989) and provoking toxin-related effects in vivo (Arciniega et al, 1991). Nevertheless, the analog holotoxins were indistinguishable from native toxin in their ability to protect mice against the effects of toxin challenge and against aerosol infection with Bordetella pertussis (Arciniega et al, 1991).…”

Reversal of the CD4+/CD8+T-Cell Ratio in Lymph Node Cells upon In Vitro Mitogenic Stimulation by Highly Purified, Water-Soluble S3-S4 Dimer of Pertussis Toxin

“…The effects of the toxin on cells of the immune system are multiple and include induction of lymphocytosis, inhibition of macrophage migration, adjuvant activity, and T-cell mitogenicity (18). A number of the biological activities of PT, such as lymphocytosis and adjuvant activity, implicate the enzymatic activity of PT in its toxicity and can be abrogated by inactivation of the S1 subunit (1,5,13). In contrast, PT-associated T-cell mitogenicity is mediated by the B oligomer (9,31,36) and appears to be independent of the enzymatic activity of the toxin, as inactivation of the S1 subunit by mutation has no effect on the mitogenic activity of PT (13,36), while alterations in the B oligomer can totally abrogate the mitogenic activity of PT (15,16,(20)(21)(22).…”