A general method for the construction of recombinant vaccinia viruses expressing multiple foreign genes

Plasmid vectors with multiple cloning sites adjacent to a vaccinia virus (VV) promoter were constructed and used to insert a protein coding sequence and a dominant selectable marker into a non-essential region of the VV genome. Recombinant viruses, selected on the basis of expression of the herpes simplex virus (HSV) thymidine kinase gene (tk), were shown to express in infected cells the model gene product, murine major histocompatibility complex (MHC) antigen H-2Kd, by cell-surface binding of antibody and by MHC-restricted recognition by cytotoxic T lymphocytes. Double recombinant VVs with insertions at two sites (in the VV tk gene and in the VV HindIII-F region) were constructed and shown to express influenza A/PR/8/34 haemagglutinin and H-2Kd antigen in addition to the HSV tk gene. The plasmids described allow the construction of recombinant VV expressing two genes of interest under the control of the same VV promoter. Such recombinant VVs can be used to study the interaction of immunologically important antigens simultaneously expressed.     

构建重组杆状病毒的几种新方法

昆虫杆状病毒作为高效的表达载体,现已广泛地用于各种外源基因的表达.但是,用传统的方法构建重组杆状病毒,存在着重组率低,纯化难及耗时长等缺点,围绕如何快速、简便、高效地构建重组杆状病毒,近几年来人们进行了一些重大的改进,包括使病毒DNA线状化以提高重组病毒的比例;在体外进行重组;同源重组和重组病毒的纯化与筛选在酵母和大肠杆菌中一次完成;使重组病毒可以形成多角体等,从而从根本上改变了传统方法中的不足;文章着重介绍了这几种新的改进方法.     

Stable Antigen Is Most Effective for Eliciting CD8+ T-Cell Responses after DNA Vaccination and Infection with Recombinant Vaccinia Virus In Vivo

The induction of strong CD8(+) T-cell responses against infectious diseases and cancer has remained a major challenge. Depending on the source of antigen and the infectious agent, priming of CD8(+) T cells requires direct and/or cross-presentation of antigenic peptides on major histocompatibility complex (MHC) class I molecules by professional antigen-presenting cells (APCs). However, both pathways show distinct preferences concerning antigen stability. Whereas direct presentation was shown to efficiently present peptides derived from rapidly degraded proteins, cross-presentation is dependent on long-lived antigen species. In this report, we analyzed the role of antigen stability on DNA vaccination and recombinant vaccinia virus (VV) infection using altered versions of the same antigen. The long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) can be targeted for degradation by N-terminal fusion to ubiquitin or, as we show here, to the ubiquitin-like modifier FAT10. Direct presentation by cells either transfected with NP-encoding plasmids or infected with recombinant VV in vitro was enhanced in the presence of short-lived antigens. In vivo, however, the highest induction of NP-specific CD8(+) T-cell responses was achieved in the presence of long-lived NP. Our experiments provide evidence that targeting antigens for proteasomal degradation does not improve the immunogenicity of DNA vaccines and recombinant VVs. Rather, it is the long-lived antigen that is superior for the efficient activation of MHC class I-restricted immune responses in vivo. Hence, our results suggest a dominant role for antigen cross-priming in DNA vaccination and recombinant VV infection.     

Effects and virulences of recombinant vaccinia viruses derived from attenuated strains that express the human T-cell leukemia virus type I envelope gene.

We constructed recombinant vaccinia viruses (RVVs) that expressed human T-cell leukemia virus type I (HTLV-I) envelope glycoproteins by using attenuated vaccinia viruses (VVs) which have much lower neurovirulence than the WR strain that is extensively used as a vector. The RVV produced from the LC16mO strain, one of the attenuated VVs, elicited a high titer of anti-HTLV-I antibody in rabbits and protected them against HTLV-I infection. The env gene was inserted into the VV hemagglutinin gene. The resultant inactivation of the hemagglutinin gene led to the attenuation of VVs, but the extent of their attenuation depended on the VV strain. The propagation of LC16mO and its RVV in rabbit brain was poorer than that of LO-1, a cloned derivative of Lister strain, and its RVV, although LC16mO replicated in other organs better than did LO-1. Taken together, these results suggest that LC16mO is a good candidate as a vector for vaccination of humans.     

A PCR-based method for manipulation of the vaccinia virus genome that eliminates the need for cloning.

