Sistemi vegetali per la produzione di antigeni virali e come sorgente di molecole immunogeniche
Author(s)
Demurtas, Olivia Costantina
Date Issued
October 28, 2010
Type
Doctoral Thesis
Abstract
This PhD work has been focused on the use of plant systems, as Nicotiana benthamiana plants and the unicellular alga Chlamydomonas reinhardtii, for the production of human pathogenic viral antigens, to develop effective vaccines and/or specific and low-cost diagnostic assays. Moreover, in this work plants have been used as a source of immunogenic molecules, able to improve therapeutic vaccine formulations developed in our laboratory.
Compared to conventional systems (bacteria, yeasts, insect and mammalian cells) actually employed for biopharmaceuticals production, plant systems offer several advantages. They are eukaryotic organisms able to generate molecules with post-translational modifications, they can grow fast, at low costs, in non-sterile conditions, and they represent a safe platform for the production of human pharmaceuticals as they don’t contain human pathogens (e.i. prions, viruses, etc.).
In the first part of this work we used plants for the production of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) antigens. By the use of a viral vector derived from the potato virus X (PVX), we produce transiently in N. benthamiana plants the best candidate for the development of SARS serological diagnostic assays, the nucleocapsid protein (N). We demonstrated its specific reactivity/antigenicity with SARS patient sera by an immunoenzymatic assay.
In addition, we were able to produce for the first time in plant the transmembrane glycoprotein (M) by agroinfiltration. Compared to other SARS-CoV antigens, this protein is less characterized because of the difficulty to produce it in a recombinant form.
Both N and M proteins obtained in plant could be employed to formulate SARS diagnostic assay and vaccines.
The second part of the work has been focused on the use of plant systems for the production of the Human Papilloma Virus type 16 (HPV-16) E7 oncoprotein. HPV-16 is the aetiological agent of cervical cancer and other tumours and the E7 protein, being involved in malignant cellular transformation, represents the ideal antigen for the development of therapeutic vaccines. Firstly we chose to express this antigen in N. benthamiana by agroinfiltration, trying to increase protein yield, compared to those previously obtained in our laboratory by infection with PVX vector, in order to purify the protein and characterize its biochemical properties. Unfortunately, the agroinfiltration technology didn’t allow the increase of the E7 protein yield, thereby we decide to express the protein in C. reinhardtii. This eukaryotic microalga shares with plants several advantages; in addition this organism is able to grow in contained conditions. This feature make this system similar to those currently used for pharmaceuticals production, hence more conformable to Good Manufacturing Practices (GMP) procedures, compared to plants.
After failure attempts of the E7 protein expression (in the mutagenized non oncogenic form E7GGG) in C. reinhardtii by nuclear transformation, also using mutated strains improved for eterologous protein expression, we decide to produce the protein in the chloroplast. Presenting a unique large chloroplast that contain about 80 genome copies, plastid transformation of C. reinhardtii is simpler and faster than plant chloroplasts transformation. Unlike nuclear transformation, this technology allowed the E7GGG protein expression in a soluble form, representing the first example of HPV antigen production in microalga. This result has been obtained using a codon-optimized E7GGG gene and modifying the transformation vector in order to place the gene under the control of a strong promoter. In the next months we will analyze in a preclinical model the biological activity of the microalga produced E7GGG protein.
In the last part of this work we focused our attention on the plant kingdom for the research of new immunomodulatory molecules, able to increase the effectiveness of therapeutic HPV-16 E7 based
vaccines. We decided to formulate vaccines based on the fusion between the E7GGG protein and the saporin protein. Saporin is an enzyme from the plant Saponaria officinalis that belongs to the Ribosome Inactivating Proteins (RIP) family, able to inhibit protein synthesis in both prokaryotic and eukaryotic cells. This protein is highly immunogenic in humans and it is also able to modulate non-specific immune responses, important for cancer therapy.
As we were not interested to saporin toxicity, we used a mutagenized form in the catalytic site (SAPKQ mutant), that could preserve the immunological properties. The toxicity abolition of the SAPKQ mutant was verified in E. coli and mammalian cells.
We realized E7GGG/SAPKQ fusion proteins that differ for the presence or absence of the saporin signal sequence and for genes orientation in the fusion (ssSAPKQ-E7GGG, SAPKQ-E7GGG, and E7GGG-SAPKQ), with the purpose of develop protein subunit vaccines (firstly produced in plant, but also produced in E. coli) and genetic vaccines.
The three different fusion proteins were expressed in E. coli, mainly in the insoluble cellular fraction.
The same proteins were obtained by PVX infection in N. benthamiana plants, but at low levels and in the insoluble fraction.
Unlike E. coli, in plant cells the saporin signal sequence was recognized, but the molecular weight of the processed protein is lower than expected. Because of low protein yields and because of necessity to clarify mechanisms involved in protein processing/degradation, it was not possible to perform vaccination experiments to evaluate the eventually increased immunogenicity of the plant produced vaccines. Nevertheless, in the next months we will be able to perform this analysis with the E. coli produced and purified fusion proteins.
Also to obtain a rapid proof of concept concerning the immunological validity of vaccines based on the fusion proteins, we elaborated DNA vaccines. After tested the fusion protein expression in mammalian cells, we used DNA constructs to vaccinate mice that, subsequently to the inoculation of tumorigenic cancer cell line expressing the E7 protein, are able to develop cancer.
Immunological results indicated that by fusion of the E7GGG gene with the mutagenized saporin it is possible to obtain in vivo an antigen able to induce a major production of total anti-E7 IgG and, more important, a major E7-specific linfocytes stimulation, than the E7GGG alone, that permit to overtake the poor immunogenicity of the E7 antigen per se. Moreover, compared to the E7GGG gene alone, the fusion proteins are able to protect more effectively from the tumour development challenged mice with tumorigenic cell line.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
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