The Infectious Diseases Directorate (IDD) consists of four research departments: Malaria, Enteric Diseases, Viral and Rickettsial Diseases, and Wound Infections. Within these departments, over 100 scientists and technicians conduct research with an annual budget exceeding $10 million per year. Infectious diseases research at NMRC had its origins in the 1960s and 1970s with work on malaria and rickettsial diseases. Research programs on enteric diseases and viral diseases were started in the early 1980s and early 1990s, respectively. Diagnostic research on biological threat agents also had its origins in IDD in the early 1990s, but was later restructured as a separate directorate at NMRC.
IDD conducts research on infectious diseases that are considered to be significant threats to our deployed sailors, marines, soldiers, and airmen. Significant threats are those that have the potential to incapacitate a large number of deployed forces over a short time period, thus hindering the ability of warfighters to accomplish their mission. The geographical distribution of a disease; the lack of an effective vaccine, treatment, or other control measures; the mode of transmission; and the historical impact during past wars are all factors that determine the importance of an infectious disease to the U.S. Military. As reflected by the departmental organization within IDD, the main infectious disease targets on which research efforts are currently focused are malaria, bacterial causes of traveler's diarrhea, dengue fever, and scrub typhus. In general, the overarching research goal in IDD is to minimize the impact of these infectious diseases by preventing infection or clinical disease. In most cases, the best approach to achieve this goal is through the development of efficacious vaccines. Therefore, most of the biomedical research in IDD is focused on vaccine discovery and testing.
IDD departments have the unique research capability of developing a new vaccine from the conceptual stage through construction, "test tube" evaluation, animal model testing, human volunteer safety and immunogenicity trials to final large-scale human volunteer field trials to prove efficacy as required for FDA licensure of a vaccine. The field testing of vaccines is made easier by IDD's close association with the Navy's overseas medical research laboratories located in Lima, Peru (Naval Medical Research Unit No. 6); Cairo, Egypt (Naval Medical Research Unit No. 3); and the Pacific region (Naval Medical Research Center-Asia and Naval Medical Research Unit No. 2) since these laboratories are located in areas of the world where the target infectious diseases are highly endemic. Scientists in IDD also work closely with their Army colleagues from the Walter Reed Army Institute of Research, which is collocated with NMRC at the modern research facility in Silver Spring, Maryland. With the direct use of recombinant DNA techniques for vaccine delivery combined with genomic and proteomic approaches to discover new vaccine components, the Navy can expect to make rapid progress in the new millennium towards controlling many of the infectious diseases that currently remain as major threats to U.S. military forces deployed around the world.
Malaria Research Department
Malaria, caused by the protozoan Plasmodium, is responsible for more suffering and death across the world than any other parasite. It is a mosquito-borne infection that kills more than 1 million people annually, most of these children under the age of 5. Even when a person survives malaria, the infection can incapacitate a victim for several weeks. Over three billion people, most living in tropical regions, are exposed to malaria, and 500-600 million clinical infections occur every year.
U.S. military forces are at great risk of developing malaria while deployed in endemic areas. In fact, more person-days were lost among U.S. military personnel due to malaria than to bullets during every military campaign fought in malaria-endemic regions during the 20th century. Malaria also is a major threat to non-military travelers, who face this infection as likely the single greatest health risk associated with travel. To address this threat, researchers within the IDD have been investigating methods to control and conquer malaria for more than two decades. This comprehensive research program is at the forefront of malaria research worldwide.
Malaria is a complex parasite with a composition of proteins 300 times more diverse than the viruses that commonly infect humans. Like viruses, the malaria parasite requires a host to live, but unlike most viruses for which vaccines exist, the parasite chronically infects its human host, avoiding the immune system and living in the blood for many years if not treated.. As a result, the parasite has evolved to co-exist with its human host, making it very difficult to stimulate the immune response effectively with a vaccine. The malaria life cycle is also more complex that the life cycle of most viruses and bacteria; in order to work effectively, it may be necessary for a vaccine to induce an equally complex immune response that attacks the parasite at multiple stages. NMRC researchers realized the need for an innovative approach if we are ever to overcome this health threat. As one important initiative, the Malaria Program played a key role in the Malaria Genome Project, a consortium that determined the entire genetic sequence of the most deadly of the four human malaria parasites, P. falciparum.
