A trial of somatic gene targeting in vivo with an adenovirus vector

We have constructed and evaluated the utility of a helper-dependent virus vector system that is derived from Human Cytomegalovirus (HCMV). This vector is based on the herpes simplex virus (HSV) amplicon system and contains the HCMV orthologs of the two cis-acting functions required for replication and packaging of HSV genomes, the complex HCMV viral DNA replication origin (oriLyt), and the cleavage packaging signal (the a sequence). The HCMV amplicon vector replicated independently and was packaged into infectious virions in the presence of helper virus. This vector is capable of delivering and expressing foreign genes in infected cells including progenitor cells such as human CD34+ cells. Packaged defective viral genomes were passaged serially in fibroblasts and could be detected at passage 3; however, the copy number appeared to diminish upon serial passage. The HCMV amplicon offers an alternative vector strategy useful for gene(s) delivery to cells of the hematopoietic lineage.     

Use of amplicon-6 vectors derived from human herpesvirus 6 for efficient expression of membrane-associated and -secreted proteins in T cells

The composite amplicon-6 vectors, which are derived from human herpesvirus 6 (HHV-6), can target hematopoietic cells. In the presence of the respective helper viruses, the amplicons are replicated by the rolling circle mechanism, yielding defective genomes of overall size 135 to 150 kb, composed of multiple repeats of units, containing the viral DNA replication origin, packaging signals, and the selected transgene(s). We report the use of amplicon-6 vectors designed for transgene expression in T cells. The selected transgenes included the green fluorescent protein marker, the herpes simplex virus type 1 glycoprotein D (gD), and the gD gene deleted in the transmembrane region (gDsec). The vectors were tested after electroporation and passage in T cells with or without helper HHV-6A superinfections. The results were as follows. (i)The vectors could be passaged both as cell-associated and as cell-free secreted virions infectious to new cells. (ii)The intact gD accumulated at the cell surface, whereas the gDsec was dispersed at internal locations of the cells or was secreted into the medium. (iii)Analyses of amplicon-6-gD expression by flow cytometry have shown significant expression in cultures with reiterated amplicons and helper viruses. The vector has spread to >60% of the cells, and the efficiency of expression per cell increased 15-fold, most likely due to the presence of concatemeric amplicon repeats. Current studies are designed to test whether amplicon-6 vectors can be used for gene therapy in lymphocytes and whether amplicon-6 vectors expressed in T cells and dendritic cells can induce strong cellular and humoral immune responses.     

The herpes simplex virus amplicon: a new eucaryotic defective-virus cloning-amplifying vector

We have employed repeat units of herpes simplex virus (HSV) defective genomes to derive a cloning-amplifying vector (amplicon) that can replicate in eucaryotic cells in the presence of standard HSV helper virus. The design of the HSV amplicon system is based on the previous observation that cotransfection of cells with helper virus DNA and seed monomeric repeat units of HSV defective genomes results in the regeneration of concatemeric defective genomes composed of multiple reiterations of the seed repeats. Cotransfection of cells with helper virus DNA and chimeric repeat units containing bacterial plasmid pKC7 DNA resulted in the generation of defective genomes composed of reiterations of the seed HSV-pKC7 repeats. These chimeric defective genomes were packaged into virus particles and could be propagated in virus stocks, with the most enriched passages containing more than 90% chimeric defective genomes. Furthermore, monomeric chimeric repeat units could be transferred back and forth between bacteria and eucaryotic cells. A derivative vector constructed so as to contain several unique restriction enzyme sites could be potentially employed in the introduction of additional viral or eucaryotic DNA sequences into eucaryotic cells.     

Role of gene 6 exonuclease in the replication and packaging of bacteriophage T7 DNA

