Development of a candidate TMV epitope display vaccine against SARS-CoV2.

Development of a candidate TMV epitope display vaccine against SARS-CoV2. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development of a candidate TMV epitope display vaccine against SARS-CoV2. Kelvin Bongani Phiri, Larry Grill This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3054976/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Essential in halting the COVID-19 pandemic caused by SARS-CoV2, it is crucial to have stable, effective, and easy-to-manufacture vaccines. We developed a potential vaccine using a tobacco mosaic virus (TMV) epitope display model presenting peptides derived from the SARS-CoV2 spike protein. The TMV-epitope fusions in laboratory tests demonstrated binding to the SARS-CoV2 polyclonal antibodies. The fusion constructs maintained critical epitopes of the SARS-CoV-2 spike protein, and two in particular spanned regions of the receptor binding domain that have mutated in the more recent SARS-CoV2 variants. This would allow for rapid modification of vaccines in response to changes in circulating variants. The TMV-peptide fusion constructs also remained stable for over 28 days when stored at temperatures between − 20 and 37°C, an ideal property when targeting developing countries. Immunogenicity studies conducted on BALB/c mice elicited robust antibody responses against SARS-CoV2. A strong IFNγ response was also observed in immunized mice. Three of the six TMV-peptide fusion constructs produced virus-neutralizing titers, as measured by a pseudovirus neutralization assay. These TMV-peptide fusion constructs can be combined to make a multivalent vaccine that could be adapted to meet changing virus variants. These findings demonstrate the development of a stable COVID-19 vaccine candidate by combining SARS-CoV2 spike protein-derived peptides presented on the surface of a TMV nanoparticle. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The pandemic that has plagued the world in the past few years has been caused by one of seven identified coronaviruses (CoVs) that can infect humans. Previously, severe acute respiratory syndrome CoV (SARS-CoV) and Middle Eastern respiratory syndrome CoV (MERS-CoV) were responsible for heightened awareness of the potential for CoVs to cause a global pandemic[ 1 ]. 2019 saw the emergence of the Wuhan strain, now called SARS-CoV2, which causes what is commonly known as coronavirus disease 2019 (COVID-19). Infection with SARS-CoV2 typically leads to high mortality and morbidity[ 2 ]. Over 6 million people have died from SARS-CoV2 infections since the pandemic started[ 3 ]. Although the mortality rate has steadily decreased since the height of the initial wave, the continued emergence of new variants hinders efforts to control the pandemic. There is still a pressing need to develop new, highly adaptable vaccines to meet the challenge posed by new viral strains as they develop. Currently, vaccines are only widely available to developed countries, leaving out an extensive reservoir of people in which the virus may spread and mutate, thus prolonging the eradication of the disease. Vaccines that are efficient, cost-effective and stable in non-refrigerated conditions would benefit society. Different types of vaccines have been developed for SARS-CoV2. These include live-attenuated (Sputnik, Russia), inactivated (Sinovac), pseudotyped-virus vectors, nucleic acid-based vaccines (Moderna, Pfizer-BioNTech), and protein subunit vaccines (Novavax). SARS-CoV2 has heralded unparalleled advancement in vaccine research and development due to the real-time sharing of research data and more available funding for research. All the vaccines and manufacturing technologies employed against COVID-19 have pros and cons. In addition to stimulating robust humoral and cellular immunity, vaccines need to cater to the speed of manufacturing scale-up, environmental stability, and delivery formulation. Adaptive mutations in the viral genome can alter the virus's pathogenic potential. This includes enhanced binding affinity to the cellular receptor ACE2 and the virus's ability to evade the immune system, thus complicating the vaccine development process. SARS-CoV2, like other RNA viruses, is prone to genetic evolution while adapting to new hosts with the development of mutations over time. Consequently, multiple variants with different characteristics compared to their ancestral strains may emerge. Throughout this pandemic, SARS-CoV2 variants have been associated with enhanced transmissibility, reduced neutralization by antibodies, the ability to evade detection, and/or decreased effectiveness of therapeutics and vaccines. Five variants: Alpha (B.1.1.7); Beta (B.1.351); Gamma (P.1); Delta (B.1.617.2); and Omicron (B.1.1.529), have been identified, and they all have mutations in the receptor binding domain of the spike protein[ 4 ]. These mutations have impacted vaccine development and efficacy. This rapid mutation rate needs to be considered when developing vaccines against SARS-CoV2. Nanoparticle (NP)-based vaccines can be delivered through various routes and elicit potent innate and adaptive immune responses in humans and animals. They are easily adapted to specific pathogens and can readily be changed in response to changing or emerging viral strains[ 6 , 7 ]. Tobacco mosaic virus (TMV) epitope display particles are one type of nanoparticle-based vaccine. This technology has previously proven clinically efficacious for non-Hodgkin's lymphoma[ 8 ] and malaria[ 9 , 10 ]. The use of plants as a manufacturing platform for vaccines and therapeutics has been well documented, and many plant-manufactured products show great promise in preclinical testing[ 11 – 13 ]. A plant-made quadrivalent influenza virus vaccine completed Phase III clinical testing recently, and it compared favorably to a commercial quadrivalent vaccine[ 14 , 15 ]. Plant viruses are harmless to animals while being effective immunogens. This characteristic has been used to study the function and maturation of antigen-presenting cells (APCs). This characteristic of plant viruses, particularly TMV, is advantageous as TMV does have adjuvanting effects when used as an epitope display particle in vaccines[ 16 ]. While one of the limitations of TMV genetic fusions is the size of the epitope that can be fused to the TMV coat protein[ 17 ], a helical linker allows larger peptides to be used[ 18 ]. The largest documented peptide successfully genetically fused to TMV has been 120aa long[ 18 ]. TMV-based vaccines are potent activators of dendritic cells[ 19 ]. TMV epitope display technology is highly adaptable and can quickly be modified to meet the rapidly changing disease landscape[ 17 , 20 , 21 ]. Preclinical trials have shown that these vaccines are efficacious[ 15 , 22 , 23 ]. To compensate for the smaller peptides displayed, TMV epitope display vaccines lend themselves easily to mixing and matching epitopes into a multivalent vaccine. This makes these vaccines adaptable to rapidly mutating viruses, such as SARS-CoV2. Here, we described the development of a candidate TMV-epitope display vaccine that displays epitopes from the SARS-CoV2 spike glycoprotein (S). This protein binds to the cellular receptor ACE2 and facilitates viral fusion to the host cells[ 24 ]. We showed that the production of the vaccine is robust and that the candidate vaccine induced high neutralizing antibody titers in mice. Methods Peptide design Peptides were designed by considering the major immunodominant protein. For SARS-CoV2, this is the S protein. B and T-cell epitopes of the S protein have been mapped and published[ 25 – 28 ]. The epitopes used in this project utilized this knowledge when designing epitopes (Table 1 ). Prediction of the secondary and tertiary structure of the S-protein was done to predict possible antigenic portions of the S protein. The length of epitopes was limited to the length of the tertiary structure in which a known epitope resides. Also, an attempt to make each peptide multi-epitopic was made without making the new peptide too long to attach to the TMV-CP. Sixteen epitopes were designed (Table 1 ) for