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Biomedical subjects

Richard E Sutton

Publications and source records attributed to Richard E Sutton.

18 recordsLinked to original sources
PubMed2006

Vaccination of mice with replication-defective human immunodeficiency virus induces cellular and humoral immunity and protects against vaccinia virus-gag challenge.

Here we describe as a potential vaccine candidate a replication-defective HIV that encodes multiple viral genes in addition to a cassette that includes both truncated cyclin T1 and an autofluorescent protein. After confirming functionality of the cyclin T1, we immunized mice intramuscularly once or twice with the replication-defective HIV vector pseudotyped with vesicular stomatitis virus (VSV) G protein (RD HIV), a plasmid encoding CMV-driven gag (gag DNA), or adenovirus gag (Ad5-gag). Capsid-specific antibody titers following RD HIV immunization were >10(6)/ml and approximately equivalent to those induced by gag DNA and Ad5-gag. Antibodies against the autofluorescent protein and VSV G were also detected. After RD HIV immunization ELISpot assays demonstrated Gag-specific interferon-gamma (IFN-gamma) SFU equivalent to that of Ad5-gag and fourfold greater than that of gag DNA. HIV polymerase-specific IFN-gamma SFU values were similar, and boosting increased both antibody titers and the IFN-gamma response. Challenge using vaccinia virus (VV)-gag demonstrated significantly lower recoverable VV for RD HIV-immunized mice compared to controls. No significant differences were observed in vaccinated mice challenged with wild-type VV. This study demonstrates the efficacy of RD HIV in conferring HIV-specific immunity and protection in mice and suggests its potential use in humans as either a prophylactic or a therapeutic vaccine.

AIDS Vaccines↗
PubMed2006

Replicative fidelity of lentiviral vectors produced by transient transfection.

Previous investigations have estimated the human immunodeficiency virus type 1 (HIV) base pair substitution rate to be approximately 10(-4) to 10(-5) per round of viral replication, and HIV has been hypothesized to be more error-prone than other retroviruses. Using a single cycle reversion assay, we unexpectedly found that the reversion rates of HIV, avian leukosis virus and Moloney murine leukemia virus were the same, within statistical error. Because both the viral enzyme reverse transcriptase (RT) and cellular RNA polymerase II (RNAP) are required for viral replication, we hypothesized that the similar reversion rates actually reflect the intrinsic error rate of RNAP, which is the enzyme common to all three retroviruses in the reversion assay. To address this possibility, HIV vectors with the U3 region replaced by a reporter reversion cassette were constructed and vector supernatant produced by transient transfection. All single integrant revertant cell lines showed the identical mutations at both long terminal repeats. This indicates that either RNAP or another cellular enzyme is responsible for these reversions, or that HIV RT only makes errors during first strand synthesis. Additionally, when HIV particles were rescued from an integrated vector as opposed to being produced by transient transfection, the reversion rate was significantly lower, suggesting that one or more factors in the virus-producing cells plays a role in the fidelity of retroviral replication. These results have implications regarding the fidelity of the transgene after transient transfection production of lentiviral vector supernatants.

Animals↗
PubMed2006

Human chromosome 2 carries a gene required for production of infectious human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV) replicates only in certain primate cells. In murine cells expressing cyclin T1, a posttranscriptional block exists such that small amounts of capsid and little infectious virus are released. This block is relieved in part by fusion with human cells. Here we have tested a panel of mouse-human somatic cell hybrids for production of infectious virus. Only those containing human chromosome 2 were permissive, which correlated with capsid production. The effect was specific to HIV in that release of murine leukemia virus was minimally affected by the presence of chromosome 2. Although expression of Vpu markedly increased capsid production in the absence of chromosome 2, it did not result in a corresponding increase in infectious HIV. The presence of chromosome 2 did not have consistent effects on the amount of unspliced viral RNA, whereas the amount of cell-associated Gag p55 was increased a fewfold. These results suggest that processing of HIV Gag can be corrected by one or more genes present on human chromosome 2 to allow production of infectious HIV from murine cells.

