- Email: [email protected]
Production and characterization of recombinant bovine herpesvirus type 5 glycoprotein D expressed in Pichia pastoris
Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347
Expression of a ScFv–E2T fusion protein in CHO-K1 cells and alfalfa transgenic plants for the selective directioning to antigen presenting cells Agustín I. Ostachuk 1,∗ , Sebastián M. Chiavenna 1 , Cristina Gómez 2 , Andrea Pecora 1 , Mariano D. Pérez-Filgueira 1 , José M. Escribano 3 , Fernando Ardila 2 , María J. Dus Santos 1 , Andrés Wigdorovitz 1 1
Instituto de Virología, CICVyA, INTA-Castelar, Buenos Aires, Argentina 2 Instituto de Genética, CICVyA, INTA-Castelar, Buenos Aires, Argentina 3 Departamento de Biotecnología, INIA, Madrid, Spain Keywords: Medicago sativa; Alfalfa; Plant expression system; Mammalian CHO-K1 cells; Recombinant protein; Single chain antibody fragment; E2 glicoprotein; Bovine viral diarrhea virus E-mail address: [email protected] (A.I. Ostachuk). Species: Ruminants; Other 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 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 0.2 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
315
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. 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¸ão 1,2 , Carlos Gil-Turnes 1,2 , Telmo Vidor 2 , Fábio P.L. Leite 1,3 1
Centro de Biotecnologia, Universidade Federal de Pelotas (UFPel), Brazil 2 Faculdade de Veterinária, UFPel, Brazil 3 Instituto de Biologia, UFPel, Brazil Keywords: Bovine herpesvirus type 5; Pichia pastoris; Recombinant vaccine E-mail address: [email protected] (L.A. Dummer).
Species: Ruminants Bovine herpesvirus type 5 (BoHV-5) is a pathogen of cattle responsible for sporadic outbreak of fatal meningoencephalitis in South America. Glycoprotein D (gD), that act in penetration into host cells, is a candidate to a recombinant vaccine since it induces a strong and persistent cellular immunity responses. Production of gD in heterologous 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 recombinant 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 24 h during 3 days and the detection of the recombinant gD (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 during 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 inactivated BoHV-5. The SDS-PAGE showed a band of approximately 50 kDa, 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 (∼38 kDa), demonstrating its antigenicity. These results suggest that the rgD expressed
316
Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347
in P. pastoris is antigenic, thus it might be used as an efficiently vaccine antigen. Immunogenicity of rgD expressed in P. pastoris will be evaluated in mice and bovine.
populations that may have importance early during an infection and in driving the acquired immune response in cattle.
doi:10.1016/j.vetimm.2008.10.225
doi:10.1016/j.vetimm.2008.10.226
Interaction of natural killer cells, monocytes and dendritic cell populations in cattle
Development of complementary diagnostic reagents for BHV-1 marker vaccines formulated with recombinant strains
Reginaldo G. Bastos 1,2,∗ , Carl Johnson 1 , Wendy C. Brown 2 , Will L Goff 1 1 Animal Disease Research Unit, USDA-ARS, Washington State University, Pullman, WA, USA 2 Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA Keywords: Dendritic cells; NK cells; Bovine
Species: Ruminants Interactions between innate immune cell populations, such as natural killer (NK) cells with monocytes/macrophages and dendritic cells (DC) have been of recent interest. We are investigating interactions of these cells from cattle and our hypothesis is that microbialexposed DC or monocyte interaction with NK cells result in the production of IFN-␥ thus contributing to the innate response to initial disease. A NKp46+ population was obtained by incubating total spleen or peripheral blood mononuclear cells with recombinant human IL15 for 2 weeks followed by depletion of B-lymphocytes, T-lymphocytes, ␥␦-T-lymphocytes and monocytes. The NKp46+ population was