Construction of a bivalent vaccine against anthrax and smallpox using the attenuated vaccinia virus KVAC103.

BACKGROUND: Anthrax and smallpox are high-risk infectious diseases, and considered as potential agents for bioterrorism. To develop an effective countermeasure for these diseases, we constructed a bivalent vaccine against both anthrax and smallpox by integrating a gene encoding protective antigen (PA) of Bacillus anthracis to the genome of the attenuated vaccinia virus strain, KVAC103. RESULTS: Immunization with this bivalent vaccine induced antibodies against both PA and vaccinia virus in a mouse model. We also observed that the efficacy of this vaccine can be enhanced by combined immunization with immunoadjuvant-expressing KVAC103. Mouse groups co-immunized with PA-expressing KVAC103 and either interleukin-15 (IL-15) or cholera toxin subunit A (CTA1)-expressing KVAC103 showed increased anti-PA IgG titer and survival rate against B. anthracis spore challenge compared to the group immunized with PA-expressing KVAC103 alone. CONCLUSIONS: We demonstrated that the attenuated smallpox vaccine KVAC103 is an available platform for a multivalent vaccine and co-immunization of immunoadjuvants can improve vaccine performance.

The prevalence and characteristics of Shiga toxin-producing Escherichia coli isolated by the enteric pathogens active surveillance network (Enter-Net) in the Republic of Korea, 2009-2018.

OBJECTIVE: Shiga toxin-producing Escherichia coli (STEC) is a water- and food-borne pathogenic agent that causes diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS), and end-stage renal disease. As the annual incidence of STEC increases, disease control is also becoming important in Korea. In this study, we aimed to analyze the incidence trends and characteristics of STEC isolated from diarrheal patients over 10 years. METHODS: From 2009 to 2018, STECs were collected by the Enteric Pathogens Active Surveillance Network (Enter-Net) and analyzed according to clinical epidemiological information (month of isolation, age, and sex of patient), O serogroup, and shiga toxin type. Shiga toxin genes (stx1 and stx2) and O serogroups of isolates were determined using multiplex PCR and an agglutination method with the available O antisera, respectively. RESULTS: A total of 418 strains were isolated over 10 years. The isolation rate according to age group and season was highest in children ≤4 years old (38.1%) and in the summer season (June to August). Among the 418 isolates, the major serogroups were divided O157 (20.3%), O103 (13.6%), O26 (7.7%), O111 (5.5%), O91 (4.3%), O108 (2.4%), and O8 (2.2%). The most frequently isolated O157 showed a lower isolation rate compared to that isolated from other developed countries. The profiles of stx genes were distinct among serogroups. In O157 and O91, stx1+stx2 was detected more frequently than either stx1 or stx2 alone. Particularly, most of the O157 (98%) isolates harbored the stx2 gene, which is an important factor in severe diseases, including HUS. In O103, O26, O111, and O108, stx1-only was more frequently present than stx2-only or stx1+stx2. CONCLUSIONS: As a result of analyzing domestic STECs collected through Enter-Net, it was confirmed that patients ≤4 years of age and in the summer months require attention, and that STEC with a serogroup of O157 is highly likely to cause diseases such as HUS. Therefore, the pathogen active surveillance network for characterization and provision of STEC isolates must be operated continuously.

Immunoproteomically identified GBAA_0345, alkyl hydroperoxide reductase subunit C is a potential target for multivalent anthrax vaccine.

