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. 2013 Apr;87(7):3752-9.
doi: 10.1128/JVI.02832-12. Epub 2013 Jan 16.

PEGylation of vesicular stomatitis virus extends virus persistence in blood circulation of passively immunized mice

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PEGylation of vesicular stomatitis virus extends virus persistence in blood circulation of passively immunized mice

Mulu Z Tesfay et al. J Virol. 2013 Apr.

Erratum in

  • J Virol. 2015 Feb;89(4):2453

Abstract

We are developing oncolytic vesicular stomatitis viruses (VSVs) for systemic treatment of multiple myeloma, an incurable malignancy of antibody-secreting plasma cells that are specifically localized in the bone marrow. One of the presumed advantages for using VSV as an oncolytic virus is that human infections are rare and preexisting anti-VSV immunity is typically lacking in cancer patients, which is very important for clinical success. However, our studies show that nonimmune human and mouse serum can neutralize clinical-grade VSV, reducing the titer by up to 4 log units in 60 min. In addition, we show that neutralizing anti-VSV antibodies negate the antitumor efficacy of VSV, a concern for repeat VSV administration. We have investigated the potential use of covalent modification of VSV with polyethylene glycol (PEG) or a function-spacer-lipid (FSL)-PEG construct to inhibit serum neutralization and to limit hepatosplenic sequestration of systemically delivered VSV. We report that in mice passively immunized with neutralizing anti-VSV antibodies, PEGylation of VSV improved the persistence of VSV in the blood circulation, maintaining a more than 1-log-unit increase in VSV genome copies for up to 1 h compared to the genome copy numbers for the non-PEGylated virus, which was mostly cleared within 10 min after intravenous injection. We are currently investigating if this increase in PEGylated VSV circulating half-life can translate to increased virus delivery and better efficacy in mouse models of multiple myeloma.

