{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,23]],"date-time":"2026-08-23T14:39:46Z","timestamp":1787495986947,"version":"build-2736575974"},"reference-count":48,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2024,12,24]],"date-time":"2024-12-24T00:00:00Z","timestamp":1734998400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100008134","name":"California State University Agricultural Research Institute","doi-asserted-by":"publisher","award":["58913"],"award-info":[{"award-number":["58913"]}],"id":[{"id":"10.13039\/100008134","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Cal Poly internal funding program FROST","award":["58913"],"award-info":[{"award-number":["58913"]}]},{"name":"Mazen Animal Health","award":["58913"],"award-info":[{"award-number":["58913"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Coronaviruses continue to disrupt health and economic productivity worldwide. Porcine epidemic diarrhea virus (PEDV) is a devastating swine disease and SARS-CoV-2 is the latest coronavirus to infect the human population. Both viruses display a similar spike protein on the surface that is a target of vaccine development. Despite the availability of commercial vaccines for both viruses, there is still a high occurrence of infections and a great need for enhanced efficacy and lower costs. We previously produced the PEDV spike protein (S) using transgenic maize, enabling a low-cost supply of the vaccine candidate. In this study, we (1) test orally delivered PEDV vaccine candidates in pigs to optimize the mucosal immune response; (2) generate the SARS-CoV-2 S1 protein in maize; and (3) perform structural characterization of the S1 protein for PEDV and SARS-CoV-2. We demonstrated high expression levels in maize of the S1 subunit of the SARS-CoV-2 spike protein, both with and without fusion to the heat-labile enterotoxin B (LTB) subunit. We found that the LTB fusion protein from both coronaviruses preferentially assembles into higher molecular weight multimers, consistent with the formation of trimers. For PEDV, administering the spike protein fused to LTB to young pigs elicited a higher level of mucosal IgAs compared to maize grain containing the S1 protein alone or controls. This suggests that fusing the coronavirus spike protein with LTB may provide better protection.<\/jats:p>","DOI":"10.3390\/v17010007","type":"journal-article","created":{"date-parts":[[2024,12,24]],"date-time":"2024-12-24T09:48:58Z","timestamp":1735033738000},"page":"7","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Effect of Fusion to the LTB Carrier Protein on Coronavirus Spike Protein Vaccine Candidates Produced in Maize"],"prefix":"10.3390","volume":"17","author":[{"given":"Erin","family":"Egelkrout","sequence":"first","affiliation":[{"name":"Applied Biotechnology Institute, California Polytechnic Tech Park, San Luis Obispo, CA 93407, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3511-3788","authenticated-orcid":false,"given":"Magdalena","family":"Maj","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, California Polytechnic State University, San Luis Obispo, CA 93407, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5691-1549","authenticated-orcid":false,"given":"Rodrigo","family":"Manjarin","sequence":"additional","affiliation":[{"name":"Department of Animal Sciences, California Polytechnic State University, San Luis Obispo, CA 93407, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gina","family":"Fake","sequence":"additional","affiliation":[{"name":"Applied Biotechnology Institute, California Polytechnic Tech Park, San Luis Obispo, CA 93407, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-1963-7671","authenticated-orcid":false,"given":"Muneaki","family":"Watanabe","sequence":"additional","affiliation":[{"name":"Applied Biotechnology Institute, California Polytechnic Tech Park, San Luis Obispo, CA 93407, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jenna","family":"Williams","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, California Polytechnic State University, San Luis Obispo, CA 93407, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nate","family":"Blanchard","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, California Polytechnic State University, San Luis Obispo, CA 93407, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John","family":"Walker","sequence":"additional","affiliation":[{"name":"Department of Statistics, California Polytechnic State University, San Luis Obispo, CA 93407, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Celine","family":"Hayden","sequence":"additional","affiliation":[{"name":"Applied Biotechnology Institute, California Polytechnic Tech Park, San Luis Obispo, CA 93407, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John","family":"Howard","sequence":"additional","affiliation":[{"name":"Applied