Naval Academy, MD, United States
Naval Academy, MD, United States

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Albrecht M.T.,Biological Defense Research Directorate | Eyles J.E.,UK Defence Science and Technology Laboratory | Eyles J.E.,Pfizer | Baillie L.W.,University of Cardiff | And 2 more authors.
FEMS Immunology and Medical Microbiology | Year: 2012

The efficacy of multi-agent DNA vaccines consisting of a truncated gene encoding Bacillus anthracis lethal factor (LFn) fused to either Yersinia pestis V antigen (V) or Y. pestis F1 was evaluated. A/J mice were immunized by gene gun and developed predominantly IgG1 responses that were fully protective against a lethal aerosolized B. anthracis spore challenge but required the presence of an additional DNA vaccine expressing anthrax protective antigen to boost survival against aerosolized Y. pestis. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Nada R.A.,Us Naval Medical Research Unit No 3 | Shaheen H.I.,Us Naval Medical Research Unit No 3 | Khalil S.B.,Us Naval Medical Research Unit No 3 | Mansour A.,Us Naval Medical Research Unit No 3 | And 7 more authors.
Journal of Clinical Microbiology | Year: 2011

Enterotoxigenic Escherichia coli (ETEC) is recognized to be a common cause of acute watery diarrhea in children from developing countries. Colonization factors (CFAs) have been identified predominantly in ETEC isolates secreting heat-stable enterotoxin (ST) or cosecreting ST with a heat-labile toxin (LT). We hypothesized that LT-only-secreting ETEC produces unique colonization factors not previously described in ST and LTSTsecreting ETEC. A set of degenerate primers based on nucleotide sequence similarities between the major structural genes of CS20 (csnA), CS18 (fotA), CS12 (cswA), and porcine antigen 987 (fasA) was developed and used to screen a collection of 266 LT-secreting ETEC isolates in which no known CFA was detected. PCRamplified products of different molecular masses were obtained from 49 (18.4%) isolates. Nucleotide sequence analysis of the PCR amplicons followed by GenBank nucleotide BLASTn analysis revealed five novel DNA sequences; translated amino acid BLASTx analysis confirmed sequence similarity to class 1b major structural proteins encoded by csnA, fotA, and fasA. Strains expressing the novel CFAs were phylotyped and analyzed using multilocus sequence typing (MLST; Achtman scheme), and the types detected were compared to those of a collection of archived global E. coli strains. In conclusion, application of the degenerate primer sets to ETEC isolates from surveillance studies increased the total number of ETEC isolates with detectable CFAs by almost 20%. Additionally, MLST analysis suggests that for many CFAs, there may be a requirement for certain genetic backgrounds to acquire and maintain plasmids carrying genes encoding CFAs. Copyright © 2011, American Society for Microbiology.

Pomerantsev A.P.,National Institute of Allergy and Infectious Diseases | Pomerantseva O.M.,Biological Defense Research Directorate | Moayeri M.,National Institute of Allergy and Infectious Diseases | Fattah R.,National Institute of Allergy and Infectious Diseases | And 2 more authors.
Protein Expression and Purification | Year: 2011

Bacillus anthracis produces a number of extracellular proteases that impact the integrity and yield of other proteins in the B. anthracis secretome. In this study we show that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), produced from the B. anthracis Ames 35 strain (pXO1 +, pXO2 -), are completely degraded at the onset of stationary phase due to the action of proteases. An improved Cre-loxP gene knockout system was used to sequentially delete the genes encoding six proteases (InhA1, InhA2, camelysin, TasA, NprB, and MmpZ). The role of each protease in degradation of the B. anthracis toxin components and ALO was demonstrated. Levels of the anthrax toxin components and ALO in the supernatant of the sporulation defective, pXO1 + A35HMS mutant strain deleted for the six proteases were significantly increased and remained stable over 24 h. A pXO1-free variant of this six-protease mutant strain, designated BH460, provides an improved host strain for the preparation of recombinant proteins. As an example, BH460 was used to produce recombinant EF, which previously has been difficult to obtain from B. anthracis. The EF protein produced from BH460 had the highest in vivo potency of any EF previously purified from B. anthracis or Escherichia coli hosts. BH460 is recommended as an effective host strain for recombinant protein production, typically yielding greater than 10 mg pure protein per liter of culture. © 2011 Elsevier Inc. All rights reserved.

