SARS-CoV-2 Spike Protein-Induced Interleukin 6 Signaling Is Blocked by a Plant-Produced Anti-Interleukin 6 Receptor Monoclonal Antibody

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, has caused more than 4.5 million deaths worldwide. Severe and fatal cases of COVID-19 are often associated with increased proinflammatory cytokine levels including interleukin 6 (IL-6) and acute respiratory distress syndrome. In this study, we explored the feasibility of using plants to produce an anti-IL-6 receptor (IL-6R) monoclonal antibody (mAb) and examined its utility in reducing IL-6 signaling in an in vitro model, which simulates IL-6 induction during SARS-CoV-2 infection. The anti-IL6R mAb (IL6RmAb) was quickly expressed and correctly assembled in Nicotiana benthamiana leaves. Plant-produced IL6RmAb (pIL6RmAb) could be enriched to homogeneity by a simple purification scheme. Furthermore, pIL6RmAb was shown to effectively inhibit IL-6 signaling in a cell-based model system. Notably, pIL6RmAb also suppressed IL-6 signaling that was induced by the exposure of human peripheral blood mononuclear cells to the spike protein of SARS-CoV-2. This is the first report of a plant-made anti-IL-6R mAb and its activity against SARS-CoV-2-related cytokine signaling. This study demonstrates the capacity of plants for producing functionally active mAbs that block cytokine signaling and implies their potential efficacy to curb cytokine storm in COVID-19 patients.

Potential for a Plant-Made SARS-CoV-2 Neutralizing Monoclonal Antibody as a Synergetic Cocktail Component

“…The variable heavy (VH) and variable light (VL) gene sequences for CA1 and CB6 [ 15 ] were codon-adapted for plant-based expression using GeneDesigner 2.0 and synthesized by Integrated DNA Technologies (IDT). The VH and VL sequences were ligated to human constant regions (kappa for the light chain and gamma for the heavy chain), cloned into a geminivirus-based plant expression vector, and transformed into Agrobacterium tumefaciens for agroinfiltration, as described previously [ 41 ]. Six-week-old Nicotiana benthamiana plants were agroinfiltrated and leaves were harvested at peak recombinant protein expression (7 days post infiltration).…”

Humanization and Expression of IgG and IgM Antibodies in Plants as Potential Diagnostic Reagents for Valley Fever

“…Primers were designed to add a plant-specific secretion signal peptide to murine variable regions of the LC and HC clones. The resulting PCR products were then digested and ligated onto a human kappa chain backbone, along with a human IgG 1 28 and a human mu chain backbone 17 , respectively, before being cloned into a plant expression vector as described 29 . Positive clones containing the human chimeric IgG and IgM constructs were transformed in Agrobacterium tumefaciens and verified by PCR.…”

“…N. benthamiana leaves expressing either the humanized 4H2 IgG or IgM were harvested and homogenized in the same extraction buffer described above for temporal analysis. The protein extract containing IgG or IgM was processed as described 29,31 , followed by Protein A (Cytiva) or human anti-mu chain (Sigma-Aldrich, Cat. No.…”

“…Western blots were performed as described previously 29 . In brief, to validate the plant-made mAbs identity, plant-made 4H2 IgG (P-4H2 IgG) or plant-made 4H2 IgM (P-4H2 IgM), were subjected to SDS-PAGE under reducing and non-reducing conditions and transferred onto polyvinylidene difluoride (PVDF, Bio-Rad) membranes.…”

A Comprehensive Review of Drug Repurposing Strategies against Known Drug Targets of COVID-19

“…The SARS-CoV-2 CPE is a 72 h assay that measures the phenotypic consequence of viral infection and cell replication [42]. SARS-CoV-2 induces cell death after 48 to 72 h of infection, and thus cell viability is an indirect measure of viral replication in vitro.…”