Biotechnology Institute, California Polytechnic Tech Park, San Luis Obispo, CA 93407, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,12,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Saif, L.J., Wang, Q., Vlasova, A.N., Jung, K., and Xiao, S. (2019). Coronaviruses. Dis. Swine, 488\u2013523.","DOI":"10.1002\/9781119350927.ch31"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2977","DOI":"10.1007\/s00705-014-2163-7","article-title":"Immunogenicity and protective efficacy of recombinant S1 domain of the porcine epidemic diarrhea virus spike protein","volume":"159","author":"Oh","year":"2014","journal-title":"Arch. Virol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1186\/s12985-015-0421-2","article-title":"Porcine epidemic diarrhea virus: An emerging and re-emerging epizootic swine virus","volume":"12","author":"Lee","year":"2015","journal-title":"Virol. J."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1038\/s41586-022-05522-2","article-title":"The WHO estimates of excess mortality associated with the COVID-19 pandemic","volume":"613","author":"Msemburi","year":"2023","journal-title":"Nature"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Shanmugaraj, B., Rattanapisit, K., Manopwisedjaroen, S., Thitithanyanont, A., and Phoolcharoen, W. (2020). Monoclonal Antibodies B38 and H4 Produced in Nicotiana benthamiana Neutralize SARS-CoV-2 in vitro. Front. Plant Sci., 11.","DOI":"10.3389\/fpls.2020.589995"},{"key":"ref_6","first-page":"28","article-title":"Expression of core neutralizing epitope gene of porcine epidemic diarrhea virus in maize","volume":"16","author":"Man","year":"2014","journal-title":"J. Agric. Sci. Technol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1016\/j.pep.2004.07.014","article-title":"High-level expression of the neutralizing epitope of porcine epidemic diarrhea virus by a tobacco mosaic virus-based vector","volume":"38","author":"Kang","year":"2004","journal-title":"Protein Expr. Purif."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2294","DOI":"10.1016\/j.vaccine.2005.01.027","article-title":"Expression of the synthetic neutralizing epitope gene of porcine epidemic diarrhea virus in tobacco plants without nicotine","volume":"23","author":"Kang","year":"2005","journal-title":"Vaccine"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Ho, T.T., Nguyen, G.T., Pham, N.B., Le, V.P., Trinh, T.B.N., Vu, T.H., Phan, H.T., Conrad, U., and Chu, H.H. (2020). Plant-derived trimeric CO-26K-equivalent epitope induced neutralizing antibodies against porcine epidemic diarrhea virus. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.02152"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"4265","DOI":"10.1002\/jmv.27881","article-title":"Receptor binding domain proteins of SARS-CoV-2 variants produced in Nicotiana benthamiana elicit neutralizing antibodies against variants of concern","volume":"94","author":"Khorattanakulchai","year":"2022","journal-title":"J. Med. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Khorattanakulchai, N., Srisutthisamphan, K., Shanmugaraj, B., Manopwisedjaroen, S., Rattanapisit, K., Panapitakkul, C., Kemthong, T., Suttisan, N., Malaivijitnond, S., and Thitithanyanont, A. (2022). A recombinant subunit vaccine candidate produced in plants elicits neutralizing antibodies against SARS-CoV-2 variants in macaques. Front. Plant Sci., 13.","DOI":"10.3389\/fpls.2022.901978"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Rattanapisit, K., Bulaon, C.J.I., Khorattanakulchai, N., Shanmugaraj, B., Wangkanont, K., and Phoolcharoen, W. (2021). Plant-produced SARS-CoV-2 receptor binding domain (RBD) variants showed differential binding efficiency with anti-spike specific monoclonal antibodies. PLoS ONE, 16.","DOI":"10.1371\/journal.pone.0253574"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Rattanapisit, K., Shanmugaraj, B., Manopwisedjaroen, S., Purwono, P.B., Siriwattananon, K., Khorattanakulchai, N., Hanittinan, O., Boonyayothin, W., Thitithanyanont, A., and Smith, D.R. (2020). Rapid production of SARS-CoV-2 receptor binding domain (RBD) and spike specific monoclonal antibody CR3022 in Nicotiana benthamiana. Sci. Rep., 10.","DOI":"10.1038\/s41598-020-74904-1"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1071","DOI":"10.1038\/s41591-021-01370-1","article-title":"Phase 1 randomized trial of a plant-derived virus-like particle vaccine for COVID-19","volume":"27","author":"Ward","year":"2021","journal-title":"Nat. Med."