Influenza

Influenza is an acute, highly contagious infectious disease caused by the orthomyxoviridae family of RNA viruses that includes influenza A, B, and C. Influenza types A and B are responsible for epidemics of respiratory illness that occur nearly every winter and are often associated with increased rates of hospitalization and death; because influenza C causes only mild illness, it lacks the public-health impact of the other influenza virus types. According to the CDC (Centers for Disease Control and Prevention), seasonal influenza results in approximately 36,000 deaths and more than 200,000 hospitalizations each year. Several serious influenza pandemics have occurred in the last 100 years, including the 1918 highly pathogenic Spanish flu, which had a mortality rate as high as 20% and caused approximately 100 million deaths worldwide. Currently, highly pathogenic avian flu is a serious concern among epidemiologists for its potential to cause another deadly pandemic.

The Southern Research Influenza Research and Development Program offers the following diverse capabilities:

• Basic research initiatives
• Antiviral in vitro compound testing
• High-throughput screening
• Evaluation in established in vivo models
• In vitro and in vivo vaccine development

Southern Research has the appropriate USDA permits to work with low and high pathogenicity human and avian influenza viruses. We provide BSL-3 containment for highly pathogenic and potentially pandemic influenza viruses, and our facilities have specially engineered features that control air flow to exceed BSL-3/ABSL-3 containment requirements. In addition, Southern Research is registered with the CDC to receive and transfer select agents in accordance with 42 CFR Part 72, including highly pathogenic avian influenza.

Influenza Basic Research Initiatives

Mechanisms of Influenza Pathogenesis and Interaction of Viruses with Host Cell Factors, James Noah, Ph.D., Diana Noah, Ph.D., William Severson, Ph.D.

This research is focused on novel avenues for antiviral discovery. During viral infection in eukaryotes, host protein kinases, polymerases, and other ATP-binding proteins play major roles in viral propagation and are broad, rational targets for interruption of the viral life cycle and modulation of the host immune response to infection. This research endeavor examines the interactions of compounds with viral proteins to determine how these compounds interfere with viral replication. It also looks to profile the activity of the entire pool of host cellular adenine nucleotide-binding proteins, including major druggable protein classes such as kinases in pathogen/host cell systems to identify new targets for antiviral therapeutics. In addition, this research will increase the understanding of the host biochemical pathways involved in the pathogen life cycle. Results will have a significant impact on the field of virology, as well as provide data for continued research. Moreover, they will comprise a powerful proteomic complement to the current human genome database.

Influenza In Vivo Animal Model Development, Diana Noah, Ph.D.

With emerging pandemic pathogens such as H5N1 and the 2009 H1N1 influenza viruses, development of vaccines and therapeutics that can elicit more potent and protective immune responses - as well as reduce the impact of future pandemic outbreaks - is essential. Successful development of such vaccines and therapeutics depends on the establishment of reliable animal models that allow for studies of efficacy and safety. Animal modeling of infectious disease also allows for global mapping of the cellular and molecular mechanisms by which these viruses infect, as well as how the vaccines against them provide protection. Influenza mouse models allow for highly reproducible initial screening of therapeutics and vaccines. Although the mouse does not demonstrate clinical parameters similar to humans, it is a first step in the animal rule required for IND filings on vaccines and therapeutics that cannot be tested for efficacy via post-treatment challenge in humans due to the high pathogenicity or risks associated with the virus and subsequent disease. The domestic ferret (Mustela putorius furo) is an excellent model for influenza virus research based on its susceptibility to infection and similarities in the pathogenesis of disease. Functional genomic analyses provide valuable knowledge about which components of the innate immune response the host activates to clear the pathogens, as well as the strategies the virus uses to defeat the immune response. Systemic approaches like these can identify innate immune signatures that may be used to assess the strength of the adaptive immune response and predict protective immunity after vaccination.

Influenza Antiviral In Vitro Compound Testing - James Noah, Ph.D.

We have validated several in vitro low- and high-throughput assays for influenza, including both seasonal and highly pathogenic avian influenza (HPAI) strains. Assays have been designed for diagnostic, antimicrobial, or vaccine evaluations. These influenza assays are crucial for identifying new compounds with antiviral properties or those that may act as molecular probes of the complex life cycle of the influenza virus. The adaptation and validation of the proposed influenza assays for HTS have the potential to vastly increase the arsenal of existing antiviral drugs available to rapidly combat an influenza epidemic or pandemic. Specific assays that have been adapted for high-throughput screening include a broad-cell-viability (cytoprotection) assay and mechanistic assays for viral neuraminidase, hemagglutinin, and M2 channel function.

As an integral element of our program, the influenza antiviral evaluation assay examines the effects of compounds at designated dose-response concentrations on cell viability. Madin Darby canine kidney (MDCK) cells are used in the assay to test the efficacy of the compounds in preventing the cytopathic effect (CPE) induced by influenza infection. Additional capabilities and features include:

• Ribavirin, Tamiflu, or Amantadine can be included in each assay as comparative positive control drugs
• Cell viability is determined 24, 48, or 72 hours later
• IC50 and IC90 values calculated by regression analysis with semi-log curve fitting
• TC50 and TC90 values are calculated, respectively
• Selectivity (therapeutic) indices (SI=TC/IC) calculated
• Combination and Synergy testing
• Generation of resistant escape mutants for target identification
• Assay development upon request

High-Throughput Screening for Influenza Antivirals

Southern Research has screened more than 2 million compounds against seasonal and avian influenza, and we maintain an active and comprehensive High-Throughput Screening program for evaluation of new compound libraries. Our capabilities include:

• Cytoprotection assays for broad antiviral identification
• Targeted assays for neuraminidase, hemagglutination, and M2 channel inhibition
• Combination, Synergy, and Time of addition assays
• Quantitative RT-PCR in A549 cells for physiological relevance

Southern Research also offers our clients established models for the evaluation of the following:

• Drug toxicology and efficacy
• Bioavailability
• Half-life
• Excretory routes
• Tissue tropism

In Vitro and In Vivo Vaccine Development

As part of our comprehensive Vaccine Development program, Southern Research offers in vitro and in vivo influenza vaccine development services for diverse virus strains, including:

• Current seasonal influenza (including H1N1 2009)
• Mouse-adapted H1N1 2009
• Highly pathogenic avian influenza viruses (mouse and ferret)
• Novel or emerging influenza viruses

• Hemagglutination-inhibition assays
• Neuraminidase-inhibition assays
• Immunological assays (ELISA, Western blot)
• Custom assays
• Immune mechanisms
• Microneutralization assays
• Clinical serology support
• Drug screening assays, including HTS
• GLP studies
• Consultation

Southern Research in vivo vaccine development capabilities include:

• Pathogenesis (small/large animal models)
• Vaccine safety/efficacy testing
• Antiviral testing