Inducible Vesicular Stomatitis Virus (VSV) L Cell Line for Packaging of Recombinant VSV

Abstract The novel pandemic betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected at least 120 million people since its identification as the cause of a December 2019 viral pneumonia outbreak in Wuhan, China1,2. Despite the unprecedented pace of vaccine development, with six vaccines already in use worldwide, the emergence of SARS-CoV-2 ‘variants of concern’ (VOC) across diverse geographic locales have prompted re-evaluation of strategies to achieve universal vaccination3. All three officially designated VOC carry Spike (S) polymorphisms thought to enable escape from neutralizing antibodies elicited during initial waves of the pandemic4–8. Here, we characterize the biological consequences of the ensemble of S mutations present in VOC lineages B.1.1.7 (501Y.V1) and B.1.351 (501Y.V2). Using a replication-competent EGFP-reporter vesicular stomatitis virus (VSV) system, rcVSV-CoV2-S, which encodes S from SARS coronavirus 2 in place of VSV-G, and coupled with a clonal HEK-293T ACE2 TMPRSS2 cell line optimized for highly efficient S-mediated infection, we determined that only 1 out of 12 serum samples from a cohort of recipients of the Gamaleya Sputnik V Ad26 / Ad5 vaccine showed effective neutralization (IC90) of rcVSV-CoV2-S: B.1.351 at full serum strength. The same set of sera efficiently neutralized S from B.1.1.7 and showed only moderately reduced activity against S carrying the E484K substitution alone. Taken together, our data suggest that control of some emergent SARS-CoV-2 variants may benefit from updated vaccines.

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Ruxolitinib and Polycation Combination Treatment Overcomes Multiple Mechanisms of Resistance of Pancreatic Cancer Cells to Oncolytic Vesicular Stomatitis Virus

Journal of Virology ◽

10.1128/jvi.00461-17 ◽

2017 ◽

Vol 91 (16) ◽

Cited By ~ 13

Author(s):

Sébastien A. Felt ◽

Gaith N. Droby ◽

Valery Z. Grdzelishvili

Keyword(s):

Cell Line ◽

Cell Lines ◽

Vesicular Stomatitis Virus ◽

Oncolytic Virus ◽

Ductal Adenocarcinoma ◽

Mechanisms Of Resistance ◽

Pdac Cell ◽

Ldl Uptake ◽

Vesicular Stomatitis ◽

Pdac Cell Line

ABSTRACT Vesicular stomatitis virus (VSV) is a promising oncolytic virus (OV). Although VSV is effective against a majority of pancreatic ductal adenocarcinoma cell (PDAC) cell lines, some PDAC cell lines are highly resistant to VSV, and the mechanisms of resistance are still unclear. JAK1/2 inhibitors (such as ruxolitinib and JAK inhibitor I) strongly stimulate VSV replication and oncolysis in all resistant cell lines but only partially improve the susceptibility of resistant PDACs to VSV. VSV tumor tropism is generally dependent on the permissiveness of malignant cells to viral replication rather than on receptor specificity, with several ubiquitously expressed cell surface molecules playing a role in VSV attachment to host cells. However, as VSV attachment to PDAC cells has never been tested before, here we examined if it was possibly inhibited in resistant PDAC cells. Our data show a dramatically weaker attachment of VSV to HPAF-II cells, the most resistant human PDAC cell line. Although sequence analysis of low-density lipoprotein (LDL) receptor (LDLR) mRNA did not reveal any amino acid substitutions in this cell line, HPAF-II cells displayed the lowest level of LDLR expression and dramatically lower LDL uptake. Treatment of cells with various statins strongly increased LDLR expression levels but did not improve VSV attachment or LDL uptake in HPAF-II cells. However, LDLR-independent attachment of VSV to HPAF-II cells was dramatically improved by treating cells with Polybrene or DEAE-dextran. Moreover, combining VSV with ruxolitinib and Polybrene or DEAE-dextran successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. IMPORTANCE Oncolytic virus (OV) therapy is an anticancer approach that uses viruses that selectively infect and kill cancer cells. This study focuses on oncolytic vesicular stomatitis virus (VSV) against pancreatic ductal adenocarcinoma (PDAC) cells. Although VSV is effective against most PDAC cells, some are highly resistant to VSV, and the mechanisms are still unclear. Here we examined if VSV attachment to cells was inhibited in resistant PDAC cells. Our data show very inefficient attachment of VSV to the most resistant human PDAC cell line, HPAF-II. However, VSV attachment to HPAF-II cells was dramatically improved by treating cells with polycations. Moreover, combining VSV with polycations and ruxolitinib (which inhibits antiviral signaling) successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. We envision that this novel triple-combination approach could be used in the future to treat PDAC tumors that are highly resistant to OV therapy.

