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B E Ivins

Publications and source records attributed to B E Ivins.

35 records · Page 2Linked to original sources
PubMed1990

Immunization against anthrax with aromatic compound-dependent (Aro-) mutants of Bacillus anthracis and with recombinant strains of Bacillus subtilis that produce anthrax protective antigen.

The safety and efficacy of five prototype, live anthrax vaccines were studied in Hartley guinea pigs and CBA/J and A/J mice. Two of the strains, Bacillus anthracis FD111 and FD112, are Aro- mutants derived by Tn916 mutagenesis of B. anthracis UM23-1. Bacillus subtilis PA1 and PA2 contain a recombinant plasmid, pPA101 or pPA102, respectively, that carries the gene from B. anthracis encoding synthesis of protective antigen (PA). The final strain, B. subtilis PA7, was isolated in this study from B. subtilis DB104 transformed with pPA101. All five strains were less virulent in guinea pigs and A/J and CBA/J mice than the toxinogenic, nonencapsulated B. anthracis veterinary vaccine Sterne strain. A/J and CBA/J inbred mice represent strains that are innately susceptible and resistant, respectively, to the Sterne strain. These differences in susceptibility are due to differences in ability to produce complement component 5. In guinea pigs, immunization with PA1 or PA2 vegetative cells or PA7 spores protected greater than or equal to 95% from an intramuscular spore challenge with the virulent, "vaccine-resistant" B. anthracis Ames strain. Strain PA2 vegetative cells and strain PA7 spores were as effective as the Sterne strain in Sterne-resistant CBA/J mice, protecting 70% of the mice from Ames strain spore challenge. Immunization with FD111 or FD112 vegetative cells fully protected guinea pigs from challenge. Immunization with FD111 cells protected up to 100% of CBA/J mice and up to 70% of A/J mice.

Animals↗
PubMed1989

Influence of body weight on response of Fischer 344 rats to anthrax lethal toxin.

Groups of Fischer 344 rats were injected intravenously with Bacillus anthracis culture supernatant containing crude anthrax toxin. Times to death of rats given identical toxin preparations varied directly with the weights of the rats (P = 0.0001). In contrast to previous reports, the data indicate that rat weight must be taken into account during in vivo assays of anthrax lethal toxin activity.

Animals↗
PubMed1988

Recent advances in the development of an improved, human anthrax vaccine.

Human anthrax vaccines currently licensed in the United States and Western Europe consist of alum-precipitated or aluminum hydroxide-adsorbed supernatant material from fermentor cultures of toxigenic, nonencapsulated strains of Bacillus anthracis. These vaccines have several drawbacks, including the need for frequent boosters, the apparent inability to protect adequately against certain strains of B. anthracis, and occasional local reactogenicity. Studies are being undertaken to develop an improved human anthrax vaccine which is safe and efficacious, and which provides long-lasting immunity. Aspects being studied include the identification of antigens and epitopes responsible for eliciting protective immunity, the mechanisms of resistance to anthrax infection, the role of specific antibody in resistance, the differences in immunity elicited by living and chemical vaccines, the potential of new adjuvants to augment immunity, and the feasibility of developing safe vaccine strains having mutationally altered toxin genes. Both living and non-living (chemical) prototype vaccines are being developed and tested.

Anthrax↗
PubMed1988

Transposon Tn916 mutagenesis in Bacillus anthracis.

Mutagenesis of Bacillus anthracis by the streptococcal tetracycline resistance transposon Tn916 is described. Tn916 was transferred from Streptococcus faecalis DS16C1 to B. anthracis VNR-1 by conjugation in a standard filter mating procedure. Tetracycline-resistant (Tcr) transconjugants were obtained at a frequency of 1.6 X 10(-8) per donor CFU. When donor and recipient cells were treated with nafcillin before conjugation, the frequency was increased nearly 10-fold. Nafcillin pretreatment of donor and recipient strains was used in all subsequent conjugation experiments. S.faecalis CG110, containing multiple chromosomal insertions of Tn916, transferred the transposon to B. anthracis VNR-1 at a frequency of 9.3 x 10(-5). A Tcr B. anthracis transconjugant, strain VNR-1-tet-1, transferred Tn916 to B. anthracis UM23-1 and Bacillus subtilis BST1 at frequencies of 2.1 x 10(-4) and 5.8 X 10(-6), respectively. The transfer of Tn916 occurred only on membrane filters, since no Tcr transconjugants were obtained when strains VNR-1-tet-1 and UM23-1 were mixed and incubated in broth culture. The presence of the Tn916-associated tetM gene in Tcr B. anthracis and B. subtilis transconjugants was confirmed in hybridization experiments by using a 5-kilobase-pair DNA fragment containing the tetM gene as a probe. Of 3,000 B. anthracis UM23-1 Tcr transconjugants tested, 21 were phenylalanine auxotrophs and 2 were auxotrophic for phenylalanine, tyrosine, and tryptophan.

