Investigating Constraints Along the Plant Secretory Pathway to Improve Production of a SARS-CoV-2 Spike Vaccine Candidate

Given the complex maturation requirements of viral glycoproteins and the challenge they often pose for expression in plants, the identification of host constraints precluding their efficient production is a priority for the molecular farming of vaccines. Building on previous work to improve viral glycoprotein production in plants, we investigated the production of a soluble SARS-CoV-2 spike comprising the ectopic portion of the glycoprotein. This was successfully transiently expressed in N. benthamiana by co-expressing the human lectin-binding chaperone calreticulin, which substantially increased the accumulation of the glycoprotein. The spike was mostly unprocessed unless the protease furin was co-expressed which resulted in highly efficient processing of the glycoprotein. Co-expression of several broad-spectrum protease inhibitors did not improve accumulation of the protein any further. The protein was successfully purified by affinity chromatography and gel filtration, although the purified product was heterogenous and the yields were low. Immunogenicity of the antigen was tested in BALB/c mice, and cellular and antibody responses were elicited after low dose inoculation with the adjuvanted protein. This work constitutes an important proof-of-concept for host plant engineering in the context of rapid vaccine development for SARS-CoV-2 and other emerging viruses.

Rational domestication of a plant-based recombinant expression system expands its biosynthetic range.

Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead and an orally active analgesic are demonstrated.

Peculiarities of the Transformation of Asteraceae Family Species: The Cases of Sunflower and Lettuce

The Asteraceae family is the largest and most diversified family of the Angiosperms, characterized by the presence of numerous clustered inflorescences, which have the appearance of a single compound flower. It is estimated that this family represents around 10% of all flowered species, with a great biodiversity, covering all environments on the planet, except Antarctica. Also, it includes economically important crops, such as lettuce, sunflower, and chrysanthemum; wild flowers; herbs, and several species that produce molecules with pharmacological properties. Nevertheless, the biotechnological improvement of this family is limited to a few species and their genetic transformation was achieved later than in other plant families. Lettuce (Lactuca sativa L.) is a model species in molecular biology and plant biotechnology that has easily adapted to tissue culture, with efficient shoot regeneration from different tissues, organs, cells, and protoplasts. Due to this plasticity, it was possible to obtain transgenic plants tolerant to biotic or abiotic stresses as well as for the production of commercially interesting molecules (molecular farming). These advances, together with the complete sequencing of lettuce genome allowed the rapid adoption of gene editing using the CRISPR system. On the other hand, sunflower (Helianthus annuus L.) is a species that for years was considered recalcitrant to in vitro culture. Although this difficulty was overcome and some publications were made on sunflower genetic transformation, until now there is no transgenic variety commercialized or authorized for cultivation. In this article, we review similarities (such as avoiding the utilization of the CaMV35S promoter in transformation vectors) and differences (such as transformation efficiency) in the state of the art of genetic transformation techniques performed in these two species.

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.

Long-Term Potato Virus X (PVX)-Based Transient Expression of Recombinant GFP Protein in Nicotiana benthamiana Culture In Vitro

Plant molecular farming has a great potential to produce valuable proteins. Transient expression technology provides high yields of recombinant proteins in greenhouse-grown plants, but every plant must be artificially agroinfiltrated, and open greenhouse systems are less controlled. Here, we propose to propagate agrobacteria-free plants with high-efficient long-term self-replicated transient gene expression in a well-controlled closed in vitro system. Nicotiana benthamiana plant tissue culture in vitro, with transient expression of recombinant GFP, was obtained through shoot induction from leaf explants infected by a PVX-based vector. The transient expression occurs in new tissues and regenerants due to the natural systemic distribution of viral RNA carrying the target gene. Gene silencing was delayed in plants grown in vitro, and GFP was detected in plants for five to six months. Agrobacteria-free, GFP-expressing plants can be micropropagated in vitro (avoiding an agroinfiltration step), “rejuvenated” through regeneration (maintaining culture for years), or transferred in soil. The mean GFP in the regenerants was 18% of the total soluble proteins (TSP) (0.52 mg/g of fresh leaf weight (FW). The highest value reached 47% TSP (2 mg/g FW). This study proposes a new method for recombinant protein production combining the advantages of transient expression technology and closed cultural systems.

Site-Specific Glycosylation of Recombinant Viral Glycoproteins Produced in Nicotiana benthamiana

There is an urgent need to establish large scale biopharmaceutical manufacturing capacity in Africa where the infrastructure for biologics production is severely limited. Molecular farming, whereby pharmaceuticals are produced in plants, offers a cheaper alternative to mainstream expression platforms, and is amenable to rapid large-scale production. However, there are several differences along the plant protein secretory pathway compared to mammalian systems, which constrain the production of complex pharmaceuticals. Viral envelope glycoproteins are important targets for immunization, yet in some cases they accumulate poorly in plants and may not be properly processed. Whilst the co-expression of human chaperones and furin proteases has shown promise, it is presently unclear how plant-specific differences in glycosylation impact the production of these proteins. In many cases it may be necessary to reproduce features of their native glycosylation to produce immunologically relevant vaccines, given that glycosylation is central to the folding and immunogenicity of these antigens. Building on previous work, we transiently expressed model glycoproteins from HIV and Marburg virus in Nicotiana benthamiana and mammalian cells. The proteins were purified and their site-specific glycosylation was determined by mass-spectrometry. Both glycoproteins yielded increased amounts of protein aggregates when produced in plants compared to the equivalent mammalian cell-derived proteins. The glycosylation profiles of the plant-produced glycoproteins were distinct from the mammalian cell produced proteins: they displayed lower levels of glycan occupancy, reduced complex glycans and large amounts of paucimannosidic structures. The elucidation of the site-specific glycosylation of viral glycoproteins produced in N. benthamiana is an important step toward producing heterologous viral glycoproteins in plants with authentic human-like glycosylation.

Producing Vaccines against Enveloped Viruses in Plants: Making the Impossible, Difficult

The past 30 years have seen the growth of plant molecular farming as an approach to the production of recombinant proteins for pharmaceutical and biotechnological uses. Much of this effort has focused on producing vaccine candidates against viral diseases, including those caused by enveloped viruses. These represent a particular challenge given the difficulties associated with expressing and purifying membrane-bound proteins and achieving correct assembly. Despite this, there have been notable successes both from a biochemical and a clinical perspective, with a number of clinical trials showing great promise. This review will explore the history and current status of plant-produced vaccine candidates against enveloped viruses to date, with a particular focus on virus-like particles (VLPs), which mimic authentic virus structures but do not contain infectious genetic material.