Truncated E2 of bovine viral diarrhea virus (BVDV) expressed in Drosophila melanogaster cells: A candidate antigen for a BVDV ELISA

Marzocca, Marina Patricia; Seki, Cristina; Giambiagi, Susana; Robiolo, Blanca; Schauer, Roland; Santos, María José Dus; Scodeller, Eduardo A.; Torre, J.L. La; Wigdorovitz, Andrés; Grigera, Pablo R.

doi:10.1016/j.jviromet.2007.03.023

Cited by 19 publications

(22 citation statements)

References 17 publications

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“…Also, sera from the same days, but belonging to 4 unvaccinated calves were used as negative controls. Briefly, U-bottom polystyrene microplates (Maxisorp, NUNC) were coated with an anti-E2 (from BVDV-1a) monoclonal antibody [14] and incubated at 4 • C ON. After blocking the plates in PBS-T, 1% skimmed milk for 1 h at 37 • C, recombinant tE2 protein was added and incubated for 1 h at 37 • C. As primary detection antibody, sera from the calves corresponding to the day of the first vaccination and to 40 dpv were used at 1:20 dilution and incubated for 1 h at 37 • C. Thereafter, peroxidase-conjugated goat anti-bovine IgG1 or IgG2 (1:1000, Serotec) were used as secondary antibody, and incubated for 1 h at 37 • C. Finally, plates were revealed using ABTS (Sigma) and optical densities (OD) were measured at 405 nm in a Multiskan Fc plate reader (Thermo Scientific).…”

Section: Immune Response After Vaccinationmentioning

confidence: 99%

Development of an APC-targeted multivalent E2-based vaccine against Bovine Viral Diarrhea Virus types 1 and 2

Pécora¹,

Malacari²,

Aguirreburualde³

et al. 2015

Vaccine

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Section: Immune Response After Vaccinationmentioning

confidence: 99%

Development of an APC-targeted multivalent E2-based vaccine against Bovine Viral Diarrhea Virus types 1 and 2

Pécora¹,

Malacari²,

Aguirreburualde³

et al. 2015

Vaccine

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“…The diagnostic sensitivity of P. pastoris expressed E2 ELISA obtained in this study was better than that reported earlier with a drosophila expressed E2 indirect ELISA which yielded 82% correlation with the virus neutralization test results and a Baculovirus expressed E2 ELISA that showed a sensitivity of 88.3%. [11,12] But it was lower than an avidity blocking ELISA using drosophila expressed E2 antigen that showed a sensitivity of 98.10%. [32] This may be explained by the reason that most ELISAs probably detect preferentially IgG isotypes, while VNT detects both IgM and IgG isotypes.…”

Section: Discussionmentioning

confidence: 97%

“…BVDV E2 protein has previously been expressed in mammalian expression system, [9,10] baculovirus expression system, [9,11] Drosophila A c c e p t e d M a n u s c r i p t 4 melanogaster system, [12] Saccharomyces cerevisiae yeast system [13] and alfalfa plants [14] . In this study, BVDV E2 protein was expressed in Pichia pastoris yeast for the first time and its potential use as a candidate antigen was evaluated in ELISA for detection of BVDV neutralizing antibodies in cattle.…”

Section: Expression Of Bovine Viral Diarrhoea Virus Envelope Glycopromentioning

confidence: 99%

Expression of Bovine Viral Diarrhea Virus Envelope Glycoprotein E2 in YeastPichia pastorisand its Application to an ELISA for Detection of BVDV Neutralizing Antibodies in Cattle

Behera

Mishra

Nema

et al. 2015

Journal of Immunoassay and Immunochemistry

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The aim of this article is to express envelope glycoprotein E2 of bovine viral diarrhea virus (BVDV) in yeast Pichia pastoris and its utility as a diagnostic antigen in ELISA. The BVDV E2 gene was cloned into the pPICZαA vector followed by integration into the Pichia pastoris strain X-33 genome for methanol-induced expression. SDS-PAGE and Western blot results showed that the recombinant BVDV E2 protein (72 kDa) was expressed and secreted into the medium at a concentration of 40 mg/L of culture under optimized conditions. An indirect ELISA was then developed by using the yeast-expressed E2 protein. Preliminary testing of 300 field cattle serum samples showed that the E2 ELISA showed a sensitivity of 91.07% and a specificity of 92.02% compared to the reference virus neutralization test. The concordance between the E2 ELISA and VNT was 91.67%. This study demonstrates feasibility of BVDV E2 protein expression in yeast Pichia pastoris for the first time and its efficacy as an antigen in ELISA for detecting BVDV neutralizing antibodies in cattle.

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“…BVDE2Fc aggregation was expected due to the non-covalent binding of Fc subunits or the interactions between E2 proteins in solution (24). The final conformation of the chimaeric molecule constitutes an important feature for E2 proteins because the structure of these molecules on the viral surface is essential for its immunogenicity (15,29). Furthermore, epitopes of the E2 protein inducing neutralising antibodies are conformational in a way highly dependent on disulphide bond formation by cysteine residues (9,39).…”

Section: Discussionmentioning

confidence: 99%

Characterisation of a new molecule based on two E2 sequences from bovine viral diarrhoea-mucosal disease virus fused to the human immunoglobulin Fc fragment

Pose

Seguí

Pérez

et al. 2021

Journal of Veterinary Research

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Introduction Proper conformational arrangement of the E2 molecules of bovine viral diarrhoea-mucosal disease virus (BVD-MDV) is crucial to obtain an effective recombinant vaccine candidate against the disease. In this study, we characterised a new molecule composed of two distinct sequences of the E2 glycoprotein of BVD-MDV and the Fc fragment of human immunoglobulin (BVDE2Fc). Materials and Methods The chimaeric protein was expressed in mammalian cell lines of different species by adenoviral transduction and purified by immobilised metal-affinity chromatography. The N-glycans were profiled by HPLC, and the BVDE2Fc immunogenicity was assessed in male mice. The antigen-antibody reactions were evaluated by ELISA. Results The MDBK cell line was selected from among five for the final production of BVDE2Fc. After purification to over 90%, the N-glycan profile showed neutral and complex oligosaccharides. The mouse immunisation induced a strong humoral response, which produced antibodies able to attach to conformational epitopes on E2 molecules, while the Fc fragment barely contributed to the immune response. Additionally, BVDE2Fc attached to antibodies from bovine sera positive to distinct BVD-MDV subtypes, whereas the loss of BVDE2Fc structure during the deglycosylation process considerably diminished those interactions. Conclusion These results demonstrate that the structure of E2 molecules arranged in tandem and attached to an Fc fragment could represent a viable design for future vaccine candidates against BVD-MD.

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Truncated E2 of bovine viral diarrhea virus (BVDV) expressed in Drosophila melanogaster cells: A candidate antigen for a BVDV ELISA

Cited by 19 publications

References 17 publications

Development of an APC-targeted multivalent E2-based vaccine against Bovine Viral Diarrhea Virus types 1 and 2

Development of an APC-targeted multivalent E2-based vaccine against Bovine Viral Diarrhea Virus types 1 and 2

Expression of Bovine Viral Diarrhea Virus Envelope Glycoprotein E2 in YeastPichia pastorisand its Application to an ELISA for Detection of BVDV Neutralizing Antibodies in Cattle

Characterisation of a new molecule based on two E2 sequences from bovine viral diarrhoea-mucosal disease virus fused to the human immunoglobulin Fc fragment

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