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The inhibition of mouse L-cell 45 S ribosomal RNA processing is a highly uv-Resistant property of vesicular stomatitis virus

Virology ◽

10.1016/0042-6822(90)90461-y ◽

1990 ◽

Vol 177 (1) ◽

pp. 75-84 ◽

Cited By ~ 1

Author(s):

Malgorzata Zan ◽

Peter Evans ◽

Jean Lucas-Lnard

Keyword(s):

Vesicular Stomatitis Virus ◽

Ribosomal Rna ◽

Rna Processing ◽

L Cell ◽

Ribosomal Rna Processing ◽

Vesicular Stomatitis

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Use of DNA-mediated gene transfer to analyze the role of H-2Ld in controlling the specificity of anti-vesicular stomatitis virus cytotoxic T cells.

Journal of Experimental Medicine ◽

10.1084/jem.157.4.1261 ◽

1983 ◽

Vol 157 (4) ◽

pp. 1261-1272 ◽

Cited By ~ 14

Author(s):

J Forman ◽

R S Goodenow ◽

L Hood ◽

R Ciavarra

Keyword(s):

T Cells ◽

Gene Transfer ◽

Cell Line ◽

Vesicular Stomatitis Virus ◽

Cytotoxic T Cells ◽

Control Cell ◽

Target Cells ◽

Effector Cells ◽

Vesicular Stomatitis ◽

Simplex Virus

Mouse thymidine kinase (tk-) C3H L (H-2k) cells transformed by the technique of DNA-mediated gene transfer with the herpes simplex virus tk gene together with the BALB/c H-2Ld gene express H-2Ld molecules indistinguishable from their counterparts on spleen cells. An established cloned cell line (8-5) was used to assess the function of the H-2Ld antigen in determining the specificity of alloreactive as well as anti-vesicular stomatitis virus (VSV) cytotoxic T cells (CTL). Both anti-H-2d and anti-H-2Ld CTL displayed a cytotoxic effect against 8-5 cells but not a control cell line transformed with the tk gene only (tk+ cells). Further evidence that 8-5 cells express H-2Ld was provided by the finding that monoclonal anti-H-2Ld but not H-2Dd antibodies blocked target cell lysis by the effector cells. Both BALB/c (H-2d) and DBA/2 (H-2d) animals generated anti-VSV CTL that lysed infected 8-5 but not tk+ cells. To further establish that H-2Ld controlled the specificity of the effector cells, a monoclonal antibody directed against H-2Ld was shown to inhibit lysis of infected 8-5 target cells. To determine whether other H-2d-encoded gene products could serve as restricting antigens for anti-VSV CTL in BALB/c animals, unlabeled VSV infected 8-5 cells were tested for their ability to block lysis of 51chromium-labeled P815 (H-2d)-infected target cells. The 8-5-VSV inhibitor cells inhibited lysis to a slightly lesser extent than unlabeled P815-VSV cells, indicating that H-2Ld plays a major if not exclusive role in restricting anti-VSV CTL in H-2d animals.

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Biochemical analysis of mutants of a macrophage cell line resistant to the growth-inhibitory activity of interferon.

The Journal of Cell Biology ◽

10.1083/jcb.98.4.1342 ◽

1984 ◽

Vol 98 (4) ◽

pp. 1342-1347 ◽

Cited By ~ 14

Author(s):

Y Nagata ◽

O M Rosen ◽

M H Makman ◽

B R Bloom

Keyword(s):

Adenylate Cyclase ◽

Cell Line ◽

Vesicular Stomatitis Virus ◽

Inhibitory Activity ◽

Macrophage Cell Line ◽

Inhibitory Effects ◽

Macrophage Cell ◽

Growth Inhibitory ◽

Growth Inhibitory Activity ◽

Vesicular Stomatitis

While a multiplicity of cellular and biochemical effects are mediated by interferons on cultured cells, the mechanisms involved in the direct growth-inhibitory activity of interferons remain problematic. We have previously found that variants in cAMP metabolism in a macrophage cell line, J774.2, were at least 50-fold less sensitive to the growth inhibitory activity of interferons (IFN) than the parental clone. To test the hypothesis that cAMP mediates the growth inhibition produced by IFN in these cells, interferon-resistant variants were selected and characterized with respect to cAMP synthesis and function. Approximately one-third of the IFN-resistant clones were found to be resistant to growth inhibition produced by cholera toxin, but not 8Br-cAMP. IFN was fully able to protect all of the interferon-resistant/choleratoxin-resistant (IFNr/CTr) clones against infection by vesicular stomatitis virus and markedly stimulated 2', 5'-oligodenylate synthetase activity. These IFNr/CTr variants were shown to have a defect in adenylate cyclase. The remaining IFN-resistant clones were fully susceptible to the growth-inhibitory effects of cholera toxin because their basal and stimulated adenylate cyclase activity is similar to that of the parental clone. IFN failed to protect these IFNr/choleratoxin sensitive clones against infection by vesicular stomatitis virus and failed to stimulate 2', 5-oligodenylate synthetase, suggesting that they have defective or deficient IFN receptors. In addition, IFN failed to increase intracellular cAMP levels in both IFNr/CTr and IFNr/choleratoxin sensitive clones. These results provide firm genetic and biochemical evidence that the growth inhibitory effects of IFN on this cell line are mediated by cAMP.

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