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1 Carl C. Icahn Center for Gene Therapy and Molecular Medicine and 2 Department of Microbiology, Mount Sinai School of Medicine, New York, New York
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We envisioned that intratumoral virus spread could be improved by syncytia-inducing VSV, which can lead to substantially improved treatment outcome. VSV normally propagates from cell to cell by virions that bud out from infected cells and become internalized by endosomes in neighboring cells, where the VSV glycoprotein (VSV/G) protein, upon acidification of the endosome, mediates fusion between the membrane of the virus and the membrane of the endosome. As a result, viral nucleocapsids are released into the cytoplasm, where they start viral replication and transcription (7) . Other viruses have evolved a different spreading mechanism that involves fusion of the infected cell with the adjacent cells, resulting in the formation of multinucleated giant cells or syncytia (10) . Plasmid-mediated expression of viral fusogenic membrane glycoproteins efficiently killed tumor cells through induction of fusion to form large multinucleated syncytia (11) . In vitro, this "bystander effect" was reported to be at least 10-fold greater than that observed with suicide gene/prodrug systems such as herpes simplex virus thymidine kinase + ganciclovir or cytosine deaminase + 5-fluorocytosine (11) . The antitumor efficacy of an oncolytic herpes simplex virus was also significantly enhanced in vivo by inclusion in its genome the COOH-terminal truncated form of the Gibbon ape leukemia virus envelope glycoprotein, which is a fusogenic membrane glycoprotein (12 , 13) . We hypothesized that expression by VSV of a fusogenic membrane glycoprotein able to induce membrane fusion at neutral pH would result in a syncytia-forming VSV with significantly increased oncolytic activity in vivo.
Membrane fusion mediated by most paramyxoviruses, such as NDV, requires two virion-associated glycoproteins, the attachment or hemagglutinin-neuraminidase protein and the fusion protein [F (10) ]. Interestingly, Sergel et al. (14) reported that a single amino acid substitution (L289A) in the heptad repeat motif (HR3) of the NDV fusion protein (NDV/F) allows this protein to induce syncytia alone, whereas the wild-type (wt) NDV/F protein requires coexpression with the hemagglutinin-neuraminidase protein. In this article, we report the generation of a novel fusogenic VSV vector, which expresses mutant (L289A) F protein from NDV, and demonstrate its superior oncolytic activity against multifocal HCC in an orthotopic and immunocompetent rat model.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasmid Construction.
To create recombinant VSV vector expressing wtNDV/F protein, the sequence encoding NDV/F (1.6 kb) was PCR amplified from pNDV/B1/F3 containing the F protein of NDV strain Hitchner B1 with a modified protease cleavage site (112R-R-Q-R-R-/F117; modified amino acids are underlined) recognized by ubiquitous proteases (15
, 16)
. PCR amplification was performed using the forward (5'-CGGCCGCTCGAGATGGGCTCCAGACCTTCTAC-3') and reverse primers (5'-AAGCTGGCTAGCTCACATTTTTGTAGTGGCTC-3') with unique restriction sites (shown in bold) XhoI and NheI, respectively. The PCR product was digested with XhoI and NheI and cloned into the full-length pVSV-XN2 plasmid (a gift from Dr. J. Rose; Yale University, New Haven, CT). The resulting plasmid was designated pVSV-NDV/F(wt). To generate recombinant VSV vector expressing mutant (L289A) F protein, the pVSV-NDV/F(wt) plasmid was modified by PCR site-directed mutagenesis. To this end, the sequence encoding the NDV/F protein was assembled from two overlapping PCR fragments. The first PCR fragment was generated by using the forward (5'-CGGCCGCTCGAGATGGGCTCCAGACCTTCTAC-3') and reverse primers (5'-GTTCCCGACTGAAGGTGCAGTTACCTGTATACC-3'). The residues introduced in the primer to change the amino acid sequence at position 289 from Leu to Ala are underlined. The second PCR fragment was generated by using the forward (5'-GGTATACAGGTAACTGCACCTTCAGTCGGGAAC-3') and reverse primers (5'-AAGCTGGCTAGCTCACATTTTTGTAGTGGCTC-3'). The two overlapping PCR fragments were joined in a third PCR, and the resulting fragment, which contained the mutant (L289A) F protein sequence, was cloned into pVSV-XN2 to generate pVSV-NDV/F(L289A). Sequencing of the plasmids was conducted in the DNA Sequencing Core Facility at Mount Sinai School of Medicine.
