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. 2020 Aug 15;21(16):5863.
doi: 10.3390/ijms21165863.

NanoBiT System and Hydrofurimazine for Optimized Detection of Viral Infection in Mice-A Novel in Vivo Imaging Platform

Natasa Gaspar  1   2   3 , Giorgia Zambito  1   2   4 , Iris J C Dautzenberg  5 , Steve J Cramer  5 , Rob C Hoeben  5 , Clemens Lowik  1   2   6 , Joel R Walker  7 , Thomas A Kirkland  7 , Thomas P Smith  7 , Wytske M van Weerden  8 , Jeroen de Vrij  9 , Laura Mezzanotte  1   2
Affiliations

NanoBiT System and Hydrofurimazine for Optimized Detection of Viral Infection in Mice-A Novel in Vivo Imaging Platform

Natasa Gaspar et al. Int J Mol Sci. .

Abstract

Reporter genes are used to visualize intracellular biological phenomena, including viral infection. Here we demonstrate bioluminescent imaging of viral infection using the NanoBiT system in combination with intraperitoneal injection of a furimazine analogue, hydrofurimazine. This recently developed substrate has enhanced aqueous solubility allowing delivery of higher doses for in vivo imaging. The small high-affinity peptide tag (HiBiT), which is only 11 amino-acids in length, was engineered into a clinically used oncolytic adenovirus, and the complementary large protein (LgBiT) was constitutively expressed in tumor cells. Infection of the LgBiT expressing cells with the HiBiT oncolytic virus will reconstitute NanoLuc in the cytosol of the cell, providing strong bioluminescence upon treatment with substrate. This new bioluminescent system served as an early stage quantitative viral transduction reporter in vitro and also in vivo in mice, for longitudinal monitoring of oncolytic viral persistence in infected tumor cells. This platform provides novel opportunities for studying the biology of viruses in animal models.

Keywords: bioluminescence imaging; hibit tag; hydrofurimazine; nanobit system; oncolytic virus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Detection of HiBiT-reporter in vitro. (A) Schematic representation of NanoBiT system applied for imaging of viral infection in vivo and in vitro. Bioluminescence in vitro imaging of HiBiT-reporter virus infection by applying the NanoBiT system (B,C). PC-3-LgBiT cells were infected with: (B) HiBiT-reporter virus or (C) with neutralized HiBiT-reporter virus by pre-incubation with intravenous immunoglobulin G (IvIg). The HiBiT-reporter virus infection was performed with several dilutions of viral stock (ranging from 4 to 0.03 MOI). The virus-exposed cells were imaged 24 h post infection by addition of substrate from the Nano-Glo Luciferase Assay System with a final concentration of 0.01 mm. (D) Signals were quantified with IVIS software. Quantification of detected signal as correlation between detected light and viral infectivity after background subtraction. Results are presented as means +SD. Data are significant different (one-way ANOVA F-value of 30.16) and the signal of infected cells is significantly different from the neutralization assay (* p-value < 0.01, ** p-value < 0.001) (E) Fluorescence microscopy of PC-3-LgBiT cells infected with different HiBiT-GFP-reporter virus dilutions (varying from 4 to 0.03 MOI). For checking the transfection rate using the GFP signal, fluorescence was detected 24 h post infection. Size of the scale bar is 2 µm.
Figure 2
Figure 2
Longitudinal bioluminescence (BLI) imaging of HiBiT-reporter virus infection dynamics in vivo in PC3-LgBiT xenografts. (A–C) Infection dynamics in nude BALB/C mice infected with 3.0 × 107 PFU of HiBiT-reporter virus. Representative infected mice were imaged at indicated time points by injecting 4.2 µmol of HFz intraperitoneally (i.p.) and monitoring the BLI signal over 43 days for the high BLI signal response group (n = 5), 20 days for the low BLI signal response group (n = 3) and 20 days for the control group (n = 4). (D) Signals were quantified at the IVIS software. Results are presented as median +SD (n = 4 for the control, n = 5 for ‘high BLI signal group’ and n = 3 for ‘low BLI signal group’). Light signals were significantly different at 20 days post infection (dpi) (F-value of 4.84) where the ‘high BLI signal group’ was statistically different from the ‘low BLI signal group’ and the control group (p-value < 0.05; p-value < 0.01). (E) Tumor volume changes over time. The volume of tumors treated with oncolytic virus was significantly lower in the group of ‘high BLI signal’ (n = 5) when compared to the groups of the control mice (n = 4) and the group with ‘low BLI signal’ (n = 3) (p-value < 0.01) at day 20.
Figure 3
Figure 3
Immunohistochemistry of PC3-LgBiT tumor sections show HiBiT-reporter virus persistence within tumor xenografts 43 days post infection. Virus infected cells assessed via hexon detection in tumor xenografts. Frozen sections of tumors injected with PBS (A) or HiBiT-reporter virus (B) were stained by immunohistochemistry for adenovirus detection with an anti-hexon antibody and an Alexa Fluor 488-labeled secondary antibody; the sections were counterstained with Hoechst in TBS. (A) Representative control section from a tumor injected with PBS. (B) A representative section from virus-infected tumors at 43 days post infection. Size of the scale bar is 100 µm.
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