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.hlife.2023.12.005","article-title":"Mucosal vaccine development for respiratory viral infections","volume":"2","author":"Lin","year":"2023","journal-title":"hLife"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2921","DOI":"10.1080\/21645515.2020.1805992","article-title":"Prospects for mucosal vaccine: Shutting the door on SARS-CoV-2","volume":"16","author":"Mudgal","year":"2020","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Karczmarzyk, K., and K\u0119sik-Brodacka, M. (2022). Attacking the intruder at the gate: Prospects of mucosal anti SARS-CoV-2 vaccines. Pathogens, 11.","DOI":"10.3390\/pathogens11020117"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e604","DOI":"10.1002\/iid3.604","article-title":"Oral and intranasal vaccines against SARS-CoV-2: Current progress, prospects, advantages, and challenges","volume":"10","author":"Kar","year":"2022","journal-title":"Immun. Inflamm. Dis."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1007\/s11240-020-01835-0","article-title":"Oral delivery of maize-produced porcine epidemic diarrhea virus spike protein elicits neutralizing antibodies in pigs","volume":"142","author":"Egelkrout","year":"2020","journal-title":"Plant Cell Tissue Organ Cult."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Maj, M., Fake, G.M., Walker, J.H., Saltzman, R., and Howard, J.A. (2021). Oral Administration of Coronavirus Spike Protein Provides Protection to Newborn Pigs When Challenged with PEDV. Vaccines, 9.","DOI":"10.3390\/vaccines9121416"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2420","DOI":"10.1016\/j.vaccine.2003.11.066","article-title":"A corn-based delivery system for animal vaccines: An oral transmissible gastroenteritis virus vaccine boosts lactogenic immunity in swine","volume":"22","author":"Lamphear","year":"2004","journal-title":"Vaccine"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1260","DOI":"10.1126\/science.abb2507","article-title":"Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation","volume":"367","author":"Wrapp","year":"2020","journal-title":"Science"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"147","DOI":"10.54846\/jshap\/941","article-title":"Measurement of neutralizing antibodies against porcine epidemic diarrhea virus in sow serum, colostrum, and milk samples and in piglet serum samples after feedback","volume":"24","author":"Clement","year":"2016","journal-title":"J. Swine Health Prod."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/j.virusres.2016.06.002","article-title":"Characterization of anti-porcine epidemic diarrhea virus neutralizing activity in mammary secretions","volume":"226","author":"Song","year":"2016","journal-title":"Virus Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e00273-17","DOI":"10.1128\/JVI.00273-17","article-title":"Cell attachment domains of the porcine epidemic diarrhea virus spike protein are key targets of neutralizing antibodies","volume":"91","author":"Li","year":"2017","journal-title":"J. Virol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1016\/j.pep.2005.07.026","article-title":"Expression of synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat-labile enterotoxin in tobacco plants","volume":"46","author":"Kang","year":"2006","journal-title":"Protein Expr. Purif."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"7499","DOI":"10.1007\/s00253-018-9110-6","article-title":"Efficacy of heat-labile enterotoxin B subunit-adjuvanted parenteral porcine epidemic diarrhea virus trimeric spike subunit vaccine in piglets","volume":"102","author":"Chang","year":"2018","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"526","DOI":"10.1089\/vim.2016.0067","article-title":"Orally administrated whole yeast vaccine against porcine epidemic diarrhea virus induced high levels of IgA response in mice and piglets","volume":"29","author":"Wang","year":"2016","journal-title":"Viral Immunol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1016\/j.jviromet.2012.12.007","article-title":"Oral administration of attenuated Salmonella typhimurium containing a DNA vaccine against rabbit haemorrhagic disease","volume":"188","author":"Qiu","year":"2013","journal-title":"J. Virol. Methods"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1136\/gut.32.1.29","article-title":"Dissociation between systemic and mucosal humoral immune responses in coeliac disease","volume":"32","author":"Arranz","year":"1991","journal-title":"Gut"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"979","DOI":"10.1111\/j.1467-7652.2012.00727.x","article-title":"Production of highly concentrated, heat-stable hepatitis B surface antigen in maize","volume":"10","author":"Hayden","year":"2012","journal-title":"Plant Biotechnol. J."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"745","DOI":"10.1038\/nbt0696-745","article-title":"High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens","volume":"14","author":"Ishida","year":"1996","journal-title":"Nat. Biotechnol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1046\/j.1365-313X.1996.10010165.x","article-title":"Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers","volume":"10","author":"Komari","year":"1996","journal-title":"Plant J."