fusion to the TMV CP. Table 1 Epitopes used in the study Name Position Epitope Sequence Mass (kDa) A 300–330 KCTLKSFTVEKGIYQTSNFRVQPTESIVRFP 24.19 B 365–395 YSVLYNSASFSTFKCYGVSPTKLNDLCFTNV 22.06 C 420–440 DYNYKLPDDFTGCVIAWNSNN 20.95 D 435–480 AWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPC 23.92 E 420–500 DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT 27.91 F 440–500 NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT 25.59 G 420–540 DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN 32.23 H 481–540 NGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN 25.18 I 475–500 AGSTPCNGVEGFNCYFPLQSYGFQPT 21.37 J 520–540 APATVCGPKKSTNLVKNKCVN 20.76 K 660–680 YECDIPIGAGICASYQTQTNS 20.82 L 660–710 YECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNN 24.02 M 990–1035 EVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLG 23.65 S21P2 709–727 PSKPSKRSFIEDLLFNKV 20.7 S14P5 552–570 TESNKKFLPFQQFGRDIA 20.72 * TMV coat protein (17.9kDa) TMV expression and virus purification The TMV coat protein (CP) gene was genetically fused at the C-terminus with the SARS-CoV2 epitopes we designed via a helical linker. The recombinant pJLTRBO plasmids were transformed into chemically competent Agrobacterium tumefaciens (GV3101)[ 29 ]. As Henderson et al . (2020)[ 30 ] indicated, transformed Agrobacterium was vacuum infiltrated into Nicotiana benthamiana plants. After 14 days, the virus was extracted from leaf tissue as described in Chapman S.N. (1998)[ 31 ] with slight modifications to the extraction buffer composition (50mM sodium acetate, 0.1% sodium metabisulfite (w/v), 0.01% BME (v/v), pH 5.0). The viral particles were further purified by sucrose density gradient centrifugation as described in Bruckman and Steinetz (2014)[ 32 ]. SDS PAGE, Western Blot and ELISA For SDS–PAGE, purified TMV virus extracts were prepared 1:1 in Laemmli sample buffer (BioRad #1610737) with beta-mercaptoethanol, boiled and run on 4–20% Mini-Protean TGX gels (BioRad #4561096) in 1× Tris/Glycine/SDS buffer and stained with BioSafe Coomassie G-250 stain (BioRad #1610787) according to manufacturer's instructions. Western blot analysis was performed as in McComb et al . (2015)[ 33 ] with the following modifications. Primary antibodies were diluted in blocking buffer at 1:3000 (Rabbit anti-SARS-CoV2 spike polyclonal, MyBioSource #MBS434243) or 1:500 (Rabbit anti-TMV polyclonal, Agdia #57400). Secondary antibodies were diluted at 1:3000 (Goat anti-Rabbit HRP, BioRad #1706515). Opti-4CN (BioRad #1708235) reagent was added for 5 minutes for detection. Images were acquired using a 5-megapixel camera. For Indirect ELISA analysis, 96-well plates were coated with SARS-CoV2 spike protein (MyBioSource #MBS1560493) or purified TMV virus in 100 mM Bicarbonate/Carbonate buffer (0.03 M Na2CO3/0.07 M NaHCO3, pH 9.6) overnight at 4°C. The primary antibodies were diluted in 5% non-fat milk in TBST and incubated at 4°C overnight. Secondary antibody dilutions were incubated at room temperature for 2hrs. O-phenylenediamine dihydrochloride (OPD) substrate (Thermo Scientific™ #34006) was used according to the manufacturer's instruction, developed for 30 min, and read at 450 nm. For serum antibody analyses, mouse serum samples were diluted 3-fold beginning at 1:100 in 5% non-fat milk in phosphate based saline. Goat anti-mouse IgG HRP (BioRad #172–1011) was used at 1:3000 for secondary antibody detection. Antibody endpoint titers were determined as the last dilution at which the OD 450 was above the cutoff. Cutoff values, determined for each dilution, were the upper prediction limit at a 95% confidence level using the Student t-distribution derived from preimmune serum samples (n = 16)[ 33 ]. Data were analyzed using Microsoft Excel. Graphs and statistical analysis between groups were done using GraphPad Prism 9.3.1. Antigenicity study All animal experiments followed IACUC standards at Pocono rabbit farms and laboratory. Seven-week-old female BALB/c mice received two subcutaneous injections two weeks apart on days 1 (50µg) and 15 (100µg) SARS-CoV2 S, TMV-(A, D, F, H, L, S21P2), wtTMV and buffer (0.01M phosphate buffer, pH7.2) + adjuvant. All samples were mixed 1:1 with AddaVax™ (InvivoGen #vac-adx-10). Serum was harvested from anaesthetized mice (five mice per group) in the bleed groups by taking blood samples from the retro-orbital sinus one day before each vaccination (days 0, 14, 28). Dot blot Analysis Dot blots were used to analyze the sera obtained from the antigenicity study in mice. Each piece of nitrocellulose was spotted, from left to right, with wtTMV, SARS-CoV2 spike protein, and phosphate buffer pH7.2. The spots were allowed to dry, and the membranes were blocked with 5% fat-free milk in TBST for 1 hr at room temperature with shaking. The membranes were then incubated in mouse sera diluted 1:500 in blocking buffer overnight at 4°C. A rabbit anti-mouse (HRP) antibody was used as the secondary antibody, and the blots were developed by incubating the blots in Clarity Western ECL Substrate (BioRad #170–5061). Visualization of the blot and imaging was done in the UVP BiospectrumAC Darkroom. IFNγ detection by ELISA To investigate the level of IFNγ production in mice, the IFNγ ELISA kit from Abcam (ab100689 IFNγ Mouse ELISA Kit) was used following the manufacturer's instructions. In short, 100µL of each recombinant Mouse IFNγ standard and sample was added into wells and incubated overnight at 4°C with gentle shaking. 100µL of biotinylated IFNγ detection antibody was added to each well after four washes and incubated for 1 hour at room temperature. 100µL of HRP-Streptavidin solution was then added and incubated for 45 minutes at room temperature, followed by 100µL of TMB One-Step Substrate Reagent to each well and incubated for 30 minutes at room temperature in the dark with gentle shaking. After incubation, 50 µL of stop solution was added to each well, and the results read at 450nm. Neutralization Neutralization titers were determined using the COVID-19 pseudovirus Neutralizing Antibody Assay (Luciferase)(Abnova #KA6152) following the manufacturer's instructions. 1x105 cells/well HEK293T-ACE2 cells in DMEM(10% FBS) were seeded in 24-well plates 5 hours before beginning the neutralization assay. In a separate microcentrifuge tube, 50µL of the diluted sample (either mouse serum or mAb) and 20µL pseudovirus expressing luciferase were mixed and incubated at room temperature for 30 minutes. The mixture was added to wells containing HEK293-ACE2 cells, and the plates were incubated at 37°C for 48 hours. Media from each well was removed, and cells were washed with 200 µL PBS. 150 µL Luciferase Cell Culture Lysis Reagent was used to harvest cells from plates, and 10 µL of this lysate was mixed with 50 µL Luciferase Assay Reagent in 96-well plates. A luminescence microplate reader was used to detect the luciferase expression. Thermal stability The thermal stability of the recombinant TMV constructs was tested by incubating 100µg samples at -20°C, 4°C, 25°C and 37°C for 28 days. On the 28th day, the samples were run on an SDS page gel and stained with Coomassie blue (BioRad), and the resultant bands were analyzed for protein degradation. Results TMV expression and virus purification Classic symptoms of TMV infection appeared within 14 days post infiltration (dpi) for all the constructs. Purified virus extracts were analyzed using SDS–PAGE, revealing the expected size for all the constructs in Table 1 . Yields of wtTMV averaged above 1mg virus/g fresh leaf weight. The average yield was calculated from five different extractions from 10 plants. The yields from the TMV-epitope fusions varied significantly. TMV-F had the lowest yield (0.3mg/g), while TMV-K L14 (0.8mg/g). There was minimal variation in the yield between extractions (Fig. 3A). Western blot with anti-TMV coat protein antibody confirmed the identity of extracted coat protein monomers and their respective sizes (Fig. 3). All six TMV-epitope constructs selected for the animal study showed cross-reactivity with polyclonal sera raised in rabbits against TMV (Fig. 3D) and SARS-CoV2 spike protein (Fig. 3E). Not surprisingly, neither the wtTMV coat protein nor SARS-CoV2 