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Charles J Arntzen - One of the best experts on this subject based on the ideXlab platform.
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Expression of Norwalk virus Capsid Protein in transgenic tobacco and potato and its oral immunogenicity in mice (edible vaccineyforeign genesyplants)
1996Co-Authors: Hugh S. Mason, J Udith, M. Ball, Xi Jiang, Mary K. Estes, Charles J ArntzenAbstract:Alternatives to cell culture systems for pro- duction of recombinant Proteins could make very safe vac- cines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the Capsid Protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in hu- mans. The Capsid Protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previ- ously recovered when the same gene was expressed in recom- binant baculovirus-infected insect cells. The Capsid Protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDSypolyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf express- ing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vac- cines are urgently needed.
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expression of norwalk virus Capsid Protein in transgenic tobacco and potato and its oral immunogenicity in mice
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Jian Jian Shi, Charles J Arntzen, Judith M. Ball, Hugh S. Mason, Xi Jiang, Mary K. EstesAbstract:Abstract Alternatives to cell culture systems for production of recombinant Proteins could make very safe vaccines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the Capsid Protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in humans. The Capsid Protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previously recovered when the same gene was expressed in recombinant baculovirus-infected insect cells. The Capsid Protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDS/polyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf expressing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vaccines are urgently needed.
Mary K. Estes - One of the best experts on this subject based on the ideXlab platform.
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the vp7 outer Capsid Protein of rotavirus induces polyclonal b cell activation
Journal of Virology, 2004Co-Authors: Sarah E Blutt, Mary K. Estes, Sue E Crawford, Kelly L Warfield, Dorothy E Lewis, Margaret E ConnerAbstract:The early response to a homologous rotavirus infection in mice includes a T-cell-independent increase in the number of activated B lymphocytes in the Peyer's patches. The mechanism of this activation has not been previously determined. Since rotavirus has a repetitively arranged triple-layered Capsid and repetitively arranged antigens can induce activation of B cells, one or more of the Capsid Proteins could be responsible for the initial activation of B cells during infection. To address this question, we assessed the ability of rotavirus and virus-like particles to induce B-cell activation in vivo and in vitro. Using infectious rotavirus, inactivated rotavirus, noninfectious but replication-competent virus, and virus-like particles, we determined that neither infectivity nor RNA was necessary for B-cell activation but the presence of the rotavirus outer Capsid Protein, VP7, was sufficient for murine B-cell activation. Preincubation of the virus with neutralizing VP7 antibodies inhibited B-cell activation. Polymyxin B treatment and boiling of the virus preparation were performed, which ruled out possible lipopolysaccharide contamination as the source of activation and confirmed that the structural conformation of VP7 is important for B-cell activation. These findings indicate that the structure and conformation of the outer Capsid Protein, VP7, initiate intestinal B-cell activation during rotavirus infection.
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Expression of Norwalk virus Capsid Protein in transgenic tobacco and potato and its oral immunogenicity in mice (edible vaccineyforeign genesyplants)
1996Co-Authors: Hugh S. Mason, J Udith, M. Ball, Xi Jiang, Mary K. Estes, Charles J ArntzenAbstract:Alternatives to cell culture systems for pro- duction of recombinant Proteins could make very safe vac- cines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the Capsid Protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in hu- mans. The Capsid Protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previ- ously recovered when the same gene was expressed in recom- binant baculovirus-infected insect cells. The Capsid Protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDSypolyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf express- ing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vac- cines are urgently needed.
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expression of norwalk virus Capsid Protein in transgenic tobacco and potato and its oral immunogenicity in mice
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Jian Jian Shi, Charles J Arntzen, Judith M. Ball, Hugh S. Mason, Xi Jiang, Mary K. EstesAbstract:Abstract Alternatives to cell culture systems for production of recombinant Proteins could make very safe vaccines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the Capsid Protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in humans. The Capsid Protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previously recovered when the same gene was expressed in recombinant baculovirus-infected insect cells. The Capsid Protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDS/polyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf expressing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vaccines are urgently needed.
