Highly ordered virus-like particles (VLPs) are often very immunogenic and have thus been widely used for high-density presentation of heterologous epitopes. Hepatitis B core antigen (HBc) produced in various non-plant expression systems, including yeast, mammalian cell cultures, vaccinia virus, and bacteria such as Escherichia coli, was found to spontaneously assemble icosahedral VLPs. Insertion of peptides at several terminal and internal positions of HBc did not interfere with the VLP formation. Furthermore, HBc-VLPs stimulated strong immune responses when delivered mucosally without adjuvant, making it an attractive epitope carrier for the development of plant-based oral vaccines, especially the ones against sex transmitted diseases (STD), because mucosal immune responses provide the first line of defenses during the invasion of pathogens. Here we describe the plant expression and VLP formation of HBc and its fusion with a common neutralizing (“Kawana”) epitope for the L2 minor capsid protein of human papillomavirus types 16 and 6 (KHBc). By using the ICON viral system, the HBc and KHBc were produced in Nicotiana benthamiana plant leaves at levels of 5% and 10% of total soluble protein, respectively, at 7 days post-inoculation. Beyond this point, necrosis occurred in the inoculated leaves, suggesting the proteins are somewhat toxic to plants. Plant-derived HBc and KHBc accumulated as full-length products as evident by Western blot, and sedimented as E. coli-derived HBc-VLPs in sucrose gradient analysis. Electron microscopy of HBc peak fractions from plant extracts further confirmed the presence of HBc- and KHBc-VLPs. The immunogenicity study is currently underway. We will immunize mice by i.p. injection and i.n. delivery of purified antigens as well as by feeding with freeze-dried leaf powder embedded in gelatin, and we will follow the serum IgG and mucosal IgA responses to the HBc and the “Kawana” epitope.
Plant-derived hepatitis B core particle as platform for mucosal delivery and presentation of foreign epitopes / Santi, L; Zhong, Huang; Kate, Gorlewski; Charles, J Arntzen; and Hugh, S Mason. - (2005). ( Plant Based Vaccines and Antibodies Prague, Czech Republic, EU ).
Plant-derived hepatitis B core particle as platform for mucosal delivery and presentation of foreign epitopes
Santi L;
2005
Abstract
Highly ordered virus-like particles (VLPs) are often very immunogenic and have thus been widely used for high-density presentation of heterologous epitopes. Hepatitis B core antigen (HBc) produced in various non-plant expression systems, including yeast, mammalian cell cultures, vaccinia virus, and bacteria such as Escherichia coli, was found to spontaneously assemble icosahedral VLPs. Insertion of peptides at several terminal and internal positions of HBc did not interfere with the VLP formation. Furthermore, HBc-VLPs stimulated strong immune responses when delivered mucosally without adjuvant, making it an attractive epitope carrier for the development of plant-based oral vaccines, especially the ones against sex transmitted diseases (STD), because mucosal immune responses provide the first line of defenses during the invasion of pathogens. Here we describe the plant expression and VLP formation of HBc and its fusion with a common neutralizing (“Kawana”) epitope for the L2 minor capsid protein of human papillomavirus types 16 and 6 (KHBc). By using the ICON viral system, the HBc and KHBc were produced in Nicotiana benthamiana plant leaves at levels of 5% and 10% of total soluble protein, respectively, at 7 days post-inoculation. Beyond this point, necrosis occurred in the inoculated leaves, suggesting the proteins are somewhat toxic to plants. Plant-derived HBc and KHBc accumulated as full-length products as evident by Western blot, and sedimented as E. coli-derived HBc-VLPs in sucrose gradient analysis. Electron microscopy of HBc peak fractions from plant extracts further confirmed the presence of HBc- and KHBc-VLPs. The immunogenicity study is currently underway. We will immunize mice by i.p. injection and i.n. delivery of purified antigens as well as by feeding with freeze-dried leaf powder embedded in gelatin, and we will follow the serum IgG and mucosal IgA responses to the HBc and the “Kawana” epitope.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
