- Email: [email protected]
Missing the target: DNAk is a dominant epitope in the humoral immune response of channel catfish (Ictalurus punctatus) to Flavobacterium columnare
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Abstracts / Fish & Shellfish Immunology 53 (2016) 58e93
The use of a second panel of antibodies enabled us to distinguish between various populations of both B and T lymphocytes, allowing the study of their specific roles during vaccination and infection. Using this panel we were able to characterise kinetics of recruitment of specific B and T lymphocyte populations to local sites after vaccination, as well as their distribution in systemic and other mucosal immune organs. Besides the characterization of local and systemic immune responses after oral DNA vaccination, using a wide array of techniques including RT-qPCR, histology, flow cytometry and in vitro macrophage-T cell cultures, we are currently studying the induction of immune memory after oral vaccination. This combination of techniques enables us to establish an unique overview of the kinetics of the immune response of carp after oral DNA vaccination against SVCV, at cell, protein and gene expression level and in a broad selection of immune-relevant organs. Together we will present the promising potential of an oral DNA vaccine against SVCV to not only induce an early local immune response, but also a systemic responses followed by long-term memory formation. Keywords: DNA vaccination, SVCV, oral vaccination, T lymphocytes, immune memory x Corresponding author. Tel.: þ31 317483708; Fax: þ31 317483962. E-mail address: [email protected] (C.WE. Embregts). * These authors have contributed equally to this work.
O-017. Missing the target: DNAk is a dominant epitope in the humoral immune response of channel catfish (Ictalurus punctatus) to Flavobacterium columnare Miles D. Lange, Benjamin H. Beck, Jason D. Brown, Bradley D. Farmer, L. Matthew Barnett, Carl D. Webster
Abstract Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry. Vaccination is considered an efficient, environmentally safe and economically sustainable alternative for control of ectoparasite infestations. Although emerging sea lice proteins have been identified recently, and have been proposed as potential targets for generating protective molecules, only a limited number of them have been evaluated in vaccine trials with unsuccessful results. On the other hand, more than 80 proteins are found in eukaryotic ribosomes. The protein P0 is essential for the assembly of the 60S ribosomal subunit and essential for cell viability. A vaccination-challenge trial with an immunogenic peptide of Rhipicephalus sanguineus protein P0 reduced survival of ticks with an overall efficacy of 90%, suggesting that it might be a promising antigen candidate for the control of ectoparasite. We have identified an immunogenic region of the ribosomal protein P0 from Caligus rogercresseyi and Lepeophtheirus salmonis that is not very conserved compared to host P0. We developed several vaccine candidates based on this peptide and produced in E. coli. These antigens were able to elicit a high specific antibody response after intraperitoneal (ip) immunization using tilapia as teleost fish model. It has been shown that ip immunization with some antigens cannot significantly reduce larval lice numbers despite enhanced specific antibody titers were raised. In this context, we have been developed for first time several tools to monitor tilapia immune response (IgT, IFN-g, IL-4, CD154) to vaccination in order to perform a more complete evaluation of the impact of the vaccinate candidates on the fish immune system. These findings will be finally validated in an immunization-challenge trial in Salmo salar. The overall results are relevant in the development of an effective vaccine against sea lice. Keywords: Sea lice, vaccine, immune response, cytokines, ribosomal protein * Corresponding author. Tel.: (53-7)2504423;. E-mail address: [email protected] (M.P. Estrada).
U.S. Department of Agriculture, Agricultural Research Service, Harry K. Dupree Stuttgart National Aquaculture Research Center, Stuttgart, AR, USA Vaccination remains a viable alternative for bacterial disease protection in fish; however additional work is required to understand the mechanisms of adaptive immunity in the channel catfish. To assess the humoral immune response to Flavobacterium columnare; a group of channel catfish were first immunized with F. columnare LV-359-01 cultured in iron-replete media, before being challenged with wild type F. columnare LV-359-01.The immunization protocol did not confer increased protection against F. columnare; however both control and immunized responders generated serum and skin IgM antibodies against F. columnare proteins. Western blot analyses of individuals from both groups showed that IgM antibodies were generated to the same 70 kDa extracellular protein, which was identified to be the bacterial chaperonin protein DNAk. Antibodies generated were cross reactive to DNAk proteins found in other gram negative bacteria. Our data suggests that DNAk is the dominant epitope in the channel catfish B-cell response to F. columnare.
