Use of a negative selectable marker for rapid selection of recombinant vaccinia virus

Vaccinia virus has been a powerful tool in molecular biology and vaccine development. The relative ease of inserting and expressing foreign genes combined with its broad host range has made it an attractive antigen delivery system against many heterologous diseases. Many different approaches have been developed to isolate recombinant vaccinia virus generated from homologous recombination; however, most are time-consuming, often requiring a series of passages or specific cell lines. Herein we introduce a rapid method for isolating recombinants using the antibiotic coumermycin and the interferon-associated PKR pathway to select for vaccinia virus recombinants. This method uses a negative selection marker in the form of a fusion protein, GyrB-PKR, consisting of the coumermycin dimerization domain of Escherichia coli gyrase subunit B fused to the catalytic domain of human PKR. Coumermycin-dependent dimerization of this protein results in activation of PKR and the phosphorylation of translation initiation factor, eIF2α. Phosphorylation of this factor leads to an inhibition of protein synthesis, and an inhibition of virus replication. In the presence of coumermycin, recombinants are isolated due to the loss of this coumermycin-sensitive gene by homologous recombination. We demonstrate that this method of selection is highly efficient and requires limited rounds of enrichment to isolate recombinant virus.     

Set of vectors for the expression of histidine-tagged proteins in vaccinia virus recombinants

Vaccinia virus expression vectors are widely used to direct the expression of proteins in eukaryotic cells. Here, we describe a new set of plasmid vectors designed for the expression of histidine-tagged proteins in the vaccinia system. To facilitate the rapid isolation of virus recombinants, the plasmids contain a viral gene (F13L) that serves as an efficient selection marker based on virus plaque phenotype. Histidine codons and restriction sites derived from pET-16b bacterial expression plasmid were included, thus facilitating the transfer of genes between E. coli and vaccinia expression plasmids. Plasmids in which the gene is placed downstream of either a strong vaccinia virus or a T7 promoter were constructed, allowing for constitutive or conditional expression, respectively, of the foreign protein.     

Plaque size phenotype as a selectable marker to generate vaccinia virus recombinants.

In this report, we provide a new method for selection of vaccinia virus recombinants expressing foreign genes. The method is based on the use of the gene encoding the viral 14,000-molecular-weight envelope protein that rescues the small-plaque-size phenotype of a vaccinia virus variant to large-plaque-size virus. Selection of recombinants is easily obtained after visual inspection of large viral plaques.     

Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants.

The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest.     

Construction of fowlpox virus vectors with intergenic insertions: expression of the beta-galactosidase gene and the measles virus fusion gene.

A DNA fragment from fowlpox virus cloned on a plasmid vector was modified to contain foreign DNA inserts within an intergenic region. In a first step, a 32-base-pair intergenic region from the fowlpox virus genome corresponding to the position of the thymidine kinase locus in the vaccinia virus genome was enlarged to 55 base pairs by site-directed mutagenesis. A unique restriction endonuclease site introduced upstream of the intergenic region was then used to insert various foreign DNA fragments. The lacZ gene encoding beta-galactosidase and the measles virus gene encoding the fusion protein were positioned downstream of two vaccinia virus p7.5 promoter elements in either a direct repeat or inverted repeat orientation. Foreign DNA inserts contained within the fowlpox virus sequence were transferred to the viral genome by homologous recombination occurring in cells infected with a fowlpox virus temperature-sensitive mutant and transfected with both wild-type viral DNA and plasmid DNA. Recombinant viruses were selected for the expression of beta-galactosidase activity by screening for blue plaques in the presence of a chromogenic substrate. Stable recombinants expressing both the lacZ gene and the unselected measles gene were obtained when the p7.5 promoter was present as an inverted repeat. However, when the p7.5 promoter was in the direct repeat orientation, viral recombinants which initially expressed both gene inserts readily deleted the lacZ gene flanked by the promoter repeat. The methods described enable precise insertion and deletion of foreign genes in the fowlpox virus genome and could be applied to other intergenic regions of the same virus as well as other poxviruses.     

