- Email: [email protected]
Adjuvanicity of CpG oligodeoxynucleotides in grouper
Abstracts / Fish & Shellfish Immunology 34 (2013) 1635–1691
O-242. In vitro neutralization of viral haemorrhagic septicemia virus (VHSV) by plasma from immunized zebrafish B. Chinchilla 1, x, E. Gómez-Casado 1,x, P. Encinas 1, A. Falco 3, A. Estepa 2, J. Coll 1, *. 1 Dpto. Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain; 2 IBMC, Universidad Miguel Hernández, Elche, Spain; 3 Cell Biology and Immunology Group, Wageningen University, Wageningen, The Netherlands
Abstract Given the increasingly use of zebrafish (Danio rerio) as a vertebrate model to study immune responses to pathogen-caused diseases and the importance of Rhabdoviruses in wild and cultured fish throughout the world, the development of new, faster and accurate molecular tools to detect zebrafish antibodies is required. Neutralizing antibodies are important for longterm protection from fish Rhabdoviruses. Current methods to estimate VHSV protein G-specific antibodies have been reported in salmonids but not yet for zebrafish, despite the growing interest in this fish species and the susceptibility of cold-acclimatized zebrafish to this virus. For this purpose, we designed a microneutralization method based on immunostaining VHSV-infected fish cell monolayers to achieve a first estimation of anti-viral antibody-like responses in individual zebrafish plasma samples. A first method used the in situ staining of cell monolayers for visual counting of focus forming units. To simplify the technique, we first fixed and permeabilized EPC cell monolayers for optimal differentiation between non-infected and infected cells. Posterior release of the stained cells in suspension for flow cytometry detection was obtained with trypsin digestion. Combination of flow cytometry with 96-well batch analysis allowed for practical and rapid management of large number of samples. Specific anti-VHSV neutralizing activity could be then estimated in small amounts of plasma samples obtained from zebrafish surviving VHSV infections. The neutralizing activity was inhibited by protein A-sepharose and rabbit antizebrafish IgM antibodies, suggesting the implication of specific IgM zebrafish antibodies. Results showed that approximately 3050 % of the zebrafish surviving 3 consecutive VHSV infections contained VHSV neutralizing antibodies when compared to wild type zebrafish This micro-method demonstrated detectable and significant VHSV neutralization titers in zebrafish surviving VHSV infections and would be useful not only to study the VHSV model, but also for similar work involving other in vitro neutralizable zebrafish pathogens. * Corresponding author. E-mail address: [email protected] (J. Coll) xThese authors have contributed equally to this work.
O-244. Adjuvanicity of CpG oligodeoxynucleotides in grouper H.C. Chuang, T.L. Ji, F.Y. Lee, S.P. Chen, N.Y. Chen, P.P. Chiou*. Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Yilan, Taiwan Abstract Synthetic unmethylated CpG oligodeoxynucleotides (ODNs) have been widely used in mammals as Toll-like receptor 9 (TLR9) agonists to trigger innate immune response or to serve as vaccine adjuvant. We have designed a serial of CpG ODNS (class A and B), and assayed their immunoregulatory effects on orange-spotted grouper (Epinephelus coioides), an economically important aquaculture species in many Asian countries. Our study showed that stimulation with synthetic class-A CpG ODNs could trigger the TLR9 pathway in grouper cells, as indicated by translocation of TLR9 and recruitment of adaptor MyD88 into the endosomes shortly upon stimulation. Immunoprecipitation assay further confirmed the interaction between TLR9 and MyD88. Compared with class-B ODN, class-A ODN preferably stimulated the production of pro-inflammatory cytokine IL-1b in grouper cells, indicating a potential role of adjuvant for this type of
1647
molecules. When incorporated a class-A ODN into a virus-like particle (VLP) vaccine against nervous necrosis virus (NNV), the titer of specific antibodies was significantly enhanced in the fish immunized with vaccine containing ODN as compared with vaccine alone. Analysis of the expression profile of T-cell markers further revealed up-regulation of CD4, CD8, TcRb and T-bet (a marker of Th1 response) in these fish. Our study indicates that class-A CpG ODN could be used as vaccine adjuvant with the benefits of promoting antibody production and likely Th1 response in grouper. * Corresponding author. E-mail address: [email protected] (P.P. Chiou)
O-246. American lobster (Homarus americanus) immune response to infection with the bumper car parasite (Anophryoides haemophila): A transcriptomic analysis K.F. Clark 1, 2, A.R. Acorn 1, S.J. Greenwood 1, 3, *. 1 AVC Lobster Science Centre, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada; 2 Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada; 3 Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada
