N-terminal truncations on L1 proteins of human papillomaviruses promote their soluble expression in Escherichia coli and self-assembly in vitro

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.

Recent Progress on the Versatility of Virus-Like Particles

“…HPV is a circular, double-stranded DNA virus, and persistent HPV infection is a major cause of cervical cancer and genital warts [86]. At present, the four marketed prophylactic HPV vaccines are based on self-assembled VLPs that contain only the L1 protein [87,88]: Gardasil (Merck), Cervarix (GSK), Gardasil-9 (Merck) and Cecolin (Innovax).…”

“…This system has excellent safety and efficacy in phase 3 clinical trials [58,59], producing the comparable levels of neutralizing antibodies as that of marketed vaccines and has recently been approved for marketing in China. Further, a nine-valent vaccine developed with E. coli has entered into phase 2 clinical trials [60].…”

Cloning and expression of Human Papilloma virus type 16 L1 capsid protein in bacteria

“…2 Consisted of at least 9 HPV subtypes, those are HPV 6,11,16,18,31,32,45,52, and 58, the high-risk group is associated with 90% of cervical cancer and 90% of genital warts. 3 Approximately 67% of cervical cancer globally was caused by HPV16 and 18. 4 The main strategy to prevent HPV infection recommended by WHO is by vaccination.…”

“…Nowadays there are three kinds of commercial prophylactic HPV vaccines, that are Gardsil, a quadrivalent vaccine containing HPV 6,11,16,18 antigens, Gardasil 9, a 9-valent vaccine containing HPV 6,11,16,18,31,33,45,52,58 antigens and a bivalent vaccine against HPV16 and 18 antigens. 3 The WHO recommends that the commercial HPV vaccine must cover at least two kinds of HPV subtypes, HPV 16 and HPV18 antigens. 5 HPV is an unenveloped virus that has a circular double-stranded DNA genome of approximately 8 kb in size.…”

“…The other promising expression system that could be developed as a production host of L1 protein is prokaryote. 11,3 Due to the limitation in providing a susceptible cell culture that can be infected by HPV, the capability of the antibody to prevent HPV infection was measured by using pseudovirion. Pseudovirion is L1 VLP that assemble DNA coding a certain reporter protein, such as green fluorescent protein (GFP).…”