DEPARTMENT OF HEALTH & HUMAN SERVICES                                        National Institutes of Health

NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES

www.niaid.nih.gov

Date: August 10, 2004

Marshall Thomas

Leonardo Concepts, Inc.

16918 Cottonwood Way

Houston, TX 77059

Dear Marshall Thomas:

Your proposal entitled, “Pneumonic plague therapeutic” including the specific aim to evaluate bacteriophage as a treatment for Yesinia pestis infection in an aersol mouse model has been found to be responsive to the NIH Guide Notice: NOT-AI-04-044 (found at http://grants1.nih.gov/grants/guide/notice-files/NOT-AI-04-044.html) and you are hereby permitted to submit the full application letter for consideration. Be advised to follow all instructions for assembling a complete application (incomplete application will be returned without review). Comprehensive instructions can be found athttp://www2.niaid.nih.gov/biodefense/bioshield.htm.  Also remember to include a copy of this letter along with your application.

Please forward the signed original of the application, five (5) paper copies, and one (1) CD with a faithful PDF file copy to:

Edward Schroder, Ph.D.

Division of Extramural Activities
National Institute of Allergy and Infectious Diseases
6700-B Rockledge Drive, Room 2216
Bethesda, MD 20892-7616
Bethesda, MD 20817-7616 (for express/courier service)
Tel:             301-435-8537      
Fax: 301-402-2638

Your application should arrive at our offices on or before September 23, 2004.  If you have any questions or require further guidance in completing your application material, please do not hesitate to contact me for clarification.

Good luck with your application.

Sincerely,

Michael Kurilla, MD-PhD

DMID, NIAID, NIH, DHHS

RESEARCH PLAN

1. Specific Aims

     The Phase II goal of this initiative is to test, and evaluate a drug therapy in small mammals that demonstrates efficacy against a category A pathogen and can be used successfully against antibiotic resistant biological weapons. Specifically, it is a drug that can successfully counter the large-scale use of an aerosolized pneumonic plague bioweapon against the civilian population, one that has been weaponized to be antibiotic resistant.

     The Strategic National Stockpile represents the repository of biodefense drugs for the United States to be used in the event of a national emergency.  Vaccine represents a proven means to guard against communicable disease but immunizing the entire country against dozens of biological agents is impractical because of the expense and the possible immunological effects.  Antibody therapy is also a proven treatment for some category A agents, but the rate of immunological complication is on the order of 10(4) versus 10(6) for vaccine.  What is more antibody proteins are often difficult to produce in very large quantities in an economical manner.  Both vaccine and antibody are readily available currently for U.S. armed forces as a means of prophylaxis and in response to attack using biological weapons on the battlefield.  Neither approach is currently available on the same scale for the civilian population.  If recent history is any guide it is the civilian population that will come under attack by biological weapons, probably in the not too distant future.  Antibiotic therapy has been wildly successful for the last 60 years against bacterial pathogen, though there is the specter of increasing antibiotic resistance, particularly in the hospital setting.  Antibiotic resistance is a primary feature of biological weapons developed during the Cold War and remains an issue of grave concern. (27) Developing new classes of antibiotics is a fabulously expensive endeavor and as a result there has been little commercial motivation to do so.  Antibiotic therapy is a mainstay of the SNS in the form of push packs that can be rushed to areas of disease outbreak in an emergency situation, however there is currently no contingency for defense against the use of biological weapons that exhibit antibiotic resistance.  The strategy of using bacteriophage therapy against antibiotic resistant strains of bacterial pathogens has been validated in previous studies. (2)(21)(34)  

     In Phase II aerosol pneumonic plague experiments will characterize empirically the efficacy of the bacteriophage drug in mice.  The experimental design will parallel that of previous in vivo tests of aerosol pneumonic plague against numerous different standard antibiotics recommended for the treatment of Y. pestis. (39) It is expected that the experiments will prove the ability of the bacteriophage drug to rescue mice 24 hours post challenge at the rate of 100%. This expectation is the measurable goal of success for Phase II trials and will constitute sufficient reason to proceed to Phase III.  A secondary goal is for the phage drug to prove even greater efficacy than the mean performance of the antibiotics at 48 hours post challenge.  The expectation that phage will show greater efficacy than the antibiotics has been borne out in previous studies with other pathogenic bacteria, and will constitute even greater evidence of the value of the phage model.  (34) The experimental design may be repeated with variations if the first study is deemed successful and valuable data can be gathered by varying parameters of the experiment.