When bacteriophage T7 gene 6 exonuclease is genetically removed from T7-infected cells, degradation of intracellular T7 DNA is observed. By use of rate zonal centrifugation, followed by either pulsed-field agarose gel electrophoresis or restriction endonuclease analysis, in the present study, the following observations were made. (1) Most degradation of intracellular DNA requires the presence of T7 gene 3 endonuclease and is independent of DNA packaging; rapidly sedimenting, branched DNA accumulates when both the gene 3 and gene 6 products are absent. (2) A comparatively small amount of degradation requires packaging and occurs at both the joint between genomes in a concatemer and near the left end of intracellular DNA; DNA packaging is only partially blocked and end-to-end joining of genomes is not blocked in the absence of gene 6 exonuclease. (3) Fragments produced in the absence of gene 6 exonuclease are linear and do not further degrade; precursors of the fragments are non-linear. (4) Some, but not most, of the cleavages that produce these fragments occur selectively near two known origins of DNA replication. On the basis of these observations, the conclusion is drawn that most degradation that occurs in the absence of T7 gene 6 exonuclease is caused by cleavage at branches. The following hypothesis is presented: most, possibly all, of the extra branching induced by removal of gene 6 exonuclease is caused by strand displacement DNA synthesis at the site of RNA primers of DNA synthesis; the RNA primers, produced by multiple initiations of DNA replication, are removed by the RNase H activity of gene 6 exonuclease during a wild-type T7 infection. Observation of joining of genomes in the absence of gene 6 exonuclease and additional observations indicate that single-stranded terminal repeats required for concatamerization are produced by DNA replication. The observed selective shortening of the left end indicates that gene 6 exonuclease is required for formation of most, possibly all, mature left ends.     

A recombinant Sendai virus is controlled by CD4+ effector T cells responding to a secreted human immunodeficiency virus type 1 envelope glycoprotein

The importance of antigen-specific CD4(+) helper T cells in virus infections is well recognized, but their possible role as direct mediators of virus clearance is less well characterized. Here we describe a recombinant Sendai virus strategy for probing the effector role(s) of CD4(+) T cells. Mice were vaccinated with DNA and vaccinia virus recombinant vectors encoding a secreted human immunodeficiency virus type 1 (HIV-1) envelope protein and then challenged with a Sendai virus carrying a homologous HIV-1 envelope gene. The primed mice showed (i) prompt homing of numerous envelope-primed CD4(+) T cell populations to the virus-infected lung, (ii) substantial production of gamma interferon, and interleukin-2 (IL-2), IL-4, and IL-5 in that site, and (iii) significantly reduced pulmonary viral load. The challenge experiments were repeated with immunoglobulin(-/-) microMT mice in the presence or absence of CD8(+) and/or CD4(+) T cells. These selectively immunodeficient mice were protected by primed CD4(+) T cells in the absence of antibody or CD8(+) T cells. Together, these results highlight the role of CD4(+) T cells as direct effectors in vivo and, because this protocol gives such a potent response, identify an outstanding experimental model for further dissecting CD4(+) T-cell-mediated immunity in the lung.     

Human herpesvirus 6 infects dendritic cells and suppresses human immunodeficiency virus type 1 replication in coinfected cultures

Human herpesvirus 6 (HHV-6) has been implicated as a cofactor in the progressive loss of CD4(+) T cells observed in AIDS patients. Because dendritic cells (DC) play an important role in the immunopathogenesis of human immunodeficiency virus (HIV) disease, we studied the infection of DC by HHV-6 and coinfection of DC by HHV-6 and HIV. Purified immature DC (derived from adherent peripheral blood mononuclear cells in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4) could be infected with HHV-6, as determined by PCR analyses, intracellular monoclonal antibody staining, and presence of virus in culture supernatants. However, HHV-6-infected DC demonstrated neither cytopathic changes nor functional defects. Interestingly, HHV-6 markedly suppressed HIV replication and syncytium formation in coinfected DC cultures. This HHV-6-mediated anti-HIV effect was DC specific, occurred when HHV-6 was added either before or after HIV, and was not due to decreased surface expression or function of CD4, CXCR4, or CCR5. Conversely, HIV had no demonstrable effect on HHV-6 replication. These findings suggest that HHV-6 may protect DC from HIV-induced cytopathicity in AIDS patients. We also demonstrate that interactions between HIV and herpesviruses are complex and that the observable outcome of dual infection is dependent on the target cell type.     

Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity.

We constructed a recombinant human immunodeficiency virus (HIV) vector to facilitate studies of virus infectivity. A drug resistance gene was inserted into a gp160- HIV proviral genome such that it could be packaged into HIV virions. The HIV genome was rendered replication defective by deletion of sequences encoding gp160 and insertion of a gpt gene with a simian virus 40 promoter at the deletion site. Cotransfection of the envelope-deficient genome with a gp160 expression vector resulted in packaging of the defective HIV-gpt genome into infectious virions. The drug resistance gene was transmitted and expressed upon infection of susceptible cells, enabling their selection in mycophenolic acid. This system provides a quantitative measure of HIV infection, since each successful infection event leads to the growth of a drug-resistant colony. The HIV-gpt virus produced was tropic for CD4+ human cells and was blocked by soluble CD4. In the absence of gp160, noninfectious HIV particles were efficiently produced by cells transfected with the HIV-gpt genome. These particles packaged HIV genomic RNA and migrated to the same density as gp160-containing virions in a sucrose gradient. This demonstrates that HIV virion formation is not dependent on the presence of a viral envelope glycoprotein. Expression of a murine leukemia virus amphotropic envelope gene in cells transfected with HIV-gpt resulted in the production of virus capable of infecting both human and murine cells. These results indicate that HIV can incorporate envelope glycoproteins other than gp160 onto particles and that this can lead to altered host range. Like HIV type 1 and vesicular stomatitis virus(HIV) pseudotypes, gp-160+ HIV-gpt did not infect murine NIH 3T3 cells that bear human CD4, confirming that these cells are blocked at an early stage of HIV infection.     