O-018. Sea lice vaccine development based on an immunogenic peptide derived from the ribosomal protein P0 Yamila Carpio 1, Janet Velazquez 1, Yeny Leal 1, Naylin Herrera 1, Claudia García 1, Jannel Acosta 1, Antonio Morales 1, Fumio Takizawa 2, Oriol Sunyer 2, Mark Fast 3, Mario Pablo Estrada 1, * tica y Biotecnología (CIGB), Habana, Cuba Centro de Ingeniería Gene School of Veterinary Medicine, University of Pennsylvania, USA 3 Atlantic Veterinary College, University of Prince Edward Island, Canada 1
2
O-019. Relationships between cell migration, adhesion, apoptosis and gene expression in free and attached peritoneal cells in turbot after administration of vaccines containing P. dicentrarchi antigen and different adjuvants Francisco Fontenla 1, Jos e M. Leiro 2, Manuel Noia 1, Iria Folgueira 1, mez-Tato 3, Bel nica Blanco-Abad 1, Antonio Go en G. Pardo 4, Vero Paulino Martínez 4, Jesús Lamas 1, * 1
Department of Cell Biology and Ecology, Spain Laboratory of Parasitology, Department of Microbiology and Parasitology, Spain 3 Department of Geometry and Topology, Spain 4 Department of Genetics, University of Santiago de Compostela, Spain 2
Abstract Intraperitoneal (i.p.) injection is the most effective method of vaccinating fish in terms of antibody production and the degree of protection induced, and most current vaccines are administered in fish via this route. Vaccines are known to generate an inflammatory reaction in the peritoneal cavity, which is mainly caused by the adjuvant. Vaccination induces bidirectional movement of cells in and out of the peritoneal cavity where free cells and cells attached to the mesothelium and forming cell-vaccine masses can be observed. However, many aspects of the cellular immune response generated in the peritoneal cavity, such as the processes that provoke those cell responses, remain unknown. The present study analyses the cell traffic, adhesion, apoptosis and changes in gene expression in free and attached peritoneal cells after administration of vaccines containing P. dicentrarchi antigen and different types of adjuvants. Turbot were injected with PBS, PBS plus P. dicentrarchi membrane antigen, microspheres of chitosan-PVMMA containing membrane antigen, or with a mixture of antigen and one the following adjuvants: Montanide Isa 763 A,
Abstracts / Fish & Shellfish Immunology 53 (2016) 58e93
The use of a second panel of antibodies enabled us to distinguish between various populations of both B and T lymphocytes, allowing the study of their specific roles during vaccination and infection. Using this panel we were able to characterise kinetics of recruitment of specific B and T lymphocyte populations to local sites after vaccination, as well as their distribution in systemic and other mucosal immune organs. Besides the characterization of local and systemic immune responses after oral DNA vaccination, using a wide array of techniques including RT-qPCR, histology, flow cytometry and in vitro macrophage-T cell cultures, we are currently studying the induction of immune memory after oral vaccination. This combination of techniques enables us to establish an unique overview of the kinetics of the immune response of carp after oral DNA vaccination against SVCV, at cell, protein and gene expression level and in a broad selection of immune-relevant organs. Together we will present the promising potential of an oral DNA vaccine against SVCV to not only induce an early local immune response, but also a systemic responses followed by long-term memory formation. Keywords: DNA vaccination, SVCV, oral vaccination, T lymphocytes, immune memory x Corresponding author. Tel.: þ31 317483708; Fax: þ31 317483962. E-mail address: [email protected] (C.WE. Embregts). * These authors have contributed equally to this work.