spike protein showed cross-reactivity with anti-S protein or anti-TMV antibodies, respectively (Fig. 3D and E) An ELISA assay was employed to detect the SARS-CoV2 peptides on the TMV CP surface. To allow detection of the fused peptides, each TMV-epitope fusion was used to coat ELISA plates, which were then probed with anti-SARS-CoV2 spike protein polyclonal antibodies. The binding affinity of the epitopes was analyzed by observing the absorbance of the different constructs in the ELISA assay. The binding affinity is directly proportional to absorbance[ 34 ]. All the constructs showed varying levels of binding affinity to the anti-SARS-CoV2 spike protein polyclonal antibodies. TMV-A, TMV-D, and TMV-S14P5 showed the highest binding affinity, while TMV-K, TMV-M and TMV-N had the lowest (Fig. 3B) Antigenicity study Vaccine Immunogenicity The ability of our candidate vaccines to promote an immune response targeting SARS-CoV2 was assessed in female BALB/c mice. On days 1 (50µg) and 15 (100µg), mice were immunized subcutaneously (s.c.) with one of the candidate vaccine constructs. The vaccine was formulated with AddaVax at a 1:1 ratio. Blood was drawn for antibody analysis on days 0, 14, and 28. The serum was tested for total IgG reactivity against SARS-CoV2 spike protein via dot-blots (Fig. 4) and ELISA, utilizing histidine-tagged S1 (S1-6His) from SARS-CoV2 as the capture antigen (Fig. 5). Dot blot analysis showed that the sera reacted to both wtTMV and SARS-CoV2 spike protein in the cases of TMV-(A, D, F, H, and L). TMV-S21P2 sera reacted only to TMV and not to SARS-CoV2 spike protein (Fig. 4). The buffer + adjuvant control did not induce cross-reactive antibodies against either wtTMV or SARS-CoV2 spike protein. ELISA analysis of preimmune serum extracted on day 0 showed no reactivity to immobilized SARS-CoV2 spike protein or TMV antigen (Fig. 5A). All mice vaccinated with the TMV-epitope fusions showed cross-reactive IgG antibodies to SARS-CoV2 spike protein by day 28 (Fig. 5A). Serum from mice vaccinated with wtTMV showed no reactivity to SARS-CoV2 spike protein, while serum from mice vaccinated with SARS-CoV2 spike protein showed no reactivity to wtTMV. SARS-CoV2 IgGs were present in all the sera from mice vaccinated with the TMV-epitope fusions on day 14 post-vaccination, and the titers increased by day 28 (Fig. 5A), except for mice vaccinated with TMV-S21P2, which showed a very low titer on both day 14 and day 28 (Fig. 5A). IFNγ detection Vaccination of mice with the TMV-epitope fusions resulted in an inflammatory response measured by the amount of IFNγ 28 dpi (Fig. 5B). IFNγ concentrations were determined by ELISA and comparing concentrations to those of known standards. Although AddaVax was used as an adjuvant, the constructs elicited a much higher IFNγ response. wtTMV also had lower IFNγ levels than the fusion constructs. Peptides A, D and H had the highest levels of IFNγ, while the other three had levels similar to that of wtTMV. Neutralization IgG titers are an essential measure of vaccine performance; however, that alone does not directly translate to protection. More important is the ability of a vaccine to elicit a neutralizing immune response that protects the host by preventing viral binding or entry into host cells. We performed a neutralization assay using a COVID-19 Pseudovirus Neutralizing Antibody Luciferase Assay (Abnova). This assay provides a highly sensitive and specific method of quantifying COVID-19 neutralizing antibodies. It consists of neutralizing antibody blockade of lentivirus pseudotyped with COVID-19 spike (S) protein with luciferase (Luc) reporter gene interacting with ACE2 expressing HEK293T cells (HEK293T-ACE2). Luc expression inside the HEK293T-ACE2 signifies viral entry and no neutralizing antibody activity. In contrast, the absence of Luc expression signifies the presence of neutralizing antibody. The Luc reporter system enables high-sensitivity measurement of antibody neutralization. Here we showed that sera from constructs TMV-A (IC 50 – 256.2), TMV-D (IC 50 – 1270.0) and TMV-H (IC 50 – 383.0) elicited a high titer of neutralizing antibodies (Fig. 5D). Figure 5D shows the neutralizing antibody titer at which IC50 is obtained. Only constructs TMV-A, TMV-D and TMV-H showed neutralization higher than 50% at any dilution. Sera from mice vaccinated with the spike protein had a higher neutralizing antibody titer than the TMV-peptide fusions. IC 50 is a crucial parameter used to evaluate the efficacy of antibodies in preventing the replication or infectivity of a specific pathogen. It represents the concentration of antibody needed to inhibit the pathogen's activity by 50%. Here Fig. 5E shows the IC50 for each of the constructs and controls. The constructs TMV-A (IC 50 – 256.2), TMV-D (IC 50 – 1270.0) and TMV-H (IC 50 – 383.0) were neutralizing. Thermal stability The three TMV-epitope fusions that elicited neutralizing antibodies in mice were stored at various temperatures. After 28 days of storage, all the samples did not show any degradation when analyzed in an SDS PAGE using Coomassie staining. Discussion In the COVID-19 vaccine response, developing countries were among the last to receive vaccines, relying on other countries for vaccine development and high-technology production. Utilizing greenhouse-grown plants to produce vaccines provides several benefits that developing countries can use. Production can be rapid, with plants yielding large amounts of the vaccine in approximately a week. The cost of building large plant growth rooms is substantially lower than the expensive production facilities for the current FDA-approved COVID-19 vaccines. In addition, the cost of extracting the vaccines from the plants is also not overly expensive. The development and production of new vaccines for outbreaks and pandemics are within the capabilities of many developing countries. The thermal stability of the plant-produced vaccines will also be advantageous for new vaccines. Several studies have shown that TMV epitope display particles are stable for long periods at 4°C[ 20 , 30 , 33 ]. This is one of the limiting factors hindering the widespread use of the highly successful mRNA vaccines currently on the market[ 35 ]. TMV epitope display vaccine technology is a step toward providing thermostable vaccines that cut down on the logistical problems posed by the need for cold-chain management. Here, we report a platform capable of producing transiently expressed recombinant antigens as TMV epitope display particles in N. benthamiana . We also show the simple purification of the recombinant TMV particles using PEG precipitation and sucrose gradient density centrifugation, which are both easily scalable. The yields obtained are relatively high and consistent over multiple extractions. This lends itself well to ease of production and scale-up. Through genetic fusion of the SARS-CoV2 antigens to the TMV particle, we augment stimulation of the immune system compared to what is achievable with peptides (SARS-CoV2 antigens) alone. Using short peptides allows for adapting a multivalent formulation in response to mutations to the SARS-CoV2 spike protein. An example is the emergence of the Brazil, South Africa, Delta, and Omicron variants that all have mutations in the spike protein RBD, an area covered by peptides D and H used in this study. Changing the sequence of just those two peptides would potentially elicit a variant-specific, protective immune response. The utility of immunity against circulating variants of SARS-CoV2, induced by the TMV-D and TMV-H constructs, can be investigated using the growing repertoire of SARS-CoV2 variant-specific pseudoviruses [ 36 , 37 ]. TMV epitope display vaccines have been shown to be stable at various temperatures[ 30 , 38 ]. Here we showed that the recombinant TMV particles we made were stable for four weeks at 0°C, 4°C, 25°C, and 37°C. One of the biggest challenges that hamstring vaccination campaigns in developing countries is maintaining the cold chain in remote areas. The areas that remain largely unvaccinated are potential reservoirs of pathogens that are the source of continued outbreaks. A vaccine that would