Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes.

A novel expression system based on coinfection of cells with two recombinant vaccinia viruses has been developed. One recombinant vaccinia virus contained the bacteriophage T7 RNA polymerase gene under control of a vaccinia virus promoter. The second recombinant vaccinia virus contained a target gene of choice flanked by bacteriophage T7 promoter and termination sequences. Maximum expression of the target gene occurred when cells were infected with 10 PFU of each recombinant virus. Although T7 RNA polymerase synthesis began shortly after infection, the target gene was not expressed until late times and was largely inhibited when DNA replication was blocked. Target gene transcripts were analyzed by agarose gel electrophoresis and had the predicted size. With this system, Escherichia coli beta-galactosidase, hepatitis B virus surface antigen, and human immunodeficiency virus envelope proteins were made. In each case, the level of synthesis was greater than had previously been obtained with the more conventional recombinant vaccinia virus expression system.

Structure and stability of mRNA synthesized by vaccinia virus-encoded bacteriophage T7 RNA polymerase in mammalian cells. Importance of the 5' untranslated leader.

We have analyzed the structure and stability of RNA synthesized by bacteriophage T7 RNA polymerase in mammalian cells. The T7 polymerase, expressed by a recombinant vaccinia virus, transcribed the Escherichia coli lacZ gene flanked by T7 promoter and terminator signals. The lacZ gene cassette was introduced into infected cells within either a transfected plasmid or a second recombinant vaccinia virus. The T7-lacZ transcripts, which had a half-life of approximately 75 minutes, represented approximately 30% of total cytoplasmic RNA after a 24 hour period. The latter estimation indicated a disparity between the levels of lacZ RNA and beta-galactosidase synthesis. Analysis of the T7 transcripts indicated that they were initiated correctly but that only 5 to 10% contained terminal cap structures, providing an explanation for the low translatability of the RNA. Since the 5' end of the T7 transcripts can form a stem-loop structure that might interfere with capping by vaccinia virus RNA guanylyltransferase, as well as ribosome binding and scanning, a similar vector lacking such sequences was constructed. In vitro experiments demonstrated that T7 RNA polymerase transcribed both templates with similar efficiency and that the RNA lacking the potential to form the stem-loop was capped more rapidly by the purified vaccinia virus enzyme. Nevertheless, when the stem-loop was removed, beta-galactosidase was not expressed in infected cells; moreover, no T7 transcripts could be detected, suggesting that the RNA was not made or more likely was degraded during or shortly after synthesis. There is previous evidence that vaccinia virus RNA guanylyltransferase is associated with the viral transcription complex, thereby allowing RNA synthesis and capping to occur concurrently. We suggest that a lack of coupling between the vaccinia viral RNA guanylyltransferase and bacteriophage T7 RNA polymerase delays capping of T7 transcripts and that, under these conditions, the 5'-terminal double-stranded stem is required to stabilize the nascent RNA against degradation. Although deletion of the 3' palindromic sequence specifying T7 transcriptional termination from the expression cassette resulted in RNA of more heterogeneous lengths, neither the apparent turnover rate nor translation of the RNAs was diminished appreciably.

Maturation of human immunodeficiency virus particles assembled from the gag precursor protein requires in situ processing by gag-pol protease.

The vaccinia virus expression system was used to determine the role of human immunodeficiency virus type 1 (HIV-1) protease in viral morphogenesis and maturation. The unprocessed p55 gag precursor polyprotein alone was assembled to form HIV-1 particles which budded from cells. The particles were spherical and immature, containing an electron-dense shell in the particle submembrane; there was no evidence of core formation. Expression of both gag and pol proteins from a recombinant containing the complete gag-pol coding sequences resulted in intracellular processing of gag-pol proteins and the production of mature particles with electron-dense cores characteristic of wild-type HIV virions. To ascertain the role of protein processing in particle maturation, the pol ORF in the gag-pol recombinant was truncated to limit expression of the pol gene to the protease domain. With this recombinant expressing p55 gag and protease, intracellular processing was observed. Some of the resultant particles were partially mature and contained processed gag protein subunits. In contrast, particle maturation was not observed when the HIV-1 protease and p55 gag were coexpressed from separate recombinants, despite evidence of intracellular gag processing. These findings suggest that HIV-1 protease must be an integral component of the full-length gag-pol precursor for optimal processing and virion maturation.