Generation of Recombinant Viruses.
For recovery experiments of recombinant VSV, the established method of reverse genetics was used (17
, 18)
. Control wtVSV not expressing a transgene was generated using the pVSV-XN2 plasmid encoding the full-length VSV cDNA. Briefly, BHK-21 cells infected previously with vaccinia virus expressing T7 RNA polymerase (vTF-7.3) were transfected with 10 µg of full-length VSV cDNA in addition to plasmids encoding VSV N, P, and L proteins by using LipofectAMINE 2000 reagent (Invitrogen, Carlsbad, CA). Two days after transfection, supernatants were filtered through a 0.22 µm filter to remove the majority of vaccinia virus and transferred onto fresh BHK-21 cells. Vaccinia virus was eliminated by plaque purification, and the titers of recombinant VSV stocks were determined by plaque assays on BHK-21 cells.
Indirect Immunofluorescence Assay.
BHK-21 cells were seeded onto plastic coverslips (Nalge Nunc, Naperville, IL) at 105 cells/6.4-cm2 plate. The following day, cells were infected with wtVSV or recombinant VSV expressing NDV fusion protein (rVSV-NDV/F) vectors at a MOI of 10. Cells were incubated at 37°C for 14 h, washed with PBS, fixed with 3% paraformaldehyde for 20 min at room temperature, and washed twice with PBS-glycine (10 mM). Coverslips were removed and incubated at 37°C for 30 min with primary antibody [mouse monoclonal antibody against NDV/F protein (Hybridoma Core, Mount Sinai School of Medicine) or VSV/G protein (VSV11-M; Alpha Diagnostic, San Antonio, TX)] at 1:100 dilutions in PBS containing 10 mM glycine and 5 mg/ml BSA (PBS-glycine-BSA). After incubation with the primary antibody, coverslips were rinsed twice with PBS-glycine and incubated at 37°C for 30 min with secondary antibody [Texas Red- or FITC-conjugated rabbit antimouse (Jackson ImmunoResearch, West Grove, PA)] at 1:50 dilutions in PBS-glycine-BSA. Coverslips were rinsed twice with PBS-glycine and examined using a fluorescence microscope.
Multicycle and One-Step Growth Curves.
BHK-21 cells were plated in 6-well plates at 5 x 105 cells/well and infected at a MOI of 10 (one-step growth) or 0.01 (multicycle growth) with wtVSV or rVSV-NDV/F vectors the following day. After infection at room temperature for 30 min, cells were washed twice with PBS to remove any unabsorbed virus, and complete medium was added. At the indicated time points after infection, 150 µl of supernatant were removed and assayed for viral RNA genome by real-time reverse transcription-PCR using specific primers as described previously (8)
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Virus Infection of Tumor Cells in Vitro.
HCC tumor cells were seeded onto glass coverslips (Nalge Nunc) at 5 x 105 cells/6.4-cm2 plate and infected with wtVSV or rVSV-NDV/F vectors at a MOI of 10. Cells were incubated at 37°C for 14 h, washed with PBS, and fixed with 3% paraformaldehyde for 20 min at room temperature. Coverslips were rinsed twice with PBS-glycine, followed by staining with H&E.
In Vitro Cytotoxicity Assay.
