15 research outputs found
Engineering Approaches in Plant Molecular Farming for Global Health
Since the demonstration of the first plant-produced proteins of medical interest, there has been significant growth and interest in the field of plant molecular farming, with plants now being considered a viable production platform for vaccines. Despite this interest and development by a few biopharmaceutical companies, plant molecular farming is yet to be embraced by ‘big pharma’. The plant system offers a faster alternative, which is a potentially more cost-effective and scalable platform for the mass production of highly complex protein vaccines, owing to the high degree of similarity between the plant and mammalian secretory pathway. Here, we identify and address bottlenecks in the use of plants for vaccine manufacturing and discuss engineering approaches that demonstrate both the utility and versatility of the plant production system as a viable biomanufacturing platform for global health. Strategies for improving the yields and quality of plant-produced vaccines, as well as the incorporation of authentic posttranslational modifications that are essential to the functionality of these highly complex protein vaccines, will also be discussed. Case-by-case examples are considered for improving the production of functional protein-based vaccines. The combination of all these strategies provides a basis for the use of cutting-edge genome editing technology to create a general plant chassis with reduced host cell proteins, which is optimised for high-level protein production of vaccines with the correct posttranslational modifications
Engineering Approaches in Plant Molecular Farming for Global Health
Since the demonstration of the first plant-produced proteins of medical interest, there has been significant growth and interest in the field of plant molecular farming, with plants now being considered a viable production platform for vaccines. Despite this interest and development by a few biopharmaceutical companies, plant molecular farming is yet to be embraced by ‘big pharma’. The plant system offers a faster alternative, which is a potentially more cost-effective and scalable platform for the mass production of highly complex protein vaccines, owing to the high degree of similarity between the plant and mammalian secretory pathway. Here, we identify and address bottlenecks in the use of plants for vaccine manufacturing and discuss engineering approaches that demonstrate both the utility and versatility of the plant production system as a viable biomanufacturing platform for global health. Strategies for improving the yields and quality of plant-produced vaccines, as well as the incorporation of authentic posttranslational modifications that are essential to the functionality of these highly complex protein vaccines, will also be discussed. Case-by-case examples are considered for improving the production of functional protein-based vaccines. The combination of all these strategies provides a basis for the use of cutting-edge genome editing technology to create a general plant chassis with reduced host cell proteins, which is optimised for high-level protein production of vaccines with the correct posttranslational modifications
Efficient In Vitro and In Vivo Activity of Glyco-Engineered Plant-Produced Rabies Monoclonal Antibodies E559 and 62-71-3.
Rabies is a neglected zoonotic disease that has no effective treatment after onset of illness. However the disease can be prevented effectively by prompt administration of post exposure prophylaxis which includes administration of passive immunizing antibodies (Rabies Immune Globulin, RIG). Currently, human RIG suffers from many restrictions including limited availability, batch-to batch inconsistencies and potential for contamination with blood-borne pathogens. Anti-rabies monoclonal antibodies (mAbs) have been identified as a promising alternative to RIG. Here, we applied a plant-based transient expression system to achieve rapid, high level production and efficacy of the two highly potent anti-rabies mAbs E559 and 62-71-3. Expression levels of up to 490 mg/kg of recombinant mAbs were obtained in Nicotiana benthamiana glycosylation mutants by using a viral based transient expression system. The plant-made E559 and 62-71-3, carrying human-type fucose-free N-glycans, assembled properly and were structurally sound as determined by mass spectrometry and calorimetric density measurements. Both mAbs efficiently neutralised diverse rabies virus variants in vitro. Importantly, E559 and 62-71-3 exhibited enhanced protection against rabies virus compared to human RIG in a hamster model post-exposure challenge trial. Collectively, our results provide the basis for the development of a multi-mAb based alternative to RIG
Plant-based production of highly potent anti-HIV antibodies with engineered posttranslational modifications
Broadly neutralising antibodies (bNAbs) against human immunodeficiency virus type 1 (HIV-1), such
as CAP256-VRC26 are being developed for HIV prevention and treatment. These Abs carry a unique
but crucial post-translational modification (PTM), namely O-sulfated tyrosine in the heavy chain
complementarity determining region (CDR) H3 loop. Several studies have demonstrated that plants
are suitable hosts for the generation of highly active anti-HIV-1 antibodies with the potential to
engineer PTMs. Here we report the expression and characterisation of CAP256-VRC26 bNAbs with
posttranslational modifications (PTM). Two variants, CAP256-VRC26 (08 and 09) were expressed
in glycoengineered Nicotiana benthamiana plants. By in planta co-expression of tyrosyl protein
sulfotransferase 1, we installed O-sulfated tyrosine in CDR H3 of both bNAbs. These exhibited similar
structural folding to the mammalian cell produced bNAbs, but non-sulfated versions showed loss
of neutralisation breadth and potency. In contrast, tyrosine sulfated versions displayed equivalent
neutralising activity to mammalian produced antibodies retaining exceptional potency against some
subtype C viruses. Together, the data demonstrate the enormous potential of plant-based systems for
multiple posttranslational engineering and production of fully active bNAbs for application in passive
immunisation or as an alternative for current HIV/AIDS antiretroviral therapy regimens.The Department of Science and Technology (DST), South African Medical Research Council - Strategic Health Innovation Partnership (SAMRC SHIP) and Council for Scientific and Industrial Research (CSIR).http://www.nature.com/srepam2021Plant Production and Soil ScienceProduction Animal Studie
Deconvoluted spectrum of intact, reduced 62-71-3 HC.
<p>The inset shows the zoomed-in LC region with theoretical molecular weights for LC and HC indicated. Detected N-linked glycoforms are shown. The N-glycan nomenclature used was from <a href="http://www.proglycan.com/" target="_blank">www.proglycan.com</a>.</p
Fifty percent end-point neutralisation activity (reciprocal titre) of E559 and 62-71-3 in a modified Rapid Fluorescent Focus Inhibition Test (RFFIT).
<p>Fifty percent end-point neutralisation activity (reciprocal titre) of E559 and 62-71-3 in a modified Rapid Fluorescent Focus Inhibition Test (RFFIT).</p
Rabies virus challenge and administration schedule of anti-rabies mAbs in Syrian hamsters.
<p>Rabies virus challenge and administration schedule of anti-rabies mAbs in Syrian hamsters.</p
Far-UV CD spectra of humanised 62-71-3 (black) and E559 (blue) mAbs.
<p>Far-UV CD spectra of humanised 62-71-3 (black) and E559 (blue) mAbs.</p
N-linked glycans on the anti-rabies mAbs.
<p>N-glycosylation profile from E559 HC and LC and from 62-71-3 HC as determined by LC-ESI-MS of glycopeptides obtained upon trypsin digestion. Numbers represent the presence of the different glyco-species in percent of total glycan. The N-glycan nomenclature used was from <a href="http://www.proglycan.com/" target="_blank">www.proglycan.com</a>.</p
SDS-PAGE analysis of purified E559 and 62-71-3 mAbs.
<p>The middle lane was a PageRuler Prestained Protein Ladder, with the sizes indicated in kDa. The numbered E559 and 62-71-3 bands (1–5) were used for further analysis.</p