Clinical and subclinical human papillomavirus (HPV) infections are one of the most common sexually transmitted infections in the world, and most sexually-active individuals are likely to be exposed to HPV infection during their lifetimes. More than 120 different human papillomavirus (HPV) types have been catalogued so far, of which more than 40 infect the epithelial lining of the anogenital tract and other mucosal areas of the body. Some types have been identified as high-oncogenic risk HPV types (HR-HPV), such as HPV-16, 18, 31. Persistent infection with HR-HPVs is established as a necessary cause of cervical cancer and is likely to be responsible for a substantial proportion of other anogenital and upper aero-digestive tract neoplasms. HPV-16 accounts for 50-60% of the cervical cancer cases in most countries, therefore a prophylactic vaccine that prevents persistent HPV-16 infection could substantially reduce the burden of HPV-related cervical disease. The papillomavirus L1 major capsid protein can self assemble into virus-like particles, or VLPs, when recombinantly expressed. VLPs mimic the true structure of the virion and they are considered to be a promising vaccine candidate. Clinical studies have shown protective effects of a HPV vaccine comprised of VLPs from HPV-6, 11, 16 and 18. The aim of this work is to produce the recombinant HPV-16 L1 major capsid protein in yeasts, focusing on the development of a cost-effective prophylactic vaccine. The facultative methylotrophic yeasts Pichia pastoris and Hansenula polymorpha have been developed as systems for heterologous gene expression. Native and codon-optimized HPV-16 L1 genes were cloned into expression vectors under the regulation of a strong methanol-inducible promoter and the constructions were used to transform competent P. pastoris and H. polymorpha. Cultures were grown to an O.D.₆₀₀ = 1.0 before the addition of methanol to induce protein expression. Aliquots were harvested after 24 and 48h for evaluation. The presence of recombinant protein in the cell lysates was confirmed by Western-blotting. Consistent L1 expression in P. pastoris was observed only with the construction containing the codon-optimized gene. Protein purification is underway. Also, we are trying to improve the expression systems to increase the levels of recombinant protein for a future large-scale production.
Supported by: FAPESP, CNPq, Fundação Butantan