Identification of vaccinia promoters by heterologous expression of hepatitis B surface antigen in mouse cells infected by recombinant vaccinia viruses

DNA fragments preceding open reading frames in a conserved segment of the vaccinia virus genome (Plucienniczak A., et al. (1985) Nucleic Acids Res. 13, 985–998) were cloned into plasmids upstream of the S gene of the hepatitis B virus encoding the surface antigen (HBsAg). Recombinant vaccinia virus obtained after insertion of these constructs into the thymidine kinase gene were used to infect mouse 1D cells. HBsAg was assayed in cellular supernatants. A strong promoter was thus identified in a 295 bp fragment preceding the coding region of the 147 kDa subunit of the vaccinia RNA polymerase.     

Expression of functional Bunyamwera virus L protein by recombinant vaccinia viruses.

A cDNA containing the complete coding sequence of the Bunyamwera virus (family Bunyaviridae) L genome segment has been constructed and cloned into two recombinant vaccinia virus expression systems. In the first, the L gene is under control of vaccinia virus P7.5 promoter; in the second, the L gene is under control of the bacteriophage T7 phi 10 promoter, and expression of the L gene requires coinfection with a second recombinant vaccinia virus which synthesizes T7 RNA polymerase. Both systems express a protein which is the same size as the Bunyamwera virus L protein and is recognized by a monospecific L antiserum. The expressed L protein was shown to be functional in synthesizing Bunyamwera virus RNA in a nucleocapsid transfection assay: recombinant vaccinia virus-infected cells were transfected with purified Bunyamwera virus nucleocapsids, and subsequently, total cellular RNA was analyzed by Northern (RNA) blotting. No Bunyamwera virus RNA was detected in control transfections, but in cells which had previously been infected with recombinant vaccinia viruses expressing the L protein, both positive- and negative-sense Bunyamwera virus S segment RNA was detected. The suitability of this system to delineate functional domains within the Bunyamwera virus L protein is discussed.     

Extracellular vaccinia virus envelope glycoprotein encoded by the A33R gene.

With the aid of three monoclonal antibodies (MAbs), a glycoprotein specifically localized to the outer envelope of vaccinia virus was shown to be encoded by the A33R gene. These MAbs reacted with a glycosylated protein that migrated as 23- to 28-kDa and 55-kDa species under reducing and nonreducing conditions, respectively. The protein recognized by the three MAbs was synthesized by all 11 orthopoxviruses tested: eight strains of vaccinia virus (including modified vaccinia virus Ankara) and one strain each of cowpox, rabbitpox, and ectromelia viruses. The observation that the protein synthesized by ectromelia virus-infected cells reacted with only one of the three MAbs provided a means of mapping the gene encoding the glycoprotein. By transfecting vaccinia virus DNA into cells infected with ectromelia virus and assaying for MAb reactivity, we mapped the glycoprotein to the A33R open reading frame. The amino acid sequence and hydrophilicity plot predicted that the A33R gene product is a type II membrane protein with two asparagine-linked glycosylation sites. Triton X-114 partitioning experiments indicated that the A33R gene product is an integral membrane protein. The ectromelia virus homolog of the vaccinia virus A33R gene was sequenced, revealing 90% predicted amino acid identity. The vaccinia and variola virus homolog sequences predict 94% identical amino acids, the latter having one fewer internal amino acid. Electron microscopy revealed that the A33R gene product is expressed on the surface of extracellular enveloped virions but not on the intracellular mature form of virus. The conservation of this protein and its specific incorporation into viral envelopes suggest that it is important for virus dissemination.     

Structural proteins of hog cholera virus expressed by vaccinia virus: further characterization and induction of protective immunity.

