By Dr. Maheswari RajasekaranNov 23 2021Reviewed by Benedette Cuffari, M.Sc.

Vaccines are currently the most effective prevention measure against coronavirus disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, currently available vaccines may not be very effective against emerging SARS-CoV-2 variants, such as the Delta strain (B.1.617).

Study: Production and Characterization of Nucleocapsid and RBD Cocktail Antigens of SARS-CoV-2 in Nicotiana benthamiana Plant as a Vaccine Candidate against COVID-19. Image Credit: Orpheus FX / Shutterstock.com

Background

There remains an urgent need to develop enhanced COVID-19 vaccines that are more effective against SARS-CoV-2 and its emerging variants. Most COVID-19 vaccines have been developed using the spike (S) protein of the wild-type SARS-CoV-2 strain, as it is known to contain neutralizing epitopes. However, mutations in the S1 domain of the S protein have been found to increase the transmissibility and virulence of new variants including the Delta variant.

The nucleocapsid (N) protein is a structural protein that plays an important role in ribonucleic acid (RNA) binding and replication. As the N protein is more conserved than the S protein and exhibits high immunogenicity, it may be a suitable COVID-19 vaccine target.

Earlier reports have demonstrated that the recombinant N protein produced in the plant N. tabacum can elicit an effective humoral response. Transient expression of recombinant proteins in plants is an effective and cost-effective system for the production of a high yield of functionally active recombinant proteins in a short duration of time.

The scientists in a recent Vaccines study utilized the plant N. benthamiana for producing the N-protein and an antigenic cocktail containing the N-protein and SARS-CoV-2 S receptor-binding domain (RBD). To this end, the researchers also investigated the potential of these products to potential to elicit an immune response against SARS-CoV-2.

N-protein and antigenic cocktail (N+RBD) were produced in the plant N. benthamiana

The N gene with Gen accession number YP_009724397 and the S gene of the SARS-CoV-2 variant RBD with GenBank accession MN985325 were expressed in N. benthamiana plants and were also de novo synthesized at Biomatik Corp.

The N and RBD sequences were modified further by adding a signal peptide to the N terminus of both the sequences, an ER retention signal sequence to the C-terminus, and FLAG epitope, which is an affinity purification tag to the C-terminus.

Two plasmids pEAQ-N and pEAQ-RBD were obtained by inserting the resulting N and RBD sequences into the pEAO binary expression vector. They were then transferred to the Agrobacterium AGL1 strain and subsequently infiltrated into the leaves of N. benthamiana. The N and RBD proteins were also co-expressed by infiltrating both the pEAQ-N and pEAQ-RBD plasmids. Endoglycosidase H (Endo H) is a recombinant glycosidase that will detect the N-glycosylation status of the plant-expressed N protein. The pGreen-Endo H plasmid was co-infiltrated with the AGL1 strain bearing the pEAQ-N to co-express it in N. benthamiana to assess the N-glycosylation status.

Characterization of the plant produced N-protein and N+RBD antigenic cocktail

Western blot analysis and the enzyme-linked immunosorbent assay (ELISA) were employed to determine the expression levels of the N protein to be 45 mg/kg of plant leaves. Purification using anti-FLAG affinity chromatography yielded 22 mg pure protein/kg of plant biomass.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting detected the N-protein molecule expressed in N. benthamiana as a doublet protein of with a molecular mass of about 48 and 24 kDa. Notably, both were detected by anti-FLAG antibody and by N protein-specific monoclonal antibodies (mAb). This confirms that both the 48 and 24 kDa forms show specificity in reacting with mABs and additionally the 24 kDa fragment also contains the epitopes for mAB recognition.

Co-expression of the N-protein and Endo H did not result in band shifting. Further, the glycan detection analysis performed using Pro-Q Emerald 300 glycoprotein staining did not detect the presence of glycans, thereby confirming that the N protein produced in N. benthamiana is not N-glycosylated.