A general method is described for altering specific genes of vaccinia virus (VV). We demonstrate and evaluate the procedure by gene inactivation, using a dominant selectable marker in conjunction with recombinant polymerase chain reaction (PCR). Primers based on the sequence of the target gene enable amplification of flanking arms and their subsequent attachment to the gpt cassette that confers resistance to mycophenolic acid. Linear PCR constructs are transfected into cells infected with wild-type vaccinia virus. Mutant viruses with gpt inserted into the target gene by homologous recombination are then selected by growth in the presence of MPA. This technique was applied to the vaccinia virus thymidine kinase gene and compared to the traditional method of constructing gpt-containing plasmids by cloning. The PCR scheme was found to be highly efficient and could theoretically be used to insert any foreign DNA element into any nonessential target gene for which partial or complete sequence information is available. The procedure can potentially be used for a wide variety of genetic modifications, including the insertion of foreign genes, with poxviruses and other DNA viruses. Genomes of microorganisms, such as bacteria and yeast that can be transformed with linear DNA, are also candidates for manipulation by this methodology.     

Expression vectors for enzyme restriction- and ligation-independent cloning for producing recombinant His-fusion proteins

In this work we have constructed two novel expression vectors, designated as pURI2 and pURI3, which enable parallel cloning of a given target gene for producing recombinant His-fusion proteins. The vectors were created using the well-known pT7-7 and pIN-III-A3 plasmids as their template. The same DNA fragment containing the His-tag, enterokinase cleavage site, and a NotI unique site, as well as keeping the HindIII unique restriction site, was introduced in both vectors. These vectors have been designed to avoid the enzyme restriction and ligation steps during the cloning. The unique NotI site was introduced to facilitate the selection of the adequate recombinant plasmid. Parallel cloning of the same polymerase chain reaction fragment can be carried out since both vectors shared the same leader sequence. The described strategy avoids tedious cloning efforts into different expression vectors and represents a highly efficient means of cloning. To validate our vectors, we have cloned one target gene in both vectors and used expression and purification techniques to obtain the recombinant target protein. We herein show that both vectors function effectively in all the required experimental steps-cloning, expression, purification, and cleavage.     

DNA cassettes containing the origin of transfer (oriT) of two broad-host-range transfer systems.

Plasmid constructs are described that carry retrievable DNA cassettes containing the origin of transfer region (oriT) from two broad-host-range plasmids. Restriction of these high copy number plasmids with any one of a variety of enzymes yields a linear DNA fragment of convenient size containing the oriT region of either pCUI or RK2. This DNA can be ligated into any vector or recombinant plasmid containing a compatible enzyme site and can be easily identified by size on an agarose gel. Any plasmid can therefore be mobilized using a number of helper strains or conjugative plasmids derived from the parental plasmids. In addition, the cassettes can be used for a variety of genetic manipulations including "selectable" linker mutagenesis.     

Multiple-cloning-site plasmids for the rapid construction of recombinant poxviruses

Plasmid vectors containing multiple cloning sites suitable for the rapid insertion of protein-coding sequences into poxviruses have been constructed. They are based on pUC plasmids and carry the thymidine kinase (TK) gene of vaccinia virus interrupted by a vaccinia virus promoter. Six unique restriction enzyme sites (BamHI, SalI/HincII, PstI, HindIII, EcoRI), located within 40 bp of vaccinia virus promoters transposed from the HindIII-F or HindIII-C fragment of the vaccinia virus genome, allow rapid insertion of foreign-protein-coding sequences into these plasmids. Such plasmids can be used to construct recombinant poxviruses expressing foreign proteins using marker-rescue recombination techniques and selection for TK negative viruses. Vaccinia viruses expressing the haemagglutinin (HA) gene of swine influenza virus, A/NJ/11/76 (H1N1), have been constructed.     

Construction of a novel set of transfer vectors to study vaccinia virus replication and foreign gene expression

Vaccinia virus (VV) is a useful expression vector for many laboratory applications. To date, approximately 60% ( approximately 120) of the VV genes remain uncharacterized. The thought of smallpox being used as a biological weapon has gained attention. In light of this, it is imperative that we continue to study the basic replication of VV, a poxvirus that is closely related to smallpox. A set of plasmid vectors were constructed to generate gene deletions (pZIPPY-NEO/GUS) in VV or for foreign gene expression (pBR-EXPRESS). The vectors contain the Escherichia coli neomycin resistance (neo) and beta-glucuronidase (gusA) genes as selectable markers to facilitate isolation of recombinant viruses. These are the first transfer vectors to use a neo/gusA selection system. We used these vectors to successfully generate a recombinant D9R deletion mutant of VV and to express E. coli lacZ gene. Results indicate that both vectors are highly suited for their designed purpose.     