A cDNA fragment covering the genomic region that encodes the structural proteins of hog cholera virus (HCV) was inserted into the tk gene of vaccinia virus. Expression studies with vaccinia virus/HCV recombinants led to identification of HCV-specific proteins. The putative HCV core protein p23 was demonstrated for the first time by using an antiserum against a bacterial fusion protein. The glycoproteins expressed by vaccinia virus/HCV recombinant migrated on sodium dodecyl sulfate-gels identically to glycoproteins precipitated from HCV-infected cells. A disulfide-linked heterodimer between gp55 and gp33 previously detected in HCV-infected cells was also demonstrated after infection with the recombinant virus. The vaccinia virus system allowed us to identify, in addition to the heterodimer, a disulfide-linked homodimer of HCV gp55. The vaccinia virus/HCV recombinant that expressed all four structural proteins induced virus-neutralizing antibodies in mice and swine. After immunization of pigs with this recombinant virus, full protection against a lethal challenge with HCV was achieved. A construct that lacked most of the HCV gp55 gene failed to induce neutralizing antibodies but induced protective immunity.     

Activation of the human immunodeficiency virus type 1 long terminal repeat by vaccinia virus.

A variety of DNA viruses are known to activate gene expression directed by the long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1). In light of the proposed use of recombinant vaccinia virus for HIV-1 vaccines, evaluation of the role of vaccinia virus in HIV-1 activation is warranted. To investigate whether vaccinia virus induces HIV LTR-directed gene expression, transient expression assays in Jurkat cells persistently infected with vaccinia virus (Jvac) using plasmid DNA containing the LTR linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were performed. CAT activity in Jvac cells was always recorded, although the level appears to fluctuate independently of virus titers. Dual intracytoplasmic staining and fluorescence-activated cell sorter analysis showed that CAT activity was expressed in the infected cells. CAT expression was not due to plasmid replication, since plasmid DNA extracted from Jvac cells 48 h after transfection was restricted only by enzymes which recognize methylated sequences, indicating a prokaryotic source for the DNA. These findings suggest that a factor(s) present in vaccinia virus-infected cells is capable of activating the LTR of HIV-1.     

Apoptin enhances the oncolytic activity of vaccinia virus in vitro

The chicken anemia virus gene that encodes apoptin, a selective killer of cancer cells, was synthe-sized and inserted into the vaccinia virus (strain L-IVP) genome. The insertion replaces a major part of the viral C11R gene that encodes viral growth factor, which is important for virulence. The recombinant virus VVdGF-ApoS24/2 was obtained by transient dominant selection using the gene of puromycin resistance as a selective marker. The expression of the apoptin gene from a synthetic early-late promoter of vaccinia virus ensured the efficient accumulation of the target protein in VVdGF-ApoS24/2-infected cells. Although recombinant apoptin carried the signal peptide of the virus growth factor at the N-end, the protein was not secreted into the culture medium. The recombinant virus VVdGF-ApoS24/2 exhibited significantly higher selective lytic activity in human cancer cell lines (A549, A431, U87MG, RD, and MCF7) than the parent strain L-IVP and its VVdGF2/6 variant with C11R deletion. These results suggest that the use of apoptin can be an efficient means of enhancing the natural anticancer activity of vaccinia virus.     

Conditional expression of foreign genes by temperature-sensitive mutants of vaccinia virus

To assess the utility of two temperature-sensitive (ts) mutant vaccinia viruses as vectors for the conditional in vitro expression of recombinant foreign genes, we have studied the kinetics of expression of foreign genes incorporated into these viruses. At nonpermissive temperature, 40 degrees C, these viruses were defective either in DNA synthesis or in virus assembly. Foreign gene expression was affected by the nature of the ts lesion and by the nature of the vaccinia promoter positioned upstream from the foreign gene. With both vector viruses, a foreign gene controlled by the p7.5 early-late promoter was expressed at both 33 degrees and 40 degrees C. With the DNA synthesis-defective vector virus, foreign gene expression controlled by the p11 DNA synthesis-dependent late promoter was inhibited at 40 degrees C, but could be turned on by shift to 33 degrees C. This ts expression system provides an alternative to use of drugs that inhibit DNA synthesis as a means for experimental manipulation of gene expression. Both vector viruses can be used with existing vaccinia virus expression technology.     