Bacillus anthracis↗
PubMed1986

Immunization studies with attenuated strains of Bacillus anthracis.

Live, attenuated strains of Bacillus anthracis lacking either the capsule plasmid pXO2, the toxin plasmid pXO1, or both were tested for their efficacy as vaccines against intravenous challenge with anthrax toxin in Fischer 344 rats and against aerosol or intramuscular challenge with virulent anthrax spores in Hartley guinea pigs. Animals immunized with toxigenic, nonencapsulated (pXO1+, pXO2-) strains survived toxin and spore challenge and demonstrated postimmunization antibody titers to the three components of anthrax toxin (protective antigen, lethal factor, and edema factor). Immunization with two nontoxigenic, encapsulated (pXO1-, pXO2+), Pasteur vaccine strains neither provided protection nor elicited titers to any of the toxin components. Therefore, to immunize successfully against anthrax toxin or spore challenge, attenuated, live strains of B. anthracis must produce the toxin components specified by the pXO1 plasmid.

Aerosols↗
PubMed1986

Cloning and expression of the Bacillus anthracis protective antigen gene in Bacillus subtilis.

The gene encoding the protective antigen (PA) moiety of the tripartite exotoxin of Bacillus anthracis was cloned from the recombinant plasmid pSE36 into Bacillus subtilis 1S53 by using the plasmid vector pUB110. Two clones, designated PA1 and PA2, were identified which produced PA in liquid cultures at levels of 20.5 to 41.9 micrograms/ml. This PA was identical to B. anthracis Sterne PA with respect to migration on sodium dodecyl sulfate-polyacrylamide gels and to Western blot antigenic reactivity. Addition of lethal factor or edema factor to PA1 and PA2 supernatants generated biologically active anthrax lethal toxin or edema-producing toxin, respectively. The recombinant plasmid in PA1 (pPA101) was 7.8 kilobases, whereas the PA2 strain plasmid (pPA102) was 6.1 kilobases. Both plasmids had deletions extending into the insert sequence but not into the DNA encoding the PA protein. Immunization with the live recombinant strains protected guinea pigs from lethal challenge with virulent B. anthracis spores, and the immunization partially or completely protected rats from intravenous challenge with anthrax lethal toxin.

Antigens, Bacterial↗
PubMed1985

Demonstration of a capsule plasmid in Bacillus anthracis.

Virulent and certain avirulent strains of Bacillus anthracis harbor a plasmid, designated pXO2, which is involved in the synthesis of capsules. Two classes of rough, noncapsulated (Cap-) variants were isolated from the capsule-producing (Cap+) Pasteur vaccine strains ATCC 6602 and ATCC 4229. One class was cured of pXO2, and the other class still carried it. Reversion to Cap+ was demonstrable only in rough variants which had retained pXO2. Proof that pXO2 is involved in capsule synthesis came from experiments in which the plasmid was transferred by CP-51-mediated transduction and by a mating system in which plasmid transfer is mediated by a Bacillus thuringiensis fertility plasmid, pXO12. Cells of Bacillus cereus and a previously noncapsulated (pXO2-) strain of B. anthracis produced capsules after the acquisition of pXO2.

Bacillus anthracis↗
PubMed1984

Immunoelectrophoretic analysis, toxicity, and kinetics of in vitro production of the protective antigen and lethal factor components of Bacillus anthracis toxin.