PubMed | Global Disease Detection Regional Center, Centers for Disease Control and Prevention, Naval Medical Research Center, Biological Defense Research Directorate and Vector Biology Research Program
Type: Journal Article | Journal: The American journal of tropical medicine and hygiene | Year: 2016

Of 49 workers at a Djiboutian abattoir, eight (16%, 95% confidence interval [CI]: 9-29) were seropositive against spotted fever group rickettsiae (SFGR), two (4%, 95% CI: 1-14) against typhus group rickettsiae, and three (6%, 95% CI: 2-17) against orientiae. One worker (9%, 95% CI: 2-38) seroconverted against orientiae during the study period. This is the first evidence of orientiae exposure in the Horn of Africa. SFGR were also identified by polymerase chain reaction in 32 of 189 (11%, 95% CI: 8-15) tick pools from 26 of 72 (36%) cattle. Twenty-five (8%, 95% CI: 6-12) tick pools were positive for Rickettsia africae, the causative agent of African tick-bite fever. Health-care providers in Djibouti should be aware of the possibility of rickettsiae infections among patients, although further research is needed to determine the impact of these infections in the country.

Zwick M.E.,Emory University | Zwick M.E.,Biological Defense Research Directorate | Joseph S.J.,Emory University | Didelot X.,University of Oxford | And 18 more authors.
Genome Research | Year: 2012

The key genes required for Bacillus anthracis to cause anthrax have been acquired recently by horizontal gene transfer. To understand the genetic background for the evolution of B. anthracis virulence, we obtained high-redundancy genome sequences of 45 strains of the Bacillus cereus sensu lato (s.l.) species that were chosen for their genetic diversity within the species based on the existing multilocus sequence typing scheme. From the resulting data, we called more than 324,000 new genes representing more than 12,333 new gene families for this group. The core genome size for the B. cereus s.l. group was ∼1750 genes, with another 2150 genes found in almost every genome constituting the extended core. There was a paucity of genes specific and conserved in any clade. We found no evidence of recent large-scale gene loss in B. anthracis or for unusual accumulation of nonsynonymous DNA substitutions in the chromosome; however, several B. cereus genomes isolated from soil and not previously associated with human disease were degraded to various degrees. Although B. anthracis has undergone an ecological shift within the species, its chromosome does not appear to be exceptional on a macroscopic scale compared with close relatives.

Albrecht M.T.,Biological Defense Research Directorate | Livingston B.D.,United Medical Systems | Livingston B.D.,MedImmune Inc. | Pesce J.T.,Biological Defense Research Directorate | And 3 more authors.
Vaccine | Year: 2012

Electroporation of DNA vaccines represents a platform technology well positioned for the development of multivalent biodefense vaccines. To evaluate this hypothesis, three vaccine constructs were produced using codon-optimized genes encoding Bacillus anthracis Protective Antigen (PA), and the Yersinia pestis genes LcrV and F1, cloned into pVAX1. A/J mice were immunized on a prime-boost schedule with these constructs using the electroporation-based TriGrid Delivery System. Immunization with the individual pDNA vaccines elicited higher levels of antigen-specific IgG than when used in combination. DNA vaccine effectiveness was proven, the pVAX-PA titers were toxin neutralizing and fully protective against a lethal B. anthracis spore challenge when administered alone or co-formulated with the plague pDNA vaccines. LcrV and F1 pVAX vaccines against plague were synergistic, resulting in 100% survival, but less protective individually and when co-formulated with pVAX-PA. These DNA vaccine responses were Th1/Th2 balanced with high levels of IFN-γ and IL-4 in splenocyte recall assays, contrary to complimentary protein Alum vaccinations displaying a Th2 bias with increased IL-4 and low levels of IFN-γ. These results demonstrate the feasibility of electroporation to deliver and maintain the overall efficacy of an anthrax-plague DNA vaccine cocktail whose individual components have qualitative immunological differences when combined. © 2012.

