Production of High-Titer Human Immunodeficiency Virus Type 1 Pseudotyped with Vesicular Stomatitis Virus Glycoprotein

Methods ◽  
1997 ◽  
Vol 12 (4) ◽  
pp. 337-342 ◽  
Author(s):  
Steven R. Bartz ◽  
Marie A. Vodicka
Keyword(s):  
Human Immunodeficiency Virus ◽  
Vesicular Stomatitis Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
High Titer ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Virus Glycoprotein ◽  
Immunodeficiency Virus ◽  
Vesicular Stomatitis

scholarly journals Attenuation of Recombinant Vesicular Stomatitis Virus-Human Immunodeficiency Virus Type 1 Vaccine Vectors by Gene Translocations and G Gene Truncation Reduces Neurovirulence and Enhances Immunogenicity in Mice

2007 ◽  
Vol 82 (1) ◽  
pp. 207-219 ◽  
Author(s):  
David Cooper ◽  
Kevin J. Wright ◽  
Priscilla C. Calderon ◽  
Min Guo ◽  
Farooq Nasar ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Immune Responses ◽  
Vesicular Stomatitis Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
N Gene ◽  
Gag Protein ◽  
Immunodeficiency Virus ◽  
Vesicular Stomatitis

ABSTRACT Recombinant vesicular stomatitis virus (rVSV) has shown great potential as a new viral vector for vaccination. However, the prototypic rVSV vector described previously was found to be insufficiently attenuated for clinical evaluation when assessed for neurovirulence in nonhuman primates. Here, we describe the attenuation, neurovirulence, and immunogenicity of rVSV vectors expressing human immunodeficiency virus type 1 Gag. These rVSV vectors were attenuated by combinations of the following manipulations: N gene translocations (N4), G gene truncations (CT1 or CT9), noncytopathic M gene mutations (Mncp), and positioning of the gag gene into the first position of the viral genome (gag1). The resulting N4CT1-gag1, N4CT9-gag1, and MncpCT1-gag1 vectors demonstrated dramatically reduced neurovirulence in mice following direct intracranial inoculation. Surprisingly, in spite of a very high level of attenuation, the N4CT1-gag1 and N4CT9-gag1 vectors generated robust Gag-specific immune responses following intramuscular immunization that were equivalent to or greater than immune responses generated by the more virulent prototypic vectors. MncpCT1-gag1 also induced Gag-specific immune responses following intramuscular immunization that were equivalent to immune responses generated by the prototypic rVSV vector. Placement of the gag gene in the first position of the VSV genome was associated with increased in vitro expression of Gag protein, in vivo expression of Gag mRNA, and enhanced immunogenicity of the vector. These findings demonstrate that through directed manipulation of the rVSV genome, vectors that have reduced neurovirulence and enhanced immunogenicity can be made.

scholarly journals Pseudotyping human immunodeficiency virus type 1 by vesicular stomatitis virus G protein does not reduce the cell-dependent requirement of Vif for optimal infectivity: functional difference between Vif and Nef

1999 ◽  
Vol 80 (11) ◽  
pp. 2945-2949 ◽  
Author(s):  
Hirofumi Akari ◽  
Tsuneo Uchiyama ◽  
Tomoharu Fukumori ◽  
Shinya Iida ◽  
A. Hajime Koyama ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Vesicular Stomatitis Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Endocytic Pathway ◽  
Functional Difference ◽  
Immunodeficiency Virus ◽  
Vesicular Stomatitis ◽  
Hiv 1

The functions of Vif and Nef in human immunodeficiency virus type 1 (HIV-1) infection have some similarities: Vif- and Nef-dependent enhancement of HIV-1 replication is cell type-specific, and defective mutations in these genes result in restricted proviral DNA synthesis in infected cells. It has recently been shown that pseudotyping HIV-1 by the envelope glycoprotein of vesicular stomatitis virus (VSV-G) targets HIV-1 entry to an endocytic pathway and suppresses the requirement of Nef for virus infectivity. In this study, we examined whether VSV-G pseudotyping suppresses the requirement of Vif for HIV-1 infectivity. It was found that pseudotyping HIV-1 by VSV-G did not compensate for the Vif function. Together with the findings that Vif does not influence virus binding/entry and virion incorporation of Env, it is concluded that Vif enhances HIV-1 infectivity at the post-entry step(s) independently of the Env function by a different mechanism to that of Nef.