Antigen cocktails were produced by infiltrating agrobacterium bearing the N protein-expressing plasmid pEAQ-N with RBD expressing plasmid pEAQ-RBD into plant leaves. The N protein and the cocktail protein N+RBD were purified using single-step anti-FLAG affinity chromatography.

SDS-PAGE and Western blot analysis of the antigenic cocktail revealed the presence of three proteins with a molecular mass of 48kDa (N protein), 36 kDa (which corresponds to glycosylated (g) RBD), and 24 kDa (N protein).

Gel filtration chromatography of the anti-FLAG affinity chromatography-purified N and N + gRBD protein additionally confirmed the presence of a double peak in the N-protein elute and the presence of triple peaks in the N+ gRBD elute. SDS-PAGE analysis of the eluted fractions showed the presence of a 48kDa full-length N-protein and in the antigen cocktail presence of 48kDa (N protein) and 36 kDa (gRBD) proteins.

The stability analysis of the 48 kDa full-length plant-produced N protein was performed by incubating it at 37 °C for 24, 48, 96, 72, and 96 hours. Incubation at 37 °C for 48 hours resulted in less than 50% degradation of the N protein. When stored at -80 °C for a six month period, the absence of degradation was observed.

Antigenic cocktail N+RBD elicited a strong immune response in mice

Immunogenicity of the plant-produced N-protein and the antigenic cocktail (N+ RBD) was tested in Balb/c male mice that were 6–7 weeks old. Mice were given intramuscular injections of 5 micrograms (mg) of N or N+ RBD adsorbed to 0.3% Alhydrogel on day 0 and day 21.

ELISA was performed on the mouse sera from the immunized mice that were collected on days 21 and 42. At a dose of 5 mg, both N and N+ RBD induced significantly high antibody titers in the immunized mice. Notably, the antigenic cocktail N+RBD elicited higher titers of antibodies when compared to the RBD or N proteins alone.

N+ gRBD exhibited neutralization activity against live SARS-CoV-2 infection in Vero E6 cells

The microneutralization test was also performed to assess the anti-SARS-CoV-2 neutralization activity of the sera of mice immunized with plant-produced and anti-FLAG affinity chromatography-purified N and N+gRBD. The neutralization activity of the mice sera against live SARS-CoV-2 infection in Vero E6 cells was evaluated.

Mice were treated with 5 μg of plant-produced N protein adsorbed to Alhydrogel. Sera were collected from these mice at day 21 after the first dose and at day 42 after the second dose and assessed for neutralizing activity. It was found that the sera collected from these mice at both time points did not exhibit neutralizing activity.

Similarly, mice were immunized with 5 μg of plant-produced N + gRBD adsorbed to Alhydrogel. Interestingly, sera collected from these mice at day 21 after the first immunization and day 42 after the second immunization exhibited neutralization activity at 1:8 and 1:128, respectively, against SARS-CoV-2.

Further, sera from mice that were administered 5 μg of plant-produced gRBD alone collected on day 42 after the second immunization exhibited neutralization activity at 1:128. A similar neutralization activity at 1:128 was shown by sera collected from mice immunized with about 2.3 μg of gRBD in combination with about 2.7 μg of N protein, which amounts to 5 μg. This suggests that the N protein may play a role in enhancing the neutralizing activity against SARS-CoV-2 in the N+gRBD cocktail antigen.

Conclusion

The emergence of new SARS-CoV-2 variants creates a need for developing new and enhanced versions of SARS-CoV-2 vaccines. The plant-produced antigen cocktail (N+RBD) that was developed in this study induced high titer antibodies that were found to exhibit potent neutralizing activity against SARS-CoV-2.

The vaccine discussed here has the potential to be developed as a future COVID-19 vaccine candidate; however, it must be validated through further investigations. Further, the N or N+RBD antigens produced in plants may also be used as diagnostic tools in SARS-CoV-2 serological tests for COVID-19 patients.