     In 1942 when Luria performed the experiments on antibiotic resistance that were later to win him the Nobel Prize for medicine, he described the factor that has come upon the medical community with a vengeance in the last two decades. Luria, working with a brand new experimental class of drugs, antibiotics, proved that starting from a single organism, mutation was an inherent feature of microbial life forms.  The new drug was penicillin, not yet introduced for widespread use.  The new drug was shown to be ineffective against an organism that it had hardly ever been exposed to before.  The mechanism of plasmid DNA was as yet not known, but the effects were proven.  Man’s latest miracle drug was not able to extinguish a bacterium that had previously been shown to exhibit sensitivity.  Antibiotic resistance and the arms race between man made drugs and bacterium were already beginning to be lost, just as the first battle had begun. Bacteriophage have been locked in this battle for billions of years.  The mutual give and take of mutation and counter mutation have probably settled down into a steady state rotation that ends with the bacteria mutating against the lytic activity of the phage, but in the process loses its envelope or some other integral functional part that confers to it the ability to propagate wildly inside a host.  Phage and bacteria have always coevolved and always will.  This is the allure of phage for treating antibiotic resistance.  It is a battle that it has always won and always will win whether man chooses to participate or not. (9)  

     The ultimate goals in Phase III will be to prove the safety of the phage drug in human trials and to build a pilot plant to determine the technologies necessary to produce large amounts of phage for stockpiling, something not done since the 1930’s in the U.S.  The proof of concept of production capability will open the door to additions to the SNS.

B. Significance

     The category A pathogens that might be used as biological weapons represent a clear and present danger.  Out of all these pathogens, antibiotic resistant pneumonic plague is the biological warfare weapon that has the potential to be one of the most devastating.  In the war game Topoff One the scenario of an aerosol pneumonic plague attack on the city of Denver, Colorado was simulated.  The war game scenario was stopped after 4 days.  Approximately 4,000 ill and 2,000 dead were tallied with the epidemic mushrooming into a global crisis.  The borders of the state of Denver had been closed to no avail, with the disease spreading to Europe and Asia.  In the words of the war game director, “ It looked like an epidemic out of control...” (12) What had started as a domestic attack had ended in a potential pandemic, a worldwide outbreak of a communicable disease that, in the case of antibiotic resistance, would have proven nearly 100% fatal without treatment.  This scenario is the exact contingency, which the phage drug is intended to counter.  

     During the Cold War the former USSR began a biological warfare program specifically to target the U.S. civilian population centers in time of war.  One aspect of this monumental effort (30,000 people) was known as Project Bonfire. This program culminated in the creation of some of the most dangerous bioweapons ever devised, one of which was pneumonic plague that was resistant to virtually every known antibiotic. (27) Though the large stockpiles have probably been destroyed, the seed stock of this very expensive effort are no doubt still in existence.  The porosity and corruption of the Russian security apparatus in the wake of their economic and political collapse are still very much in evidence today. 

     Antibiotic resistance plasmids are naturally occurring across a wide spectrum of bacterial diseases and Y. pestis is no different.  In Madagascar a case of resistant plague was discovered in 1995, one that was resistant to multiple drugs, including all the drugs recommended for prophylaxis and therapy against plague. (5) The isolate carried the resistance genes in a plasmid and was able to conjugate to other Y. pestis isolates.   Without a response to antibiotic resistance in Y. pestis, be it natural or man made, the eventuality of a communicable disease outbreak of a kind not seen in over a century is a very real possibility.  Achieving the goals of this initiative will greatly enhance public health in this regard.  

     Historical Pandemic that destroyed nations and civilizations were pneumonic in nature.  Pneumonic Plague spreads rapidly and is nearly 100% fatal without treatment.  The Plague of Justinian in the sixth century depopulated much of the Mediterranean Basin and effectively ended Roman Civilization.  Christianity emerged as a political force to fill the power vacuum left in its wake.  The Black Death began in Italy in the 1340's and laid waste to a third of Europe.  Before it was over Feudalism was broken, and disaffection with the church led to the Renaissance and the birth of Science.  In each case society was radically transformed, socially and politically.  Civilizations that lose 50% or more of their populations in a plague event suffer functional and psychological collapse.  The societies are converted or taken over with relative ease.  The old society, the old belief systems are no longer of value if they cannot protect you.  Historically after such an event new religions arise to replace old ones.  Complex societies literally fall apart and reform into something different or split into more than one new entity be it tribe or state.  The impact of Yersinia pestis upon human history is perhaps greater than all other factors save geography and climate.  