Navy Malaria Program
The primary objective of the Navy Malaria Program is to develop a vaccine that kills the parasite during its first few days of development in the liver, before it breaks out into the blood. If this approach is successful, it will prevent the clinical manifestation of malaria, which occurs only in conjunction with blood stage infection and not with the liver stage. Such a vaccine would benefit deployed military personnel as well as travelers and other non-immune populations. At the same time, the program is investigating vaccines that would target blood stage infection to limit the severity of symptoms associated with this stage. Both liver and blood stage vaccines, if deployed in endemic areas, could alleviate much of the suffering caused by this parasite in tropical countries.
The program’s strategy is based on the idea that protective immunity will depend on using multiple antigens to induce both cellular and humoral immunity. We are characterizing several promising Plasmodium antigens in a variety of antigen delivery platforms, including plasmid DNA, vaccinia vectors, adenoviral vectors, Venezuelan equine encephalitis replicons, and recombinant proteins. We are testing heterologous prime-boost immunization strategies to find effective combinations of these antigens and vectors. We are using genomic and proteomic strategies to identify novel proteins with potential as vaccine antigens. We are also investigating the protective potential of attenuated malaria parasites.
The Malaria Program’s activities range from discovery research, in which we try to understand the nature of protective immunity, to clinical trials of candidate vaccines, carried out in our clinical trials center on the campus of the National Naval Medical Center. Successful vaccines can be transitioned to testing in field settings, with collaborating institutions in Africa, Asia, and South America. The Malaria Program also benefits from the Navy’s overseas laboratories, which allow study of the epidemiology of parasite in its native habitat, and also help to coordinate field testing of novel vaccines and drugs.
Infectious diarrhea has historically been a substantial cause of morbidity for deployed U.S. military personnel and continues to the present day in those deployed in the global war on terrorism. Pathogenic bacteria, including Campylobacter, enterotoxigenic Escherichia coli (ETEC), and Shigella, are principal causative agents. These pathogens are also major causes of travelers’ diarrhea in civilian populations and endemic diarrheal diseases in young children in resource-limited regions of the globe. While acute infections resolve on their own in three to five days, half of those infected report decrease in job performance and one in ten will go on to develop post-infectious irritable bowel syndrome. The Enteric Diseases Department’s research program is centered on the development of effective countermeasures to prevent or abate bacterial diarrhea, with most efforts aimed at vaccine research and development.
Research efforts for Campylobacter and ETEC include studies of molecular pathogenesis, antigen discovery, and vaccine development and clinical trials. In addition, the department’s Clinical Trials Branch works closely with the Walter Reed Army Institute of Research (WRAIR) Division of Bacterial and Rickettsial Diseases in the clinical evaluation of new Shigella vaccine candidates.
The department is organized into four closely integrated branches: Molecular Biology, Immunology, Biochemistry, and Clinical Trials. Principal investigators within the program work with a number of extramural academic, industry, and government partners to achieve the goal of developing new-generation vaccines against bacterial diarrhea. In the realm of clinical trials, the WRAIR/NMRC facilities afford access to the WRAIR Pilot Bioproduction Facility for scale-up and manufacture of investigational vaccines and with a state-of-the-art outpatient clinical trials center where Phase I safety and immunogenicity trials are conducted.
Enterotoxigenic E. coli
Most Escherichia coli are normal inhabitants of our environment and gastrointestinal tracts and do not cause disease. However, a number of different so-called diarrheagenic E. coli have the ability to colonize the gastrointestinal tract and cause diarrhea in humans and animals. The most common is enterotoxigenic E. coli (ETEC), which causes disease by attaching to the intestinal lining through specialized projections called fimbriae. Once attached, ETEC multiply and produce toxins that stimulate an outpouring of intestinal fluids, which can cause diarrhea and consequent dehydration. NMRC researchers are developing vaccines that would block attachment and toxin activity, interrupting the infection at its earliest stages, which should reduce the number and severity of ETEC diarrhea cases.