remain viable and efficient over a long period would help resolve this issue. With COVID-19, most vaccines require stringent cold temperatures of at least 4°C while the mRNA (Moderna and Pfizer) have even more rigid temperature requirements to maintain viability[ 39 , 40 ]. We selected the S protein as it is the dominant target for neutralizing response in COVID-19 infection[ 27 , 41 ]. We used short peptides because the full-length S protein of other coronaviruses has been implicated in the enhancement of viral infection or pulmonary toxicity in other studies [ 42 ]. While all the candidate vaccines injected into mice elicited a strong IgG response against SARS-CoV2 spike glycoprotein, not all candidate vaccines elicited a neutralizing antibody response. Three of the eight candidates in the mouse trials elicited high neutralizing antibody titers. Although in vitro assays to detect neutralizing antibodies cannot completely predict in vivo protection afforded by a vaccine, such assays are helpful for screening vaccine candidates. This suggests a multivalent formulation containing these three candidates would elicit a strong, protective immune reaction in humans[ 43 ]. As with previous TMV epitope display vaccines and TMV-based VLPs, the presence of the TMV particle has been shown to elicit a potent cellular and humoral immune response [ 44 ]. We suggest that this formulation would perform similarly, which is important for SARS-CoV2 as both arms of the immune system are essential for viral clearance and protection. We also observed high levels of the inflammatory marker IFNγ in mice immunized with TMV-A, TMV-D and TMV-H. These constructs also elicited the highest neutralization levels, suggesting that the selected peptides may contain both B and T-cell epitopes. The peptides A, B and H may elicit a robust T-cell response that leads to the activation of the cellular arm of the immune system[ 16 ]. The correlation between the levels of IFNγ observed in serum from mice immunized with TMV-A, TMV-D and TMV-H and the IC50s seen in the same sera suggest that the IFNγ response observed is SARS-CoV2 mainly specific. Since the start of the COVID-19 pandemic, vaccine development has accelerated at a very high rate. Vaccines using different platforms have emerged, and even more technologies are being developed. These technologies include vaccines based on inactivated viruses, nucleic acid (mRNA and DNA), recombinant spike proteins (including those presented as VLPs), as well as replication-competent or incompetent virus vectors[ 40 ][ 45 ][ 39 ]. Recently, utilization of the 1018 CpG or AS02 adjuvant with a plant-based NP vaccine elicited high neutralizing antibody titers in humans [ 17 ]. As such, we used Adavax, an adjuvant in the same class of adjuvants (squalene-based, oil-in-water nano-emulsion). The successful production of neutralizing antibodies provided here shows the utility of plant-based vaccine technology. When coupled with positive safety data, it suggests that NP vaccines could play a role in combating SARS-CoV2 by eliciting a robust immune response. Abbreviations SARS-CoV2: Severe acute respiratory syndrome coronavirus 2; COVID: coronaviral disease; TMV: Tobacco mosaic virus; NAb: Neutralizing antibody; S: spike; RBD: receptor binding domain; IC50: Half-maximal inhibitory concentration. Declarations Acknowledgements Not applicable. Authors' contributions K.P. and L.G. designed the research; K.P. performed the research; all analyzed data; and K.P. and L.G. wrote the paper, which was edited by all. The author (s) read and approved the final manuscript. Funding Gift from Dr S Glanville Availability of data and materials Not applicable. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests Author details Henry E. Riggs School of Applied Life Sciences, Keck Graduate Institute, Claremont, CA 91711, USA. References Philip V. Coronavirus biology and replication : implications for SARS- . Nat Rev Microbiol [Internet]. 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Modified tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications. Virology. 2006;348:475–88. Werner S, Marillonnet S, Hause G, Klimyuk V, Gleba Y. Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A. Proc Natl Acad Sci U S A [Internet]. 2006;103:17678–83. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1635023&tool=pmcentrez&rendertype=abstract Kemnade JO, Seethammagari M, Collinson-Pautz M, Kaur H, Spencer DM, McCormick AA. Tobacco mosaic virus efficiently targets DC uptake, activation and antigen-specific T cell responses in vivo. Vaccine [Internet]. 2014;32:4228–33. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0264410X14005891 McCormick AA, Palmer KE. Genetically engineered Tobacco mosaic virus as nanoparticle vaccines. Expert Rev Vaccines. 2008;7:33–41. McCormick A a, Corbo T a, Wykoff-Clary S, Nguyen L V, Smith ML, Palmer KE, et al. TMV-peptide fusion vaccines induce cell-mediated immune responses and tumor protection in two murine models. Vaccine [Internet]. 2006 [cited 2012 Sep 22];24:6414–23. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16860441 Wu L. Expression of foot-and-mouth disease virus epitopes in tobacco by a tobacco mosaic virus-based vector*1. Vaccine [Internet]. 2003 [cited 2012 Sep 26];21:4390–8. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0264410X03004286 Chandler GL. Influenza Hemagglutinin Expression in Nicotiana tabacum and Nicotiana benthamiana. Baylor University; 2007. Chakraborti S, Prabakaran P, Xiao X, Dimitrov DS. The SARS coronavirus S glycoprotein receptor binding domain: Fine mapping and functional characterization. Virol J. 2005;2:1–10. Poh CM, Carissimo G, Wang B, Amrun SN, Lee CYP, Chee RSL, et al. Two linear epitopes on the SARS-CoV-2 spike protein that elicit neutralizing antibodies in COVID-19 patients. Nat Commun [Internet]. Springer US; 2020;11. Available from: http://dx.doi.org/10.1038/s41467-020-16638-2 Ahmed SF, Quadeer AA. Preliminary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies. 2020; Maghsood F, Shokri MR, Jeddi-Tehrani M, Rahvar MT, Ghaderi A, Salimi V, et al. Identification of immunodominant epitopes on nucleocapsid and spike proteins of the SARS-CoV-2 in Iranian COVID-19 patients. Pathog Dis. 2022;80:1–10. He Y, Li J, Du L, Yan X, Hu G, Zhou Y, et al. Identification and characterization of novel neutralizing epitopes in the receptor-binding domain of SARS-CoV spike protein : Revealing the critical antigenic determinants in inactivated SARS-CoV vaccine. 2006;24:5498–508. Lindbo JA. TRBO : A High-Efficiency Tobacco Mosaic Virus. Breakthr Technol. 2007;145:1232–40. Henderson EA, Tam CC, Cheng LW, Ngono AE, Nguyen AV, Shresta S, et al. Investigation of the immunogenicity of Zika glycan loop. Virol J. Virology Journal; 2020;17:1–15. Chapman S. Tobamovirus isolation and RNA extraction. In: Foster G., Taylor S., editors. Plant Virol Protoc [Internet]. Totowa, New Jersey: Humana Press Inc; 1998 [cited 2013 Nov 8]. p. 123–30. Available from: http://link.springer.com/protocol/10.1385/0-89603-385-6:123 Bruckman MA, Steinmetz NF. Chemical Modification of the Inner and Outer Surfaces of Tobacco Mosaic Virus (TMV). 2014;173–85. Available from: https://link.springer.com/10.1007/978-1-62703-751-8_13 McComb RC, Ho C, Bradley KA, Grill LK, Martchenko M. Presentation of peptides from Bacillus anthracis protective antigen on Tobacco Mosaic Virus as an epitope targeted anthrax vaccine. Vaccine [Internet]. Elsevier Ltd; 2015;33:6745–51. Available from: http://dx.doi.org/10.1016/j.vaccine.2015.10.075 Bobrovnik SA. Determination of antibody affinity by ELISA. Theory. J Biochem Biophys Methods. 2003;57:213–36. Rosa SS, Prazeres DMF, Azevedo AM, Marques MPC. mRNA vaccines manufacturing: Challenges and bottlenecks. Vaccine [Internet]. 2021;39:2190–200. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0264410X21003194 Kalkeri R, Cai Z, Lin S, Farmer J, Kuzmichev Y V., Koide F. Sars-cov-2 spike pseudoviruses: A useful tool to study virus entry and address emerging neutralization escape phenotypes. Microorganisms. 