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1291","DOI":"10.1128\/jb.168.3.1291-1301.1986","article-title":"The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA","volume":"168","author":"Hood","year":"1986","journal-title":"J. Bacteriol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"699","DOI":"10.1007\/s12155-012-9288-x","article-title":"Enhanced Expression Levels of Cellulase Enzymes Using Multiple Transcription Units","volume":"6","author":"Egelkrout","year":"2013","journal-title":"BioEnergy Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1056\/NEJMoa2035389","article-title":"Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine","volume":"384","author":"Baden","year":"2021","journal-title":"N. Engl. J. Med."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1920","DOI":"10.1056\/NEJMoa2022483","article-title":"An mRNA vaccine against SARS-CoV-2\u2014Preliminary report","volume":"383","author":"Jackson","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"4877","DOI":"10.1038\/s41467-022-32588-3","article-title":"In situ structure and dynamics of an alphacoronavirus spike protein by cryo-ET and cryo-EM","volume":"13","author":"Huang","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1186\/s13567-021-00971-5","article-title":"Stable trimer formation of spike protein from porcine epidemic diarrhea virus improves the efficiency of secretory production in silkworms and induces neutralizing antibodies in mice","volume":"52","author":"Masuda","year":"2021","journal-title":"Vet. Res."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"828","DOI":"10.1016\/j.vaccine.2024.01.020","article-title":"Immunogenicity and protective efficacy of a trimeric full-length S protein subunit vaccine for porcine epidemic diarrhea virus","volume":"42","author":"Guo","year":"2024","journal-title":"Vaccine"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"4385","DOI":"10.1016\/j.vaccine.2004.01.073","article-title":"Immunogenicity of recombinant LT-B delivered orally to humans in transgenic corn","volume":"22","author":"Tacket","year":"2004","journal-title":"Vaccine"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"821","DOI":"10.1586\/erv.10.88","article-title":"Plant-made immunogens and effective delivery strategies","volume":"9","author":"Paul","year":"2010","journal-title":"Expert Rev. Vaccines"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"607","DOI":"10.1038\/nm0598-607","article-title":"Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato","volume":"4","author":"Tacket","year":"1998","journal-title":"Nat. Med."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/S0168-1656(99)00107-8","article-title":"Production of recombinant subunit vaccines: Protein immunogens, live delivery systems and nucleic acid vaccines","volume":"73","author":"Liljeqvist","year":"1999","journal-title":"J. Biotechnol."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Harutyunyan, S., Neuhauser, I., Mayer, A., Aichinger, M., Szij\u00e1rt\u00f3, V., Nagy, G., Nagy, E., Girardi, P., Malinoski, F.J., and Henics, T. (2020). Characterization of shigetec, a novel live attenuated combined vaccine against shigellae and etec. Vaccines, 8.","DOI":"10.3390\/vaccines8040689"},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Peng, X., Zhang, R., Wang, C., Yu, F., Yu, M., Chen, S., Fan, Q., Xi, Y., and Duan, G.E. (2019). coli enterotoxin LtB enhances vaccine-induced anti-H. pylori protection by promoting leukocyte migration into gastric mucus via inflammatory lesions. Cells, 8.","DOI":"10.3390\/cells8090982"},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Solis-Andrade, K.I., Gonzalez-Ortega, O., Govea-Alonso, D.O., Comas-Garcia, M., and Rosales-Mendoza, S. (2022). Production and purification of LTB-RBD: A potential antigen for mucosal vaccine development against SARS-CoV-2. Vaccines, 10.","DOI":"10.3390\/vaccines10101759"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1098","DOI":"10.1016\/j.vaccine.2022.01.025","article-title":"Oral subunit SARS-CoV-2 vaccine induces systemic neutralizing IgG, IgA and cellular immune responses and can boost neutralizing antibody responses primed by an injected vaccine","volume":"40","author":"Pitcovski","year":"2022","journal-title":"Vaccine"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/1\/7\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T16:59:18Z","timestamp":1760115558000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/1\/7"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,12,24]]},"references-count":48,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,1]]}},"alternative-id":["v17010007"],"URL":"https:\/\/doi.org\/10.3390\/v17010007","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,12,24]]}}}