Animals↗
PubMed2005

HIV-based vectors and angiogenesis following rabbit hindlimb ischemia.

BACKGROUND: Numerous medical and surgical options exist for the treatment of vessel ischemia, which some patients fail or cannot tolerate. These investigations were designed to determine the effects of lentiviral-delivered vascular endothelial-derived growth factor (VEGF) and angiopoietin-2 (Ang-2) on collateralization in a rabbit model of hindlimb ischemia. MATERIALS AND METHODS: Self-inactivating human immunodeficiency virus (HIV)-based vectors were constructed encoding VEGF or Ang-2, co-transfected with vesicular stomatitis virus glycoprotein (VSV G) into 293T cells, and vector supernatants (1 x 10(8) IU/ml after concentration) were harvested. New Zealand white rabbits had ligation of either the right or left external iliac artery and excision of the ipsilateral femoral artery. Ten days later, empty, VEGF, or VEGF+Ang-2 vector supernatant was injected intramuscularly (IM) into the ipsilateral thigh. Ankle systolic blood pressure (SBP) ratios were recorded and venous blood samples collected on postoperative days (POD) 10, 25, and 40. On POD 40, run-off angiography was performed to measure vessel collateralization. Capillary density was determined by thin sectioning of muscle. RESULTS: A significant increase was noted in SBP in the VEGF-treated animals over time. Capillary density was not elevated despite significantly increased large vessel collateralization in rabbits receiving VEGF, which was counteracted by Ang-2. Antibodies against vector components were detected in exposed serum. CONCLUSIONS: Arterial collateralization and SBP increased significantly following VEGF vector administration, which was reversed by the Ang-2 vector. Development of antibody against VSV G can limit repeated injections of vector. Future experiments will involve the addition of other pro-angiogenic factors, repeated vector administration, and alternative routes of vector delivery.

Angiopoietin-2↗
PubMed2005

Lentivirus-mediated transduction of PKR into CD34(+) hematopoietic stem cells inhibits HIV-1 replication in differentiated T cell progeny.

Previous studies from this laboratory evaluated the role of p68 kinase (PKR) in the control of HIV-1 replication via retrovirus-mediated gene transfer. PKR was studied because it is a key component of the interferon (IFN)-associated innate antiviral defense pathway in mammalian cells. In this study, CD34(+) hematopoietic stem cells (HSC) were transduced with an HIV-1-based lentiviral vector encoding the PKR transgene (pHIV-PIB) and cultured under conditions that support in vitro differentiation. With high-titer pseudotyped vector stocks, the histogram suggests 100% transduction of the HSC because the cells were blasticidin resistant. Analysis of transduced cells by hybridization revealed an average proviral vector copy number of 1.8 and 2.1 copies of vector sequence per cell. Increased PKR expression and activity (phosphorylation of eukaryotic initiation factor 2alpha [eIF2alpha]) were demonstrated in PKR-transduced, differentiated HSC. There was minimal reduction in cell viability and no induction of apoptosis after transduction of PKR. HSC transduced with the pHIV-PIB lentiviral vector demonstrated normal differentiation into CD34-derived T cell progeny. Two days after HIV-1 infection, lentivirus-mediated transduction of PKR inhibited HIV-1 replication by 72% in T cell progeny compared with cells transduced with the empty vector control (pHIV-IB). By days 5 and 7 post-HIV-1 infection, the surviving PKR-transduced cells were protected from HIV-1 infection, as evidenced by a decrease in p24 antigen expression of at least two orders of magnitude. Our results demonstrate that PKR can be effectively delivered to HSC by a lentiviral vector and can protect CD34-derived T cell progeny from HIV-1 infection. These results provide support for application of the innate antiviral defense pathway in a gene therapy setting to the treatment of HIV-1 infection.

Antigens, CD34↗
PubMed2005

Expression of glucose transporter 1 confers susceptibility to human T-cell leukemia virus envelope-mediated fusion.

Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus identified and causes both adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-1-associated myelopathy, among other disorders. In vitro, HTLV-1 has an extremely broad host cell tropism in that it is capable of infecting most mammalian cell types, although at the same time viral titers remain relatively low. Despite years of study, only recently has a bona fide candidate cellular receptor, glucose transporter 1 (glut-1), been identified. Although glut-1 was shown to bind specifically to the ectodomain of HTLV-1 and HTLV-2 envelope glycoproteins, which was reversible with small interfering RNA directed against glut-1, cellular susceptibility to HTLV upon expression of glut-1 was not established. Here we show that expression of glut-1 in relatively resistant MDBK cells conferred increased susceptibility to both HTLV-1- and HTLV-2-pseudotyped particles. glut-1 also markedly increased syncytium formation in MDBK cells after exposure to HTLV-1. Another assay also demonstrated HTLV-1 envelope-cell fusion in the presence of glut-1. Taken together, these results provide additional evidence that glut-1 is a receptor for HTLV.

Animals↗
PubMed2004

Identification of synthetic endothelial cell-specific promoters by use of a high-throughput screen.

Transcriptional targeting is a desirable property for many gene transfer applications. Because endothelial cells line most blood vessels, they are attractive candidates for the introduction of therapeutic gene products. As a proof-of-concept study, we attempted to identify a synthetic, endothelial cell-specific promoter by use of a high-throughput screen involving self-inactivating (SIN) human immunodeficiency virus type 1 (HIV-1)-based vectors. Select duplex oligodeoxynucleotides recognized by transcription factors and located 5' of endothelial cell-specific mRNA transcripts were randomly ligated and cloned upstream of a minimal ICAM-2 promoter driving enhanced green fluorescent protein (eGFP) in a SIN HIV-1-based vector. Vesicular stomatitis virus G protein-pseudotyped particles were prepared from a library of >10(6) vector recombinants and used to transduce an endothelial cell line. The highest eGFP expressers were repeatedly sorted, and the synthetic promoters were recovered and retested by a luciferase reporter. Several promoters were active and specific to endothelial cells of varied species, with high selectivity indexes and inducibility under hypoxia-mimetic conditions. One in particular was then introduced back into a SIN HIV-1-based vector to confirm its endothelial cell activity and specificity. This study suggests that SIN vectors may be used in a high-throughput manner to identify tissue-specific promoters of high activity, with potential applications for both transcriptional targeting and gene transfer.

Antigens, CD↗
PubMed2004

The role of phenotyping and replication capacity in anti-HIV therapeutics.

The management of antiretroviral-experienced and -naive HIV-infected patients is becoming increasingly complex with the emergence of multidrug-resistant viruses and with the inter-relationships between numerous virological, immunological and therapeutic factors. In addition to standard surrogate markers of peripheral CD4+ T-cell counts and plasma viral loads, viral resistance phenotype, replication capacity and genotype assays serve as objective measures of viral properties, which presumably reflect, to a certain degree, what is occurring in vivo. This review explores the principles behind, and the basic science and clinical evidence supporting the application of viral resistance phenotypic assays and replication capacity assays in the therapeutic management of HIV disease.

Acquired Immunodeficiency Syndrome↗
PubMed2003

Genetic footprinting of the HIV co-receptor CCR5: delineation of surface expression and viral entry determinants.

Human immunodeficiency virus type 1 (HIV-1) utilizes CD4 as a primary receptor for viral entry and any of several 7-transmembrane chemokine receptors, including CCR5, as a co-receptor. Previous studies have demonstrated that multiple extracellular domains (ECDs) of CCR5 contribute to co-receptor function; here we applied genetic footprinting to CCR5 to confirm and extend those investigations. In genetic footprinting, a duplex oligonucleotide is inserted into the DNA sequence of interest by use of either a bacterial transposase or retroviral integrase. Here, CCR5 mutants were analyzed in bulk for their ability to be expressed on the recipient cell surface and to mediate viral entry of R5 HIV isolates. Most of the approximately 150 CCR5 mutants were not expressed on the cell surface. Of those remaining, 8 were specifically reduced or absent after macrophage (M)-tropic HIV infection, confirming a critical role of ECDs three (extracellular loop 2 or ECL2) and possibly four (ECL3) in viral entry. Mutational and functional analyses of ECD4 (ECL3) suggest it is under severe topological constraint for CCR5 surface expression and are consistent with it contributing to co-receptor function.

