Viral Zoonoses

Antivirals and vaccines against arenavirus infections

Michael J. Buchmeier, UC Irvine

Abstract:
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

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

Nipah and Hendra virus entry and budding

Benhur Lee, UC Los Angeles

Abstract:
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.

Novel approaches for the development of live and inactivated viral vaccines

David Clark, UC Davis/David Brault, Centers for Disease Control

Abstract:
Currently, no vaccines exist for a significant number of historically important viral pathogens and newly emerging viral zoonotic diseases. In part, this situation results from the incompatibility of existing methodologies with the challenges posed by a wide array of emerging viruses. The extent of attenuation mediated by approaches such as serial passaging of viruses, viral recombination and directed molecular evolution is unpredictable and this characteristic contributes to the lengthy production times for many vaccines made using these strategies. Likewise, a number of vaccines constructed using these methodologies have demonstrated poor safety profiles due to the inherent uncertainty in the number of attenuation determinants introduced. In this study we aim to further define a novel strategy to intuitively construct live-attenuated viral (LAV) vaccines applicable to the development of vaccines to a broad array of acute viral diseases. Our approach is based on the manipulation of host strategies for the regulation of protein translation. We hypothesize that the use of rare codons as attenuation determinants for vaccine candidates will produce more stably attenuated viruses that can be rationally engineered with predictable replication phenotypes within host cells. Moreover, we have demonstrated that these substitutions can be introduced into various places in viral genomes, unlike amino acid substitutions, which are conventionally employed to attenuate viruses. We have also designed a second strategy to further improve the safety of vaccines that restricts the replication of vaccine viruses in tissues that are responsible for engendering disease pathology. By introducing the target sequences for tissue-specific miRNAs into the viral genome, we have demonstrated the ability to specifically block infection of target cells expressing the cognate miRNA, while permitting normal unaltered levels of replication in the cell populations required for the induction of a protective immune response. Since this strategy is compatible with the modification of existing vaccines to improve their safety profiles, this work is broadly applicable to the design of live viral vaccines