Host range restricted, non-replicating vaccinia virus vectors as vaccine candidates.

Three model systems were used to demonstrate the immunogenicity of highly attenuated and replication-defective recombinant MVA. (1) Intramuscular inoculation of MVA-IN-Fha/np induced humoral and cell-mediated immune responses in mice and protectively immunized them against a lethal respiratory challenge with influenza virus. Intranasal vaccination was also protective, although higher doses were needed. (2) In rhesus macaques, an immunization scheme involving intramuscular injections of MVA-SIVenv/gag/pol greatly reduced the severity of disease caused by an SIV challenge. (3) In a murine cancer model, immunization with MVA-beta gal prevented the establishment of tumor metastases and even prolonged life in animals with established tumors. These results, together with previous data on the safety of MVA in humans, suggest the potential usefulness of recombinant MVA for prophylactic vaccination and therapeutic treatment of infectious diseases and cancer.

Molecular cloning and analysis of the chromosomal region 26A of Drosophila melanogaster.

Region 26A of the second chromosome of Drosophila melanogaster has been extensively characterized at the genetic level. We report here the cloning of virtually the entire 26A region via a bidirectional chromosome walk. Deletion and translocation breakpoints in the 26A interval have been localized at the molecular level by both chromosomal in situ hybridization and Southern analysis. The locations of the genetically defined loci in this chromosomal region have also been correlated with transcriptional units mapped onto the DNA of the proximal region of the chromosomal walk. The position of the alpha-glycerophosphate dehydrogenase (alpha-Gpdh) gene in 26A5-7 has been confirmed and a putative transcriptional unit for the beta-galactosidase-1 (beta-Gal-1) gene has been identified in the 26A7-9 interval.

Use of vaccinia virus vectors to study the synthesis, intracellular localization, and action of the human immunodeficiency virus trans-activator protein.

A recombinant vaccinia virus that expresses the human immunodeficiency virus (HIV) trans-activator (tat) gene was constructed. The tat polypeptide migrated anomalously with an apparent molecular mass of 14 kDa on a sodium dodecyl sulfate-polyacrylamide gel and reacted with polyclonal anti-tat serum. The tat protein was localized predominantly in the cell nucleus despite the absence of other HIV proteins or intranuclear HIV DNA. Additional recombinant vaccinia viruses that contain the Escherichia coli chloramphenicol acetyltransferase (CAT) gene under control of an early vaccinia promoter were constructed. Insertion of the HIV trans-activator-responsive (tar) sequence at the precise start of the CAT mRNA decreased CAT expression slightly. Trans-activation of vaccinia virus-encoded tarCAT failed to occur when CV-1 or HeLa cells were coinfected with the recombinant vaccinia virus expressing tat or when a HeLa cell line containing stably integrated copies of tat was used for infection, indicating the absence of transcriptional or translational effects under these conditions.

A soluble recombinant polypeptide comprising the amino-terminal half of the extracellular region of the CD4 molecule contains an active binding site for human immunodeficiency virus.

Infection of helper T lymphocytes by human immunodeficiency virus is initiated by a specific interaction of the viral envelope glycoprotein with CD4, an integral membrane glycoprotein of the target cell. We have adapted a vaccinia virus-based mammalian cell expression system to produce variants of the CD4 molecule for structure-function studies. In this report we demonstrate that a truncated 180-amino acid fragment representing approximately the N-terminal half of the extracellular region of CD4 is found primarily in soluble form in the extracellular medium. Epitope analysis with a panel of anti-CD4 murine monoclonal antibodies indicates that the fragment reacts with those antibodies known to block the interaction between CD4 and the human immunodeficiency virus envelope glycoprotein but reacts poorly or not at all with those antibodies that do not block this interaction. We also show that the fragment forms a specific complex with a soluble form of gp120, the CD4-binding subunit of the viral envelope glycoprotein. These results indicate that this soluble CD4 fragment contains an active binding site for human immunodeficiency virus.

Transfer of the inducible lac repressor/operator system from Escherichia coli to a vaccinia virus expression vector.