Amino Acid Sequence↗
PubMed2003

Development of an HIV-based cDNA expression cloning system.

Expression cloning of cDNAs is a powerful tool with which to identify genes based on their specific functional properties. Here we describe the development of a cDNA library transfer system based on the human immunodeficiency virus type-1 (HIV). This system represents an improvement over current oncoretroviral cDNA expression systems in terms of target cell range and the inclusion of a selectable marker. By use of a simple packaging system, we were able to produce high-titer vector stocks from HIV vector-based cDNA libraries and demonstrate highly efficient cDNA expression cloning in three model experiments. First, HOS TK(-) cells, which are null for thymidine kinase (TK) expression, were transduced with an HIV-based cDNA library derived from primary human foreskin fibroblasts (HFFs) and functionally selected for TK expression. In a second experiment, hypoxanthine guanine phosphoribosyltransferase-1-deficient (HPRT(-)) fibroblasts were transduced with a T cell (PM1) line-derived cDNA library and selected for HPRT expression. Both TK (frequency 1 in 5.0 x 10(4)) and HPRT (frequency 1 in 2.0 x 10(4)) cDNAs were readily isolated from these HIV-based cDNA libraries. As a third example, we demonstrated the ability of this vector system to allow functional cDNA library screens to be performed in primary, mitotically inactive cell types. Using senescent HFFs as a target cell population, we were able to isolate SV40 large T antigen cDNA-containing clones (frequency 1 in 2.5 x 10(4)) based on their ability to overcome the senescence-induced block to cell proliferation. Thus, this system can be used to clone relatively low-abundance cDNAs based upon their expression. Because of the ability of HIV-based vectors to transduce primary and nondividing cells efficiently, this vector system will further broaden the range of cell types in which expression cloning studies can be performed.

Base Sequence↗
PubMed2003

Characterization and detection of artificial replication-competent lentivirus of altered host range.

Replication-competent lentivirus (RCL) may be generated during the production phase or subsequently after introduction of a lentiviral vector into target cells, potentially by homologous or nonhomologous recombination. Because most gene transfer of HIV-based vectors involves the use of high-titer vesicular stomatitis virus (VSV) G-pseudotyped particles, one particular concern would be the generation of an RCL of altered host range, i.e., one that has incorporated the VSV G envelope in cis configuration. We report here on the artificial generation and properties of such a virus, including its detection after biological amplification. Viral spread, beginning with a very low inoculum, takes several weeks in culture and is characterized by "autoinfection," resulting in multiple proviral copies per cell, higher levels of viral gene expression, and eventual cell death. After this initial amplification step, the RCL is easily detectable by standard p24 assay or by "marker-rescue" assay. For the latter, a 293T-based cell line that has an integrated replication-defective provirus encoding alkaline phosphatase (AP) was used and mobilization of AP-containing virus was detected by transduction of naïve cells. Replication-defective virus was not amplified nor detected, demonstrating assay specificity. These results suggest that these artificial RCLs of broad host range have slightly different biological properties compared to wild-type HIV but still spread and are readily detectable.

Alkaline Phosphatase↗
PubMed2003

Genetic therapy for HIV/AIDS.

Despite the tremendous success of highly active antiretroviral treatment (HAART) introduced nearly 8 years ago for the treatment of human immunodeficiency virus (HIV), innovative therapies, including gene transfer approaches, are still required for nearly half of the general patient population. A number of potential gene therapeutic targets for HIV have been identified and include both viral and cellular genes essential for viral replication. The diverse methods used to inhibit viral replication comprise RNA-based strategies such as ribozymes, RNA decoys, antisense messenger RNAs and small interfering RNA (siRNA) molecules. Other potential anti-HIV genes include dominant negative viral proteins, intracellular antibodies, intrakines and suicide genes, all of which have had a modicum of success in vitro. Cellular targets include CD4+ T cells, macrophages and their progenitors. The greatest gene transfer efficiency has been achieved using retroviral or, more recently, lentiviral vectors. A limited number of Phase I clinical trials suggest that the general method is safe. It is proposed that a national network for HIV gene therapy (similar to the AIDS Clinical Trial Groups) may be the best way to determine which approaches should proceed clinically.

