2010 Annual Report 1a.Objectives (from AD-416) 1. Identify swine influenza virus strain specific antigenic epitopes to support the development of serological assays for surveillance in swine. 2. Compare pathogenesis and transmissibility of selected isolates of the wild-type novel A/HINI virus from animals (e.g., Canadian, Chilean and Argentinean viruses) and correlate with genetic and antigenic changes. 3. Determine efficacy of conventional inactivated swine influenza vaccines against emerging isolates of pandemic A/H1Nl from animals (e.g., Canadian, Chilean and Argentinean viruses). 4. Evaluate a reverse genetics-derived modified live vaccine in pigs and other susceptible animal hosts against pandemic A/H1N1. 5. Evaluate an adenovirus-vectored influenza virus vaccine in pigs. 1b.Approach (from AD-416) 1. Conduct immunological investigations of influenza A virus components that lead to immune responses against specific epitopes that may enable serological surveillance for the 2009 A/H1N1 in swine and determine whether heterologous immunity against endemic swine influenza viruses interferes with serological surveillance methods. 2. Conduct an animal study to determine the pathogenesis and transmissibility of selected isolates of the wild-type novel A/HINI virus from animals and correlate with genetic and antigenic changes. 3. Conduct an animal study to determine the efficacy of conventional inactivated swine influenza vaccines against emerging isolates of pandemic A/H1Nl from animals. 4. Conduct an animal study utilizing a reverse genetics-derived modified live vaccine in pigs and other susceptible animal hosts against pandemic A/H1N1. 5. Conduct an animal study evaluating an adenovirus-vectored H1N1 influenza virus vaccine in pigs. 3.Progress Report A postdoctoral research associate and a temporary full-time veterinary medical officer were recruited to meet the objectives to be completed at the National Animal Disease Center (NADC). Two Specific Cooperative Agreements (SCA) were established to further meet the objectives. An SCA with Iowa State University (ISU) and with University of Maryland (UMD) were developed and fully executed. Initial project planning and experimental designs have been established with the SCA cooperators and initial phases of the agreements have been initiated. Monoclonal antibodies to differentiate the hemagglutinin protein of pandemic H1N1 from endemic swine influenza viruses were generated at the UMD and preliminary testing on panels of viruses has been completed. Additional antibodies are in development. Three in vivo studies have been completed to evaluate inactivated vaccines (developed at the National Animal Disease Center-ARS, Ames, IA, or commercially available) and modified live virus vaccines (developed at the UMD) against the pandemic H1N1 in pigs and/or mice. An initial animal study investigating humoral and cellular immune responses against influenza A viruses (including the 2009 A/H1N1 virus) in swine has been completed. 4.Accomplishments 1. Demonstrated heterologous (cross-protective) cellular immune response against the 2009 A/H1N1 pandemic virus in pigs vaccinated with a human-like delta-cluster H1N1 virus. Humoral (serum antibody) and cellular (activated immune cells from whole blood) immune responses to inactivated swine influenza virus (SIV) vaccine were evaluated and compared by ARS scientists at the National Animal Disease Center in Ames, Iowa and their collaborators at Iowa State University as part of a Specific Cooperative Agreement. Pigs vaccinated with an inactivated (killed) SIV vaccine were shown to have strong cell mediated immune responses against the same virus and lower but significant cross-protective responses to the 2009 pandemic virus, especially in the lymphocytes involved with immune memory (e.g., CD4+CD8+ T cell subset). This is significant as the swine virus contained a distinctly different hemagglutinin (H1) protein molecule compared to the pandemic H1N1 virus. A standard antibody test detected humoral antibody responses only to the swine virus. In contrast, the cross-reactive cellular responses to the pandemic virus detected by flow cytometry suggested that the cellular assay was more sensitive and that the cellular immune system potentially responds to a wider range of virus molecules than what is detected by the standard antibody assay. This cellular immunity measurement research tool may provide a better method for predicting cross protection against newly emerging viruses in the absence of antibodies recognizing a novel emerging virus. 2. Demonstrated modifications in the influenza virus polymerase complex (the virus replication machinery) of a swine-like triple reassortant influenza virus that generated a series of attenuated or modified live vaccines against the 2009 pandemic H1N1. The development of effective pandemic virus vaccines for use in multiple species would minimize the impact of future flu pandemics. ARS scientists at the National Animal Disease Center in Ames, Iowa, and collaborators at the University of Maryland as part of a Specific Cooperative Agreement, generated and tested the safety and the efficacy of a modified-live H1N1 vaccine in both mice and pigs. Vaccination of pigs with the modified-live H1N1 vaccine candidate resulted in sterilizing immunity following experimental challenge with the 2009 H1N1 pandemic virus. This research highlights the potential of modified-live vaccines for humans and livestock. These results can be used by regulatory agencies and scientists developing improved influenza vaccines for swine.