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ABSTRACT: The envelope proteins (env) of simian immunodeficiency virus (SIV) and HIV type 1 assemble to form noncovalently associated oligomers in the endoplasmic reticulum. After cleavage in a Golgi compartment, oligomeric env complexes are transported to the surface of infected cells, where incorporation into budding virions can occur. Difficulties in obtaining adequate quantities of virions retaining env, as well as the unstable nature and hydrophobicity of the oligomer, may account for the absence of previous biophysical studies to determine the oligomeric valency of membrane-associated env. The aim of this study was to evaluate the oligomeric state of SIV env before membrane-fusion activation. Virion-associated env, obtained by crosslinking and detergent extraction, and non-crosslinked secreted env ectodomain (recombinant gp140) were purified by lentil-lectin chromatography and gel filtration as single predominant species. Sedimentation equilibrium-derived mass values for both forms of SIV env were close to those predicted for trimeric assemblies. Determination of the mass of individual molecules by scanning transmission electron microscopy confirmed that SIV virion-associated env and gp140 formed largely homogeneous populations of trimers. Furthermore, a triangular or tri-lobed morphology was clearly visualized in a subset of the trimers.
Proceedings of the National Academy of Sciences 01/2002; 98(26):14877-82. · 9.68 Impact Factor
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ABSTRACT: We have demonstrated previously the oncolytic effects of a systemically delivered, replicating vaccinia virus. To enhance the tumor specificity of this vector, we have developed a combined thymidine kinase-deleted (TK-) and vaccinia growth factor-deleted (VGF-) vaccinia virus and investigated its properties in vitro and in vivo. The gene for enhanced green fluorescent protein (EGFP) was inserted into the TK locus of a VGF- vaccinia virus by homologous recombination creating a double-deleted mutant vaccinia virus (vvDD-GFP). Infection of resting and dividing NIH3T3 cells with vvDD-GFP yielded reduced viral recovery compared with wild-type (WT), TK-, or VGF- viruses from resting cultures but equivalent virus recovery from dividing cultures. Eight days after nude mice were injected i.p. with 10(7) plaque-forming units (pfu) of WT, TK-, VGF-, or vvDD-GFP vaccinia virus, tissues and tumor were harvested for viral titer determination. No virus was recovered from the brains of mice injected with vvDD-GFP compared with the other viruses, which ranged from 130 to 28,000 pfu/mg protein; however, equivalent amounts were recovered from tumor. There was no toxicity from vvDD-GFP because nude mice receiving 10(8) pfu of IP vvDD-GFP lived >100 days, whereas mice receiving WT, VGF-, or TK- virus had median survivals of only 6, 17, and 29 days, respectively. Similar results were seen when 10(9) pfu of vvDD-GFP were given. Nude mice bearing s.c. murine colon adenocarcinoma (MC38) had significant tumor regression after treatment with 10(9) pfu of systemic (i.p.) vvDD-GFP compared with control (mean tumor size, 180.71 +/- 35.26 mm(3) versus 2796.79 +/- 573.20 mm(3) 12 days after injection of virus). Our data demonstrate that a TK- and VGF- mutant vaccinia virus is significantly attenuated in resting cells in vitro and demonstrates tumor-specific replication in vivo. It is a promising vector for use in tumor-directed gene therapy, given its enhanced safety profile, tumor selectivity, and the oncolytic effects after systemic delivery.
Cancer Research 12/2001; 61(24):8751-7. · 7.86 Impact Factor
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ABSTRACT: Vaccinia virus is being investigated as a replicating vector for tumor-directed gene therapy. However, the majority of cancer patients have preformed immunologic reactivity against vaccinia virus, as a result of smallpox vaccination, which may limit its use as a vector. The Yaba-like disease (YLD) virus was investigated here as an alternative, replicating poxvirus for cancer gene therapy. We have demonstrated that the YLD virus does not cross-react with vaccinia virus antibodies, and it replicates efficiently in human tumor cells. YLD virus can be expanded and purified to high titer in CV-1 cells under conditions utilized for vaccinia virus. The YLD virus RNA polymerase was able to express genes regulated by a synthetic promoter designed for use in orthopoxviruses. We sequenced the YLD virus TK gene and created a shuttle plasmid, which allowed the recombination of the green fluorescent protein (GFP) gene into the YLD virus. In a murine model of ovarian cancer, up to 38% of cells in the tumor expressed the GFP transgene 12 days after intraperitoneal virus delivery. YLD virus has favorable characteristics as a vector for cancer gene therapy, and this potential should be explored further.
