The objectives of the proposed research are (1) to manufacture and perform phase 1 clinical testing of an alphavirus replicon particle vaccine previously shown to be effective for preventing disease and mortality after challenge with a homologous strain of Marburg virus in guinea pigs and nonhuman primates, (2) to evaluate in vitro immune assays as correlates of protective immunity, (3) to evaluate the effects of changes in the replicon particle surface coat on immunogenicity, (4) to design, construct and perform preclinical evaluations of bivalent alphavirus replicon particle vaccines using heterologous strains of Marburg virus, and (5) to manufacture and perform phase 1 clinical testing of a bivalent alphavirus replicon particle vaccine containing genes from two heterologous strains of Marburg virus. Marburg virus, a causative pathogen of viral hemorrhagic fever, is infectious by the aerosol route, can be grown to high titer, and can be stabilized by Iyophylization, making it an important biowarfare/bioterrorism agent. The glycoprotein gene from one strain of Marburg virus, which has been identified as a protective immunogen in both rodent and primate challenge models, will be inserted into self-replicating RNAs (replicons) derived from attenuated strains of Venezuelan equine encephalitis (VEE). The replicons will be packaged into single-cycle, propagation- defective, virus-Iike replicon particles (VRP) using a VEE glycoprotein coat that confers dendritic cell tropism. GMP manufacture and clinical testing in healthy volunteers will be performed using a study design based on previous experience with a VRP vaccine for HIV. To identify correlates of protective immunity, animals will be immunized with graded doses of Marburg VRP vaccines carrying different VEE glycoprotein surface coats, challenged with a lethal dose of Marburg virus, and protective efficacy after immunization will be correlated with pre-challenge immune responses (virus neutralization in vitro, ELISA, ADCC, ELISPOT). The same immunological assays will be performed with samples obtained during clinical trials. Bivalent VRP vaccines, expressing glycoprotein genes from two heterologous Marburg virus strains, will be constructed and used to immunize nonhuman primates. Immunogenicity and efficacy will be compared in animals immunized with bivalent or monovalent VRP vaccines. The most effective bivalent Marburg VRP vaccine strategy will be selected for GMP manufacture and clinical testing.