Anthrax is caused by the spore-forming bacterium Bacillus anthracis, which has been used as a weapon for bioterrorism. Although current vaccines are effective, they involve prolonged dose regimens and often cause adverse reactions. High rates of mortality associated with anthrax have made the development of an improved vaccine a top priority. To identify novel vaccine candidates, we applied an immunoproteomics approach. Using sera from convalescent guinea pigs or from human patients with anthrax, we identified 34 immunogenic proteins from the virulent B. anthracis H9401. To evaluate vaccine candidates, six were expressed as recombinant proteins and tested in vivo. Two proteins, rGBAA_0345 (alkyl hydroperoxide reductase subunit C) and rGBAA_3990 (malonyl CoA-acyl carrier protein transacylase), have afforded guinea pigs partial protection from a subsequent virulent-spore challenge. Moreover, combined vaccination with rGBAA_0345 and rPA (protective antigen) exhibited an enhanced ability to protect against anthrax mortality. Finally, we demonstrated that GBAA_0345 localizes to anthrax spores and bacilli. Our results indicate that rGBAA_0345 may be a potential component of a multivalent anthrax vaccine, as it enhances the efficacy of rPA vaccination. This is the first time that sera from patients with anthrax have been used to interrogate the proteome of virulent B. anthracis vegetative cells.

Monoclonal antibody against the poly-gamma-D-glutamic acid capsule of Bacillus anthracis protects mice from enhanced lethal toxin activity due to capsule and anthrax spore challenge.

BACKGROUND: The poly-gamma-D-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT. METHODS: To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with K(d) values of 0.8 microM and 2.6 microM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed. RESULTS: Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen. GENERAL SIGNIFICANCE: Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.

Bacillus anthracis genomic DNA enhances lethal toxin-induced cytotoxicity through TNF-α production.

BACKGROUND: Bacillus anthracis is the etiological agent of anthrax. Lethal toxin (LT) produced by B. anthracis is a well-known key virulence factor for anthrax because of its strong cytotoxic activity. However, little is known about the role of B. anthracis genomic DNA (BAG) in anthrax pathogenesis. RESULTS: We examined the effect of BAG on TNF-α production and LT-mediated cytotoxicity during B. anthracis spore infection in mouse macrophage cell lines (RAW264.7 cells and J774A.1) and BALB/c mice. Infection of RAW264.7 cells with B. anthracis spores induced TNF-α expression in a multiplicity of infection (MOI)-dependent manner, and this enhancement was attenuated by the toll-like receptor (TLR) 9 inhibitor oligodeoxynucleotide (ODN)2088. BAG led to TNF-α expression in a dose- and time-dependent manner when applied to RAW264.7 cells. TNF-α expression induced by BAG was reduced by either pretreatment with TLR9 inhibitors (ODN2088 and chloroquine (CQ)) or transfection with TLR9 siRNA. Furthermore, BAG-induced TNF-α production in TLR9(+/+) macrophages was completely abrogated in TLR9(-/-) macrophages. BAG enhanced the phosphorylation of mitogen-activated protein kinases (MAPK), and BAG-induced TNF-α expression was attenuated by pretreatment with MAPK inhibitors. A reporter gene assay and confocal microscopy demonstrated that BAG increased NF-κB activation, which is responsible for TNF-α expression. Treatment with BAG alone showed no cytotoxic activity on the macrophage cell line J774A.1, whereas LT-mediated cytotoxicity was enhanced by treatment with BAG or TNF-α. Enhanced LT-induced lethality was also confirmed by BAG administration in mice. Furthermore, LT plus BAG-mediated lethality was significantly recovered by administration of Infliximab, an anti-TNF-α monoclonal antibody. CONCLUSIONS: Our results suggest that B. anthracis DNA may contribute to anthrax pathogenesis by enhancing LT activity via TLR9-mediated TNF-α production.

Complete genome sequence of Bacillus anthracis H9401, an isolate from a Korean patient with anthrax.

Bacillus anthracis H9401 (NCCP 12889) is an isolate from a Korean patient with gastrointestinal anthrax. The whole genome of H9401 was sequenced. It is a circular chromosome containing 5,480 open reading frames (ORFs) and two plasmids, pXO1 containing 202 ORFs and pXO2 containing 110 ORFs. H9401 shows high pathogenicity and genome sequence similarity to Ames Ancestor.