identified as CD3− , TcR1− CD2+/− and CD8+ and was able to produce IFN-␥ in response to exogenous IL-12/IL-18. CD13+ DC and CD172a+ monocytes were obtained by positive selection from total spleen or peripheral blood mononuclear cells for determining the role of soluble factors and cell-to-cell contact in the interaction with NK cells. CD13+ DC or CD172a+ monocytes were incubated for 24 hr with heat-killed Mycobacterium bovis BCG (HK-BCG) either in the presence or absence of exogenous IL-18 or recombinant bovine CD40L. NKp46+ cells were then added and co-cultured for 24 hr in trans-well and cell-to-cell formats. The supernatants were then checked by ELISA for the presence of IFN-␥. CD172a+ cells exposed to HK-BCG-induced IFN-␥ production from NKp46+ cells. The interaction was CD40L independent but this T-cell signal significantly enhanced the interaction (P < 0.05). Moreover, the production of IFN-␥ was completely suppressed by the presence of the p38 MAPK inhibitor SB203580, demonstrating the involvement of innate NF-B pathway in the interaction. In contrast, BCG-exposed CD13+ DC obtained from the spleen failed to induce IFN-␥ production from NKp46+ cells regardless of cell-to-cell contact or addition of CD40L or in the presence of IL-18. We and others have recently demonstrated that CD13+ DC represent an immature phenotype. Therefore, we incubated CD13+ DC for 72 h in the presence of IL-4, GM-CSF and Flt3 ligand prior to interaction with BCG and NK cells. Despite maturation, splenic DC maintained unique characteristics compared to monocytes. In conclusion, we provide evidence of interaction of innate immune cell
Daniel M. Pérez Filgueira 1,2 , Maria A. Palacios 1 , Carlos Palacios 1 , Alejandra Romera 1,2,∗ , José M. Escribano 3,4 , Ana M. Sadir 1,2 1
Instituto de Virología, CICVyA, INTA-Castelar, Buenos Aires, Argentina 2 Consejo Nacional de Investigaciones Científicas y Técnicas, (CONICET), Argentina 3 Departamento de Biotecnología, INIA-Madrid, Spain 4 Alternative Gene Expression S.A. (Algenex), Madrid, Spain Keywords: BHV-1 marker vaccines; Diagnosis; Recombinant proteins; Insect expression system E-mail address: [email protected] (A. Romera).
Species: Other; Ruminants Marker vaccines against Bovine Herpes Virus type 1 (BHV-1) lacking non-essential structural proteins are widely utilized around the world. Our group has developed a recombinant BHV-1 strain lacking of glycoprotein E (gE) and including -galactosidase gene as a replication reporter (BHV-1gEgal). Currently, an additional strain is being developed following the same strategy but including the E2 gene from the Bovine viral diarrhea virus (BVDV) and the -Gus gene as a replication reporter instead. Here, we present the development of diagnostic reagents to be used as complement for vaccines formulated with these BHV1 recombinant strains. Different recombinant versions of gE have been produced using an insect larvae-baculovirus expression strategy or a bacterial system. A truncated gE gene (gEc) – without signal peptide and transmembrane domains – was cloned into a baculovirus vector (pFastMel1) to produce the mel-gE-BAC recombinant baculovirus which allowed glycosylation of the recombinant gEc (rgEc). Trichoplusia ni larvae were inoculated with mel-gE-BAC and processed at 72 h. post-inoculation. A recombinant protein with the expected electrophoretic mobility was readily detected by Western blot in total protein extracts from infected larvae. Crude extracts containing rgEc were used to coat ELISA plates and BHV-1 positive bovine serum samples as well as positive and negative controls included in a commercial assay were used to set up optimal conditions for rgEc-based ELISA. Results from a limited number of samples showed that the rgEc-based ELISA was able to detect gE-specific antibodies in sera from cattle infected with BHV-1, showing no reaction against negative samples. However, results using samples from immunized cattle – using coventional or marker vaccines – proved more variable, in a similar way to those obtained with a commercial ELISA. In order to obtain a confirmatory assay, an immunoblotting test has also been assayed using other
