ARENAVIRUSES/HANTAVIRUSES
Development of a broad spectrum vaccine
for Arenavirus infection
Michael J. Buchmeier
Abstract:
Arenaviruses cause chronic infections of rodents indigenous to Europe, Africa, Asia and the Americas. Arenavirus infections of humans are common and may be severe. The mechanisms that have evolved to ensure chronic infection and intergenerational survival of the arenaviruses in rodents are complex and their elucidation over the last 60 years has led to a deeper understanding of immunology and immunopathology. There is presently only one vaccine for arenavirus infection; the live attenuated Junin candid 1 vaccine. In a double blind study in 6500 male agricultural workers in the endemic area, the vaccine was found to be effective and without adverse effects. The Candid 1 vaccine is licensed as an investigational new drug in the U.S. This project builds upon that experience to define the elements necessary to confer protection by using advanced bioinformatics and in vivo vaccination and evaluation strategies to determine the elements necessary for and responses to broad spectrum arenavirus vaccination. Work in the first project year has focused on epitope prediction and confirmation, and has defined several epitopes shared among arenaviruses.
The broad aims of this project have not changed substantially during the past project period, however the availability of complete sequence for the Candid 1 strain of JUNV has allowed acceleration of the pace of the predictive aspects of the peptide analysis. With the shortened funding time line of 4 years this will allow us to proceed with the advanced aims more rapidly than projected in the original application.
Receptor use by pathogenic new world Arenaviruses
Paula M. Cannon
Abstract:
The clade B New World arenaviruses contain several severe human pathogens, including the causative agents of Argentine, Bolivian and Venezuelan hemorrhagic fevers. Limited information is available about the molecular biology of these viruses. We are interested in the entry step of the viral life cycle, whereby the surface glycoprotein (GP) of these viruses binds to a cellular receptor and triggers fusion between the virus and the host cell membrane. At present, the cellular receptor(s) used by the pathogenic clade B viruses is unknown. Furthermore, the domains in GP that are involved in binding to the receptor and triggering fusion are also unknown. A greater understanding of these functional components would greatly facilitate the development of therapeutics targeted specifically to this stage of the viral life cycle. We are using a variety of approaches to identify the clade B cell surface receptor(s) including genetic complementation cloning of receptor-defective cells and GP-receptor immunoprecipitations. Simultaneously, we are using mutagenesis and binding assays to define domains within GP that are needed to bind to the receptor, and thereby identify a minimal receptor-binding domain within GP
The Structural basis for Arenavirus neutralization
Michael J. Buchmeier
Abstract:
Arenavirus hemorrhagic fevers pose important natural risk of epidemic potential in the new and old world and are potential agents of bioterrorism. In recognition of this these viruses have been assigned as category A agents. Among the known arenaviruses, only Junin virus, the agent of Argentine hemorrhagic fever has been shown to be amenable to treatment by intervention with human convalescent sera. This project will characterize the structural and mechanistic basis of arenavirus neutralization by antibodies, and seek alternative antiviral compounds to be used in tandem with neutralizing antibodies to control arenavirus infections. We will capitalize on recent studies that have provided a cryo EM structure of three arenaviruses, LCMV, Tacaribe, and Pichinde viruses, and we will extend the investigation to Junin virus using the Candid 1 vaccine strain.
Recently developed three-dimensional reconstructions of native and fusion-activated arenaviruses will be used to study the structural basis for antibody-mediated neutralization of viral surface proteins. We will characterize the binding specificity of neutralizing antibodies developed for arenavirus glycoproteins and map antibody binding sites to three dimensional reconstructions of Old World and New World arenavirus glycoprotein complexes using electron cryomicroscopy (Cryo-EM) and image analysis. The effects of antibody binding of specific, pre-determined regions of the glycoprotein will be evaluated both in terms of their direct effect on glycoprotein structure and obstruction or facilitation of receptor-GP1 interactions and membrane fusion. Preliminary data also suggest the virion core undergoes a substantial conformational shift in response to acidic and osmotic triggers of fusion activation, and antibodies will be screened for internal and external effects of binding. In this way it will be possible to correlate the effects of monoclonal antibody binding on viral attachment and fusion to specific sequence and structural determinants on the virion surface, and to facilitate rational design of antiviral treatment cocktails acting both intracellularly and at the level of receptor binding and entry.