2021;9. Chen M, Zhang XE. Construction and applications of sars-cov-2 pseudoviruses: A mini review. Int J Biol Sci. 2021;17:1574–80. Royal JM, Simpson CA, McCormick AA, Phillips A, Hume S, Morton J, et al. Development of a sars-cov-2 vaccine candidate using plant-based manufacturing and a tobacco mosaic virus-like nanoparticle. Vaccines. 2021;9:1–17. Aggarwal A, Mehta S, Gupta D, Sheikh S, Pallagatti S, Singh R, et al. Safety & effectiveness of COVID-19 vaccines: A narrative review. JIndian J Med Res [Internet]. 2022;155:91–104. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23144490 Rahman MM, Masum MHU, Wajed S, Talukder A. A comprehensive review on COVID-19 vaccines: development, effectiveness, adverse effects, distribution and challenges. VirusDisease [Internet]. Springer India; 2022;33:1–22. Available from: https://doi.org/10.1007/s13337-022-00755-1 Tsai TI, Khalili JS, Gilchrist M, Waight AB, Cohen D, Zhuo S, et al. ACE2-Fc fusion protein overcomes viral escape by potently neutralizing SARS-CoV-2 variants of concern. Antiviral Res. 2022;199. Rahman M, Irmler M, Keshavan S, Introna M, Beckers J, Palmberg L, et al. Differential Effect of SARS-CoV-2 Spike Glycoprotein 1 on Human Bronchial and Alveolar Lung Mucosa Models: Implications for Pathogenicity. Viruses. 2021;13:1–18. Banik S, Mansour AA, Suresh RV, Wykoff-Clary S, Malik M, McCormick AA, et al. Development of a Multivalent Subunit Vaccine against Tularemia Using Tobacco Mosaic Virus (TMV) Based Delivery System. PLoS One [Internet]. 2015;10:1–22. Available from: http://dx.plos.org/10.1371/journal.pone.0130858 Shivprasad S, Pogue GP, Lewandowski DJ, Hidalgo J, Donson J, Grill LK, et al. Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors. Virology. 1999;255:312–23. Sukhikh GT, Priputnevich T V., Ogarkova DA, Pochtovyi AA, Kustova DD, Zlobin VI, et al. Sputnik Light and Sputnik V Vaccination Is Effective at Protecting Medical Personnel from COVID-19 during the Period of Delta Variant Dominance. Vaccines. 2022;10:1–11. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3054976","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":210523751,"identity":"7b9aef26-0f2a-4466-8461-7cfd535dd9e0","order_by":0,"name":"Kelvin Bongani Phiri","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYBACCQYehgMMBjYGDMxAHg8JWtJI1AIEhw3APKK0SM7uPXi4ouC8sbw7A+ODt21EaJGWOZdw8IzBbTPDwwzMhnOJ0SInkWNwsMHgto1hMwObNC8JWs6BtLD/JkqLNETLATN5ZgY2ZqK0SM45A9KSbGzAzNgsOeccEVokbvcYf2z4Y2c4v//wwQ9vyojQAowYCDA4wNhAjHokLfLEahgFo2AUjIKRBwAMIDJj0Rcq6wAAAABJRU5ErkJggg==","orcid":"","institution":"Keck Graduate Institute","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Kelvin","middleName":"Bongani","lastName":"Phiri","suffix":""},{"id":210523752,"identity":"affc7b00-15e2-4e9d-bee0-e48de14a5b93","order_by":1,"name":"Larry Grill","email":"","orcid":"","institution":"Keck Graduate Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Larry","middleName":"","lastName":"Grill","suffix":""}],"badges":[],"createdAt":"2023-06-12 20:44:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3054976/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3054976/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":39104089,"identity":"c2d83d2f-1bb1-4775-b5b1-c5e08eaf877e","added_by":"auto","created_at":"2023-06-26 18:03:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":445413,"visible":true,"origin":"","legend":"\u003cp\u003eLandscape of amino acid mutations within the SARS-CoV2 RBD. Alignment of the RBD residues SARS-CoV2 variants is shown, highlighting key residues critical for binding by antibodies and ACE2. The grey bars at the top of the diagram represent two of the peptides used in this study in relation to the RBD and the mutations they cover. The chart was adapted from Yi (2022)[5]\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3054976/v1/f8de7f4f5dc5ee070cc21461.png"},{"id":39104090,"identity":"bc4e1930-7f7d-419f-9c20-11ecf7bcbaf3","added_by":"auto","created_at":"2023-06-26 18:03:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":150831,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of injection and bleed schedule. The immunization regime and dosing schedule in BALB/c mice vaccinated with 50μg of recombinant TMV formulated with AddaVax™ is depicted. All vaccine formulations were administered as a 100μL dose subcutaneously (s.c.). The preimmune bleed was done a day before the 1\u003csup\u003est\u003c/sup\u003e dose on day 0. The second bleed was done on day 14, prior to administering the booster. Day 28 was the final bleed. The vaccination groups are shown in the table.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3054976/v1/a9716749709f8951b5f2a424.png"},{"id":39106151,"identity":"a8ec9c1b-50c1-4424-98a1-c23160efd3e7","added_by":"auto","created_at":"2023-06-26 18:19:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":243773,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of purified TMV-epitope constructs. A shows a comparison of the average virus yield per construct. Purified recombinant TMV was quantified using absorbance at 280nm, and the yield was calculated relative to fresh plant matter. (B) ELISA results showing antigenicity of the TMV-epitope constructs. A 96-well plate was coated with purified TMV-epitope. Rabbit anti-SARS-CoV2 spike polyclonal antibody was used as the primary antibody, and the ELISA was developed using OPD. The images show analysis of the TMV-epitope constructs using SDS PAGE (C). The Western blots were carried out using either anti-TMV (D); or anti-SARS-CoV2 S (E) polyclonal antibody. The blots were loaded as follows: Lane 1: Prestained molecular weight marker (BioRad). Lane 2: TMV-A, Lane 3 TMV-D, Lane 4: TMV-F, Lane 5: TMV-H, Lane 6: TMV-H, Lane 7: TMV-L, Lane 8: TMV-S21P2, Lane 9: SARS-CoV2 spike protein, Lane 10: Ladder.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3054976/v1/3a3aa927514864eb9e9b0810.png"},{"id":39104092,"identity":"472e168e-49ee-41b6-bedf-af767d458e4b","added_by":"auto","created_at":"2023-06-26 18:03:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":448139,"visible":true,"origin":"","legend":"\u003cp\u003eDot blot analysis of sera obtained from vaccinated mice. Three items were spotted on each piece of Nitrocellulose membrane. From left to right, it was wtTMV, SARS-CoV2 spike protein and phosphate buffer pH7.2. The membrane was then dried, blocked, and incubated with a 1:500 dilution of the indicated mouse serum in blocking buffer. After washing, the antibody bound to the membrane was detected using horseradish-peroxidase conjugated secondary antibodies and Clarity Western Substrate (BioRad). The blots were visualized using the UVP Biospectrum darkroom.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3054976/v1/679d743db3a882940fdf592b.png"},{"id":39104095,"identity":"00fbc64e-fe25-46cd-a218-dced9be93953","added_by":"auto","created_at":"2023-06-26 18:03:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":276705,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Shows antibody quantification using endpoint titers against SARS-CoV2 Spike protein using sera from mice immunized with wtTMV, TMV-A, TMV-D, TMV-F, TMV-H, TMV-L and TMV-S21P2 at 14 and 28 dpi. SARS-CoV2 coated ELISA plates were incubated with serum samples taken at various points during the vaccination schedule (preimmune—day 0, two weeks following vaccination—day 14, two weeks following the first bleed—day 28. Reciprocal IgG endpoint titers are plotted as geometric means with error bars from the \"Serum Harvested\" vaccination groups. (B) shows the estimated IFNγ concentrations (pg/mL) in mice are 28 days post-inoculation. Sera from mice was used to coat wells in an ELISA plate and probed with an anti-IFNγ antibody. The concentration of IFNγ in the mice sera was extrapolated from a standard curve generated from known IFNγ concentrations. (C) shows the percentage neutralization of sera taken 28 dpi. Neutralization was carried out using a pseudovirus assay (Abnova). The IC50 (D) was calculated from the neutralization assay, and the IC50 dilution titers are shown in (E). All the data analysis was done using GraphPad Prism.