Expression of a ScFv–E2T fusion protein in CHO-K1 cells and alfalfa transgenic plants for the selective directioning to antigen presenting cells Agustín I. Ostachuk 1,∗ , Sebastián M. Chiavenna 1 , Cristina Gómez 2 , Andrea Pecora 1 , Mariano D. Pérez-Filgueira 1 , José M. Escribano 3 , Fernando Ardila 2 , María J. Dus Santos 1 , Andrés Wigdorovitz 1 1
Instituto de Virología, CICVyA, INTA-Castelar, Buenos Aires, Argentina 2 Instituto de Genética, CICVyA, INTA-Castelar, Buenos Aires, Argentina 3 Departamento de Biotecnología, INIA, Madrid, Spain Keywords: Medicago sativa; Alfalfa; Plant expression system; Mammalian CHO-K1 cells; Recombinant protein; Single chain antibody fragment; E2 glicoprotein; Bovine viral diarrhea virus E-mail address: [email protected] (A.I. Ostachuk). Species: Ruminants; Other 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 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 0.2 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
315
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. 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¸ão 1,2 , Carlos Gil-Turnes 1,2 , Telmo Vidor 2 , Fábio P.L. Leite 1,3 1
Centro de Biotecnologia, Universidade Federal de Pelotas (UFPel), Brazil 2 Faculdade de Veterinária, UFPel, Brazil 3 Instituto de Biologia, UFPel, Brazil Keywords: Bovine herpesvirus type 5; Pichia pastoris; Recombinant vaccine E-mail address: [email protected] (L.A. Dummer).
Species: Ruminants Bovine herpesvirus type 5 (BoHV-5) is a pathogen of cattle responsible for sporadic outbreak of fatal meningoencephalitis in South America. Glycoprotein D (gD), that act in penetration into host cells, is a candidate to a recombinant vaccine since it induces a strong and persistent cellular immunity responses. Production of gD in heterologous 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 recombinant 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 24 h during 3 days and the detection of the recombinant gD (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 during 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 inactivated BoHV-5. The SDS-PAGE showed a band of approximately 50 kDa, 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 (∼38 kDa), demonstrating its antigenicity. These results suggest that the rgD expressed
316
Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347
in P. pastoris is antigenic, thus it might be used as an efficiently vaccine antigen. Immunogenicity of rgD expressed in P. pastoris will be evaluated in mice and bovine.
populations that may have importance early during an infection and in driving the acquired immune response in cattle.
doi:10.1016/j.vetimm.2008.10.225
doi:10.1016/j.vetimm.2008.10.226
Interaction of natural killer cells, monocytes and dendritic cell populations in cattle
Development of complementary diagnostic reagents for BHV-1 marker vaccines formulated with recombinant strains
Reginaldo G. Bastos 1,2,∗ , Carl Johnson 1 , Wendy C. Brown 2 , Will L Goff 1 1 Animal Disease Research Unit, USDA-ARS, Washington State University, Pullman, WA, USA 2 Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA Keywords: Dendritic cells; NK cells; Bovine
Species: Ruminants Interactions between innate immune cell populations, such as natural killer (NK) cells with monocytes/macrophages and dendritic cells (DC) have been of recent interest. We are investigating interactions of these cells from cattle and our hypothesis is that microbialexposed DC or monocyte interaction with NK cells result in the production of IFN-␥ thus contributing to the innate response to initial disease. A NKp46+ population was obtained by incubating total spleen or peripheral blood mononuclear cells with recombinant human IL15 for 2 weeks followed by depletion of B-lymphocytes, T-lymphocytes, ␥␦-T-lymphocytes and monocytes. The NKp46+ population was identified as CD3− , TcR1− CD2+/− and CD8+ and was able to produce IFN-␥ in response to exogenous