     Phage therapy is nearly as old as the science of microbiology.  It is still used today in the Russia, Poland, and Georgia to treat naturally occurring bacterial diseases that we treat in the west using antibiotics, as well as the treatment of antibiotic resistant organisms.  Felix D’ Herelle successfully treated bubonic plague victims using bacteriophage therapy. (26) 

     Bacteriophage attach to the plague bacillus and enter the cell to reproduce 200 or more copies of itself in 25 minutes.  The phage lyse the cell wall and release the daughter cells that collide with more host bacteria in the blood stream.  A small dose has a magnified effect.  If each of the 200 daughter cells infects a plague bacillus there will be 40,000 new phage, then 8 million, then 1.6 billion at the end of the 4^th cycle. (7) The phage are self limiting, if no host bacteria are available for reproduction they are cleared from the blood stream in approximately one week by the reticuloendothelial system, predominantly collected in the spleen.  The phage drug does not interact with eukaryotic cells, only those prokaryotic cells that are the narrow range of its host bacterial strain.  These phage are so specific in fact that their interactions with one host and lack of lytic activity in another function as the definition of where one strain ends and another begins.  

 C. Relevant Experience

     Dr. Stephen D. Prior will be the Principal Investigator project director for the proposed research project.  Dr. Richard M. Carlton will be acting as Co-Principal Investigator.  Dr. Biswajit Biswas will collaborate as a laboratory investigator. Dr. Hank Lockman will participate as a consultant.  Marshall Thomas will participate as the project leader and research assistant.  Their qualifications are listed below.

Principal Investigator

     Dr. Steven Pryor of Exponential Biotherapies, Inc. has served as the companies Chief Scientific Officer, Dr. Prior is the Research Director, National Security Health Policy Center and the Field Director of the Critical Incident Analysis Group. He advises the U.S. government on defense against bioterrorism and was formerly the President and CEO of Dynport, LLC.

Co-Principal Investigator  

     Dr. Richard M. Carlton of Exponential Therapies, Inc. is the Company’s President and a Director.  Dr. Carlton was previously the President of Clinical Trials Consulting, Inc., a firm that facilitated technology transfer and clinical trials for academia and industry.  In 1990, he founded the Center for Research in Anxiety Disorders, a clinical research site for new investigative drugs. 

Co-Investigator-Collaborator

     Dr. Biswajit Biswas of Exponential Biotherapies, Inc. is the Science Director of their laboratories and has worked intensively for the last nine years perfecting techniques for phage propagation and use in experimentation.  He is skilled in producing species that have efficacy against antibiotic resistant organisms and has published multiple papers on the subject working with his colleagues at EBI.

Consultant

     Dr. Hank A. Lockman of the Battelle Memorial Institute was hired specifically to run their program for testing aerosol pathogens in small mammals.  He was formerly a member of the faculty at Ohio State University.  The Battelle Memorial Institute began operation in 1929 and is the site of the longest running continual experimental facility in the U.S.  They have 16,000 employees and did $2.7 billion in scientific consulting last year.

Project Leader/Research Associate

     Marshall Thomas of Leonardo Concepts Inc. was a member of the Hanta Virus Team at Texas Tech University. Previously he was employed by McDonnell Douglas SSC (NASA) and has worked for the Lunar and Planetary Institute (USRA)   

D. Experimental Design and Methods     

     Bacteriophage that are capable of lysing bacteria in vitro have been successfully used to treat mice that were experimentally infected with, e.g., Bacillus cereus, Escherichia coli, and Salmonella typhimurium.  Phage therapy is an alternative to the use of antibiotics or vaccination and may augment both of the former modalities.  A virulent bacteriophage that lyses Y. pestis in vitro will be tested in mice.  Mice will be challenged with a lethal aerosol dose of Y. pestis and then treated with phage to determine if the phage are active in vivo, post exposure prophylaxis.

     Adult mice (50% male and 50% female) will be obtained from a USDA approved vendor and quarantined prior to the start of the study.  To determine the efficacy of the bacteriophage therapy, mice will be exposed to an aerosol containing virulent Yersinia pestis and then treated with phage that will be administered by intraperitoneal injection (IP).  Mice will be passed through an airlock into a class III-biosafety cabinet and loaded into a nose only exposure system.  As mice are placed into the exposure chamber, they will be provided with filtered fresh air until the exposures begin.  Aerosols will be produced from aqueous suspensions of Y. pestis in a 3-jet or 6-jet Collison nebulizer and delivered to the mice via a nose-only system.  The targeted aerosol challenge dose will be approximately 1 LD50.  A sample of the aerosol will be collected in an all-glass impinger for the duration of each exposure.  Impinger suspensions will be assayed to determine colony-forming units (CFU) per ml as per Battelle Standard Operating Procedure (SOP) Number MREF.X-054.  The volume of the test atmosphere sampled through impingers will be used to calculate the concentration of Y. pestis in the test atmosphere.  The mass-median aerodynamic diameters for Y. pestis aerosols may be determined with an Aerodynamic Particle Sizer spectrometer or, alternatively, by a cascade impactor.  Previously published values for mouse tidal volumes and minute ventilation will be used to determine the duration of the aerosol challenge.  All exhausts will be filtered through HEPA filters before exhausting into the class III-biosafety cabinet and then double HEPA-filtered again before exiting the building.  The mice will be decontaminated and removed form the class III cabinet as per Battelle SOP.  The mice in this study will be challenged over 4 days with approximately 50 phage treated and 20 untreated control mice challenged on each day.  Details on aerosol exposures are contained in Battelle SOP. 