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3054976/v1/6b8b3846e8a2fc11a7dfc67c.png"},{"id":39104767,"identity":"291c7832-fc67-49ea-9926-722aca2f717f","added_by":"auto","created_at":"2023-06-26 18:11:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":272379,"visible":true,"origin":"","legend":"\u003cp\u003eCoomassie-stained SDS PAGE showing the stability of the three TMV-epitope fusions that elicited neutralizing antibodies in mice after storage at -20°C, 4°C, 25°C and 37°C for 28°C days.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3054976/v1/30ae34e6b57f68f4ede4e60d.png"},{"id":39283270,"identity":"92005919-5671-43ba-a6fe-34a7536a5932","added_by":"auto","created_at":"2023-06-29 08:44:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1557353,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3054976/v1/56308320-0a41-4839-8507-6c57494d03e3.pdf"},{"id":39104769,"identity":"6710c4ce-8944-4838-b162-ce7c09838e82","added_by":"auto","created_at":"2023-06-26 18:11:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":996674,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-3054976/v1/756e8706618d775dd4644256.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of a candidate TMV epitope display vaccine against SARS-CoV2.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe pandemic that has plagued the world in the past few years has been caused by one of seven identified coronaviruses (CoVs) that can infect humans. Previously, severe acute respiratory syndrome CoV (SARS-CoV) and Middle Eastern respiratory syndrome CoV (MERS-CoV) were responsible for heightened awareness of the potential for CoVs to cause a global pandemic[\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. 2019 saw the emergence of the Wuhan strain, now called SARS-CoV2, which causes what is commonly known as coronavirus disease 2019 (COVID-19). Infection with SARS-CoV2 typically leads to high mortality and morbidity[\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]. Over 6\u0026nbsp;million people have died from SARS-CoV2 infections since the pandemic started[\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eAlthough the mortality rate has steadily decreased since the height of the initial wave, the continued emergence of new variants hinders efforts to control the pandemic. There is still a pressing need to develop new, highly adaptable vaccines to meet the challenge posed by new viral strains as they develop. Currently, vaccines are only widely available to developed countries, leaving out an extensive reservoir of people in which the virus may spread and mutate, thus prolonging the eradication of the disease. Vaccines that are efficient, cost-effective and stable in non-refrigerated conditions would benefit society. Different types of vaccines have been developed for SARS-CoV2. These include live-attenuated (Sputnik, Russia), inactivated (Sinovac), pseudotyped-virus vectors, nucleic acid-based vaccines (Moderna, Pfizer-BioNTech), and protein subunit vaccines (Novavax). SARS-CoV2 has heralded unparalleled advancement in vaccine research and development due to the real-time sharing of research data and more available funding for research. All the vaccines and manufacturing technologies employed against COVID-19 have pros and cons. In addition to stimulating robust humoral and cellular immunity, vaccines need to cater to the speed of manufacturing scale-up, environmental stability, and delivery formulation.\u003c/p\u003e\n\u003cp\u003eAdaptive mutations in the viral genome can alter the virus\u0026apos;s pathogenic potential. This includes enhanced binding affinity to the cellular receptor ACE2 and the virus\u0026apos;s ability to evade the immune system, thus complicating the vaccine development process. SARS-CoV2, like other RNA viruses, is prone to genetic evolution while adapting to new hosts with the development of mutations over time. Consequently, multiple variants with different characteristics compared to their ancestral strains may emerge. Throughout this pandemic, SARS-CoV2 variants have been associated with enhanced transmissibility, reduced neutralization by antibodies, the ability to evade detection, and/or decreased effectiveness of therapeutics and vaccines. Five variants: Alpha (B.1.1.7); Beta (B.1.351); Gamma (P.1); Delta (B.1.617.2); and Omicron (B.1.1.529), have been identified, and they all have mutations in the receptor binding domain of the spike protein[\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. These mutations have impacted vaccine development and efficacy. This rapid mutation rate needs to be considered when developing vaccines against SARS-CoV2.\u003c/p\u003e\n\u003cp\u003eNanoparticle (NP)-based vaccines can be delivered through various routes and elicit potent innate and adaptive immune responses in humans and animals. They are easily adapted to specific pathogens and can readily be changed in response to changing or emerging viral strains[\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. Tobacco mosaic virus (TMV) epitope display particles are one type of nanoparticle-based vaccine. This technology has previously proven clinically efficacious for non-Hodgkin\u0026apos;s lymphoma[\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e] and malaria[\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]. The use of plants as a manufacturing platform for vaccines and therapeutics has been well documented, and many plant-manufactured products show great promise in preclinical testing[\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. A plant-made quadrivalent influenza virus vaccine completed Phase III clinical testing recently, and it compared favorably to a commercial quadrivalent vaccine[\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. Plant viruses are harmless to animals while being effective immunogens. This characteristic has been used to study the function and maturation of antigen-presenting cells (APCs). This characteristic of plant viruses, particularly TMV, is advantageous as TMV does have adjuvanting effects when used as an epitope display particle in vaccines[\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eWhile one of the limitations of TMV genetic fusions is the size of the epitope that can be fused to the TMV coat protein[\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e], a helical linker allows larger peptides to be used[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. The largest documented peptide successfully genetically fused to TMV has been 120aa long[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. TMV-based vaccines are potent activators of dendritic cells[\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. TMV epitope display technology is highly adaptable and can quickly be modified to meet the rapidly changing disease landscape[\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. Preclinical trials have shown that these vaccines are efficacious[\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. To compensate for the smaller peptides displayed, TMV epitope display vaccines lend themselves easily to mixing and matching epitopes into a multivalent vaccine. This makes these vaccines adaptable to rapidly mutating viruses, such as SARS-CoV2.\u003c/p\u003e\n\u003cp\u003eHere, we described the development of a candidate TMV-epitope display vaccine that displays epitopes from the SARS-CoV2 spike glycoprotein (S). This protein binds to the cellular receptor ACE2 and facilitates viral fusion to the host cells[\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. We showed that the production of the vaccine is robust and that the candidate vaccine induced high neutralizing antibody titers in mice.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003ePeptide design\u003c/p\u003e\n\u003cp\u003ePeptides were designed by considering the major immunodominant protein. For SARS-CoV2, this is the S protein. B and T-cell epitopes of the S protein have been mapped and published[\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. The epitopes used in this project utilized this knowledge when designing epitopes (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Prediction of the secondary and tertiary structure of the S-protein was done to predict possible antigenic portions of the S protein. The length of epitopes was limited to the length of the tertiary structure in which a known epitope resides. Also, an attempt to make each peptide multi-epitopic was made without making the new peptide too long to attach to the TMV-CP. Sixteen epitopes were designed (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) for fusion to the TMV CP.