HCC tumor cells were plated in 24-well plates at 2.5 x 105 cells/well and infected with wtVSV or rVSV-NDV/F vectors at a MOI of 0.01 the following day. Cell viability was measured at the indicated time points after infection using Cell Proliferation Kit I (Roche, Indianapolis, IN). All cell viability results are expressed as percentage of viable cells compared with mock-infected control at each time point.
Animal Studies.
All procedures involving animals were approved by and performed according to guidelines of the Institutional Animal Care and Use Committee of the Mount Sinai School of Medicine. Six-week-old male Buffalo rats were purchased from Harlan (Indianapolis, IN) and housed in a specific pathogen-free environment under standard conditions. To establish multifocal HCC lesions within the liver, 107 syngeneic McA-RH7777 rat HCC cells (in a 1-ml suspension) were infused into the portal vein. Fifteen days after tumor cell implantation, the development of multifocal hepatic tumors was confirmed, and 1.3 x 107 plaque-forming units of rVSV-NDV/F vectors or PBS control (in a 1-ml suspension) were administered via the hepatic artery. To evaluate virus-induced syncytia formation in tumor tissues, tumor-bearing rats were sacrificed 1 day after VSV vector infusion, and the livers were subjected to histopathological analysis. In addition, groups of animals infused with VSV vectors or buffer control were followed for survival, which was checked daily in all animals.
Histology and Immunohistochemistry.
Livers were fixed in 4% paraformaldehyde overnight and then paraffin-embedded. Five-µm thin sections were subjected to either H&E staining for histological analysis or immunohistochemical staining using a monoclonal antibody against VSV/G protein (Alpha Diagnostic). Immunohistochemistry sections were counterstained with hematoxylin.
Statistical Analyses.
For comparison of individual time points, unpaired t test was applied to determine statistical difference. Survival curves of animals were plotted according to the Kaplan-Meier method, and statistical significance in different treatment groups was compared using the log-rank test. Results and graphs were obtained using the GraphPad Prism 3.0 program (GraphPad Software, San Diego, CA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. We chose this cloning strategy for two reasons: first, the expression level of the NDV/F protein would be similar to levels achieved for endogenous VSV/G protein; and second, relatively low levels of expression were expected to be sufficient to induce cell fusion with neighboring cell membranes. Reverse genetics was used to generate rVSV-NDV/F(L289A) and rVSV-NDV/F(wt) as described previously (17
, 18)
. The latter virus would not be able to form syncytia and is therefore an excellent control virus. Expression of the NDV/F protein was monitored in infected BHK-21 cells by indirect immunofluorescence assay, using monoclonal antibodies specific for NDV/F protein (Fig. 1B)
. As a control, expression of VSV/G protein was detected in both wtVSV- and rVSV-NDV/F-infected cells by using a monoclonal antibody specific for VSV/G protein. In contrast, NDV/F protein expression was clearly localized to the cell surface and cytoplasm in BHK-21 cells infected with rVSV-NDV/F, but not in those infected with wtVSV.
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. Both rVSV-NDV/F viruses grew very similarly and reached approximately the same titer at the same time points, indicating that there were no differences in viral replication and spread. The replication kinetics of wtVSV appeared to be slightly faster, but all three viruses reached a similar titer at 24 h after infection. We also performed a one-step growth curve by infecting BHK-21 cells at a MOI of 10 to analyze a single cell cycle of viral replication (Fig. 2B)
. All three viruses showed approximately the same viral titer at all four time points. Therefore, incorporation of F protein into the genome of VSV did not appear to have any major impact on viral life cycle and viral yield.
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. At 14 h after infection, large multinucleated syncytia were observed for all three tumor cells infected by rVSV-NDV/F(L289A). In contrast, the same tumor cells infected with wtVSV or control rVSV-NDV/F(wt) lacked this phenotype.