Abstract The bumper car parasite (Anophryoides haemophila) infects American lobster (Homarus americanus) in the wild as well as causing major losses of lobsters maintained in commercial holding facilities. Expression of >14,500 H. americanus hepatopancreatic genes were monitored during an A. haemophila infection challenge in order to discover molecular mechanisms involved in the lobster immune response. One hundred and forty-five genes were found to be differentially expressed during infection. For many genes, this study is the first to link their expression to an immune response to a known lobster pathogen. Several of the genes have previously been linked to crustacean or invertebrate immune response including: several antilipopolysaccharide factor isoforms (ALFHa), acute phase serum amyloid protein A (SAA), a serine protease inhibitor, a toll-like receptor, several haemocyanin subunits, phagocyte signaling-impaired protein, vitelline membrane outer layer protein-1, trypsin, and a C-type lectin receptor. Microarray results were verified using RT-qPCR and agreement was good between the two methods. The expression of six ALFHa isoforms were monitored via microarray where ALFHa-1, ALFHa-2, ALFHa-4 and ALFHa-6 were differentially expressed while ALFHa-3 and ALFHa7 were not. RTqPCR analysis confirmed that ALFHa-1, ALFHA-2 and ALFHa-4 expression increased during infection with a peak at 5-7 weeks for ALFHa-1 and 10 weeks for ALFHa-2 and ALFHa-4. This suggests that different ALFHa isoforms are temporally expressed during A. haemophila infection. Importantly, these results provide further evidence that different ALFHa isoforms have more significant roles in responding to A. haemophila infection. Significant increases in SAA gene expression were also found, corroborating previous findings of increased SAA expression during A. viridans infections; highlighting the importance of SAA as a marker of H. americanus immune activation and potential indicator of H. americanus health. * Corresponding author. E-mail address: [email protected] (S.J. Greenwood)
O-408. Identification of novel lobster (Homarus americanus) immune mediators utilized in response to the Gram-positive bacterium Aerococcus viridans var. homari K.F. Clark 1, 2, *, A.R. Acorn 1, 2, S.J. Greenwood 1, 3. 1 AVC Lobster Science Centre, University of Prince Edward Island, Charlottetown, PEI, Canada;
O-242. In vitro neutralization of viral haemorrhagic septicemia virus (VHSV) by plasma from immunized zebrafish B. Chinchilla 1, x, E. Gómez-Casado 1,x, P. Encinas 1, A. Falco 3, A. Estepa 2, J. Coll 1, *. 1 Dpto. Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain; 2 IBMC, Universidad Miguel Hernández, Elche, Spain; 3 Cell Biology and Immunology Group, Wageningen University, Wageningen, The Netherlands
Abstract Given the increasingly use of zebrafish (Danio rerio) as a vertebrate model to study immune responses to pathogen-caused diseases and the importance of Rhabdoviruses in wild and cultured fish throughout the world, the development of new, faster and accurate molecular tools to detect zebrafish antibodies is required. Neutralizing antibodies are important for longterm protection from fish Rhabdoviruses. Current methods to estimate VHSV protein G-specific antibodies have been reported in salmonids but not yet for zebrafish, despite the growing interest in this fish species and the susceptibility of cold-acclimatized zebrafish to this virus. For this purpose, we designed a microneutralization method based on immunostaining VHSV-infected fish cell monolayers to achieve a first estimation of anti-viral antibody-like responses in individual zebrafish plasma samples. A first method used the in situ staining of cell monolayers for visual counting of focus forming units. To simplify the technique, we first fixed and permeabilized EPC cell monolayers for optimal differentiation between non-infected and infected cells. Posterior release of the stained cells in suspension for flow cytometry detection was obtained with trypsin digestion. Combination of flow cytometry with 96-well batch analysis allowed for practical and rapid management of large number of samples. Specific anti-VHSV neutralizing activity could be then estimated in small amounts of plasma samples obtained from zebrafish surviving VHSV infections. The neutralizing activity was inhibited by protein A-sepharose and rabbit antizebrafish IgM antibodies, suggesting the implication of specific IgM zebrafish antibodies. Results showed that approximately 3050 % of the zebrafish surviving 3 consecutive VHSV infections contained VHSV neutralizing antibodies when compared to wild type zebrafish This micro-method demonstrated detectable and significant VHSV neutralization titers in zebrafish surviving VHSV infections and would be useful not only to study the VHSV model, but also for similar work involving other in vitro neutralizable zebrafish pathogens. * Corresponding author. E-mail address: [email protected] (J. Coll) xThese authors have contributed equally to this work.