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEpitopes used in the study\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eName\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePosition\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEpitope Sequence\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMass (kDa)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e300\u0026ndash;330\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKCTLKSFTVEKGIYQTSNFRVQPTESIVRFP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e24.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e365\u0026ndash;395\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYSVLYNSASFSTFKCYGVSPTKLNDLCFTNV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e420\u0026ndash;440\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDYNYKLPDDFTGCVIAWNSNN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e435\u0026ndash;480\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23.92\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e420\u0026ndash;500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e440\u0026ndash;500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e420\u0026ndash;540\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e481\u0026ndash;540\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e475\u0026ndash;500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAGSTPCNGVEGFNCYFPLQSYGFQPT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eJ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e520\u0026ndash;540\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAPATVCGPKKSTNLVKNKCVN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e660\u0026ndash;680\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYECDIPIGAGICASYQTQTNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e660\u0026ndash;710\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e24.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e990\u0026ndash;1035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23.65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS21P2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e709\u0026ndash;727\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePSKPSKRSFIEDLLFNKV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS14P5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e552\u0026ndash;570\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTESNKKFLPFQQFGRDIA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e* TMV coat protein (17.9kDa)\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003eTMV expression and virus purification\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe TMV coat protein (CP) gene was genetically fused at the C-terminus with the SARS-CoV2 epitopes we designed via a helical linker. The recombinant pJLTRBO plasmids were transformed into chemically competent \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e (GV3101)[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. As Henderson \u003cem\u003eet al\u003c/em\u003e. (2020)[\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e] indicated, transformed Agrobacterium was vacuum infiltrated into \u003cem\u003eNicotiana benthamiana\u003c/em\u003e plants. After 14 days, the virus was extracted from leaf tissue as described in Chapman S.N. (1998)[\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e] with slight modifications to the extraction buffer composition (50mM sodium acetate, 0.1% sodium metabisulfite (w/v), 0.01% BME (v/v), pH 5.0). The viral particles were further purified by sucrose density gradient centrifugation as described in Bruckman and Steinetz (2014)[\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eSDS PAGE, Western Blot and ELISA\u003c/p\u003e\n\u003cp\u003eFor SDS\u0026ndash;PAGE, purified TMV virus extracts were prepared 1:1 in Laemmli sample buffer (BioRad #1610737) with beta-mercaptoethanol, boiled and run on 4\u0026ndash;20% Mini-Protean TGX gels (BioRad #4561096) in 1\u0026times; Tris/Glycine/SDS buffer and stained with BioSafe Coomassie G-250 stain (BioRad #1610787) according to manufacturer\u0026apos;s instructions. Western blot analysis was performed as in McComb \u003cem\u003eet al\u003c/em\u003e. (2015)[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e] with the following modifications. Primary antibodies were diluted in blocking buffer at 1:3000 (Rabbit anti-SARS-CoV2 spike polyclonal, MyBioSource #MBS434243) or 1:500 (Rabbit anti-TMV polyclonal, Agdia #57400). Secondary antibodies were diluted at 1:3000 (Goat anti-Rabbit HRP, BioRad #1706515). Opti-4CN (BioRad #1708235) reagent was added for 5 minutes for detection. Images were acquired using a 5-megapixel camera. For Indirect ELISA analysis, 96-well plates were coated with SARS-CoV2 spike protein (MyBioSource #MBS1560493) or purified TMV virus in 100 mM Bicarbonate/Carbonate buffer (0.03 M Na2CO3/0.07 M NaHCO3, pH 9.6) overnight at 4\u0026deg;C. The primary antibodies were diluted in 5% non-fat milk in TBST and incubated at 4\u0026deg;C overnight. Secondary antibody dilutions were incubated at room temperature for 2hrs. O-phenylenediamine dihydrochloride (OPD) substrate (Thermo Scientific\u0026trade; #34006) was used according to the manufacturer\u0026apos;s instruction, developed for 30 min, and read at 450 nm. For serum antibody analyses, mouse serum samples were diluted 3-fold beginning at 1:100 in 5% non-fat milk in phosphate based saline. Goat anti-mouse IgG HRP (BioRad #172\u0026ndash;1011) was used at 1:3000 for secondary antibody detection. Antibody endpoint titers were determined as the last dilution at which the OD\u003csub\u003e450\u003c/sub\u003e was above the cutoff. Cutoff values, determined for each dilution, were the upper prediction limit at a 95% confidence level using the Student t-distribution derived from preimmune serum samples (n\u0026thinsp;=\u0026thinsp;16)[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. Data were analyzed using Microsoft Excel. Graphs and statistical analysis between groups were done using GraphPad Prism 9.3.1.\u003c/p\u003e\n\u003cp\u003eAntigenicity study\u003c/p\u003e\n\u003cp\u003eAll animal experiments followed IACUC standards at Pocono rabbit farms and laboratory. Seven-week-old female BALB/c mice received two subcutaneous injections two weeks apart on days 1 (50\u0026micro;g) and 15 (100\u0026micro;g) SARS-CoV2 S, TMV-(A, D, F, H, L, S21P2), wtTMV and buffer (0.01M phosphate buffer, pH7.2)\u0026thinsp;+\u0026thinsp;adjuvant. All samples were mixed 1:1 with AddaVax\u0026trade; (InvivoGen #vac-adx-10). Serum was harvested from anaesthetized mice (five mice per group) in the bleed groups by taking blood samples from the retro-orbital sinus one day before each vaccination (days 0, 14, 28).\u003c/p\u003e\n\u003cp\u003eDot blot Analysis\u003c/p\u003e\n\u003cp\u003eDot blots were used to analyze the sera obtained from the antigenicity study in mice. Each piece of nitrocellulose was spotted, from left to right, with wtTMV, SARS-CoV2 spike protein, and phosphate buffer pH7.2. The spots were allowed to dry, and the membranes were blocked with 5% fat-free milk in TBST for 1 hr at room temperature with shaking. The membranes were then incubated in mouse sera diluted 1:500 in blocking buffer overnight at 4\u0026deg;C. A rabbit anti-mouse (HRP) antibody was used as the secondary antibody, and the blots were developed by incubating the blots in Clarity Western ECL Substrate (BioRad #170\u0026ndash;5061). Visualization of the blot and imaging was done in the UVP BiospectrumAC Darkroom.\u003c/p\u003e\n\u003cp\u003eIFN\u0026gamma; detection by ELISA\u003c/p\u003e\n\u003cp\u003eTo investigate the level of IFN\u0026gamma; production in mice, the IFN\u0026gamma; ELISA kit from Abcam (ab100689 IFN\u0026gamma; Mouse ELISA Kit) was used following the manufacturer\u0026apos;s instructions. In short, 100\u0026micro;L of each recombinant Mouse IFN\u0026gamma; standard and sample was added into wells and incubated overnight at 4\u0026deg;C with gentle shaking. 