Effect of Fusion to the LTB Carrier Protein on Coronavirus Spike Protein Vaccine Candidates Produced in Maize

Overview

Journal Viruses

Publisher MDPI

Date 2025 Jan 25

PMID 39861796

Authors

Erin Egelkrout

Magdalena Maj

Rodrigo Manjarin

Gina Fake

Muneaki Watanabe

Jenna Williams

Nate Blanchard

John Walker

Celine Hayden

John Howard

Affiliations

Soon will be listed here.

Abstract

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.

References

Wrapp D, Wang N, Corbett K, Goldsmith J, Hsieh C, Abiona O . Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020; 367(6483):1260-1263. PMC: 7164637. DOI: 10.1126/science.abb2507. View

Peng X, Zhang R, Wang C, Yu F, Yu M, Chen S . Enterotoxin LtB Enhances Vaccine-Induced Anti- Protection by Promoting Leukocyte Migration into Gastric Mucus via Inflammatory Lesions. Cells. 2019; 8(9). PMC: 6770474. DOI: 10.3390/cells8090982. View

Jackson L, Anderson E, Rouphael N, Roberts P, Makhene M, Coler R . An mRNA Vaccine against SARS-CoV-2 - Preliminary Report. N Engl J Med. 2020; 383(20):1920-1931. PMC: 7377258. DOI: 10.1056/NEJMoa2022483. View

OMahony S, Arranz E, Barton J, Ferguson A . Dissociation between systemic and mucosal humoral immune responses in coeliac disease. Gut. 1991; 32(1):29-35. PMC: 1379209. DOI: 10.1136/gut.32.1.29. View

Kang T, Han S, Yang M, Jang Y . Expression of synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat-labile enterotoxin in tobacco plants. Protein Expr Purif. 2005; 46(1):16-22. DOI: 10.1016/j.pep.2005.07.026. View

Maj M, Fake G, Walker J, Saltzman R, Howard J . Oral Administration of Coronavirus Spike Protein Provides Protection to Newborn Pigs When Challenged with PEDV. Vaccines (Basel). 2021; 9(12). PMC: 8706244. DOI: 10.3390/vaccines9121416. View

Kang T, Kim Y, Jang Y, Yang M . Expression of the synthetic neutralizing epitope gene of porcine epidemic diarrhea virus in tobacco plants without nicotine. Vaccine. 2005; 23(17-18):2294-7. DOI: 10.1016/j.vaccine.2005.01.027. View

Rattanapisit K, Shanmugaraj B, Manopwisedjaroen S, Purwono P, Siriwattananon K, Khorattanakulchai N . Rapid production of SARS-CoV-2 receptor binding domain (RBD) and spike specific monoclonal antibody CR3022 in Nicotiana benthamiana. Sci Rep. 2020; 10(1):17698. PMC: 7573609. DOI: 10.1038/s41598-020-74904-1. View

Tacket C, Mason H, Losonsky G, Clements J, Levine M, Arntzen C . Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nat Med. 1998; 4(5):607-9. DOI: 10.1038/nm0598-607. View

10.

Kang T, Kang K, Kim J, Kwon T, Jang Y, Yang M . High-level expression of the neutralizing epitope of porcine epidemic diarrhea virus by a tobacco mosaic virus-based vector. Protein Expr Purif. 2004; 38(1):129-35. DOI: 10.1016/j.pep.2004.07.014. View

11.

Hood E, Helmer G, Fraley R, Chilton M . The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol. 1986; 168(3):1291-301. PMC: 213636. DOI: 10.1128/jb.168.3.1291-1301.1986. View

12.

Tacket C, Pasetti M, Edelman R, Howard J, Streatfield S . Immunogenicity of recombinant LT-B delivered orally to humans in transgenic corn. Vaccine. 2004; 22(31-32):4385-9. DOI: 10.1016/j.vaccine.2004.01.073. View

13.

Solis-Andrade K, Gonzalez-Ortega O, Govea-Alonso D, Comas-Garcia M, Rosales-Mendoza S . Production and Purification of LTB-RBD: A Potential Antigen for Mucosal Vaccine Development against SARS-CoV-2. Vaccines (Basel). 2022; 10(10). PMC: 9609574. DOI: 10.3390/vaccines10101759. View

14.

Guo W, Wang C, Song X, Xu H, Zhao S, Gu J . Immunogenicity and protective efficacy of a trimeric full-length S protein subunit vaccine for porcine epidemic diarrhea virus. Vaccine. 2024; 42(4):828-839. DOI: 10.1016/j.vaccine.2024.01.020. View

15.

Msemburi W, Karlinsky A, Knutson V, Aleshin-Guendel S, Chatterji S, Wakefield J . The WHO estimates of excess mortality associated with the COVID-19 pandemic. Nature. 2022; 613(7942):130-137. DOI: 10.1038/s41586-022-05522-2. View

16.

Wang X, Wang Z, Xu H, Xiang B, Dang R, Yang Z . Orally Administrated Whole Yeast Vaccine Against Porcine Epidemic Diarrhea Virus Induced High Levels of IgA Response in Mice and Piglets. Viral Immunol. 2016; 29(9):526-531. DOI: 10.1089/vim.2016.0067. View

17.

Li C, Li W, Lucio De Esesarte E, Guo H, van den Elzen P, Aarts E . Cell Attachment Domains of the Porcine Epidemic Diarrhea Virus Spike Protein Are Key Targets of Neutralizing Antibodies. J Virol. 2017; 91(12). PMC: 5446644. DOI: 10.1128/JVI.00273-17. View

18.

Huang C, Draczkowski P, Wang Y, Chang C, Chien Y, Cheng Y . In situ structure and dynamics of an alphacoronavirus spike protein by cryo-ET and cryo-EM. Nat Commun. 2022; 13(1):4877. PMC: 9388967. DOI: 10.1038/s41467-022-32588-3. View

19.

Egelkrout E, Hayden C, Fake G, Keener T, Arruda P, Saltzman R . Oral delivery of maize-produced porcine epidemic diarrhea virus spike protein elicits neutralizing antibodies in pigs. Plant Cell Tissue Organ Cult. 2020; 142(1):79-86. PMC: 7212245. DOI: 10.1007/s11240-020-01835-0. View

20.

Pitcovski J, Gruzdev N, Abzach A, Katz C, Ben-Adiva R, Brand-Shwartz M . 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. Vaccine. 2022; 40(8):1098-1107. PMC: 8768024. DOI: 10.1016/j.vaccine.2022.01.025. View