Extraction, purification and characterization of the plant-produced HPV16 subunit vaccine candidate E7 GGG

Buyel, Johannes F.; Bautista, James A.; Fischer, Rainer; Yusibov, Vidadi

doi:10.1016/j.jchromb.2011.11.010

Cited by 33 publications

(22 citation statements)

References 46 publications

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“…Up to now, purification of the E7 or E7GGG proteins from plants has been described only for fusion forms with carrier polypeptides such as bacterial lichenase [26], [36], or HPV L1 and E6 proteins [37]. We purified the E7GGG- FLAG protein by affinity chromatography on anti-FLAG M2 affinity resin (see Materials and Methods).…”

Section: Resultsmentioning

confidence: 99%

A Chlamydomonas-Derived Human Papillomavirus 16 E7 Vaccine Induces Specific Tumor Protection

et al. 2013

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BackgroundThe E7 protein of the Human Papillomavirus (HPV) type 16, being involved in malignant cellular transformation, represents a key antigen for developing therapeutic vaccines against HPV-related lesions and cancers. Recombinant production of this vaccine antigen in an active form and in compliance with good manufacturing practices (GMP) plays a crucial role for developing effective vaccines. E7-based therapeutic vaccines produced in plants have been shown to be active in tumor regression and protection in pre-clinical models. However, some drawbacks of in whole-plant vaccine production encouraged us to explore the production of the E7-based therapeutic vaccine in Chlamydomonas reinhardtii, an organism easy to grow and transform and fully amenable to GMP guidelines.Methodology/Principal FindingsAn expression cassette encoding E7GGG, a mutated, attenuated form of the E7 oncoprotein, alone or as a fusion with affinity tags (His6 or FLAG), under the control of the C. reinhardtii chloroplast psbD 5′ UTR and the psbA 3′ UTR, was introduced into the C. reinhardtii chloroplast genome by homologous recombination. The protein was mostly soluble and reached 0.12% of total soluble proteins. Affinity purification was optimized and performed for both tagged forms. Induction of specific anti-E7 IgGs and E7-specific T-cell proliferation were detected in C57BL/6 mice vaccinated with total Chlamydomonas extract and with affinity-purified protein. High levels of tumor protection were achieved after challenge with a tumor cell line expressing the E7 protein.ConclusionsThe C. reinhardtii chloroplast is a suitable expression system for the production of the E7GGG protein, in a soluble, immunogenic form. The production in contained and sterile conditions highlights the potential of microalgae as alternative platforms for the production of vaccines for human uses.

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Section: Resultsmentioning

confidence: 99%

A Chlamydomonas-Derived Human Papillomavirus 16 E7 Vaccine Induces Specific Tumor Protection

et al. 2013

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“…Evidence of high-yield expression of H5 haemagglutinin-derived VLPs via transient expression [44] 10 million vaccine dose "rapid fire" milestone by Medicago Inc. in DARPA Blue Angel programme [50] Human clinical trial of plant-made H5N1 vaccine candidate [47] HA-only VLPs produced for H7N9 outbreak virus [54] Phase 1 trials of H1N1pdm and HPAI H5N1 HA-derived plant-made products [48] Testing of plant-made engineered soluble trimeric HA (H1N1pdm) in mice [58] Adjuvanting of monomeric H1N1pdm HA with SiO 2 and bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) [59] Emergency response influenza vaccine candidates made in South Africa [43] Conjugation of plant-made HA to TMV particles and successful testing in mice [60] Elicitation of neutralising Ab with elastin-like polypeptide fused with stabilised soluble trimer-forming H5N1 HA [61] Presentation of M2e epitope on surface of rTMV virions elicits protective immunity to homologous and heterologous challenge in mice [64] Papillomaviruses Proof of efficacy of a plant-made Cottontail rabbit and Rabbit oral papillomavirus vaccines [74,75] High yields of HPV-16 L1 and VLPs via agroinfiltration-mediated transient expression or via transplastomic expression [76,77] Transplastomic expression of capsomere-forming HPV-16 L1 fused with Escherichia coli LTB as a built-in adjuvant [78] Successful expression of HPV-8 and Bovine papillomavirus L1 VLPs in plants [79,80] Co-expression of HPV-16 L1 with E coli LTB and oral immunisation elicits increased intestinal mucosal IgA responses to L1 [90] High-yield plant transient expression of chimaeric L1::L2 VLPs and proof of increased breadth of immune response [91] rPVX CP fusion with L2 108-120 epitope yields well and elicits high-titre anti-L2 protein sera in mice [97] Plant production, scale-up and protective efficacy in mouse model of therapeutic E7GGG-LicKM fusion protein vaccine [85][86][87] Plant expressed HPV-16 L1 with C-terminal string of E6 and E7 T-cell epitopes is viable prophylactic/therapeutic vaccine candidate [98] Production and proof of efficacy in mice of soluble E7GGG therapeutic vaccine in transplastomic Chlamydomonas reinhardtii [100] Proof of yield increase and effi...…”