Use of electroporation to construct isogenic mutants of Haemophilus ducreyi.

Little is known about the genetics of Haemophilus ducreyi, the etiologic agent of chancroid. To develop a method for constructing isogenic mutants of this organism that could be utilized in pathogenesis-related studies, electroporation techniques were evaluated as a means of introducing DNA into this organism. Electroporation of the plasmid shuttle vector pLS88 into H. ducreyi yielded approximately 10(6) antibiotic-resistant transformants per microgram of plasmid DNA. Studies of the feasibility of moving mutated genes into H. ducreyi were initiated by using NotI linker insertion and mini-Tn10kan mutagenesis techniques to introduce insertion mutations into cloned H. ducreyi genes encoding cell envelope antigens. In the former case, a gene encoding chloramphenicol acetyltransferase was then inserted into the NotI linker site created in the cloned H. ducreyi gene. The recombinant Escherichia coli strains containing these mutated plasmids no longer expressed the homologous H. ducreyi cell envelope antigens, as evidenced by their lack of reactivity with monoclonal antibody probes for these H. ducreyi proteins. Subsequent electroporation of both circular and linearized forms of plasmids carrying these mutated H. ducreyi genes into the homologous wild-type strain of H. ducreyi yielded antibiotic-resistant transformants which also lacked reactivity with the cell envelope antigen-specific monoclonal antibodies. Southern blot analysis confirmed that homologous recombination had occurred in these monoclonal antibody-unreactive transformants, resulting in the replacement of the wild-type allele with the mutated allele. Allelic exchange was most efficient when linear DNA molecules were used for electroporation. These results indicate that electroporation methods can be utilized to construct isogenic mutants of H. ducreyi.     

Isolation of cis-acting vaccinia virus DNA fragments promoting the expression of herpes simplex virus thymidine kinase by recombinant viruses.

Recombinant TK- vaccinia viruses containing the pBR322 sequence inserted in either orientation within the coding sequence of the viral thymidine kinase gene were constructed. They were characterized by genomic analysis, hybridization studies, reversion to wild-type virus by in vivo recombination, and rescue from their genomes of plasmids which contained all or parts of the pBR322 sequence. TK- cells were infected with one of these recombinant viruses and then transfected with pools of chimeric plasmids composed of a cloned herpes simplex virus thymidine kinase gene which contained upstream inserts of different vaccinia DNA fragments prepared by restriction or sonication. Recombination between homologous pBR322 sequences within infected cells generated selectable recombinant viruses in which expression of the herpes simplex virus thymidine kinase gene was promoted by the upstream vaccinia insert. These viruses were characterized by genomic analysis, hybridization, and in vivo or in vitro phosphorylation of (5-[125I]deoxycytidine as a specific assay for the expressed herpes simplex virus thymidine kinase. Vaccinia DNA inserts were isolated conveniently for transfer to bacteria by rescuing appropriate plasmids from the genome of recombinant viruses. The sequence of 100 nucleotides adjacent to the upstream region of the herpes simplex virus gene was determined in nine different inserts measuring 0.17 to 1.07 kilobase pairs.     

Genetic requirements for homologous recombination in Autographa californica nucleopolyhedrovirus

It is known that baculovirus infection promotes high-frequency recombination between its genomes and plasmid DNA during the construction of recombinant viruses for foreign gene expression. However, little is known about the viral genes necessary to promote homologous recombination (HR). We developed an assay to identify viral genes that are necessary to stimulate HR. In this assay, we used two plasmids containing extensive sequence homology that yielded a visible and quantifiable phenotype if HR occurred. The plasmids contained the green fluorescent protein gene (gfp) that was mutated at either the N or the C terminus and a viral origin of DNA replication. When the plasmids containing these mutant gfp genes were transfected into insect cells alone or together, few green fluorescent protein (GFP)-positive cells were observed, confirming that the host cell machinery alone was not able to promote high levels of HR. However, if viral DNA or viral genes involved in DNA replication were cotransfected into cells along with the mutant gfp-containing plasmids, a dramatic increase in GFP-positive cells was observed. The viral genes ie-1, ie-2, lef-7, and p35 were found to be important for efficient HR in the presence of all other DNA replication genes. However, ie-1 and ie-2 were sufficient to promote HR in the absence of other viral genes. Recombination substrates lacking a viral origin of replication had similar genetic requirements for recombination but were less dependent on ie-1. Interestingly, even though HR was stimulated by the presence of a viral origin of DNA replication, virally stimulated HR could proceed in the presence of the DNA synthesis inhibitor aphidicolin.     

