Ebola virus disease (EVD) or Ebola hemorrhagic fever (EHF) is an extremely virulent disease that leads to death in 25 to 90 percent of cases, and may be caused by any of four of the five known ebola viruses: Bundibugyo virus (BDBV), Ebola virus (EBOV), Sudan virus (SUDV), and Taï Forest virus (TAFV, formerly and more commonly Côte d’Ivoire Ebola virus (Ivory Coast Ebolavirus, CIEBOV)). EVD is a viral hemorrhagic fever (VHF), and is clinically nearly indistinguishable from Marburg virus disease (MVD).
Outbreaks of the fast-moving virus, which spreads via the blood or other bodily fluids of an infected person, have occurred in Uganda and the Democratic Republic of Congo in recent years. Ebola, and its close relative, Marburg virus, are both filoviruses. The research project will seek to advance treatments for these, as well as the Lassa, Junín and Machupo arenaviruses. No FDA-approved treatments exist for any of these pathogens.
According to Wikipedia, manifestation of Ebola fever begins with a sudden onset of an influenza-like stage characterized by general malaise, fever with chills, arthralgia, myalgia, and chest pain. Nausea is accompanied by abdominal pain, diarrhea, and vomiting. Respiratory tract symptoms include pharyngitis with sore throat, cough, dyspnea, and hiccups. The central nervous system involvement is manifested by development of severe headaches, agitation, confusion, fatigue, depression, seizures, and sometimes coma. Development of hemorrhagic symptoms is indicative of a negative prognosis, but contrary to popular belief, hemorrhage does not lead to hypovolemia and is not the cause of death (total blood loss is low except during labor). Instead, death occurs due to multiple organ dysfunction syndrome (MODS) due to fluid redistribution, hypotension, disseminated intravascular coagulation, and focal tissue necroses.
Bleeding from mucous membranes and puncture sites is reported in 40–50% of cases, while maculopapular rashes are evident in approximately 50% of cases. Sources of bleeds include hematemesis, hemoptysis, melena, and aforementioned bleeding from mucous membranes (gastroinestinal tract, nose, vagina and gingiva). Diffuse bleeding, however, is rare, and is usually exclusive to the gastrointestinal tract.
The National Institutes of Health (NIH) has awarded a five-year, $28 million grant to establish a new center for excellence to find an antibody “cocktail” to fight two types of viruses that cause severe hemorrhagic fever, including the Ebola virus. The project involves researchers from 15 institutions, including Kartik Chandran, Ph.D., Associate Professor, Department of Microbiology & Immunology Harold and Muriel Block Faculty Scholar in Virology and Associate Professor, Department of Biochemistry Jonathan Lai, Ph.D., at Albert Einstein College of Medicine of Yeshiva University in New York. Einstein will receive approximately $4 million of the total grant.
The project will be led by Erica Ollmann Saphire, Ph.D., professor at The Scripps Research Institute (TSRI). “It’s a global collaboration,” says Dr. Saphire. “Everyone in the field got on the same page to collaborate on a set of definitive experiments.”
The Saphire’s lab’s research focus is on structural studies of viral hemorrhagic fever pathogenesis. Her team studies viruses with compact genomes of only 4-7 genes. Consequently, each protein is critical, many are obligated to perform multiple functions, and some actually rearrange their structures to achieve those new functions. As a result, these few polypeptides accomplish a surprisingly complex set of biological functions: immune evasion, receptor recognition, cell entry, transcription, translation, assembly and exit. She notes that viruses with limited genomes also offer a well-defined landscape of possible protein-protein interactions, which together comprise the totality of their life cycle. By systematically analyzing the structures and functions of each protein the virus encodes (as we set out to do for the Ebola and Lassa viruses in particular), the researchers gain fundamental insights into the biology of entry, immune evasion, and assembly, and can decipher the collaborative roles of these proteins in pathogenesis.
Advancing Therapies Against Ebola
Monoclonal antibodies, a type of protein that can bind to and disable specific substances in the body, are currently thought to be the most effective form of antiviral treatment for Ebola and related viruses. In the last year, researchers have identified specific antibodies and cocktails of antibodies that effectively protect against Ebola virus and Lassa virus in animal models and have pioneered approaches to develop new antiviral antibodies and to assess their effectiveness.