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Figures

Fig 1
Fig 1
VSV is rapidly neutralized by normal human serum. VSV-GFP was incubated with normal human serum (a and d), anti-IgM antibody (ab)-treated human serum (b), heat-treated (56°C for 30 min) human serum (a), or medium as a control, and then it was used to infect Vero cells. (c) In vivo stability of VSV in naïve mice after i.v. injection. (d) Time course experiments in which virus was incubated with serum for 5 min, 10 min, 15 min, 30 min, 60 min, 120 min, or 240 min, followed by virus titer determination on Vero cells.
Fig 2
Fig 2
VSV-immune serum negates the oncolytic efficacy of i.v. VSV. (a) BALB/c mice (4 to 6 weeks old) were immunized by i.v. injection of VSV (1 × 108 TCID50s/mouse). Two weeks after immunization, the serum titer was determined using neutralization assay. Mice with similar serum titers were selected, and MPC11 subcutaneous tumors were implanted by injecting 5 × 106 MPC11 cells subcutaneously into immunized and control naïve mice. When tumors reached an average volume of 100 mm3, mice were randomly divided into three groups: group 1 was immunized and i.v. injected with no virus (PBS only; n = 8), group 2 was immunized and i.v. injected with VSV–mIFN-β virus (2.5 × 108 TCID50s/mouse; n = 9), and group 3 was nonimmunized (naïve) and i.v. injected with VSV–mIFN-β (2.5 × 108 TCID50s/mouse; n = 5). Tumor size (b) and survival data (c) were collected. In panel b, at day 12 in the PBS group, there is no error bar, as only one mouse was left for measurement.
Fig 3
Fig 3
PEGylation of VSV. Different amounts of PEG5000 activated by succinimidyl (Jenkem Technology, Beijing, China) (a) or FSL-PEG2000 (KODE Biotech, Auckland, New Zealand) (b) were added for 5 × 108 TCID50s of VSV. All conjugation reactions were performed at 25°C with gentle agitation. The reactions were stopped by 15 min incubation at 4°C as described by O'Riordan et al. (45). Unreacted PEG and reaction by-products were eliminated by buffer exchange over a Micro-Bio Spin P-30 chromatography column (Bio-Rad) equilibrated with 100 mM potassium PBS (pH 7.4). As a control, VSV with the same PBS buffer but without PEG or FSL-PEG was incubated under a similar condition and processed in the same manner as the conjugated virus. PEGylation status was assessed by in vitro infectivity on Vero cells. The fluorescent (top) and phase-contrast (bottom) images taken at 24 h postinfection are shown for each condition.
Fig 4
Fig 4
PEGylation provides protection of VSV from serum neutralization both in vitro and in vivo. (a) For in vitro experiments, 150 μl of VSV-immune serum diluted 1:15,000 was incubated with 1 × 107 TCID50s of VSV PEGylated with the indicated concentrations of PEG or FSL-PEG or unmodified GFP-expressing VSV for 1 h at 37°C. As a control, virus was incubated with medium. The mix was plated on Vero cells to determine virus infectivity. Pictures were taken at 18 h postinfection and indicate that PEGylation confers some level of virus protection from serum neutralization. Both PEG and FSL-PEG experiments were performed at the same time. (b) In vivo studies show the anti-VSV titers recovered in serum harvested from mice that were immunized by passive transfer of VSV-specific antibody. Twofold dilutions of serum were injected to different mouse groups, and at 24 h postinjection, serum was harvested and titers were determined on Vero cells. (c) VSV genome copy numbers after i.v. injection of VSV with or without PEGylation in the presence of passively transferred neutralizing antibodies (1:100 dilutions). Blood RNA samples harvested at 10 and 60 min were subjected to quantitative RT-PCR with primers specific to the VSV N gene to determine virus genome copy numbers. Results show that PEGylation significantly improved the persistence of virus RNA in the blood circulation at 10 min in the presence of neutralizing antibodies (VSV versus VSV PEG, P = 0.0330; VSV versus FSL-PEG, P = 0.0001), whereas the difference was not significant at 60 min postinjection (VSV versus VSV-PEG, P = 0.9610; VSV versus VSV-FSL-PEG, P = 0.0617).
Fig 5
Fig 5
PEGylation of VSV increases virus accumulation in tumor. Four subcutaneous MPC11 tumor-bearing BALB/c mice per group in the presence of passively transferred VSV-specific immune (Imm) serum were i.v. injected with 1 × 108 TCID50s/mouse unmodified VSV, PEG-VSV, or FSL-PEG-VSV. Control mice were treated with 200 μl of potassium PBS. At 4 days after virus injection, mice were sacrificed and total RNA was isolated from tumor and analyzed for VSV N genome copy number using qPCR.
Fig 6
Fig 6
PEGylation of VSV reduces virus-induced toxicity. Hepatotoxicity assessment was performed by blood chemistry analysis to determine serum ALP and ALT levels at day 3 after virus injection in BALB/c mice. ALP (a) and ALT (b) levels were significantly increased in animals treated with unmodified VSV compared to control animals or animals treated with PEGylated VSV, whereas animals treated with PEGylated VSV had lower levels of ALP and ALT which were not significantly different from those of control animals treated with PBS.

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References

    1. Cook R. 2008. Economic and clinical impact of multiple myeloma to managed care. J. Manag. Care Pharm. 14(7 Suppl):19–25 - PMC - PubMed
    1. Edwards CM, Zhuang J, Mundy GR. 2008. The pathogenesis of the bone disease of multiple myeloma. Bone 42:1007–1013 - PMC - PubMed
    1. Hideshima T, Mitsiades C, Tonon G, Richardson PG, Anderson KC. 2007. Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets. Nat. Rev. 7:585–598 - PubMed
    1. Kyle RA, Rajkumar SV. 2004. Multiple myeloma. N. Engl. J. Med. 351:1860–1873 - PubMed
    1. Kyle RA, Rajkumar SV. 2009. Treatment of multiple myeloma: a comprehensive review. Clin. Lymphoma Myeloma 9:278–288 - PMC - PubMed

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