Abstract
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.
Highlights
The absence of suitable infrastructure to produce vaccines for emerging viruses leaves most developing countries vulnerable and unable to respond appropriately to emerging disease threats
The native viral leader sequence was replaced with the tissue plasminogen activator leader sequence (TPA) to promote translocation of the antigen into the secretory pathway
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 (Binley et al, 2002; Margolin et al, 2020c)
Summary
The absence of suitable infrastructure to produce vaccines for emerging viruses leaves most developing countries vulnerable and unable to respond appropriately to emerging disease threats. Plant-Produced SARS-CoV-2 Spike to facilitate local production is almost entirely absent in Africa (Margolin et al, 2020a) This has culminated in an unacceptable reliance on high income countries and charitable initiatives, such as COVAX, to support the roll out of vaccines to prevent COVID-19 (Adepoju, 2021). In the absence of widespread immunity to SARS-CoV-2, uncontrolled viral circulation has given rise to novel variants which rapidly predominate in vulnerable populations (Tegally et al, 2021a,b) In the extreme, these variants may partly undermine natural immunity from previous infections or even vaccine-elicited immunity (Cele et al, 2021; Madhi et al, 2021), pre-existing immunity in either scenario still confers considerable clinical benefit. The emergence of novel viral variants adds to the burden of morbidity and mortality in resource-limited areas where vaccine coverage is limited and could potentially jeopardize global efforts to contain the pandemic if vaccine-elicited immunity does not confer adequate cross-protection (Fontanet et al, 2021)
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