Retargeting vesicular stomatitis virus using measles virus envelope glycoproteins

Abstract

Oncolytic vesicular stomatitis virus (VSV) has potent antitumor activity, but infects a broad range of cell types. Here, we used the measles virus (MV) hemagglutinin (H) and fusion (F) envelope glycoproteins to redirect VSV entry and infection specifically to tumor-associated receptors. Replication-defective VSV, deleted of its glycoprotein gene (VSVΔG), was pseudotyped with MV-F and MV-H displaying single-chain antibodies (scFv) specific for epidermal growth factor receptor (EGFR), folate receptor (FR), or prostate membrane-specific antigen (PSMA). Viral titers were ∼10(5) PFU/ml, but could be concentrated to 10(7) PFU/ml. Immunoblotting confirmed incorporation of the MV-H-scFv and MV-F into functional VSV virions. Although VSV-G was able to infect all tumor cell lines tested, the retargeted VSV infected only cells that expressed the targeted receptor. In vivo specificities of the EGFR-, FR-, and PSMA-retargeted VSV were assessed by intratumoral injection into human tumor xenografts. Analysis of green fluorescent protein reporter gene expression indicated that VSV infection was restricted to receptor-positive tumors. In summary, we have demonstrated for the first time that VSV can be efficiently retargeted to different cellular receptors using the measles display technology, yielding retargeted VSV vectors that are highly specific for tumors that express the relevant receptor.

FIG. 1.

(A) Schematic representation of the protocol for pseudotyping VSVΔG with MV or VSV glycoproteins. The recovered supernatant contains VSVΔG-FH or VSVΔG-G vectors that can infect target cells and express viral proteins, but cannot produce viral progeny due to deletion of the VSV-G gene from the viral genome. (B) Titers of VSV pseudotyped with MV F/H glycoproteins bearing parental MV-H and F proteins or with truncated cytoplasmic tails (MV-HΔ24 or MV-FΔ30). Viral titers were determined on Vero-αHis cells. Results show representative data from two experiments.

FIG. 2.

Titers of retargeted VSV vectors. VSV were pseudotyped with VSV-G, MV-F alone, or a combination of MV-F and MV-H with and without an scFv, and titers were determined on Vero-αHis cells. Results show average of four independent experiments.

FIG. 3.

Immunochemical analysis of VSV pseudotypes. Viral supernatants were purified, and proteins were fractionated by SDS-PAGE. MV and VSV proteins were detected with polyclonal anti-MV or anti-VSV antibodies.

FIG. 4.

Retargeted VSV pseudotypes preferentially transduced receptor-positive cells. (A) Photographs of SKOV3.ip.1, KAS 6/1, PC3, and PC3-PSMA cells at 24 hr post transduction by retargeted VSV pseudotypes (MOI 3.0). GFP signal was observed under an epifluorescence microscope. (B) Quantitation of the numbers of VSV-transduced GFP-expressing cells at 24 hr post infection (MOI 0.1). Data show the average of three independent experiments.

FIG. 5.

Human neuronal cells were not transduced by αFR- or αPSMA-retargeted VSVΔG pseudotypes (MOI 1.0). Representative photographs of GFP-expressing cells taken at 48 hr post transduction under an epifluorescence microscope (100× magnification) are shown.

FIG. 6.

Specificity of retargeted VSV pseudotypes was retained in vivo. Subcutaneous SKOV3.ip.1, KAS 6/1, PC3, or PC3-PSMA tumors were injected intratumorally with one dose of 10⁶ retargeted VSV vectors. Tumors were harvested 48 hr later, and GFP signals were detected using a fluorescence microscope. Representative images are shown (100× magnification). Color images available online at www.liebertonline.com/hum