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, were killed by the fusogenic VSV-NDV/F(L289A) much more efficiently than by the control nonfusogenic rVSV-NDV/F(wt) vector or wtVSV. In fact, fusogenic VSV killed most cells within 36 h, at which time point 50–75% of the control vector-infected cells were still viable. On the other hand, rat McA-RH7777 cells tend to shed from the growth surface before achieving confluence, and the degree of viral killing through syncytia induction, which is known to be cell density dependent (11)
, was not as extensive as that seen in the human HCC cell lines, and statistical significance was achieved only 48 h postinfection (Fig. 4C)
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, top panel). Immunohistochemical staining of consecutive sections demonstrated abundant expression of VSV/G protein that localized at the plasma membrane of individual tumor cells but surrounded the multinucleated structure in animals infused with the control and fusogenic virus, respectively (Fig. 5A
, bottom panel).
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and neighboring hepatic parenchyma (Fig. 5C)
were examined. The liver histology was completely normal, and, in particular, no evidence of inflammatory cell infiltrate or syncytia formation of hepatocytes was found.
Prolonged Survival of Multifocal HCC-Bearing Rats Treated with Fusogenic versus Control VSV.
To assess the potential of fusogenic VSV as an improved oncolytic agent, rats bearing more than 10 visible HCC tumors in their livers with sizes ranging from 1 to 10 mm in diameter were randomly assigned to a single infusion with 1.3 x 107 plaque-forming units of either rVSV-NDV/F(L289A) (n = 11), rVSV-NDV/F(wt) (n = 9), or PBS (n = 9) via the hepatic artery, and survival was followed (Fig. 6)
. Buffer-treated rats started to die of tumor progression in 5 more days, and all of them expired at 18 days (median survival, 10 days). In contrast, rats treated with control rVSV-NDV/F(wt) survived for up to 24 days (median survival, 18 days), which was a significant improvement over the PBS group (P = 0.0016). In the fusogenic rVSV-NDV/F(L289A) treatment group, animals survived for up to 31 days, and median survival was further extended to 23 days (P < 0.0001 versus PBS). In pairwise comparison with the nonfusogenic control virus rVSV-NDV/F(wt), survival of rVSV-NDV/F(L289A)-treated animals was further improved (P = 0.0078).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we provide conclusive evidence that fusogenic VSV displays increased oncolytic activity over its nonfusogenic counterpart, which translated into highly significant prolongation of animal survival (P = 0.0078, log-rank test). This enhanced oncolytic activity of fusogenic VSV was achieved after a single administration of the virus, and the tumor burden at the time of treatment (15 days after tumor cell implantation) was quite extensive, resembling the actual situation in HCC patients with end-stage multifocal disease. In addition, the orthotopic locale of our tumor model more realistically recapitulates the liver environment and results in a locally aggressive, spontaneously metastatic tumor. On the basis of these considerations, the observed prolongation of median survival from 18 to 23 days posttreatment conferred by fusogenic VSV over its nonfusogenic counterpart is indeed impressive.
This benefit may be attributable to various factors. Our in vitro analysis has shown that fusogenic VSV kills tumor cells much more efficiently than wtVSV or nonfusogenic control viruses. The process of syncytial formation is known to involve a significant bystander cell killing effect through recruitment of adjacent untransduced cells into the evolving syncytium (11) . In addition, fusogenic VSV kills tumor cells through both replication with subsequent cell lysis and direct cell membrane fusion, and these two cytolytic mechanisms may have produced a synergistic effect that has facilitated the spread of fusogenic VSV in the solid tumor tissue in vivo. Both limited dispersion of viral particles and antiviral immunity are known contributing factors to the relative inefficiency of viral spread of current nonfusogenic oncolytic viruses within a solid tumor mass in vivo (20 , 21) . It is possible that by inducing cell-cell fusion, the virus may have propagated more efficiently in vivo because several steps in the life cycle of VSV, including viral budding and virus endocytosis, are bypassed. Fusogenic VSV may also have avoided humoral immune mechanisms of virus neutralization because syncytia-forming viruses can spread from cell to cell within the solid tumor mass that is refractory to the presence of neutralizing antibodies (10) .
