Production of a Bacteria-like Particle Vaccine Targeting Rock Bream (Oplegnathus fasciatus) Iridovirus Using Nicotiana benthamiana.

Viral diseases are extremely widespread infections that change constantly through mutations. To produce vaccines against viral diseases, transient expression systems are employed, and Nicotiana benthamiana (tobacco) plants are a rapidly expanding platform. In this study, we developed a recombinant protein vaccine targeting the major capsid protein (MCP) of iridovirus fused with the lysine motif (LysM) and coiled-coil domain of coronin 1 (ccCor1) for surface display using Lactococcus lactis. The protein was abundantly produced in N. benthamiana in its N-glycosylated form. Total soluble proteins isolated from infiltrated N. benthamiana leaves were treated sequentially with increasing ammonium sulfate solution, and recombinant MCP mainly precipitated at 40-60%. Additionally, affinity chromatography using Ni-NTA resin was applied for further purification. Native structure analysis using size exclusion chromatography showed that recombinant MCP existed in a large oligomeric form. A minimum OD600 value of 0.4 trichloroacetic acid (TCA)-treated L. lactis was required for efficient recombinant MCP display. Immunogenicity of recombinant MCP was assessed in a mouse model through enzyme-linked immunosorbent assay (ELISA) with serum-injected recombinant MCP-displaying L. lactis. In summary, we developed a plant-based recombinant vaccine production system combined with surface display on L. lactis.

Plant-based, adjuvant-free, potent multivalent vaccines for avian influenza virus via Lactococcus surface display.

Influenza epidemics frequently and unpredictably break out all over the world, and seriously affect the breeding industry and human activity. Inactivated and live attenuated viruses have been used as protective vaccines but exhibit high risks for biosafety. Subunit vaccines enjoy high biosafety and specificity but have a few weak points compared to inactivated virus or live attenuated virus vaccines, especially in low immunogenicity. In this study, we developed a new subunit vaccine platform for a potent, adjuvant-free, and multivalent vaccination. The ectodomains of hemagglutinins (HAs) of influenza viruses were expressed in plants as trimers (tHAs) to mimic their native forms. tHAs in plant extracts were directly used without purification for binding to inactivated Lactococcus (iLact) to produce iLact-tHAs, an antigen-carrying bacteria-like particle (BLP). tHAs BLP showed strong immune responses in mice and chickens without adjuvants. Moreover, simultaneous injection of two different antigens by two different formulas, tHAH5N6 + H9N2 BLP or a combination of tHAH5N6 BLP and tHAH9N2 BLP, led to strong immune responses to both antigens. Based on these results, we propose combinations of plant-based antigen production and BLP-based delivery as a highly potent and cost-effective platform for multivalent vaccination for subunit vaccines.

A multi-epitope plant-made chimeric protein (LTBentero) targeting common enteric pathogens is immunogenic in mice.

KEY MESSAGE: A plant-based multiepitopic protein (LTBentero) containing epitopes from ETEC, S. typhimurium, and V. parahaemolyticus was produced in plants cells and triggered systemic and intestinal humoral responses in immunized mice. Around 200 million people suffer gastroenteritis daily and more than 2 million people die annually in developing countries due to such pathologies. Vaccination is an alternative to control this global health issue, however new low-cost vaccines are needed to ensure proper vaccine coverage. In this context, plants are attractive hosts for the synthesis and delivery of subunit vaccines. Therefore, in this study a plant-made multiepitopic protein named LTBentero containing epitopes from antigens of enterotoxigenic E. coli, S. typhimurium, and V. parahaemolyticus was produced and found immunogenic in mice. The LTBentero protein was expressed in tobacco plants at up to 5.29 µg g-1 fresh leaf tissue and was deemed immunogenic when administered to BALB/c mice either orally or subcutaneously. The plant-made LTBentero antigen induced specific IgG (systemic) and IgA (mucosal) responses against LTB, ST, and LptD epitopes. In conclusion, multiepitopic LTBentero was functionally produced in plant cells, being capable to trigger systemic and intestinal humoral responses and thus it constitutes a promising oral immunogen candidate in the fight against enteric diseases.

