Effect of leaf position and days post-infiltration on transient expression of colorectal cancer vaccine candidate proteins GA733-Fc and GA733-FcK in Nicotiana benthamiana plant.

Immunization with thetumor-associated antigen GA733 glycoprotein, which is highly expressed in colorectal cancer, is considered to be a promising strategy for cancer prevention and treatment. We cloned a fusion gene of GA733 and immunoglobulin Fc fragment (GA733-Fc), and that of GA733-Fc and an endoplasmic reticulum retention motif (GA733-FcK) into the Cowpea mosaic virus (CPMV)-based transient plant expression vector, pEAQ-HT. Agrobacterium tumefaciens (LBA4404) transformed with the vectors pEAQ-HT-GA733-Fc and pEAQ-HT-GA733-FcK was infiltrated into the leaves of Nicotiana benthamiana plants. To optimize harvesting of leaf to express therapeutic glycoproteins both spatially and temporally, protein expression levels at various leaf positions (top, middle, and base) and days post-infiltration (dpi) were investigated. The GA733-Fc and GA733-FcK genes were detected in leaves at 1-10 dpi using PCR. As assessed by western blot, GA733-Fc and GA733-FcK were expressed at the highest levels in the top leaf position at 5 dpi, and GA733-FcK was expressed more than GA733-Fc. The proteins were successfully purified from infiltrated N. benthamiana leaves using protein A affinity chromatography. ELISA verified that an anti-GA733 antibody recognized both purified proteins. Thus, a functional GA733-Fc colorectal cancer vaccine protein can be transiently expressed using a CPMV virus-based vector, with an optimized expression time and leaf position post-infiltration.

Plant-made dengue virus-like particles produced by co-expression of structural and non-structural proteins induce a humoral immune response in mice.

Dengue virus (DENV) is an emerging threat causing an estimated 390 million infections per year. Dengvaxia, the only licensed vaccine, may not be adequately safe in young and seronegative patients; hence, development of a safer, more effective vaccine is of great public health interest. Virus-like particles (VLPs) are a safe and very efficient vaccine strategy, and DENV VLPs have been produced in various expression systems. Here, we describe the production of DENV VLPs in Nicotiana benthamiana using transient expression. The co-expression of DENV structural proteins (SP) and a truncated version of the non-structural proteins (NSPs), lacking NS5 that contains the RNA-dependent RNA polymerase, led to the assembly of DENV VLPs in plants. These VLPs were comparable in appearance and size to VLPs produced in mammalian cells. Contrary to data from other expression systems, expression of the protein complex prM-E was not successful, and strategies used in other expression systems to improve the VLP yield did not result in increased yields in plants but, rather, increased purification difficulties. Immunogenicity assays in BALB/c mice revealed that plant-made DENV1-SP + NSP VLPs led to a higher antibody response in mice compared with DENV-E domain III displayed inside bluetongue virus core-like particles and a DENV-E domain III subunit. These results are consistent with the idea that VLPs could be the optimal approach to creating candidate vaccines against enveloped viruses.

Covalent protein display on Hepatitis B core-like particles in plants through the in vivo use of the SpyTag/SpyCatcher system.

Virus-like particles (VLPs) can be used as nano-carriers and antigen-display systems in vaccine development and therapeutic applications. Conjugation of peptides or whole proteins to VLPs can be achieved using different methods such as the SpyTag/SpyCatcher system. Here we investigate the conjugation of tandem Hepatitis B core (tHBcAg) VLPs and the model antigen GFP in vivo in Nicotiana benthamiana. We show that tHBcAg VLPs could be successfully conjugated with GFP in the cytosol and ER without altering VLP formation or GFP fluorescence. Conjugation in the cytosol was more efficient when SpyCatcher was displayed on tHBcAg VLPs instead of being fused to GFP. This effect was even more obvious in the ER, showing that it is optimal to display SpyCatcher on the tHBcAg VLPs and SpyTag on the binding partner. To test transferability of the GFP results to other antigens, we successfully conjugated tHBcAg VLPs to the HIV capsid protein P24 in the cytosol. This work presents an efficient strategy which can lead to time and cost saving post-translational, covalent conjugation of recombinant proteins in plants.

Co-expression of human calreticulin significantly improves the production of HIV gp140 and other viral glycoproteins in plants.

Plant molecular farming (PMF) is rapidly gaining traction as a viable alternative to the currently accepted paradigm of producing biologics. While the platform is potentially cheaper and more scalable than conventional manufacturing systems, expression yields and appropriate post-translational modifications along the plant secretory pathway remain a challenge for certain proteins. Viral fusion glycoproteins in particular are often expressed at low yields in plants and, in some cases, may not be appropriately processed. Recently, however, transiently or stably engineering the host plant has shown promise as a strategy for producing heterologous proteins with more complex maturation requirements. In this study we investigated the co-expression of a suite of human chaperones to improve the production of a human immunodeficiency virus (HIV) type 1 soluble gp140 vaccine candidate in Nicotiana benthamiana plants. The co-expression of calreticulin (CRT) resulted in a dramatic increase in Env expression and ameliorated the endoplasmic reticulum (ER) stress response - as evidenced by lower transcript abundance of representative stress-responsive genes. The co-expression of CRT similarly improved accumulation of glycoproteins from Epstein-Barr virus (EBV), Rift Valley fever virus (RVFV) and chikungunya virus (CHIKV), suggesting that the endogenous chaperone machinery may impose a bottleneck for their production. We subsequently successfully combined the co-expression of human CRT with the transient expression of human furin, to enable the production of an appropriately cleaved HIV gp140 antigen. These transient plant host engineering strategies are a promising approach for the production of high yields of appropriately processed and cleaved viral glycoproteins.

