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Although most HIV-1 infections worldwide result from heterosexual transmission, most vaccine candidates have focused on induction of systemic immunity and protection. We hypothesized that combining systemic priming with mucosal boosting would induce mucosal immunity that would protect from intravaginal challenge. Macaques were primed systemically with recombinant vaccinia viruses and boosted mucosally using inactivated SHIV89.6 plus adjuvant. Other animals received protein boosts with adjuvant alone. Priming and boosting induced antiviral IgG and IgA antibodies. Such antibodies were induced to a lesser degree in animals receiving boosts alone. Anti-SHIV T cell responses were induced only in the prime-boost animals. Immunized animals and controls were challenged intravaginally with SHIV89.6 and significant reductions in proviral and viral RNA loads were observed in the prime-boost animals. The boost-only animals did not have significant viral load reductions. These data suggest that cellular immunity was required for protection from intravaginal challenge. This immunization regimen provides a promising lead for vaccine development. [Copyright &y& Elsevier]