Expression and active testing of VP7 from GCRV (Grass carp reovirus) fused with cholera toxin B subunit in rice calli.

Grass carp reovirus (GCRV) is one of the most serious pathogens threatening grass carp (Ctenopharyngodon idellus) production and results in high mortality in China. VP7 from GCRV is involved in viral infection and could be suitable for developing vaccines for the control of GCRV infection. To obtain a genetically engineered vaccine and a plant-based oral vaccine and to evaluate their immune efficacy as an oral vaccine against GCRV, cholera toxin B subunit (CTB) of Vibrio cholerae fused to VP7 (CTB-VP7) was transformed into BL21(DE3) for expression. SDS-PAGE and Western blotting showed that the purified CTB-VP7 fusion protein (rCTB-VP7) was approximately 49.0 kDa. Meanwhile, CTB-VP7 was transformed into rice callus cells by Agrobacterium tumefaciens-mediated gene transformation. CTB-VP7 was integrated into the nuclear genome by PCR, and mRNA transcripts of CTB-VP7 were detected. ELISA and Western blot analyses revealed that the CTB-VP7 fusion protein (CTB-VP7) could be expressed in rice callus lines. The level of expression was determined to be 1.54% ±â€¯0.43 of the total soluble protein. CTB-VP7 showed a binding affinity for monosialoganglioside(GM1), a receptor for CTB. CTB-VP7 showed a higher affinity towards GM1 compared to rCTB-VP7. CTB-VP7 bonded to GM1 with different affinities under different temperatures. Maximum binding of CTB-VP7 to GM1 was reported to occur within 2 h at 37 °C, and approximately half of the binding affinity remained at 25 °C. Our results suggest that CTB-VP7 could be produced in rice calli, increasing the possibility that edible plants can be employed in mucosal vaccines for protection against GCRV in aquaculture.

Plant-based vaccines for Alzheimer's disease.

Alzheimer's disease (AD) is one of the major causes of chronic and progressive cognitive decline, with the pathological hallmarks of senile plaques and neurofibrillary tangles. Amyloid ß peptide (Aß) is the main component of senile plaques, and the pathological load of Aß in the brain has been shown to be a marker of the severity of AD. To prevent the accumulation of plaques, novel and safer plant-based vaccine strategies have been suggested. In this review, we summarize the results of plant vaccines against Aß.

Plant synthetic GP4 and GP5 proteins from porcine reproductive and respiratory syndrome virus elicit immune responses in pigs.

MAIN CONCLUSION: We demonstrated successful overexpression of porcine reproductive and respiratory syndrome virus (PRRSV)-derived GP4D and GP5D antigenic proteins in Arabidopsis. Pigs immunized with transgenic plants expressing GP4D and GP5D proteins generated both humoral and cellular immune responses to PRRSV. Porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS, the most economically significant disease affecting the swine industry worldwide. However, current commercial PRRSV vaccines (killed virus or modified live vaccines) show poor efficacy and safety due to concerns such as reversion of virus to wild type and lack of cross protection. To overcome these problems, plants are considered a promising alternative to conventional platforms and as a vehicle for large-scale production of recombinant proteins. Here, we demonstrate successful production of recombinant protein vaccine by expressing codon-optimized and transmembrane-deleted recombinant glycoproteins (GP4D and GP5D) from PRRSV in planta. We generated transgenic Arabidopsis plants expressing GP4D and GP5D proteins as candidate antigens. To examine immunogenicity, pigs were fed transgenic Arabidopsis leaves expressing the GP4D and GP5D antigens (three times at 2-week intervals) and then challenged with PRRSV at 6-week post-initial treatment. Immunized pigs showed significantly lower lung lesion scores and reduced viremia and viral loads in the lung than pigs fed Arabidopsis leaves expressing mYFP (control). Immunized pigs also had higher titers of PRRSV-specific antibodies and significantly higher levels of pro-inflammatory cytokines (TNF-α and IL-12). Furthermore, the numbers of IFN-γ+-producing cells were higher, and those of regulatory T cells were lower, in GP4D and GP5D immunized pigs than in control pigs. Thus, plant-derived GP4D and GP5D proteins provide an alternative platform for producing an effective subunit vaccine against PRRSV.