IL-12/IL-18. CD13+ DC and CD172a+ monocytes were obtained by positive selection from total spleen or peripheral blood mononuclear cells for determining the role of soluble factors and cell-to-cell contact in the interaction with NK cells. CD13+ DC or CD172a+ monocytes were incubated for 24 hr with heat-killed Mycobacterium bovis BCG (HK-BCG) either in the presence or absence of exogenous IL-18 or recombinant bovine CD40L. NKp46+ cells were then added and co-cultured for 24 hr in trans-well and cell-to-cell formats. The supernatants were then checked by ELISA for the presence of IFN-␥. CD172a+ cells exposed to HK-BCG-induced IFN-␥ production from NKp46+ cells. The interaction was CD40L independent but this T-cell signal significantly enhanced the interaction (P < 0.05). Moreover, the production of IFN-␥ was completely suppressed by the presence of the p38 MAPK inhibitor SB203580, demonstrating the involvement of innate NF-B pathway in the interaction. In contrast, BCG-exposed CD13+ DC obtained from the spleen failed to induce IFN-␥ production from NKp46+ cells regardless of cell-to-cell contact or addition of CD40L or in the presence of IL-18. We and others have recently demonstrated that CD13+ DC represent an immature phenotype. Therefore, we incubated CD13+ DC for 72 h in the presence of IL-4, GM-CSF and Flt3 ligand prior to interaction with BCG and NK cells. Despite maturation, splenic DC maintained unique characteristics compared to monocytes. In conclusion, we provide evidence of interaction of innate immune cell
Daniel M. Pérez Filgueira 1,2 , Maria A. Palacios 1 , Carlos Palacios 1 , Alejandra Romera 1,2,∗ , José M. Escribano 3,4 , Ana M. Sadir 1,2 1
Instituto de Virología, CICVyA, INTA-Castelar, Buenos Aires, Argentina 2 Consejo Nacional de Investigaciones Científicas y Técnicas, (CONICET), Argentina 3 Departamento de Biotecnología, INIA-Madrid, Spain 4 Alternative Gene Expression S.A. (Algenex), Madrid, Spain Keywords: BHV-1 marker vaccines; Diagnosis; Recombinant proteins; Insect expression system E-mail address: [email protected] (A. Romera).
Species: Other; Ruminants Marker vaccines against Bovine Herpes Virus type 1 (BHV-1) lacking non-essential structural proteins are widely utilized around the world. Our group has developed a recombinant BHV-1 strain lacking of glycoprotein E (gE) and including -galactosidase gene as a replication reporter (BHV-1gEgal). Currently, an additional strain is being developed following the same strategy but including the E2 gene from the Bovine viral diarrhea virus (BVDV) and the -Gus gene as a replication reporter instead. Here, we present the development of diagnostic reagents to be used as complement for vaccines formulated with these BHV1 recombinant strains. Different recombinant versions of gE have been produced using an insect larvae-baculovirus expression strategy or a bacterial system. A truncated gE gene (gEc) – without signal peptide and transmembrane domains – was cloned into a baculovirus vector (pFastMel1) to produce the mel-gE-BAC recombinant baculovirus which allowed glycosylation of the recombinant gEc (rgEc). Trichoplusia ni larvae were inoculated with mel-gE-BAC and processed at 72 h. post-inoculation. A recombinant protein with the expected electrophoretic mobility was readily detected by Western blot in total protein extracts from infected larvae. Crude extracts containing rgEc were used to coat ELISA plates and BHV-1 positive bovine serum samples as well as positive and negative controls included in a commercial assay were used to set up optimal conditions for rgEc-based ELISA. Results from a limited number of samples showed that the rgEc-based ELISA was able to detect gE-specific antibodies in sera from cattle infected with BHV-1, showing no reaction against negative samples. However, results using samples from immunized cattle – using coventional or marker vaccines – proved more variable, in a similar way to those obtained with a commercial ELISA. In order to obtain a confirmatory assay, an immunoblotting test has also been assayed using other