{"doi":"10.1111/pbi.13993","title":"Oral booster vaccine antigen—Expression of full‐length native <scp>SARS‐CoV</scp>‐2 spike protein in lettuce chloroplasts","abstract":"Current vaccines continue to save lives during the pandemic but do not prevent virus transmission. Unfortunately, fully vaccinated individuals with repeated boosters also get infected, and breakthrough infections have peak viral loads similar to unvaccinated individuals and transmit SARS-CoV-2 in household settings, with or without symptoms (Singanayagam et al., 2022). AI studies of vaccine-resistant mutations in >2.2 million SARS-CoV-2 genomes show that the mutation frequency correlates strongly with the vaccination rates in Europe and America and predicts a complementary transmission pathway, vaccine-breakthrough or antibody-resistant mutations, like those in Omicron (Wang et al., 2021). Amidst the emergence of new SARS-CoV-2 variants like the omicron strain resistant to current vaccines, with higher rates of transmissibility, it is prudent to consider additional affordable measures to minimize viral transmission and infection. Airborne-lifetime-weighed volume of saliva droplets in healthy subjects is 3–5 orders of magnitude higher than breath droplets, and speaking four words transmits more virus than 1 h of maskless breathing (Shen et al., 2022). Oral epithelial cells are enriched in ACE2 receptors and GM1 coreceptors, thereby facilitating viral entry (Daniell et al., 2022a). Therefore, one approach recently developed involved debulking SARS-CoV-2 or other oral viruses in saliva using virus-trap proteins via chewing gums to minimize self-infection and transmission (Daniell et al., 2022a,2022b). Most importantly, proteins bioencapsulated in the chewing gum are stable and fully functional for several years when stored at ambient temperature, thereby making them affordable by the elimination of complex fermenter-based manufacturing processes, expensive cold storage, transportation, and other costs associated with current vaccines. Indeed, this is the first engineered therapeutic protein approved by FDA free of protein purification or cold chain, and clinical trials are in progress to evaluate SARS-CoV-2 infection and transmission (Daniell et al., 2022b). Medicago recently reported transient expression of full-length spike protein in tobacco and the development of an adjuvanted injectable vaccine after purification of VLPs (Hager et al., 2022). The native full-length spike protein is challenging to express due to degradation and therefore required modifications of certain amino acids or the addition of tags to enhance stability (Ward et al., 2021). Therefore, most of the early publications in this field expressed the receptor-binding domain (RBD) and observed effective immunization and protection in animal models. Interestingly, higher neutralization and affinity to ACE2 have been reported for aglycosylated RBD than glycosylated version (Mamedov et al., 2021). While several boosters are needed for continued protection, recent studies show that heterologous boosters (mRNA vaccine boosted with inactivated or nonreplicating adenovirus vaccine—Sputnik, Oxford Astro-Zeneca, or Sinopharm) offered better immunity and protection (Larkin, 2022). Therefore, in this study, we expressed full-length CTB-Spike fusion protein in chloroplasts to facilitate oral delivery, for the eventual development of a cold chain-free heterologous mucosal booster vaccine. The nucleotide sequence of S-gene (Wuhan-Hu-1 (NC_045512.2) strain) encoding full-length native spike protein (Figure 1a) was codon optimized based on the hierarchy of chloroplast psbA gene. In this codon optimization process, among 1273 amino acids, 314 codons, including 105 rare codons, were replaced (Figure S1). The full-length synthetic S-gene was subcloned downstream of cholera toxin-B (CTB) subunit encoding nucleotide sequence with the hinge (GPGP) and furin cleavage site (RRKR) and was inserted into the marker-free chloroplast vector pLsLF-MF, and the expression cassette is regulated by the psbA promoter/5'UTR and 3'UTR (Figure S2). Lettuce leaf bombarded with pLsLF-MF-CTB-Spike exp","journal":"Plant Biotechnology Journal","year":2022,"id":265604,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":11,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9528,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":684166,"name":"Shina Lin","orcid":null,"position":1,"is_corresponding":false},{"id":651022,"name":"Smruti K. Nair","orcid":"0000-0001-8986-8874","position":2,"is_corresponding":false},{"id":684165,"name":"Yao Shi","orcid":null,"position":3,"is_corresponding":false},{"id":290765,"name":"Henry Daniell","orcid":"0000-0003-4485-1176","position":4,"is_corresponding":false},{"id":651346,"name":"Rahul Singh","orcid":"0000-0001-9271-2037","position":0,"is_corresponding":true}],"reference_count":11,"raw_metadata":null,"created_at":"2026-07-19T00:26:49.862989Z","pmid":"36577691","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}