Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: rescue of replication-competent virus progeny and packaging of amplicon vectors

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors contain only approximately 1% of the 152-kb HSV-1 genome, and consequently, replication and packaging into virions depends on helper functions. These helper functions have been provided conventionally by a helper virus, usually a replication-defective mutant of HSV-1, or more recently, by a set of five cosmids that overlap and represent the genome of HSV-1 deleted for DNA cleavage/packaging signals (pac). In the absence of pac signals, potential HSV-1 genomes that are reconstituted from the cosmids via homologous recombination are not packageable. The resulting amplicon stocks are, therefore, virtually free of contaminating helper virus. To simplify this packing system, the HSV-1 genome was cloned and maintained stably as a single-copy, F plasmid-based bacterial artificial chromosome in E. coli. Such a plasmid containing the HSV-1 genome deleted for the pac signals (fHSV delta pac) did not generate replication-competent progeny virus on transfection into mammalian cells, but rather, it was able to support the packaging of cotransfected amplicon DNA that contained a functional pac signal. The resulting amplicon vector stocks had titers of up to 10(7) transducing units per milliliter of culture medium and efficiently transduced neural cells in the rat brain, as well as hepatocytes in the rat. The capacity of generating infectious and replication-competent HSV-1 progeny following transfection into mammalian cells was restored after insertion of a pac signal into fHSV delta pac.     

A novel herpesvirus amplicon system for in vivo gene delivery

For gene therapy approaches to succeed, improved vector systems are needed that combine a large carrying capacity with high transduction efficiency in vivo. Towards this goal, we have developed a novel herpes simplex virus (HSV) amplicon vector, pHE, which contains an HSV-1 replication origin (ori S) and packaging sequence that permit vector replication and packaging into HSV-1 capsids. The vector also contains the Epstein-Barr virus (EBV) unique latent replication origin (ori P) sequence and a modified EBNA-1 gene to allow the vector to be maintained as an episome in transfected E5 helper cells. This system allows for efficient packaging of high-titer vector since the E5 cells are first selected for the presence of the pHE vector before helper virus infection. The infectious pHE vector has efficient transgene expression in a variety of human cell lines in vitro. Stereotactic injection of pHE vector supernatant into the rat brain resulted in high, localized reporter gene expression. Finally, the pHE vector could carry a stable 21 kb DNA payload into HSV virions. This pHE vector system should have a broad range of gene transfer applications.     

A novel packaging system for the generation of helper-free oncolytic MVM vector stocks

MVM-based autonomous parvoviral vectors have been shown to target the expression of heterologous genes in neoplastic cells and are therefore of interest for cancer gene therapy. The traditional method for production of parvoviral vectors requires the cotransfection of vector and helper plasmids into MVM-permissive cell lines, but recombination between the cotransfected plasmids invariably gives rise to vector stocks that are heavily contaminated with wild-type MVM. Therefore, to minimise recombination between the vector and helper genomes we have utilised a cell line in which the MVM helper functions are expressed inducibly from a modified MVM genome that is stably integrated into the host cell chromosome. Using this MVM packaging cell line, we could reproducibly generate MVM vector stocks that contained no detectable helper virus.     

Complementation of helper-dependent adenoviral vectors: size effects and titer fluctuations

The complementation of adenoviral vectors with large deletions in the viral genome was studied. The helper adenovirus used to complement these vectors contains a partial deletion of the packaging signal and the E1 region substituted by the lacZ gene. The effect of vector size on packaging efficiency was analysed in 293 cells using decreasingly shorter vectors expressing GFP from a CMV enhancer-beta-actin promoter. Vectors with longer genomes propagated more efficiently than shorter ones. Vectors containing only the packaging signal and the ITRs of Ad5, having all the viral genes replaced with unrelated sequences packaged as efficiently as vectors of the same size containing adenoviral DNA instead of exogenous DNA. The amounts of helper and vector produced in coinfected 293 cells exhibited the typical cycling fluctuation observed during serial propagation of a virus with defective interfering particles.     