Novel strategies to target Arenaviruses
Juan Carlos de la Torre
Abstract:
We have developed a reverse genetics system for the prototypic Arenavirus LCMV. We can now investigate the cis-acting signals and trans-acting factors involved in the control of Arenavirus RNA synthesis, assembly and budding. These advances allow us to propose developing novel antiviral strategies to combat Lassa fever virus (LFV) and other hemorrhagic fever (HF) arenaviruses by targeting two essential steps of the Arenavirus life cycle: 1) RNA synthesis mediated by the Arenavirus polymerase, and 2) viral budding mediated by the Arenavirus small RING finger Z protein.
Our overall hypothesis is that targeting these key steps of the Arenavirus life cycle will limit virus spread, thus providing the host immune system with a window of opportunity to mount an effective response to control these infections without development of disease symptoms.
Our specific aims are:
- Use of aminoglycosides to inhibit RNA synthesis mediated by the Arenavirus polymerase. The interaction of the virus polymerase with the genome promoter is a first and necessary step to initiate virus replication and gene expression of negative strand RNA viruses. We hypothesize that aminoglycosides can target the RNA structure of the Arenavirus promoter. We will screen aminoglycoside-based libraries to identify candidates with antiviral activity against LCMV first in cell-based assays and subsequently in animal models. The conservation of the Arenavirus genome promoter predicts that aminoglycosides with anti-LCMV activity will also inhibit LFV and other HF arenaviruses.
- Inhibition of Arenavirus budding. We have identified the Arenavirus Z protein as the driving force of virus budding, and shown that LFV Z budding activity is mediated by L domain motifs PPPY and PTAP. We will test the hypothesis that Z interaction, via its L domain motifs, with members of the endosomal/multivesicular body (MVB) cellular pathway is required for virus budding. We will use tandem affinity purification (TAP) method combined with mass spectrometry procedures to identify Z-interacting cell proteins. The functional significance of Z-interacting cellular proteins in Arenavirus budding will be investigated using RNAi approaches and functional assays of budding. We will develop biochemical and fluorescence resonance energy transfer (FRET)-based assays to screen combinatorial chemical libraries to identify small molecules capable of inhibiting Z-host cell protein interactions. Inhibitors will be tested for their antiviral activity first in cell-based culture assays and subsequently animal models.
Lassa virus receptor binding and host cell pathology
Michael Oldstone
Abstract:
In fatal Lassa fever (LF), an unchecked viremia leads to progressive signs and symptoms of hemorrhagic disease and shock. Since death occurs in the absence of extensive inflammation and tissue destruction, the fatal disease is most likely caused by virus-induced changes in host cell function, rather than by immunopathology. The molecular analysis of the virus-host cell interaction is therefore of great importance to understand the mechanisms of the disease and for the development of novel therapeutic strategies to treat critically ill patients. The interaction of a virus with its cellular receptors is a fundamental aspect of the virus-host cell relationship and a key determinant for the tissue tropism and the disease potential of a virus. Based on its pivotal importance for normal cell homeostasis and function, the cellular receptor of Lassa fever virus (LFV), a-dystroglycan (a-DG), is of particular interest regarding virus-induced cytopathology. In the host cell, a-DG provides an essential molecular link between the extracellular matrix (ECM) and the cytoskeleton. The glycoprotein (GP) of LFV binds a-DG with high affinity and efficiently competes with the interaction of a-DG with ECM proteins. Binding of LFVGP therefore likely interferes with the normal function of this important cellular receptor, contributing to virus-induced host cell dysfunction. Using a combination of biochemical and cell biological techniques, our study aims at the identification of a-DG-associated host cell proteins whose interactions, activation states, and cellular localization are changed by LFVGP binding. Since a-DG and its known binding partners are ubiquitously expressed cellular proteins, candidates identified by our study represent likely crucial targets of LFV critically involved in the pathogenesis of fatal LF. a-DG-associated proteins and signaling pathways that are affected by LFVGP will therefore be evaluated as potential targets for anti-viral therapy.