Highly efficient induction of protective immunity by a vaccinia virus vector defective in late gene expression

Vaccinia viruses defective in the essential gene coding for the enzyme uracil DNA glycosylase (UDG) do not undergo DNA replication and do not express late genes in wild-type cells. A UDG-deficient vaccinia virus vector carrying the tick-borne encephalitis (TBE) virus prM/E gene, termed vD4-prME, was constructed, and its potential as a vaccine vector was evaluated. High-level expression of the prM/E antigens could be demonstrated in infected complementing cells, and moderate levels were found under noncomplementing conditions. The vD4-prME vector was used to vaccinate mice; animals receiving single vaccination doses as low as 10(4) PFU were fully protected against challenge with high doses of virulent TBE virus. Single vaccination doses of 10(3) PFU were sufficient to induce significant neutralizing antibody titers. With the corresponding replicating virus, doses at least 10-fold higher were needed to achieve protection. The data indicate that late gene expression of the vaccine vector is not required for successful vaccination; early vaccinia virus gene expression induces a potent protective immune response. The new vaccinia virus-based defective vectors are therefore promising live vaccines for prophylaxis and cancer immunotherapy.     

The envelope G3L protein is essential for entry of vaccinia virus into host cells

The vaccinia virus G3L/WR079 gene encodes a conserved protein with a predicted transmembrane domain. Our proteomic analyses of vaccinia virus revealed that G3L protein is incorporated into intracellular mature virus; however, the function of G3L protein in the vaccinia virus life cycle has not been investigated. In this study, a recombinant vaccinia virus, viG3L, expressing G3L protein under IPTG (isopropyl-beta-d-thiogalactopyranoside) regulation was constructed. Under permissive conditions when G3L protein was expressed, the vaccinia virus life cycle proceeded normally, resulting in plaque formation in BSC40 cells. In contrast, under nonpermissive conditions when G3L protein expression was repressed, no plaques were formed, showing that G3L protein is essential for vaccinia virus growth in cell cultures. In infected cells when G3L protein was not expressed, the formation of intracellular mature virus (IMV) and cell-associated enveloped virus occurred normally, showing that G3L protein is not required for virion morphogenesis. IMV particles containing (G3L(+)) or lacking (G3L(-)) G3L protein were purified and were found to be indistinguishable on microscopic examination. Both G3L(+) and G3L(-) IMV bound to HeLa cells; however, G3L(-) IMV failed to enter the cells, showing that G3L protein is required for IMV penetration into cells. Finally, G3L protein was required for fusion of the infected cells under low-pH treatment. Thus, our results provide direct evidence that G3L is an essential component of the vaccinia virus fusion complex, in addition to the previously reported A28, H2, L5, A21, and A16 proteins.     

Mutations in active-site residues of the uracil-DNA glycosylase encoded by vaccinia virus are incompatible with virus viability.

