Antivirals and vaccines against arenavirus infections
Michael J. Buchmeier, UC Irvine
Arenaviruses are rodent-borne pathogens that cause significant morbidity and mortality in humans. Pathogenic arenaviruses include Lassa (LASV), lymphocytic choriomeningitis (LCMV), Junin (JUNV), Machupo (MACV), Guanarito (GTOV), Sabia (SABV) and Whitewater Arroyo (WWAV) viruses. Following human infection with the Old World arenaviruses LCMV or LASV, cellular immunity plays a pivotal role in viral clearance and protective immunity. Therefore, it is important to develop sensitive reagents to measure the cell-mediated immune response in the context of human infection or vaccine studies. The identification of HLA-restricted epitopes is required to develop assays that can be used to determine the quality of immune responses, define correlates of protection and immunopathology, and ultimately guide the selection of candidate vaccines. This project utilizes bioinformatic predictions to identify candidate epitopes, in vitro binding assays and in vivo immunogenicity studies in transgenic mice to validate vaccine candidates and determine if they are protective against viral challenge.
Targeting the entry pathway of New World arenaviruses for anti-viral therapeutics
Paula M. Cannon, University of Southern California
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
Nipah and Hendra virus entry and budding
Benhur Lee, UC Los Angeles
Nipah and Hendra viruses are lethal paramyxoviruses that require high levels of containment. This project uses a Vesicular Stomatitis Virus (VSV) reporter platform to study the structural virology of the hanipaviral fusion cascade and develop high throughput assays to detect neutralizing and/or cross-reactive antibodies for diagnostics, surveillance, and vaccine development at less than BSL4 levels. The underlying rationale for the proposed studies is that serum neutralization assays and structural virology studies done with our VSV-rluc (Renilla luciferase) reporter pseudotypes will provide biologically relevant information more efficiently and economically than using their live viral counterparts. To accomplish our stated objectives, we propose the following specific aims: (1) to gain a supramolecular nanoscale understanding of the henipavirus fusion cascade using cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) to visualize Nipah virus F and G oligomers pseudotyped onto VSV; (2) to study relevant serum samples from the Global Viral Forecasting Initiative using our henipavirus VSV pseudotypes; and (3) to use a beta-lactamase-Nipah matrix based assay for sensitive, specific and high throughput analysis of native henipavirus entry and budding at BSL2 conditions.
Molecular mechanism of zoonotic prion transmission
Christina Sigurdson, UC San Diego
Prion disorders are infectious, fatal neurological diseases in humans and animals caused by misfolded and aggregated prion protein. Some diseases are zoonotic, as nearly 200 cases of variant Creutzfeldt-Jakob disease (vCJD) in humans was believed to be incited by consumption of bovine spongiform encephalopathy (BSE)-infected cattle, and human-to human transmissions are now occurring through blood transfusions. Since the year 2000, chronic wasting disease (CWD) in our North American deer and elk has been detected in states from Utah to New York, and is transmitted naturally among animals at an alarmingly high rate. Hunters have consumed CWD-infected deer and elk, and whether this exposure will ultimately cause prion infection remains unclear. We aim to determine the molecular basis for the species barrier between cervid CWD and humans. Efficient conversion of the normal host cellular prion, PrPC, by the incoming misfolded prion, PrPSc, requires structural similarity between the two isoforms. Prion transmission between species depends on both the incoming strain and the homology between PrPC and PrPSc sequences, with particular residue positions having a strong influence on conversion. We hypothesize that a small variable loop region of PrP is a key interaction site in prion conversion that can influence prion transmission between species. The major goal of this project is to assess the role of the loop region on the species barrier between humans and deer/elk using in vivo experimental models. To accomplish this goal, we propose to do the following: 1. Develop and characterize a new transgenic mouse model expressing the human prion protein, but with the loop sequence of the elk. 2. Inoculate CWD prions into mice expressing human-elk chimeric PrP or human PrP to determine susceptibility to CWD infection. 3. Characterize the resulting strains arising from the CWD infections and determine whether the chimeric prions are infectious for the human PrP expressing mice. As new prion diseases emerge in livestock and wild ruminants, understanding the contribution of the primary prion structure in species barriers will enable us to predict the risk of transmission to humans and other animals. In addition, we expect that knowledge of the key residues engaged during prion conversion will advance the rational design of therapeutics and prevention strategies for prion diseases.
A functional IVIG aptamer agonist for West Nile Virus infections
Edouard Cantin, Beckman Research Institute at the City of Hope
Intravenous immune globulin (IVIG) therapy has shown potent anti-inflammatory activities and has proven to be beneficial in the treatment of variety of autoimmune and inflammatory diseases. We reported recently that fatal herpes simplex virus encephalitis (HSE) results from hyper-inflammatory responses in the CNS and we showed that high dose IVIG protected susceptible mice, because it potently suppressed inflammation and induced regulatory cells. Substantial evidence now implicates immune pathology in encephalitis caused by flaviviruses such as West Nile virus (WNV) and Japanese encephalitis virus (JEV). IVIG protection of susceptible mice from WNV encephalitis is thought to involve virus neutralization. However, based on results in the HSE model we expect high dose IVIG suppression of pathogenic inflammatory responses in flavivirus infections will be protective and facilitate virus clearance without bystander immune pathology. The overall goals of the project are to demonstrate that the anti-inflammatory activity of IVIG can protect mice humanized for immunity from WNV encephalitis and to produce nucleic acid molecules that can functionally substitute for IVIG – IVIG aptamers. An in vitro PCR based procedure termed SELEX will be used to derive a library of short oligonucleotides termed aptamers that can assume 3D structures compatible in some instances with binding to specific ligands or receptors on cells. In this project, aptamers capable of binding to DC-SIGN-Fc, the receptor on dendritic cells (DC) that interacts initially with IVIG, will be selected from the library and subjected to successive rounds of amplification and selection to evolve aptamers showing specific high affinity binding to DC-SIGN-Fc in vitro. The selected aptamers will be screened for their ability to induce a signature cytokine secretion profile comparable to IVIG when added to DC cultures that express DC-SIGN. Aptamers with the desired biological activity profile will then be compared to IVIG for their ability to transform DCs into rDCs that can protect adoptively transferred humanized mice from encephalitis. IVIG aptamer agonists have several advantages compared to IVIG including being a homogeneous, chemically well defined, highly specific product that is cost effective to produce as a large scale GMP product.