TOMAVAC plant development
A binary pART27 vector bearing the custom synthesised S1 gene (Fig. 1) of SARS-CoV-2, driven by CaMV 35S viral promoter, was constructed and transformed into A. tumefaciens strain LB440424. In fruit transformation of tomatoes (Solanum lycopersicum cv. Bella-Rossa) was carried out by modified methods25,26. Transgenic plant development and characterisation, genomic DNA isolation, PCR and qPCR analyses, including relative expression and qPCR-based transgene copy number identification, were carried out as described in our previous work27, optimising for tomatoes. S1-specific protein synthesis in transgenic plants was determined using Western blot and ELISA (Supplementary methods).
TOMAVAC feeding in mice
Seventy-five (n=75) 20-day-old outbred ICR CD-1 were kept in plastic cages and fed between 7-8 am and 4-5 pm daily on a standard diet (Supplementary methods) adaptation. After serum NAbs analysis and initial verification of the non-infected status of the mice (sacrificing n=3), 72 healthy mice (n=72) were divided into three equal groups (n=24/group, males and females). Groups were 1) the TOMAVAC group, force-fed with 1 ml homogenate of transformation event 4; 2) the untransformed tomato group, force-fed 1 ml homogenate of original cv. Bella Rossa®; and 3) a control group fed only with a standard diet. Seven days-spaced two-dose force-feeding with tomatoes was performed using a syringe before every morning feeding (Fig.3a). For the analyses of NAbs, blood samples were taken on day 14 (n=18, six mice/group), day 28 (n=18, six mice/group), day 42 (n=18, six mice/group), and day 56 (n=12, four mice/group). Mice experiments were conducted with an ethics board approval (No3/30062022) and in accordance with the US National Institutes of Health Guidelines for the Care and Use of Laboratory Animals28. Animal experiments were conducted at the Drug Standardisation Scientific Centre, Pharmaceutical Institute, Tashkent, Uzbekistan (Supplementary Protocol).
Determination of NAbs titre and their neutralising ability
The RBD-specific serum IgG was assessed in three different groups using the SARS-CoV-2 (2019-nCoV) Spike RBD Antibody Titre Assay Kit (Mouse) according to the manufacturer's instructions (Krishgen Biosystems, Cerritos, California, USA). The neutralising activity of RBD-specific antibodies was determined in blood serum on days 42 and 56 post-vaccination using the SARS-CoV-2 surrogate Virus Neutralization Test (sVNT) Kit according to the manufacturer's instructions (GenScript, Piscataway, New Jersey, USA).
Proof-of-concept human consumption study
A volunteer group of 14 healthy adults 30-52 years of age has given written consent to participate in human consumption, a proof-of-concept experiment. After the ethical committee approved the study (No4/07072002), volunteers were asked to be randomly grouped into two groups: 1) invited to consume TOMAVAC (n=7) before dining and 2) keeping usual everyday dining (n=7). Both groups were instructed not to have any other vaccination during the experiments (Supplementary Protocol). All procedures were explained clearly to all participants, and their approval of using collected data for “research purposes” was obtained. Participants were informed about blood test results but de-identified for reporting purposes.
Briefly, the experimental group consumed 50 g of TOMAVAC on an empty stomach once a day for three consecutive days, 20-30 minutes before dining. Blood samples were taken from all participants before consumption (day 0) and on days 7, 14, and 21. The NAbs levels were tested at the Institute of Immunology and Human Genomics, Uzbekistan, using the automatic chemiluminescence immunoassay analyser MAGLUMI series, according to the manufacturer's instructions for human sera (Snibe Diagnostic, Pingshan, China).
Statistical analysis
Expression and ELISA data in tomatoes were analysed using ordinary one-way ANOVA with Turkey’s multiple comparisons. For mice data, after checking the normal distribution using the Shapiro-Wilk test or data normalisation using Log10 transformation, ordinary one-way ANOVA analysis was used with recommended Turkey’s multiple comparisons. Non-parametric Kruskal Wallis (KW) one-way ANOVA was also applied to determine abnormal data's statistical significance and seek additional statistical evidence to judge ordinary ANOVA findings. In the human study, due to data abnormality, the nonparametric Friedman’s ANOVA test, followed by Dunn’s multiple comparisons, was performed comparing the initial NAbs mean ranks to the repeated measurement mean ranks of NAbs from days 7, 14 and 21. Data analysis and visualisation were performed using GraphPad Prism version 8.0.1 for Windows (www.graphpad.com; GraphPad Software, San Diego, California, USA). A threshold of p<0.05 was considered significant (Supplementary Tables 7 and 8).
Ethics approval of the research
The Centre of Genomics and Bioinformatics ethics boards, Uzbekistan, has approved the TOMAVAC studies (No2/30032022 and No4/07072022) and results (No3/30062022 and No5/05092022).
Data availability
Individual participant data underlying the results reported in this Article will be made available upon request of the corresponding authors. Researchers who provide a scientifically sound proposal will be allowed to access the de-identified individual participant data with a signed data access agreement.
References
24. Holsters, M. et al. Transfection and transformation of Agrobacterium tumefaciens. Mol. Gen. Genet.163, 181–187 (1978).
25. Yasmeen, A. et al. In planta transformation of tomato. Plant Mol. Biol. Report.27, 20–28 (2009).
26. Orzaez, D., Mirabel, S., Wieland, W. H. & Granell, A. Agroinjection of tomato fruits. A tool for rapid functional analysis of transgenes directly in fruit. Plant Physiol.140, 3–11 (2006).
27. Abdurakhmonov, I. Y. et al. Phytochrome RNAi enhances major fibre quality and agronomic traits of the cotton Gossypium hirsutum L. Nat. Commun.5, 3062 (2014).
28. Guide for the Care and Use of Laboratory Animals. (The National Academies Press USA, 2011).