Section: Hepatitis B Virusmentioning

confidence: 99%

Plant-based vaccines against viruses

Rybicki

2014

Virol J

133

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Plant-made or “biofarmed” viral vaccines are some of the earliest products of the technology of plant molecular farming, and remain some of the brightest prospects for the success of this field. Proofs of principle and of efficacy exist for many candidate viral veterinary vaccines; the use of plant-made viral antigens and of monoclonal antibodies for therapy of animal and even human viral disease is also well established. This review explores some of the more prominent recent advances in the biofarming of viral vaccines and therapies, including the recent use of ZMapp for Ebolavirus infection, and explores some possible future applications of the technology.

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“…The combination of two or more of these methods is also possible. For example, both immobilized metal-ion affinity chromatography (IMAC) and gel filtration methods were used to purify a candidate vaccine destined to treat the human papillomavirus (HPV16) with high purity up to 99% and yield up to 0.1 g per 1 kg plant biomass (Buyel et al, 2012). The recombinant construct contained the subunit E7 GGG of HPV16, fused to hexa-His-and KDEL-tagged variants of the bacterial lichenase.…”

Section: Chromatography Methodsmentioning

confidence: 99%

Molecular farming on rescue of pharma industry for next generations

Moustafa

Makhzoum

Trémouillaux-Guiller

2015

Critical Reviews in Biotechnology

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Recombinant proteins expressed in plants have been emerged as a novel branch of the biopharmaceutical industry, offering practical and safety advantages over traditional approaches. Cultivable in various platforms (i.e. open field, greenhouses or bioreactors), plants hold great potential to produce different types of therapeutic proteins with reduced risks of contamination with human and animal pathogens. To maximize the yield and quality of plant-made pharmaceuticals, crucial factors should be taken into account, including host plants, expression cassettes, subcellular localization, post-translational modifications, and protein extraction and purification methods. DNA technology and genetic transformation methods have also contributed to great parts with substantial improvements. To play their proper function and stability, proteins require multiple post-translational modifications such as glycosylation. Intensive glycoengineering research has been performed to reduce the immunogenicity of recombinant proteins produced in plants. Important strategies have also been developed to minimize the proteolysis effects and enhance protein accumulation. With growing human population and new epidemic threats, the need for new medications will be paramount so that the traditional pharmaceutical industry will not be alone to answer medication demands for upcoming generations. Here, we review several aspects of plant molecular pharming and outline some important challenges that hamper these ambitious biotechnological developments.

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Extraction, purification and characterization of the plant-produced HPV16 subunit vaccine candidate E7 GGG

Cited by 33 publications

References 46 publications

A Chlamydomonas-Derived Human Papillomavirus 16 E7 Vaccine Induces Specific Tumor Protection

A Chlamydomonas-Derived Human Papillomavirus 16 E7 Vaccine Induces Specific Tumor Protection

Plant-based vaccines against viruses

Molecular farming on rescue of pharma industry for next generations

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