title: Refining HPV 16 L1 purification from E. coli: reducing endotoxin contaminations and their impact on immunogenicity.
abstract: HPV 16 L1 capsomeres purified from Escherichia coli represent a promising and potentially cost-effective alternative to the recently licensed VLP-based vaccines for the prevention of cervical cancer. However, recombinant protein preparations from bacteria always bear the risk of contaminating endotoxins which are highly toxic in humans and therefore have to be eliminated from vaccine preparations. In this study, we measured the LPS concentration at various stages of the purification of HPV 16 L1 from E. coli and determined that it enhances the immunogenicity of HPV 16 VLPs and capsomeres. We confirmed the immunogenicity of the L1 capsomeres in TLR4(-/-) mice without the enhancing effect of the LPS and then elaborated a suitable protocol using Triton X-114 phase separation for the removal of LPS without any significant protein loss or influence on the structural integrity of the particles. The LPS-free capsomeres purified from E. coli induced neutralizing L1-specific antibodies. Our results demonstrate the excellent potential of capsomeres as an economically interesting alternative vaccine to prevent cervical cancer that could be made available in developing countries.
authors.affiliation: German Cancer Research Center, Department of Genome Modifications and Carcinogenesis, 69120 Heidelberg, Germany. l.schaedlich@dkfz.de
authors.forename: Lysann
authors.initials: L
authors.lastname: Schädlich
authors.forename: Tilo
authors.initials: T
authors.lastname: Senger
authors.forename: Carsten J
authors.initials: CJ
authors.lastname: Kirschning
authors.forename: Martin
authors.initials: M
authors.lastname: Müller
authors.forename: Lutz
authors.initials: L
authors.lastname: Gissmann
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D002460:Cell Line; D004340:Drug Contamination / Q000517:prevention & control; D004731:Endotoxins / Q000276:immunology / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology; D005260:Female; D052162:Human papillomavirus 16 / Q000235:genetics / Q000276:immunology*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000276:immunology / Q000517:prevention & control / Q000821:virology; D053918:Papillomavirus Vaccines / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D002583:Uterine Cervical Neoplasms / Q000276:immunology / Q000517:prevention & control / Q000821:virology
chemical_list: D000914:Antibodies, Viral; D036022:Capsid Proteins; D004731:Endotoxins; D009856:Oncogene Proteins, Viral; D053918:Papillomavirus Vaccines; D011994:Recombinant Proteins; C044101:endotoxin, Escherichia coli; C063317:L1 protein, Human papillomavirus type 16