title: The large-scale production of an artificial influenza virus-like particle vaccine in silkworm pupae.
abstract: We successfully established a mass production system for an influenza virus-like particle (VLP) vaccine using a synthetic H5 hemagglutinin (HA) gene codon-optimized for the silkworm. A recombinant baculovirus containing the synthetic gene was inoculated into silkworm pupae. Four days after inoculation, the hemagglutination titer in homogenates from infected pupae reached a mean value of 0.8 million hemagglutination units (HAU), approximately 2,000 μg HA protein per pupa, more than 50-fold higher than that produced with an embryonated chicken egg. VLPs ranging from 30 nm to 300 nm in diameter and covered with a large number of spikes were detected in the homogenates. The spikes were approximately 14 nm long, similar to an authentic influenza HA spike. Detailed electron micrographs indicated that the VLP spike density was similar to that of authentic influenza virus particles. The results clearly show that the expression of a single HA gene can efficiently produce VLPs in silkworm pupae. When chickens were immunized with the pupae homogenate, the hemagglutination inhibition titer in their sera reached values of 2,048-8,192 after approximately 1 month. This is the first report demonstrating that a large amount of VLP vaccine could be produced by single synthetic HA gene in silkworm pupae. Our system might be useful for future vaccine development against other viral diseases.
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: 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: The Institute of Biological Resources, 893-2, Nakayama, Nago, Okinawa 905-0004, Japan.
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Hirose
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: Baculotechnologies Co., Ltd., 2217-16, Hayashi-cho, Takamatsu, Kagawa 761-0301, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Majima
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: Division of Gastroenterology and Hepatology, Nihon University School of Medicine, 30-1, Oyaguchi-kamicho, Itabshi-ku, Tokyo 173-8610, Japan.
authors.forename: Toshikatsu
authors.initials: T
authors.lastname: Shibata
authors.affiliation: Faculty of Veterinary Medicine, Bogor Agricultural University IPB, JL Agatis Kampus IPB Darmaga, Bogor 16680, Indonesia.
authors.forename: Okti Nadia
authors.initials: ON
authors.lastname: Poetri
authors.affiliation: Faculty of Veterinary Medicine, Bogor Agricultural University IPB, JL Agatis Kampus IPB Darmaga, Bogor 16680, Indonesia.
authors.forename: Retno D
authors.initials: RD
authors.lastname: Soejoedono
authors.affiliation: Faculty of Veterinary Medicine, Bogor Agricultural University IPB, JL Agatis Kampus IPB Darmaga, Bogor 16680, Indonesia.
authors.forename: Ni L P Ika
authors.initials: NL
authors.lastname: Mayasari
authors.affiliation: Faculty of Veterinary Medicine, Bogor Agricultural University IPB, JL Agatis Kampus IPB Darmaga, Bogor 16680, Indonesia.
authors.forename: Srihadi
authors.initials: S
authors.lastname: Agungpriyono
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: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D016367:Baculoviridae / Q000235:genetics / Q000254:growth & development*; D001709:Biotechnology / Q000379:methods*; D012831:Bombyx; D002645:Chickens; D006385:Hemagglutination Inhibition Tests; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics*; D007252:Influenza Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D008854:Microscopy, Electron; D009975:Orthomyxoviridae / Q000235:genetics; D011679:Pupa; D011994:Recombinant Proteins / Q000235:genetics; D013678:Technology, Pharmaceutical / Q000379:methods*; D014611:Vaccination / Q000379:methods; D058425:Vaccines, Virus-Like Particle / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000648:ultrastructure
chemical_list: D000914:Antibodies, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D011994:Recombinant Proteins; D058425:Vaccines, Virus-Like Particle; C109963:hemagglutinin, avian influenza A virus
keywords: Baculovirus; H5N1 influenza virus; Vaccine; Virus-like particle