19 Aug 2008
research publications and abstracts are listed in ascending order, starting in
1983. Each listed Bruce E. Ivins as a lead or contributing author.
This section features publications from 1983 through 1990.
1. Mikesell P, Ivins BE, Ristroph JD, Dreier TM. Evidence for
plasmid-mediated toxin production in Bacillus anthracis. Infect Immun.
Abstract: 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
2. Ristroph JD, Ivins BE. Elaboration of
Bacillus anthracis antigens in a new, defined culture medium. Infect Immun.
Abstract: 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.
3. Ezzell JW, Ivins BE, Leppla SH.
Immunoelectrophoretic analysis, toxicity, and kinetics of in vitro production of
the protective antigen and lethal factor components of Bacillus anthracis toxin.
Infect Immun. 45(3):761-7, 1984
Abstract: 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.
4. Green BD,
Battisti L, Koehler TM, Thorne CB, Ivins BE. Demonstration of a capsule
plasmid in Bacillus anthracis. Infect Immun. 49(2):291-7, 1985.
Abstract: 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.
5. Ivins BE,
Ezzell JW Jr, Jemski J, Hedlund KW, Ristroph JD, Leppla SH. Immunization studies
with attenuated strains of Bacillus anthracis. Infect Immun. 52(2):454-8,
Abstract: 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.
6. Ivins BE,
Welkos SL. Cloning and expression of the Bacillus anthracis protective antigen
gene in Bacillus subtilis. Infect Immun. 54(2):537-42, 1986.
Abstract: 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.
7. Ivins BE, Welkos SL, Knudson GB, Leblanc DJ.
Transposon Tn916 mutagenesis in Bacillus anthracis. Infect Immun.
Abstract: 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.
8. Ivins BE, Welkos SL.. Recent advances in the
development of an improved, human anthrax vaccine. Eur J Epidemiol.
Abstract: 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.
9. Ivins BE,
Ristroph JD, Nelson GO. Influence of body weight on response of Fischer 344 rats
to anthrax lethal toxin. Appl Environ Microbiol. 55(8):2098-100, 1989.
Abstract: 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.
10. Ivins BE, Welkos SL, Knudson GB, Little SF.
Immunization against anthrax with aromatic compound-dependent (Aro-) mutants of
Bacillus anthracis and with recombinant strains of Bacillus subtilis that
produce anthrax protective antigen. Infect Immun. 58(2):303-8, 1990.
Abstract: 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.
click to continue to 11-20 of Ivins' publications.