Tomato-made edible COVID-19 vaccine TOMAVAC induces neutralizing IgGs in the blood sera of mice and humans

Abstract

Plant-based edible vaccines that provide two-layered protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outweigh the currently used parenteral types of vaccines, which predominantly cause a systemic immune response. Here, we engineered and selected a transgenic tomato genotype (TOMAVAC) that stably synthesized an antigenic S1 protein of SARS-CoV-2. Two-course spaced force-feeding of mice with ≈5.4 μg/ml TOMAVAC increased up to 16-fold the synthesis of RBD-specific NAbs in blood serum and the significant induction of S-IgA in intestinal lavage fluid. In a surrogate virus neutralization test, TOMAVAC-induced NAbs had 15-25% viral neutralizing activity. The results suggested early evidence of the immunogenicity and protectivity of TOMAVAC against the coronavirus disease 2019 (COVID-19) infection. Furthermore, we observed a positive trend of statistically significant 1.2-fold (average of +42.28 BAU/ml) weekly increase in NAbs in the volunteers' serum relative to the initial day. No severe side effects were observed, preliminarily supporting the safety of TOMAVAC. With the completion of future large-scale studies, higher-generation TOMAVAC should be a cost-effective, ecologically friendly, and widely applicable novel-generation COVID-19 vaccine, providing two-layered protection against SARS-CoV-2.

Keywords: Edible vaccine; RBD-specific neutralizing antibodies; SARS-CoV-2; efficiency; safety; viral neutralizing activity.

Figure 1

The S1 gene sequence of SARS-CoV-2 was used to construct a binary vector: (A) sequence alignment and (B) a scheme of vector construction used for tomato transformation.

Figure 2

Molecular evaluation of transformed tomatoes. (A) PCR analysis: M – GeneRuler 50 bp DNA Ladder (Thermo Fisher, Waltham, MA, United States); 1–8 – transformed tomato plant DNA; Pl – S1 plasmid DNA (a positive control); Mix – Master mix without DNA (a negative control); C – a negative untransformed tomato plant DNA control. (B, G) Western blot quantification of expressed protein in transgenic tomatoes. RBD protein (ZF-UZ-VAC200123,28 with 61.7 kDa) and untransformed tomatoes were used as positive and negative controls (C, E) Relative expression analysis of transformed T1-generation tomato plants and tissues. The untransformed tomato was used as a negative control. (D, F) ELISA analysis of transformed T₁-generation tomato plants: leaf tissue (1), unripened green tomato (2), ripened red tomato (3), and control tissues from untransformed tomato (2, 4, and 6). Different letters designate significant differences at the p ≤ 0.05 level of one-way ANOVA or Kruskal-Wallis one-way nonparametric ANOVA (Supplementary Tables 1, 3), with similar letters reporting nonsignificant differences. Refer to Supplementary Figures S1–S3 for the original gel images for (A, B, G), cropped for presentation purposes.

Figure 3

Titer and neutralizing activity of RBD-specific serum IgG in mice after oral vaccination. (A) – animal experiment scheme; (B) – titer on day 14 postvaccination; (C) – titer on day 28 postvaccination; (D) – titer on day 42 postvaccination; (E) – titer on day 56 postvaccination; (F) – the neutralizing activity of NAbs on day 42 postvaccination; and (G) – neutralizing activity on day 56 postvaccination. Statistical significance level (**–≤0.01, and ***–≤0.001) from ordinary one-way ANOVA; Kruskal–Wallis (KW) one-way ANOVA, KW*–≤0.05, KW**–≤0.01, KW***–≤0.001, Supplementary Table 7).

Figure 4

Titer of RBD-specific mucosal S-IgA in mice after oral vaccination. (A) – titer on day 14 post-vaccination; (B) – titer on day 28 post-vaccination; (C) – titer on day 42 post-vaccination; (D) – titer on day 56 post-vaccination. Statistical significance level (*–≤0.05, **–≤0.01, ***–≤0.001 and ****–≤0.0001) from ordinary one-way ANOVA; Kruskal-Wallis (KW) one-way ANOVA, KW*–≤0.05, KW**–≤0.01, KW***–≤0.001, Supplementary Table 8).