- Email: [email protected]
Innate and adaptive immune responses of catfish antigen-presenting cells to live attenuated Edwardsiella ictaluri vaccines
Abstracts / Fish & Shellfish Immunology 53 (2016) 58e93
antigens that were selected based on an in silico antigen discovery approach. Thus, the proteome of AS strain A449 was analyzed by an antigen discovery platform and its proteins consequently ranked by their predicted ability to evoke protective immune response against AS. Fourteen proteins were prepared in 3 different experimental subunit vaccine combinations and used to vaccinate rainbow trout by intraperitoneal (i.p.) injection. We tested the proteins for their ability to elicit antibody production and protection. Thus, fish were exposed to virulent AS 7 weeks post-vaccination by applying a novel, multi-puncture challenge method. The immune response in fish was evaluated following vaccination and challenge by measuring antibody levels and recording survival. The control group showed 56 % mortality whereas the groups of fish vaccinated with experimental subunit vaccines exhibited significantly lower mortalities (17-30 %). These results imply that in silico-predicted protective protein antigens of AS have significant protective properties and should be considered for further validation as potential candidates for a subunit vaccine against furunculosis. Keywords: Furunculosis, subunit vaccines, rainbow trout, Aeromonas salmonicida, challenge x Corresponding author. Tel.: þ45 22243861. E-mail address: [email protected] (M.H. Marana). * These authors have contributed equally to this work.
O-014. Innate and adaptive immune responses of catfish antigen-presenting cells to live attenuated Edwardsiella ictaluri vaccines Adef Kordon 1, *, Hossam Abdelhamed 1, *, Wes A. Baumgartner 2, Attila Karsi 1, Lesya M. Pinchuk 1, § 1 Department of Basic Science, Collage of Veterinary Medicine, Mississippi State University, Starkville, MS, 39762, United States 2 Department of Pathobiology and Population Medicine, Collage of Veterinary Medicine, Mississippi State University, Starkville, MS, 39762, United States
Abstract Efficacious live attenuated Edwardsiella ictaluri vaccines are expected to deliver the antigen to antigen-presenting cells (APCs) that can elicit potent protective innate and adaptive immune responses resulting in the killing of the infected targets or inducing the killing mechanisms in the infected cells. T cell-inducing vaccines must aim to deliver the antigen to APCs so that it can be presented on MHC molecules to the specific T cells. Dendritic cells (DCs) are the most powerful APCs that bridge, control and regulate innate and adaptive immune responses in vertebrates. Recently, we have identified the presence of the Langerhan-like cells containing Birbeck granules in the hemopoietic tissues of channel catfish by the following approaches: flow cytometry, immunohistochemistry, and transmission electron microscopy. Langerin/CD207-positive cells have been detected in catfish spleen and anterior kidney but not in peripheral blood and skin by using mAbs to human Langerin/CD207. These results lay a foundation for our present study that is to determine the vaccine- induced productive innate and adaptive immune responses through catfish APCs, DCs, macrophages, and B cells. Currently, we are evaluating the possible role of Langerin/CD207-positive cells in the protective immunity to E. ictaluri vaccines by immunohistochemistry. The function of professional phagocytes, macrophages, to produce nitric oxide in response to live attenuated vaccines are being evaluated by assessing the nitric oxide synthase gene expression levels by real-time PCR. In addition, to characterize the effects of live attenuated vaccines, we are evaluating T cell-mediated immunity that controls function of APCs and by assessing the gene expression levels of IFNg in peripheral blood, anterior kidney, and spleen of catfish. Finally, the effects of B cell-mediated immunity, B cell-specific gene expression, and immunoglobulin M titers are being determined to live attenuated E. ictaluri vaccines. We suggest that current research will provide the optimal vaccination regime and important basic knowledge on the mechanisms of innate and adaptive immune responses against the most effective vaccines.
63
Keywords: Edwardsiella ictaluri, Live Attenuated Vaccine, Antigen-Presenting Cells, Dendritic Cells, Catfish x Corresponding author. Tel.: (662) 325 1436; Fax.: (662) 325 1031. E-mail address: [email protected] (L.M. Pinchuk). * These authors have contributed equally to this work.