Packaging Cells Based on Inducible Gene Amplification for the Production of Adeno-Associated Virus Vectors

Although vectors based on adeno-associated virus (AAV) offer several unique advantages, their usage has been hampered by the difficulties encountered in vector production. In this report, we describe a new AAV packaging system based on inducible amplification of integrated helper and vector constructs containing the simian virus 40 (SV40) replication origin. The packaging and producer cell lines developed express SV40 T antigen under the control of the reverse tetracycline transactivator system, which allows inducible amplification of chromosomal loci linked to the SV40 origin. Culturing these cells in the presence of doxycycline followed by adenovirus infection resulted in helper and vector gene amplification as well as higher vector titers. Clonal producer cell lines generated vector titers that were 10 times higher than those obtained by standard methods, with approximately 10⁴ vector particles produced per cell. These stocks were free of detectable replication-competent virus. The lack of a transfection step combined with the reproducibility of stable producer lines makes this packaging method ideally suited for the large-scale production of vector stocks for human gene therapy.     

Transfer of the full-length dystrophin-coding sequence into muscle cells by a dual high-capacity hybrid viral vector with site-specific integration ability

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, making it a potential target for gene therapy. There is, however, a scarcity of vectors that can accommodate the 14-kb DMD cDNA and permanently genetically correct muscle tissue in vivo or proliferating myogenic progenitors in vitro for use in autologous transplantation. Here, a dual high-capacity adenovirus-adeno-associated virus (hcAd/AAV) vector with two full-length human dystrophin-coding sequences flanked by AAV integration-enhancing elements is presented. These vectors are generated from input linear monomeric DNA molecules consisting of the Ad origin of replication and packaging signal followed by the recently identified AAV DNA integration efficiency element (p5IEE), the transgene(s) of interest, and the AAV inverted terminal repeat (ITR). After infection of producer cells with a helper Ad vector, the Ad DNA replication machinery, in concert with the AAV ITR-dependent dimerization, leads to the assembly of vector genomes with a tail-to-tail configuration that are efficiently amplified and packaged into Ad capsids. These dual hcAd/AAV hybrid vectors were used to express the dystrophin-coding sequence in rat cardiomyocytes in vitro and to restore dystrophin synthesis in the muscle tissues of mdx mice in vivo. Introduction into human cells of chimeric genomes, which contain a structure reminiscent of AAV proviral DNA, resulted in AAV Rep-dependent targeted DNA integration into the AAVS1 locus on chromosome 19. Dual hcAd/AAV hybrid vectors may thus be particularly useful to develop safe treatment modalities for diseases such as DMD that rely on efficient transfer and stable expression of large genes.     

Human herpesvirus 7: antigenic properties and prevalence in children and adults.

The recent isolation of human herpesvirus 7 (HHV-7) from activated CD4+ T lymphocytes of a healthy individual raises questions regarding the prevalence of this virus in humans and its immunological relationship to previously characterized human herpesviruses. We report that HHV-7 is a ubiquitous virus which is immunologically distinct from the highly prevalent T-lymphotropic HHV-6. Thus, (i) only two of six monoclonal antibodies to HHV-6 cross-reacted with HHV-7-infected cells, (ii) Western immunoblot analyses of viral proteins revealed different patterns for HHV-6- and HHV-7-infected cells, (iii) tests of sequential serum samples from children revealed seroconversion to HHV-6 without concomitant seroconversion to HHV-7, and (iv) in some instances HHV-7 infection occurred in the presence of high titers of HHV-6 antibodies, suggesting the lack of apparent protection of children seropositive for HHV-6 against subsequent infection with HHV-7. On the basis of the analyses of sera from children and adults it can be concluded that HHV-7 is a prevalent human herpesvirus which, like other human herpesviruses, infects during childhood. The age of infection appears to be somewhat later than the very early age documented for HHV-6.     

