Plant Molecular Farming

An effective prophylactic vaccine is urgently needed to protect against SARS-CoV-2 infection. The viral spike, which mediates entry into cells by interacting with the host angiotensin-converting enzyme 2, is the primary target of most vaccines in development. These vaccines aim to elicit protective immunity against the glycoprotein by use of inactivated virus, vector-mediated delivery of the antigen in vivo, or by direct immunization with the purified antigen following expression in a heterologous system. These approaches are mostly dependent on the growth of mammalian or insect cells, which requires a sophisticated infrastructure that is not generally available in developing countries due to the incumbent costs which are prohibitive. Plant-based subunit vaccine production has long been considered as a cheaper alternative, although low expression yields and differences along the secretory pathway to mammalian cells have posed a challenge to producing certain viral glycoproteins. Recent advances that have enabled many of these constraints to be addressed include expressing the requisite human proteins in plants to support the maturation of the protein of interest. In this study we investigated these approaches to support the production of a soluble and putatively trimeric SARS-CoV-2 spike mimetic in Nicotiana benthamiana via transient Agrobacterium-mediated expression. The co-expression of human calreticulin dramatically improved the accumulation of the viral spike, which was barely detectable in the absence of the co-expressed accessory protein. The viral antigen was efficiently processed even in the absence of co-expressed furin, suggesting that processing may have occurred at the secondary cleavage site and was mediated by an endogenous plant protease. In contrast, the spike was not efficiently processed when expressed in mammalian cells as a control, although the co-expression of furin improved processing considerably. This study demonstrates the feasibility of molecular engineering to improve the production of viral glycoproteins in plants, and supports plant-based production of SARS-CoV-2 spike-based vaccines and reagents for serological assays.

The Tier 1 select agent Burkholderia pseudomallei is the causative agent of melioidosis, a global pathogen and a major cause of pneumonia and sepsis for which no licensed vaccines currently exist. Previous work has shown the potential for Burkholderia capsular polysaccharide (CPS) to be used as a vaccine antigen but the T-cell independent nature of the immune response to this molecule requires the use of this polysaccharide as a glycoconjugate for vaccination. Recent studies have focussed on the use of Crm197 (a non-toxic mutant protein derived from diphtheria toxin) as the carrier but there are concerns regarding its potential to cause interference with other vaccines containing Crm197. Therefore research with alternative carrier proteins would be beneficial. In this study, CPS was isolated from the non-pathogenic B. thailandensis strain E555. This was chemically conjugated to Crm197, or Tandem Core™ virus-like particles (TCVLP) consisting of hepatitis B core protein, which is the first documented use of VLPs in melioidosis vaccine development. Analysis of CPS-specific IgG antibody titres showed that mice vaccinated with the Crm197 conjugate generated significantly higher titres than the mice that received TCVLP-CPS but both conjugate vaccines were able to protect mice against intraperitoneal B. pseudomallei strain K96243 challenges of multiple median lethal doses.

We describe the antiviral activity of plant lectins with specificity for different glycan structures against the severe acute respiratory syndrome coronavirus (SARS-CoV) and the feline infectious peritonitis virus (FIPV) in vitro. The SARS-CoV emerged in 2002 as an important cause of severe lower respiratory tract infection in humans, and FIPV infection causes a chronic and often fatal peritonitis in cats. A unique collection of 33 plant lectins with different specificities were evaluated. The plant lectins possessed marked antiviral properties against both coronaviruses with EC50 values in the lower microgram/ml range (middle nanomolar range), being non-toxic (CC50) at 50–100 μg/ml. The strongest anti-coronavirus activity was found predominantly among the mannose-binding lectins. In addition, a number of galactose-, N-acetylgalactosamine-, glucose-, and N-acetylglucosamine-specific plant agglutinines exhibited anti-coronaviral activity. A significant correlation (with an r-value of 0.70) between the EC50 values of the 10 mannose-specific plant lectins effective against the two coronaviruses was found. In contrast, little correlation was seen between the activity of other types of lectins. Two targets of possible antiviral intervention were identified in the replication cycle of SARS-CoV. The first target is located early in the replication cycle, most probably viral attachment, and the second target is located at the end of the infectious virus cycle.

CRISPR/Cas9 systems have been widely used in functional genomics and crop genetic improvement, but the protospacer adjacent motif (PAM) sequence NGG of Streptococcus pyogenes Cas9 (SpCas9) limits its targeting scope. To expand the targetable genomic loci, several other Cas proteins and Cas9 variants with different PAM specificities have been developed (Li et al. , 2019), such as Cpf1 (Cas12a) with T‐rich PAM, and SpCas9 VQR and VRER variants with non‐canonical NGA and NGCA PAM sequence, respectively. Recently, two engineered SpCas9 variants, xCas9 3.7 and Cas9‐NG, expand the PAM recognition site to NG and show function efficiency in mammalian cells (Hu et al. , 2018; Nishimasu et al. , 2018). Although a few tests of the two variants have been reported in plants (Ge et al. , 2019; Li et al. , 2019), there are still not enough comparative data on their efficiencies on genome and base editing in plants. Furthermore, to reduce the possible off‐target effects, it is necessary to develop a new Cas9 variant with both enhanced specificity (eCas9) and altered PAM site such as NG. The eCas9 variant has neutralization of positive charges in the nt‐groove that can remarkably decrease off‐target (Slaymaker et al. , 2016). In this study, we developed a serial of variants including a new eCas9‐NG and investigated the editing activities of those variants (xCas9, Cas9‐NG and eCas9‐NG) with expended target scope in genome editing, cytosine base editors including CBE4, xCas9n‐CBE, Cas9n‐NG‐CBE and eCas9n‐NG‐CBE, adenine base editors including ABE7.10, xCas9n‐ABE, Cas9n‐NG‐ABE and eCas9n‐NG‐ABE in transgenic rice.

Geminiviruses are a family of plant viruses with circular single‐stranded DNA genomes. They have been deconstructed by researchers for multiple biotechnological applications, including protein expression, gene silencing and genome editing, in plants (Lozano‐Durán, 2016). Under the deconstructed virus strategy, the coat protein and movement protein genes were removed from the geminiviruses, while the sequences required for replication were retained. The replicons replicate after delivery to plant cells and increase the copy number of carried DNA; this leads to high levels of gene expression (Lozano‐Durán, 2016). Although a few geminiviral replicon‐based vectors have been used in gene targeting (Baltes et al. , 2014; Cermak et al. , 2015; Wang et al. , 2017), the list of DNA replicon‐based vectors is still limited in plants, especially for food crops. In this study, we developed sweet potato leaf curl virus (SPLCV) replicon‐based expression vectors. We tested the efficiency of these vectors in CRISPR‐Cas‐mediated RNA targeting and gene editing by using Nicotiana benthamiana as model plant.