Overview--combination vaccines and simultaneous administration. Past, present, and future.
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Communicable Disease Control↗
Biomedical subjects
D L Burns
Publications and source records attributed to D L Burns.
Localization of the promoter for the ptl genes of Bordetella pertussis, which encode proteins essential for secretion of pertussis toxin.
The ptl locus of Bordetella pertussis, which encodes proteins necessary for the secretion of pertussis toxin into the extracellular medium, is located directly downstream from the ptx locus, which encodes the structural subunits of the toxin. We have found that the ptx promoter is essential for expression of the ptl genes. A strain of B. pertussis which lacked only the ptx promotor region but which retained all other portions of the ptx-ptl region did not produce PtlF. Moreover, insertion of a functional ptx promoter from B. pertussis at the 5' end of the ptx region of Bordetella bronchiseptica resulted in the production of PtlF in B. bronchiseptica, a species which normally does not produce PtlF. These results suggest that the ptx operon is larger than originally proposed and contains both the ptx and ptl genes.
Bacterial Proteins↗
Detection and subcellular localization of three Ptl proteins involved in the secretion of pertussis toxin from Bordetella pertussis.
The ptl locus of Bordetella pertussis contains eight open reading frames which are predicted to encode proteins (PtlA to PtlH) that are essential for secretion of pertussis toxin from the bacterium and which are members of a family of transport proteins found in other types of bacteria. We have detected PtlE, PtlF, and PtlG in immunoblots of extracts of B. pertussis by using antibodies raised to fusion proteins consisting of maltose-binding protein and the individual Ptl proteins. These proteins have apparent molecular weights similar to those predicted by DNA sequence analysis. Cell fractionation studies indicated that all three Ptl proteins are associated with the membranes of B. pertussis, suggesting that the Ptl proteins form a gate or channel which facilitates transport of pertussis toxin. Cell extracts of other Bordetella spp. were probed with antibodies to Ptl proteins for the presence of these transport proteins. Neither Bordetella parapertussis nor Bordetella bronchiseptica contained detectable levels of PtlE or PtlF. This lack of detectable Ptl protein may provide an explanation for previous observations which indicated that introduction of the genes encoding pertussis toxin subunits from B. pertussis into other Bordetella spp. results in production of the toxin but not secretion of the toxin.
Bordetella↗
Molecular characterization of an operon required for pertussis toxin secretion.
Mutants of Bordetella pertussis which are defective in secretion of pertussis toxin were isolated and characterized. The region of the B. pertussis chromosome identified by mutagenesis as playing a role in transport of pertussis toxin was sequenced. Analysis of this region revealed eight open reading frames, seven of which predict a protein exhibiting homology with one of the VirB proteins of Agrobacterium tumefaciens, which are involved in the transport of the T-DNA molecule across bacterial and plant membranes. Thus a set of accessory proteins are most likely involved in the secretion of pertussis toxin, and these proteins appear to be members of a family of proteins involved in the secretion of macromolecules from bacteria.
Agrobacterium tumefaciens↗
Antibodies to Bordetella pertussis adenylate cyclase toxin in neonatal and maternal sera.
To investigate the high prevalence among infants of antibodies to Bordetella pertussis adenylate cyclase toxin (ACT), cord-blood sera were examined for antibodies to ACT, filamentous hemagglutinin (FHA) and pertussis toxin (PT) using immunoblot analysis. Antibodies reactive with ACT were the most prevalent in neonatal sera. Similar reactivity of IgG with ACT was found in each sample of a given neonatal-maternal pair, yet IgM reactive with ACT was virtually absent in neonatal sera, suggesting that antibodies to ACT are maternally derived. Antibodies to ACT might come from infection or childhood vaccination of the mothers since pertussis vaccines from all US manufacturers elicited antibodies to ACT in mice. Alternatively, these antibodies may have been elicited by a cross-reactive antigen such as Escherichia coli alpha-hemolysin, since all of the neonatal and maternal sera contained antibodies reactive with alpha-hemolysin.
Adenylate Cyclase Toxin↗
Binding of pertussis toxin to lipid vesicles containing glycolipids.
