In vitro expression studies.

The plasmid constructs were transfected into EPC cells and the encoded proteins were subsequently detected using specific primary antibodies: Polyclonal antibody rabbit anti-G5II Iag52B (PAb RαIag), monoclonal mouse I. multifiliis (I-ag) specific antibody MAb G3–61, monoclonal antibody VHSV G specific antibody IP1H3 (MAb IP1H3), respectively. The pcDNA3.1-Iag52A (or B) plasmids encoded I-ags encoding their own signal peptides as well as a glycosylphosphatidylinositol (GPI) glycolipid anchor, herby enabling the proteins to attach to the membrane. In the cassette constructs pcDNA3.0-CassL-Iag52A (or B) and pcDNA3.1-CassN-IAG52A (or B) the core I-ag region is flanked by the signal peptide and trimerization- and transmembrane region from the VHSV G protein, respectively. However, the two cassettes differ as CassL includes a linear VHSV G epitope for the MAb IP1H3, whereas this region has been omitted in the CassN construction. The derived pVax-CassL-Iag52A (or B)-3C3d represents secreted I-ag fusion variants including three copies of C3d.