Cis- and trans-acting elements of the Escherichia coli lac operon were transferred to vaccinia virus and used to regulate gene expression. A recombinant virus that constitutively expresses a modified lac repressor gene (lacI) was constructed. We calculated that each infected cell contained approximately 2 x 10(7) active repressor molecules (and 1-2 x 10(4) copies of the vaccinia virus genome). A strong vaccinia-virus late promoter was modified by insertion of the lac operator (lacO) at various positions. The ability of each modified promoter to regulate expression of beta-galactosidase was tested by transient assays in cells infected with wild-type or lacI-containing vaccinia virus. Placement of the lacO just downstream of the conserved TAAAT sequence of a late promoter was consistent with a minimal effect on basal expression and good repressibility, whereas basal expression was severely inhibited when lacO overlapped or preceded the TAAAT motif. A single recombinant vaccinia virus containing lacI and the beta-galactosidase gene under control of the optimal lacO promoter was constructed. In the absence of inducer, cells infected with this double recombinant virus synthesized little or no detectable beta-galactosidase. Addition of isopropyl beta-D-thiogalactoside restored expression to greater than 20% of the unrepressed level. This inducible vector system has potential applications for expression of heterologous and homologous genes.

Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system.

A recombinant vaccinia virus that directs the synthesis of bacteriophage T7 RNA polymerase provides the basis for the expression of genes that are regulated by T7 promoters in mammalian cells. The T7 transcripts, which account for as much as 30% of the total cytoplasmic RNA at 24 hr after infection, are largely uncapped. To improve the translatability of the uncapped RNA, the encephalomyocarditis virus (EMCV) untranslated region (UTR) was inserted between the T7 promoter and the chloramphenicol acetyltransferase (CAT) gene. Experiments with a reticulocyte extract demonstrated that the EMCV UTR conferred efficient and cap-independent translatability to CAT RNA synthesized in vitro by T7 RNA polymerase. In cells infected with recombinant vaccinia viruses containing the T7 promoter-regulated CAT gene, the EMCV UTR increased the amount of CAT RNA on polyribosomes. The polyribosome-derived CAT RNA, which contained the EMCV UTR, was translated in vitro in a cap-independent fashion as well. Use of the EMCV UTR significantly enhanced the vaccinia/T7 hybrid expression system as it resulted in a 4- to 7-fold increase in total CAT activity. A further approximately 2-fold improvement was achieved by incubating the cells in hypertonic medium, which favors the translation of uncapped picornavirus RNA over cellular mRNAs. With this newly modified expression system, CAT was the predominant protein synthesized by infected cells and within 24 hr accounted for greater than 10% of the total cell protein.

Roles of vaccinia virus in the development of new vaccines.

Vaccinia virus is an efficient expression vector with broad host range infectivity and large DNA capacity. This vector has been particularly useful for identifying target antigens for humoral and cell-mediated immunity. With increased levels of gene expression, obtained either with stronger vaccinia promoters or through incorporation of the bacteriophage T7 RNA polymerase gene into the vaccinia genome, proteins may be synthesized in mammalian cells for use as subunit vaccines. For use as a live recombinant vaccine, efforts are being made to attenuate vaccinia virus further, either by inactivating genes contributing to virulence or by introducing human lymphokine genes into the vaccinia genome.

Regulated expression of foreign genes in vaccinia virus under the control of bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor.

The gene encoding bacteriophage T7 RNA polymerase (T7gene1) was placed under the control of regulatory elements from the Escherichia coli lac operon to construct an inducible vaccinia virus expression system consisting entirely of prokaryotic transcriptional machinery. Regulated expression of T7 RNA polymerase was necessary to construct a stable recombinant vaccinia virus harboring a T7 promoter; otherwise, uncontrolled expression led to interference with endogenous virus replication. To this end, the gene encoding the repressor protein of the lac operon was fused to a viral early/late promoter so that it was expressed constitutively, and the lac operator was interposed between a viral major late promoter and T7gene1. Greater than 99% repression of T7 RNA polymerase, which was relieved approximately 80-fold in the presence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), was obtained. An expression cassette containing a T7 promoter-controlled beta-galactosidase reporter gene was recombined into a different region of the viral genome containing T7gene1. A stable, double recombinant virus was isolated and grown to a high titer. In the absence of inducer, beta-galactosidase expression was substantially repressed. Addition of increasing amounts of IPTG induced expression of beta-galactosidase to the point of suppression of viral replication. This hybrid vaccinia virus system (Vac/Op/T7) has potential applications for the efficient bioproduction of a wide variety of gene products.