LTB-Syn: a recombinant immunogen for the development of plant-made vaccines against synucleinopathies.

MAIN CONCLUSION: A recombinant antigen targeting α-synuclein was produced in the plant cell rendering an immunogenic protein capable to induce humoral responses in mice upon oral administration. Synucleinopathies are neurodegenerative diseases characterized by the abnormal accumulation of α-synuclein (α-Syn, a 140 amino acid protein that normally plays various neurophysiologic roles) aggregates. Parkinson's disease (PD) is the synucleinopathy with the highest epidemiologic impact and although its etiology remains unknown, α-Syn aggregation during disease progression pointed out α-Syn as target in the development of immunotherapies. Herein a chimeric protein, comprising the B subunit of the enterotoxin from enterotoxigenic Escherichia coli and α-Syn epitopes, was expressed in the plant cell having the potential to induce humoral responses following oral immunization. This approach will serve as the basis for the development of oral plant-based vaccines against PD with several potential advantages such as low cost, easy scale-up during production, and easy administration.

Expression and assembly of cholera toxin B subunit and domain III of dengue virus 2 envelope fusion protein in transgenic potatoes.

The rates of mosquito-transmitted dengue virus infection in humans have increased in tropical and sub-tropical areas. Domain III of dengue envelope protein (EDIII) is involved in cellular receptor binding and induces serotype-specific neutralizing antibodies. EDIII fused to the B subunit of Vibrio cholera (CTB-EDIII) was expressed in potatoes to develop a plant-based vaccine against dengue virus type 2. CTB-EDIII fused to an endoplasmic reticulum (ER) retention signal, SEKDEL, was introduced into potatoes by A. tumefaciens-mediated gene transformation. The integration of the CTB-EDIII fusion gene into the nuclear genome of transgenic plants was confirmed by genomic DNA polymerase chain reaction (PCR), and mRNA transcripts of CTB-EDIII were detected. CTB-EDIII fusion protein was expressed in potato tubers and assembled into a pentameric form capable of binding monosialotetrahexosylganglioside (GM1). The level of expression was determined to be ∼0.005% of total soluble protein in potato tubers. These results suggest that dengue virus antigen could be produced in potatoes, raising the possibility that edible plants are employed in mucosal vaccines for protection against dengue infection.

Production and immunogenicity of Actinobacillus pleuropneumoniae ApxIIA protein in transgenic rice callus.

Actinobacillus pleuropneumoniae is a major etiological agent that is responsible for swine pleuropneumonia, a highly contagious respiratory infection that causes severe economic losses in the swine production industry. ApxIIA is one of the virulence factors in A. pleuropneumoniae and has been considered as a candidate for developing a vaccine against the bacterial infection. A gene encoding an ApxIIA fragment (amino acids 439-801) was modified based on a plant-optimized codon and constructed into a plant expression vector under the control of a promoter and the 3' UTR of the rice amylase 3D gene. The plant expression vector was introduced into rice embryogenic callus (Oryza sativa L. cv. Dongjin) via particle bombardment-mediated transformation. The integration and transcription of the ApxIIA439-801 gene were confirmed by using genomic DNA PCR amplification and Northern blot analysis, respectively. The synthesis of ApxIIA439-801 antigen protein in transgenic rice callus was confirmed by western blot analysis. The concentration of antigen protein in lyophilized samples of transgenic rice callus was 250 µg/g. Immunizing mice with protein extracts from transgenic plants intranasally elicited secretory IgA. These results demonstrate the feasibility of using a transgenic plant to elicit immune responses against A. pleuropneumoniae.