Seroepidemiologic evidence of Q fever and associated factors among workers in veterinary service laboratory in South Korea.

The incidence of Q fever has rapidly increased in South Korea since 2015. This study was undertaken to investigate the seroprevalence and seroreactivity of Q fever and the risk factors associated with its seroprevalence among workers in the veterinary service laboratory (VSL) in South Korea. This seroepidemiologic study was conducted in a total of 661 human subjects out of 1,328 subjects working in 50 VSL existing in South Korea between July 15 and July 29, 2019. Data were collected by administering survey questionnaires and by analyzing collected blood samples to determine the presence of antibodies against Coxiella burnetii. The seroprevalence and seroreactivity of C. burnetii infection were determined based on serum titers as (phase II IgG ≥1:256 and/or IgM ≥1:16) and (phase II IgG ≥1:16 and/or IgM ≥1:16) as determined by indirect immunofluorescent assay. Work, work environment, behavioral risk and protective factors associated with seroprevalence of Q fever were assessed by employing multivariable logistic regression analysis. Among the 661, the seroprevalence and seroreactivity of C. burnetii infection were 7.9% and 16.0%, respectively. Multivariate logistic regression analysis showed the risk factors significantly associated with seroprevalence were the antemortem inspection of cattle, goats, or sheep (APR (adjusted prevalence ratio), 2.52; 95% CI, 1.23-4.70)), animal blood splashed into or around eyes (APR, 2.24; 95% CI, 1.04-4.41), and contact with animals having Q fever (APR, 6.58; 95% CI, 3.39-10.85) during the previous year. This study suggests the need for precautions when contact with cattle, goats, or sheep is expected, especially during the antemortem inspection, when dealing with C. burnetii infected animals, or when there is a risk of ocular contact with animal derivatives. Therefore, we recommend the consistent use of appropriate personal protective equipment and other protective measures including PPE treatment and washing of body surfaces after work to prevent C. burnetii infections among VSL staff in South Korea.

The poly-γ-D-glutamic acid capsule of Bacillus anthracis enhances lethal toxin activity.

The poly-γ-D-glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis, which causes a highly lethal infectious disease. The PGA capsule disguises B. anthracis from immune surveillance and allows its unimpeded growth in the host. The PGA capsule recently was reported to be associated with lethal toxin (LT) in the blood of experimentally infected animals (M. H. Cho, et al., Infect. Immun. 78:387-392, 2010). The effect of PGA, either alone or in combination with LT, on macrophages, which play an important role in the progression of anthrax disease, has not been thoroughly investigated. In this study, we investigated the effect of PGA on LT cytotoxicity using the mouse macrophage cell line J774A.1. PGA produced a concentration-dependent enhancement of the cytotoxicity of LT on J774A.1 cells through an enhancement in the binding and accumulation of protective antigen to its receptors. The increase of LT activity was confirmed using Western blot analysis, which showed that the combination of PGA and LT produced a greater degree of degradation of mitogen-activated protein kinase kinases and an increased level of the activation of the proform of caspase-1 to its processed form compared to the effects of LT alone. In addition, mice that received a tail vein injection of both PGA and LT had a significantly increased rate of death compared to that of mice injected with LT alone. PGA had no effect when added to cultures or administered to mice in the absence of LT. These results emphasize the importance of PGA in the pathogenesis of anthrax infection.

Emerging high-level ciprofloxacin-resistant Salmonella enterica serovar typhi haplotype H58 in travelers returning to the Republic of Korea from India.

