Expression of Human Papillomavirus Type 16 L1 Protein inEscherichia coli:Denaturation, Renaturation, and Self-Assembly of Virus-like Particlesin Vitro

Zhang, Wei; Carmichael, Jeremy; Ferguson, Jackie; Inglis, Stephen; Ashrafian, Houman; Stanley, Margaret

doi:10.1006/viro.1998.9050

Cited by 62 publications

(88 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…HPV vaccines are particularly needed in developing countries, and subcapsid particles (pentameric capsomeres) may be an economically advantageous alternative to VLPs especially in medically underfunded areas, since they can be readily purified after expression in Escherichia coli (6,23,51) and are very stable (27,33). Immunization with papillomavirus L1 capsomeres triggers neutralizing antibodies (13,38,50) and protects against experimental challenge in the canine oral papillomavirus model (50).…”

mentioning

confidence: 99%

Human Papillomavirus Type 16 L1 Capsomeres Induce L1-Specific Cytotoxic T Lymphocytes and Tumor Regression in C57BL/6 Mice

Öhlschläger

Osen

Dell

et al. 2003

J Virol

View full text Add to dashboard Cite

show abstract

mentioning

confidence: 99%

Human Papillomavirus Type 16 L1 Capsomeres Induce L1-Specific Cytotoxic T Lymphocytes and Tumor Regression in C57BL/6 Mice

Öhlschläger

Osen

Dell

et al. 2003

J Virol

View full text Add to dashboard Cite

show abstract

“…Although early attempts to use bacteria for producing papillomavirus L1 protein vaccines were unsuccessful due to poor immunogenicity or inefficient expression (1,9,13,28,29), recent studies have shown that the HPV type 11 (HPV-11) and HPV-16 L1 proteins can be expressed in Escherichia coli in a capsomeric form that assembles into VLPs in vitro (6,17). Capsomeres of HPV-11 L1 react with conformation-specific antibodies, including neutralizing monoclonal antibodies, and induce neutralizing antibodies in rabbits (21).…”

mentioning

confidence: 99%

Immunization with a Pentameric L1 Fusion Protein Protects against Papillomavirus Infection

Yuan

Estes

Chen

et al. 2001

J Virol

125

105

View full text Add to dashboard Cite

The prophylactic papillomavirus vaccines currently in clinical trials are composed of viral L1 capsid protein that is synthesized in eukaryotic expression systems and purified in the form of virus-like particles (VLPs). To evaluate whether VLPs are necessary for effective vaccination, we expressed the L1 protein as a glutathione S-transferase (GST) fusion protein in Escherichia coli and assayed its immunogenic activity in an established canine oral papillomavirus (COPV) model that previously validated the efficacy of VLP vaccines. The GST-COPV L1 fusion protein formed pentamers, but these capsomere-like structures did not assemble into VLPs. Despite the lack of VLP formation, the GST-COPV L1 protein retained its native conformation as determined by reactivity with conformation-specific anti-COPV antibodies. Most importantly, the GST-COPV L1 pentamers completely protected dogs from high-dose viral infection of their oral mucosa. L1 fusion proteins expressed in bacteria represent an economical alternative to VLPs as a human papillomavirus vaccine.Genital human papillomavirus (HPV) infection is a common sexually transmitted disease that is the primary cause of cervical cancer, resulting in approximately 400,000 deaths per year worldwide (30). An effective vaccine against HPV infection would potentially prevent the development of most human cervical dysplasias and carcinomas (4). In addition, a vaccine would also reduce the cost (estimated at $6 billion annually in the United States) of screening and treating premalignant cervical disease (16).Since HPV cannot replicate in other animal species, evaluation of potential HPV vaccines requires the use of related animal papillomaviruses. The mucosotropic, oncogenic canine oral papillomavirus (COPV) closely mimics the biology of HPV, and the capsid proteins of COPV are closely related to those of HPV, making COPV a relevant and accepted animal model for testing the efficacy of prophylactic vaccine candidates (2,19,26,27).The L1 capsid protein of papillomaviruses self-assembles into virus-like particles (VLPs) when expressed in insect cells (11,14) or yeast (12,23). These L1 VLPs are morphologically similar to virions, being comprised of 72 pentamers (i.e., capsomeres) of L1 arranged in a Tϭ7 icosahedral lattice, but lacking the L2 capsid protein and the viral genome. Previous studies have shown that immunization with purified VLPs protects against experimental papillomavirus infection in rabbits (5, 8), cows (15), and dogs (27). Conformational epitopes on VLPs appear critical for the induction of neutralizing immunoglobulin G (IgG) and for successful vaccination, since denatured L1 protein fails to generate neutralizing antibodies or protect against experimental infection (10,18,27).Although early attempts to use bacteria for producing papillomavirus L1 protein vaccines were unsuccessful due to poor immunogenicity or inefficient expression (1, 9, 13, 28, 29), recent studies have shown that the HPV type 11 (HPV-11) and HPV-16 L1 proteins can be expressed in Escherichia col...

