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

Abstract

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

Rapid expression of COVID-19 proteins by transient expression in tobacco

“…Other recent reports (Diego-Martin et al, 2020;Makatsa et al, 2020;Margolin et al, 2020;Rattanapisit et al, 2020) indicate similar results. The rapid and high level of expression makes this an attractive system for larger scale production for research, or development of tests or of vaccines.…”

“…We successfully purified recombinant spike and Nucleocapsid SARS-CoV-2 proteins from N. benthamiana at a concentration comparable to other studies (Diego-Martin et al, 2020;Makatsa et al, 2020;Margolin et al, 2020;Rattanapisit et al, 2020). The spike ectodomain was purified in both monomeric and trimeric forms, suggesting that it was folding properly.…”

“…We were surprised that a tobacco codon optimized spike construct expressed at lower levels than the mammalian codon optimized version, but several other reports expressing mammalian proteins in tobacco have yielded similar results (Maclean et al, 2007;Margolin et al, 2020;Mortimer et al, 2012). In contrast, however, the tobacco codon-optimized RBD construct produced more protein than the mammalian codon-optimized version.…”

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

“…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 approaches 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 . Three leading plant biotechnology companies, Medicago Inc. (Canada), Ibio Inc. (USA) and Kentucky BioProcessing Inc. (USA), have already announced the successful production of candidate virus-like particle vaccines against SARS-CoV-2.…”

N-Glycosylation of the SARS-CoV-2 Receptor Binding Domain Is Important for Functional Expression in Plants

“…CRT, on the other hand, did not associate with the SARS-CoV spike protein revealing different requirements of viral glycoproteins for lectin chaperone-mediated protein folding. In N. benthamiana , overexpression of human CRT increased the overall yield of several recombinant viral glycoproteins, including HIV-1 Env ( Margolin et al, 2020a ) and the ectodomain of the SARS-CoV-2 spike protein ( Margolin et al, 2020c ) and attenuated ER stress responses associated with viral glycoprotein expression. These findings underscore the importance of N-glycosylation and N-glycan-dependent quality control processes for recombinant protein production and reveal limitations that have to be addressed to make plant-based expression platforms such as N. benthamiana more attractive for economic production ( Dicker and Strasser, 2015 ).…”