Viral hemorrhagic septicemia virus glycoprotein production in tobacco.

Viral hemorrhagic septicemia virus (VHSV) causes mortality in numerous marine and freshwater fish species resulting in heavy losses in fish farming. The glycoprotein gene of VHSV was fused with the cholera toxin B subunit (CTB) and expressed transiently in leaf tissues of Nicotiana benthamiana via the agroinfiltration method. The glycoprotein gene was divided into two parts to improve assembly of CTB fusion proteins (CTB-VHSV99-235 and CTB-VHSV258-417). Production of CTB fusion proteins was confirmed in the agroinfiltrated leaf tissue by western blot analysis. The plant-produced CTB fusion proteins showed biological activity to GM1-ganglioside, a receptor for biologically active CTB, on GM1-ELISA. The expression level of the CTB-VHSV fusion proteins was 0.86% (CTB-VHSV99-235) and 0.93% (CTB-VHSV258-417) of total proteins in agroinfiltrated leaf tissue, as determined by GM1-ELISA. These results suggest that Agrobacterium-mediated transient expression of CTB fusion antigens of VHSV is a rapid and convenient method and demonstrate the feasibility of using agroinfiltrated plant leaf tissues expressing CTB-fusion antigens as a plant-based vaccine to prevent VHSV infection.

Expression of an immunogenic LTB-based chimeric protein targeting Zaire ebolavirus epitopes from GP1 in plant cells.

KEY MESSAGE: An antigenic protein targeting two epitopes from the Zaire ebolavirus GP1 protein was expressed in plant cells rendering an antigen capable of inducing humoral responses in mouse when administered subcutaneously or orally. The 2014 Ebola outbreak made clear that new treatments and prophylactic strategies to fight this disease are needed. Since vaccination is an intervention that could achieve the control of this epidemic disease, exploring the production of new low-cost vaccines is a key path to consider; especially in developing countries. In this context, plants are attractive organisms for the synthesis and delivery of subunit vaccines. This study aimed at producing a chimeric protein named LTB-EBOV, based on the B subunit of the Escherichia coli heat-labile enterotoxin as an immunogenic carrier and two epitopes from the Zaire ebolavirus GP1 protein recognized by neutralizing antibodies. The LTB-EBOV protein was expressed in plant tissues at levels up to 14.7 µg/g fresh leaf tissue and proven to be immunogenic in BALB/c mice when administered by either subcutaneous or oral routes. Importantly, IgA and IgG responses were induced following the oral immunization. The potential use of the plant-made LTB-EBOV protein against EBOV is discussed.

Towards the development of an oral vaccine against porcine cysticercosis: expression of the protective HP6/TSOL18 antigen in transgenic carrots cells.

MAIN CONCLUSION: The Taenia solium HP6/TSOL18 antigen was produced in carrot cells, yielding an immunogenic protein that induced significant protection in an experimental murine model against T. crassiceps cysticercosis when orally administered. This result supports the potential of HP6/TSOL18-carrot as a low-cost anti-cysticercosis vaccine candidate. Cysticercosis is a zoonosis caused by Taenia solium that can be prevented by interrupting the parasite life cycle through pig vaccination. Several injectable vaccine candidates have been reported, but the logistic difficulties and costs for its application limited its use in nationwide control programs. Oral plant-based vaccines can deal with this limitation, because of their easy administration and low cost. A stable expression of the HP6/TSOL18 anti-T. solium cysticercosis protective antigen in carrot calli transformed with an optimized transgene is herein reported. An antigen accumulation up to 14 µg g(-1) of dry-weight biomass was achieved in the generated carrot lines. Mouse immunization with one of the transformed calli induced both specific IgG and IgA anti-HP6/TSOL18 antibodies. A statistically significant reduction in the expected number of T. crassiceps cysticerci was observed in mice orally immunized with carrot-made HP6/TSOL18, in a similar extent to that obtained by subcutaneous immunization with recombinant HP6/TSOL18 protein. In this study, a new oral plant-made version of the HP6/TSOL18 anti-cysticercosis vaccine is reported. The vaccine candidate should be further tested against porcine cysticercosis.