Campylobacter jejuni is a relatively newly identified pathogen, yet it has been a focus of the Navy enterics research program since its importance was first recognized. A food-borne pathogen, Campylobacter jejuni causes a more severe disease than ETEC, causing high fever, severe stomach cramps, headache, and joint pains in addition to diarrhea. NMRC’s Enteric Diseases Department has identified many surface structures of the bacteria, found how it invades human cells, and characterized many aspects of the immune response. This work continues with the use of comparative genomics, expression arrays, and studies to try to better understand the protective immune response, all of which will enable researchers to develop an effective vaccine.
Viral and Rickettsial Diseases Department
The Viral and Rickettsial Diseases Department is divided into two divisions. The primary focus of the Viral Diseases Division is on the development of a vaccine to prevent dengue fever; the main objective of the Rickettsial Diseases Division is to develop a scrub typhus vaccine.
Viral Diseases Division
Because of the historic and current military impact of dengue virus infections, the key focus of the Viral Diseases Division is on the development of a vaccine to prevent dengue fever. The major thrust is on utilizing novel vaccine technologies such as DNA vaccines and agile vaccine platforms of recombinant adeno- and Venezuelan equine encephalitis viruses. Development of a phase 1 clinical trial in humans based on demonstrated safety and efficacy in pre-clinical testing utilizing a DNA-based dengue-1 vaccine has proceeded with the submission of an investigational new drug application (IND) to the FDA with plans to begin enrollment in early 2006. Second-generation vaccine approaches are in pre-clinical investigation. One way the department plans to enhance the immunogenicity of the DNA vaccine is through the development of molecular adjuvants.
A critical corollary to vaccine development efforts is the exploration of novel and improved measures for immunological protection against dengue infection. This work is critical to the evaluation of vaccine candidates as well as to the understanding of the beneficial and detrimental aspects of the human immune response to natural dengue infections. Dendritic cells, natural targets of dengue viruses, have been used to establish a useful in vitro system that has the potential to bridge immunological studies with many aspects of vaccine development.
An additional focus of the department is to develop and evaluate field-deployable diagnostic assays for the rapid and accurate detection of dengue virus (antigen or viral RNA) and dengue-specific antibodies in human clinical samples using innovative immunological or molecular approaches.
Rickettsial Diseases Division
Rickettsiae are arthropod-transmitted bacteria that cause a number of diseases capable of debilitating deployed military personnel. Four of the diseases (murine typhus, epidemic typhus, scrub typhus, and trench fever) have had major historical impacts on military operations during wartime. Numerous episodes of rickettsia-like illnesses have also been encountered in the past few years in peacetime military operations. Rickettsial diseases are re-emerging infectious diseases, causing significant medical problems for military personnel deployed throughout the world.
Vaccine development: Currently, the primary objective of the Rickettsial Diseases Division (RDD) program is to develop a scrub typhus vaccine. Different genetically engineered subunit vaccine candidates (recombinant proteins, DNA vaccine, and VRP particles) have been identified in a mouse lethal challenge model. One of these candidates will be selected for pre-clinical trials in FY08.
Diagnosis: RDD has developed quantitative real-time PCR (qPCR) assays for identification and detection of various rickettsial pathogens in clinical specimens and various pathogen vectors. The validation studies of the Rickettsia-specific and the O. tsutsugamushi-specific assays have been initiated in order to obtain sufficient data to request FDA 510k approval. The validation of a rapid, hand held assay for scrub typhus is almost finished and the request for FDA 510k approval will be filed in the spring of 2007. Similar rapid assays for other rickettsial diseases are under development.
