Compact, Synthetic, Vaccinia Virus Early/Late Promoter for Protein Expression

Chakrabarti, Sekhar; Sisler, Jerry R.; Moss, Bernard

doi:10.2144/97236st07

Cited by 358 publications

(292 citation statements)

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“…rVV that expresses group M consensus (CON6) rgp120 (11) was generated as described previously (19). Briefly, a DNA fragment encoding CON6 gp120 was produced by introducing stop codons after the gp120 cleavage site (REKR) by PCR and was cloned into a transfer vector, pSC65 vector (from Bernard Moss) at SalI and KpnI restriction enzyme sites (3). BSC-1 cells were seeded at 2 ϫ 10 5 in each well in a 6-well plate and were infected with wild-type vaccinia virus (WR) at a multiplicity of infection of 0.1 PFU/cell, and 2 h after infection pSC65-derived plasmids containing CON6 env genes were transfected into the VV-infected cells by using Lipofectamine 2000 based on the protocol recommended by the manufacturer (Invitrogen, Carlsbad, Calif.).…”

Section: Methodsmentioning

confidence: 99%

Immunogenicity of Constrained Monoclonal Antibody A32-Human Immunodeficiency Virus (HIV) Env gp120 Complexes Compared to That of Recombinant HIV Type 1 gp120 Envelope Glycoproteins

Liao

Alam

Mascola

et al. 2004

J Virol

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One strategy for the generation of broadly reactive neutralizing antibodies (NA) against human immunodeficiency virus type 1 (HIV-1) primary isolates is to use immunogens that have constrained HIV-1 envelope gp120 conformations reflective of triggered envelope on the surface of virions. A major change in gp120 following binding to CD4 is the enhanced exposure of the CCR5 binding site. One inducer of CCR5 binding site epitopes on gp120 is the human anti-gp120 monoclonal antibody, A32. We have made cross-linked A32-rgp120 89.6 and A32-rgp120 BaL complexes and have compared their immunogenicities to those of uncomplexed recombinant gp120 BaL (rgp120 BaL ) and rgp120 89.6 . A32-rgp120 89.6 and A32-rgp120 BaL complexes had stable induced CCR5 binding site expression compared to that of uncomplexed rgp120s. However, the A32-rgp120 complexes had similar capacities in guinea pigs for induction of NA against HIV-1 primary isolates versus that of rgp120 alone. A32-rgp120 89.6 induced antibodies that neutralized 6 out of 11 HIV-1 isolates, while rgp120 89.6 alone induced antibodies that neutralized 4 out of 11 HIV-1 isolates. A32-rgp120 BaL complexes induced antibodies that neutralized 4 out of 14 HIV-1 isolates while, surprisingly, non-cross-linked rgp120 BaL induced antibodies that neutralized 9 out of 14 (64%) HIV-1 isolates. Thus, stable enhanced expression of the coreceptor binding site on constrained gp120 is not sufficient for inducing broadly neutralizing anti-HIV-1 NA. Moreover, the ability of HIV-1 rgp120 BaL to induce antibodies that neutralized ϳ60% of subtype B HIV-1 isolates warrants consideration of using HIV-1 BaL as a starting point for immunogen design for subtype B HIV-1 experimental immunogens.The design of immunogens that will neutralize a broad spectrum of human immunodeficiency virus type 1 (HIV-1) primary isolates is a high priority for development of a practical HIV-1 vaccine. Following binding of virion gp120 to cellular CD4, the HIV-1 envelope undergoes conformational changes that result in exposure of the coreceptor binding site leading to virion-host cell fusion (1, 24).One potential strategy for inducing broadly reactive neutralizing antibodies (NA) is to construct immunogens that are constrained and reflect wild-type fusion intermediate Env forms, with the hope of stably exposing conserved immunogenic epitopes that otherwise would not be readily available for antibody induction. An alternative strategy for selection of Env immunogens is to select the best envelopes from among many screened for their ability to induce antibodies that broadly neutralize HIV-1 primary isolates.In this work we describe the immunogenicity of recombinant HIV-1 gp120s complexed with the CD4 mimic, monoclonal antibody (MAb) A32. Like CD4, MAb A32 induces expression of the CCR5 binding site on rgp120, but unlike CD4, MAb A32 does not bind at the CD4 binding site (26). Thus, MAb A32 has a potential advantage over CD4 in a constrained Env complex in that A32-rgp120 complexes have exposed CD4 binding sites. Here we show ...