In Korea, typhoid fever is a rare disease due to improved living standards. However, typhoid fever remains a major burden in developing countries and regions, such as India and Southeast Asia. In this study, we isolated Salmonella Typhi (S. Typhi) from eight patients with typhoid fever who were travelers returning from India. The strains isolated were characterized by antimicrobial susceptibility profiling and whole-genome sequencing (WGS) analysis. All strains were resistant to nalidixic acid and azithromycin. Among them, four isolates were highly resistant to ciprofloxacin (MIC ≥32 µg/ml); these strains have not been confirmed in Korea PulseNet DB. According to WGS, the ciprofloxacin-resistant strains belong to the global dominant multidrug-resistant (MDR) haplotype H58 (SNP glpA C1047T, SptP protein Q185* (premature stop codon)) and do not harbor the MDR plasmid. H58-associated SNPs in membrane and metabolism genes, including yhdA, yajI, hyaE, tryE, rlpB and metH, are present. Additionally, phylogenetic analysis assigned the H58 strains to sublineage II, whereas the non-H58 strains are closely related to haplotype H50. The presence of high-level ciprofloxacin-resistant S. Typhi haplotype H58 in Korea was first confirmed as due to influx from overseas via travelers. This study provides information about intercontinental drug-resistant transmission between countries and suggests that travelers need to be careful about personal hygiene.

Bacillus anthracis capsule activates caspase-1 and induces interleukin-1beta release from differentiated THP-1 and human monocyte-derived dendritic cells.

The poly-gamma-d-glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis, which causes a highly lethal infection. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Recently, efforts have been made to include PGA as a component of anthrax vaccine; however, the innate immune response of PGA itself has been poorly investigated. In this study, we characterized the innate immune response elicited by PGA in the human monocytic cell line THP-1, which was differentiated into macrophages with phorbol 12-myristate 13-acetate (PMA) and human monocyte-derived dendritic cells (hMoDCs). PGA capsules were isolated from the culture supernatant of either the pXO1-cured strain of B. anthracis H9401 or B. licheniformis ATCC 9945a. PGA treatment of differentiated THP-1 cells and hMoDCs led to the specific extracellular release of interleukin-1beta (IL-1beta) in a dose-dependent manner. Evaluation of IL-1beta processing by Western blotting revealed that cleaved IL-1beta increased in THP-1 cells and hMoDCs after PGA treatment. Enhanced processing of IL-1beta directly correlated with increased activation of its upstream regulator, caspase-1, also known as IL-1beta-converting enzyme (ICE). The extracellular release of IL-1beta in response to PGA was ICE dependent, since the administration of an ICE inhibitor prior to PGA treatment blocked induction of IL-1beta. These results demonstrate that B. anthracis PGA elicits IL-1beta production through activation of ICE in PMA-differentiated THP-1 cells and hMoDCs, suggesting the potential for PGA as a therapeutic target for anthrax.

Traveller-associated high-level ciprofloxacin-resistant Salmonella enterica Serovar Kentucky in the Republic of Korea.

OBJECTIVE: Salmonella enterica serotype Kentucky ST198 (S. Kentucky) is frequently associated with human infections and has been identified in travellers to North Africa, South Asia, Europe, and North America. The antimicrobial resistance of this serotype to multiple drugs, including ciprofloxacin (CIP), is a growing concern. However, little information is available regarding the occurrence and characterization of S. Kentucky in Korea. METHODS: To investigate the characteristics and possible origin of these infections, we characterized highly CIP-resistant S. Kentucky isolates collected through a national surveillance program. Single-nucleotide polymorphisms (SNPs) were identified by whole-genome sequencing (WGS), and genome sequencing was performed to investigate the genetic relationships and resistance mechanisms of the isolates. RESULTS: Ten CIP-resistant S. Kentucky strains were isolated from diarrheal patients in Korea from 2008 to 2017. The travel histories of the patients indicated that seven had returned from Southeast Asia. WGS of all the isolates revealed the presence of Salmonella genomic island 1 (SGI1) and substitutions in the gyrA and parC genes, which are known to confer resistance to CIP. A multilocus sequence type (MLST) analysis revealed that the isolates belonged to ST198, which has been prevalent in Europe and Africa. Furthermore, a phylogenetic analysis showed that all 10 isolates shared close genetic relationships. CONCLUSIONS: We report the identification of S. Kentucky in Korea through long-term surveillance. International travel, especially to Southeast Asia, has been a major risk factor for human infections of CIP-resistant S. Kentucky in Korea.