Journal of Virology 12/2001; 75(21):10300-8. · 5.40 Impact Factor
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ABSTRACT: Two mechanisms have been proposed for the intracellular movement of enveloped vaccinia virus virions: rapid actin polymerization and microtubule association. The first mechanism is used by the intracellular pathogens Listeria and Shigella, and the second is used by cellular vesicles transiting from the Golgi network to the plasma membrane. To distinguish between these models, two recombinant vaccinia viruses that express the B5R membrane protein fused to enhanced green fluorescent protein (GFP) were constructed. One had Tyr(112) and Tyr(132) of the A36R membrane protein, which are required for phosphorylation and the nucleation of actin tails, conservatively changed to Phe residues; the other had the A36R open reading frame deleted. Although the Tyr mutant was impaired in Tyr phosphorylation and actin tail formation, digital video and time-lapse confocal microscopy demonstrated that virion movement from the juxtanuclear region to the periphery was saltatory with maximal speeds of >2 microm/s and was inhibited by the microtubule-depolymerizing drug nocodazole. Moreover, this actin tail-independent movement was indistinguishable from that of a control virus with an unmutated A36R gene and closely resembled the movement of vesicles on microtubules. However, in the absence of actin tails, the Tyr mutant did not induce the formation of motile, virus-tipped microvilli and had a reduced ability to spread from cell to cell. The deletion mutant was more severely impaired, suggesting that the A36R protein has additional roles. Optical sections of unpermeabilized, B5R antibody-stained cells that expressed GFP-actin and were infected with wild-type vaccinia virus revealed that all actin tails were associated with virions on the cell surface. We concluded that the intracellular movement of intracellular enveloped virions occurs on microtubules and that the motile actin tails enhance extracellular virus spread to neighboring cells.
Journal of Virology 12/2001; 75(23):11651-63. · 5.40 Impact Factor
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ABSTRACT: Marker rescue experiments demonstrated that the genetic lesion of a previously isolated vaccinia virus temperature-sensitive mutant which forms multilayered envelope structures with lucent interiors and foci of viroplasm with dense centers mapped to the A30L open reading frame. A single base change, resulting in a nonconservative Ser-to-Phe substitution at residue 17, was associated with degradation of the A30L protein at elevated temperatures.
Journal of Virology 12/2001; 75(22):11222-6. · 5.40 Impact Factor
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ABSTRACT: Molluscum contagiosum virus, a human poxvirus that causes persistent small benign skin tumors, encodes a variety of putative immune defense proteins. Three such proteins, MC51L, MC53L, and MC54L, have 20 to 35% amino acid sequence identities with human interleukin-18 (hIL-18)-binding protein (hIL-18BP), a naturally occurring antagonist of the proinflammatory cytokine IL-18. We previously demonstrated that seven amino acids within the immunoglobulin-like domain of hIL-18BP were important for high-affinity binding to hIL-18. Model building indicated that MC54L, which has been shown to bind hIL-18, contains five of the seven amino acids at corresponding positions in its immunoglobulin-like domain, the exceptions being the conservative substitution of isoleucine for a leucine and the nonconservative substitution of valine for a phenylalanine. We found that individual alanine substitutions for these six identical or highly conserved amino acids of MC54L caused changes in affinity and binding free energy for hIL-18 that were quantitatively similar to those produced by mutagenesis of hIL-18BP. Furthermore, when the nonconserved valine of MC54L was mutated to phenylalanine, making it more like hIL-18BP, its affinity for hIL-18 increased more than 10-fold. In addition, the carboxyl-terminal half of MC54L, which has no similarity with hIL-18BP, was dispensable for hIL-18 binding. Thus, despite their relatively low overall sequence identity, MC54L and hIL-18BP have similar hIL-18 binding sites and functional epitopes. On the other hand, MC51L and MC53L have nonconservative substitutions of three to six of the seven critical amino acids of hIL-18BP and neither protein bound hIL-18, suggesting that they may interact with unidentified ligands.