Zhang J.,Vaxin Inc | Jex E.,Vaxin Inc | Jex E.,Southern Research Institute | Feng T.,Vaxin Inc | And 6 more authors.
Clinical and Vaccine Immunology | Year: 2013

Bacillus anthracis is the causative agent of anthrax, and its spores have been developed into lethal bioweapons. To mitigate an onslaught from airborne anthrax spores that are maliciously disseminated, it is of paramount importance to develop a rapid-response anthrax vaccine that can be mass administered by nonmedical personnel during a crisis. We report here that intranasal instillation of a nonreplicating adenovirus vector encoding B. anthracis protective antigen could confer rapid and sustained protection against inhalation anthrax in mice in a single-dose regimen in the presence of preexisting adenovirus immunity. The potency of the vaccine was greatly enhanced when codons of the antigen gene were optimized to match the tRNA pool found in human cells. In addition, an adenovirus vector encoding lethal factor can confer partial protection against inhalation anthrax and might be coadministered with a protective antigen-based vaccine. Copyright © 2013, American Society for Microbiology. All Rights Reserved.

Brenneman K.E.,Biological Defense Research Directorate | Brenneman K.E.,Ohio State University | Doganay M.,Erciyes University | Akmal A.,Biological Defense Research Directorate | And 6 more authors.
FEMS Immunology and Medical Microbiology | Year: 2011

Bacillus anthracis, the causative agent of anthrax, produces a tripartite toxin composed of two enzymatically active subunits, lethal factor (LF) and edema factor (EF), which, when associated with a cell-binding component, protective antigen (PA), form lethal toxin and edema toxin, respectively. In this preliminary study, we characterized the toxin-specific antibody responses observed in 17 individuals infected with cutaneous anthrax. The majority of the toxin-specific antibody responses observed following infection were directed against LF, with immunoglobulin G (IgG) detected as early as 4 days after the onset of symptoms in contrast to the later and lower EF- and PA-specific IgG responses. Unlike the case with infection, the predominant toxin-specific antibody response of those immunized with the US anthrax vaccine absorbed and UK anthrax vaccine precipitated licensed anthrax vaccines was directed against PA. We observed that the LF-specific human antibodies were, like anti-PA antibodies, able to neutralize toxin activity, suggesting the possibility that they may contribute to protection. We conclude that an antibody response to LF might be a more sensitive diagnostic marker of anthrax than to PA. The ability of human LF-specific antibodies to neutralize toxin activity supports the possible inclusion of LF in future anthrax vaccines. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.

Walper S.A.,U.S. Navy | Anderson G.P.,Center for Bio Molecular Science and Engineering | Lee P.A.B.,Nova Research Inc. | Glaven R.H.,Nova Research Inc. | And 6 more authors.
PLoS ONE | Year: 2012

Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs) were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors.

Dragan A.I.,University of Maryland Baltimore County | Albrecht M.T.,Biological Defense Research Directorate | Pavlovic R.,University of Maryland Baltimore County | Keane-Myers A.M.,Biological Defense Research Directorate | Geddes C.D.,University of Maryland Baltimore County
Analytical Biochemistry | Year: 2012

Rapid presymptomatic diagnosis of Bacillus anthracis at early stages of infection plays a crucial role in prompt medical intervention to prevent rapid disease progression and accumulation of lethal levels of toxin. To detect low levels of the anthrax protective antigen (PA) exotoxin in biological fluids, we have developed a metal-enhanced fluorescence (MEF)-PA assay using a combination of the MEF effect and microwave-accelerated PA protein surface absorption. The assay is based on a modified version of our "rapid catch and signal" (RCS) technology previously designed for the ultra-fast and sensitive analysis of genomic DNA sequences. Technologically, the proposed MEF-PA assay uses standard 96-well plastic plates modified with silver island films (SiFs) grown within the wells. It is shown that the fluorescent probe, covalently attached to the secondary antibody, plays a crucial role of indicating complex formation (i.e., shows a strong MEF response to the recognition event). Microwave irradiation rapidly accelerates PA deposition onto the surface ("rapid catch"), significantly speeding up the MEF-PA assay and resulting in a total assay run time of less than 40 min with an analytical sensitivity of less than 1 pg/ml PA. © 2012 Elsevier Inc. All rights reserved.

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