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.

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.

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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.

Heterologous glycoprotein production relies on host glycosylation‐dependent folding. When the biosynthetic machinery differs from the usual expression host, there is scope to remodel the assembly pathway to enhance glycoprotein production. Here we explore the integration of chaperone coexpression with glyco‐engineering to improve the production of a model HIV‐1 envelope antigen. Calreticulin was coexpressed to support protein folding together with Leishmania major STT3D oligosaccharyltransferase, to improve glycan occupancy, RNA interference to suppress the formation of truncated glycans, and Nicotiana benthamiana plants lacking α1,3‐fucosyltransferase and β1,2‐xylosyltransferase was used as an expression host to prevent plant‐specific complex N‐glycans forming. This approach reduced the formation of undesired aggregates, which predominated in the absence of glyco‐engineering. The resulting antigen also exhibited increased glycan occupancy, albeit to a slightly lower level than the equivalent mammalian cell‐produced protein. The antigen was decorated almost exclusively with oligomannose glycans, which were less processed compared with the mammalian protein. Immunized rabbits developed comparable immune responses to the plant‐produced and mammalian cell‐derived antigens, including the induction of autologous neutralizing antibodies when the proteins were used to boost DNA and modified vaccinia Ankara virus‐vectored vaccines. This study demonstrates that engineering glycosylation‐directed folding offers a promising route to enhance the production of complex viral glycoproteins in plants.

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15An effective prophylactic vaccine is urgently needed to protect against SARS-CoV-2 16 infection. The viral spike, which mediates entry into cells by interacting with the host 17 angiotensin-converting enzyme 2, is the primary target of most vaccines in development. 18These vaccines aim to elicit protective immunity against the glycoprotein by use of 19 inactivated virus, vector-mediated delivery of the antigen in vivo, or by direct immunization 20 with the purified antigen following expression in a heterologous system. These approaches 21 are mostly dependent on the growth of mammalian or insect cells, which requires a 22 mammalian cells despite including a modified site for improved processing [17]. Co-126 expression of furin did however improve processing, yielding the expected cleavage product, 127 which increased in intensity with higher levels of furin expression. 128 129 130 131 132 133 134 135 136 137 138 139

The crystal structure and polymorphism of the well-known aminoazobenzene, N,N-dimethyl-4-aminoazobenzene (DAB) are studied; a second polymorph of DAB reported and the relationship between the two polymorphs studied using differential scanning calorimetry and by evaluating their lattice energies and absorption spectra. Without significantly strong intermolecular interactions present in the two forms of DAB, a balance between conformational strain and a cumulative π-orbital overlapping effect results in the existence of the enantiotropically related polymorphs and their alternative packing arrangements. The UV/vis spectra of the two polymorphs are different, thus illustrating the significance of small structural changes on the colour of a material, which could behave differently under particular sensing conditions. DAB has been used primarily as an acid/base indicator solution and a dye molecule and the implications of the polymorphic relationships established here are discussed with respect to the range of applications this molecule finds as a sensor and for its incorporation into various functional films and devices