Journal of Virology 11/2001; 75(20):9947-54. · 5.40 Impact Factor
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ABSTRACT: The wrapping of intracellular mature vaccinia virions by modified trans-Golgi or endosomal cisternae to form intracellular enveloped virions is dependent on at least two viral proteins encoded by the B5R and F13L open reading frames. B5R is a type I integral membrane glycoprotein, whereas F13L is an unglycosylated, palmitylated protein with a motif that is conserved in a superfamily of phospholipid-metabolizing enzymes. Microscopic visualization of the F13L protein was achieved by fusing it to the enhanced green fluorescent protein (GFP). F13L-GFP was functional when expressed by a recombinant vaccinia virus in which it replaced the wild-type F13L gene or by transfection of uninfected cells with a plasmid vector followed by infection with an F13L deletion mutant. In uninfected or infected cells, F13L-GFP was associated with Golgi cisternae and post-Golgi vesicles containing the LAMP 2 late endosomal-lysosomal marker. Association of F13L-GFP with vesicles was dependent on an intact phospholipase catalytic motif and sites of palmitylation. The B5R protein was also associated with LAMP2-containing vesicles when F13L-GFP was coexpressed, but was largely restricted to Golgi cisternae in the absence of F13L-GFP or when the F13L moiety was mutated. We suggest that the F13L protein, like its human phospholipase D homolog, regulates vesicle formation and that this process is involved in intracellular enveloped virion membrane formation.
Journal of Virology 09/2001; 75(16):7528-42. · 5.40 Impact Factor
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ABSTRACT: The previously uncharacterized A30L gene of vaccinia virus has orthologs in all vertebrate poxviruses but no recognizable nonpoxvirus homologs or functional motifs. We determined that the A30L gene was regulated by a late promoter and encoded a protein of approximately 9 kDa. Immunoelectron microscopy of infected cells indicated that the A30L protein was associated with viroplasm enclosed by crescent and immature virion membranes. The A30L protein was also present in mature virions and was partially released by treatment with a nonionic detergent and reducing agent, consistent with a location in the matrix between the core and envelope. To determine the role of the A30L protein, we constructed a stringent conditional lethal mutant with an inducible A30L gene. In the absence of inducer, synthesis of viral early and late proteins occurred but the proteolytic processing of certain core proteins was inhibited, suggesting an assembly block. Inhibition of virus maturation was confirmed by electron microscopy. Under nonpermissive conditions, we observed aberrant large, dense, granular masses of viroplasm with clearly defined margins; viral crescent membranes that appeared normal except for their location at a distance from viroplasm; empty immature virions; and an absence of mature virions. The data indicated that the A30L protein is needed for vaccinia virus morphogenesis, specifically the association of the dense viroplasm with viral membranes.
Journal of Virology 08/2001; 75(13):5752-61. · 5.40 Impact Factor
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ABSTRACT: The vaccinia virus A22R gene encodes a protein that is homologous to the bacterial enzyme RuvC and specifically cleaves and resolves four-way DNA Holliday junctions into linear duplex products. To investigate the role of the vaccinia virus Holliday junction resolvase during an infection, we constructed two recombinant viruses: vA22-HA, which has a short C-terminal epitope tag appended to the A22R open reading frame, and vA22i, in which the original A22R gene is deleted and replaced by an inducible copy. Polyacrylamide gel electrophoresis and Western blot analysis of extracts and purified virions from cells infected with vA22-HA revealed that the resolvase was expressed after the onset of DNA replication and incorporated into virion cores. vA22i exhibited a conditional replication defect. In the absence of an inducer, (i) viral protein synthesis was unaffected, (ii) late-stage viral DNA replication was reduced, (iii) most of the newly synthesized viral DNA remained in a branched or concatemeric form that caused it to be trapped at the application site during pulsed-field gel electrophoresis, (iv) cleavage of concatemer junctions was inhibited, and (v) virion morphogenesis was arrested at an immature stage. These data indicated multiple roles for the vaccinia virus Holliday junction resolvase in the replication and processing of viral DNA into unit-length genomes.