Reverse genetics system for Hantaviruses
Stephen St Jeor
Abstract:
Hantaviruses are epizootic viruses belonging to the family Bunyaviridae. Hantaviruses have evolved with specific rodent hosts. In humans, infection can result in severe complications and a high rate of mortality. Worldwide there are over 100,000 clinical cases per year of hantavirus disease. The primary syndrome caused by hantaviruses in the New World is Hantavirus Pulmonary Syndrome (HPS), which has a mortality rate of app. 45%. In South America, because of the diverse rodent species, there are several distinct hantavirus types which appear to vary in their ability to cause disease. Andes virus, a South American hantavirus, which causes HPS in humans has also been implicated in human to human transmission. Recently, an animal model was described in which Andes virus causes a pulmonary syndrome in Syrian Hamsters but Sin Nombre virus, another New World hantavirus, fails to produce pulmonary disease in hamsters but produces HPS in humans. The approach we have used to identify markers associated with hostrange and pathogenicity of these viruses is as follows:
- The development of reassortants between New World hantaviruses. In our initial studies following dual infection of cells with ANDV and SNV, 8.9% of 337 progeny plaques contained reassortants. The monoploid reassortants contained the S and L segments of SNV and ANDV M segment. Recombinants are analyzed to identify their host range, cell tropism, and growth characteristics.
- The use of a reverse genetics system for hantaviruses. To identify markers associated with pathogenicity and to be able to mutate specific regions in the virus, we are developing a reverse genetics system for hantaviruses, using a similar approach to that described for influenza virus. To date we have successfully cloned the entire S and, M segment of Andes virus and have partial clones of the L segment. The open reading frames of the S and M segments have also been cloned. Additionally, we have cloned the S and M segments of SNV, and expressed the nucleocapsid and glycoproteins of these viruses. We have a number of hantavirus strains from South America which vary from non pathogenic to highly pathogenic strains and differ in the rodent reservoir they infect. These strains will be used to identify potential regions associated with pathogenicity. Related to these projects, we have developed an ELISA assay using recombinant antigens to differentiate between various hantavirus strains. This is being tested in Argentina at INEVH in Pergamino Argentina. To identify functions of the nuclear protein of the virus, in collaboration with Roberto Guzman at the University of Kansas, we are examining the structure of the N protein RNA binding site as part of an overall plan to associate structure with function.
Therapeutic human monoclonal antibodies for treatment
of Hantavirus cardiopulmonary syndrome
Jason W. Botten
Abstract:
Sin Nombre (SNV) virus and Andes virus (ANDV) are the primary etiologic agents of hantavirus cardiopulmonary syndrome (HCPS) in North America and South America, respectively. Despite improved patient care since the initial outbreak of HCPS in 1993, the mortality rate remains high at 30-50% for these two pathogens. An effective vaccine for the prevention of HCPS in the Americas has not been developed and treatment is limited to supportive care at present. The goal of this project is to develop a new treatment for HCPS patients using human monoclonal antibodies to improve patient survival rates.
Recent studies of ANDV- and SNV-infected patients have demonstrated that mild disease is significantly associated with high levels of antiviral neutralizing (Nt) antibodies at the time of hospital admission, while low Nt antibody titers correlate with severe disease and high mortality. In rodent models of hantavirus infection, maternally-acquired or passively transferred antibodies are sufficient to prevent infection and/or ameliorate disease. Based upon these observations, we hypothesize that passive administration of Nt antibodies may be an effective therapeutic intervention to reduce/prevent morbidity and mortality due to HCPS disease resulting from infection with SNV and ANDV. Our project proposes to identify SNV- and ANDV-specific antibodies capable of Nt these viruses, both in vitro and in vivo, through the use of phage display library technology. We currently have two phage display libraries that were developed using mRNA from bone marrow-derived lymphocytes obtained from SNV- and ANDV-immune individuals. Each library will be screened for antibodies capable of Nt SNV or ANDV through in vitro assays and in vivo challenge studies. Such antibodies would be excellent candidates as potential passive immunoprophylactic or immunotherapeutic reagents and could be utilized in human trials to address treatment efficacy. High affinity, non-Nt antibodies identified through these studies will also be valuable reagents in diagnostic assays involving ANDV and SNV.