O-017. Missing the target: DNAk is a dominant epitope in the humoral immune response of channel catfish (Ictalurus punctatus) to Flavobacterium columnare Miles D. Lange, Benjamin H. Beck, Jason D. Brown, Bradley D. Farmer, L. Matthew Barnett, Carl D. Webster
Abstract Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry. Vaccination is considered an efficient, environmentally safe and economically sustainable alternative for control of ectoparasite infestations. Although emerging sea lice proteins have been identified recently, and have been proposed as potential targets for generating protective molecules, only a limited number of them have been evaluated in vaccine trials with unsuccessful results. On the other hand, more than 80 proteins are found in eukaryotic ribosomes. The protein P0 is essential for the assembly of the 60S ribosomal subunit and essential for cell viability. A vaccination-challenge trial with an immunogenic peptide of Rhipicephalus sanguineus protein P0 reduced survival of ticks with an overall efficacy of 90%, suggesting that it might be a promising antigen candidate for the control of ectoparasite. We have identified an immunogenic region of the ribosomal protein P0 from Caligus rogercresseyi and Lepeophtheirus salmonis that is not very conserved compared to host P0. We developed several vaccine candidates based on this peptide and produced in E. coli. These antigens were able to elicit a high specific antibody response after intraperitoneal (ip) immunization using tilapia as teleost fish model. It has been shown that ip immunization with some antigens cannot significantly reduce larval lice numbers despite enhanced specific antibody titers were raised. In this context, we have been developed for first time several tools to monitor tilapia immune response (IgT, IFN-g, IL-4, CD154) to vaccination in order to perform a more complete evaluation of the impact of the vaccinate candidates on the fish immune system. These findings will be finally validated in an immunization-challenge trial in Salmo salar. The overall results are relevant in the development of an effective vaccine against sea lice. Keywords: Sea lice, vaccine, immune response, cytokines, ribosomal protein * Corresponding author. Tel.: (53-7)2504423;. E-mail address: [email protected] (M.P. Estrada).
U.S. Department of Agriculture, Agricultural Research Service, Harry K. Dupree Stuttgart National Aquaculture Research Center, Stuttgart, AR, USA Vaccination remains a viable alternative for bacterial disease protection in fish; however additional work is required to understand the mechanisms of adaptive immunity in the channel catfish. To assess the humoral immune response to Flavobacterium columnare; a group of channel catfish were first immunized with F. columnare LV-359-01 cultured in iron-replete media, before being challenged with wild type F. columnare LV-359-01.The immunization protocol did not confer increased protection against F. columnare; however both control and immunized responders generated serum and skin IgM antibodies against F. columnare proteins. Western blot analyses of individuals from both groups showed that IgM antibodies were generated to the same 70 kDa extracellular protein, which was identified to be the bacterial chaperonin protein DNAk. Antibodies generated were cross reactive to DNAk proteins found in other gram negative bacteria. Our data suggests that DNAk is the dominant epitope in the channel catfish B-cell response to F. columnare.
O-018. Sea lice vaccine development based on an immunogenic peptide derived from the ribosomal protein P0 Yamila Carpio 1, Janet Velazquez 1, Yeny Leal 1, Naylin Herrera 1, Claudia García 1, Jannel Acosta 1, Antonio Morales 1, Fumio Takizawa 2, Oriol Sunyer 2, Mark Fast 3, Mario Pablo Estrada 1, * tica y Biotecnología (CIGB), Habana, Cuba Centro de Ingeniería Gene School of Veterinary Medicine, University of Pennsylvania, USA 3 Atlantic Veterinary College, University of Prince Edward Island, Canada 1
2
O-019. Relationships between cell migration, adhesion, apoptosis and gene expression in free and attached peritoneal cells in turbot after administration of vaccines containing P. dicentrarchi antigen and different adjuvants Francisco Fontenla 1, Jos e M. Leiro 2, Manuel Noia 1, Iria Folgueira 1, mez-Tato 3, Bel nica Blanco-Abad 1, Antonio Go en G. Pardo 4, Vero Paulino Martínez 4, Jesús Lamas 1, * 1
Department of Cell Biology and Ecology, Spain Laboratory of Parasitology, Department of Microbiology and Parasitology, Spain 3 Department of Geometry and Topology, Spain 4 Department of Genetics, University of Santiago de Compostela, Spain 2
Abstract Intraperitoneal (i.p.) injection is the most effective method of vaccinating fish in terms of antibody production and the degree of protection induced, and most current vaccines are administered in fish via this route. Vaccines are known to generate an inflammatory reaction in the peritoneal cavity, which is mainly caused by the adjuvant. Vaccination induces bidirectional movement of cells in and out of the peritoneal cavity where free cells and cells attached to the mesothelium and forming cell-vaccine masses can be observed. However, many aspects of the cellular immune response generated in the peritoneal cavity, such as the processes that provoke those cell responses, remain unknown. The present study analyses the cell traffic, adhesion, apoptosis and changes in gene expression in free and attached peritoneal cells after administration of vaccines containing P. dicentrarchi antigen and different types of adjuvants. Turbot were injected with PBS, PBS plus P. dicentrarchi membrane antigen, microspheres of chitosan-PVMMA containing membrane antigen, or with a mixture of antigen and one the following adjuvants: Montanide Isa 763 A,