Biological Defense Research Directorate

Naval Academy, MD, United States

Biological Defense Research Directorate

Naval Academy, MD, United States
SEARCH FILTERS
Time filter
Source Type

News Article | April 25, 2017
Site: www.eurekalert.org

VIDEO:  Scientists and physicians at University of California San Diego School of Medicine, working with colleagues at the U.S. Navy Medical Research Center (NMRC), Texas A&M University, a San Diego-based biotech... view more Scientists and physicians at University of California San Diego School of Medicine, working with colleagues at the U.S. Navy Medical Research Center - Biological Defense Research Directorate (NMRC-BDRD), Texas A&M University, a San Diego-based biotech and elsewhere, have successfully used an experimental therapy involving bacteriophages -- viruses that target and consume specific strains of bacteria -- to treat a patient near death from a multidrug-resistant bacterium. The therapeutic approach, which has been submitted to a peer-reviewed journal, is scheduled to be featured in an oral presentation tomorrow at the Centennial Celebration of Bacteriophage Research at the Institute Pasteur in Paris by Biswajit Biswas, MD, one of the case study's co-authors and chief of the phage division in the Department ?Genomics and Bioinformatics at NMRC-BDRD. April 27 is Human Phage Therapy Day, designated to mark 100 years of clinical research launched by Felix d'Herelle, a French-Canadian microbiologist at Institute Pasteur who is credited with co-discovering bacteriophages with British bacteriologist Frederick Twort. Authors say the case study could be another catalyst to developing new remedies to the growing global threat of antimicrobial resistance, which the World Health Organization estimates will kill at least 50 million people per year by 2050. Based on the success of this case, in collaboration with NMRC, UC San Diego is exploring options for a new center to advance research and development of bacteriophage-based therapies. "When it became clear that every antibiotic had failed, that Tom could die, we sought an emergency investigational new drug application from the FDA to try bacteriophages," said lead author Robert "Chip" Schooley, MD, professor of medicine, chief of the Division of Infectious Diseases in the UC San Diego School of Medicine and primary physician on the case. "To our knowledge, he is the first patient in the United States with an overwhelming, systemic infection to be treated with this approach using intravenous bacteriophages. From being in a coma near death, he's recovered well enough to go back to work. Of course, this is just one patient, one case. We don't yet fully understand the potential -- and limitations -- of clinical bacteriophage therapy, but it's an unprecedented and remarkable story, and given the global health threat of multidrug-resistant organisms, one that we should pursue." The story begins in late-2015. Tom Patterson, PhD, a 69-year-old professor in the Department of Psychiatry at UC San Diego School of Medicine, and his wife, Steffanie Strathdee, PhD, chief of the Division of Global Public Health in the Department of Medicine, were spending the Thanksgiving holiday in Egypt when Patterson became ill, wracked by abdominal pain, fever, nausea, vomiting and a racing heartbeat. Local doctors diagnosed pancreatitis -- inflammation of the pancreas -- but standard treatment didn't help. Patterson's condition worsened and he was medevacked to Frankfurt, Germany Dec. 3, 2015, where doctors discovered a pancreatic pseudocyst, a collection of fluid around the pancreas. The fluid was drained and the contents cultured. Patterson had become infected with a multidrug-resistant strain of Acinetobacter baumannii, an opportunistic and often deadly pathogen. The bacterium has proved particularly problematic in hospital settings and in the Middle East, with many injured veterans and soldiers returning to the U.S. with persistent infections. Initially, the only antibiotics with any effect proved to be a combination of meropenem, tigecycline and colistin, a drug of last resort because it often causes kidney damage, among other side effects. Patterson's condition stabilized sufficiently for him to be airlifted Dec. 12, 2015, from Germany to the Intensive Care Unit (ICU) at Thornton Hospital at UC San Diego Health. Upon arrival, it was discovered that his bacterial isolate had become resistant to all of these antibiotics. At Thornton Hospital, now part of Jacobs Medical Center, Patterson began to recover, moving from the ICU to a regular ward. But the day before scheduled discharge to a long-term acute care facility, an internal drain designed to localize his infection and keep it at bay slipped, spilling bacteria into his abdomen and bloodstream. Patterson immediately experienced septic shock. His heart began racing. He could not breathe. He became feverish and would subsequently fall into a coma that would last for most of the next two months. He was, in effect, dying. "That's a period of my life I don't remember," recalled Patterson. "There was so much pain that it's almost beyond your ability to cope. I'm happy not to remember." Strathdee, his wife, is no stranger to the terrors of disease. As an