title: Expression of codon optimized major capsid protein (L1) of human papillomavirus type 16 and 18 in Pichia pastoris; purification and characterization of the virus-like particles.
abstract: The major capsid protein (L1) of human papillomaviruses (HPV) expressed in heterologous systems assembles into virus-like particles (VLPs). We report cloning and expression of codon optimized HPV L1 genes of the two high-risk HPV types 16 and 18 in methylotropic yeast, Pichia pastoris. The VLPs produced in P. pastoris were subjected to three step purification method involving density gradient centrifugations and size exclusion chromatography. The enriched VLPs were characterized using conformation-specific monoclonal antibodies in ELISA and by transmission electron microscopy. Mice immunized with a bivalent HPV16 and HPV18 VLPs developed high serum antibody titers to both HPV types that persisted for 190 days post vaccination. Serum of mice immunized with the HPV-VLP preparations could neutralize homologous pseudoviruses in an in vitro assays. Our results demonstrate that the L1 proteins expressed in P. pastoris fold properly as evidenced by assembly into VLPs and induction of type-specific neutralizing antibody response in mice. This work constitutes a step towards developing an alternate production platform for generating an affordable HPV vaccine to meet the needs of developing countries.
authors.affiliation: Research & Development Centre, Indian Immunologicals Limited, Rakshapuram, Gachibowli, Hyderabad 500032, Andhra Pradesh, India.
authors.forename: N
authors.initials: N
authors.lastname: Hanumantha Rao
authors.forename: P
authors.initials: P
authors.lastname: Baji Babu
authors.forename: L
authors.initials: L
authors.lastname: Rajendra
authors.forename: R
authors.initials: R
authors.lastname: Sriraman
authors.forename: Yuk-Ying S
authors.initials: YY
authors.lastname: Pang
authors.forename: John T
authors.initials: JT
authors.lastname: Schiller
authors.forename: V A
authors.initials: VA
authors.lastname: Srinivasan
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood / Q000276:immunology; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D057809:HEK293 Cells; D052162:Human papillomavirus 16 / Q000235:genetics* / Q000276:immunology; D052161:Human papillomavirus 18 / Q000235:genetics* / Q000276:immunology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D030361:Papillomavirus Infections / Q000517:prevention & control; D053918:Papillomavirus Vaccines / Q000276:immunology*; D010843:Pichia / Q000235:genetics*; D058425:Vaccines, Virus-Like Particle / Q000276:immunology*
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D036022:Capsid Proteins; D009856:Oncogene Proteins, Viral; D053918:Papillomavirus Vaccines; D058425:Vaccines, Virus-Like Particle; C063317:L1 protein, Human papillomavirus type 16; C480565:L1 protein, Human papillomavirus type 18