Abstract
Human papillomavirus (HPV) is the causative agent in genital warts and nearly all cervical, anogenital, and oropharyngeal cancers. Nine HPV types (6, 11, 16, 18, 31, 33, 45, 52, and 58) are associated with about 90% of cervical cancers and 90% of genital warts. HPV neutralization by vaccine-elicited neutralizing antibodies can block viral infection and prevent HPV-associated diseases. However, there is only one commercially available HPV vaccine, Gardasil 9, produced from Saccharomyces cerevisiae that covers all nine types, raising the need for microbial production of broad-spectrum HPV vaccines. Here, we investigated whether N-terminal truncations of the major HPV capsid proteins L1, improve their soluble expression in Escherichia coli. We found that N-terminal truncations promoted the soluble expression of HPV 33 (truncated by 10 amino acids [aa]), 52 (15 aa), and 58 (10 aa). The resultant HPV L1 proteins were purified in pentamer form and extensively characterized with biochemical, biophysical, and immunochemical methods. The pentamers self-assembled into virus-like particles (VLPs) in vitro, and 3D cryo-EM reconstructions revealed that all formed T = 7 icosahedral particles having 50–60-nm diameters. Moreover, we formulated a nine-valent HPV vaccine candidate with aluminum adjuvant and L1 VLPs from four genotypes used in this study and five from previous work. Immunogenicity assays in mice and non-human primates indicated that this HPV nine-valent vaccine candidate elicits neutralizing antibody titers comparable to those induced by Gardasil 9. Our study provides a method for producing a nine-valent HPV vaccine in E. coli and may inform strategies for the soluble expression of other vaccine candidates.
Highlights
Human Papillomavirus (HPV) is a small, non-enveloped epitheliotropic DNA virus associated with multiple human diseases, such as benign verrucae vulgares, Wei et al Emerging Microbes & Infections (2018)7:160 condylomata acuminata, and malignancies of the cervix, vulva, anus, and penis[1,2]
We found that truncation of several N-terminal residues could improve the solubility of Human papillomavirus (HPV) L1 proteins[35,36], and showed that such N-terminal modification offers a feasible strategy to improve the solubility of HPV L1 proteins in E. coli for HPV 6, 11, 16, and 1835,37
Protein expression of full-length and a series of N-terminally truncated HPV 33, 45, 52, and 58 L1 proteins were determined by SDS-PAGE and western blotting (WB) using cell lysates from E. coli. (Fig. 1a); we compared these with the five HPV types previously reported (Fig. S3a, S3b)
Summary
Human Papillomavirus (HPV) is a small, non-enveloped epitheliotropic DNA virus associated with multiple human diseases, such as benign verrucae vulgares (common wart), Wei et al Emerging Microbes & Infections (2018)7:160 condylomata acuminata (genital warts), and malignancies of the cervix, vulva, anus, and penis[1,2]. 200 HPV types have been identified to date, with over 15 high-risk types closely linked to cervical cancer and other epithelial tumors[3,4]. Cervical cancer is one of the most common types of female cancers, of which 88% are caused by HPV types 16, 18, 31, 33, 45, 52, and 584–6. HPV 33, 45, 52, and 58 account for 15% of cervical cancers worldwide[7,8]. HPV 6 and HPV 11, which are considered as low-risk HPV subtypes, are usually not connected with cancer but are responsible for more than 90% of genital warts[9]. There are currently three prophylactic HPV vaccines on the market: Gardasil (Merck, Sharp and Dohme; Hoddesdon, UK), a quadrivalent vaccine containing HPV 6, 11, 16, and 18 antigens[10]; Gardasil 9
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