O-015. Attenuated Infectious Hematopoietic Necrosis Virus (IHNV) with rearranged gene order as potential vaccine Ronan N. Rouxel 1, Carolina Tafalla 1, 2, Emilie M erour 1, Esther Leal 2, St ephane Biacchesi 1, $, Michel Bremont 1, * Paris-Saclay, 78350, Jouy-en-Josas, France VIM, INRA, Universite Centro de Investigacion en Sanidad Animal (CISA-INIA), Valdeolmos, 28130, Madrid, Spain 1
2
The genome of the Infectious Hematopoietic Necrosis Virus (IHNV), a salmonid Novirhabdovirus has been engineered to modify the gene order and to evaluate the impact on a possible attenuation of the virus in vitro and in vivo. By reverse genetics, eight recombinant IHNV (rIHNV), termed NxGy according to the respective position of the nucleoprotein (N) and glycoprotein (G) genes along the genome, have been recovered. All rIHNV have been fully characterized for their cytopathic effect, kinetics of replication, profile of viral gene transcription and their induced-immune response potential in fish. These rIHNV are stable through up to ten passages in cell culture. Following bath immersion administration of the various rIHNV to juvenile trout, some of the rIHNV were clearly attenuated (N2G3, N2G4, N3G4 and N4G1). Position of the N gene is one of the most critical features correlated to the level of viral attenuation. The recombinant virus N2G3 induced a strong antibody response in immunized fish and conferred 86% of protection against wild-type IHNV challenge in trout, thus representing a promising live-attenuated vaccine candidate. Keywords: reverse genetics, IHNV, trout, attenuation, vaccine * Corresponding author. Tel.: þ33 6 88 97 63 33. E-mail address: [email protected] (M. Bremont). $ phane Biacchesi. E-mail address: [email protected] Speaker: Ste
O-016. Characterization of local, systemic and mucosal immune responses after oral DNA vaccination against spring viremia of carp virus Carmen WE. Embregts *,§, Geert F. Wiegertjes, Maria Forlenza * Cell Biology and Immunology Group, Wageningen University, The Netherlands DNA vaccination through i.m. injection of DNA plasmid encoding the G protein of SVCV (SVCV-G) is very successful in carp but this method of vaccine delivery is not practical. Alternatively, oral vaccination would be highly preferred due to its stress-free, labour-extensive and easy delivery. On the downside, oral vaccines are often less efficacious when compared to traditional injection vaccines and little is known about their activation of immune mechanisms. In an effort to generate an oral DNA vaccine against SVCV, carp of 2 g were orally vaccinated three times with alginate microspheres containing either 8 ug pcDNA3 plasmid, 8 ug pcDNA3-SVCVG plasmid, or PBS. Owing to the availability of several carp leukocytespecific monoclonal antibodies, we revealed the early recruitment of specific populations of leukocytes to the intestine and spleen following oral vaccination. The combined use of cell type-specific antibodies and an antibody against the SVCV-G protein allowed us for the first time, to identify the sites and kinetics of expression of the SVCV-G protein, and to concomitantly recognize the cell types involved in antigen uptake and processing.
antigens that were selected based on an in silico antigen discovery approach. Thus, the proteome of AS strain A449 was analyzed by an antigen discovery platform and its proteins consequently ranked by their predicted ability to evoke protective immune response against AS. Fourteen proteins were prepared in 3 different experimental subunit vaccine combinations and used to vaccinate rainbow trout by intraperitoneal (i.p.) injection. We tested the proteins for their ability to elicit antibody production and protection. Thus, fish were exposed to virulent AS 7 weeks post-vaccination by applying a novel, multi-puncture challenge method. The immune response in fish was evaluated following vaccination and challenge by measuring antibody levels and recording survival. The control group showed 56 % mortality whereas the groups of fish vaccinated with experimental subunit vaccines exhibited significantly lower mortalities (17-30 %). These results imply that in silico-predicted protective protein antigens of AS have significant protective properties and should be considered for further validation as potential candidates for a subunit vaccine against furunculosis. Keywords: Furunculosis, subunit vaccines, rainbow trout, Aeromonas salmonicida, challenge x Corresponding author. Tel.: þ45 22243861. E-mail address: [email protected] (M.H. Marana). * These authors have contributed equally to this work.
