2011 Annual Report 1a.Objectives (from AD-416) Vaccination against Classical Swine Fever Virus (CSFV) with live-attenuated viruses, although efficacious, do not allow for the differentiation from vaccinated infected animals. Current CSFV subunit marker vaccines utilizing recombinant E2 envelope protein provides protection, but not until 15 days post inoculation. ARS, PIADC and INIA will develop an adjuvant-free antigen-delivery system utilizing subunit CSF vaccine based on the glycoprotein E2 fused to the adjuvant molecule antigen-presenting cells (APC) expressed in insect larvae. The objectives of this project are: 1. Express recombinants CSFV E2 proteins as a fusion construct with APCH I in the baculovirus system using insects as expression vector. 2. Evaluate the immunogenicity of the construct in terms of kinetics in the elicitation of anti-E2 antibodies and protection against the virulent challenge in swine. 1b.Approach (from AD-416) 1. Different versions of a fusion protein containing the CDSFV-E2/APCH-I construct will be designed, produced and purified. INIA will design the constructs, conduct baculovirus development, protein production and purification and conduct antigenic assessment. 2. Purified proteins will be assessed for their ability to induce anti-E2 antibody response. The immune response will be evaluated via ELISA and by the CSFV neutralizing response in vivo at ARS, PIADC. 3.Progress Report The genetic constructs that enable the expression of CSFV E2 proteins of strain Brescia as a fusion protein with the single chain monoclonal antibody was assembled. The construct was then expressed in baculovirus. The expression of the fusion protein was detected in extracts of insect cells infected with the recombinant baculovirus. Fusion protein was further purified by affinity chromatography based in specific proteins tag incorporated in the genetic construct. Purified proteins are now ready to be assessed in their immunogenicity and protective effect in swine. This project was monitored through email and telephone exchange.