ABSTRACT: BACKGROUND: An isolate originally labeled Bacillus megaterium CDC 684 was found to contain both pXO1 and pXO2, was non-hemolytic, sensitive to gamma-phage, and produced both the protective antigen and the poly-D-glutamic acid capsule. These phenotypes prompted Ezzell et al.,(J. Clin. Microbiol. 28:223) to reclassify this isolate to Bacillus anthracis in 1990. RESULTS: We demonstrate that despite these B. anthracis features, the isolate is severely attenuated in a guinea pig model. This prompted whole genome sequencing and closure. The comparative analysis of CDC 684 to other sequenced B. anthracis isolates and further analysis reveals: a) CDC 684 is a close relative of a virulent strain, Vollum A0488; b) CDC 684 defines a new B. anthracis lineage (at least 51 SNPs) that includes 15 other isolates; c) the genome of CDC 684 contains a large chromosomal inversion that spans 3.3 Mbp; d) this inversion has caused a displacement of the usual spatial orientation of the origin of replication (ori) to the termination of replication (ter) from 180o in wild-type B. anthracis to 120o in CDC 684 and e) this isolate also has altered growth kinetics in liquid media. CONCLUSIONS: We propose two alternative hypotheses explaining the attenuated phenotype of this isolate. Hypothesis 1 suggests that the skewed ori / ter relationship in CDC 684 has altered its DNA replication and/or transcriptome processes resulting in altered growth kinetics and virulence capacity. Hypothesis 2 suggests that one or more of the single nucleotide polymorphisms in CDC 684 has altered the expression of a regulatory element or other genes necessary for virulence.

Background.@nbsp; Postexposure prophylaxis of inhalational anthrax requires prolonged antibiotic therapy or antibiotics and vaccination. The duration of treatment for established anthrax is controversial, because retained spores may germinate and cause disease after antibiotics are discontinued. Using rhesus macaques, we determined whether a short course of antibiotic treatment, as opposed to prophylaxis, could effectively treat inhalational anthrax and prevent disease caused by the germination of spores after discontinuation of antibiotics. Methods.@nbsp; Two groups of 10 rhesus macaques were exposed to an aerosol dose of Bacillus anthracis spores. Animals in group 1 received ciprofloxacin prophylaxis beginning 1-2 h after exposure. Those in group 2 began receiving ciprofloxacin after becoming bacteremic, and treatment was continued for 10 days. When each group 2 animal completed 10 days of therapy, the prophylactic antibiotic was discontinued in the paired group 1 animal. Results.@nbsp; In group 1 (prophylaxis), no deaths occurred during antibiotic treatment, but only 2 (20%) of 10 animals survived after antibiotics were discontinued. In contrast, in group 2 (treatment), 3 deaths occurred during antibiotic treatment, but all 7 animals (100%) alive after 10 days of therapy survived when antibiotics were discontinued. Conclusions.@nbsp; In the treatment of inhalational anthrax, the prolonged course of antibiotics required to achieve prophylaxis may not be necessary to prevent anthrax that results from the germination of retained spores after the discontinuation of antibiotics.

The inhaled form of Bacillus anthracis infection may be fatal to humans. The current standard of care for inhalational anthrax postexposure prophylaxis is ciprofloxacin therapy twice daily for 60 days. The potent in vitro activity of oritavancin, a semisynthetic lipoglycopeptide, against B. anthracis (minimum inhibitory concentration against Ames strain, 0.015 microg/ml) prompted us to test its efficacy in a mouse aerosol-anthrax model. In postexposure prophylaxis dose-ranging studies, a single intravenous (i.v.) dose of oritavancin of 5, 15 or 50 mg/kg 24 h after challenge with 50 to 75 times the median lethal dose of Ames strain spores provided 40, 70, and 100% proportional survival, respectively, at 30 days postchallenge. Untreated animals died within 4 days of challenge whereas 90% of control animals receiving ciprofloxacin at 30 mg/kg intraperitoneally twice daily for 14 days starting 24 h after challenge survived. Oritavancin demonstrated significant activity postsymptom development: a single i.v. dose of 50 mg/kg administered 42 h after challenge provided 56% proportional survival at 30 days. In a preexposure prophylaxis study, a single i.v. dose of 50 mg/kg oritavancin administered 1, 7, 14, or 28 days before lethal challenge protected 90, 100, 100, and 20% of mice at 30 days; mice treated with ciprofloxacin 24 h or 24 and 12 h before challenge all died within 5 days. Efficacy in pre- and postexposure models of inhalation anthrax together with a demonstrated low propensity to engender resistance promote further study of oritavancin pharmacokinetics and efficacy in non-human primate models.