Risk Assessment by serology study: RDD assessed the risk of tick-borne rickettsial diseases for military personnel. We determined that the prevalence of antibodies specific for human granulocytotropic anaplasmosis (HGA) agent Anaplasma phagocytophilum and spotted fever group rickettsiae (SFGR) for 10,000 military personnel was 0.1 percent and 6.0 percent, respectively. These results show that military personnel are no more likely to be infected with A. phagocytophilum or SFGR than non-military personnel living in the United States.
Critical reagent program or homeland security forensic reagent program: RDD maintains one of the largest collections of various rickettsial pathogens in the world.
We provide qPCR primers, probes, and standards for qPCR assays and reagents, both recombinant proteins (e.g. Kp r56) and whole cell antigens derived from cultures, for serological assays to overseas laboratories (NAMRU-3, AFRIMS, NAMRU-2, NMRCD) and the DHS.
Comparative genomics and proteomics: Rickettsial pathogens often exhibit different virulence or different sensitivity to therapeutic drugs. Based on the whole genome sequence of R. prowazekii, we constructed the first rickettsial microarray with all predicted ORFs. Genomic compositions of virulent strain and attenuated strain were studied by co-hybridization on this DNA microarray. The genome sequence of O. tsutsugamushi (Karp strain) is more than 90 percent completed and will be used to compare with those of drug resistant strains in the future. We have used 2D gel electrophoresis and LC-MS/MS to compare the global expression profiles of different rickettsial pathogens under various growth conditions and to characterize post-translational modifications of virulence factors. Taken together, these studies have provided significant information towards the elucidation of the mechanisms of virulence and may facilitate the discovery of new drug targets and vaccine candidates.
Wound Infections Department
The Wound Infections Department (WID) was established in September 2011 in response to the Navy Surgeon General’s call for research activities that will provide direct benefit to our wounded warfighters. The primary mission of the WID is to develop and evaluate novel and alternative treatment and prevention strategies for skin and soft tissue infections (SSTIs) associated with multidrug-resistant organisms, which have increasingly afflicted U.S. military members wounded in combat.
An important objective of the WID is to conduct immunologic studies to identify and better characterize correlate(s) of protection for skin and soft tissue infections, which may become critical elements for developing protective vaccines in the future. The WID continues to collaborate with Department of Defense investigators at the Uniformed Services University of the Health Sciences to study human immunologic response to SSTI due to organisms such as Staphylococcus aureus, including methicillin-resistant Staphylocccus aureus (MRSA). New collaborations include research investigators in the Regenerative Medicine Department, NMRC, and Walter Reed National Military Medical Center, providing microbiology/bacteriology services in support of studies by clinical investigators at these facilities. In addition, the WID has established a partnership with research investigators at the Walter Reed Army Institute of Research on the development of an appropriate animal model for studying wound bacterial infections.
Skin and Soft Tissue Infections
Immunology of methicillin resistant Staphylococcus aureus skin infections—Skin and soft tissue infections (SSTIs) in service members, especially those that are caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), can adversely affect military training and combat missions. Currently, virtually nothing is understood about the changes in humoral immunity that occur during the course of an active SSTI or how humoral immunity changes over time. In collaboration with clinical and research investigators at the Uniformed Services University of the Health Sciences, we are conducting studies that will (1) examine the association between the occurrence of SSTI and pre-exposure/pre-infection antibodies and (2) prospectively examine human humoral and cell mediated responses to CA-MRSA SSTIs in a closed military population.
Novel treatment model for Acinetobacter baumannii skin and soft tissue infections—The overall pattern in both military and civilian hospitals is that infections due to multi-drug resistant organisms (MDROs), such as Acinetobacter baumannii, are increasingly common in hospitalized populations. These multi-drug resistant infections increase morbidity and mortality and require broad-spectrum antimicrobial coverage, which in turn may contribute to increasing prevalence of MDROs. The failure of current treatment protocols requires new strategies for combatting and studying wound infections. Our close collaboration with the Walter Reed Army Institute of Research Bacterial Rickettsial Disease-Wound Infection Department has led to the development of a patent-pending murine excision model in which novel antimicrobials can be evaluated from multiple quantitative/qualitative and microbiological/wound healing endpoints throughout the course of aggressive MDRO skin and soft tissue infection.