Journal of Virology 08/2001; 75(14):6460-71. · 5.40 Impact Factor
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D H Barouch,
S Santra,
M J Kuroda,
J E Schmitz,
R Plishka,
A Buckler-White,
A E Gaitan,
R Zin,
J H Nam,
L S Wyatt,
M A Lifton,
C E Nickerson, B Moss,
D C Montefiori,
V M Hirsch,
N L Letvin
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ABSTRACT: Since cytotoxic T lymphocytes (CTLs) are critical for controlling human immunodeficiency virus type 1 (HIV-1) replication in infected individuals, candidate HIV-1 vaccines should elicit virus-specific CTL responses. In this report, we study the immune responses elicited in rhesus monkeys by a recombinant poxvirus vaccine and the degree of protection afforded against a pathogenic simian-human immunodeficiency virus SHIV-89.6P challenge. Immunization with recombinant modified vaccinia virus Ankara (MVA) vectors expressing SIVmac239 gag-pol and HIV-1 89.6 env elicited potent Gag-specific CTL responses but no detectable SHIV-specific neutralizing antibody (NAb) responses. Following intravenous SHIV-89.6P challenge, sham-vaccinated monkeys developed low-frequency CTL responses, low-titer NAb responses, rapid loss of CD4+ T lymphocytes, high-setpoint viral RNA levels, and significant clinical disease progression and death in half of the animals by day 168 postchallenge. In contrast, the recombinant MVA-vaccinated monkeys demonstrated high-frequency secondary CTL responses, high-titer secondary SHIV-89.6-specific NAb responses, rapid emergence of SHIV-89.6P-specific NAb responses, partial preservation of CD4+ T lymphocytes, reduced setpoint viral RNA levels, and no evidence of clinical disease or mortality by day 168 postchallenge. There was a statistically significant correlation between levels of vaccine-elicited CTL responses prior to challenge and the control of viremia following challenge. These results demonstrate that immune responses elicited by live recombinant vectors, although unable to provide sterilizing immunity, can control viremia and prevent disease progression following a highly pathogenic AIDS virus challenge.
Journal of Virology 07/2001; 75(11):5151-8. · 5.40 Impact Factor
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ABSTRACT: We produced an infectious vaccinia virus that expressed the B5R envelope glycoprotein fused to the enhanced green fluorescent protein (GFP), allowing us to visualize intracellular virus movement in real time. Previous transfection studies indicated that fusion of GFP to the C-terminal cytoplasmic domain of B5R did not interfere with Golgi localization of the viral protein. To determine whether B5R-GFP was fully functional, we started with a B5R deletion mutant that made small plaques and inserted the B5R-GFP gene into the original B5R locus. The recombinant virus made normal-sized plaques and acquired the ability to form actin tails, indicating reversal of the mutant phenotype. Moreover, immunogold electron microscopy revealed that both intracellular enveloped virions (IEV) and extracellular enveloped virions contained B5R-GFP. By confocal microscopy of live infected cells, we visualized individual fluorescent particles, corresponding to IEV in size and shape, moving from a juxtanuclear location to the periphery of the cell, where they usually collected prior to association with actin tails. The fluorescent particles could be seen emanating from cells at the tips of microvilli. Using a digital camera attached to an inverted fluorescence microscope, we acquired images at 1 frame/s. At this resolution, IEV movement appeared saltatory; in some frames there was no net movement, whereas in others movement exceeded 2 microm/s. Further studies indicated that IEV movement was reversibly arrested by the microtubule-depolymerizing drug nocodazole. This result, together with the direction, speed, and saltatory motion of IEV, was consistent with a role for microtubules in intracellular transport of IEV.
Journal of Virology 06/2001; 75(10):4802-13. · 5.40 Impact Factor
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ABSTRACT: This unit describes the maintenance of cell lines used with vaccinia virus, both in monolayer cultures and in suspension. The suspended cell culture is then used in the preparation of vaccinia virus stocks. The preparation of chick embryo fibroblasts (CEF) is also presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Additionally, support protocols are presented for the titration of standard and MVA vaccinia virus stocks.
