Expression of a ScFv–E2T Fusion Protein in CHO-K1 Cells and Alfalfa Transgenic Plants for the Selective Directioning to Antigen Presenting Cells

Abstract

Bovine viral diarrhea virus (BVDV), a pestivirus of the Flaviviridae family, is an important cause of mortality, morbidity and economical losses of cattle with a worldwide distribution. Subunit vaccines provide the opportunity to develop safe vaccines. However, the challenge is to generate a protective immune response to a cost affordable for veterinary applications. One alternative to solve this problem is to increase the vaccine immunogenicity. A central event in the development of an adaptive immune response is the presentation of the antigens to CD4+ T cells from antigen presenting cells (APCs). In consequence, a way to obtain a more intense specific immune response is to increase the number of MHC-peptide complexes on the surface of APCs. This would be possible by directioning antigens to APCs, fusing them to specific antibodies against APCs´ surface markers. In this work, we report the development of a fusion protein between a single chain antibody fragment (ScFv), recognizing a conserved region of MHC class II molecules, and a truncated form of the E2 glicoprotein (E2T) from BVDV, without its transmembrane domain. The expression systems chosen to express these proteins were mammalian cells (CHO-K1) and plants (Medicago sativa, alfalfa). E2T and ScFv-E2T fusion protein were expressed transiently and stably in CHO-K1 cells, as evidenced by Western blot and ELISA using a monoclonal antibody against E2. They were purified by IMAC (Ion Metal Affinity Chromatography) from cell culture supernatants, resulting in an average final yield of 0.2 mg/L. Alfalfa transgenic plants were generated by infection of petioles with recombinant Agrobacterium tumefaciens, which contained the appropriate gene cloned in a pCAR vector downstream of CsVMV promoter, from Cassava vein mosaic virus. Plants were analyzed for the expression of the proteins by ELISA, and many of them resulted positive. Binding of fusion proteins to the surface of peripheral blood mononuclear cells (PBMCs) from cattle, pig, horse, guinea pig, sheep and goat was studied by flow cytometry. Our results demonstrate that ScFv-E2T expressed in CHO-K1 cells was capable of binding to the surface of PBMCs more intensively than E2T, thus supporting the use of this strategy for delivering vaccine antigens to APCs. Studies comparing the immune responses elicited by both proteins are currently being conducted using a guinea pig experimental model.