Host Cell Entry Mechanisms for New World Arenaviruses
Kenneth Bradley
Abstract:
Arenaviruses are rodent-borne enveloped RNA viruses classified as potential bioterrorism agents by the Centers for Disease Control and Prevention. Importantly, five members of the arenavirus family are capable of causing severe hemorrhagic fevers in humans, and are considered Category A bioterrorism agents because of the combination of high morbidity/mortality rates and airborne transmission. A major factor governing viral tropism is the nature of the cellular receptor(s), and interactions between viral glycoproteins (GPs) and their receptors are important targets for antiviral agents. Previously, ?-dystroglycan (?DG) and transferrin receptor 1 (TfR1) were identified as receptors used by pathogenic arenaviruses. However, recent studies indicate that at least one other receptor is used by the clade B New World arenaviruses, which
include four of the five human pathogenic strains. Here, we propose two specific aims with the goal of elucidating arenavirus-host interactions and identifying small molecule inhibitors of these processes. First, we will perform somatic cell genetic screens to identify additional clade B receptor(s), as well as other host factors that are involved in arenavirus entry. Second, we will develop a high throughput chemical genetic screen to identify small molecules that affect this stage of the arenavirus life cycle. Such inhibitors may also serve as lead compounds for therapeutic development. The research proposed here represents a synergistic approach to these questions, in which the arenavirus-specific research tools developed in the Cannon lab, will be leveraged against the somatic cell and chemical genetic systems developed by the Bradley lab.
Development of Poxvirus-based vectoras as vaccines against biodefense threat agent
Bertram Jacobs
Abstract:
The long-term goal of this research is to develop an effective and safe human vaccine against potential biological warfare agents such as Lassa virus using attenuated yet highly immunogenic vaccinia vaccine vectors. The immediate goal is to provide a proof of concept that highly attenuated yet highly immunogenic modified vaccinia virus vectors can induce a protective immune response in mice against LCMV, a model hemorrhagic fever virus. This research will not only pave the way for future pre-clinical research towards development of vaccines against hemorrhagic fever viruses of import to biodefense and emerging infectious diseases, such as Lassa fever virus and Junin virus, but will demonstrate the feasibility of using this novel vaccine platform to rapidly generate vaccine candidates to newly emergent agents, either natural or genetically engineered. The vectors developed within our laboratory, present the advantage of retaining the ability of wild-type vaccinia virus to induce a robust and long lasting immune response after a single immunization, unlike some of the more attenuated Modified Ankara Vaccinia (MVA) or NYVAC viruses. At the same time, we have accumulated evidence that, unlike wild type vaccinia viruses that make up the current smallpox vaccine, the vectors we produced will not result in the severe and frequent complications that make widespread smallpox vaccination inadvisable for the general population and contraindicated for immunocompromised individuals.
The large genome of vaccinia viruses enables easy insertion and expression of complete antigens from other organisms, potential resulting in very successful vaccines, as was the vaccinia-based rabies vaccine for wildlife. Although the multimammate rat is the natural reservoir for Lassa virus, the mouse, naturally susceptible to Lymphocytic Chorio-Meningitis Virus (LCMV), another arenavirus, is a more practical model.
We will therefore use mice to test the efficacy of two novel vaccine platforms in protecting against lethal challenge with LCMV. We will use two highly attenuated, yet highly immunogenic mutants of vaccinia virus
(VV) to express the LCMV glycoprotein GPC. Immunogenicity of these vaccines will be compared to wtVV expressing GPC and to the replication defective VV, MVA, also expressing glycoprotein C. Induction of total antibodies, neutralizing antibodies and LCMV GPC specific T cells will be evaluated. Vaccinated animals will be challenged with lethal doses of LCMV to determine if these vaccines can protect against disease in this relevant animal model.