Current protocols in protein science / editorial board, John E. Coligan ... [et al.] 06/2001; Chapter 5:Unit5.12.
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ABSTRACT: The vaccinia virus expression system differs from others in that transcription occurs in the cytoplasm of mammalian cells rather than in the nucleus. As a vector, vaccinia virus has a number of useful characteristics, including a capacity that permits cloning large fragments of foreign DNA (20+ kbp) with retention of infectivity, a wide host range, a relatively high level of protein synthesis, and "appropriate" transport, secretion, processing, and posttranslational modifications as dictated by the primary structure of the expressed protein and the cell type used. This overview discusses the life cycle of the vaccinia virus along with effects of vaccinia infection. The vaccinia vector expression system is described along with specific steps for expressing genes using these vectors. Important safety considerations are also presented.
Current protocols in protein science / editorial board, John E. Coligan ... [et al.] 06/2001; Chapter 5:Unit5.11.
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ABSTRACT: The human interleukin (IL)-18-binding protein (hIL-18BP) is a naturally occurring antagonist of IL-18, a proinflammatory cytokine that is related to IL-1beta and has an important role in defense against microbial invaders. As its name implies, the hIL-18BP binds to IL-18 with high affinity and prevents the interaction of IL-18 with its receptor. We genetically modified the C terminus of hIL-18BP by appending a 15-amino acid biotinylation recognition site and a six-histidine tag and then performed site-directed mutagenesis to determine the functional epitopes that mediate efficient binding to IL-18. The mutated IL-18BPs were secreted from mammalian cells, captured by metal affinity chromatography, biotinylated in situ, eluted, and immobilized on streptavidin-coated chips. Using surface plasmon resonance, we identified seven amino acids of hIL-18BP which, when changed individually to alanine, caused an 8-750-fold decrease in binding affinity, largely because of increased off-rates. These seven amino acids localized to the predicted beta-strand c and d of hIL-18BP immunoglobulin-like domain, and most had hydrophobic side chains. Just two amino acids, tyrosine 97 and phenylalanine 104, contributed approximately 50% of the binding free energy. Information obtained from these studies could contribute to the design of molecular antagonists of IL-18 for treatment of inflammatory diseases.
Journal of Biological Chemistry 06/2001; 276(20):17380-6. · 4.77 Impact Factor
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ABSTRACT: The B1 gene of vaccinia virus encodes a serine/threonine protein kinase that is expressed early after infection. Under nonpermissive conditions, temperature-sensitive mutants (ts2 and ts25) that map to B1 fail to efficiently replicate viral DNA. Our goal was to extend studies on the function of B1 by determining if the kinase is required for intermediate or late gene expression, two events that ordinarily depend on viral DNA replication. First, we established that early viral gene expression occurred at the nonpermissive temperature. By using a transfection procedure that circumvents the viral DNA replication requirement, we found that reporter genes regulated by an intermediate promoter were transcribed only under conditions permissive for expression of active B1. To assay late gene expression, the T7 RNA polymerase gene was inserted into the genome of ts25 to form ts25/T7. A DNA replication-independent late gene transcription system was established by cotransfecting plasmids containing T7 promoter-driven late gene transcription factors and a late promoter reporter gene into ts25/T7-infected cells. Late genes, unlike intermediate genes, were expressed at the nonpermissive temperature. Last, we showed that overexpression of B1 stimulated intermediate but inhibited late gene expression in cells infected with wild-type virus.
Journal of Virology 06/2001; 75(9):4048-55. · 5.40 Impact Factor
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ABSTRACT: This unit first describes how to infect cells with vaccinia virus and then transfect them with a plasmid-transfer vector to generate a recombinant virus. Methods are also presented for purifying vaccinia virus and for isolating viral DNA, which can be used during transfection. Also presented are selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented.
Current protocols in protein science / editorial board, John E. Coligan ... [et al.] 06/2001; Chapter 5:Unit5.13.