The kinetics of Bacillus anthracis toxin production in culture and its lethal activity in rats, mice, and guinea pigs were investigated. Lethal toxin activity was produced in vitro throughout exponential growth at essentially identical rates in both encapsulated virulent and nonencapsulated avirulent strains. The two toxin proteins which produce lethality when in combination, lethal factor (LF) and protective antigen (PA), could be quantitated directly from culture fluids by rocket immunoelectrophoresis. Using purified preparations of these proteins, we determined that a combination of 8 micrograms of LF and 40 micrograms of PA was required for a maximal rate of killing (39 to 40 min) in Fischer 344 rats (250 to 300 g). Conversely, a minimum of 0.6 microgram of LF and 3 micrograms of PA was required for lethality. The 50% lethal dose for Hartley guinea pigs was 50 micrograms of LF and 250 micrograms of PA, and for Swiss mice it was 2.5 micrograms of LF and 12.5 micrograms of PA. Analyses classically reserved for enzyme kinetic studies were used to study the kinetics of lethal activity in the rat model after intravenous injection of LF-PA mixtures. The amounts of LF and PA which were required to give half the rate of killing (i.e., double the minimum time to death) were 1.2 and 5.8 micrograms, respectively. A theoretical minimum time to death was determined to be 38 min. A third anthrax toxin component, edema factor, was shown to inhibit lethal toxin activity. Edema factor could not be quantitated by rocket immunoelectrophoresis because the protein did not form distinct precipitin bands with available antisera.

Animals↗
PubMed1983

Synthesis and secretion of the plasmid-coded heat-labile enterotoxin of Escherichia coli in Vibrio cholerae.

Both cholera toxin and heat-labile enterotoxin were made and secreted into culture supernatants by Vibrio cholerae containing the enterotoxin plasmid pCG86. Several regulatory mutations in V. cholerae that increased or decreased the synthesis of cholera toxin did not affect production of heat-labile enterotoxin. In contrast, a mutation in V. cholerae that interfered with the secretion of cholera toxin also decreased the secretion of heat-labile enterotoxin, indicating that they are processed by a common secretory pathway. Vibrio cholerae should be useful as a model system for analyzing the secretion of true extracellular proteins by Gram-negative bacteria.

Bacterial Toxins↗
PubMed1983

Evidence for plasmid-mediated toxin production in Bacillus anthracis.

Large-molecular-weight plasmids were isolated from virulent and avirulent strains of Bacillus anthracis. Each strain contained a single plasmid species unique from the others with respect to molecular weight. Bacterial strains were cured of their resident extrachromosomal gene pools by sequential passage of cultures at 42.5 degrees C. Coincidental to the curing of plasmids was a loss of detectable lethal toxin and edema-producing activities and a dramatic decrease in lethal factor and protective antigen serological activities. The involvement of these plasmids in the production of toxin was firmly established by transformation of heat-passaged cells with plasmid DNA purified from the parent strain. The ability to produce parent strain levels of toxin was restored, and the plasmid DNA similar in molecular weight to that isolated from the parent was reisolated in all transformants examined. The exact role these plasmids play in the production of toxin remains to be elucidated. Two additional strains of B. anthracis, designated Pasteur vaccine strains, were examined for the ability to produce toxin and for the presence of plasmid DNA. Both strains were found to be nontoxigenic and contained no detectable plasmid elements. It is therefore likely that we, like Pasteur, cured B. anthracis strains of temperature-sensitive plasmids which code for toxin structural or regulatory proteins.

Bacillus anthracis↗
PubMed1983

Elaboration of Bacillus anthracis antigens in a new, defined culture medium.

Improved culture conditions and a new, completely synthetic medium (R medium) were developed to facilitate the production of Bacillus anthracis holotoxin antigens. Levels of these antigens up to fivefold greater than the highest previously reported values were recovered with the described system. Cultures of Sterne, V770-NP1-R, and Vollum 1B strains of B. anthracis were monitored for growth, pH change, glucose utilization, supernatant protein concentration, lethal toxin activity, and protease activity.

Antigens, Bacterial↗
PubMed1981

Factors affecting phaeomelanin production by a melanin-producing (mel) mutant of Vibrio cholerae.