Breakage--reunion and copy choice mechanisms of recombination between short homologous sequences.

To study recombination between short homologous sequences in Escherichia coli we constructed plasmids composed of the pBR322 replicon, M13 replication origin and a recombination unit inserted within and inactivating a gene encoding chloramphenicol resistance. The unit was composed of short direct repeats (9, 18 or 27 bp) which flanked inverted repeats (0, 8 or 308 bp) and a gene encoding kanamycin resistance. Recombination between direct repeats restored a functional chloramphenicol resistance gene, and could be detected by a simple phenotype test. The plasmids replicated in a double-stranded form, using the pBR322 replicon, and generated single-stranded DNA when the M13 replication origin was activated. The frequency of chloramphenicol-resistant cells was low (10(-8)-10(-4] when no single-stranded DNA was synthesized but increased greatly (to 100%) after induction of single-stranded DNA synthesis. Recombination between 9 bp direct repeats entailed no transfer of DNA from parental to recombinant plasmids, whereas recombination between 18 or 27 bp repeats entailed massive transfer. The presence or length of inverted repeats did not alter the pattern of DNA transfer. From these results we propose that direct repeats of 9 bp recombine by a copy choice process, while those greater than or equal to 18 bp can recombine by a breakage-reunion process. Genome rearrangements detected in many organisms often occur by recombination between sequences less than 18 bp, which suggests that they may result from copy choice recombination.     

Directional and direct cloning strategy for high-throughput generation of recombinant baculoviruses

The baculovirus expression vector system (BEVS) has become one of the most widely used systems for routine protein expression. We have developed an improved strategy to clone foreign genes directionally and directly into the baculovirus genome vector via a one-step procedure to generate recombinant viruses in a week. In this work, we constructed a host strain Escherichia coli DH10BacHB1.1, which contains the modified baculovirus shuttle genome vector pHBMBacmid1.1 for the cloning vector. The treated PCR products of foreign genes were ligated with the Bsu36I-digested vector. Then Spodoptera frugiperda (Sf9) cells were transfected directly with the ligation mixture. Using this method, the DsRed fluorescence protein and mannanase genes have been cloned in the baculovirus genome and expressed in the Sf9 cells. This strategy not only provides a means for high-throughput construction of recombinant baculoviruses, but also offers an idea of constructing other large plasmids and DNA virus-based expression vectors.     

Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression.

A method is described for the production of recombinant adeno-associated virus (AAV) stocks that contain no detectable wild-type helper AAV. The recombinant viruses contained only the terminal 191 nucleotides of the AAV chromosome bracketing a nonviral marker gene. trans-Acting AAV functions were provided by a helper DNA in which the terminal 191 nucleotides of the AAV chromosome were substituted with adenovirus terminal sequences. Although the helper DNA did not appear to replicate, it expressed AAV functions at a substantially higher level than did DNA molecules that contained neither AAV nor adenovirus termini. Since the recombinant viruses with AAV termini contained no sequence homology to the helper DNA, no wild-type AAV was generated by homologous recombination within infected cells. Since the terminal region of the AAV chromosome is required for replication and encapsidation, only recombinant DNAs were amplified and packaged into AAV virions. When human cells were infected at a high multiplicity with a recombinant virus carrying a drug resistance marker gene, approximately 70% of the infected cells gave rise to colonies stably expressing the marker. The recombinant virus gene was then used to generate drug-resistant human cell lines subsequent to infection. These cells contained stably integrated copies of the recombinant viral DNA which could be excised, replicated, and encapsidated by infection with wild-type AAV plus adenovirus. Thus, AAV gene expression is not required for normal integration of an infecting DNA containing AAV termini.     