O-244. Adjuvanicity of CpG oligodeoxynucleotides in grouper H.C. Chuang, T.L. Ji, F.Y. Lee, S.P. Chen, N.Y. Chen, P.P. Chiou*. Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Yilan, Taiwan Abstract Synthetic unmethylated CpG oligodeoxynucleotides (ODNs) have been widely used in mammals as Toll-like receptor 9 (TLR9) agonists to trigger innate immune response or to serve as vaccine adjuvant. We have designed a serial of CpG ODNS (class A and B), and assayed their immunoregulatory effects on orange-spotted grouper (Epinephelus coioides), an economically important aquaculture species in many Asian countries. Our study showed that stimulation with synthetic class-A CpG ODNs could trigger the TLR9 pathway in grouper cells, as indicated by translocation of TLR9 and recruitment of adaptor MyD88 into the endosomes shortly upon stimulation. Immunoprecipitation assay further confirmed the interaction between TLR9 and MyD88. Compared with class-B ODN, class-A ODN preferably stimulated the production of pro-inflammatory cytokine IL-1b in grouper cells, indicating a potential role of adjuvant for this type of
1647
molecules. When incorporated a class-A ODN into a virus-like particle (VLP) vaccine against nervous necrosis virus (NNV), the titer of specific antibodies was significantly enhanced in the fish immunized with vaccine containing ODN as compared with vaccine alone. Analysis of the expression profile of T-cell markers further revealed up-regulation of CD4, CD8, TcRb and T-bet (a marker of Th1 response) in these fish. Our study indicates that class-A CpG ODN could be used as vaccine adjuvant with the benefits of promoting antibody production and likely Th1 response in grouper. * Corresponding author. E-mail address: [email protected] (P.P. Chiou)
O-246. American lobster (Homarus americanus) immune response to infection with the bumper car parasite (Anophryoides haemophila): A transcriptomic analysis K.F. Clark 1, 2, A.R. Acorn 1, S.J. Greenwood 1, 3, *. 1 AVC Lobster Science Centre, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada; 2 Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada; 3 Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada
Abstract The bumper car parasite (Anophryoides haemophila) infects American lobster (Homarus americanus) in the wild as well as causing major losses of lobsters maintained in commercial holding facilities. Expression of >14,500 H. americanus hepatopancreatic genes were monitored during an A. haemophila infection challenge in order to discover molecular mechanisms involved in the lobster immune response. One hundred and forty-five genes were found to be differentially expressed during infection. For many genes, this study is the first to link their expression to an immune response to a known lobster pathogen. Several of the genes have previously been linked to crustacean or invertebrate immune response including: several antilipopolysaccharide factor isoforms (ALFHa), acute phase serum amyloid protein A (SAA), a serine protease inhibitor, a toll-like receptor, several haemocyanin subunits, phagocyte signaling-impaired protein, vitelline membrane outer layer protein-1, trypsin, and a C-type lectin receptor. Microarray results were verified using RT-qPCR and agreement was good between the two methods. The expression of six ALFHa isoforms were monitored via microarray where ALFHa-1, ALFHa-2, ALFHa-4 and ALFHa-6 were differentially expressed while ALFHa-3 and ALFHa7 were not. RTqPCR analysis confirmed that ALFHa-1, ALFHA-2 and ALFHa-4 expression increased during infection with a peak at 5-7 weeks for ALFHa-1 and 10 weeks for ALFHa-2 and ALFHa-4. This suggests that different ALFHa isoforms are temporally expressed during A. haemophila infection. Importantly, these results provide further evidence that different ALFHa isoforms have more significant roles in responding to A. haemophila infection. Significant increases in SAA gene expression were also found, corroborating previous findings of increased SAA expression during A. viridans infections; highlighting the importance of SAA as a marker of H. americanus immune activation and potential indicator of H. americanus health. * Corresponding author. E-mail address: [email protected] (S.J. Greenwood)
O-408. Identification of novel lobster (Homarus americanus) immune mediators utilized in response to the Gram-positive bacterium Aerococcus viridans var. homari K.F. Clark 1, 2, *, A.R. Acorn 1, 2, S.J. Greenwood 1, 3. 1 AVC Lobster Science Centre, University of Prince Edward Island, Charlottetown, PEI, Canada;