The binding of pertussis toxin and its B oligomer to lipid vesicles containing glycosphingolipids was studied. Both pertussis toxin and the B oligomer bound to lipid vesicles containing ganglioside GD1a. Binding of pertussis toxin to these vesicles decreased upon treatment of the vesicles with neuraminidase, suggesting that sialic acid residues are important for efficient binding of the toxin to GD1a.
Carbohydrate Sequence↗
Immunochemical localization of a region of chaperonin-60 important for productive interaction with chaperonin-10.
An IgG1 monoclonal antibody (mAb 54G8) which binds to both Bordetella pertussis chaperonin-60 (cpn60) and Escherichia coli cpn60 (GroEL) was produced. mAb 54G8 as well as Fab fragments prepared from this antibody were found to abolish the ability of chaperonin-10 (cpn10, GroES) to inhibit the ATPase activity of both B. pertussis cpn60 and E. coli cpn60. Electron microscopy was used to localize the binding site of the monoclonal antibody on the B. pertussis cpn60 molecule. In the absence of the antibody, the B. pertussis molecule exhibited the tetradecameric structure typical of cpn60. Both end views (showing 7-fold symmetry of the face of the molecule) and side views were evident. When mAb 54G8 was bound, B. pertussis cpn60 molecules appeared to be cross-linked so that they formed long chains. Only side views of the molecules were seen in these long chains. When B. pertussis cpn60 complexed with Fab fragments of mAb 54G8 was examined, chains were no longer observed. Instead, side views of B. pertussis cpn60 were often seen with Fab fragments extending from the ends of the molecule. These data indicate that mAb 54G8 appears to bind at or near the end of the B. pertussis cpn60 molecule and that binding of mAb 54G8 at this location affects the ability of cpn10 to productively interact with cpn60, most likely either by sterically blocking the binding of cpn10, by affecting the conformation of cpn60 in such a way that it no longer binds cpn10, or by inhibiting proper transduction of the effects of cpn10 binding.
Adenosine Triphosphatases↗
Interaction of pertussis toxin with cells and model membranes.
The interaction of pertussis toxin (PT) with cells and model membranes was investigated by examining PT-induced intoxication of Chinese hamster ovary cells and by studying the binding of PT and its subunits to phospholipid vesicles. Since certain bacterial toxins require an acidic environment for efficient interaction with membranes and subsequent entry into the cell, the requirement for an acidic environment for PT action was examined. PT, unlike bacterial toxins such as diphtheria toxin, did not require an acidic environment for efficient intoxication of Chinese hamster ovary cells. Potential modes by which PT might interact with biological membranes were studied by examining the binding of PT to a model membrane system. PT was found to be capable of interacting with phospholipid vesicles, however, efficient binding of the toxin to the vesicles occurred only in the presence of both ATP and reducing agent. The A subunit portion of the toxin bound preferentially to the vesicles while little binding of the B oligomer portion of PT to the model membranes was observed. Isolated A subunit, in the absence of the B oligomer, also bound to the vesicles with optimal binding occurring in the presence of reducing agent. After cleavage of the A subunit by trypsin, probably at Arg-181, Arg-182, and/or Arg-193, large fragments which lacked the C-terminal portion of the A subunit of PT no longer associated with the lipid vesicles. These results suggest that the A subunit of PT can interact directly with a lipid matrix and, if freed from the constraints imposed by the B oligomer, may be capable of interacting with cellular membranes.
Animals↗
Myocardial infarction caused by cardiac microvasculopathy in a patient with the primary antiphospholipid syndrome.