Molecular aspects of gene transfer and foreign DNA acquisition in prokaryotes with regard to safety issues

Horizontal gene transfer (HGT) is part of prokaryotic life style and a major factor in evolution. In principle, any combinations of genetic information can be explored via HGT for effects on prokaryotic fitness. HGT mechanisms including transformation, conjugation, transduction, and variations of these plus the role of mobile genetic elements are summarized with emphasis on their potential to translocate foreign DNA. Complementarily, we discuss how foreign DNA can be integrated in recipient cells through homologous recombination (HR), illegitimate recombination (IR), and combinations of both, site-specific recombination, and the reconstitution of plasmids. Integration of foreign DNA by IR is very low, and combinations of IR with HR provide intermediate levels compared to the high frequency of homologous integration. A survey of studies on potential HGT from various transgenic plants indicates very rare transfer of foreign DNA. At the same time, in prokaryotic habitats, genes introduced into transgenic plants are abundant, and natural HGT frequencies are relatively high providing a greater chance for direct transfer instead of via transgenic plants. It is concluded that potential HGT from transgenic plants to prokaryotes is not expected to influence prokaryotic evolution and to have negative effects on human or animal health and the environment.     

A new method for constructing NotI linking and boundary libraries using a restriction trapper.

We have developed a novel method for constructing NotI linking and boundary libraries using a modified "solid-supported ligation primer" (restriction trapper). The restriction trapper could be used to purify the DNA fragments with a specific restriction enzyme cutting site(s) at their ends. The method uses a ligation and recutting reaction with double-stranded DNA ends of a hairpin-shaped oligolinker which is connected covalently to the surface of the latex beads. Selectivity is based on the specificity of the restriction enzyme for its recognition site, resulting in efficient purification. We applied this technique to the construction of high-quality NotI linking and NotI boundary libraries, which contain almost all the NotI sites of the genome and, in addition, are free of illegitimately ligated clones.     

A new cloning system for Bacillus subtilis comprising elements of phage, plasmid and transposon vectors

A new cloning system for Bacillus subtilis was devised which makes use of a combination of Tn917-containing phage SP beta derivatives and Tn917-containing Escherichia coli-B. subtilis shuttle plasmids. This system allows the initial cloning of genes in single copy, via 'prophage transformation', with a selection for complementation of mutational defects in B. subtilis hosts and permits subsequent transfer of the cloned material by homologous recombination to low-copy and high-copy vectors that replicate in both B. subtilis and E. coli. Because cloned sequences are adjacent to pB322-derived DNA in the recombinant phages, inserts can also be 'rescued' directly from the phage DNA after digestion with appropriate restriction enzymes, circularization of the fragments by ligation and transformation of an E. coli recipient. Two genomic libraries of B. subtilis chromosomal Sau3A-generated partial-digest fragments in the size ranges of 5-8 kb and 8-10 kb were constructed and screened for the complementation of mutations aroI906, cysA14, dal-1, glyB133, metC3, purA16, purB33, thrA5, trpC2 and recE4. In all cases, specialized transducing phages carrying inserts that complemented the selected markers were recovered. Inserts complementing the dal-1 and trpC2 mutations could be transferred from recombinant phages to Tn917-containing plasmids by homologous recombination without in vitro subcloning. Another insert complementing the purB33 mutation was rescued directly into E. coli from a recombinant phage DNA.     

Marker rescue of adeno-associated virus (AAV) capsid mutants: a novel approach for chimeric AAV production

Marker rescue, the restoration of gene function by replacement of a defective gene with a normal one by recombination, has been utilized to produce novel adeno-associated virus (AAV) vectors. AAV serotype 2 (AAV2) clones containing wild-type terminal repeats, an intact rep gene, and a mutated cap gene, served as the template for marker rescue. When transfected alone in 293 cells, these AAV2 mutant plasmids produced noninfectious AAV virions that could not bind heparin sulfate after infection with adenovirus dl309 helper virus. However, the mutation in the cap gene was corrected after cotransfection with AAV serotype 3 (AAV3) capsid DNA fragments, resulting in the production of AAV2/AAV3 chimeric viruses. The cap genes from several independent marker rescue experiments were PCR amplified, cloned, and then sequenced. Sequencing results confirmed not only that homologous recombination occurred but, more importantly, that a mixed population of AAV chimeras carrying 16 to 2,200 bp throughout different regions of the type 3 cap gene were generated in a single marker rescue experiment. A 100% correlation was observed between infectivity and the ability of the chimeric virus to bind heparin sulfate. In addition, many of the AAV2/AAV3 chimeras examined exhibited differences at both the nucleotide and amino acid levels, suggesting that these chimeras may also exhibit unique infectious properties. Furthermore, AAV helper plasmids containing these chimeric cap genes were able to function in the triple transfection method to generate recombinant AAV. Together, the results suggest that DNA from other AAV serotypes can rescue AAV capsid mutants and that marker rescue may be a powerful, yet simple, technique to map, as well as develop, chimeric AAV capsids that display different serotype-specific properties.