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

Abstract

Summary 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.

Calreticulin co-expression supports high level production of a recombinant SARS-CoV-2 spike mimetic in Nicotiana benthamiana

“…Modification of the SARS-CoV-2 spike sequence for heterologous expression A soluble spike antigen (S∆TM), consisting of both the S1 and S2 subunits but lacking the transmembrane and cytoplasmic domains, was designed for optimal expression and processing in plants ( Figure 1A). The modified spike included an optimized cleavage sequence (RRRRRR) to promote processing by furin, as previously reported for the HIV envelope glycoprotein when produced in both plants and mammalian cells [13,18]. The synthesized sequence was also human codon optimized for dual expression in human and plant cells, resulting in a 56% GC content.…”

“…Additional constraints, such as the host chaperone machinery or differences in the plant oligosaccaryltransferase complex, may also complicate the production of, and glycosylation of, certain viral glycoproteins in planta [11][12][13]. Recently, it has been proposed that engineering the plant secretory pathway to support the production of viral glycoproteins could improve their accumulation in plants, and support their maturation during synthesis [9].…”

“…The co-expression of human chaperones has recently been reported to improve the accumulation of a range of envelope viral glycoproteins in N. benthamiana, including the HIV envelope glycoprotein and the Epstein-Barr gp350 glycoprotein, which are possibly amongst the most complex proteins produced in a plant expression system to date [13]. This approach was also combined with the expression of human furin to support proteolytic processing in planta, where this would not otherwise occur [13].…”

Prospects for SARS-CoV-2 diagnostics, therapeutics and vaccines in Africa

“…Moreover, the Biovac Institute in South Africa has the capacity for bacterial fermentation and the required infrastructure for downstream processing, and a new plant-based production facility (Cape Bio Pharms) is currently generating S1 protein derivatives as reagents. Although the spike glycoprotein is heavily glycosylated and needs to be expressed in a more complex expression host to ensure appropriate post-translational modifications, both mammalian cells and plants would be suitable to produce both spike and nucleocapsid, and novel approaches to enhance recombinant glycoprotein production in plants have also been developed in South Africa 47 .…”

“…Together with MVA and NYVAC, the avipoxvirus vectors ALVAC (attenuated canarypox virus) and FWPV are probably more realistic targets for rapid clinical development, as they have also undergone testing in humans, and ALVAC is already licensed for several veterinary applications 97 Plant-based vaccine protein production is an emerging technology that is well-suited to resource-limited areas given the capacity of the system for rapidly scalable production, the low manufacturing costs and the less sophisticated infrastructure requirements than mammalian expression systems 98 . The platform is well established to produce diverse classes of recombinant proteins, and recent advances in expression technologies and molecular engineering have also enabled improvements in glycoprotein production in plants 47,99 . Encouragingly, a preliminary pilot study suggests that these appro aches can be applied to produce the SARS-CoV-2 spike in Nicotiana benthamiana plants, warranting further testing of the recombinant antigen in preclinical vaccine immunogenicity models 100 .…”