infectious disease epidemiologist and director of the UC San Diego Global Health Institute, she has worked around the world, from India to Afghanistan to Mexico, trying to lower HIV infection and mortality rates. "There came a point when he was getting weaker and weaker, and I didn't want to lose him. I wasn't ready to let him go and so I held his hand and said, 'Honey, they're doing everything they can and there's nothing that can kill this bug, so if you want to fight, you need to fight. Do you want me to find some alternative therapies? We can leave no stone unturned.'" Tom recalled the moment: "I vaguely remember you saying, 'do you want me to try or not because it's going to be a tough time and it's not certain that it will work.' I remember squeezing your hand, but it was just a flash in the whole process." Strathdee began doing research. A colleague mentioned a friend had traveled to Tblisi, Georgia to undergo "phage therapy" for a difficult condition and had been "miraculously cured." Strathdee had learned of bacteriophages while she was a student, but they were not part of mainstream medical doctrine. She turned to strangers in the phage research community and to her colleague Chip Schooley for help. Bacteriophages are ubiquitous viruses, found wherever bacteria exist. It's estimated there are more than 1031 bacteriophages on the planet. That's ten million trillion trillion, more than every other organism on Earth, including bacteria, combined. Each is evolved to infect a specific bacterial host in order to replicate -- without affecting other cells in an organism. The idea of using them therapeutically is not new. Described a century ago, phage therapy was popular in the 1920s and 1930s to treat multiple types of infections and conditions, but results were inconsistent and lacked scientific validation. The emergence of antibiotics in the 1940s pushed phage therapy aside, except in parts of Eastern Europe and the former Soviet Union, where it remained a topic of active research. With dwindling options, Strathdee, Schooley and colleagues went looking for help. They found many researchers willing to help. Three teams possessed suitable phages that were active against Patterson's particular bacterial infection: the Biological Defense Research Directorate of the NMRC in Frederick, MD; the Center for Phage Technology at Texas A&M University; and AmpliPhi, a San Diego-based biotech company specializing in bacteriophage-based therapies. A research team at San Diego State University, headed by microbial ecologist Forest Rowher, PhD, purified the phage samples for clinical use. With emergency approval from the Food and Drug Administration, each source provided phage strains to UC San Diego doctors to treat Patterson, with no guarantee that any of the strains would actually work. "That's one of the remarkable things to come out of this whole experience," said Schooley, "the incredible and rapid collaboration among folks scattered around the world. It was a desperate time and people really stepped up." Phage therapy is typically administered topically or orally. In Patterson's case, the phages were introduced through catheters into his abdominal cavity and intravenously to address a broader, systemic infection, which had not been done in the antibiotic era in the U.S. "That makes them more effective," said Schooley. "The action is at the interface of the patient and the organism." With tweaking and adjustments -- his physicians were learning on the fly -- Patterson began to improve. He emerged from his coma within three days of the start of IV phage therapy. "Tom woke up, turned to his daughter and said, 'I love you'," recalled Schooley. Patterson was soon weaned off of the respirator and blood pressure drugs. "As a treating doctor, it was a challenge," said Schooley. "Usually you know what the dosage should be, how often to treat. Improving vital signs is a good way to know that you're progressing, but when you're doing it for the first time, you don't have anything to compare it to. "A lot was really worked out as we went along, combining previous literature, our own intuition about how these phages would circulate and work and advice from people who had been thinking about this for a long time." By the time Patterson was airlifted to Thornton Hospital at UC San Diego Health, he was in dire straits. His abdomen had swelled, distended by the pseudocyst teeming with multi-drug resistant A. baumaunnii. His white blood cell count had soared -- a sign of rampant infection. Doctors tried various combinations of antibiotics. He developed respiratory failure and hypotension that required ventilation and recurrent emergency treatment. He became increasingly delirious. When he lapsed into a coma in mid-January, he was essentially being kept alive on life support. Eventually Schooley said there were no antimicrobial agents left to try. Strathdee recalled colleagues wondering aloud if she was prepared for Tom to die. She wasn't. Bacteriophage therapy began March 15, 2016, with a cocktail of four phages provided by Texas A&M and the San Diego-based biotech company AmpliPhi, pumped through catheters into the pseudocyst. If the treatment didn't kill him, Patterson's medical team planned to