Antiviral Immunotoxin Against Bovine herpesvirus-1: Targeted Inhibition of Viral Replication and Apoptosis of Infected Cell.

Jian Xu,Xiaoyu Feng,Bo Jiang,Munir Iqbal,Yunhong Cai,Joshua E Sealy,Xiufen Huang,Yongqing Li,Xiaoyang Li,Jing Wu,Xixi Zhang

doi:10.3389/fmicb.2018.00653

Abstract

Bovine herpesvirus 1 (BoHV-1) is a highly contagious viral pathogen which causes infectious bovine rhinotracheitis in cattle worldwide. Currently, there is no antiviral prophylactic treatment available capable of mitigating the disease impact and facilitating recovery from latent infection. In this study, we have engineered a novel recombinant anti-BoHV-1 immunotoxin construct termed “BoScFv-PE38” that consists of a single-chain monoclonal antibody fragment (scFv) fused with an active domain of Pseudomonas exotoxin A as a toxic effector (PE38). The recombinant BoScFv-PE38 immunotoxin expressed in a prokaryotic expression system has specific binding affinity for BoHV-1 glycoprotein D (gD) with a dissociation constant (Kd) of 12.81 nM and for BoHV-1 virus particles with a Kd value of 97.63 nM. We demonstrate that the recombinant BoScFv-PE38 is internalized into MDBK cell compartments that inhibit BoHV-1 replication with a half-maximal inhibitory concentration (IC50) of 4.95 ± 0.33 nM and a selective index (SI) of 456 ± 31. Furthermore, the BoScFv-PE38 exerted a cytotoxic effect through the induction of ATP and ammonia, leading to apoptosis of BoHV-1-infected cells and the inhibition of BoHV-1 replication in MDBK cells. Collectively, we show that BoScFv-PE38 can potentially be employed as a therapeutic agent for the treatment of BoHV-1 infection.

Highlights

Bovine herpesvirus-1 (BoHV-1) belongs to the Herpesviridae family in the Alphaherpesvirinae subfamily (Muylkens et al, 2007) and is an economically important pathogen that causes infectious bovine rhinotracheitis (IBR) in cattle (Rola et al, 2017; Thakur et al, 2017)
The western blot analysis showed that both the recombinant glycoprotein D (gD) protein produced in E. coli. and the wild type gD protein derived from Bovine herpesvirus 1 (BoHV-1) virus had specific binding affinity for purified BoScFv-PE38 immunotoxin (Figures 2C,D)
The results showed that BoScFv-PE38 immunotoxin selectively internalized into the Madin-Darby bovine kidney (MDBK) cells infected with BoHV-1 rather than negative control cells (Figure 4A), indicating that BoScFv-PE38 immunotoxin had efficient capability to target and internalize BoHV-1 in MDBK cells

Summary

Introduction

Bovine herpesvirus-1 (BoHV-1) belongs to the Herpesviridae family in the Alphaherpesvirinae subfamily (Muylkens et al, 2007) and is an economically important pathogen that causes infectious bovine rhinotracheitis (IBR) in cattle (Rola et al, 2017; Thakur et al, 2017). Treatment of viral infections with currently available synthetic drugs possess several deficiencies including toxicity and resistance (Spiess et al, 2016; Khandelwal et al, 2017; Wambaugh et al, 2017), there is urgency for new and improved antivirals. Immunotoxins against a variety of viruses have been developed, including singlestranded RNA viruses infecting humans, such as HIV, PCV, rabies virus, and herpesvirus, HCMV, EBV and HSV-2 (Mareeva et al, 2010; Chatterjee et al, 2012; Spiess et al, 2017). Immunotoxins, that are chimeric proteins consisting of the antigen-binding fragment (Fab) of an antibody conjugated to a toxin molecule, have shown promise in targeted delivery of antiviral toxins to virus infected cells (Margolis et al, 2016; Spiess et al, 2016). The target molecule is the major element within the immunotoxin and plays a vital role in targeting virus-infected cells

Methods

Results

Conclusion