scholarly journals Expression and processing of human immunodeficiency virus type 1 gp160 using the vesicular stomatitis virus New Jersey serotype vector system

2009 ◽  
Vol 90 (5) ◽  
pp. 1135-1140 ◽  
Author(s):  
Kunyu Wu ◽  
Gyoung Nyoun Kim ◽  
C. Yong Kang
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Vesicular Stomatitis Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Vector System ◽  
Immunodeficiency Virus ◽  
Vesicular Stomatitis ◽  
Hiv 1

The Indiana serotype of vesicular stomatitis virus (VSVIND), but not the New Jersey serotype (VSVNJ), has been widely used as a gene expression vector. In terms of prime–boost-based vaccine strategies, it would be desirable to use two different VSV serotypes to avoid immunity against the priming viral vector. Here, we report that we have applied the VSVNJ vector system for expression of the env gene of human immunodeficiency virus type 1 (HIV-1). The HIV-1 env gene was inserted into the VSVNJ vector system at two different sites: between the P and M genes (NP-gp160-MGL) and between the G and L genes (NPMG-gp160-L). The HIV-1 env gene product, gp160, was efficiently expressed and processed in cells infected with either of these two recombinant VSV–HIV-1gp160 viruses. In this study, we have investigated the applicability of the VSVNJ vector system for foreign gene expression.

scholarly journals Human Immunodeficiency Virus Type 1 Vectors with Alphavirus Envelope Glycoproteins Produced from Stable Packaging Cells

2005 ◽  
Vol 79 (3) ◽  
pp. 1765-1771 ◽  
Author(s):  
Blair L. Strang ◽  
Yasuhiro Takeuchi ◽  
Thomas Relander ◽  
Johan Richter ◽  
Ranbir Bailey ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Semliki Forest Virus ◽  
High Titer ◽  
Hematopoietic Stem ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Alphavirus glycoproteins have broad host ranges. Human immunodeficiency virus type 1 (HIV-1) vectors pseudotyped with their glycoproteins could extend the range of tissues that can be transduced in both humans and animal models. Here, we established stable producer cell lines for HIV vectors pseudotyped with alphavirus Ross River virus (RRV) and Semliki Forest virus (SFV) glycoproteins E2E1. RRV E2E1-stable clones could routinely produce high-titer pseudotyped vectors for at least 5 months. SFV E2E1-stable clones, however, produced relatively low titers. We examined the properties of RRV E2E1-pseudotyped vectors [HIV-1(RRV)] and compared them with amphotropic murine leukemia virus Env- and vesicular stomatitis virus glycoprotein G-pseudotyped vectors. HIV-1(RRV) displayed a number of characteristics which would be advantageous in ex vivo and in vivo experiments, including resistance to inactivation by heat-labile components in fresh human sera and thermostability at 37°C. Upon single-step concentration by ultracentrifugation of HIV-1(RRV), we could achieve vector stocks with titers up to 6 × 107 IU/ml. HIV-1(RRV) efficiently transduced cells from several different species, including murine primary dendritic cells, but failed to transduce human and murine T cells as well as human hematopoietic stem cells (HSC). These results indicate that HIV-1(RRV) could be used in a number of applications including animal model experiments and suggest that expression of RRV cellular receptors is limited or absent in certain cell types such as T cells and human HSC.

scholarly journals High-Titer Human Immunodeficiency Virus Type 1-Based Vector Systems for Gene Delivery into Nondividing Cells

1998 ◽  
Vol 72 (11) ◽  
pp. 8873-8883 ◽  
Author(s):  
Hideki Mochizuki ◽  
Joan P. Schwartz ◽  
Koichi Tanaka ◽  
Roscoe O. Brady ◽  
Jakob Reiser
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
High Titer ◽  
Vector System ◽  
Coding Region ◽  
Immunodeficiency Virus ◽  
Env Protein ◽  
Hiv 1