Generation of high-titer defective HSV-1 vectors using an IE 2 deletion mutant and quantitative study of expression in cultured cortical cells

Vectors based on herpes simplex virus type 1 (HSV-1) show promise for gene transfer into mammalian cells because of their wide host range, efficient infection and ability to deliver genes to nondividing cells. Defective HSV-1 vectors, or amplicons, are plasmid vectors which are unable to propagate on their own but contain specific HSV-1 sequences that, in the presence of helper virus, support DNA replication and subsequent packaging into virus particles. We compared three replication-incompetent HSV-1 mutants (KOS strain 5dl1.2, strain 17 D30EBA, KOS strain d120) as the helper virus for packaging the prototype defective HSV-1 vector, pHSVlac, which uses the HSV-1 immediate-early (1E) 4/5 promoter to regulate expression of the Escherichia coli lacZ gene. Use of 5dl1.2, which contains a deletion in the IE 2 gene, consistently produced virus stocks that contained a high level of vector, undetectable levels of wild-type HSV-1 and a ratio of vector to helper greater than 1. Virus stocks prepared using 5dl1.2 were superior to those prepared using helper viruses that harbor a deletion in the IE 3 gene, either D30EBA or dl20, and supported more efficient gene transfer than possible with previously published procedures. Lactate dehydrogenase efflux assays in rat cortical cultures showed that 5dl1.2 was no more cytotoxic than either D30EBA or dl20, despite the expression of more viral genes. Rat cortical cultures infected with pHSVlac packaged with either 5dl1.2 or D30EBA were used to quantify the stability of vector expression. Our results show a decrease in the number of cells with detectable levels of beta-galactosidase to 30% of peak levels after one week, irrespective of the helper virus used. However, simultaneous superinfection with 5dl1.2, but not with either D30EBA or dl20, produced a transient increase in the number of cells expressing beta-galactosidase. Superinfection with 5dl1.2 at 9 days after gene transfer increased the number of cells expressing detectable beta-galactosidase back to peak levels, most probably because of reactivation of the IE 4/5 promoter in pHSVlac. These results thus provide the first quantitative demonstration of long-term persistence of defective HSV-1 vectors in neurons.     

Evidence that deficient IFN-gamma production is a biological basis of herpes simplex virus type-2 neurovirulence

Although immune response control of herpes simplex virus (HSV) has been well demonstrated, numerous HSV-2 strains are neurovirulent in immunocompetent mice. Using an RNase protection assay and an ELISA, we found that HSV-2-infected mice exhibited a deficient IFN-gamma response, an inability to clear virus, and eventual death. An HSV-based amplicon vector expressing mouse IFN-gamma was constructed and packaged into HSV-1-helper virus (HSV(pIFN-gamma)). In mice treated with HSV(pIFN-gamma), (i) the LD50 of HSV-2(G) increased 5000-fold, (ii) intracerebral IFN-gamma expression increased 10-fold, and (iii) HSV titer rapidly decreased. We suggest that the deficient IFN-gamma response is a basis for HSV-2 neurovirulence in mice.     

Study of herpes simplex virus maturation during a synchronous wave of assembly

Production of an infectious herpes simplex virus (HSV) particle requires sequential progression of maturing virions through a series of complex assembly events. Capsids must be constructed in the nucleus, packaged with the viral genome, and transported to the nuclear periphery. They then bud into the nuclear membrane to acquire an envelope, traffic through the cytoplasm, and are released from the cell. Most of these phenomena are very poorly defined, and no suitable model system has previously been available to facilitate molecular analyses of genomic DNA packaging, capsid envelopment, and intracellular virion trafficking. We report the development of such an assay system for HSV type 1 (HSV-1). Using a reversible temperature-sensitive mutation in capsid assembly, we have developed conditions in which an accumulated population of immature capsids can be rapidly, efficiently, and synchronously chased to maturity. By assaying synchronized scaffold cleavage, DNA packaging, and acquisition of infectivity, we have demonstrated the kinetics with which these events occur. Kinetic and morphological features of intranuclear and extranuclear virion trafficking have similarly been examined by indirect immunofluorescence microscopy and electron microscopy. This system should prove a generally useful tool for the molecular dissection of many late events in HSV-1 biogenesis.     

Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus

Recently, efficient and long-term in vivo gene transfer by recombinant adeno-associated virus type 2 (rAAV) vectors has been demonstrated in a variety of tissues. Further improvement in vector titer and purity will expedite this in vivo exploration and provide preclinical information required for use in human gene therapy. In an effort to obtain higher titers, we constructed a novel AAV helper plasmid which utilizes translational control of AAV Rep genes (J. Li et al., J. Virol. 71:5236-5243, 1997). To address the issue of purity, in this study we report the first rAAV production method which is completely free of adenovirus (Ad) helper virus. The new production system uses a plasmid construct which contains a mini-Ad genome capable of propagating rAAV in the presence of AAV Rep and Cap genes. This construct is missing some of the early and most of the late Ad genes and is incapable of producing infectious Ad. Transfection of 293 cells with the new mini-Ad helper and AAV packaging plasmids results in high-titer rAAV vectors with yields greater than 1,000 transducing units, or 10(5) viral particles per cell. When rAAV vectors were produced by using this production scheme and compared to traditional heat-inactivated rAAV preparations in vitro and in vivo, we observed transduction equivalent to or better than normal levels. The complete removal of infectious Ad from AAV production should facilitate a better understanding of immune response to AAV vectors in vivo, eliminate the need for developing replication-competent Ad assays, and provide a more defined reagent for clinical use.     

The a sequence is dispensable for isomerization of the herpes simplex virus type 1 genome

The herpes simplex virus type 1 (HSV-1) genome consists of two components, L (long) and S (short), that invert relative to each other during productive infection to generate four equimolar isomeric forms of viral DNA. Recent studies have indicated that this genome isomerization is the result of DNA replication-mediated homologous recombination between the large inverted repeat sequences that exist in the genome, rather than site-specific recombination through the terminal repeat a sequences present at the L-S junctions. However, there has never been an unequivocal demonstration of the dispensability of the latter element for this process using a recombinant virus whose genome lacks a sequences at its L-S junctions. This is because the genetic manipulations required to generate such a viral mutant are not possible using simple marker transfer, since the cleavage and encapsidation signals of the a sequence represent essential cis-acting elements which cannot be deleted outright from the viral DNA. To circumvent this problem, a simple two-step strategy was devised by which essential cis-acting sites like the a sequence can be readily deleted from their natural loci in large viral DNA genomes. This method involved initial duplication of the element at a neutral site in the viral DNA and subsequent deletion of the element from its native site. By using this approach, the a sequence at the L-S junction was rendered dispensable for virus replication through the insertion of a second copy into the thymidine kinase (TK) gene of the viral DNA; the original copies at the L-S junctions were then successfully deleted from this virus by conventional marker transfer. The final recombinant virus, HSV-1::L-S(delta)a, was found to be capable of undergoing normal levels of genome isomerization on the basis of the presence of equimolar concentrations of restriction fragments unique to each of the four isomeric forms of the viral DNA. Interestingly, only two of these genomic isomers could be packaged into virions. This restriction was the result of inversion of the L component during isomerization, which prevented two of the four isomers from having the cleavage and encapsidation signals of the a sequence in the TK gene in a packageable orientation. This phenomenon was exploited as a means of directly measuring the kinetics of HSV-1::L-S(delta)a genome isomerization. Following infection with virions containing just the two packaged genomic isomers, all four isomers were readily detected at a stage in infection coincident with the onset of DNA replication, indicating that the loss of the a sequence at the L-S junction had no adverse effect on the frequency of isomerization events in this virus. These results therefore validate the homologous recombination model of HSV-1 genome isomerization by directly demonstrating that the a sequence at the L-S junction is dispensable for this process. The strategy used to remove the a sequence from the HSV-1 genome in this work should be broadly applicable to studies of essential cis-acting elements in other large viral DNA molecules.     

Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo

A replicon vaccine vector system was developed from an attenuated strain of Venezuelan equine encephalitis virus (VEE). The replicon RNA consists of the cis-acting 5' and 3' ends of the VEE genome, the complete nonstructural protein gene region, and the subgenomic 26S promoter. The genes encoding the VEE structural proteins were replaced with the influenza virus hemagglutinin (HA) or the Lassa virus nucleocapsid (N) gene, and upon transfection into eukaryotic cells by electroporation, these replicon RNAs directed the efficient, high-level synthesis of the HA or N proteins. For packaging of replicon RNAs into VEE replicon particles (VRP), the VEE capsid and glycoproteins were supplied in trans by expression from helper RNA(s) coelectroporated with the replicon. A number of different helper constructs, expressing the VEE structural proteins from a single or two separate helper RNAs, were derived from attenuated VEE strains Regeneration of infectious virus was not detected when replicons were packaged using a bipartite helper system encoding the VEE capsid protein and glycoproteins on two separate RNAs. Subcutaneous immunization of BALB/c mice with VRP expressing the influenza HA or Lassa virus N gene (HA-VRP or N-VRP, respectively) induced antibody responses to the expressed protein. After two inoculations of HA-VRP, complete protection against intranasal challenge with influenza was observed. Furthermore, sequential immunization of mice with two inoculations of N-VRP prior to two inoculations of HA-VRP induced an immune response to both HA and N equivalent to immunization with either VRP construct alone. Protection against influenza challenge was unaffected by previous N-VRP immunization. Therefore, the VEE replicon system was characterized by high-level expression of heterologous genes in cultured cells, little or no regeneration of plaque-forming virus particles, the capability for sequential immunization to multiple pathogens in the same host, and induction of protective immunity against a mucosal pathogen.     