Chimeric retroviral helper virus and picornavirus IRES sequence to eliminate DNA methylation for improved retroviral packaging cells

Most retroviral packaging cell lines were established by a helper virus plasmid cotransfected with a separate plasmid encoding a selection marker. Since this selection marker coexisted in trans with the helper virus sequence, helper virus gene expression could be inactivated by host DNA methylation despite selection for the cotransfected selection marker. We have reported that DNA methylation could occur in the long terminal repeat (LTR) region of helper virus in vector producer cells (VPC) in up to 2% of the population per day (W. B. Young, G. L. Lindberg, and C. J. Link, Jr., J. Virol. 74:3177-3187, 2000). To overcome host cell DNA methylation that suppresses viral gene expression, we constructed a chimeric retroviral helper virus, pAM3-IRES-Zeo, that contains Moloney murine leukemia virus as a helper virus and a picornavirus internal ribosome entry site (IRES) sequence followed by a Zeocin selection marker at the 3' end of the env sequence. This pAM3-IRES-Zeo permitted selection for intact and functional helper virus in transfected cells without subcloning. By selection with Zeocin, a mixed population of pAM3-IRES-Zeo-transfected NIH3T3 cells (AMIZ cells) was maintained with little or no DNA methylation of the helper virus 5' LTR. The high level of pAM3-IRES-Zeo gene expression resulted in no detectable vector superinfection and in high vector titers (2 x 10(6) to 1.5 x 10(7) CFU/ml) after introduction of a retroviral vector. When Zeocin selection was withdrawn from AMIZ cells, methylation of the 5' LTR increased from 17 to 36% of the population during 67 days of continuous culture and the cells became susceptible to superinfection. During this period, gene expression of pAM3-IRES-Zeo decreased and vector titer production was reduced to 2 x 10(4) CFU/ml. These data demonstrate an important role of DNA methylation in the genetic instability of VPC. The chimeric helper virus allows the establishment of a mixed population of packaging cells capable of high-level and sustained vector production without cloning procedures.     

Superinfection rescue of an integrated defective polyomavirus genome.

We have characterized the viral sequences integrated in a polyomavirus-transformed mouse cell line, Py-3T3 (clone Py-6), and followed their excision and packaging upon superinfection. The polyomavirus sequences contained in Py-6 cells are present as a single insert of nonidentical tandem copies which includes, in addition to a normal middle T-antigen-coding region, some very rearranged sequences. Infection of Py-6 cells with polyomavirus strains encoding a normal large T antigen leads to the reproducible recovery in the resulting viral stock of specific defective viral genomes. The defective genomes contain a wild-type coding region for middle and small T antigens and intact viral origin and enhancer sequences. The remainder of the viral genome is rearranged or lost, so that there is no capacity to code for large T antigen or viral capsid proteins. The recovered defective sequences are also found integrated in Py-6 genomic DNA. Presumably, in infections of Py-6 cells, large T antigen, provided by the superinfecting virus, amplifies and excises the integrated viral sequences. The superinfecting helper virus must also produce viral capsids for packaging of the defective viral DNA and thus provides a means to shuttle the defective sequences from the mouse cells into other hosts, such as rat cells. In the latter host, the defective sequences are able to induce transformation.     

Viral Vectors for Gene Delivery and Expression in the CNS

Viral vectors have emerged as an important tool for manipulating gene expression in the adult mammalian brain. The adult brain is composed largely of nondividing cells, and therefore DNA viruses have become the vehicle of choice for neurobiologists interested in somatic gene transfer. Recombinant viral vectors based upon adenovirus or herpes simplex virus have been created in which a gene essential for viral replication is removed and a gene of interest is inserted in the viral genome. While this eliminates pathogenicity due to viral replication, retention of viral genes and continued expression of these genes may limit the potential of the current generation of vectors. Defective viral vectors represent a different approach, in which only viral recognition signals are used to allow packaging of foreign DNA into a viral coat while eliminating the possibility of viral gene expression within target cells. The defective HSV vector has been used to transfer genes into the adult rat brain. This vector has also been used for analysis of the preproenkephalin promoterin vivo,and important regions of this promoter have been identified using this technique. A modification ofin situPCR has been developed as an adjunctive tool for sensitively documenting the presence of vector DNA within target cells duringin vivopromoter studies. Finally, the adenoassociated virus vector has been used as the first fully defective DNA viral vector, which also eliminates any contamination by helper viruses. This vector can transfer genes into the mammalian brain and has shown significant behavioral recovery in a rodent model of Parkinson's disease. Future work will undoubtedly result in still more diverse and improved vectors; however, these studies have documented the importance of viral vectors to both basic neurobiology and the potential treatment of neurologic disease.     

Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells.

Herpes simplex virus type 1 (HSV-1) plasmid vectors have promise for genetic intervention in the brain, but several problems caused by the helper virus have compromised their utility. To develop a helper virus-free packaging system for these vectors, the DNA cleavage/packaging signals were deleted from a set of cosmids that represents the HSV-1 genome. Following cotransfection into cells, this modified cosmid set supported replication and packaging of vector DNA. However, in the absence of the DNA cleavage/packaging signals, the HSV-1 genome was not packaged, and consequently vector stocks were free of detectable helper virus. In the absence of helper virus, the vectors efficiently infected rat neural cells in culture or in the brain with minimal cytopathic effects. beta-galactosidase-positive cells were observed for at least 1 month in vivo, and vector DNA persisted for this period. This system may facilitate studies on neuronal physiology and potential therapeutic applications.     

Productive infection of primary macrophages with human herpesvirus 7

Here we demonstrate replication of human herpesvirus 7 (HHV-7), a T-lymphotropic virus, in macrophages. Productive replication was lost after 2 weeks, but HHV-7 DNA was detected up to 1 month after infection. Thus, macrophages become infected by HHV-7 and might play an important role as a viral reservoir, as has been demonstrated for human immunodeficiency virus type 1.     

Human CD4+ T cell response to human herpesvirus 6

Following primary infection, human herpesvirus 6 (HHV-6) establishes a persistent infection for life. HHV-6 reactivation has been associated with transplant rejection, delayed engraftment, encephalitis, muscular dystrophy, and drug-induced hypersensitivity syndrome. The poor understanding of the targets and outcome of the cellular immune response to HHV-6 makes it difficult to outline the role of HHV-6 in human disease. To fill in this gap, we characterized CD4 T cell responses to HHV-6 using peripheral blood mononuclear cell (PBMC) and T cell lines generated from healthy donors. CD4(+) T cells responding to HHV-6 in peripheral blood were observed at frequencies below 0.1% of total T cells but could be expanded easily in vitro. Analysis of cytokines in supernatants of PBMC and T cell cultures challenged with HHV-6 preparations indicated that gamma interferon (IFN-γ) and interleukin-10 (IL-10) were appropriate markers of the HHV-6 cellular response. Eleven CD4(+) T cell epitopes, all but one derived from abundant virion components, were identified. The response was highly cross-reactive between HHV-6A and HHV-6B variants. Seven of the CD4(+) T cell epitopes do not share significant homologies with other known human pathogens, including the closely related human viruses human herpesvirus 7 (HHV-7) and human cytomegalovirus (HCMV). Major histocompatibility complex (MHC) tetramers generated with these epitopes were able to detect HHV-6-specific T cell populations. These findings provide a window into the immune response to HHV-6 and provide a basis for tracking HHV-6 cellular immune responses.     

Induction of T-cell apoptosis by human herpesvirus 6.

The mechanisms of cell death in CD4+ T cells mediated by human herpesvirus 6 (HHV-6) were investigated. The frequency of cell death in the human CD4+ T-cell line JJHAN, which had been inoculated with HHV-6 variant A or B, appeared to be augmented by tumor necrosis factor alpha (TNF-alpha). Agarose gel electrophoresis of DNA from HHV-6-inoculated cells showed DNA fragmentation in multiples of the oligonucleosome length unit. The degree of DNA fragmentation increased when HHV-6-inoculated cells were cultured in the presence of TNF-alpha. Flow cytometry and Scatchard analysis of TNF receptors revealed an increase in the number of the p55 form of TNF receptors on JJHAN cells after HHV-6 inoculation. It also appeared that treatment with anti-Fas monoclonal antibody (MAb) induced marked apoptosis in HHV-6-inoculated cells. Transmission electron microscopy showed characteristics of apoptosis, such as chromatin condensation and fragmentation of nuclei, but virus particles were hardly detected in apoptotic cells. Two-color flow cytometric analysis using anti-HHV-6 MAb and propidium iodide revealed that DNA fragmentation was present predominantly in uninfected cells but not in productively HHV-6-infected cells. In addition, JJHAN cells incubated with UV light-irradiated and ultracentrifuged culture supernatant of HHV-6-infected cells appeared to undergo apoptosis. The present study demonstrated that both HHV-6 variants A and B induce apoptosis in CD4+ T cells by indirect mechanisms, as reported recently in human immunodeficiency virus type 1 infection.