title: Quantitative analysis of the yield of avian H7 influenza virus haemagglutinin protein produced in silkworm pupae with the use of the codon-optimized DNA: A possible oral vaccine.
abstract: In this study, we aimed to quantitatively compare the increased production of three H7 influenza virus-like particle (VLP) haemagglutinin (HA) with the use of a codon-optimized single HA gene in silkworm pupae. Recombinant baculovirus (Korea H7-BmNPV) could produce 0.40 million HA units per pupa, corresponding to 1832μg protein. The yield of the HA produced in larva was estimated to be approximately 0.31 million HA units per larva, and there were no significant differences between the three HA proteins. We could establish efficient recovery system of HA production in larvae and pupae with the use of three cycles sonication methods. Next, we compared yields of HA proteins from three different H7 and two H5 recombinant baculoviruses based on the amount of mRNA synthesized in BmN cells, suggesting that mRNA synthesis may be also a useful indicator for the production of HA. Based on HA titres from four recombinants, the yield of HA had a great influence on the codon-optimized effect and the characteristics of the viral HA gene. The recombinant containing codon optimized HA DNA of A/tufted duck/Fukushima/16/2011 (H5N1) did produce more than one million HA units, although another recombinant including of the wild H5N1 strain failed to show HA activity. Electron microscopy revealed the presence of large VLP and small HA particle in the heavy and light fractions. The purified VLPs reacted with the authentic anti-H7 antibodies and the antibodies prepared after immunization with the VLP H7 antigen. Also H5 and H7VLPs could produce HI antibody in chickens and mice with oral immunization. The antibodies elicited with oral immunization were confirmed in fluorescent antibody analysis and western blotting in Korea H5-BmNPV and H7HA-BmNPV recombinant infected BmN cells. Taken together, these findings provided important insights into future oral vaccine development.
authors.affiliation: The Institute of Biological Resources, 893-2, Nakayama, Nago, Okinawa 905-0004, Japan. Electronic address: rnerome_ibr@train.ocn.ne.jp.
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Nerome
authors.affiliation: The Institute of Biological Resources, 893-2, Nakayama, Nago, Okinawa 905-0004, Japan.
authors.forename: Sayaka
authors.initials: S
authors.lastname: Matsuda
authors.affiliation: The Institute of Biological Resources, 893-2, Nakayama, Nago, Okinawa 905-0004, Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Maegawa
authors.affiliation: Equine Research Institute, Japan Racing Association, 321-4, Tokami-cho, Utsunomiya, Tochigi 320-0856, Japan.
authors.forename: Shigeo
authors.initials: S
authors.lastname: Sugita
authors.affiliation: Division of Microbiology, Nihon University School of Medicine, 30-1, Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan.
authors.forename: Kazumichi
authors.initials: K
authors.lastname: Kuroda
authors.affiliation: National Institute of Advanced Science and Technology (AIST), 1-8-31, Midorigaoka, Ikeda, Osaka 563-8577, Japan.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Kawasaki
authors.affiliation: The Institute of Biological Resources, 893-2, Nakayama, Nago, Okinawa 905-0004, Japan.
authors.forename: Reiko
authors.initials: R
authors.lastname: Nerome
mesh_terms: D000284:Administration, Oral; D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis*; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D012831:Bombyx / Q000235:genetics* / Q000378:metabolism; D002645:Chickens; D003062:Codon; D005260:Female; D015870:Gene Expression; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000276:immunology; D057916:Influenza A Virus, H7N1 Subtype / Q000235:genetics / Q000276:immunology*; D007252:Influenza Vaccines / Q000008:administration & dosage / Q000096:biosynthesis*; D007814:Larva / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009976:Orthomyxoviridae Infections / Q000276:immunology / Q000517:prevention & control* / Q000821:virology; D011679:Pupa / Q000235:genetics / Q000378:metabolism; D014614:Vaccines, Synthetic; D058425:Vaccines, Virus-Like Particle / Q000008:administration & dosage / Q000096:biosynthesis*
chemical_list: D000914:Antibodies, Viral; D003062:Codon; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D014614:Vaccines, Synthetic; D058425:Vaccines, Virus-Like Particle; C109963:hemagglutinin, avian influenza A virus
keywords: Avian influenza virus; Oral vaccine; Recombinant antigen; Vaccine development; Virus-like particles