inject the Navy's phages intravenously, flooding his bloodstream to reach the infection raging throughout his body. As far as Patterson's doctors knew, such treatment had never been tried before. On March 17, the Navy phages were injected intravenously. There were fears about endotoxins naturally produced by the phages. No one knew what to expect, but Patterson tolerated the treatment well -- indeed there were no adverse side effects -- and on March 19, he suddenly awoke and recognized his daughter. "One of NMRC's goals with respect to bacteriophage science has been providing military members infected with multidrug-resistant organisms additional antimicrobial options so we were experienced and well-positioned to provide an effective phage cocktail for Dr. Patterson," said Theron Hamilton, PhD, head of Genomics and Bioinformatics at the Navy's Biological Defense Research Directorate. "Obviously, we are thrilled with the outcome and hope this case increases awareness of the possibility of applying phage therapy to tough cases like this one." Subsequent treatment, however, would not be easy. The learning curve was steep and unmarked. There were bouts of sepsis -- a life-threatening complication caused by massive infection. Despite improvement, Patterson's condition remained precarious. Doctors discovered that the bacterium eventually developed resistance to the phages, what Schooley would characterize as "the recurring Darwinian dance," but the team compensated by continually tweaking treatment with new phage strains -- some that the NMRC had derived from sewage -- and antibiotics. In early May, Patterson was taken off of antibiotics. After June 6, there was no evidence of A. baumannii in his body. He was discharged home August 12, 2016. Recovery has not been entirely smooth and steady. There have been setbacks unrelated to the phages. A formerly robust man, Patterson had been fed intravenously for months in the hospital and had lost 100 pounds, much of it muscle. He has required intense physical rehabilitation to regain strength and movement. "It's not like in the movies where you just wake up from a coma, look around and pop out of bed," Patterson said. "You discover that your body doesn't work right anymore." He said he could feel parts of his brain coming back alive. Nonetheless, Patterson described the experience as miraculous. Even comatose, when he often wrestled with imagined demons, he recalled hearing and recognizing voices and realizing that beyond his darkness, there was life and hope. And beyond him, he hopes his experience will translate into new treatments for others: "The phage therapy has really been a miracle for me, and for what it might mean that millions of people who may be cured from multidrug-resistant infections in the future. It's been sort of a privilege." Schooley said Patterson was lucky. His wife was a trained scientist and determined to find a remedy -- and they both worked at UC San Diego School of Medicine: "He was fortunate to be in a place that had all of the resources and courage necessary to support him while this innovative therapy was developed, which was essentially a home brew cocktail of viruses to be given to a desperately ill individual. I think a lot of other places would have hesitated. I think the response that he had clinically has been very gratifying and speaks to the strength of a multidimensional medical center with all of the pieces you need." Still, Schooley said any broad, future approved application of phage therapy faces fundamental challenges unlike past treatments. "What the FDA is used to saying is 'This is an antibiotic. We know what its structure is and how you can give it to multiple people.' With bacteriophage therapy, the FDA would be dealing with an approach in which doctors would have to develop phage cocktails for each patient tailored to their infecting organisms. It's the ultimate personalized medicine." The good news, Schooley said, is that new molecular tools, robotics and other advances make personalized medicine possible in a way it wasn't 10 or 15 years ago. "Then, it would have been impossible to contemplate. There's still much research to be done, but I think there are going to be a lot of clinical applications where this approach may be very beneficial to patients." Derived from the Greek words meaning "bacteria eater," bacteriophages are ancient and abundant -- found on land, in water, within any form of life harboring their target. According to Rowher at San Diego State University and colleagues in their book Life in Our Phage World, phages cause a trillion trillion successful infections per second and destroy up to 40 percent of all bacterial cells in the ocean every day. Thousands of varieties of phage exist, each evolved to infect only one type or a few types of bacteria. Like other viruses, they cannot replicate by themselves, but must commandeer the reproductive machinery of bacteria. To do so, they attach to a bacterium and insert their genetic material. Lytic phages then destroy the cell, splitting it open to release new viral particles to continue the process. As such, phages could be considered the only "drug"' capable of multiplying; when their job is done, they are excreted by the body.