Transient host range selection for genetic engineering of modified vaccinia virus Ankara

Recombinant vaccinia viruses are extremely valuable tools for research in molecular biology and immunology. The extension of vaccinia vector technology to replication-deficient and safety-tested virus strains such as modified vaccinia virus Ankara (MVA) have made this versatile eukaryotic expression system even more attractive for basic and clinical research. Here, we report on easily obtaining recombinant MVA using stringent growth selection on rabbit kidney RK-13 cells. We describe the construction and use of new MVA vector plasmids that carry an expression cassette of the vaccinia virus host range gene, K1L, as a transient selectable marker. These plasmids allow either stable insertion of additional recombinant genes into the MVA genome or precisely targeted mutagenesis of MVA genomic sequences. Repetitive DNA sequences flanking the K1L gene were designed to remove the marker gene from the viral genome by homologous recombination under nonselective growth conditions. The convenience of this new selection technique is demonstrated by isolating MVA recombinants that produce green fluorescent protein and by generating MVA deletion mutants.     

Attenuated modified vaccinia virus Ankara can be used as an immunizing agent under conditions of preexisting immunity to the vector

A problem associated with the use of vaccinia virus recombinants as vaccines is the existence of a large human population with preexisting immunity to the vector. Here we showed that after a booster with attenuated recombinant modified vaccinia virus Ankara (rMVA), higher humoral and cellular immune responses to foreign antigens (human immunodeficiency virus type 1 Env and beta-galactosidase) were found in mice preimmunized with rMVA than in mice primed with the virulent Western Reserve strain and boosted with rMVA. This enhancement correlated with higher levels of expression of foreign antigens after the booster.     

Defective Vaccinia Virus as a Biologically Safe Tool for the Overproduction of Recombinant Human Secretory Proteins

We have described recently the construction of a defective vaccinia virus (VV) lacking the essential D4R open reading frame and have shown furthermore the selection of a complementing cell line providing the essential D4R gene product. The D4R gene belongs to the group of early transcribed vaccinia genes preventing a virus defective in D4R from entering into the intermediate and late phase of replication under noncomplementing conditions. Here we show that this property, which is unique among the group of so called nonreplicating poxviruses, is helpful for the production of (secretable) recombinant human proteins. Recombinant VV based on a D4R-defective parental strain expressing cDNAs coding for the human blood coagulation factors VII and XI produced significantly more recombinant protein than the corresponding recombinants based on wild-type VV. Moreover, the complementing cell line RK-D4R-44.20 was a more effective production cell system for both vD4 and wild-type VV recombinants compared to wild-type RK-13 cells. Surprisingly, recombinant human factor VII was more efficiently produced with the defective vaccinia recombinant even under noncomplementing conditions, suggesting that persistence of the early phase of vaccinia replication in combination with a delayed host shutoff is advantageous for the overproduction of certain recombinant proteins using the VV expression system.     

人癌胚抗原-重组痘苗病毒的构建和制备

痘苗病毒的基因组庞大,结构复杂而特殊,不可能将外源基因直接插入它的基因组,必须利用一种特殊的痘苗病毒质粒,才能构建成功重组痘苗病毒．在分析了痘苗病毒质粒ｐＪ１２０〔含有我国天花疫苗－痘苗病毒天坛株７６１的启动子和胸苷激酶（ｔｈｙｍｉｄｉｎｅｋｉｎａｓｅ,简称ＴＫ基因）,及含有人癌胚抗原（ｃａｒｃｉｎｏｅｍｂｒｙｎｉｃａｎｔｉｇｅｎ,简称ＣＥＡ）ｃＤＮＡ全序列的质粒ｐ９１０２３Ｂ－ｃｅａ－１７结构的基础上,设计出三步法构建了重组疫苗病毒质粒ｐＪ－ＣＥＡ．经酶切及ＰＣＲ鉴定ｐＪ－ＣＥＡ中ＣＥＡｃＤ－ＮＡ的存在,进一步用同源重组方法构建了表达人ＣＥＡ的重组痘苗病毒,并以人体成纤维细胞作为宿主细胞,对ＣＥＡ－重组痘苗病毒进行了大量培养．再次证实痘苗病毒是良好的真核表达载体,可以高效而准确地表达细胞膜糖蛋白ＣＥＡ．     

