Stop codon mutagenesis for homogenous expression of human papillomavirus L1 protein in Escherichia coli

Wang, Daning; Fan, Fei; Li, Zhihai; Liu, Xinlin; Song, Shuo; Wei, Shuangping; He, Maozhou; Lin, Yahua; Li, Zhongyi; Wei, Minxi; Yu, Hai; Gu, Ying; Xia, Ningshao

doi:10.1016/j.pep.2017.03.003

Cited by 7 publications

(8 citation statements)

References 55 publications

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“…The results from particle analyses for HPV 6, 11, 16, 18, and 31 L1 VLP (Fig. S 5 ) were consistent with previous results 34 – 36 . Overall, the L1 VLPs of nine HPV types qualified for inclusion in the manufactured HPV vaccine.…”

Section: Resultssupporting

confidence: 91%

“…The morphology of HPV 33, 45, 52, and 58 VLPs was determined by negative staining TEM. Similar to the E. coli -generated L1 VLPs for the 5 HPV types determined previously (HPV 6, 11, 16, 18, and 31) 34 – 36 , particles for HPV 33, 45, 52, and 58 VLPs appeared to be irregularly spherical and heterogeneous in size (Fig. 4a–d , upper left); this was also observed in the unstained, vitrified L1 VLPs (Fig.…”

Section: Resultssupporting

confidence: 82%

“…All of the L1 proteins in the 9-valent vaccine had the desired characteristics, with similar secondary structure composition, robust thermal stability, and the ability to self-assemble into a VLPs in vitro. Purified HPV 33, 45, 52, and 58 L1 VLPs showed variable particle size but with a mean of ~55 nm, similar to that previously reported 34 , 46 , 47 . The heterogeneous sizes of the HPV L1 VLPs among the different types may be caused by the different amino acid sequences of the N-terminal domains, which are important for the assembly of HPV L1 VLPs 27 , 32 .…”

Section: Discussionsupporting

confidence: 86%

“…Further research found that the 5′-end of the HPV L1 gene contained an element that could negatively affect the expression of L1 proteins in human epithelial cells 33 , and showed that point mutations or deletions in the inhibitory element to inactivate the effector could improve the production of L1 proteins 24 . However, the exact location of this regulatory region might vary with different HPV types, with maximum protein expression levels achieved with different N-terminal truncations for HPV 6, 11, 16, 18, and 31 34 – 36 . Therefore, it is necessary to investigate the effects of the N-terminal region on L1 expression for different HPV types for the successful and efficient manufacturing of an E. coli -based HPV vaccine.…”

Section: Introductionmentioning

confidence: 99%

“…In our previous work, we successfully produced high-potency VLP vaccine candidates of HPV 6, 11, 16, 18, and 31 using E. coli 34 – 36 , among which the HPV 16/18 bivalent vaccine and HPV 6/11 bivalent vaccine have been in phase 3 and phase 2 clinical trials, respectively. The L1 proteins were produced through non-fusion expression, and harbored N-terminal deletions (except for HPV 31) that rendered them still capable of self-assembling into T = 7 particles.…”

Section: Introductionmentioning

confidence: 99%

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

Wei

Wang

et al. 2018

Emerging Microbes & Infections

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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.

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Section: Resultssupporting

confidence: 91%

Section: Resultssupporting

confidence: 82%

Section: Discussionsupporting

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Wei

Wang

et al. 2018

Emerging Microbes & Infections

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Characterization of an Escherichia coli -derived human papillomavirus type 16 and 18 bivalent vaccine

Gu¹,

Wei

Wang

et al. 2017

Vaccine

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Rational design of a multi-valent human papillomavirus vaccine by capsomere-hybrid co-assembly of virus-like particles

et al. 2020

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The capsid of human papillomavirus (HPV) spontaneously arranges into a T = 7 icosahedral particle with 72 L1 pentameric capsomeres associating via disulfide bonds between Cys175 and Cys428. Here, we design a capsomere-hybrid virus-like particle (chVLP) to accommodate multiple types of L1 pentamers by the reciprocal assembly of single C175A and C428A L1 mutants, either of which alone encumbers L1 pentamer particle self-assembly. We show that co-assembly between any pair of C175A and C428A mutants across at least nine HPV genotypes occurs at a preferred equal molar stoichiometry, irrespective of the type or number of L1 sequences. A nine-valent chVLP vaccine-formed through the structural clustering of HPV epitopes-confers neutralization titers that are comparable with that of Gardasil 9 and elicits minor cross-neutralizing antibodies against some heterologous HPV types. These findings may pave the way for a new vaccine design that targets multiple pathogenic variants or cancer cells bearing diverse neoantigens.

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Stop codon mutagenesis for homogenous expression of human papillomavirus L1 protein in Escherichia coli

Cited by 7 publications

References 55 publications

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

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

Characterization of an Escherichia coli -derived human papillomavirus type 16 and 18 bivalent vaccine

Rational design of a multi-valent human papillomavirus vaccine by capsomere-hybrid co-assembly of virus-like particles

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