O-014. Innate and adaptive immune responses of catfish antigen-presenting cells to live attenuated Edwardsiella ictaluri vaccines Adef Kordon 1, *, Hossam Abdelhamed 1, *, Wes A. Baumgartner 2, Attila Karsi 1, Lesya M. Pinchuk 1, § 1 Department of Basic Science, Collage of Veterinary Medicine, Mississippi State University, Starkville, MS, 39762, United States 2 Department of Pathobiology and Population Medicine, Collage of Veterinary Medicine, Mississippi State University, Starkville, MS, 39762, United States
Abstract Efficacious live attenuated Edwardsiella ictaluri vaccines are expected to deliver the antigen to antigen-presenting cells (APCs) that can elicit potent protective innate and adaptive immune responses resulting in the killing of the infected targets or inducing the killing mechanisms in the infected cells. T cell-inducing vaccines must aim to deliver the antigen to APCs so that it can be presented on MHC molecules to the specific T cells. Dendritic cells (DCs) are the most powerful APCs that bridge, control and regulate innate and adaptive immune responses in vertebrates. Recently, we have identified the presence of the Langerhan-like cells containing Birbeck granules in the hemopoietic tissues of channel catfish by the following approaches: flow cytometry, immunohistochemistry, and transmission electron microscopy. Langerin/CD207-positive cells have been detected in catfish spleen and anterior kidney but not in peripheral blood and skin by using mAbs to human Langerin/CD207. These results lay a foundation for our present study that is to determine the vaccine- induced productive innate and adaptive immune responses through catfish APCs, DCs, macrophages, and B cells. Currently, we are evaluating the possible role of Langerin/CD207-positive cells in the protective immunity to E. ictaluri vaccines by immunohistochemistry. The function of professional phagocytes, macrophages, to produce nitric oxide in response to live attenuated vaccines are being evaluated by assessing the nitric oxide synthase gene expression levels by real-time PCR. In addition, to characterize the effects of live attenuated vaccines, we are evaluating T cell-mediated immunity that controls function of APCs and by assessing the gene expression levels of IFNg in peripheral blood, anterior kidney, and spleen of catfish. Finally, the effects of B cell-mediated immunity, B cell-specific gene expression, and immunoglobulin M titers are being determined to live attenuated E. ictaluri vaccines. We suggest that current research will provide the optimal vaccination regime and important basic knowledge on the mechanisms of innate and adaptive immune responses against the most effective vaccines.
63
Keywords: Edwardsiella ictaluri, Live Attenuated Vaccine, Antigen-Presenting Cells, Dendritic Cells, Catfish x Corresponding author. Tel.: (662) 325 1436; Fax.: (662) 325 1031. E-mail address: [email protected] (L.M. Pinchuk). * These authors have contributed equally to this work.
O-015. Attenuated Infectious Hematopoietic Necrosis Virus (IHNV) with rearranged gene order as potential vaccine Ronan N. Rouxel 1, Carolina Tafalla 1, 2, Emilie M erour 1, Esther Leal 2, St ephane Biacchesi 1, $, Michel Bremont 1, * Paris-Saclay, 78350, Jouy-en-Josas, France VIM, INRA, Universite Centro de Investigacion en Sanidad Animal (CISA-INIA), Valdeolmos, 28130, Madrid, Spain 1
2
The genome of the Infectious Hematopoietic Necrosis Virus (IHNV), a salmonid Novirhabdovirus has been engineered to modify the gene order and to evaluate the impact on a possible attenuation of the virus in vitro and in vivo. By reverse genetics, eight recombinant IHNV (rIHNV), termed NxGy according to the respective position of the nucleoprotein (N) and glycoprotein (G) genes along the genome, have been recovered. All rIHNV have been fully characterized for their cytopathic effect, kinetics of replication, profile of viral gene transcription and their induced-immune response potential in fish. These rIHNV are stable through up to ten passages in cell culture. Following bath immersion administration of the various rIHNV to juvenile trout, some of the rIHNV were clearly attenuated (N2G3, N2G4, N3G4 and N4G1). Position of the N gene is one of the most critical features correlated to the level of viral attenuation. The recombinant virus N2G3 induced a strong antibody response in immunized fish and conferred 86% of protection against wild-type IHNV challenge in trout, thus representing a promising live-attenuated vaccine candidate. Keywords: reverse genetics, IHNV, trout, attenuation, vaccine * Corresponding author. Tel.: þ33 6 88 97 63 33. E-mail address: [email protected] (M. Bremont). $ phane Biacchesi. E-mail address: [email protected] Speaker: Ste
O-016. Characterization of local, systemic and mucosal immune responses after oral DNA vaccination against spring viremia of carp virus Carmen WE. Embregts *,§, Geert F. Wiegertjes, Maria Forlenza * Cell Biology and Immunology Group, Wageningen University, The Netherlands DNA vaccination through i.m. injection of DNA plasmid encoding the G protein of SVCV (SVCV-G) is very successful in carp but this method of vaccine delivery is not practical. Alternatively, oral vaccination would be highly preferred due to its stress-free, labour-extensive and easy delivery. On the downside, oral vaccines are often less efficacious when compared to traditional injection vaccines and little is known about their activation of immune mechanisms. In an effort to generate an oral DNA vaccine against SVCV, carp of 2 g were orally vaccinated three times with alginate microspheres containing either 8 ug pcDNA3 plasmid, 8 ug pcDNA3-SVCVG plasmid, or PBS. Owing to the availability of several carp leukocytespecific monoclonal antibodies, we revealed the early recruitment of specific populations of leukocytes to the intestine and spleen following oral vaccination. The combined use of cell type-specific antibodies and an antibody against the SVCV-G protein allowed us for the first time, to identify the sites and kinetics of expression of the SVCV-G protein, and to concomitantly recognize the cell types involved in antigen uptake and processing.