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antigenic mapping of the recombinant norwalk virus Capsid Protein using monoclonal antibodies
Virology, 1996Co-Authors: M. E. Hardy, Noritoshi Kitamoto, Laura J White, Tomoyuki Tanaka, Judith M. Ball, Xi Jiang, Mary K. EstesAbstract:Abstract Norwalk virus (NV) is the prototype strain of a group of noncultivatable caliciviruses that infect humans and cause outbreaks of epidemic acute nonbacterial gastroenteritis. The NV virion is composed of 180 copies of a single structural Protein that, when expressed in insect cells infected with a recombinant baculovirus, assembles into empty recombinant Norwalk virus-like particles (rNV VLPs) which are morphologically and antigenically similar to native NV. We have begun to dissect the antigenic structure of the rNV particles using monoclonal antibodies made to the rNV VLPs. Ten MAbs made to rNV particles were characterized for their reactivity as detector antibodies by ELISA, as capture antibodies in an ELISA to detect NV in stools, by Western blot, and by immunoprecipitation. Seven of the MAbs recognize discontinuous epitopes, requiring the rNV Capsid Protein to remain at least partially folded, while the other three recognize continuous epitopes. Eight of the MAbs map to the C-terminal half of the Capsid Protein as they react by Western blot and by immunoprecipitation with a 32K trypsin cleavage product of the full-length 58K Capsid Protein, suggesting that the C-terminal half of the Capsid Protein may contain the immunodominant epitopes. The three MAbs that recognize continuous epitopes map to the extreme C terminus of the Capsid Protein, between amino acids 457 and 530, in a region that is relatively conserved among different human calicivirus Capsid Proteins. These MAbs which were assigned into three antigenic groups will be useful as tools to further dissect the structural and antigenic topography of the NV virion, and as unlimited reagents to detect NV in diagnostic assays.
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Specific proteolytic cleavage of recombinant Norwalk virus Capsid Protein.
Journal of Virology, 1995Co-Authors: M. E. Hardy, Laura J White, Judith M. Ball, Mary K. EstesAbstract:Norwalk virus (NV) causes epidemic outbreaks of acute nonbacterial gastroenteritis in humans. The NV Capsid is made up of a single Protein, and expression of the Capsid Protein in baculovirus recombinants results in spontaneous assembly of the Protein into virus-like particles (X. Jiang, M. Wang, D. Y. Graham, and M. K. Estes, J. Virol. 66:6527-6532, 1992). We have investigated whether the NV Capsid Protein undergoes a specific proteolytic cleavage. Recombinant NV (rNV) particles were digested with trypsin to determine if a specific cleavage occurred. A predominant band with a molecular weight of approximately 32,000 (32K Protein) was observed when trypsin-treated rNV was electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. Determination of the N-terminal sequence of this band showed that a trypsin-specific cleavage occurred at amino acid residue 227. Early studies identified two Proteins with molecular weights of 59,000 and 30,000 (59K and 30K Proteins) in the stool of NV-infected volunteers that were reactive with postinfection antiserum. (H. B. Greenberg, J. R. Valdesuso, A. R. Kalica, R. G. Wyatt, V. J. McAuliffe, A. Z. Kapikian, and R. M. Chanock, J. Virol. 37:994-999, 1981). We hypothesized that the 32K rNV cleavage product might be analogous to the 30K soluble Protein detected in stools of NV-infected volunteers. Immunoprecipitation of soluble Protein from these stool extracts with a rabbit polyclonal antiserum made against rNV, and Western blot detection with a mouse polyclonal antiserum made against rNV, revealed a single band with an apparent molecular weight of 30,000 that migrated similarly to the trypsin cleavage product observed in vitro. The N terminus of this band was identical to that of the 32K cleavage product of rNV Capsid Protein. These data show that the 30K Protein in stool is produced by specific cleavage of the NV Capsid Protein in vivo. Trypsin cleavage of isolated soluble rNV 58K Capsid Protein and of assembled particles showed that only soluble 58K Capsid Protein is susceptible to cleavage. The presence of a large amount of soluble Capsid Protein may influence the immune response to or pathogenicity of NV infections.
Anette Schneemann - One of the best experts on this subject based on the ideXlab platform.
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Capsid Protein synthesis from replicating rna directs specific packaging of the genome of a multipartite positive strand rna virus
Journal of Virology, 2005Co-Authors: Arno P Venter, Neel K Krishna, Anette SchneemannAbstract:Flock house virus (FHV) is a bipartite, positive-strand RNA insect virus that enCapsidates its two genomic RNAs in a single virion. It provides a convenient model system for studying the principles underlying the copackaging of multipartite viral RNA genomes. In this study, we used a baculovirus expression system to determine if the uncoupling of viral Protein synthesis from RNA replication affected the packaging of FHV RNAs. We found that neither RNA1 (which encodes the viral replicase) nor RNA2 (which encodes the Capsid Protein) were packaged efficiently when Capsid Protein was supplied in trans from nonreplicating RNA. However, Capsid Protein synthesized in cis from replicating RNA2 packaged RNA2 efficiently in the presence and absence of RNA1. These results demonstrated that Capsid Protein translation from replicating RNA2 is required for specific packaging of the FHV genome. This type of coupling between genome replication and translation and RNA packaging has not been observed previously. We hypothesize that RNA2 replication and translation must be spatially coordinated in FHV-infected cells to facilitate retrieval of the viral RNAs for enCapsidation by newly synthesized Capsid Protein. Spatial coordination of RNA and Capsid Protein synthesis may be key to specific genome packaging and assembly in other RNA viruses.
