The production and selection of infectious vaccinia virus recombinants expressing foreign genes was facilitated by the construction of plasmid vectors. These vectors contain all or part of the vaccinia virus thymidine kinase (TK) gene interrupted by multiple unique restriction endonuclease sites placed adjacent to the TK promoter or another promoter translocated within the TK gene. The insertion of a continuous coding sequence for a foreign protein at one of the unique restriction endonuclease sites juxtaposes the transcriptional start site of a vaccinia promoter and the translational start site of a foreign gene. After transfection of vaccinia virus-infected cells with such plasmids, homologous recombination occurs between the vaccinia virus sequences flanking the chimeric gene and the same sequences within the virus genome. Recombinants formed in this manner have the chimeric gene inserted within the body of the vaccinia virus TK gene under control of a vaccinia virus promoter. Since recombinants have an interrupted TK gene, they are selected on the basis of their TK-phenotype and then checked for the presence and expression of the foreign gene. Infectious recombinant viruses expressing the procaryotic enzyme chloramphenicol acetyltransferase were constructed to optimize the system. The absence of chloramphenicol acetyltransferase activity in uninfected cells or in cells infected with wild-type vaccinia virus and the availability of a sensitive and quantitative enzyme assay allowed an estimation of the relative strengths of various promoter constructs. The expression of chloramphenicol acetyltransferase was detected within 1 h after infection of cells with recombinant virus, reflecting the early nature of the promoters used.

Foreign DNA was inserted into two nonessential regions ofthe vaccinia virus genome by homologous recombination in cells infected with virus and transfected with plasmids containing the foreign DNA elements flanked by vaccinia virus DNA. Thymidine kinase-negative (TK-) recombinants were selected after inserting foreign DNA into the coding region of the TK gene ofwild-type vaccinia virus; TKV recombinants were selected after inserting the herpesvirus TK gene into TK-mutants of vaccinia virus. For TKV expression, it was necessary to insert a 275-basepair DNA fragment containing the initiation site and sequences upstream of an early vaccinia virus transcript next to the coding sequences of the herpesvirus gene. The unique ability of the herpesvirus TK to phosphorylate "MI-labeled deoxycytidine provided independent confirmation of gene expression. These studies demonstrate the use of vaccinia virus as a selectable cloning and expression vector, confirm the map location of the vaccinia virus TK gene, and provide initial information regarding the location of vaccinia virus transcriptional regulatory sequences.Several virus groups, including the papovaviruses (1-3), papillomaviruses (4), adenoviruses (5,6), and retroviruses (7,8) In considering the development of vaccinia virus as an expression vector, the following biological characteristics ofthis unique agent must be taken into account (9, 10): a large [180-kilobase (kb)] genome, a lack ofinfectivity ofisolated viral DNA, the packaging ofviral enzymes necessary for transcription within the infectious particle, the probability that vaccinia virus has evolved its own transcriptional regulatory sequences, and the cytoplasmic site of virus transcription and replication. Initially, the major technical problems involved insertion of DNA into the large genome, efficient expression of heterologous genes, and selection of recombinant virus.Insertion of DNA into the vaccinia virus genome can be accomplished by homologous recombination in vivo. Because vaccinia virus DNA by itself is not infectious, intact virus and calcium phosphate-precipitated viral DNA (11, 12) or plasmids containing viral sequences (13) are added in succession. By using plasmids, it is possible to perform the majority of manipulations in vitro except for the final step of transfection.Presumably MATERIALS AND METHODS Preparation of DNA. Recombinant plasmids were prepared from pBR328 (16) or pUC7 (a gift ofJ. Viera and J. Messing) and purified as described by Birnboim and Doly (17). DNA fragments were isolated from agarose gels by electrophoresis onto or by binding to powdered glass (19).Marker Rescue. Two hours after infection of TK-143 cells (20) with TK' or TK-vaccinia virus WR (0.01-0.05 plaqueforming unit/cell), calcium phosphate-precipitated plasmid DNA was added (13). For isolation of TK' recombinants, amethopterin-containing selective medium was added at 6 hr and cells were harvested at 48 hr after infection (13). For isolation of TK-mutants, selection with bromodeoxyuridine at 25 Ag/ml wa...

Potential live vaccines against hepatitis B virus have been produced. The coding sequence for hepatitis B virus surface antigen (HBsAg) has been inserted into the vaccinia virus genome under control of vaccinia virus early promoters. Cells infected with these vaccinia virus recombinants synthesize and excrete HBsAg and vaccinated rabbits rapidly produce antibodies to HBsAg.

Inoculation of rabbits and mice with a vaccinia-rabies glycoprotein recombinant (V-RG) virus resulted in rapid induction of high concentrations of rabies virus-neutralizing antibodies and protection from severe intracerebral challenge with several strains of rabies virus. Protection from virus challenge also was achieved against the rabies-related Duvenhage virus but not against the Mokola virus. Effective immunization by V-RG depended on the expression of a rabies glycoprotein that registered proline rather than leucine as the eighth amino acid from its NH2 terminus (V-RGpro8). A minimum dose required for effective immunization of mice was 104 plaque-forming units of V-RGpro8 virus. fi-propiolactone-inactivated preparations of V-RGpro8 virus also induced high levels of rabies virus-neutralizing antibody and protected mice against intracerebral challenge with street rabies virus. VRGpro8 virus was highly effective in priming mice to generate a secondary rabies virus-specific cytotoxic T-lymphocyte response following culture of lymphocytes with either ERA or PM strains of rabies virus.