The D4R gene of vaccinia virus encodes a functional uracil-DNA glycosylase that is essential for viral viability (D. T. Stuart, C. Upton, M. A. Higman, E. G. Niles, and G. McFadden, J. Virol. 67:2503-2513, 1993), and a D4R mutant, ts4149, confers a conditional lethal defect in viral DNA replication (A. K. Millns, M. S. Carpenter, and A. M. DeLange, Virology 198:504-513, 1994). The mutant ts4149 protein was expressed in vitro and assayed for uracil-DNA glycosylase activity. Less than 6% of wild-type activity was observed at permissive temperatures, but the ts4149 protein was completely inactive at the nonpermissive temperature. Mutagenesis of the ts4149 gene back to wild type (Arg-179-->Gly) restored full activity. The ts4149 protein was considerably reduced in lysates of cells infected at the permissive temperature, and its activity was undetectable, even in the presence of the uracil glycosylase inhibitor protein, which inhibits the host uracil-DNA glycosylases but not that of vaccinia virus. Thus the ts4149 protein is thermolabile, correlating uracil removal with vaccinia virus DNA replication. Three active-site amino acids of the vaccinia virus uracil-DNA glycosylase were mutated (Asp-68-->Asn, Asn-120-->Val, and His-181-->Leu), producing proteins that were completely defective in uracil excision but still retained the ability to bind DNA. Each mutated D4R gene was transfected into vaccinia virus ts4149-infected cells in order to assess the recombination events that allowed virus survival at 40 degrees C. Genetic analysis and sequencing studies revealed that the only viruses to survive were those in which recombination eliminated the mutant locus. We conclude that the uracil cleavage activity of the D4R protein is essential for its function in vaccinia virus DNA replication, suggesting that the removal of uracil residues plays an obligatory role.     

Expression in recombinant vaccinia virus of the equine herpesvirus 1 gene encoding glycoprotein gp13 and protection of immunized animals.

The equine herpesvirus 1 (EHV-1) gene encoding glycoprotein 13 (gp13) was cloned into the hemagglutinin (HA) locus of vaccinia virus (Copenhagen strain). Expression of the gp13 gene was driven by the early/late vaccinia virus H6 promoter. Metabolically radiolabeled polypeptides of approximately 47 and 44 kilodaltons and 90 kilodaltons (glycosylated form) were precipitated with both polyclonal and gp13-specific monoclonal antibodies. Presentation of gp13 on the cytoplasmic membrane of cells infected with the recombinant gp13 vaccinia virus was demonstrated by immunofluorescence of unfixed cells. Inoculation of the recombinant gp13 vaccinia virus into guinea pigs induced neutralizing antibodies to both EHV-1 and vaccinia virus. Hamsters vaccinated with the recombinant gp13 vaccinia virus survived a lethal challenge with the hamster-adapted Kentucky strain of EHV-1. These results indicate that expression in vaccinia virus vectors of EHV-1 gp13, the glycoprotein homolog of herpes simplex virus gC-1 and gC-2, pseudorabies virus gIII, and the varicella-zoster virus gpV may provide useful vaccine candidates for equine herpesvirus infections.     

Immunogenicity and safety of defective vaccinia virus lister: comparison with modified vaccinia virus Ankara

Potent and safe vaccinia virus vectors inducing cell-mediated immunity are needed for clinical use. Replicating vaccinia viruses generally induce strong cell-mediated immunity; however, they may have severe adverse effects. As a vector for clinical use, we assessed the defective vaccinia virus system, in which deletion of an essential gene blocks viral replication, resulting in an infectious virus that does not multiply in the host. The vaccinia virus Lister/Elstree strain, used during worldwide smallpox eradication, was chosen as the parental virus. The immunogenicity and safety of the defective vaccinia virus Lister were evaluated without and with the inserted human p53 gene as a model and compared to parallel constructs based on modified vaccinia virus Ankara (MVA), the present "gold standard" of recombinant vaccinia viruses in clinical development. The defective viruses induced an efficient Th1-type immune response. Antibody and cytotoxic-T-cell responses were comparable to those induced by MVA. Safety of the defective Lister constructs could be demonstrated in vitro in cell culture as well as in vivo in immunodeficient SCID mice. Similar to MVA, the defective viruses were tolerated at doses four orders of magnitude higher than those of the wild-type Lister strain. While current nonreplicating vectors are produced mainly in primary chicken cells, defective vaccinia virus is produced in a permanent safety-tested cell line. Vaccines based on this system have the additional advantage of enhanced product safety. Therefore, a vector system was made which promises to be a valuable tool not only for immunotherapy for diseases such as cancer, human immunodeficiency virus infection, or malaria but also as a basis for a safer smallpox vaccine.