     Mice will be treated b.i.d. with 10(9) or 10(11) plaque forming units (PFU) per mouse.  The first dose will be administered to groups of mice at 1, 6, 12, 24, and 48 hours after exposure, and subsequently at approximately 12 hour intervals after the first dose of phage (table 1, pg.14).  Phage will be administered IP and treatments will continue for 7 consecutive days following the first dose.  At 2 and 5 days after the initiation of phage therapy, 5 mice from each group will be exsanguinated for quantitative bacterial and phage titers of the blood.   The remainder of the mice will be observed for 14 days post challenge and then survivors will be euthanized.

     Previous tests involving aerosolized pneumonic plague tests and the post exposure prophylaxis of antibiotics have laid a good framework for comparisons to be drawn between phage and standard antibiotics.  The ability of phage to work against antibiotic resistant forms of Y. pestis is a factor that goes far beyond the simple comparisons that might be drawn relating to survivability of experimental animals.  With this in mind, it is only necessary for phage therapy to be equivalent to the mean performance of standard antibiotic treatment for Y. pestis to be judged a success.

     In one study of pneumonic plague against standard antibiotic treatment, Y. pestis strain CO92 was used with an LD50 at 2.3 x 10(4) CFU (Guyton’s formula). (39) Antibiotics of 20mg/kg were administered every 6 hours for 5 days, after waiting 24 and 42 hours respectively.  Ciprofloxacin at standard dosage proved most effective in prophylaxis and rescue of 100% of the mice at 24 hours and 60% rescue at 42 hours from 100 LD50.  After 48 hours only 25% were survivors. The factor of antibiotic resistance is difficult to apply without empirical results, but suffice it to say the outcome would have been much worse.  Should bacteriophage therapy rescue comparable numbers as antibiotics, and under similar study conditions than the best performing antibiotic, then the new drug must be viewed as an unqualified success.  In a separate study using baboons in place of mice one of the best performing antibiotics was streptomycin, which did not yield good results if the blood concentration of plague was greater than 4 x 10(4) per cm3. (42) The ability of bacteriophage to rescue plague victims at these blood specimen concentrations may be determined with some degree of certainty in the proposed experiment.  The specter of death by sepsis due to release of endotoxins and exotoxins may preclude rescue from this degree of infection.  The sensitivity of mice is considerably more to plague than human or baboon and there will have to be some statistical adjustment made to state such a finding with any degree of confidence.  Oral phage preparations may be desirable to treat cases of such high blood saturation by plague.  These questions lay the groundwork for future experiments to increase our understanding and expand our arsenal. 

 Phase III

     Phase II experimental studies of aerosol pneumonic plague challenge in mice will prove empirically that the phage drug works equally as well or better than standard antibiotic therapy against Y. pestis.  Finally in Phase III the human safety trials will validate the safety issue and the pilot plant project will investigate the technologies of materials handling and materials processing that a batch process for the new phage drug will entail.  

E. Human Subjects

No human subjects are involved in this aspect of the project.  An IND is scheduled for FDA review of human safety trials to be conducted in Phase III.

F. Vertebrate Animals

Vertebrate animals will be used in this study.  The animal standards documents will be supplied at the time of award or before. 

G. Consultants

Dr. Hank A. Lockman of the Battelle Memorial Institute will conduct the aerosol pneumonic plague experiments on mice. Dr. Lockman is a former member of the faculty at Ohio State University and was hired expressly by Battelle for his degree of expertise at aerosol pathogen testing in small mammals.  The Battelle Memorial Institute is the premier private entity for NBC testing and the only entity totally capable of performing these experiments outside of USAMRIID.

 H. Contractual Arrangements

Exponential Biotherapies Inc., and Leonardo Concepts Inc., have entered into a contractual agreement concurrent with the STTR guidelines. 

Marshall Gregory Thomas