Co-infection With Chromosomally-Located bla CTX-M-14 and Plasmid-Encoding bla CTX-M-15 in Pathogenic Escherichia coli in the Republic of Korea.

The emergence of third-generation cephalosporin resistance in Escherichia coli is increasing at an alarming rate in many countries. Thus, the aim of this study was to analyze co-infecting bla CTX-M-producing pathogenic E. coli isolates linked to three school outbreaks. Among 66 E. coli isolates, 44 were identified as ETEC O25, an ETEC isolate serotype was O2, and the other 21 were confirmed as EAEC O44. Interestingly, six patients were co-infected with EAEC O44 and ETEC O25. For these isolates, molecular analysis [antibiotic susceptibility testing, identification of the ß-lactamase gene, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE)] was performed for further characterization. In addition, the transmission capacity of bla CTX-M genes was examined by conjugation experiments. Whole-genome sequencing (WGS) was performed on representative EAEC O44 and ETEC O25 isolates associated with co-infection and single-infection. All isolates were resistant to cefotaxime and ceftriaxone. All EAEC isolates carried the bla CTX-M-14 gene and all ETEC isolates the bla CTX-M-15 gene, as detected by multiplex PCR and sequencing analysis. Sequence type and PFGE results indicated three different patterns depending on the O serotype. WGS results of representative isolates revealed that the ETEC O25 strains harbored bla CTX-M-15 located on IncK plasmids associated with the Δbla TEM-bla CTX-M-15-orf477 transposon. The representative EAEC O44 isolates carried bla CTX-M-14 on the chromosome, which was surrounded by the ISEcp1-bla CTX-M-14-IS903 transposon. To the best of our knowledge, this is the first report of co-infection with chromosomally located bla CTX-M-14 and plasmid-encoding bla CTX-M-15 in pathogenic E. coli. Our findings indicate that resistance genes in clinical isolates can spread through concurrent combinations of chromosomes and plasmids.

The role of international travellers in the spread of CTX-M-15-producing Shigella sonnei in the Republic of Korea.

BACKGROUND: Multidrug-resistant Shigella isolates have recently emerged as a serious public health threat worldwide. In particular, overseas travel is a risk factor for acquisition of antimicrobial-resistant Shigella strains. To explore the role of travel in the spread of cefotaxime-resistant Shigella sonnei in Korea, we screened 751 Shigella spp. isolates from 2007 to 2016 through the National Surveillance system, and 28 cephalosporin-resistant S. sonnei isolates were identified. METHODS: For cephalosporin-resistant S. sonnei isolates, epidemiological and molecular analyses (plasmid structure analysis, pulsed-field gel electrophoresis (PFGE) and high-quality single-nucleotide polymorphisms (hqSNPs) based on whole-genome sequencing (WGS)) were conducted to investigate the source of infection and transmission route. RESULTS: Among the 28 cefotaxime-resistant S. sonnei strains, 18 were isolated from travellers returning from Asia, including Vietnam (n=11). Molecular analysis of 18 blaCTX-M-type isolates revealed that 15 contain CTX-M-15; 50% of isolates from domestic patients contain CTX-M-14. Analysis of the genetic environments of the blaCTX-M-14 and blaCTX-M-15 genes revealed different genetic organization surrounding the blaCTX-M genes. Additionally, PFGE and hqSNP results suggested a large phylogenetic distance between the S. sonnei isolates related to overseas travel and those acquired domestically in Korea. CONCLUSION: Our study data demonstrates that two prevalent blaCTX-M genes, blaCTX-M-14 and blaCTX-M-15, have been circulating in S. sonnei in Korea over the last 10 years. Recently, international travellers are at a high risk for acquisition of CTX-M-15-producing S. sonnei in Korea.

