Neutralization of SARS-CoV-2 by neutralizing monoclonal antibodies of plant origin

In a recent study published in the journal Vaccinesresearchers have developed plant-based monoclonal antibodies (mAbs) against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

The coronavirus disease 2019 (COVID-19) pandemic has been wreaking havoc for more than two years and has had a negative impact on global health and economy. Although more than 11.4 billion COVID-19 vaccines have been administered worldwide so far, and despite the effectiveness of vaccines against serious diseases, the persistence of COVID-19 infections with the continued emergence of novel mutants of SARS-CoV-2 underpins the development of new therapeutic and prophylactic measures.

Monoclonal antibodies (mAbs) are biological therapeutic agents used to treat various conditions, including cancers and infectious diseases. Neutralizing monoclonal antibodies (nAbs) against SARS-CoV-2 have been isolated from convalescent individuals and humanized mice. Most catches inhibit SARS-CoV-2 infection by binding to the viral spike (S) protein, abrogating its ability to engage with the host cell receptor.

Plants could serve as an alternative system for mAb synthesis, and as eukaryotic systems, they exhibit post-translational modifications. Additionally, protein production in plants could be more affordable and safer with easier scalability than conventional methods.

Study: Potential of a Plant Made CoV-SARS-2 Neutralizing Monoclonal Antibody as a Synergistic Cocktail Component. Image Credit: SINITAR/Shutterstock

About the study

In the current study, the researchers developed two plant traps and evaluated their effectiveness. CA1 and CB6 antibody light (VL) and heavy (VH) gene variable sequences were codon matched for expression in the plant system and synthesized by integrated deoxyribonucleic acid (DNA) (IDT) technologies. These sequences were ligated to constant regions of human antibodies, cloned into an expression vector and transformed into Agrobacterium tumefaciens.

Leaves of Nicotiana benthamiana the plants were harvested seven days after agroinfiltration. Antibodies were extracted from leaves, and protein A affinity chromatography was performed. After purification, the plant-made antibodies were evaluated by western blot analysis and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE).

Temporal antibody expression was analyzed using a sandwich enzyme immunoassay (ELISA). Antibody binding to the receptor binding domain (RBD) of SARS-CoV-2 strain WA1 was determined by ELISA. A focus formation test (FFA) was performed to study the neutralizing power of the antibodies.

Results

The authors found that the antibodies in plants were very homogeneous, and their purity was comparable to that of control antibodies produced from mammalian cells. Temporal expression analysis revealed that CA1 expression reached peak levels, namely, 157.4 pg per gram fresh leaf weight (FLW), seven days after agro-infiltration. Similarly, CB6 expression peaked at 141.6 pg/g FLW after seven days. The dissociation constants (KD) were 0.04938 nM (CA1) and 0.1274 nM (CB6). Antibodies made by the plant neutralized strain WA1 with a half-maximal inhibitory concentration (IC50) of 9.29 nM for CA1 and 0.93 nM for CB6.

Additionally, the SARS-CoV-2 Delta variant was also neutralized by CB6 (IC50: 0.75 nM) and CA1 (89.87 nM). A competitive ELISA revealed that none of the antibodies made by the plants compete with the mAb CR3022 (synthesized in the hybridomas) to bind to the RBD. Moreover, no binding of CB6 to the RBD was observed when CA1 was already bound to the RBD, indicating that the two catches are in competition for the binding of the RBD. In addition, two additional hybridoma-produced mAbs (11D7 and 3C4) were used to characterize plant-produced antibodies. The researchers noted an overlapping epitope for 3C4 and CR3022. In contrast, CB6 exhibited binding to RBD already bound to 11D7 or 3C4.

Next, the synergistic potential of plant CB5 made with 11D7 and 3C4 to neutralize SARS-CoV-2 was investigated. FFAs were generated with CB6 when combined with 3C4 or 11D7, or both. Neutralization data were analyzed with Loewe and Highest Single Agent (HSA) models using SynergyFinder to predict synergy at IC20, THIS30, and CI50. The researchers noted that the predicted neutralization values ​​at any inhibitory concentration were lower than the observed neutralization, indicating neutralization synergy between CB6, 11D7 and 3C4 as a double or triple mAb cocktail.

conclusion

Researchers have shown that the expression of mAbs CA1 and CB6 reaches peak accumulation levels seven days after gene delivery in N. benthamiana, which are higher than the levels seen for previous anti-SARS-CoV-2 antibodies made from plants. Notably, CR3022 mAb, synthesized in the plant in parallel experiments, lacked neutralizing activity.

CA1 and CB6 antibodies neutralized SARS-CoV-2 WA1 strain and Delta variant. Nonetheless, the reduced neutralizing potency of CA1 against the Delta variant indicated that CA1 would likely exhibit further reduction in neutralizing activity against emerging variants. Additionally, synergistic neutralization was observed when plant-made CB6 was used in combination with other hybridoma-produced mAbs.

Journal reference:

  • Potential of a Plant Made CoV-SARS-2 Neutralizing Monoclonal Antibody as a Synergistic Cocktail Component. Jugler, C.; Sun, H.; Grill, F.; Kibler, K.; Esqueda, A.; Lai, H.; Li, Y.; Lake, D.; Chen, Q. Vaccines 2022, DOI: https://doi.org/10.3390/vaccines10050772, https://www.mdpi.com/2076-393X/10/5/772

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