News Article | April 26, 2017
Site: www.sciencedaily.com

Scientists and physicians at University of California San Diego School of Medicine, working with colleagues at the U.S. Navy Medical Research Center -- Biological Defense Research Directorate (NMRC-BDRD), Texas A&M University, a San Diego-based biotech and elsewhere, have successfully used an experimental therapy involving bacteriophages -- viruses that target and consume specific strains of bacteria -- to treat a patient near death from a multidrug-resistant bacterium. The therapeutic approach, which has been submitted to a peer-reviewed journal, is scheduled to be featured in an oral presentation at the Centennial Celebration of Bacteriophage Research at the Institute Pasteur in Paris by Biswajit Biswas, MD, one of the case study's co-authors and chief of the phage division in the Department ?Genomics and Bioinformatics at NMRC-BDRD. April 27 is Human Phage Therapy Day, designated to mark 100 years of clinical research launched by Felix d'Herelle, a French microbiologist at Institute Pasteur who is credited with co-discovering bacteriophages with British bacteriologist Frederick Twort. Authors say the case study could be another catalyst to developing new remedies to the growing global threat of antimicrobial resistance, which the World Health Organization estimates will kill at least 50 million people per year by 2050. Based on the success of this case, in collaboration with NMRC, UC San Diego is exploring options for a new center to advance research and development of bacteriophage-based therapies. "When it became clear that every antibiotic had failed, that Tom could die, we sought an emergency investigational new drug application from the FDA to try bacteriophages," said lead author Robert "Chip" Schooley, MD, professor of medicine, chief of the Division of Infectious Diseases in the UC San Diego School of Medicine and primary physician on the case. "To our knowledge, he is the first patient in the United States with an overwhelming, systemic infection to be treated with this approach using intravenous bacteriophages. From being in a coma near death, he's recovered well enough to go back to work. Of course, this is just one patient, one case. We don't yet fully understand the potential -- and limitations -- of clinical bacteriophage therapy, but it's an unprecedented and remarkable story, and given the global health threat of multidrug-resistant organisms, one that we should pursue." The story begins in late-2015. Tom Patterson, PhD, a 69-year-old professor in the Department of Psychiatry at UC San Diego School of Medicine, and his wife, Steffanie Strathdee, PhD, chief of the Division of Global Public Health in the Department of Medicine, were spending the Thanksgiving holiday in Egypt when Patterson became ill, wracked by abdominal pain, fever, nausea, vomiting and a racing heartbeat. Local doctors diagnosed pancreatitis -- inflammation of the pancreas -- but standard treatment didn't help. Patterson's condition worsened and he was medevacked to Frankfurt, Germany Dec. 3, 2015, where doctors discovered a pancreatic pseudocyst, a collection of fluid around the pancreas. The fluid was drained and the contents cultured. Patterson had become infected with a multidrug-resistant strain of Acinetobacter baumannii, an opportunistic and often deadly pathogen. The bacterium has proved particularly problematic in hospital settings and in the Middle East, with many injured veterans and soldiers returning to the U.S. with persistent infections. Initially, the only antibiotics with any effect proved to be a combination of meropenem, tigecycline and colistin, a drug of last resort because it often causes kidney damage, among other side effects. Patterson's condition stabilized sufficiently for him to be airlifted Dec. 12, 2015, from Germany to the Intensive Care Unit (ICU) at Thornton Hospital at UC San Diego Health. Upon arrival, it was discovered that his bacterial isolate had become resistant to all of these antibiotics. At Thornton Hospital, now part of Jacobs Medical Center, Patterson began to recover, moving from the ICU to a regular ward. But the day before scheduled discharge to a long-term acute care facility, an internal drain designed to localize his infection and keep it at bay slipped, spilling bacteria into his abdomen and bloodstream. Patterson immediately experienced septic shock. His heart began racing. He could not breathe. He became feverish and would subsequently fall into a coma that would last for most of the next two months. He was, in effect, dying. "That's a period of my life I don't remember," recalled Patterson. "There was so much pain that it's almost beyond your ability to cope. I'm happy not to remember." Strathdee, his wife, is no stranger to the terrors of disease. As an infectious disease epidemiologist and director of the UC San Diego Global Health Institute, she has worked around the world, from India to Afghanistan to Mexico, trying to lower HIV infection and mortality rates. "There came a point when he was getting weaker and weaker, and I didn't want to lose him. I wasn't ready to let him go and so I held his hand and said, 'Honey, they're doing