Development of BCG as a live recombinant vector system: potential use as an HIV vaccine.

Bacille Calmette-Guèrin (BCG), a live attenuated tubercle bacillus, is currently the most widely used vaccine in the world. Because of its unique characteristics, including low toxicity, adjuvant potential, and long-lasting immunity, BCG represents a novel vaccine vehicle with which to deliver protective antigens of multiple pathogens. We have developed episomal and integrative expression vectors employing regulatory sequences of major BCG heat shock proteins for stable maintenance and expression of foreign antigens in BCG vaccine strains (22). Shuttle plasmids capable of autonomous replication in Escherichia coli and BCG were constructed with a DNA cassette containing a minimal replicon derived from the Mycobacterium fortuitum plasmid pAL5000. Efficient and stable chromosomal integration of recombinant plasmids into BCG was achieved using a DNA segment containing the mycobacteriophage L5 attachment site and integrase coding sequence. Using the BCG hsp60 and hsp70 stress gene promoters, we were able to express Escherchia coli beta-galactosidase to levels in excess of 10% of total cell protein. The major antigens of HIV-1 gag, pol, and env were also stably expressed using our vector systems. The recombinant BCG elicited long-lasting humoral and cellular immune responses to these antigens in mice. Antibody responses to beta-galactosidase using as few as 200 colony-forming units were detected 6 weeks after immunization, and titers (1:30,000) were sustained for more than 10 weeks. Cellular immune responses, of both cytotoxic T cell (CTL) and helper T lymphocytes, were detected to beta-galactosidase. CTL responses were also induced to the HIV-1 envelope protein. Thus, we have demonstrated stable recombinant antigen expression, processing, and presentation using our recombinant BCG vector system. This live recombinant vector system shows promise as a universally applicable and safe vaccine vehicle for protection against various infectious diseases.

Demonstration of in vitro infection of chimpanzee hepatocytes with hepatitis C virus using strand-specific RT/PCR.

Analysis of hepatitis C virus (HCV) replication has been hampered due to the difficulty encountered in in vitro cultivation of the virus in conventional tissue culture systems. In this study, primary chimpanzee hepatocyte cultures maintained in a serum-free medium formulation were susceptible to in vitro infection with HCV. In order to document infection, two new methods of reverse transcription/polymerase chain reaction were developed that permit accurate distinction between positive and negative strand HCV RNA. One method relied upon the use of a tagged cDNA primer, while the second method employed a thermostable reverse transcriptase. Following inoculation of chimpanzee hepatocytes with HCV, intracellular positive and negative strand HCV RNA were detectable 4 days postinfection and throughout the remainder of the experimental period, 25 days. Analysis of HCV-inoculated baboon hepatocytes revealed a total absence of negative strand HCV RNA, while residual positive strand RNA from the inoculum could be detected for up to 11 days. The in vitro replication of HCV RNA in chimpanzee hepatocytes could be suppressed by alpha-interferon. This system should be amenable to the study of HCV replication, antiviral compounds, and the development of neutralization assays.

Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase.

DNA coding for bacteriophage T7 RNA polymerase was ligated to a vaccinia virus transcriptional promoter and integrated within the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and stably expressed T7 RNA polymerase in mammalian cells. Target genes were constructed by inserting DNA segments that code for beta-galactosidase or chloramphenicol acetyltransferase into a plasmid with bacteriophage T7 promoter and terminator regions. When cells were infected with the recombinant vaccinia virus and transfected with plasmids containing the target genes, the latter were expressed at high levels. Chloramphenicol acetyltransferase activity was 400-600 times greater than that observed with conventional mammalian transient-expression systems regulated either by the enhancer and promoter regions of the Rous sarcoma virus long terminal repeat or by the simian virus 40 early region. The vaccinia/T7 hybrid virus forms the basis of a simple, rapid, widely applicable, and efficient mammalian expression system.

Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells.