“Our consortium represents an unprecedented soup-to-nuts effort to develop antibody therapeutics against hemorrhagic fever viruses,” says Dr. Chandran. “These products, when translated into clinical practice, will provide a much needed pre- or post-exposure therapy against some of the world’s most lethal viruses.”
Dr. Chandran will receive $2 million of the NIH total grant and serve as co-director of the mechanistic virology core for this project, along with Yoshihiro Kawaoka, Ph.D., D.V.M., at the University of Wisconsin–Madison. “Our core will support all individual research projects within the consortium by assessing how well each antibody works and the mechanism by which it blocks infection. This will help us design antibody cocktails with the best chance of working in animals,” says Dr. Chandran. His lab at Einstein carries out both basic and translational research on Ebola virus and identified an essential receptor that Ebola uses to enter cells and cause infection.
Dr. Chandran’s lab has two overarching goals. First, the team seeks to make a ‘molecular movie’ of the process by which the highly pathogenic Ebola and Marburg filoviruses gain entry into the cytoplasm of host cells, where all the ‘goodies’ for viral multiplication are located. Second, they seek to exploit this knowledge to develop new anti-filovirus therapeutics.
Dr. Chandran notes that the filovirus entry mechanism is unusually complex, consisting of multiple steps in which the virus interacts with and co-opts distinct host cell molecules, and is itself structurally transformed as a result, and that over the last few years his lab and others have found that filovirus entry is profoundly dependent upon the cellular endocytic pathway.
“First as a postdoc, and then as a faculty member at Einstein, I have helped to identify endosomal host factors that are critical for filovirus entry and potential targets for antiviral therapy,” He notes on his Einstein Webpage. “In 2005, I showed that the endosomal cysteine proteases cathepsins B and L are required for viral entry and act by cleaving the viral glycoprotein. Very recently, grad students Tony Wong and Emily Miller in my lab have made a remarkable discovery. They have shown that the Niemann-Pick C1 protein, a highly studied cholesterol transporter in lysosomes, is an indispensable host factor for filovirus entry.
The discovery of a central role for NPC1 in filovirus entry promises to revolutionize our understanding of the filovirus infection cycle, in vivo viral pathogenesis, and the ecology and natural history of filovirus infections. Not least, NPC1 provides a new target for the development of anti-filovirus therapeutics.”
Dr. Lai, an expert in antibody technologies at Einstein, will focus on discovering new immunotherapeutic candidates against the Sudan Ebola virus and Marburg virus. “We aim to fill critical gaps in the antibody pipeline against these specific filoviruses,” said Dr. Lai, associate professor of biochemistry, who will receive approximately $2 million of the grant.” More ambitiously, we will seek to develop antibodies that can target more than one filovirus species, or ‘broadly neutralizing antibodies.’ In other viruses, such as HIV-1 and influenza, such antibodies are critical for fighting or weakening infection. However, no broadly neutralizing antibodies exist against Ebola or Marburg.”
Dr. Lai observes that the objective of the research is to understand principles governing molecular recognition by proteins and antibodies, with the long-term goal of developing new research tools and therapies. Students and post-doctoral fellows can expect to gain expertise in new and traditional biochemical techniques including phage display (library design, synthesis, and screening), protein expression and purification, structural analysis by circular dichroism and X-ray crystallography, and viral neutralization assays. His lab is currently engaged in two lines of research:
1. Antibody Recognition Explored by Phage Display. Antibody phage display has emerged as a powerful alternative to hybridoma technology for the generation of monoclonal antibodies and analysis of their interactions with antigens. It is now possible to select high-affinity antibodies against virtually any antigen from phage libraries that bear tailored diversity elements encoded by synthetic DNA (“synthetic antibodies”). This approach obviates the requirement for animal immunization, greatly reducing the labor and cost of antibody production. Selective enrichment of high-affinity binders from phage antibody libraries under controlled conditions enhances the reliability of output antibodies, and permits selection of binding with user-specified stringency. The expression of antibody domains on the surface of bacteriophage was first reported nearly two decades ago, but only recently have synthetic libraries (where diversity is not borne from natural source repertoires) become sophisticated enough for general use. The Lai lab is developing and testing new synthetic antibody technologies to produce therapeutic, diagnostic, or research agents. Their strategy involves two aspects: first, use of high-throughput mutagenesis to interrogate physicochemical parameters of high-affinity antibody-antigen interactions; and second, utilization of the information obtained from these studies to engineer new synthetic libraries directed against targets that have resisted traditional antibody isolation methods.