everything they can and there's nothing that can kill this bug, so if you want to fight, you need to fight. Do you want me to find some alternative therapies? We can leave no stone unturned.'" Tom recalled the moment: "I vaguely remember you saying, 'do you want me to try or not because it's going to be a tough time and it's not certain that it will work.' I remember squeezing your hand, but it was just a flash in the whole process." Strathdee began doing research. A colleague mentioned a friend had traveled to Tblisi, Georgia to undergo "phage therapy" for a difficult condition and had been "miraculously cured." Strathdee had learned of bacteriophages while she was a student, but they were not part of mainstream medical doctrine. She turned to strangers in the phage research community and to her colleague Chip Schooley for help. Bacteriophages are ubiquitous viruses, found wherever bacteria exist. It's estimated there are more than 1031 bacteriophages on the planet. That's ten million trillion trillion, more than every other organism on Earth, including bacteria, combined. Each is evolved to infect a specific bacterial host in order to replicate -- without affecting other cells in an organism. The idea of using them therapeutically is not new. Described a century ago, phage therapy was popular in the 1920s and 1930s to treat multiple types of infections and conditions, but results were inconsistent and lacked scientific validation. The emergence of antibiotics in the 1940s pushed phage therapy aside, except in parts of Eastern Europe and the former Soviet Union, where it remained a topic of active research. With dwindling options, Strathdee, Schooley and colleagues went looking for help. They found many researchers willing to help. Three teams possessed suitable phages that were active against Patterson's particular bacterial infection: the Biological Defense Research Directorate of the NMRC in Frederick, MD; the Center for Phage Technology at Texas A&M University; and AmpliPhi, a San Diego-based biotech company specializing in bacteriophage-based therapies. A research team at San Diego State University, headed by microbial ecologist Forest Rowher, PhD, purified the phage samples for clinical use. With emergency approval from the Food and Drug Administration, each source provided phage strains to UC San Diego doctors to treat Patterson, with no guarantee that any of the strains would actually work. "That's one of the remarkable things to come out of this whole experience," said Schooley, "the incredible and rapid collaboration among folks scattered around the world. It was a desperate time and people really stepped up." Phage therapy is typically administered topically or orally. In Patterson's case, the phages were introduced through catheters into his abdominal cavity and intravenously to address a broader, systemic infection, which had not been done in the antibiotic era in the U.S. "That makes them more effective," said Schooley. "The action is at the interface of the patient and the organism." With tweaking and adjustments -- his physicians were learning on the fly -- Patterson began to improve. He emerged from his coma within three days of the start of IV phage therapy. "Tom woke up, turned to his daughter and said, 'I love you'," recalled Schooley. Patterson was soon weaned off of the respirator and blood pressure drugs. "As a treating doctor, it was a challenge," said Schooley. "Usually you know what the dosage should be, how often to treat. Improving vital signs is a good way to know that you're progressing, but when you're doing it for the first time, you don't have anything to compare it to. "A lot was really worked out as we went along, combining previous literature, our own intuition about how these phages would circulate and work and advice from people who had been thinking about this for a long time." By the time Patterson was airlifted to Thornton Hospital at UC San Diego Health, he was in dire straits. His abdomen had swelled, distended by the pseudocyst teeming with multi-drug resistant A. baumaunnii. His white blood cell count had soared -- a sign of rampant infection. Doctors tried various combinations of antibiotics. He developed respiratory failure and hypotension that required ventilation and recurrent emergency treatment. He became increasingly delirious. When he lapsed into a coma in mid-January, he was essentially being kept alive on life support. Eventually Schooley said there were no antimicrobial agents left to try. Strathdee recalled colleagues wondering aloud if she was prepared for Tom to die. She wasn't. Bacteriophage therapy began March 15, 2016, with a cocktail of four phages provided by Texas A&M and the San Diego-based biotech company AmpliPhi, pumped through catheters into the pseudocyst. If the treatment didn't kill him, Patterson's medical team planned to inject the Navy's phages intravenously, flooding his bloodstream to reach the infection raging throughout his body. As far as Patterson's doctors knew, such treatment had never been tried before. On March 17, the Navy phages were injected intravenously. There were fears about endotoxins naturally produced by the phages. No one knew what