Immunogenicity of a secreted, C-terminally truncated, form of bovine viral diarrhea virus E2 glycoprotein as a potential candidate in subunit vaccine development

Abstract

Both current live, attenuated, and killed virus vaccines for bovine viral diarrhea virus (BVDV) have their limitations. Here, we report the development of a BVDV subunit vaccine by (i) the expression of a secreted form of a recombinant E2 glycoprotein using BHK21 cells and (ii) determination of the immune responses in mice. The E2 glycoprotein was modified by deletion of the C-terminal transmembrane anchor domain and fusion to a V5 epitope tag. This allowed detection using anti-V5 monoclonal antibodies together with simple purification of the expressed, secreted, form of E2 from the cell media. Furthermore, we genetically fused green fluorescent protein (GFP) linked to E2 via a Thosea asigna virus 2A (T2A) ribosome skipping sequence thereby creating a self-processing polyprotein [GFP-T2A-BVDV-E2trunk-V5], producing discrete [GFP-T2A] and [E2trunk-V5] translation products: GFP fluorescence acts, therefore, as a surrogate marker of E2 expression, BALB/c mice were inoculated with [E2trunk-V5] purified from cell media and both humoral and cellular immune responses were observed. Our antigen expression system provides, therefore, both (i) a simple antigen purification protocol together with (ii) a feasible strategy for further, large-scale, production of vaccines.

Immune protection induced by E2 recombinant glycoprotein of bovine viral diarrhea virus in a murine model

“…The efficacy of these BVDV vaccine candidates has been evaluated in animal models such as goats, guinea pigs, cattle, and mice models with promising results. Depending on the study, the protection conferred against BVDV infection was evidenced by a reduction in clinical signs such as lymphopenia and lack of pyrexia, reduction in viral shedding, antibody neutralization titers, and an increase in CD4 + and CD8 + lymphocytes along with the cytokine levels for humoral and cellular immune responses ( 12 , 44 – 49 ).…”

The 2A Story: The End of the Beginning

“…Although not a full-proof solution, a GSG linker used in conjunction with a furin recognition site (e.g -furin-GSG-2A-, -GSG-furin-GSG-2A-) reportedly enhanced cleavage efficiency via increased exposure of the -RRKR-site [32,44,64]. The "unwanted" tag may remain-recognised by monoclonal antibodies, peptide epitopes can characterise, purify, and localise proteins of interest in vitro and in vivo [65,66]. Commercial antibodies have been raised against the consensus 2A sequence, which thus serves as a useful target for identifying 2A-tagged proteins in biochemical assays-validated research applications include ICC, immunofluorescence, IP, and Western Blot [67,68].…”

“…In this "proof-of-principle" study, truncated G glycoprotein was detected throughout the exocytic pathway and secreted efficiently from BHK cells into the cell media. In a follow-on study we evaluated the immunogenicity of a secreted, C-terminally truncated form of bovine viral diarrhoea virus (BVDV) E2 glycoprotein in mice [66]. BVDV is the causative agent of one of the most widespread and economically important…”