Expression of CphB- and CphE-type cyanophycinases in cyanophycin-producing tobacco and comparison of their ability to degrade cyanophycin in plant and plant extracts.

Increasing the arginine (Arg) content in plants used as feed or food is of interest, since the supplementation of food with conditionally essential Arg has been shown to have nutritional benefits. An increase was achieved by the expression of the Arg-rich bacterial storage component, cyanophycin (CGP), in the chloroplast of transgenic plants. CGP is stable in plants and its degradation into ß-aspartic acid (Asp)-Arg dipeptides, is solely catalyzed by bacterial cyanophycinases (CGPase). Dipeptides can be absorbed by animals even more efficiently than free amino acids (Matthews and Adibi 1976; Wenzel et al. 2001). The simultaneous production of CGP and CGPase in plants could be a source of ß-Asp-Arg dipeptides if CGP degradation can be prevented in planta or if dipeptides are stable in the plants. We have shown for the first time that it is possible to co-express CGP and CGPase in the same plant without substrate degradation in planta by transient expression of the cyanobacterial CGPase CPHB (either in the plastid or cytosol), and the non-cyanobacterial CGPase CPHE (cytosol) in CGP-producing Nicotiana tabacum plants. We compared their ability to degrade CGP in planta and in crude plant extracts. No CGP degradation appeared prior to cell homogenization independent of the CGPase produced. In crude plant extracts, only cytosolic CPHE led to a fast degradation of CGP. CPHE also showed higher stability and in vitro activity compared to both CPHB variants. This work is the next step to increase Arg in forage plants using a stable, Arg-rich storage protein.

Stable production of cyanophycinase in Nicotiana benthamiana and its functionality to hydrolyse cyanophycin in the murine intestine.

Food supplementation with the conditionally essential amino acid arginine (Arg) has been shown to have nutritional benefits. Degradation of cyanophycin (CGP), a peptide polymer used for nitrogen storage by cyanobacteria, requires cyanophycinase (CGPase) and results in the release of ß-aspartic acid (Asp)-Arg dipeptides. The simultaneous production of CGP and CGPase in plants could be a convenient source of Arg dipeptides. Different variants of the cphB coding region from Thermosynechococcus elongatus BP-1 were transiently expressed in Nicotiana benthamiana plants. Translation and enzyme stability were optimized to produce high amounts of active CGPase. Protein stability was increased by the translational fusion of CGPase to the green fluorescent protein (GFP) or to the transit peptide of the small subunit of RuBisCO for peptide production in the chloroplasts. Studies in mice showed that plant-expressed CGP fed in combination with plant-made CGPase was hydrolysed in the intestine, and high levels of ß-Asp-Arg dipeptides were found in plasma, demonstrating dipeptide absorption. However, the lack of an increase in Asp and Arg or its metabolite ornithine in plasma suggests that Arg from CGP was not bioavailable in this mouse group. Intestinal degradation of CGP by CGPase led to low intestinal CGP content 4 h after consumption, but after ingestion of CGP alone, high CGP concentrations remained in the large intestine; this indicated that intact CGP was transported from the small to the large intestine and that CGP was resistant to colonic microbes.

Tobacco as platform for a commercial production of cyanophycin.

Cyanophycin (CP) is a proteinogenic polymer that can be substituted for petroleum in the production of plastic compounds and can also serve as a source of valuable dietary supplements. However, because there is no economically feasible system for large-scale industrial production, its application is limited. In order to develop a low-input system, CP-synthesis was established in the two commercial Nicotiana tabacum (N. tabacum) cultivars 'Badischer Geudertheimer' (BG) and 'Virginia Golta' (VG), by introducing the cyanophycin-synthetase gene from Thermosynecchococcus elongatus BP-1 (CphATe) either via crossbreeding with transgenic N. tabacum cv. Petit Havana SR1 (PH) T2 individual 51-3-2 or by agrobacterium-mediated transformation. Both in F1 hybrids (max. 9.4% CP/DW) and T0 transformants (max. 8.8% CP/DW), a substantial increase in CP content was achieved in leaf tissue, compared to a maximum of 1.7% CP/DW in PH T0 transformants of Hühns et al. (2008). In BG CP, yields were homogenous and there was no substantial difference in the variation of the CP content between primary transformants (T0), clones of T0 individuals, T1 siblings and F1 siblings of hybrids. Therefore, BG meets the requirements for establishing a master seed bank for continuous and reliable CP-production. In addition, it was shown that the polymer is not only stable in planta but also during silage, which simplifies storage of the harvest prior to isolation of CP.