We developed a stringently regulated expression system for mammalian cells that uses (i) the RNA polymerase, phi 10 promoter, and T phi transcriptional terminator of bacteriophage T7; (ii) the lac repressor, lac operator, rho-independent transcriptional terminators and the gpt gene of Escherichia coli; (iii) the RNA translational enhancer of encephalomyocarditis virus; and (iv) the genetic background of vaccinia virus. In cells infected with the recombinant vaccinia virus, reporter beta-galactosidase synthesis was not detected in the absence of inducer. An induction of at least 10,000- to 20,000-fold occurred upon addition of isopropyl beta-D-thiogalactopyranoside or by temperature elevation from 30 to 37 degrees C using a temperature-sensitive lac repressor. Regulated synthesis of the secreted and highly glycosylated human immunodeficiency virus 1 envelope protein gp120 was also demonstrated. Yields of both proteins were approximately 2 mg per 10(8) cells in 24 hr. Plasmid transfer vectors for cloning and expression of complete or incomplete open reading frames in recombinant vaccinia viruses are described.

Purification of a soluble hepatitis E open reading frame 2-derived protein with unique antigenic properties.

The second open reading frame (ORF2) of hepatitis E virus (HEV) is predicted to encode a 73-kDa capsid protein (1). When full-length ORF2 was expressed in insect cells (Spodoptera frugiperda (Sf9)) using a recombinant baculovirus, two distinct HEV polypeptides were observed: a full-length insoluble 73-kDa protein, and a soluble 56.5-kDa protein. Following purification and sequence analysis, it was determined that the 56.5-kDa protein was derived from endoproteolytic cleavage site that was between the Thr and Ala residues located at amino acids 111 and 112 in the ORF2 sequence with the carboxy terminus corresponding to residue 636 of the ORF2 sequence. Comparative ELISA data using human acute-phase antisera demonstrated that the 56.5-kDa protein served as a highly reactive antigen in detecting anti-HEV antibodies. These data suggest that the 56.5-kDa protein may serve as a particularly useful antigen for both diagnostic and vaccine purposes.

Binding region for human immunodeficiency virus (HIV) and epitopes for HIV-blocking monoclonal antibodies of the CD4 molecule defined by site-directed mutagenesis.

The binding region for human immunodeficiency virus (HIV) and epitopes for a panel of HIV-blocking anti-CD4 monoclonal antibodies of the CD4 molecule were defined by using in vitro site-directed mutagenesis. Codons for two amino acid residues (Ser-Arg) were inserted at selected positions within the region encoding the first and second immunoglobulin-like domains of CD4. A vaccinia virus-based expression system was used to produce soluble full-length extracellular CD4 fragments containing the insertions. The mutant proteins were tested for direct binding to soluble gp120 (the CD4-binding subunit of the viral envelope glycoprotein) and to a series of HIV-blocking anti-CD4 monoclonal antibodies. Impaired gp120 binding activity resulted from insertions after amino acid residues 31, 44, 48, 52, 55, and 57 in the first immunoglobulin-like domain. The epitopes for two HIV-blocking monoclonal antibodies, OKT4A and OKT4D, were also mapped in the gp120-binding region in the first domain. Insertions after amino acid residues 21 and 91 in the first domain had no effect on gp120 binding but impaired the binding of OKT4E, suggesting that this antibody recognizes a discontinuous epitope not directly involved in gp120 binding. Moderate impairment of gp120 binding resulted from the insertion after amino acid residues 164 in the second immunoglobulin-like domain, where the epitopes for monoclonal antibodies MT151 and OKT4B were also mapped.

In vitro propagation and production of hepatitis E virus from in vivo-infected primary macaque hepatocytes.

Hepatitis E virus (HEV) is responsible for sporadic cases as well as large epidemics of acute viral hepatitis in many developing countries. The nucleotide sequence of HEV appears to be unique among known viruses and thus may represent a prototype human pathogen in a novel class of single-stranded, positive-sense RNA viruses. To facilitate further studies of the biology of HEV, a tissue culture system using a serum-free medium formulation has been developed to propagate the virus in vitro. Hepatocytes were isolated from livers of cynomolgus macaques experimentally infected with a HEV (Burma strain) inoculum and maintained in long-term cultures. Using a highly strand-specific RT-PCR assay, both the positive-sense and the negative replicative strands of HEV RNA were detected in these hepatocytes throughout the course of the experiments. Positive-strand genomic RNA was also detected in the culture medium, suggesting the production and secretion of HEV virus particles. The virus particles were successfully concentrated 200-fold from the medium using ultrafiltration, and they could be observed by immunoelectron microscopy using anti-HEV-positive immune serum. These results demonstrate the capacity of this hepatocyte culture system to replicate HEV in vitro, thus providing an experimental means to study the replicative process of the virus.