2. Dissecting Mechanisms of Viral Membrane Fusion. The envelope glycoproteins of membrane-bound viruses such as HIV-1, influenza, and ebolavirus all catalyze viral entry into host cells using essentially the same mechanism. Central to this mechanism are well-timed conformational changes of the envelope glycoprotein that result in formation of a six-helix bundle hemifusion intermediate. Formation of this hemifusion intermediate provides the driving force for fusion of the virus and host cell membranes. Small molecules, peptides, or proteins that bind viral envelope glycoproteins and prevent formation of the hemifusion intermediate have been used clinically as antiviral therapies. In addition, antibodies arising from natural infection (or other sources) that prevent the formation of the hemifusion intermediate are able to effectively neutralize the virus, suggesting that conformational mimicry of viral glycoprotein in the prefusion states may serve as an avenue for vaccine development. Using synthetic antibody technologies coupled with traditional biophysical and biochemical approaches, we seek to understand details of the viral membrane fusion process and which steps along the pathway are susceptible to inhibition by antibodies. Information gained from these studies will pave the way for structure-based vaccine design.
Dr. Lai received the $300,000 Arnold and Mabel Beckman Foundation’s 2009 Young Investigator Award, which allowed him Dr. Lai to continue his innovative research on human immunodeficiency virus (HIV) and antibody-mediated immunity over a three-year period.
“It is very exciting to be working with a large group of talented scientists, including those here at Einstein,” Dr. Lai observes. “Together, we have a chance to make a real impact on prevention of these diseases.”
In addition to those named above, the other institutions and scientists on the NIH National Institute of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Translational Research (CETR) program grant, number U19AI109762, include: Gary Kobinger, Ph.D., at Public Health Agency of Canada, John Dye, Ph.D., at U.S. Army Medical Research Institute of Infectious Diseases, Leslie Lobel, M.D., Ph.D., at Ben Gurion University, Julius Lutwama, Ph.D., at Uganda Virus Research Institute, Robert Garry, Ph.D., and James Robinson, M.D., at Tulane University, Thomas Geisbert, Ph.D., at University of Texas Medical Branch, and Gene Olinger at NIAID. Biopharmaceutical companies on the grant include Mapp Biopharmaceutical (Larry Zeitlin), Zalgen Labs (Luis Branco) and Cangene (Cory Nykiforuk).
Albert Einstein College of Medicine of Yeshiva University is one of the nation’s premier centers for research, medical education and clinical investigation. During the 2013-2014 academic year, Einstein is home to 734 M.D. students, 236 Ph.D. students, 106 students in the combined M.D./Ph.D. program, and 353 postdoctoral research fellows. The College of Medicine has more than 2,000 full-time faculty members located on the main campus and at its clinical affiliates. In 2013, Einstein received more than $155 million in awards from the NIH. This includes the funding of major research centers at Einstein in diabetes, cancer, liver disease, and AIDS. Other areas where the College of Medicine is concentrating its efforts include developmental brain research, neuroscience, cardiac disease, and initiatives to reduce and eliminate ethnic and racial health disparities. Its partnership with Montefiore Medical Center, the University Hospital and academic medical center for Einstein, advances clinical and translational research to accelerate the pace at which new discoveries become the treatments and therapies that benefit patients. Through its extensive affiliation network involving Montefiore, Jacobi Medical Center -Einstein’s founding hospital, and five other hospital systems in the Bronx, Manhattan, Long Island and Brooklyn, Einstein runs one of the largest residency and fellowship training programs in the medical and dental professions in the United States. For more information, visit:
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