Samples of human plasma from anthrax vaccine adsorbed (AVA, BioThraxtrade mark)-vaccinated individuals were used to demonstrate passive protection of A/J mice from a lethal challenge with the Sterne strain of anthrax bacteria. The maximum concentration of human anti-protective antigen IgG in mouse sera 24h after injection of 260mug of anti-PA IgG was 134mug/ml, declining to 91mug/ml at 72h (half-life=101.7h). Mice showed significant survival (p</=0.001) after injection of serial dilutions up to 1:4 of the standard plasma and challenged with 100 LD(50). Similarly, mice injected with the standard anti-AVA plasma and challenged up to 5 days post-treatment also survived (p</=0.001). Using a cohort of human plasma to measure passive protection, the best correlation between passive protection and an in vitro assay was found to be with the quantitative toxin neutralization assay (minimum fold increase in odds of survival: 2.71, p=0.0062). These results demonstrate a reliable in vivo neutralization method that correlates with standard in vitro measures of neutralizing antibody levels in plasma from individuals vaccinated with the standard anthrax vaccine. This analytical method may provide additional opportunities to compare the efficacy of improved anthrax vaccines with the licensed vaccine.

An anthrax-spore aerosol infection mouse model was developed as a first test of in vivo efficacy of antibiotics identified as active against Bacillus anthracis. Whole-body, LD50 aerosol challenge doses in a range of 1.9 x10(3) to 3.4 x 10(4) with spores of the fully virulent Ames strain were established for three inbred and one outbred mouse strain (A/J, BALB/c, C57BL and Swiss Webster). The BALB/c strain was further developed as a model for antibiotic efficacy. Time-course microbiological examination of tissue burdens in mice after challenge showed that spores could remain dormant in the lungs while vegetative cells disseminated to the mediastinal lymph nodes and then to the spleen accompanied with bacteremia. For antibiotic efficacy studies, BALB/c mice were challenged with 50-100 LD50 of spores followed by intraperitoneal (i.p.) injection of either ciprofloxacin 30 mg/kg (q12h), or doxycycline 40 mg/kg (q6h). A control group was treated with PBS q6h. Treatment was begun 24 h after challenge in groups of 10 mice for 14 or 21 days. The PBS-treated control mice all succumbed (10/10) to inhalation anthrax infection within 72 h. Sixty-day survival rates for ciprofloxacin and doxycycline-treated groups were 8/10, 9/10 for 14-day treatment and 10/10, 7/10 for 21-day treatment. Delayed treatment with ciprofloxacin initiated 36- and 48-h postexposure resulted in 80% survival and was statistically no different than early postexposure (24 h) treatment. This mouse model correlates closely with clinical observations of inhalational anthrax in humans and with earlier antibiotic studies in the non-human primate inhalational-anthrax model.

The serological response and efficacy of Bacillus anthracis recombinant protective antigen (rPA) vaccines formulated with aluminum hydroxide adjuvant, either with or without formaldehyde, were evaluated in rabbits. Rabbits that had been injected with a single dose of 25mug of rPA adsorbed to 500mug of aluminum in aluminum hydroxide gel (Alhydrogel) had a significantly higher quantitative anti-rPA IgG ELISA titers (p<0.0001) and toxin neutralizing antibody (TNA) assay titers (p<0.0001) than rabbits tested at the next lowest concentration of aluminum (158mug). Rabbits injected with two doses of 50mug of rPA formulated with 500mug of aluminum also had significantly higher serological responses, as measured by a quantitative anti-rPA IgG ELISA (p<0.0001) and TNA assay (p<0.0001), than sera from rabbits injected with a rPA vaccine formulated without adjuvant. Short-term protection against an aerosol spore challenge (448 LD(50)), however, was not significantly different between the two groups (12/12 and 11/12, respectively). Rabbits injected with a single dose of 50mug of rPA formulated with 500mug of aluminum and 0.2% formaldehyde had significantly higher ELISA (p<0.0001) and TNA assay (p<0.0001) titers than rabbits that had been injected with a rPA vaccine formulated with adjuvant but without formaldehyde. Short-term protection against a 125 LD(50) parenteral spore challenge, however, was not significantly different between the two groups (14/24 and 9/24, respectively; p=0.2476). Under the conditions tested in the rabbit animal model, significantly higher serological responses were observed in rabbits that had been injected with rPA formulated with aluminum hydroxide gel adjuvant and formaldehyde. However, differences in short-term efficacy were not observed.