Isolation of vaccinia MVA recombinants using the viral F13L gene as the selective marker

Modified vaccinia Ankara (MVA) is a highly attenuated vaccine vector that has an excellent vaccine safety record. Also, as a eukaryotic gene expression vector, MVA can be used in a biosafety level 1 setup, in contrast to more virulent vaccinia virus strains. Isolation of recombinant MVA involves repeated plaquing of the virus and is burdensome because virus plaques are slow to develop and difficult to recognize. To facilitate the generation of MVA recombinants, we have developed a cloning system for MVA based on the selection of the viral F13L gene. Deletion of F13L in MVA produced a small plaque phenotype and a reduction in extracellular virus formation, indicating a severe block in cell-to-cell spread. When using the F13L knockout virus as the parental virus, reintroduction of the F13L gene in the original locus was used as an efficient selection for the isolation of virus recombinants. The selection procedure can be done entirely in the permissive baby hamster kidney (BHK)-21 cell line, does not require plaque isolation, and rendered close to 100% recombinant virus.     

Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge.

cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge.     

Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques.

We constructed a plasmid coexpression vector that directs the insertion of a foreign gene of interest together with the Escherichia coli beta-galactosidase (beta gal) gene into the thymidine kinase (TK) locus of the vaccinia virus genome. Tissue culture cells that had been infected with vaccinia virus were transfected with a plasmid vector containing a foreign gene. TK- recombinants could be selected by a plaque assay on TK- cells in the presence of 5-bromodeoxyuridine and distinguished from spontaneous TK- mutants by the addition of a beta-gal indicator to the agarose overlay. Plaques that expressed beta-gal stained dark blue within several hours at 37 degrees C. Alternatively, TK- selection could be eliminated, and recombinant plaques could be readily identified solely by their blue color. The reverse procedure, in which the starting virus expresses beta-gal (i.e., forms blue plaques) and the desired recombinant has deleted the entire beta-gal gene (i.e., forms white plaques), is another alternative. Each protocol was tested by constructing vaccinia virus recombinants that express hepatitis B virus surface antigen.     

A selectable and excisable marker system for the rapid creation of recombinant poxviruses

Background

Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a number of vector candidates for the treatment of a variety of human diseases. The development of recombinant poxviruses often involves the genomic insertion of a selectable marker for purification and selection purposes. The use of marker genes however inevitably results in a vector that contains unwanted genetic information of no therapeutic value.

Methodology/Principal Findings

Here we describe an improved strategy that allows for the creation of marker-free recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM) system incorporates a unique fusion marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent removal of the marker. We have defined and characterized this new methodological tool by insertion of a foreign gene into vaccinia virus, with the subsequent removal of the selectable marker. We then analyzed the importance of loxP orientation during Cre recombination, and show that the SEM system can be used to introduce site-specific deletions or inversions into the viral genome. Finally, we demonstrate that the SEM strategy is amenable to other poxviruses, as demonstrated here with the creation of an ectromelia virus recombinant lacking the EVM002 gene.

Conclusion/Significance

The system described here thus provides a faster, simpler and more efficient means to create clinic-ready recombinant poxviruses for therapeutic gene therapy applications.     

Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors.