We and others have shown previously that wtVSV primarily induces tumor cell apoptosis in vivo (22 , 23) , which is mediated by the VSV matrix protein and by another viral component that has yet to be identified (24) . Cell death by apoptosis is a highly ordered process, which escapes detection by the immune system and is actually immunosuppressive (25) . In contrast, cancer cell death through syncytia formation mediated by fusogenic VSV is a cytopathic effect that can lead to tumor necrosis in vivo, which, in contrast to apoptotic cell death, may result in highly efficient immune activation (26) . Fusing tumor cells release large numbers of tumor-derived, exosome-like vesicles, which may be associated with an effective release of tumor antigens, their efficient transfer into immature dendritic cells, and subsequent cross-presentation to T cells to generate systemic antitumor immune responses (27 , 28) . Therefore, fusogenic VSV-mediated oncolysis may combine very effective local effects with the potential to be a highly immunogenic method of cytotoxic gene therapy. In the present study, we have focused on the use of fusogenic VSV as an effective and safe oncolytic agent, which will permit more detailed mechanistic studies in our immunocompetent animal tumor model in the future.
Although we detected large necrotic regions within multiple tumors, which led to significant prolongation of animal survival, there were no long-term survivors. Repeated intrahepatic artery infusions would likely result in further improved survival over a single injection. Although VSV administration through the hepatic artery is technically not feasible in the rat, percutaneous catheterization of the hepatic artery for local-regional therapy of liver tumors is commonly used in the clinic, thereby potentially allowing readministration of the oncolytic virus before onset of humoral immune response. Moreover, the ability to express foreign sequence of at least 4.0 kb makes it possible to insert more than one foreign gene into the VSV genome (29) . In this regard, recombinant VSV vectors encoding for the interleukin-4 cytokine or the herpes simplex virus thymidine kinase suicide gene were generated and reported to exhibit considerably more oncolytic activity than a control virus expressing green fluorescent protein (30) . Recently published data (31 , 32) presented the generation of IFN-inducing VSV variants or recombinant VSV vectors expressing IFN-ß with greatly improved therapeutic indices over wtVSV after systemic delivery. These and other transgenes can be integrated into fusogenic VSV vectors to further increase their oncolytic potential.
Uncontrolled induction of syncytia formation by fusogenic viruses raises potential safety concerns (12 , 13) . In the case of VSV, expression of viral and any accompanying foreign genes depends directly on viral RNA replication (33) . Therefore, it was anticipated that fusogenic VSV would be compromised in its ability to induce fusion in normal cells because expression of an F protein is unlikely to result in a better ability of VSV to escape the innate IFN response. Indeed, hepatic artery infusion of fusogenic VSV in rats bearing multifocal HCC lesions in their livers resulted in extensive syncytia formation within tumors but caused no damage to surrounding hepatocytes. Thus, the fusogenic VSV vector can be developed into effective and safe therapeutic agents for HCC and other types of cancer in the future.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Savio L. C. Woo, Carl C. Icahn Center for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1496, New York, NY 10029. Phone: (212) 659-8260; Fax: (212) 803-6740; E-mail: savio.woo{at}mssm.edu
Received 12/ 2/03. Revised 1/23/04. Accepted 2/20/04.
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REFERENCES |
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5' orientation, and the open reading frames of the major VSV proteins are indicated. The top of the figure shows the wtVSV vector, which was used for cloning the NDV/F sequence in the noncoding region of the G protein gene. B, immunofluorescence of NDV/F protein expression from rVSV-NDV/F vectors (original magnification, x20). VSV/G protein expression is seen in both rVSV-NDV/F- and wtVSV-infected BHK-21 cells (bottom panels). In contrast, NDV/F protein expression is seen only in rVSV-NDV/F-infected cells (top panels).