ABSTRACT Previously we designed novel pseudotyped high-titer replication defective human immunodeficiency virus type 1 (HIV-1) vectors to deliver genes into nondividing cells (J. Reiser, G. Harmison, S. Kluepfel-Stahl, R. O. Brady, S. Karlsson, and M. Schubert, Proc. Natl. Acad. Sci. USA 93:15266–15271, 1996). Since then we have made several improvements with respect to the safety, flexibility, and efficiency of the vector system. A three-plasmid expression system is used to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells with a defective packaging construct, a plasmid coding for a heterologous envelope (Env) protein, and a vector construct harboring a reporter gene such as neo, ShlacZ (encoding a phleomycin resistance/β-galactosidase fusion protein), HSA (encoding mouse heat-stable antigen), or EGFP (encoding enhanced green fluorescent protein). The packaging constructs lack functional Vif, Vpr, and Vpu proteins and/or a large portion of the Env coding region as well as the 5′ and 3′ long terminal repeats, the Nef function, and the presumed packaging signal. Using G418 selection, we routinely obtained vector particles pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) with titers of up to 8 × 107 CFU/μg of p24, provided that a functional Tat coding region was present in the vector. Vector constructs lacking a functional Tat protein yielded titers of around 4 × 106 to 8 × 106 CFU/μg of p24. Packaging constructs with a mutation within the integrase (IN) core domain profoundly affected colony formation and expression of the reporter genes, indicating that a functional IN protein is required for efficient transduction. We explored the abilities of other Env proteins to allow formation of pseudotyped HIV-1 particles. The rabies virus and Mokola virus G proteins yielded high-titer infectious pseudotypes, while the human foamy virus Env protein did not. Using the improved vector system, we successfully transduced contact-inhibited primary human skin fibroblasts and postmitotic rat cerebellar neurons and cardiac myocytes, a process not affected by the lack of the accessory proteins.

scholarly journals High-Level Primary CD8+ T-Cell Response to Human Immunodeficiency Virus Type 1 Gag and Env Generated by Vaccination with Recombinant Vesicular Stomatitis Viruses

2002 ◽  
Vol 76 (6) ◽  
pp. 2730-2738 ◽  
Author(s):  
Karl Haglund ◽  
Ingrid Leiner ◽  
Kristen Kerksiek ◽  
Linda Buonocore ◽  
Eric Pamer ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Primary Response ◽  
Vector System ◽  
Primary Immune Response ◽  
Immunodeficiency Virus ◽  
Vesicular Stomatitis ◽  
Vesicular Stomatitis Viruses

ABSTRACT We investigated the primary cellular immune responses to human immunodeficiency virus type 1 (HIV-1) Env and Gag proteins elicited by recombinant vesicular stomatitis viruses (rVSVs). The primary response to Env peaked 5 to 7 days after intraperitoneal vaccination, at which time 40% of CD8+ cells were Env tetramer positive and activated (CD62LLo). These freshly isolated cells actively lysed target cells pulsed with the p18-I10 peptide and secreted gamma interferon and tumor necrosis factor alpha after stimulation with the Env p18-I10 peptide. The primary response to Env elicited by rVSVs was sixfold higher than that elicited by recombinant vaccinia viruses (rVVs) at 5 days postvaccination. An intranasal route of vaccination with VSV-Env also elicited a strong primary response to Env. The primary immune response to Gag elicited by rVSV peaked 7 days after vaccination, at which time 3% of CD8+ cells were Gag tetramer positive and CD62LLo and functional by intracellular cytokine staining. This response was eightfold higher than that elicited by rVV expressing Gag. VSV-GagEnv, which expresses both Gag and Env from a single recombinant, also induced strong cytotoxic T-lymphocyte (CTL) responses to both Env and Gag. Our quantitative analyses illustrate the potency of the VSV vector system in CTL induction.

scholarly journals Phosphorothioate 2′ Deoxyribose Oligomers as Microbicides That Inhibit Human Immunodeficiency Virus Type 1 (HIV-1) Infection and Block Toll-Like Receptor 7 (TLR7) and TLR9 Triggering by HIV-1

