Recombinant Infectious Bursal Disease Virus Carrying Hepatitis C Virus Epitopes

Abstract

The delivery of foreign epitopes by a replicating nonpathogenic avian infectious bursal disease virus (IBDV) was explored. The aim of the study was to identify regions in the IBDV genome that are amenable to the introduction of a sequence encoding a foreign peptide. By using a cDNA-based reverse genetics system, insertions or substitutions of sequences encoding epitope tags (FLAG, c-Myc, or hepatitis C virus epitopes) were engineered in the open reading frames of a nonstructural protein (VP5) and the capsid protein (VP2). Attempts were also made to generate recombinant IBDV that displayed foreign epitopes in the exposed loops (P BC and P HI ) of the VP2 trimer. We successfully recovered recombinant IBDVs expressing c-Myc and two different virus-neutralizing epitopes of human hepatitis C virus (HCV) envelope glycoprotein E in the VP5 region. Western blot analyses with anti-c-Myc and anti-HCV antibodies provided positive identification of both the c-Myc and HCV epitopes that were fused to the N terminus of VP5. Genetic analysis showed that the recombinants carrying the c-Myc/HCV epitopes maintained the foreign gene sequences and were stable after several passages in Vero and 293T cells. This is the first report describing efficient expression of foreign peptides from a replication-competent IBDV and demonstrates the potential of this virus as a vector. (26), has been used as a therapeutic agent without any toxicity in clinical trials with patients suffering from acute and chronic hepatitis C virus (HCV) infections (2,8). IBDV belongs to the genus Avibirnavirus of the Birnaviridae family, and its genome consists of two segments of double-stranded RNA (11). The smaller segment, B, encodes VP1, a 97-kDa multifunctional protein with polymerase and capping enzyme activities (21, 27). The larger segment, A, encodes a 110-kDa precursor polyprotein in a single large open reading frame (ORF) which is cotranslationally processed by the viral protease VP4 (4, 24) into the VP2 precursor (pVP2), VP3, and VP4. pVP2 is further processed by several proteolytic cleavages at its C terminus for conversion into mature VP2 (10). Segment A also encodes VP5, a 17-kDa nonstructural (NS) protein, in a small ORF partly preceding and overlapping the polyprotein ORF. VP5 is dispensable for viral replication in vitro and in vivo (29), which makes it a prime candidate for the construction of marked vaccines carrying deletions. These marked vectors could be easily distinguished from the wild-type virus and could also trigger a specific cellular immune response in the host species. The available structural data for VP2 (7,14,17) reveal that the protein is folded into three different domains (base B, shell S, and projection P). Expression of VP2 by itself leads to dodecahedral subviral particles (SVPs) containing 20 VP2 trimers (6) and exposing the four loops of the P domain (named P BC , P DE , P FG , and P HI ). Here, we explore the possibility of displaying foreign epitopes stably in recombinant IBDV by either inserting or replacing sequences ...

Critical Role for Voltage-Dependent Anion Channel 2 in Infectious Bursal Disease Virus-Induced Apoptosis in Host Cells via Interaction with VP5

“…IBDV is capable of replicating in multiple types of cells, including chicken B cells (25), CEF and DF-1 cells (37), and Vero and HEK293T cells (33). To determine if IBDV induces a CPE in DF-1 cells, we infected cells of this cell line with the Lx strain of this virus at an MOI of 10.…”

The Clinically Validated Viral Superinfection Therapy (SIT) Platform Technology May Mitigate Severe Cases of COVID-19 Infections

“…Since it was already demonstrated that SIT generates strong anti-IBDV antibody response, it is our hypotheses that incorporation of COVID-19 specific epitopes into the IBDV capsid will be able to provide coronavirus specific immune responses, which will synergize with the SIT technology. Therefore, we will incorporate the genetic sequence for COVID-19's spike epitopes into the R903/78 vector and recombinant IBDV vectors carrying COVID-19 spike epitopes will be generated (Upadhyay, et al, 2011). These vectors will be evaluated for their ability to generate immune responses against COVID-19.…”

“…An attenuated vaccine strain of IBDV was already successfully administered to resolve infections induced by two completely different viruses, the hepatitis B (DNA) and C (RNA) viruses (HBV/HCV) in acute and decompensated chronic hepatitis patients (Csatary, et al, 1998) (Csatary, et al, 1999). Importantly, this virus is also a potential vaccine vector drug candidate, since a recombinant IBDV was previously generated that displays exogenous viral peptides from a replication competent IBDV (Upadhyay, et al, 2011). The epidemiological efficacy of a similar strategy to SIT using attenuated vaccine viruses that stimulate the production of endogenous interferon of the host had already been tested in pivotal large scale clinical trials 2 as described below.…”

Critical Roles of Glucocorticoid-Induced Leucine Zipper in Infectious Bursal Disease Virus (IBDV)-Induced Suppression of Type I Interferon Expression and Enhancement of IBDV Growth in Host Cells via Interaction with VP4

“…IBDV is capable of replicating in multiple types of cells, including chicken B cells (33), CEF and DF-1 cells (34), and Vero and HEK293T cells (35). To determine if the IBDV Lx strain could replicate in HEK293T cells, we infected this cell line with the virus at an MOI of 10 and examined the viral growth with immunofluorescence antibody assay (IFA).…”