Revealing the attenuating mutations of F. tularensis                              LVS

Recombinant Antigen-based Assays for Flavivirus                              Serodiagnosis and                              Surveillance

Identification and Inhibition of Cytokines Induced                              During OHFV Infection

Cell Wall Proteins in Bacillus anthracis as
                      Vaccines

Rational Design and Optimization of New Live-                              attenuated Vaccines for                              Alphaviral Enciphalitides

Nodavirus-based RNA Replicon Vaccines for                              Tick-borne Encephalitis                               Virus

Antiviral Agents as Therapy for SARS

Typhus Group Rickettsial Antigens Recognized by                              CD8+ T Lymphocytes

Rational Design and Optimization of New Live-attenuated Vaccines for Alphaviral Encephalitides

Institution: Louisiana State University Health Science Center (LSUHSC), Shreveport, LA

Principal Investigator: Katherine Ryman, Ph.D.

Co-Investigator: William B. Klimstra, Ph.D.- LSUHSC, Shreveport, LA

Mentors/Consultant:
a) Adolfo García-Sastre, Ph.D. – Mount Sinai School of Medicine, New York, NY
b) Dennis J. O’Callaghan, Ph.D. – LSUHSC, Shreveport, LA
c) Michael D. Parker, Ph.D. – U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD
d) Scott C. Weaver, Ph.D. – University of Texas Medical Branch, Galveston, TX

Expected Product: An attenuated alphavirus vaccine.

Description: Eastern equine encephalitis virus (EEEV), an alphavirus in the family Togaviridae, is classified in Category B of the NIH Priority Pathogens List. It is also listed as a high consequence livestock pathogen by the USDA because it is highly lethal for humans and equines, and because effective vaccines and therapies are lacking for EEEV. The formalin-inactivated vaccine strain of EEEV is not suitable for wide-scale human use due to poor immunogenicity and possible residual virulence. Clearly, alternative strategies for vaccine production are required. Our long-term goal is to develop a live-attenuated virus vaccine with sufficient degree of attenuation to be safe for human populations. The objective of the proposed research is the rational design of attenuated strains via the selective deletion (or disabling) of innate immune evasion properties. This is based on the hypothesis that EEEV possesses mechanism(s) to antagonize the interferon alpha/beta (IFN-a/ß) response elicited by infected dendritic cells (DCs) which can be disabled to attenuate the virus and enhance the immune response.

Using the non-pathogenic alphavirus Sindbis (SB) as a “baseline” for IFN-a/ß sensitivity, we will determine which of the IFN-a/ß-mediated responses that suppress SB replication do not do so for EEEV. We will infect cultures of primary myeloid bone marrow derived DCs (BMDCs). The use of these cells is particularly important as they are representative of the cells targeted by alphaviruses in vivo following subcutaneous inoculation. By infecting BMDCs with chimeric replicon particles in which either the SB or the EEEV genome is encapsidated in SB structural proteins, we will for the first time be able to dissociate SB versus EEEV replication events from the role of the non-structural proteins (nsPs) from attachment/entry steps and thereby ensure equal delivery of RNA genomes to each cell. We have compelling evidence that the nsPs and/or cis-acting sequences in the viral RNA encode the IFN-a/ß resistance of the virulent alphaviruses. Therefore, in Aim 1 we will determine whether individual EEEV proteins or cis-acting elements alter replicative capability and IFN-a/ß sensitivity. In Aim 2, we will characterize the effects of targeted mutations in the EEEV genome on antagonism/evasion of the IFN-a/ß-mediated response, focusing on the following pivotal steps in the IFN-a/ß antiviral pathway: i) the activity of antiviral effector mechanisms, particularly PKR; ii) IFN-a/ß induction; and iii) IFN-a/ß signaling.

We anticipate that these studies will allow the identification and disablement of EEEV-encoded product(s) that antagonize/resist IFN-a/ß activity. Our long-term goal is the rational design of attenuated alphavirus strains with sufficient degree of attenuation to be safe for human populations via the selective inactivation of innate immune evasion properties.