Plant crude extracts containing oligomeric hemagglutinins protect chickens against highly Pathogenic Avian Influenza Virus after one dose of immunization.

Highly pathogenic avian influenza viruses (HPAIV) have been responsible for causing several severe outbreaks across the world. To protect poultry farms and to prevent the possible spread of new influenza pandemics, vaccines that are both efficacious and low-cost are in high demand. We produced stable, large hemagglutinin H5 oligomers in planta by the specific interaction between S•Tag and S•Protein. H5 oligomers combined via S•Tag::S•Protein interaction in plant crude extracts induced strong humoral immune responses, strong neutralizing antibody responses, and resistance in chickens after challenge with a wild type HPAIV H5 virus strain. In all three parameters, plant crude extracts with H5 oligomers induced better responses than crude extracts containing trimers. The neutralizing antibodies induced by by two-dose and one dose immunization with an adjuvanted crude extract containing H5 oligomer protected vaccinated chickens from two lethal H5N1 virus strains with the efficiency of 92% and 100%, respectively. Following housing vaccinated chickens together with ten non-immunized chickens, only one of these chickens had detectable levels of the H5N1 virus. To facilitate the easy storage of a candidate vaccine, the H5 oligomer crude extracts were mixed with adjuvants and stored for 3.5 and 5.5 months at 4 °C, and chickens were immunized with these crude extracts. All these vaccinated chickens survived after a lethal H5N1 virus challenge. H5 oligomer crude extracts are comparable to commercial vaccines as they also induce strong virus-neutralizing immune responses following the administration of a single dose. The cost-effective production of plant crude extract vaccine candidates and the high stability after long-term storage will enable and encourage the further exploration of this technology for veterinary vaccine development.

Engineering of a plant-produced virus-like particle to improve the display of the Plasmodium falciparum Pfs25 antigen and transmission-blocking activity of the vaccine candidate.

Malaria kills around 409,000 people a year, mostly children under the age of five. Malaria transmission-blocking vaccines work to reduce malaria prevalence in a community and have the potential to be part of a multifaceted approach required to eliminate the parasites causing the disease. Pfs25 is a leading malaria transmission-blocking antigen and has been successfully produced in a plant expression system as both a subunit vaccine and as a virus-like particle. This study demonstrates an improved version of the virus-like particle antigen display molecule by eliminating known protease sites from the prior A85 variant. This re-engineered molecule, termed B29, displays three times the number of Pfs25 antigens per virus-like particle compared to the original Pfs25 virus-like particle. An improved purification scheme was also developed, resulting in a substantially higher yield and improved purity. The molecule was evaluated in a mouse model and found to induce improved transmission-blocking activity at lower doses and longer durations than the original molecule.

The immunogenicity of plant-based COE-GCN4pII protein in pigs against the highly virulent porcine epidemic diarrhea virus strain from genotype 2.

Porcine epidemic diarrhea virus (PEDV) is a serious infectious causative agent in swine, especially in neonatal piglets. PEDV genotype 2 (G2) strains, particularly G2a, were the primary causes of porcine epidemic diarrhea (PED) outbreaks in Vietnam. Here, we produced a plant-based CO-26K-equivalent epitope (COE) variant from a Vietnamese highly virulent PEDV strain belonging to genotype 2a (COE/G2a) and evaluated the protective efficacy of COE/G2a-GCN4pII protein (COE/G2a-pII) in piglets against the highly virulent PEDV G2a strain following passive immunity. The 5-day-old piglets had high levels of PEDV-specific IgG antibodies, COE-IgA specific antibodies, neutralizing antibodies, and IFN-γ responses. After virulent challenge experiments, all of these piglets survived and had normal clinical symptoms, no watery diarrhea in feces, and an increase in their body weight, while all of the negative control piglets died. These results suggest that the COE/G2a-pII protein produced in plants can be developed as a promising vaccine candidate to protect piglets against PEDV G2a infection in Vietnam.