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs act as immune adjuvants, improving the response elicited by a coadministered vaccine. Combining CpG ODN with anthrax vaccine adsorbed (AVA, the licensed human vaccine) increases the speed, magnitude, and avidity of the resultant antibody response. IgG Abs against anthrax protective antigen (PA) protect mice, guinuea pigs, and rhesus macaques from infection.

Prevention of inhalational anthrax after Bacillus anthracis spore exposure requires a prolonged course of antibiotic prophylaxis. In response to the 2001 anthrax attack in the United States, approximately 10,000 people were offered 60 days of antibiotic prophylaxis to prevent inhalational anthrax, but adherence to this regimen was poor. We sought to determine whether a short course of antibiotic prophylaxis after exposure could protect non-human primates from a high-dose spore challenge if vaccination was combined with antibiotics. Two groups of 10 rhesus macaques were exposed to approximately 1,600 LD50 of spores by aerosol. Both groups were given ciprofloxacin by orogastric tube twice daily for 14 days, beginning 1-2 h after exposure. One group also received three doses of the licensed human anthrax vaccine (anthrax vaccine adsorbed) after exposure. In the ciprofloxacin-only group, four of nine monkeys (44%) survived the challenge. In contrast, all 10 monkeys that received 14 days of antibiotic plus anthrax vaccine adsorbed survived (P = 0.011). Thus postexposure vaccination enhanced the protection afforded by 14 days of antibiotic prophylaxis alone and completely protected animals against inhalational anthrax. These data provide evidence that postexposure vaccination can shorten the duration of antibiotic prophylaxis required to protect against inhalational anthrax and may impact public health management of a bioterrorism event.

The next-generation human anthrax vaccine developed by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) is based upon purified Bacillus anthracis recombinant protective antigen (rPA) adsorbed to aluminum hydroxide adjuvant (Alhydrogel). In addition to being safe, and effective, it is important that such a vaccine be fully characterized. Four major protein isoforms detected in purified rPA by native PAGE during research and development were reduced to two primary isoforms in bulk material produced by an improved process performed under Good Manufacturing Practices (GMP). Analysis of both rPA preparations by a protein-isoaspartyl-methyl-transferase assay (PIMT) revealed the presence of increasing amounts of iso-aspartic acid correlating with isoform content and suggesting deamidation as the source of rPA charge heterogeneity. Additional purification of GMP rPA by anion exchange chromatography separated and enriched the two principal isoforms. The in vitro and in vivo biological activities of each isoform were measured in comparison to the whole GMP preparation. There was no significant difference in the biological activity of each isoform compared to GMP rPA when analyzed in the presence of lethal factor using a macrophage lysis assay. Vaccination with the two individual isoforms revealed no differences in cytotoxicity neutralization antibody titers when compared to the GMP preparation although one isoform induced more anti-PA IgG antibody than the GMP material. Most importantly, each of the two isoforms as well as the whole GMP preparation protected 90-100% of rabbits challenged parenterally with 129 LD(50) of B. anthracis Ames spores. The equivalent biological activity and vaccine efficacy of the two isoforms suggests that further processing to separate isoforms is unnecessary for continued testing of this next-generation anthrax vaccine.

Long-term protection of rabbits that had been vaccinated with two doses of a recombinant protective antigen (rPA) vaccine was examined against an aerosol spore challenge with the Ames isolate of Bacillus anthracis at 6 and 12 months. At 6 months after the primary injection, survival was 74.1%(20/27) with quantitative ELISA titer of 22.3mug of anti-rPA IgG per millilitre and toxin neutralizing antibody (TNA) assay titer of 332. At 12 months after the primary injection, only 37.5%(9/24) of the rabbits were protected with quantitative ELISA titer of 19.8mug of anti-rPA IgG per millilitre and TNA assay titer of 286. There was a significant loss of protection (p=0.0117) and a significant difference in survival curves (p=0.0157) between the 6- and 12-month groups. When ELISA or TNA assay titer, gender, and challenge dose were entered into a forward logistic regression model, week 26 ELISA titer (p=0.0236) and week 13 TNA assay titer (p=0.0147) for the 6-month group, and week 26 ELISA titer (p=0.0326) and week 8 TNA assay titer (p=0.0190) for the 12-month group, were significant predictors of survival. Neither gender nor challenge dose were identified as having a statistically significant effect on survival. Booster vaccinations with rPA may be required for the long-term protection of rabbits against anthrax.