2010 ◽  
Vol 54 (10) ◽  
pp. 4064-4073 ◽  
Author(s):  
Joseph A. Fraietta ◽  
Yvonne M. Mueller ◽  
Duc H. Do ◽  
Veronica M. Holmes ◽  
Mary K. Howett ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Mononuclear Cells ◽  
High Titer ◽  
Target Cells ◽  
Continuous Exposure ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Topical microbicides may prove to be an important strategy for preventing human immunodeficiency virus type 1 (HIV-1) transmission. We examined the safety and efficacy of sequence-nonspecific phosphorothioate 2′ deoxyribose oligomers as potential novel microbicides. A short, 13-mer poly(T) phosphorothioate oligodeoxynucleotide (OPB-T) significantly inhibited infection of primary peripheral blood mononuclear cells (PBMC) by high-titer HIV-1Ba-L and simian immunodeficiency virus mac251 (SIVmac251). Continuous exposure of human vaginal and foreskin tissue explants to OPB-T showed no toxicity. An abasic 14-mer phosphorothioate 2′ deoxyribose backbone (PDB) demonstrated enhanced anti-HIV-1 activity relative to OPB-T and other homo-oligodeoxynucleotide analogs. When PDB was used to pretreat HIV-1, PDB was effective against R5 and X4 isolates at a half-maximal inhibitory concentration (IC50) of <1 μM in both PBMC and P4-R5 MAGI cell infections. PDB also reduced HIV-1 infectivity following the binding of virus to target cells. This novel topical microbicide candidate exhibited an excellent in vitro safety profile in human PBMC and endocervical epithelial cells. PDB also retained activity in hydroxyethylcellulose gel at pH 4.4 and after transition to a neutral pH and was stable in this formulation for 30 days at room temperature. Furthermore, the compound displayed potent antiviral activity following incubation with a Lactobacillus strain derived from normal vaginal flora. Most importantly, PDB can inhibit HIV-1-induced alpha interferon production. Phosphorothioate 2′ deoxyribose oligomers may therefore be promising microbicide candidates that inhibit HIV-1 infection and also dampen the inflammation which is critical for the initial spread of the virus.

scholarly journals Identification and Characterization of UK-201844, a Novel Inhibitor That Interferes with Human Immunodeficiency Virus Type 1 gp160 Processing

2007 ◽  
Vol 51 (10) ◽  
pp. 3554-3561 ◽  
Author(s):  
Wade S. Blair ◽  
Joan Cao ◽  
Lynn Jackson ◽  
Judith Jimenez ◽  
Qinghai Peng ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Activity ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Laboratory Strain ◽  
Immunodeficiency Virus ◽  
Vesicular Stomatitis ◽  
Hiv 1

ABSTRACT More than 106 compounds were evaluated in a human immunodeficiency virus type 1 (HIV-1) high-throughput antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (UK-201844). UK-201844 exhibited antiviral activity against HIV-1 NL4-3 in MT-2 and PM1 cells, with 50% effective concentrations of 1.3 and 2.7 μM, respectively, but did not exhibit measurable antiviral activity against the closely related HIV-1 IIIB laboratory strain. UK-201844 specifically inhibited the production of infectious virions packaged with an HIV-1 envelope (Env), but not HIV virions packaged with a heterologous Env (i.e., the vesicular stomatitis virus glycoprotein), suggesting that the compound targets HIV-1 Env late in infection. Subsequent antiviral assays using HIV-1 NL4-3/IIIB chimeric viruses showed that HIV-1 Env sequences were critical determinants of UK-201844 susceptibility. Consistent with this, in vitro resistant-virus studies revealed that amino acid substitutions in HIV-1 Env are sufficient to confer resistance to UK-201844. Western analysis of HIV Env proteins expressed in transfected cells or in isolated virions showed that UK-201844 inhibited HIV-1 gp160 processing, resulting in the production of virions with nonfunctional Env glycoproteins. Our results demonstrate that UK-201844 represents the prototype for a unique HIV-1 inhibitor class that directly or indirectly interferes with HIV-1 gp160 processing.

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