Oral Immunization With Plant-Based Vaccine Induces a Protective Response Against Infectious Bursal Disease.

Infectious bursal disease virus (IBDV) is the etiological agent of an immunosuppressive and highly contagious disease that affects young birds causing important economic losses in the poultry industry worldwide. We have previously developed a plant-based vaccine candidate for infectious bursal disease (IBD) that is able to protect against infection with IBDV when administered through intramuscular (im) route. Given that oral vaccination is non-invasive and stimulates the immunity of the mucosal gastrointestinal surface, the initial site of contact and entry of IBDV, the aim of this work was to study if our immunogen was also able to elicit a protective immune response when orally administered. We demonstrated that 85% of the animals that received two oral doses of the vaccine formulation and all animals that were orally boosted after an im prime scheme developed virus neutralizing antibodies and were protected against IBDV infection, evidenced by the bursa/body weight (BB) ratio, absence of T-cell infiltration, and low viral load in bursa. Although mild to moderate bursal damage was observed in some of these animals, these lesions were not as severe as the ones observed in challenged control groups, which also presented signs of acute inflammation, bursal atrophy, T-cell infiltration, and absence of viral clearance. These results show that two immunizations with our recombinant immunogen are able to induce a specific and protective immune response in chicken against IBDV when orally administered in a prime/boost scheme or when the oral boost follows an im prime scheme. In conclusion, our oral plant-based vaccine candidate could represent a viable alternative to conventional vaccines and is of great interest to the poultry industry.

Immunogenicity Studies of Plant-Produced SARS-CoV-2 Receptor Binding Domain-Based Subunit Vaccine Candidate with Different Adjuvant Formulations.

Due to the rapid transmission of the coronavirus disease 2019 (COVID-19) causing serious public health problems and economic burden, the development of effective vaccines is a high priority for controlling the virus spread. Our group has previously demonstrated that the plant-produced receptor-binding domain (RBD) of SARS-CoV-2 fused with Fc of human IgG was capable of eliciting potent neutralizing antibody and cellular immune responses in animal studies, and the immunogenicity could be improved by the addition of an alum adjuvant. Here, we performed a head-to-head comparison of different commercially available adjuvants, including aluminum hydroxide gel (alum), AddaVax (MF59), monophosphoryl lipid A from Salmonella minnesota R595 (mPLA-SM), and polyinosinic-polycytidylic acid (poly(I:C)), in mice by combining them with plant-produced RBD-Fc, and the differences in the immunogenicity of RBD-Fc with different adjuvants were evaluated. The specific antibody responses in terms of total IgG, IgG1, and IgG2a subtypes and neutralizing antibodies, as well as vaccine-specific T-lymphocyte responses, induced by the different tested adjuvants were compared. We observed that all adjuvants tested here induced a high level of total IgG and neutralizing antibodies, but mPLA-SM and poly (I:C) showed the induction of a balanced IgG1 and IgG2a (Th2/Th1) immune response. Further, poly (I:C) significantly increased the frequency of IFN-γ-expressing cells compared with control, whereas no significant difference was observed between the adjuvanted groups. This data revealed the adjuvants' role in enhancing the immune response of RBD-Fc vaccination and the immune profiles elicited by different adjuvants, which could prove helpful for the rational development of next-generation SARS-CoV-2 RBD-Fc subunit vaccines. However, additional research is essential to further investigate the efficacy and safety of this vaccine formulation before clinical trials.