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ABSTRACT: After a recombinant vaccinia virus is made, its DNA and protein products can be analyzed in several ways. Protocols are provided in this unit for identification of the recombinant virus by PCR (with verification of correct insertion of the DNA by Southern blotting) and by dot-blot hybridization. Also, when antibodies are available, protein expression can be analyzed by immunological methods detailed here such as dot blotting with an antibody, immunoblotting and/or immunoprecipitation. In addition, immunostaining can be used for identification of recombinant plaques as well as for determination of the purity of a recombinant virus stock. All of the protocols in this unit can be used for characterization of modified vaccinia virus Ankara (MVA) recombinant viruses.
Current protocols in protein science / editorial board, John E. Coligan ... [et al.] 06/2001; Chapter 5:Unit5.14.
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ABSTRACT: This unit describes a transient cytoplasmic expression system that relies on the synthesis of the bacteriophage T7 RNA polymerase in the cytoplasm of mammalian cells. A gene of interest is inserted into a plasmid such that it comes under the control of the T7 RNA polymerase promoter (p(T7)). Using liposome-mediated transfection, this recombinant plasmid is introduced into the cytoplasm of cells infected with vTF7-3, a recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase. During incubation, the gene of interest is transcribed with high efficiency by T7 RNA polymerase. For large-scale work, protocols are provided for insertion of the p(T7)-regulated gene into a second recombinant vaccinia virus by homologous recombination and subsequent coinfection with vTF7-3 into cells grown in suspension or for direct transfection into OST7-1 cells (a stable cell line that constitutively expresses the T7 RNA polymerase). Expressed protein is then analyzed by pulse-labeling and purified. One new development to this vaccinia virus/T7 RNA polymerase hybrid expression system described here is the VOTE inducible expression system, which eliminates the need to use two recombinant viruses or a special cell line.
Current protocols in protein science / editorial board, John E. Coligan ... [et al.] 06/2001; Chapter 5:Unit5.15.
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ABSTRACT: This unit describes a transient cytoplasmic expression system that relies on the synthesis of the bacteriophage T7 RNA polymerase in the cytoplasm of mammalian cells. A gene of interest is inserted into a plasmid such that it comes under the control of the T7 RNA polymerase promoter (p(T7)). Using liposome-mediated transfection, this recombinant plasmid is introduced into the cytoplasm of cells infected with vTF7-3, a recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase. During incubation, the gene of interest is transcribed with high efficiency by T7 RNA polymerase. For large-scale work, protocols are provided for insertion of the p(T7)-regulated gene into a second recombinant vaccinia virus by homologous recombination and subsequent coinfection with vTF7-3 into cells grown in suspension or for direct transfection into OST7-1 cells (a stable cell line that constitutively expresses the T7 RNA polymerase). Expressed protein is then analyzed by pulse-labeling and purified. One new development to this vaccinia virus/T7 RNA polymerase hybrid expression system described here is the VOTE inducible expression system, which eliminates the need to use two recombinant viruses or a special cell line.
Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] 06/2001; Chapter 16:Unit16.19.
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R R Amara,
F Villinger,
J D Altman,
S L Lydy,
S P O'Neil,
S I Staprans,
D C Montefiori,
Y Xu,
J G Herndon,
L S Wyatt, [......],
P L Earl,
J M Smith,
H L Ma,
B D Grimm,
M L Hulsey,
J Miller,
H M McClure,
J M McNicholl, B Moss,
H L Robinson
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ABSTRACT: Heterologous prime/boost regimens have the potential for raising high levels of immune responses. Here we report that DNA priming followed by a recombinant modified vaccinia Ankara (rMVA) booster controlled a highly pathogenic immunodeficiency virus challenge in a rhesus macaque model. Both the DNA and rMVA components of the vaccine expressed multiple immunodeficiency virus proteins. Two DNA inoculations at 0 and 8 weeks and a single rMVA booster at 24 weeks effectively controlled an intrarectal challenge administered 7 months after the booster. These findings provide hope that a relatively simple multiprotein DNA/MVA vaccine can help to control the acquired immune deficiency syndrome epidemic.
Science 05/2001; 292(5514):69-74. · 31.20 Impact Factor