In a previous study we isolated melanin-producing (mel) mutants of Vibrio cholerae and demonstrated that production of melanin during growth on solid media was stimulated by L-tyrosine and L-cysteine. In the studies reported here we analyzed factors that affected melanin production in liquid media and determined the abilities of radioactively labeled amino acids to serve as precursors for the formation of melanin by V. cholerae. Radioactivity from L-cysteine and from L-tyrosine was preferentially incorporated into partially purified melanin, providing further evidence for production of phaeomelanin by V. cholerae. Cuprous ions stimulated production of melanin by V. cholerae in defined liquid medium, but melanin formation was inhibited by high concentrations of L-cysteine or thiouracil. The inhibition of melanin formation by sulfhydryl compounds is most likely due to their ability to bind copper ions that are essential for tyrosinase activity.

Copper↗
PubMed1980

Isolation and characterization of melanin-producing (mel) mutants of Vibrio cholerae.

Vibrio cholerae strain Htx-3, a hypertoxinogenic mutant of V. cholerae 569B Inaba, produces a dark brown pigment under certain growth conditions, whereas strain 569B does not. We investigated the biochemical basis for this pigment production and the possible relationships between pigmentation and other phenotypic properties in V. cholerae. After mutagenesis of V. cholerae 569B with N-methyl-N'-nitro-N-nitrosoguanidine, 28 independently derived pigment-forming (mel) mutants were isolated and characterized. The mel mutants frequently differed from wild type in toxinogenicity or motility and occasionally differed in other phenotypic traits. Individual mel mutants differed from wild type both in the amount of toxin produced and in the growth conditions optimal for toxin production. It has not yet been established whether multiple phenotypic changes in individual mel mutants represent pleiotropic effects of single mutations or induction of multiple mutations by N-methyl-N'-nitro-N-nitrosoguanidine or both. Production of pigment by mel mutants occurred at temperatures from 22 to 40 degrees C, was inhibited by anaerobiosis, and was stimulated by supplementation of growth media with the amino acid precursors of melanin (l-phenylalanine, l-tyrosine, or l-tyrosine plus l-cysteine). The pigment possessed several other properties reported for microbial melanins. We conclude that a biochemical pathway for melanin production is present in V. cholerae, that this pathway cannot be fully expressed in wild-type strain 569B, and that mutations in the gene(s) which we have designated mel can permit hyperproduction of melanin under appropriate conditions.

Cholera Toxin↗
PubMed1978

Interaction of Chlamydia psittaci with mouse peritoneal macrophages.

L-cell-grown Chlamydia psittaci elementary bodies (EB) were rapidly phagocytized by mouse peritoneal macrophages in vitro. However, the intracellular fate of chlamydiae in macrophages appeared to be dependent on the multiplicity of infection (MOI), i.e., the EB-to-macrophage ratio, and the treatment of the EB. At an MOI of 1:1 or less, survival is maximal, and growth and multiplication of live, untreated chlamydiae did occur. In contrast, at a high MOI (100:1), survival of chlamydiae is reduced, as confirmed by release of 3H-labeled nucleic acid into the supernatant. At the high MOI, macrophage damage occurred that resulted in significant release of the lactic dehydrogenase, beginning 2 h postinfection. This immediated macrophage cytotoxicity as abolished by pretreatment of EB with heat (5 min at 56 degrees C) and was reduced about 50% by coating EB with homologous antibody. Pretreatment of the chlamydia with heat or opsonizing antibody provides increased uptake of EB by macrophages but may contribute to increased destruction of these obligate intracellular pathogens in professional phagocytic cells.

Animals↗
PubMed1978

Response of C3H/HeJ and C3H/HeN mice and their peritoneal macrophages to the toxicity of Chlamydia psittaci elementary bodies.

Intravenous injection of toxic doses of Chlamydia psittaci elementary bodies into endotoxin-responsive C3H/HeN mice or endotoxin-nonresponsive C3H/HeJ mice resulted in essentially identical time intervals to death. Inoculation of monolayer cultures of thioglycolate-stimulated peritoneal macrophages from the two strains of mice with 250 elementary bodies per macrophage resulted in immediate host cell toxicity, although the C3H/HeJ macrophages were somewhat less sensitive to elementary body toxicity than were the C3H/HeN macrophages.

Animals↗