Current treatment options for multi-drug resistant organisms (MDROs) are becoming more limited. Photodynamic therapy (PDT) is a novel and rapidly expanding approach that shows incredible promise for the treatment multi-drug resistant infections. It involves the combination of a dye called a photosensitizer (PS) and visible light. After exposure to visible light, the formation of excited oxygen singlets or free radicals occurs. The cytotoxic effects arise from their potential to react with nucleic acids, proteins, or cell membranes. PDT can provide targeted therapy against pathogens through the selection and/or design of PS agents that can preferentially enter and bind microbial organisms while they are minimally taken up by mammalian cells. Photodynamic therapy has been employed in blood components pathogen reduction and treatment of periodontitis and infectious keratitis. We hypothesize that blue light and riboflavin will lead to pathogen inactivation and killing in skin and soft tissue infections. If demonstrated to achieve antibacterial activity, this method may be developed as an adjunct or alternate therapy for MDRO infected combat-related wounds.
Phage Therapy for Wound Infections
Treatment of wound infections from combat injuries includes the use of systemic and/or local antibiotics. Increasing prevalence of multi-drug resistant Acinetobacter baumannii requires the development of alternative strategies. Bacteriophage (phage) therapy has been explored; however, the field lacks significant pre-clinical and clinical data for employing phage as a therapy for wound infections. In collaboration with the Biological Defense Research Directorate, the Wound Infections Department submitted a research proposal to the Military Infectious Diseases Research Program to identify, test, and develop relevant phage to potentially treat Acinetobacter baumannii wound infections in humans.
The Wound Infections Department (WID) established the Bacteriology Division, which now has the capability of performing basic bacteriology and antimicrobial susceptibility to support research studies in the NMRC enterprise.
Bacteriology Support Services
The WID provides basic bacteriology services in support of an NMRC Regenerative Medicine Department study on the use of an anti-inflammatory medication—Celebrex®—to prevent heterotopic ossification (HO). The purpose of this study is to evaluate a standard-of-care medication and assess its ability to lessen both the incidence and severity of HO. The WID is integral in this process by isolating, identifying, and providing information about the strains of bacteria that are associated with wounds that lead to HO.
In collaboration with LCDR Hamilton Tilley at NAMRU-6 (Peru), the WID has made preparations to study the antibiotic resistance over time associated with hospital-acquired wound infections. There is a need to optimize the management of wound infections in order to decrease morbidity and improve patient outcomes. Our ability to optimally manage infections is inhibited by the challenge of determining whether a patient has a true infection or only colonization with these bacteria. Consequently, these patients receive empiric broad-spectrum antibiotics, increasing their risk for developing multi-drug resistant pathogens and adverse events that may be associated with antimicrobial toxicities and side effects. Basic knowledge of the epidemiology of antibiotic-resistant organism services can provide critical information important for the treatment and transmission control of these nosocomial pathogens.
Wound Infections Diagnostics
In collaboration with the Trauma Infectious Disease Outcomes Study (TIDOS) at the Uniformed Services University of the Health Sciences and Walter Reed Army Institute of Research, we have submitted research proposals to evaluate molecular methods for diagnosing combat-associated wound infections. Infection is a recognized complication following combat-associated traumatic injury, and patients who sustain more severe injuries are at a higher risk of infection. The current practice of diagnosing wound infections primarily relies on orthopedic or general surgeons’ clinical assessment of these wounds based on their visual appearance and intra-operative bacterial cultures obtained post-debridement. The application of molecular diagnostic methods in the clinical care of traumatic wounds has not been evaluated. Furthermore, the performance of this novel method, compared with established diagnostic modalities, has not been assessed. This constitutes an important capability gap in the diagnosis of trauma-associated wound infections.