Treatment of experimental Staphylococcus epidermidis endophthalmitis with oral trovafloxacin

In this report, a replication-defective herpes simplex virus type 1 (HSV-1) vector has been employed to deliver the Escherichia coli LacZ and HSV thymidine kinase (HSVtk) genes to six human ovarian carcinoma cell lines and the efficacy of gene transfer compared to that of adenoviral vectors in vitro. The transduction efficiency of the LacZ-containing virus TOZ.1 was evaluated qualitatively and quantitatively following infection of the different ovarian cancer cell lines. The therapeutic ability of the HSV-T3 vector, which contains the HSVtk gene, was additionally investigated in comparison to the AdCMVHSVTK. Our results show that HSV-1-mediated gene transfer is quantitatively superior to adenoviral vector in five of the six ovarian cancer cell lines at a 100-fold lower dose in vitro. Our preliminary studies suggest that HSV-1 may be a promising alternative vector for ovarian cancer gene therapy.     

Toward highly efficient cell-type-specific gene transfer with retroviral vectors displaying single-chain antibodies

Recently, we constructed retroviral vector particles derived from spleen necrosis virus (SNV) that display a single-chain antibody (scA) on the viral surface. By transient transfection protocols, we showed that such particles are competent for infection and cell type specific. Efficient infection was dependent on the presence of wild-type envelope, although wild-type SNV was not infectious on target cells (T.-H. T. Chu and R. Dornburg, J. Virol. 69:2659-2663, 1995; T.-H. T. Chu, I. Martinez, W. C. Sheay, and R. Dornburg, Gene Ther. 1:292-299, 1994). In this study, stable packaging lines were constructed and detailed biological and biochemical studies were performed. Chimeric scA-envelope fusion proteins were expressed as efficiently as wild-type envelope and were stable over a period of at least 6 h. Only a fully functional wild-type envelope could act as a helper for efficient virus penetration. The ratio of wild-type envelope protein to chimeric envelope protein appears to determine the efficiency of infection. Virus titers of targeting vectors obtained from stable packaging lines were as high as 10(4) CFU/ml. A 25-fold concentration of vector virus stocks resulted in a 200-fold increase in virus titers (up to 10(6) CFU/ml). These data indicate that an inhibitor of infection was (at least partially) removed by the concentration protocol. Our data show that this technology has several variables for further improvements and, therefore, has the potential to become a powerful tool for cell-type-specific in vivo human gene therapy.     

A helper-independent adenovirus vector with E1, E3, and fiber deleted: structure and infectivity of fiberless particles

The adenovirus (Ad) fiber protein largely determines viral tropism through interaction with specific cell surface receptors. This molecule may also be involved in virion assembly or maturation, as some previously characterized fiber mutants were defective for processing of viral structural proteins. We previously described packaging cell lines that express Ad type 5 (Ad5) fiber and can complement the temperature-sensitive Ad fiber mutant H5ts142. We have now used these packaging cells to construct a new adenoviral vector (Ad5.betagal.DeltaF) with E1, E3, and L5 (fiber) deleted and analyzed the fiber null phenotype. Ad5.betagal.DeltaF growth was completely helper independent, and fiberless particles were produced by a single final round of growth in 293 cells. Cryoelectron microscopic studies and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the structure and composition of these particles was nearly identical to those of first-generation Ad vectors. As expected, fiberless particles had reduced infectivity on epithelial cells, but they retained the ability to infect monocytic cells via an integrin-dependent pathway. These studies provide a novel approach to developing retargeted Ad gene therapy vectors.     