Antiphospholipid antibodies occur in various clinical states, including the primary antiphospholipid syndrome. Clinical features in these conditions appear to be caused by vasculopathy associated with the presence of these antibodies. We report the case of a patient with primary antiphospholipid syndrome who experienced cardiac necrosis secondary to myocardial microvasculopathy in the absence of vasculitis. This case demonstrates unequivocally that noninflammatory myocardial microvasculopathy occurs in the primary antiphospholipid syndrome per se without any clinical or immunologic signs of systemic lupus erythematosus or other disease process. The histopathologic findings in the skin and myocardial biopsies showed a noninflammatory vasculopathy characterized by bland thrombi and lack of infiltration of the vessel wall by inflammatory cells. Ultrastructural examination of the myocardial biopsy confirmed the vascular thrombosis and endothelial activation and showed no deposits in basement membranes. The patient survived after appropriate treatment. Evidence presented here supports the concept that the vasculopathy in the antiphospholipid syndrome is distinct from other types of vascular occlusions seen in systemic lupus erythematosus. We suggest that myocardial biopsy can be crucial in showing an underlying myocardial ischemic process despite "normal" findings on coronary angiography. Results of the biopsy hastened the decision to use potentially lifesaving plasmapheresis and anticoagulation therapy in this patient.
Adult↗
Bordetella heat-labile toxin causes release of radioactivity from smooth muscle cells labeled with [14C]arachidonic acid.
The effect of Bordetella heat-labile toxin (HLT) on cultured vascular or tracheal smooth cells labeled with [14C]arachidonic acid for 3 h was examined. At 37 degrees C, in the presence of Ca2+, HLT induced the release of radioactive substances from the cells in a dose-dependent manner but HLT had no effect on release of radioactivity from cells at 0 degrees C or in the absence of Ca2+. After cells were exposed to HLT, a 2-h lag period occurred before release of radioactivity was detected. The substances released from cells by HLT were identified as arachidonic acid and phosphatidylcholine.
Animals↗
Recent advances in the development of pertussis vaccines.
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Bacterial Outer Membrane Proteins↗
Pertussis toxin and target eukaryotic cells: binding, entry, and activation.
Pertussis toxin, a protein virulence factor produced by Bordetella pertussis, is composed of an A protomer and a B oligomer. The A protomer consists of a single polypeptide, termed the S1 subunit, which disrupts transmembrane signaling by ADP-ribosylating eukaryotic G-proteins. The B oligomer, containing five polypeptides, binds to cell receptors (most likely containing carbohydrate) and delivers the S1 subunit. Current knowledge suggests that expression of ADP-ribosyltransferase activity in target eukaryotic cells arises after 1) nucleotides and membrane lipids allosterically promote the release of the S1 subunit; and 2) the single disulfide bond in the S1 subunit is reduced by reductants such as glutathione. This model suggests conditions for the proper use of the toxin as an experimental reagent.
Animals↗
Heat-labile toxin from Bordetella parapertussis induces contraction of smooth muscle cells in culture.
The ability of Bordetella heat-labile toxin (HLT) to contract various types of cells in culture was examined. HLT from B. parapertussis induced contraction of cultured smooth muscle cells from trachea, intestine, uterus and vas deferens as well as from aorta. The time required for contraction decreased as the dose of B. parapertussis HLT increased from 3 to 100 MID/ml. Upon exposure of cells to concentrations of toxin greater than 100 MID/ml, at least 2 hr was required for contraction. HLT from B. parapertussis did not affect cultured cardiac or skeletal muscle cells within 8 hr after the exposure to HLT (100 MID/ml). No effect on other types of primary culture cells or established cells such as Chinese hamster ovary (CHO) cells has been described. These data indicate that the primary target cells for HLT might be smooth muscle cells.
Animals↗
Properties of pertussis toxin B oligomer assembled in vitro from recombinant polypeptides produced by Escherichia coli.
The subunits that make up the pentameric B oligomer of pertussis toxin (S2, S3, S4, and S5) were individually synthesized as recombinant polypeptides in Escherichia coli, isolated as insoluble inclusion bodies, and assembled into a multimeric form in vitro by spontaneous association following treatment with a chaotropic agent, reduction, and reoxidation. The recombinant B multimer, purified by fetuin-Sepharose affinity chromatography, contained all four of the individual subunits and possessed the mitogenic and hemagglutinating activities characteristic of the native B oligomer. Immunization of mice with the recombinant B oligomer elicited antibodies that neutralized pertussis toxin in vitro and, moreover, provided protection in vivo against the leukocytosis-promoting activity of the toxin. These results demonstrate the potential for assembly of complex multimeric proteins from recombinant DNA-derived polypeptides and provide a novel means for production of an acellular pertussis vaccine component.