Acquired Immunodeficiency Syndrome↗
PubMed2003

Rodent models for HIV-1 infection and disease.

The development of a predictive, small animal model for human immunodeficiency virus type 1 (HIV-1) disease would greatly facilitate the analysis of many aspects of viral infection, pathogenesis and treatment. While numerous small animal models exist which emulate various aspects of HIV-1 infection and/or disease in humans, none of these models support robust HIV-1 replication within the context of an intact immune system. Despite this major limitation, these models have helped to elucidate different aspects of HIV-1 pathogenesis in humans. Moreover, recent advances regarding the underlying nature of the blocks to viral replication in non-human cells have raised the possibility that rodents may be engineered to support HIV-1 infection. This review will focus on recent attempts to develop a rodent model for HIV-1 disease, and will also describe currently available systems for studying HIV-1.

Animals↗
PubMed2003

Development of a mouse model for HIV/AIDS.

A small animal model would be very valuable for HIV/AIDS vaccine testing, investigating HIV pathophysiology, and exploring anti-HIV therapeutics. Unfortunately, HIV does not replicate in mouse cells. Provision of mouse cells with human CD4, CCR5 and cyclin T1 (cycT1) has uncovered a block to HIV assembly or release. Since mouse-human cell fusions allow viral replication, mouse cells lack at least one critical factor that permits completion of the viral life cycle. To identify this factor(s) we are employing 2 similar genetic approaches. Each cell line of a panel of monochromosomal mouse-human somatic cell hybrids was individually transduced with an HIV vector encoding both cycT1 and blasticidin resistance (HIV-CIB). Each was then transfected with vesicular stomatitis virus (VSV) G protein and measurable virus was recovered from only the hybrid-containing chromosome 2. This was verified with an M-tropic envelope and was shown to be specific to HIV. In addition, the amount of p24 release from that hybrid was substantially greater than that from the parent. A second cell line expressing chromosome 2 had a similar phenotype. CycT1 has been introduced into one chromosome 2 line to monitor the spread of HIV. In a related but separate approach, an entire collection of approximately 500 mouse-human microcell hybrids was transduced with HIV-CIB and broken down into manageable pools. Virus was similarly recovered as above from a few of the pools. Those pools were then broken down to clones and several cell clones have been identified that allow virus release. Revertants that no longer have the human chromosome are now being tested for loss of phenotype. Clones will then be tested for ability to support both HIV replication and Gag processing. Human chromosomal content of the clones of greatest interest will be determined by STS content analysis. Results from the 2 approaches are expected to be in agreement and may provide direction for an expression cloning approach.

Animals↗
PubMed2002

Lentiviral vectors for gene delivery into cells.

Human immunodeficiency virus type I (HIV) is the etiologic agent of acquired immunodeficiency syndrome or AIDS. Vectors based upon HIV have been in use for over a decade. Beginning in 1996, with the demonstration of improved pseudotyping using vesicular stomatitis virus (VSV) G protein along with transduction of resting mammalian cells, a series of improvements have been made in these vectors, making them both safer and more efficacious. Taking a cue from vector development of murine leukemia virus (MLV), split coding and self-inactivating HIV vectors now appear quite suitable for phase I clinical trials. In parallel, a number of pre-clinical efficacy studies in animals have demonstrated the utility of these vectors for various diseases processes, especially neurodegenerative and hematopoietic illnesses. These vectors are also appropriate for the study of other viruses (specifically of viral entry) and investigation of the HIV replicative cycle, along with straightforward transgene delivery to target cells of interest. Vectors based upon other lentiviruses have shown similar abilities and promise. Although concerns remain, particularly with regards to detection and propagation of replication-competent lentivirus, it is almost certain that these vectors will be introduced into the clinic within the next 3-5 years.

Animals↗