Full text

Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347 315
Expression of a ScFv–E2T fusion protein in CHO-K1 cells
and alfalfa transgenic plants for the selective direction-
ing to antigen presenting cells
Agustín I. Ostachuk1,∗, Sebastián M. Chiavenna1, Cristina
Gómez2, Andrea Pecora1, Mariano D. Pérez-Filgueira1,
José M. Escribano3, Fernando Ardila2, María J. Dus
Santos1, Andrés Wigdorovitz1
1Instituto de Virología, CICVyA, INTA-Castelar, Buenos Aires,
Argentina
2Instituto de Genética, CICVyA, INTA-Castelar, Buenos Aires,
Argentina
3Departamento de Biotecnología, INIA, Madrid, Spain
Keywords: Medicago sativa; Alfalfa; Plant expression sys-
tem; Mammalian CHO-K1 cells; Recombinant protein;
Single chain antibody fragment; E2 glicoprotein; Bovine
viral diarrhea virus
E-mail address: aostachuk@cnia.inta.gov.ar (A.I. Ostachuk).
Species: Ruminants; Other
Bovine viral diarrhea virus (BVDV), a pestivirus of the
Flaviviridae family, is an important cause of mortality, mor-
bidity and economical losses of cattle with a worldwide
distribution. Subunit vaccines provide the opportunity to
develop safe vaccines. However, the challenge is to gen-
erate a protective immune response to a cost affordable
for veterinary applications. One alternative to solve this
problem is to increase the vaccine immunogenicity. A cen-
tral event in the development of an adaptive immune
response is the presentation of the antigens to CD4+T cells
from antigen presenting cells (APCs). In consequence, a
way to obtain a more intense specific immune response
is to increase the number of MHC–peptide complexes on
the surface of APCs. This would be possible by direction-
ing antigens to APCs, fusing them to specific antibodies
against APCs’ surface markers. In this work, we report the
development of a fusion protein between a single chain
antibody fragment (ScFv), recognizing a conserved region
of MHC class II molecules, and a truncated form of the
E2 glicoprotein (E2T) from BVDV, without its transmem-
brane domain. The expression systems chosen to express
these proteins were mammalian cells (CHO-K1) and plants
(Medicago sativa, alfalfa). E2T and ScFv–E2T fusion proteins
were expressed transiently and stably in CHO-K1 cells, as
evidenced by Western blot and ELISA using a monoclonal
antibody against E2. They were purified by IMAC (Ion Metal
Affinity Chromatography) from cell culture supernatants,
resulting in an average final yield of 0.2mg/L. Alfalfa trans-
genic plants were generated by infection of petioles with
recombinant Agrobacterium tumefaciens, which contained
the appropriate gene cloned in a pCAR vector downstream
of CsVMV promoter, from Cassava vein mosaic virus. Plants
were analyzed for the expression of the proteins by ELISA,
and many of them resulted positive. Binding of fusion pro-
teins to the surface of peripheral blood mononuclear cells
(PBMCs) from cattle, pig, horse, guinea pig, sheep and goat
was studied by flow cytometry. Our results demonstrate
that ScFv–E2T expressed in CHO-K1 cells was capable of
binding to the surface of PBMCs more intensively than
E2T, thus supporting the use of this strategy for delivering
vaccine antigens to APCs. Studies comparing the immune
responses elicited by both proteins are currently being con-
ducted using a guinea pig experimental model.
doi:10.1016/j.vetimm.2008.10.224
Production and characterization of recombinant bovine
herpesvirus type 5 glycoprotein D expressed in Pichia
pastoris
Luana A. Dummer 1,∗, Leandro Q. Nizoli 1, Andréa S.R.
Rocha 1, Carina M. Moraes 1, Fabricio R. Conceic¸ão1,2, Car-
los Gil-Turnes1,2, Telmo Vidor2, Fábio P.L. Leite1,3
1Centro de Biotecnologia, Universidade Federal de Pelotas
(UFPel), Brazil
2Faculdade de Veterinária, UFPel, Brazil
3Instituto de Biologia, UFPel, Brazil
Keywords: Bovine herpesvirus type 5; Pichia pastoris;
Recombinant vaccine
E-mail address: dummer@gmail.com (L.A. Dummer).
Species: Ruminants
Bovine herpesvirus type 5 (BoHV-5) is a pathogen of
cattle responsible for sporadic outbreak of fatal menin-
goencephalitis in South America. Glycoprotein D (gD), that
act in penetration into host cells, is a candidate to a recom-
binant vaccine since it induces a strong and persistent
cellular immunity responses. Production of gD in heterolo-
gous systems should be able to keep the protein solubility
and authentic immugenicity. The yeast Pichia pastoris is
an expression system that supports this requirement. We
used the expression vector pPICZ␣B, which is integrative,
inducible with methanol and allows the secretion of recom-
binant protein to medium. The gD gene was amplified
through PCR using 2% of DMSO and cloned into pPICZ␣B
to generate the pPICZ␣B/gD. This plasmid was linearized
and then transformed into P. pastoris strain KM71H (MutS)
by electroporation. Recombinant strains were selected by
Zeocin resistance in YPDS plates and the expression was
detected by Colony Dot Blot in BMMY plates. The induction
was performed by addiction of 1% of methanol every 24h
during 3 days and the detection of the recombinantgD (rgD)
was performed with monoclonal antibody (MAb) anti-his
tag. One positive colony was selected, inoculated in BMGY
and incubated 24 h at 28◦C in shaking flask. The cells were
harvested and resuspended in 1/5 volume BMMY to induce
the expression. Methanol was added to 1% every 24 h dur-
ing 2 days. The cells were harvested and the supernatant
saturated overnight at 4◦C with 80% of ammonium sulfate.
The precipitated was obtained by centrifugation, dialyzed
overnight into PBS pH 7.4 and the rgD characterized by
SDS-PAGE and Western Blot with serum of mice and bovine
vaccinated with inactivatedBoHV-5. The SDS-PAGE showed
a band of approximately 50kDa, which suggest that the
rgD was glycosilated. Serum of mice and bovine vaccinated
with inactivated BoHV-5 recognized the glycosilated rgD
(∼50 kDa) and unglycosilated (∼38kDa), demonstrating its
antigenicity. These results suggest that the rgD expressed