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Section: Methodsmentioning

confidence: 99%

Immunogenicity of Constrained Monoclonal Antibody A32-Human Immunodeficiency Virus (HIV) Env gp120 Complexes Compared to That of Recombinant HIV Type 1 gp120 Envelope Glycoproteins

Liao

Alam

Mascola

et al. 2004

J Virol

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“…The fcy1 gene was ligated into pCB02311, resulting in pMP-CD, in which the fcy1 gene is driven by the strong synthetic early late promoter for vaccinia. 40 To create the new virus vvDD-CD, CV-1 cells were transfected with the shuttle plasmid pMP-CD, and then infected with virus vSC20 that contains a genetic deletion in the vgf locus. 25 Recombinant VV was subsequently isolated from the cells by mycophenolic acid selection, and the final viral construct was confirmed with DNA sequencing of viral genomic DNA.…”

Section: Recombinant Vvmentioning

confidence: 99%

Oncolytic virotherapy for ovarian carcinomatosis using a replication-selective vaccinia virus armed with a yeast cytosine deaminase gene

et al. 2007

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In this study, we assessed the ability of a highly tumor-selective oncolytic vaccinia virus armed with a yeast cytosine deaminase gene to infect and lyse human and murine ovarian tumors both in vitro and in vivo. The virus vvDD-CD could infect, replicate in and effectively lyse both human and mouse ovarian cancer cells in vitro. In two different treatment schedules involving either murine MOSEC or human A2780 ovarian carcinomatosis models, regional delivery of vvDD-CD selectively targeted tumor cells and ovarian tissue, effectively delaying the development of either tumor or ascites and leading to significant survival advantages. Oncolytic virotherapy using vvDD-CD in combination with the prodrug 5-fluorocytosine conferred an additional long-term survival advantage upon tumor-bearing immunocompetent mice. These findings demonstrate that a tumor-selective oncolytic vaccinia combined with gene-directed enzyme prodrug therapy is a highly effective strategy for treating advanced ovarian cancers in both syngeneic mouse and human xenograft models. Given the biological safety, tumor selectivity and oncolytic potency of this armed oncolytic virus, this dual therapy merits further investigation as a promising new treatment for metastatic ovarian cancer.

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“…The following recombinant viruses expressing ␤-galactosidase (␤GAL) under the control of the indicated promoters were used: vMJ343 (synthetic strong early promoter), 64 WR-G8R (natural intermediate), 65 vSC56 (synthetic strong early/late), 66 vTFCLZ-1 (p11 late promoter) and VV-GFPS65T, 67 which expresses GFP under the control of a synthetic strong early/late promoter. Virus stocks were prepared on CV-1 cells titered on CV-1 or BS-C-1 monolayers according to standard procedures.…”

Section: Virusmentioning

confidence: 99%

Poxvirus as a vector to transduce human dendritic cells for immunotherapy: abortive infection but reduced APC function

et al. 2000

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Compact, Synthetic, Vaccinia Virus Early/Late Promoter for Protein Expression

Cited by 358 publications

References 1 publication

Immunogenicity of Constrained Monoclonal Antibody A32-Human Immunodeficiency Virus (HIV) Env gp120 Complexes Compared to That of Recombinant HIV Type 1 gp120 Envelope Glycoproteins

Immunogenicity of Constrained Monoclonal Antibody A32-Human Immunodeficiency Virus (HIV) Env gp120 Complexes Compared to That of Recombinant HIV Type 1 gp120 Envelope Glycoproteins

Oncolytic virotherapy for ovarian carcinomatosis using a replication-selective vaccinia virus armed with a yeast cytosine deaminase gene

Poxvirus as a vector to transduce human dendritic cells for immunotherapy: abortive infection but reduced APC function

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