Hugh S. Mason - One of the best experts on this subject based on the ideXlab platform.
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Expression of Norwalk virus Capsid Protein in transgenic tobacco and potato and its oral immunogenicity in mice (edible vaccineyforeign genesyplants)
1996Co-Authors: Hugh S. Mason, J Udith, M. Ball, Xi Jiang, Mary K. Estes, Charles J ArntzenAbstract:Alternatives to cell culture systems for pro- duction of recombinant Proteins could make very safe vac- cines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the Capsid Protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in hu- mans. The Capsid Protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previ- ously recovered when the same gene was expressed in recom- binant baculovirus-infected insect cells. The Capsid Protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDSypolyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf express- ing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vac- cines are urgently needed.
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expression of norwalk virus Capsid Protein in transgenic tobacco and potato and its oral immunogenicity in mice
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Jian Jian Shi, Charles J Arntzen, Judith M. Ball, Hugh S. Mason, Xi Jiang, Mary K. EstesAbstract:Abstract Alternatives to cell culture systems for production of recombinant Proteins could make very safe vaccines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the Capsid Protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in humans. The Capsid Protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previously recovered when the same gene was expressed in recombinant baculovirus-infected insect cells. The Capsid Protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDS/polyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf expressing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vaccines are urgently needed.
Xi Jiang - One of the best experts on this subject based on the ideXlab platform.
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Expression of Norwalk virus Capsid Protein in transgenic tobacco and potato and its oral immunogenicity in mice (edible vaccineyforeign genesyplants)
1996Co-Authors: Hugh S. Mason, J Udith, M. Ball, Xi Jiang, Mary K. Estes, Charles J ArntzenAbstract:Alternatives to cell culture systems for pro- duction of recombinant Proteins could make very safe vac- cines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the Capsid Protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in hu- mans. The Capsid Protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previ- ously recovered when the same gene was expressed in recom- binant baculovirus-infected insect cells. The Capsid Protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDSypolyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf express- ing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vac- cines are urgently needed.
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expression of norwalk virus Capsid Protein in transgenic tobacco and potato and its oral immunogenicity in mice
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Jian Jian Shi, Charles J Arntzen, Judith M. Ball, Hugh S. Mason, Xi Jiang, Mary K. EstesAbstract:Abstract Alternatives to cell culture systems for production of recombinant Proteins could make very safe vaccines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the Capsid Protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in humans. The Capsid Protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previously recovered when the same gene was expressed in recombinant baculovirus-infected insect cells. The Capsid Protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDS/polyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf expressing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vaccines are urgently needed.
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antigenic mapping of the recombinant norwalk virus Capsid Protein using monoclonal antibodies
Virology, 1996Co-Authors: M. E. Hardy, Noritoshi Kitamoto, Laura J White, Tomoyuki Tanaka, Judith M. Ball, Xi Jiang, Mary K. EstesAbstract:Abstract Norwalk virus (NV) is the prototype strain of a group of noncultivatable caliciviruses that infect humans and cause outbreaks of epidemic acute nonbacterial gastroenteritis. The NV virion is composed of 180 copies of a single structural Protein that, when expressed in insect cells infected with a recombinant baculovirus, assembles into empty recombinant Norwalk virus-like particles (rNV VLPs) which are morphologically and antigenically similar to native NV. We have begun to dissect the antigenic structure of the rNV particles using monoclonal antibodies made to the rNV VLPs. Ten MAbs made to rNV particles were characterized for their reactivity as detector antibodies by ELISA, as capture antibodies in an ELISA to detect NV in stools, by Western blot, and by immunoprecipitation. Seven of the MAbs recognize discontinuous epitopes, requiring the rNV Capsid Protein to remain at least partially folded, while the other three recognize continuous epitopes. Eight of the MAbs map to the C-terminal half of the Capsid Protein as they react by Western blot and by immunoprecipitation with a 32K trypsin cleavage product of the full-length 58K Capsid Protein, suggesting that the C-terminal half of the Capsid Protein may contain the immunodominant epitopes. The three MAbs that recognize continuous epitopes map to the extreme C terminus of the Capsid Protein, between amino acids 457 and 530, in a region that is relatively conserved among different human calicivirus Capsid Proteins. These MAbs which were assigned into three antigenic groups will be useful as tools to further dissect the structural and antigenic topography of the NV virion, and as unlimited reagents to detect NV in diagnostic assays.