to expect, but Patterson tolerated the treatment well -- indeed there were no adverse side effects -- and on March 19, he suddenly awoke and recognized his daughter. "One of NMRC's goals with respect to bacteriophage science has been providing military members infected with multidrug-resistant organisms additional antimicrobial options so we were experienced and well-positioned to provide an effective phage cocktail for Dr. Patterson," said Theron Hamilton, PhD, head of Genomics and Bioinformatics at the Navy's Biological Defense Research Directorate. "Obviously, we are thrilled with the outcome and hope this case increases awareness of the possibility of applying phage therapy to tough cases like this one." Subsequent treatment, however, would not be easy. The learning curve was steep and unmarked. There were bouts of sepsis -- a life-threatening complication caused by massive infection. Despite improvement, Patterson's condition remained precarious. Doctors discovered that the bacterium eventually developed resistance to the phages, what Schooley would characterize as "the recurring Darwinian dance," but the team compensated by continually tweaking treatment with new phage strains -- some that the NMRC had derived from sewage -- and antibiotics. In early May, Patterson was taken off of antibiotics. After June 6, there was no evidence of A. baumannii in his body. He was discharged home August 12, 2016. Recovery has not been entirely smooth and steady. There have been setbacks unrelated to the phages. A formerly robust man, Patterson had been fed intravenously for months in the hospital and had lost 100 pounds, much of it muscle. He has required intense physical rehabilitation to regain strength and movement. "It's not like in the movies where you just wake up from a coma, look around and pop out of bed," Patterson said. "You discover that your body doesn't work right anymore." He said he could feel parts of his brain coming back alive. Nonetheless, Patterson described the experience as miraculous. Even comatose, when he often wrestled with imagined demons, he recalled hearing and recognizing voices and realizing that beyond his darkness, there was life and hope. And beyond him, he hopes his experience will translate into new treatments for others: "The phage therapy has really been a miracle for me, and for what it might mean that millions of people who may be cured from multidrug-resistant infections in the future. It's been sort of a privilege." Schooley said Patterson was lucky. His wife was a trained scientist and determined to find a remedy -- and they both worked at UC San Diego School of Medicine: "He was fortunate to be in a place that had all of the resources and courage necessary to support him while this innovative therapy was developed, which was essentially a home brew cocktail of viruses to be given to a desperately ill individual. I think a lot of other places would have hesitated. I think the response that he had clinically has been very gratifying and speaks to the strength of a multidimensional medical center with all of the pieces you need." Still, Schooley said any broad, future approved application of phage therapy faces fundamental challenges unlike past treatments. "What the FDA is used to saying is 'This is an antibiotic. We know what its structure is and how you can give it to multiple people.' With bacteriophage therapy, the FDA would be dealing with an approach in which doctors would have to develop phage cocktails for each patient tailored to their infecting organisms. It's the ultimate personalized medicine." The good news, Schooley said, is that new molecular tools, robotics and other advances make personalized medicine possible in a way it wasn't 10 or 15 years ago. "Then, it would have been impossible to contemplate. There's still much research to be done, but I think there are going to be a lot of clinical applications where this approach may be very beneficial to patients." Derived from the Greek words meaning "bacteria eater," bacteriophages are ancient and abundant -- found on land, in water, within any form of life harboring their target. According to Rowher at San Diego State University and colleagues in their book Life in Our Phage World, phages cause a trillion trillion successful infections per second and destroy up to 40 percent of all bacterial cells in the ocean every day. Thousands of varieties of phage exist, each evolved to infect only one type or a few types of bacteria. Like other viruses, they cannot replicate by themselves, but must commandeer the reproductive machinery of bacteria. To do so, they attach to a bacterium and insert their genetic material. Lytic phages then destroy the cell, splitting it open to release new viral particles to continue the process. As such, phages could be considered the only "drug"' capable of multiplying; when their job is done, they are excreted by the body.


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.


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.


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.

Loading Biological Defense Research Directorate collaborators
Loading Biological Defense Research Directorate collaborators