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

Giorgia Zambito,Thomas A. Kirkland,Joel R. Walker,Clemens Lowik,Jeroen de Vrij,Steve J. Cramer,Iris J. C. Dautzenberg,Rob C. Hoeben,Wytske M. van Weerden,Thomas P. Smith,Laura Mezzanotte,Natasa Gaspar

doi:10.3390/ijms21165863

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.

Highlights

The development of novel strategies for pre-clinical and clinical imaging of viral replication and dissemination is of crucial importance [1]
The approaches established within this study will have a direct application to various studies where sensitive detection is needed for a better understanding of infection dynamics in vivo, making it a preferable detection method especially for viruses not tolerating accommodation of large inserts or are at the limit of their packaging ability
In order to determine the functionality and the expression stability of the HiBiT tag from the NanoLuc binary technology (NanoBiT) system, a series of cell lines were infected with the oncolytic reporter virus (HAdV-5-DELTA-24-RGD-GFP-T2A-HiBiT)

Summary

Introduction

The development of novel strategies for pre-clinical and clinical imaging of viral replication and dissemination is of crucial importance [1]. A recombinant virus genetically engineered to express a luciferase produces light from infected cells after substrate (luciferin) administration, which can be detected using a sensitive, charged-coupled device (CCD) camera This approach has been used in animal models for several viruses, e.g., Dengue virus, Herpes simplex virus 1 (HSV-1), Sindbis virus, Vaccina virus, Influenza virus and oncolytic viruses (OVs) [3,4,5,6,7,8,9]. A new generation of imaging tools is expected to improve in vivo analysis over current methods, which are often not suitable for imaging the therapeutic efficacy of particular types of OVs. Here we demonstrate the power of our system for viral tracking over the course of 6 weeks post infection using an oncolytic adenovirus as model virus. The approaches established within this study will have a direct application to various studies where sensitive detection is needed for a better understanding of infection dynamics in vivo, making it a preferable detection method especially for viruses not tolerating accommodation of large inserts or are at the limit of their packaging ability

Expression of HiBiT and Preserved Virulence of the Reporter Virus In Vitro

Sensitive Longitudinal In Vivo monitoring of HiBiT-Reporter Oncolytic Virus

Discussion

Cell Lines and Cell Culture Conditions

Lentiviral Vector Construction and Cell Transduction

Reporter Virus Construction

In Vitro BLI and FLI

Neutralization Assay

In Vivo Bioluminescence

Immunohistochemistry

Statistical Analysis