Mycophenolic acid, an inhibitor of purine metabolism, was shown to block the replication of vaccinia virus in normal cell lines. This observation led to the development of a dominant one-step plaque selection system, based on expression of the Escherichia coli gpt gene, for the isolation of recombinant vaccinia viruses. Synthesis of xanthine-guanine phosphoribosyltransferase enabled only the recombinant viruses to form large plaques in a selective medium containing mycophenolic acid, xanthine, and hypoxanthine. To utilize the selection system efficiently, we constructed a series of plasmids that contain the E. coli gpt gene and allow insertion of foreign genes into multiple unique restriction endonuclease sites in all three reading frames between the translation initiation codon of a strong late promoter and synthetic translation termination sequences. The selection-expression cassette is flanked by vaccinia virus DNA that directs homologous recombination into the virus genome. The new vectors allow high-level expression of complete or partial open reading frames and rapid construction of recombinant viruses by facilitating the cloning steps and by simplifying their isolation. The system was tested by cloning the E. coli beta-galactosidase gene; in 24 h, this enzyme accounted for approximately 3.5% of the total infected-cell protein.     

The live vector approach—viruses

Recombinant DNA technology has made it possible to express foreign genes in viruses and bacteria. This has led to the idea of engineering live attenuated vaccines to express protective antigens from foreign pathogens. Vaccinia virus, the smallpox vaccine, has been used to ploneer this approach. Several hundred recombinants have been recorded in the literature and many of them have been shown to protect animals against challenge with the appropriate pathogen. Although mixed results have been obtained with experiments in humans and primates, and there is some concern over complications associated with vaccination, recent advances in our under-standing of the basic biology of vaccinia virus should allow these difficulties to be overcome. Whatever the final outcome of proposals to use vaccinia recombinants in humans, vaccinia has proved to be a valuable general purpose expression system and is particularly useful for studying cellular immunity. The success of field trials with a vaccinia recombinant expressing the rabies virus glycoprotein may lead to widespread use of vaccinia recombinants as animal vaccines. Therefore it seems likely that vaccinia recombinants will continue to play a useful part in the development of new vaccines for infectious disease.This paper was presented at the IUMS Symposium on New Developments in Diagnosis and Control of Infectious Diseases held in conjunction with the Eighth International Congress of Virology, Berlin, Germany, 24–31 August 1990.     

Fowlpox virus recombinants expressing the envelope glycoprotein of an avian reticuloendotheliosis retrovirus induce neutralizing antibodies and reduce viremia in chickens.

Eight stable fowlpox virus (FPV) recombinants which express the envelope glycoprotein of the spleen necrosis virus (SNV) strain of reticuloendotheliosis virus (REV), an avian retrovirus, were constructed. These recombinants differ in the genomic location of the inserted genes, in the orientation of the insert relative to flanking viral sequences, and in the promoter used to drive expression of the env gene. Of these variables, promoter strength seems to be the most crucial. The P7.5 promoter of vaccinia virus, which is commonly used in the construction of both vaccinia virus and FPV recombinants, resulted in lower levels of expression of the envelope antigen in infected chicken cells compared with a strong synthetic promoter, as determined by immunofluorescence and enzyme-linked immunosorbent assay. Two peptides encoded by the env gene, the 21-kDa transmembrane peptide and a 62-kDa precursor, were detected by immunoprecipitation of labeled proteins from cells infected with recombinant FPVs, using monoclonal antibodies against REV. These peptides comigrated with those precipitated from REV-infected cells. One of the recombinants (f29R-SNenv) was used for vaccination of 1-day-old chickens. Vaccinated chicks developed neutralizing antibodies to SNV more rapidly than did unvaccinated controls following SNV challenge and were protected against both viremia and the SNV-induced runting syndrome.     

Vaccinia and ectromelia recombinant viruses, causing an infection, characteristic for ectromelia, in mice

Ten recombinants between the viruses of vaccinia and ectromelia were isolated that cause the ectromelia virus specific lesions in mice. The structure of recombinant viral genomes, the efficiency of viral propagation in mice, the nature of lesions induced by viruses have been studied. Eight of obtained recombinants have a DNA insertion originating from the right end of ectromelia viral genome, nine recombinants have an insertion originating from the left end, seven recombinants possess both insertions. The latter recombinants have more pronounced pathogenicity for mice. Both revealed regions are supposed to define the specific pathogenicity of ectromelia virus for mice.