Enhancement of the immune responses of mice to Bacillus anthracis protective antigen by CIA07 combined with alum.

Anthrax is an acute zoonotic disease caused by infection with Bacillus anthracis. B. anthracis spores are highly resistant to environmental degradation and are used as a biological weapon. In this study, we investigated the adjuvant activity of CIA07 to anthrax protective antigen (PA). A/J mice were immunized intraperitoneally once, or twice with a 4-week interval, with recombinant PA alone or combined with alum, CpG1826, or CIA07 as adjuvant, and serum anti-PA IgG antibody responses were measured 4 weeks after each immunization. All three adjuvants significantly enhanced anti-PA IgG antibody titer 4 weeks after the priming and boosting immunizations, and alum gave the highest titer. In order to evaluate the adjuvant activity of CIA07 in the presence of alum, Balb/c mice were immunized 3 times at 1-week intervals with PA in combination with alum, CIA07 or alum plus CIA07, and the immune responses were assessed 2 weeks after the third immunization. The serum anti-PA IgG antibody titer of the CIA07-treated group was 14-fold higher than the group given PA alone, and the coadministration of CIA07 with alum further increased the titer 3.5-fold (P < 0.05). The toxin neutralizing activity of the sera from the mice given the combination of CIA07 and alum was 109-times higher than the animals given PA alone. The mice given CIA07 plus alum also showed a marked increase in the number of IFN-gamma-, IL-2-, and IL-4-producing CD4(+) T cells among their splenocytes. These data suggest the potential of CIA07 in combination with alum as an adjuvant for the development of a potent anthrax vaccine.

Construction of Luminescence- and Fluorescence-Tagged Burkholderia pseudomallei for Pathogen Tracking in a Mouse Model.

Molecular imaging is a powerful method for tracking various infectious disease-causing pathogens in host organisms. Currently, a dual molecular imaging method that can provide temporal and spatial information on infected hosts at the organism, organ, tissue, and cellular levels simultaneously has not been reported for Burkholderia pseudomallei, a high-risk pathogen that causes melioidosis. In this study, we have established an experimental method that provides spatiotemporal information on infected hosts using luminescent and fluorescent dual-labeled B. pseudomallei. Using this method, we visualized B. pseudomallei infection at the organism, organ, and tissue levels in a BALB/c mouse model by detecting its luminescence and fluorescence. The infection of B. pseudomallei at the cellular level was also visualized by its emitted fluorescence in infected macrophage cells. This method could be an extremely useful and applicable tool to study the pathogenesis of B. pseudomallei-related infectious diseases.

Draft Genome Sequence of the First South Korean Clinical Isolate of Burkholderia pseudomallei, H0901.

We report here the draft genome sequence of Burkholderia pseudomallei H0901. This strain was isolated in 2003 from the first melioidosis patient in South Korea.

Determination of neurotoxin gene expression in Clostridium botulinum type A by quantitative RT-PCR.

Real time reverse transcription (RT)-PCR was used to quantify the expression of the botulinum neurotoxin type A (BoNT/A) gene (cntA) by normalization with the expression of 16S rRNA. The method were confirmed by monitoring the mRNA levels of cntA during growth in five type A strains. In all but one of the strains the expression of cntA mRNA was maximal in the late exponential phase, and approximately 35-fold greater than in the early exponential phase. The concentration of the extracellular BoNT/A complex detected by ELISA was highest in stationary phase. Sodium nitrite and sorbic acid completely inhibited growth at 20 ppm and 4 mg ml-1, respectively. CntA expression became lower in proportion to the concentration of sorbic acid, and this reduction was confirmed by mouse bioassay. Our results show that real time RT-PCR can be used to quantify levels of C. botulinum type A neurotoxin transcripts and to assess the effects of food additives on botulinal risk.

Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis.

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.