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, 9 (3), e90508
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Attenuated and Protease-Profile Modified Sendai Virus Vectors as a New Tool for Virotherapy of Solid Tumors

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Attenuated and Protease-Profile Modified Sendai Virus Vectors as a New Tool for Virotherapy of Solid Tumors

Martina Zimmermann et al. PLoS One.

Abstract

Multiple types of oncolytic viruses are currently under investigation in clinical trials. To optimize therapeutic outcomes it is believed that the plethora of different tumor types will require a diversity of different virus types. Sendai virus (SeV), a murine parainfluenza virus, displays a broad host range, enters cells within minutes and already has been applied safely as a gene transfer vector in gene therapy patients. However, SeV spreading naturally is abrogated in human cells due to a lack of virus activating proteases. To enable oncolytic applications of SeV we here engineered a set of novel recombinant vectors by a two-step approach: (i) introduction of an ubiquitously recognized cleavage-motive into SeV fusion protein now enabling continuous spreading in human tissues, and (ii) profound attenuation of these rSeV by the knockout of viral immune modulating accessory proteins. When employing human hepatoma cell lines, newly generated SeV variants now reached high titers and induced a profound tumor cell lysis. In contrast, virus release from untransformed human fibroblasts or primary human hepatocytes was found to be reduced by about three log steps in a time course experiment which enables the cumulation of kinetic differences of the distinct phases of viral replication such as primary target cell infection, target cell replication, and progeny virus particle release. In a hepatoma xenograft animal model we found a tumor-specific spreading of our novel recombinant SeV vectors without evidence of biodistribution into non-malignant tissues. In conclusion, we successfully developed novel tumor-selective oncolytic rSeV vectors, constituting a new tool for virotherapy of solid tumors being ready for further preclinical and clinical development to address distinct tumor types.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation of recombinant Sendai virus variants.
(A) Schematic representation of SeV genomes and the protein sequence of the SeV-F protein cleavage-site of newly generated SeV virus variants. At the 3′-end all variants encode a reporter gene for enhanced green fluorescent protein (EGFP). In contrast to the P and F gene wild-type variant (D52), in all SeV Fmut variants the wild-type F protein cleavage-site (VPQSR) was replaced by the oligobasic cleavage-site of Newcastle disease virus F protein (RRQKR). The seven wild-type P gene encoded proteins are a result of multiple open reading frames (ORF; C′, C, Y1, Y2) and RNA editing, respectively, leading to a frame shift and thus to the V or W ORF's. For attenuation in non-malignant cell types, different mutations were introduced in C (ORFs; C′-, C-, Y1-, Y2) or V/W-ORFs. For the Vko variants (no V and no W proteins), mutations of the editing-site within the P frame were introduced without changing the amino acid sequence of the P protein. Thus, P protein but no truncated P protein variants (V and W proteins) can be synthesized. For the C protein deficient variants (Cko: no C′ and C proteins; Yko: no Y1 and Y2 proteins), inserted point mutations are marked by numbers from 1-5 (1: M1T, 2: L5stop, 3: L11stop, 4: M24T, 5: M30T; numbers refer to amino acid position in C/Y-frame whereas 1 refers to the initiator methionine of the C protein). (B) Schematic overview of functional ORFs from the P gene of different SeV variants. The SeV P-gene wild-type expression pattern is shown for D52 and Fmut (1.+2.).
Figure 2
Figure 2. Growth kinetics of newly generated SeV variants in Vero producer cells.
Growth kinetics of six different recombinant SeV viruses over a 96×107 TCID50 was inserted for better orientation.
Figure 3
Figure 3. Growth kinetics and spreading of newly generated SeV variants in different cell types.
(A+B) Growth kinetics of six different recombinant SeV viruses over a 96×107 TCID50 is depicted. (A) Malignant human hepatoma cells PLC/PRF/5, Hep3B and HuH7. (B) Non-malignant MRC-5 fibroblasts and primary human hepatocytes (PHH) from three different donors. (C+D) Detection of EGFP reporter protein expression over a 72 h observation period by fluorescence microscopy as a surrogate marker for viral replication and spread to neighboring cells. Size bar: 200 µm. (C) Infection of PLC/PRF/5 hepatoma cells. (D) Infection of MRC-5 human fibroblast cells.
