The rapid spread of HIV-1 underscores the urgent need to develop an effective vaccine. Using modified vaccinia Ankara (MVA) as a vector, we designed and constructed a multigenic candidate vaccine against a recombinant C/B' subtype of HIV-1 that is dominant in southwest China. Five HIV-1 genes (gag, pol, DeltaV2env, tat, and nef) were introduced into 2 separate regions of the MVA genome using modified single- and dual-promoter insertion vectors. Recombinant MVA was selected by immunofluorescence double-staining and foci purification. The end product is a single recombinant MVA, termed ADMVA, that expresses HIV-1 DeltaV2Env and fusion proteins Gag-Pol and Nef-Tat. By in vitro analyses, all expected HIV-1 proteins were expressed in infected chicken embryo fibroblasts and various human cell lines. Additionally, 2 sequential intramuscular injections of 10(6) 50% tissue infectious culture dose (TCID50) of ADMVA into BALB/c and B6 x B10 mice elicited broad cell-mediated immune responses against all 5 viral proteins as determined by interferon-gamma enzyme immunospot assays. The number of spot-forming cells was in the range of 200 to 800 per million splenocytes, and both CD4 and CD8 T-cell responses were detected. Moreover, high serum titers (>1:20,000) of antibodies against HIV-1 gp120 were also elicited. The magnitude of immune responses correlated with the dose of ADMVA, and the vaccine caused no overt adverse consequences, up to 10(7) TCID50 per injection. ADMVA has since been advanced into clinical trials. A phase 1 study has been completed, and a prime-boost with ADVAX (see accompanying article) is now underway.