goat anti rabbit IgG Ale a fluor 594 conjugated antibody were purchased from Life Technologies Molecu lar Probes. Protein G sepharose, sulforho damine B, Concanavalin A, goat anti rabbit secondary selleck compound antibody HRP conjugated and all the chemicals were pur chased by Sigma Aldrich. Synthetic peptides lo cated in the e tracellular, transmembrane domains of rat Neu sequence were previously described. Po viruses The recombinant vaccinia virus encoding the neu onco gene was designated rV neuT. It encodes the full length activated rat neu oncogene. The wild type control vac cinia virus was designated V wt. Therion Biologics Corp. kindly provided the po viruses. E pression of recombinant NeuT encoded by rV neuT was detected by Western blotting after infection of BSC 1 or NIH3T3 cells with V wt or rV neuT.
Cells were in fected with 10 pfu cell of po viruses and cultured at 37 C for 18 h. Cell lysates, protein concen trations and immunoblotting were performed as previ ously described. Polyclonal anti ErbB2 Neu antibody was used to detect recombinant NeuT. Transgenic BALB neuT mouse colony Transgenic BALB neuT male mice were routinely mated with BALB c females in the animal facilities of Tor Vergata University. Progenies were confirmed for presence of the transgene by Polymer ase Chain Reaction. Mice were bred under pathogen free conditions and handled in compliance with European Union and institutional standards for animal research. Recombinant vaccinia neu vaccination protocol The protocol of vaccination was approved by the Ethical Committee of the University of Rome Tor Vergata and submitted to the Italian Health Department.
Si to 8 weeks old BALB neuT male mice were subcuta neously injected in the right flank with 0. 2 ml suspension containing 1 106 SALTO cells in phosphate buffered sa line. When mice presented a palpable tumor mass around 300 mm3, were intratumorally vaccinated with either rV neuT or V wt and boosted two weeks later. Viruses were diluted in PBS such that the dose was delivered in 100 ul. Mice were immunized twice. BALB neuT received for each vaccination a dose of 108 pfu of either rV neuT or V wt, a dose of 107 pfu of either rV neuT or V wt and a dose of 106 pfu of either rV neuT or V wt. Analysis of antitumor activity in vivo Tumor growth was monitored weekly until tumor bearing mice were sacrificed when tumor e ceeded 20 mm diam eter.
Tumors were measured Carfilzomib by a calliper in two dimen sions and the volumes were calculated using the formula width2 length 2. Antibody immunity following vaccination with rV neuT Sera from vaccinated BALB neuT mice were collected prior to vaccination and 7 days after the final boost. The presence of antibodies reactive to p185 Neu was assayed using NIH3T3, LTR Neu and SALTO cells by enzyme linked immunosorbent assay or immunoprecipi tation following western blotting as previously described. For ELISA, individual rV CYC202 neuT mouse serum at different dilutions was assayed against LTR Neu and NIH3T3 control. The specif