Bovine leukemia virus (BLV) infection of rabbits is a tractable model system to evaluate vaccination strategies against lymphotropic retroviruses, which represent a global human health problem. We have previously developed genetically simplified BLV structural gene vector (SGV) that replicates BLV structural and enzymatic genes independently of BLV regulatory and accessory genes. Results of a 20-month study in a rabbit model demonstrated that BLV SGV induces an antiviral immunological response and lacks pathogenicity. Here, these chronically infected-BLV SGV rabbits are assessed in a proof-of-principle study of preventative vaccination against challenge with pathogenic BLV. This study commences 24 months after BLV SGV inoculation and proceeds for an additional 20 months. The previously characterized BLV SGV rabbits and age-matched control rabbits were challenged with 1 x 10(8) fetal lamb kidney/BLV producer cells. BLV SGV rabbits seroconverted upon BLV challenge, but did not progress to BLV infection nor clinical disease. By contrast, naive rabbits became infected and succumbed to lymphotropic disease. Our findings provide proof-of-principle that chronic infection with BLV SGV induces protection against BLV infection. The data indicate that SGV based on HTLV or HIV is a promising approach against lymphotropic disease by human retroviruses.