We developed the method to efficiently construct recombinant vaccinia viruses based on LC16m8Delta strain that can replicate in mammalian cells but is still safe in human. Immunization in a prime-boost strategy using DNA and LC16m8Delta expressing SIV Gag elicited 7-30-fold more IFN-gamma-producing T cells in mice than that using DNA and non-replicating vaccinia DIs recombinant strain. As the previous study on the DNA-prime and recombinant DIs-boost anti-SIV vaccine showed protective efficacy in the macaque model [Someya K, Ami Y, Nakasone T, Izumi Y, Matsuo K, Horibata S, et al. Induction of positive cellular and humoral responses by a prime-boost vaccine encoded with simian immunodeficiency virus gag/pol. J Immunol 2006;176(3):1784-95], LC16m8Delta would have potential as a better recombinant viral vector for HIV vaccine.