Animals↗
Human serologic response to envelope-associated proteins and adenylate cyclase toxin of Bordetella pertussis.
The human serologic response to several envelope-associated proteins and adenylate cyclase toxin of Bordetella pertussis was examined using immunoblot techniques. Antigens recognized by sera from individuals with culture-confirmed pertussis and by sera from infants immunized with three doses of conventional whole-cell pertussis vaccine included a 63,000-Da protein that was shown to be antigenically related to a mycobacterial heat-shock protein. A 29,000-Da protein reacted with sera from convalescent individuals, whereas a 91,000-Da protein reacted with sera from vaccinated individuals. Antibodies to adenylate cyclase toxin were common in sera from individuals diagnosed with pertussis. B. pertussis lipooligosaccharide was also recognized by antibodies in some of these sera. These data suggest that some of these antigens may play a role in immunity to pertussis.
Adenylate Cyclase Toxin↗
Contribution of the B oligomer to the protective activity of genetically attenuated pertussis toxin.
An enzymatically deficient recombinant S1 subunit, in which Arg-9 was replaced by Lys, was combined with native B oligomer to form a mutant holotoxin molecule. This molecule exhibited decreased leukocytosis-promoting and histamine-sensitizing activities compared with those of the native toxin, supporting the view that the B oligomer is not responsible for these activities. The protective activity of this genetically attenuated pertussis toxin was compared with that of B oligomer alone. The mutant pertussis toxin and B oligomer were similarly capable of protecting mice against a respiratory infection with Bordetella pertussis, suggesting that the B oligomer makes a significant contribution to the protection afforded by the genetically attenuated holotoxin.
Animals↗
Purification and immunological characterization of a GroEL-like protein from Bordetella pertussis.
A GroEL-like protein from Bordetella pertussis was purified. This protein was found to have the tetradecameric subunit structure typical of the GroEL family of proteins and to contain epitopes similar to those of other members of this family, including a human GroEL-like protein. Active immunization of neonatal mice with the B. pertussis GroEL-like protein provided little protection against an aerosol challenge with B. pertussis. Antibodies to this protein were elicited in mice by a standard diphtheria-tetanus-pertussis (DTP) vaccine but not by an experimental acellular pertussis vaccine. Since the Bordetella GroEL-like protein was found to contain epitopes similar to those on the mammalian analog, the potential exists that vaccination with standard DTP vaccines may induce antibodies which react with the mammalian GroEL analog.
Animals↗
Monoclonal antibodies against the enzymatic subunit of both pertussis and cholera toxins.
A synthetic peptide corresponding to amino acids 6-17 of the A subunit of pertussis toxin was synthesised and used for the immunization of Balb/c mice and the subsequent production of monoclonal antibodies (MAbs). This peptide contains a region of eight amino acids which is homologous to a region in the cholera toxin A subunit. The properties of two of the resultant MAbs are described. Both of the antibodies (CP7-3003F7, an IgG3 and CP7-3004G6X1, an IgG1) react in an ELISA with the peptide and with intact pertussis toxin, pertussis toxin A subunit and cholera toxin A subunit, but do not react significantly with pertussis toxin B subunit, intact cholera toxin, or cholera toxin B subunit. Competition ELISA assays in which the peptide, the intact toxins and the toxin subunits were compared with respect to their ability to inhibit the binding of the MAbs to peptide-coated ELISA plates demonstrated that only pertussis toxin A subunit was as active, on a molar basis, as the peptide. Western blot analyses of the holotoxins confirmed that both MAbs were reactive only with the toxin A subunits. The MAbs were unable to neutralize the activity of cholera toxin or pertussis toxin in a Chinese hamster ovary (CHO) cell assay. Both were also unable to neutralize either the ADP-ribosylation activity or the NAD-glycohydrolase activity of the pertussis toxin A subunit. The significance of these results with respect to the role of this conserved site in the activity of these two toxins is discussed.
Amino Acid Sequence↗