Plant-Based COVID-19 Vaccines: Current Status, Design, and Development Strategies of Candidate Vaccines.

The prevalence of the coronavirus disease 2019 (COVID-19) pandemic in its second year has led to massive global human and economic losses. The high transmission rate and the emergence of diverse SARS-CoV-2 variants demand rapid and effective approaches to preventing the spread, diagnosing on time, and treating affected people. Several COVID-19 vaccines are being developed using different production systems, including plants, which promises the production of cheap, safe, stable, and effective vaccines. The potential of a plant-based system for rapid production at a commercial scale and for a quick response to an infectious disease outbreak has been demonstrated by the marketing of carrot-cell-produced taliglucerase alfa (Elelyso) for Gaucher disease and tobacco-produced monoclonal antibodies (ZMapp) for the 2014 Ebola outbreak. Currently, two plant-based COVID-19 vaccine candidates, coronavirus virus-like particle (CoVLP) and Kentucky Bioprocessing (KBP)-201, are in clinical trials, and many more are in the preclinical stage. Interim phase 2 clinical trial results have revealed the high safety and efficacy of the CoVLP vaccine, with 10 times more neutralizing antibody responses compared to those present in a convalescent patient's plasma. The clinical trial of the CoVLP vaccine could be concluded by the end of 2021, and the vaccine could be available for public immunization thereafter. This review encapsulates the efforts made in plant-based COVID-19 vaccine development, the strategies and technologies implemented, and the progress accomplished in clinical trials and preclinical studies so far.

Plant-Expressed Receptor Binding Domain of the SARS-CoV-2 Spike Protein Elicits Humoral Immunity in Mice.

The current 15-month coronavirus disease-19 (COVID-19) pandemic caused by SARS-CoV-2 has accounted for 3.77 million deaths and enormous worldwide social and economic losses. A high volume of vaccine production is urgently required to eliminate COVID-19. Inexpensive and robust production platforms will improve the distribution of vaccines to resource-limited countries. Plant species offer such platforms, particularly through the production of recombinant proteins to serve as immunogens. To achieve this goal, here we expressed the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein in the glycoengineered-tobacco plant Nicotiana benthamiana to provide a candidate subunit vaccine. This recombinant RBD elicited humoral immunity in mice via induction of highly neutralizing antibodies. These findings provide a strong foundation to further advance the development of plant-expressed RBD antigens for use as an effective, safe, and inexpensive SARS-CoV-2 vaccine. Moreover, our study further highlights the utility of plant species for vaccine development.

Plant-Derived Trimeric CO-26K-Equivalent Epitope Induced Neutralizing Antibodies Against Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) is a causative agent of a highly infectious disease with a high mortality rate, especially in newborn piglets in Asian countries resulting in serious economic loss. The development of a rapid, safe, effective and cost-efficient vaccine is crucial to protect pigs against PEDV infection. The COE antigen is regarded to be a major target for subunit vaccine development against PEDV infection. The naturally assembled COE protein forms a homotrimeric structure. In the present study, we successfully produced a trimeric COE protein as a native structure by fusion with the C-terminal isoleucine zipper trimerization (GCN4pII) motif in Nicotiana benthamiana, with a high expression level shown via semi-quantified Western blots. Trimeric COE protein was purified via immobilized metal affinity chromatography (IMAC), and its trimeric structure was successfully demonstrated by a cross-linking reaction, and a native PAGE gel. A crude extract containing the COE trimer was used for evaluating immunogenicity in mice. After 1 and 2 booster immunizations, the crude extract containing trimeric COE elicited elevated PEDV-specific humoral responses, as demonstrated by ELISA and Western blot analyses. Notably, a virus-neutralizing antibody assay indicated that the neutralization activities of sera of mice vaccinated with the crude extract containing COE-GCN4pII were similar to those of mice vaccinated with a commercial vaccine. These results suggest that crude extract containing trimeric COE is a promising plant-based subunit vaccine candidate for PEDV prevention.