show abstract

“…These parameters are difficult to regulate in BEVS . Secondly, the E. coli expression system offers a high yield of proteins, and the expressed proteins are easy to purify and recover , Zhang et al 1998). The well-studied denaturation and refolding mechanism also provides a solution to overcome the solubility problem for some recombinant proteins, especially those which originate from eukaryotic hosts using the E. coli expression system, may result in insoluble proteins (Zeltins 2013).…”

Section: Escherichia Coli Expression Systemmentioning

confidence: 99%

Engineering of virus-like particles for alternative vaccine candidate targeting a hypervariable peptide antigen element

Anggraeni¹

View full text Add to dashboard Cite

A promising alternative to replace the current egg-or cell culture-based technology for vaccine production from live viruses is virus-like particle (VLP) technology based on a microbial platform. VLPs are macromolecular assemblies of viral capsid proteins, which have been shown to tolerate insertion of antigen modules via genetic recombinant technology, yielding modular VLPs. Many studies on modular VLPs presume that when a peptide antigen element is taken out from the intact proteins and then modularised on VLPs, it is unable to fold into its native structure. However, until now, presentation of a peptide antigen element on a VLP and the impact of the display strategy to present the antigen element on the quality of the resulting antibodies (i.e. the ability of the antibodies to recognise the intact protein) are not fully understood. This thesis aims to understand the underlying fundamentals regarding modularisation of peptide antigen elements on VLPs for induction of high-quality antibodies. A hypervariable receptor-binding domain, Helix 190 (H190), from the hemagglutinin protein of influenza A virus was used as a model for modularisation on VLPs from murine polyomavirus (MuPyV) VP1 protein. Four major findings are presented. Firstly, two display strategies, i.e. arraying of H190 in tandem repeats and the use of helix promoter elements, were shown to display H190 in its immunogenic form equally. However, modularisation using tandem repeat display induced antibodies of a higher quality than modularisation using helix promoter elements.Secondly, the quality of antibodies induced by the tandem repeat display bearing two copies of H190 was optimum, thus no significant improvement was observed following the use of adjuvant or increasing the copy number of H190. Additionally, the increase in the copy number of H190 was shown to reduce the assembly capability and solubility of modular VP1 in an environment that was optimised for wild-type VP1. Thirdly, this thesis shows the novel finding in the use of flanking ionic elements to stabilise VLP precursors, termed as capsomeres, bearing two copies of H190 containing a hydrophobic stretch, which caused aggregation. Fourthly, the first steps towards obtaining the atomic crystal structure of presented H190 on a modular protein were performed, i.e. a mild and satisfactory laboratory process was developed to achieve high-purity modular VP1 capsomeres, unattainable using previously established expression and purification process of wild-type MuPyV VP1. This thesis shows a step forward towards understanding the presentation of a peptide antigen element on a VLP that enables induction of highquality antibodies, and towards VLP engineering to manipulate the aggregation and solubility of modular VP1. VLP technology based on a microbial platform presented here is iii a potentially safe and effective alternative vaccine candidate that targets a hypervariable peptide antigen element. The speed of the microbial platform allows a rapid response to the hypervariability of the pe...

show abstract

Expression of Human Papillomavirus Type 16 L1 Protein inEscherichia coli:Denaturation, Renaturation, and Self-Assembly of Virus-like Particlesin Vitro

Cited by 62 publications

References 35 publications

Human Papillomavirus Type 16 L1 Capsomeres Induce L1-Specific Cytotoxic T Lymphocytes and Tumor Regression in C57BL/6 Mice

Human Papillomavirus Type 16 L1 Capsomeres Induce L1-Specific Cytotoxic T Lymphocytes and Tumor Regression in C57BL/6 Mice

Immunization with a Pentameric L1 Fusion Protein Protects against Papillomavirus Infection

Engineering of virus-like particles for alternative vaccine candidate targeting a hypervariable peptide antigen element

Contact Info

Product

Resources

About