Sequences in pol are required for transfer of human foamy virus-based vectors

A series of vectors with heterologous genes was constructed from HSRV1, an infectious clone of human foamy virus (HFV), and transfected into baby hamster kidney cells to generate stably transfected vector cell lines. Two cis-acting sequences were required to achieve efficient rescue by helper virus. The first element was located at the 5' end upstream of position 1274 of the proviral DNA. Interestingly, a mutation in the leader sequence which decreased the ability to dimerize in vitro inhibited transfer by helper HFV. A second element that was important for vector transfer was located in the pol gene between positions 5638 and 6317. Constructs lacking this element were only poorly transferred by helper HFV, even though their RNA was produced in the vector cell lines. This finding rules out the possibility that the observed lack of transfer was due to RNA instability. A minimal vector containing only these two elements could be successfully delivered by helper HFV, confirming that all essential cis-acting sequences were present. The presence of a sequence described as a second polypurine tract in HFV was not necessary for transfer. Our data identified the minimal sequence requirements for HFV vector transfer for the development of useful vector systems.     

Regulated expression of a Sindbis virus replicon by herpesvirus promoters

We describe the use of herpesvirus promoters to regulate the expression of a Sindbis virus replicon (SINrep/LacZ). We isolated cell lines that contain the cDNA of SINrep/LacZ under the control of a promoter from a herpesvirus early gene which requires regulatory proteins encoded by immediate-early genes for expression. Wild-type Sindbis virus and replicons derived from this virus cause death of most vertebrate cells, but the cells discussed here grew normally and expressed the replicon and beta-galactosidase only after infection with a herpesvirus. Vero cell lines in which the expression of SINrep/LacZ was regulated by the herpes simplex virus type 1 (HSV-1) infected-cell protein 8 promoter were generated. One Vero cell line (V3-45N) contained, in addition to the SINrep/LacZ cDNA, a Sindbis virus-defective helper cDNA which provides the structural proteins for packaging the replicon. Infection of V3-45N cells with HSV-1 resulted in the production of packaged SINrep/LacZ replicons. HSV-1 induction of the Sindbis virus replicon and packaging and spread of the replicon led to enhanced expression of the reporter gene, suggesting that this type of cell could be used to develop sensitive assays to detect herpesviruses. We also isolated a mink lung cell line that was transformed with SINrep/LacZ cDNA under the control of the promoter from the human cytomegalovirus (HCMV) early gene UL45. HCMV carries out an abortive infection in mink lung cells, but it was able to induce the SINrep/LacZ replicon. These results, and those obtained with an HSV-1 mutant, demonstrate that this type of signal amplification system could be valuable for detecting herpesviruses for which a permissive cell culture system is not available.     

Heart function in patients with chronic glomerulonephritis and mildly to moderately impaired renal function. An echocardiographic study

We recently described a novel nonviral/viral vector for gene transfer, the plasmovirus (Noguiez-Hellin P, Robert-le Meur M, Laune S, et al. C R Acad Sci Paris, Sciences de la Vie. 1996;319:45-50; Noguiez-Hellin P, Robert-le Meur M, Salzmann J-L, et al. Proc Natl Acad Sci USA. 1996;93:4175-4180). Plasmoviruses are plasmids capable of expressing all the viral genes required for generating infectious particles and packaging a defective genome containing a transgene. Transfected as plasmids, plasmoviruses transform the transduced cells into packaging cells that release infectious replication-defective retrovirus vectors (RV) containing a transgene, which are capable of infecting nearby cells. We previously showed that such a vector can efficiently "propagate" the transgene after transfection. Here we examine in greater detail the different steps of plasmovirus replication in vitro in human (143 B TK-) and murine (NIH 3T3 TK-) cells. Molecular-biological analysis revealed plasmovirus-coded protein expression starting from 24 hours post-transfection, followed by the detection of infectious RV 48 hours post-transfection. The gag proteins were correctly processed in the released particles. Electron microscopic analysis revealed typical type C particles. Nonintegrated plasmovirus DNA was not toxic for the cells and could be detected for at least 14 days post-transfection. While the transfected gag gene and the transgene could also be detected throughout this period, we observed that env-coded proteins decreased after 72 hours post-transfection. Nevertheless, the production of RV resulted in the propagation of the transgene in the culture, with stable integration of plasmovirus proviral DNA into the host genome of infected cells. We show that this propagation results in a major improvement in therapeutic efficacy using an HSV1-TK transgene and ganciclovir treatment, when compared to that of plasmovirus constructs that cannot propagate. Altogether, these results demonstrate the functionality of this gene transfer method and suggest that improvements in the vector design enhance its efficacy.