2011 Annual Report 1a.Objectives (from AD-416) Evaluate swine influenza virus strain specific antigenic epitopes that might enable development of serological assays for pandemic H1N1 surveillance in swine. Evaluate selected antigenic epitopes and genetic factors of swine influenza virus that may lead to heterologous immunity elicited by influenza vaccines to enable development of improved vaccines with broader protective immunity against novel emerging influenza strains. 1b.Approach (from AD-416) 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. Collaborate in conducting animal studies 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.Progress Report A study of T-cell responses to a killed SIV vaccine, using flow cytometry-based in vitro assays was completed and published. T-cell immune responses were analyzed during a subsequent vaccination-challenge study that investigated a phenomenon of enhanced pneumonia in pigs that are primed with an SIV vaccine and infected with pandemic H1N1 influenza (pH1N1). In vitro assays to quantify priming of various T cells in all vaccinated and control animals were completed. Data are currently being analyzed and compiled. Work was initiated to express SIV proteins using a baculovirus expression vector. This system was adopted primarily to make virus-like particles (VLPs), which are comprised of only three viral proteins (HA, NA, M1). VLPs representing different SIV and pandemic H1N1 strains of interest will be tested as non-replicating vaccine antigens. Due to their well-defined and simple composition these constructs will be valuable in ongoing efforts to define protective and non-protective epitopes in the event of heterologous infection. Matrix protein of the novel 2009 A/H1N1 virus was evaluated as a potential diagnostic target for differentiating pigs exposed to the pandemic virus versus contemporary swine influenza isolates. Such a diagnostic test is against one of the most highly conserved proteins of influenza virus, the matrix (M1) protein. Although the M1 protein is not a structural protein, antibodies to M1 are produced during an influenza infection. We identified antigenic sites unique in the novel 2009 A/H1N1 virus matrix (M1) protein based on virus sequences. Three putative antigenic sites with maximum amino acid dissimilarity to non-pandemic swine H1N1 strains were selected for analysis in an immunoassay designed to differentiate exposure to the pandemic H1N1 strains from previously circulating swine influenza strains. Cross-reactivity at varying levels was found with antisera against several contemporary swine influenza strains containing the traditional North American matrix gene found in nearly all strains of swine influenza widely circulating in North American prior to the emergence of the 2009 A/H1N1 pandemic virus. These data indicate such a diagnostic approach is not possible and will be fraught with varying degrees of cross-reactivity and loss of specificity as the virus of interest undergoes antigenic drift. Progress is monitored via email and conference calls.