This study is a multi-center, double-blind, placebo-controlled, Phase I study to evaluate the safety, reactogenicity, tolerability, and immunogenicity of MVA-BN-YF in Flavivirus-naïve healthy male and non-pregnant female adult subjects. There are six dose groups in this study. Subjects who have never received a licensed or investigational smallpox vaccine will be randomized to Groups 1-5 and vaccine administration and follow-up will be conducted in a double-blinded fashion. Subjects who have previously received two, 1 x 108 TCID50 doses of MVA-BN between 19 and 45 days apart by SC or IM routes will be enrolled in Group 6 and will be dosed open-label. Since this is a first in human, phase I study, a sentinel cohort will be utilized. The first two subjects (1st sentinel group) one at each clinical site will be randomized to Group 2 or 3 and vaccinated with MVA-BN-YF with or without ISA 720. Subjects and study personnel will be blinded as to whether ISA 720 was administered. Subjects will be monitored for safety for one day, and if no pre-defined halting rule is met (Section 9.5.1) then two additional subjects (2nd sentinel group) one at each clinical site will be assigned to the group the previous subject was not assigned to. These subjects will be vaccinated and monitored as above. A total of 4 sentinel subjects will be vaccinated. Primary objectives are assessment of the safety, tolerability, and reactogenicity of MVA-BN-YF vaccine administered with or without ISA 720; comparison of the safety, tolerability, and reactogenicity of MVA-BN-YF vaccine administered with or without ISA 720 with YF-VAX and MVA-BN. Secondary objectives are: assessment of the immunogenicity against the MVA-BN backbone and Yellow Fever virus (YF) antigen insert of MVA-BN-YF with and without ISA 720 as assessed by kinetics of the immune responses, seroconversion rates, and peak Geometric Mean Titer (GMT); assessment of the impact of previous MVA-BN vaccination on peak immune responses to YF antigen in MVA-BN-YF; comparison of the peak immunogenicity against YF antigen of 1 or 2 doses of MVA-BN-YF with or without ISA 720 with YF-VAX; comparison of the peak immunogenicity against the MVA-BN backbone of 1 or 2 doses of MVA-BN-YF with or without ISA 720 with MVA-BN; assessment of durability of immune response to YF antigen and MVA-BN at 6 months after 2nd vaccination or placebo administration.