Rice-derived SARS-CoV-2 glycoprotein S1 subunit vaccine elicits humoral and cellular immune responses

Abstract

Since 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19, has been spreading and mutating globally despite the expedited approval of many commercial vaccines. Therefore, developing safe, effective and affordable vaccines remains essential to meet the global demand, particularly in developing countries. Transgenic plants have emerged as a promising platform to express recombinant proteins for pharmaceutical and vaccine applications. Two binary vectors, pCAMBIA1300Gt1-S1 and pCAMBIA1300Actin-S1, containing distinct promoters, were constructed and transformed into rice via Agrobacterium. Overall, 56 independent transgenic rice lines were regenerated. Expression analysis revealed that the rice-derived S1 (rS1) protein could be expressed in pGt1::S1 transgenic rice seeds. rS1 protein expression levels reached up to 282 μg/g dry weight, with S1 gene insertion having no effect on grain size and weight. The rS1 protein exhibited a high affinity for human angiotensin-converting enzyme 2 (ACE2) in vitro. Moreover, the immunogenicity of purified rS1 protein co-administered with various adjuvants demonstrated that mice vaccinated with Alum-adjuvant rS1 generated enhanced humoral immune responses with high serum IgG, IgG1 and neutralizing antibody levels. Salmonella Typhimurium flagellin (FliC)-adjuvanted rS1 elicited stronger S1-specific IgG2a levels, promoted splenocyte proliferation and induced mixed Th1/Th2/Th17 cytokine responses. This was evidenced by increased proportions of antigen-specific interferon (IFN)-γ, interleukin-4 (IL-4) and IL-17A-positive CD4⁺ T lymphocytes, suggesting its potential to induce both humoral and cellular immune responses. These findings suggest that rS1 protein offers a promising approach for affordable COVID-19 subunit vaccine production, and this strategy can be universally applied to other viral vaccines.

Keywords: SARS‐CoV‐2; humoral and cellular immune responses; immunogenicity; rice‐derived S1 protein; subunit vaccine; transgenic rice.

Figure 1

Genetic construction and detection of rS1 protein. (a) Schematic diagram of the recombinant S1 antigen design. Gt1_pro: Gt1 Promoter, a rice seed storage protein glutelin gene promoter; Kozak sequence: Sequence enhancing translation initiation; Gt1_sp: Gt1 Signal peptide; Codon‐optimized S1 gene: Sequence adapted for expression in rice; KDEL: Endoplasmic reticulum retention signal; NOS_ter: Nopaline synthase gene terminator; Act_pro: Actin promoter, a constitutive promoter. (b) Expression of the rS1 protein in T₂ transgenic rice seeds. Total protein from T₂ generation transgenic rice seeds was extracted for rS1 protein identification using Western blot analysis. Hsp protein was used as the internal control for gene expression normalization. M: Protein marker; WT: Non‐transgenic control. (c) N‐glycosylation modification of the rS1 protein. M: Protein marker; 1: T₂‐2 Transgenic rice seed extract; 2: T₂‐2 Transgenic rice seed extract treated with PNGase F. (d) Mass spectrometry analysis of the rS1 protein. Matched peptides in the S1 protein are highlighted in Bold Red. (e) Representative diagram of homozygous seeds from T₂ transgenic rice.

Figure 2

Quantification and purification of rS1 protein. Developing seeds (T₃ generation) were collected 15 days after flowering (DAF) to analyse the transcription levels of the S1 gene. Total protein was extracted from mature T₃ generation rice seeds and the rS1 protein concentration in the extract was measured using a quantitative Western blot assay. (a) mRNA levels of the S1 gene in different transgenic lines and the non‐transgenic control (WT). (b) rS1 concentration in T₃ generation transgenic rice seeds. (c) Thousand‐grain weight (TGW) of T₃ generation transgenic rice seeds and the WT. (d) SDS‐PAGE of purified rS1 protein. (e) Western blot results of purified rS1 protein. M: Protein marker; 1: Extract from T₃ transgenic seeds; 2–3: Purified rS1 protein; 4: Extract from non‐transgenic control (WT). (f) ACE2 binding activity of the S1 proteins. rS1 (glyc): purified rS1 protein; rS1 (deglyc): rS1 protein treatment with PNGase F; S1 (HEK293): S1 protein expressed in HEK293 cells.

Figure 3

Humoral immune responses induced by rS1 protein. (a) Schematic illustration of the immunization and sampling procedure. Mice were intramuscularly immunized with three doses of rS1, rS1 + FliC, rS1 + Alum or PBS. Serum samples were collected on day 12 after the second and third immunisations to analyse antibody immune responses. (b) S1‐specific IgG titres in the serum on day 12 after the second and third immunisations. (c) S1‐specific IgG subtypes (IgG1, IgG2a) titres in serum on day 12 after the third immunization. (d) Neutralizing antibody titres in serum on day 12 after the third immunization. S1‐specific IgG, IgG1 and IgG2a titres were analysed using log10‐transformed data. Neutralizing antibody titres were analysed using log2‐transformed data. The data were presented as mean ± SEM from six mice per group. *P < 0.05, **P < 0.01 and ***P < 0.001 were considered statistically significant.

Figure 4

Cellular immune responses induced by rS1 protein. Mice were intramuscularly immunized with three doses of rS1, rS1 + FliC, rS1 + Alum or PBS. Spleens were harvested 2 weeks after the last vaccination and splenocytes were isolated and treated using 5 μg/mL of rS1 protein to assess cellular immune responses. (a) Expression levels of Th1, Th2 and Th17 cytokines (IFN‐γ, TNF‐α, IL‐4, IL‐10 and IL‐17A) measured using qRT‐PCR. (b) Secretion levels of Th1, Th2 and Th17 cytokines (IFN‐γ, TNF‐α, IL‐4, IL‐10 and IL‐17A) in cell supernatants measured using ELISA. (c) Splenocyte proliferation measured using a BrdU‐based ELISA kit. (d) IFN‐γ and IL‐4 secretion by splenic lymphocytes stimulated with S1 determined using ELISpot. The data were presented as mean ± SEM from six mice per group. *P < 0.05, **P < 0.01 and ***P < 0.001 were considered statistically significant.

Figure 5

Cytokine‐producing CD4⁺ and CD8⁺ T cell responses. Splenocytes from vaccinated mice were harvested 2 weeks after last immunization and restimulated with rS1 protein for 6 h. Cytokine‐producing CD4⁺ and CD8⁺ T cell responses were detected by intracellular staining and quantified by flow cytometry. (a) IFN‐γ⁺, IL‐4⁺ and IL‐17A⁺ CD4⁺ T cells quantified within the CD4⁺ T cell population. (b) IFN‐γ⁺, IL‐4⁺ and IL‐17A⁺ CD8⁺ T cells quantified within the CD8⁺ T cell population. The results were expressed as the percentage of positive cells. The data were presented as mean ± SEM from six mice per group. *P < 0.05, **P < 0.01 and ***P < 0.001 were considered statistically significant.