Figure 4
Figure 4. Comparison of attainable peak SeV titers during replication in different cell types.
The mean peak titer (sum of the infectious virus particles determined via TCID50 for super natant and lysate) over a period of 96 h was determined including all experiments with the investigated three hepatoma cell lines (HuH7, Hep3B, PLC/PRF/5) and compared to the achievable peak titer in non-malignant cells (MRC-5 or PHH). Numbers over the columns display fold-changes between the mean of all hepatoma cells compared to either MRC-5 or PHH. The dotted line shows the amount of inoculated viral particles during the initial infection (5×103 TCID50). Data represent mean and SEM.
Figure 5
Figure 5. Attenuated SeV variants in primary human hepatocytes (PHH).
PHH from three different donors were infected with SeV D52 and all six recombinant SeV variants (MOI of 0.1). (A) Exemplarily chosen pictures of PHH 72 hpi for seven different recombinant viruses (detection of GFP by fluorescence microscopy). Bar represents 200 µm. For donor 1, the analysis of the SeV FmutC/YtermVko variant was not done (n.d.). (B) 72 h after the inoculation with the different viruses, infected cells were counted in three randomly chosen areas and calculated as a ratio of all cells in the same area. Data are shown in mean and SD of three independent experiments in triplicates.
Figure 6
Figure 6. Quantification of cellular viability in a dose and time-dependent manner.
Infection experiments with Vero and PLC/PRF/5 (at MOIs of 1 and 0.1) cells were performed with all variants of the newly generated Sendai viruses as well as the wild type variant SeV D52. Cellular viability was investigated via CytoTox-Glo™ assay at different time points post infection (0 h, 24 hpi, 48 hpi, 72 hpi, 96 hpi). The assay was performed in triplicates and repeated three times; data are shown as mean and SEM. SeV 1 (green line): SeV D52, SeV 2: SeV Fmut, SeV 3: SeV FmutVko, SeV 4: SeV FmutCko, 5: SeV FmutYko, 6: SeV FmutCkoVko, 7: SeV FmutC/YtermVko, TX: Triton X-100 (positive control for the induction of a maximum grade, chemically-mediated destruction of test cells).
Figure 7
Figure 7. Cytolytic capacity of recombinant SeV particles in different cell types.
Cell growth and cell lysis in different cell types with SeV D52 and six recombinant SeV Fmut variants (black bars: MOI 0.01, grey bars: MOI 0.1). (A+C) Analysis of cell mass by sulforhodamine B (SRB) assay 72 hpi in (A) hepatoma and (C) non-malignant MRC-5 cells or PHH. All values are shown in relation to uninfected control cells (mock). (B+D) Analysis of LDH release of infected cells relative to LDH release of uninfected control cells (mock) as a surrogate marker of cellular membrane integrity 72 hpi. Results represent mean and SD of three independent experiments in triplicates, for PHH two different donors, in triplicates each.
Figure 8
Figure 8. Intratumoral spread of recombinant SeV particles in vivo in a PLC/PRF/5 hepatoma xenograft model.
Recombinant SeV variants (SeV D52, SeV Fmut, SeV FmutCkoVko, SeV FmutC/YtermVko, 1×107 TCID50/100 µl) were injected intratumorally in tumors of a PLC/PRF/5 hepatoma xenograft mouse model. (A) 48 h post injection the mice were sacrificed, tumors were removed and one quarter of each tumor was fixed and embedded in paraffin. Virus spread was investigated applying an anti-GFP antibody for immunohistochemistry analysis (brown color). Bar represents 100 µm. (B) Indicator cultures (Vero cells) were infected with lysates from frozen tissue sections (one quarter of tumor, liver, spleen, heart, lung, each). Early primary infections (24-72 hpi) were observed and single infected cells were counted as initial virus particles (VP). Fluorescence microscopy pictures of the infected Vero cells were taken 72 hpi. Shown are representative picture for each animal. Bar represents 400 µm.
Figure 9
Figure 9. The amount of infectious virus particles in tumor tissue was quantified with the TCID50 method.
4850 titration (mean and standard deviation of two (D52) or three analyzed tumors of each group).

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This work was supported by grants from the Deutsche Forschungsgemeinschaft SFB 773 (B3) and BI 669/3-6, and from the Federal Ministry for Education and Research of Germany (BMBF 01GU0506 & 01GU0806). The authors acknowledge additional support by the "Deutsche Forschungsgemeinschaft and Open Access Publishing Fund of Tuebingen University". The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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