100\u0026micro;L of biotinylated IFN\u0026gamma; detection antibody was added to each well after four washes and incubated for 1 hour at room temperature. 100\u0026micro;L of HRP-Streptavidin solution was then added and incubated for 45 minutes at room temperature, followed by 100\u0026micro;L of TMB One-Step Substrate Reagent to each well and incubated for 30 minutes at room temperature in the dark with gentle shaking. After incubation, 50 \u0026micro;L of stop solution was added to each well, and the results read at 450nm.\u003c/p\u003e\n\u003cp\u003eNeutralization\u003c/p\u003e\n\u003cp\u003eNeutralization titers were determined using the COVID-19 pseudovirus Neutralizing Antibody Assay (Luciferase)(Abnova #KA6152) following the manufacturer\u0026apos;s instructions. 1x105 cells/well HEK293T-ACE2 cells in DMEM(10% FBS) were seeded in 24-well plates 5 hours before beginning the neutralization assay. In a separate microcentrifuge tube, 50\u0026micro;L of the diluted sample (either mouse serum or mAb) and 20\u0026micro;L pseudovirus expressing luciferase were mixed and incubated at room temperature for 30 minutes. The mixture was added to wells containing HEK293-ACE2 cells, and the plates were incubated at 37\u0026deg;C for 48 hours. Media from each well was removed, and cells were washed with 200 \u0026micro;L PBS. 150 \u0026micro;L Luciferase Cell Culture Lysis Reagent was used to harvest cells from plates, and 10 \u0026micro;L of this lysate was mixed with 50 \u0026micro;L Luciferase Assay Reagent in 96-well plates. A luminescence microplate reader was used to detect the luciferase expression.\u003c/p\u003e\n\u003cp\u003eThermal stability\u003c/p\u003e\n\u003cp\u003eThe thermal stability of the recombinant TMV constructs was tested by incubating 100\u0026micro;g samples at -20\u0026deg;C, 4\u0026deg;C, 25\u0026deg;C and 37\u0026deg;C for 28 days. On the 28th day, the samples were run on an SDS page gel and stained with Coomassie blue (BioRad), and the resultant bands were analyzed for protein degradation.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTMV expression and virus purification\u003c/p\u003e\n\u003cp\u003eClassic symptoms of TMV infection appeared within 14 days post infiltration (dpi) for all the constructs. Purified virus extracts were analyzed using SDS\u0026ndash;PAGE, revealing the expected size for all the constructs in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Yields of wtTMV averaged above 1mg virus/g fresh leaf weight. The average yield was calculated from five different extractions from 10 plants. The yields from the TMV-epitope fusions varied significantly. TMV-F had the lowest yield (0.3mg/g), while TMV-K L14 (0.8mg/g). There was minimal variation in the yield between extractions (Fig.\u0026nbsp;3A). Western blot with anti-TMV coat protein antibody confirmed the identity of extracted coat protein monomers and their respective sizes (Fig.\u0026nbsp;3). All six TMV-epitope constructs selected for the animal study showed cross-reactivity with polyclonal sera raised in rabbits against TMV (Fig.\u0026nbsp;3D) and SARS-CoV2 spike protein (Fig.\u0026nbsp;3E). Not surprisingly, neither the wtTMV coat protein nor SARS-CoV2 spike protein showed cross-reactivity with anti-S protein or anti-TMV antibodies, respectively (Fig.\u0026nbsp;3D and E)\u003c/p\u003e\n\u003cp\u003eAn ELISA assay was employed to detect the SARS-CoV2 peptides on the TMV CP surface. To allow detection of the fused peptides, each TMV-epitope fusion was used to coat ELISA plates, which were then probed with anti-SARS-CoV2 spike protein polyclonal antibodies. The binding affinity of the epitopes was analyzed by observing the absorbance of the different constructs in the ELISA assay. The binding affinity is directly proportional to absorbance[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. All the constructs showed varying levels of binding affinity to the anti-SARS-CoV2 spike protein polyclonal antibodies. TMV-A, TMV-D, and TMV-S14P5 showed the highest binding affinity, while TMV-K, TMV-M and TMV-N had the lowest (Fig.\u0026nbsp;3B)\u003c/p\u003e\n\u003cp\u003eAntigenicity study\u003c/p\u003e\n\u003cp\u003eVaccine Immunogenicity\u003c/p\u003e\n\u003cp\u003eThe ability of our candidate vaccines to promote an immune response targeting SARS-CoV2 was assessed in female BALB/c mice. On days 1 (50\u0026micro;g) and 15 (100\u0026micro;g), mice were immunized subcutaneously (s.c.) with one of the candidate vaccine constructs. The vaccine was formulated with AddaVax at a 1:1 ratio. Blood was drawn for antibody analysis on days 0, 14, and 28. The serum was tested for total IgG reactivity against SARS-CoV2 spike protein via dot-blots (Fig.\u0026nbsp;4) and ELISA, utilizing histidine-tagged S1 (S1-6His) from SARS-CoV2 as the capture antigen (Fig.\u0026nbsp;5). Dot blot analysis showed that the sera reacted to both wtTMV and SARS-CoV2 spike protein in the cases of TMV-(A, D, F, H, and L). TMV-S21P2 sera reacted only to TMV and not to SARS-CoV2 spike protein (Fig.\u0026nbsp;4). The buffer\u0026thinsp;+\u0026thinsp;adjuvant control did not induce cross-reactive antibodies against either wtTMV or SARS-CoV2 spike protein.\u003c/p\u003e\n\u003cp\u003eELISA analysis of preimmune serum extracted on day 0 showed no reactivity to immobilized SARS-CoV2 spike protein or TMV antigen (Fig. 5A). All mice vaccinated with the TMV-epitope fusions showed cross-reactive IgG antibodies to SARS-CoV2 spike protein by day 28 (Fig. 5A). Serum from mice vaccinated with wtTMV showed no reactivity to SARS-CoV2 spike protein, while serum from mice vaccinated with SARS-CoV2 spike protein showed no reactivity to wtTMV. SARS-CoV2 IgGs were present in all the sera from mice vaccinated with the TMV-epitope fusions on day 14 post-vaccination, and the titers increased by day 28 (Fig. 5A), except for mice vaccinated with TMV-S21P2, which showed a very low titer on both day 14 and day 28 (Fig. 5A).\u003c/p\u003e\n\u003cp\u003eIFN\u0026gamma; detection\u003c/p\u003e\n\u003cp\u003eVaccination of mice with the TMV-epitope fusions resulted in an inflammatory response measured by the amount of IFN\u0026gamma; 28 dpi (Fig.\u0026nbsp;5B). IFN\u0026gamma; concentrations were determined by ELISA and comparing concentrations to those of known standards. Although AddaVax was used as an adjuvant, the constructs elicited a much higher IFN\u0026gamma; response. wtTMV also had lower IFN\u0026gamma; levels than the fusion constructs. Peptides A, D and H had the highest levels of IFN\u0026gamma;, while the other three had levels similar to that of wtTMV.\u003c/p\u003e\n\u003cp\u003eNeutralization\u003c/p\u003e\n\u003cp\u003eIgG titers are an essential measure of vaccine performance; however, that alone does not directly translate to protection. More important is the ability of a vaccine to elicit a neutralizing immune response that protects the host by preventing viral binding or entry into host cells. We performed a neutralization assay using a COVID-19 Pseudovirus Neutralizing Antibody Luciferase Assay (Abnova). This assay provides a highly sensitive and specific method of quantifying COVID-19 neutralizing antibodies. It consists of neutralizing antibody blockade of lentivirus pseudotyped with COVID-19 spike (S) protein with luciferase (Luc) reporter gene interacting with ACE2 expressing HEK293T cells (HEK293T-ACE2). Luc expression inside the HEK293T-ACE2 signifies viral entry and no neutralizing antibody activity. In contrast, the absence of Luc expression signifies the presence of neutralizing antibody. The Luc reporter system enables high-sensitivity measurement of antibody neutralization. Here we showed that sera from constructs TMV-A (IC\u003csub\u003e50\u003c/sub\u003e \u0026ndash; 256.2), TMV-D (IC\u003csub\u003e50\u003c/sub\u003e \u0026ndash; 1270.0) and TMV-H (IC\u003csub\u003e50\u003c/sub\u003e \u0026ndash; 383.0) elicited a high titer of neutralizing antibodies (Fig. 5D).\u003c/p\u003e\n\u003cp\u003eFigure 5D shows the neutralizing antibody titer at which IC50 is obtained. Only constructs TMV-A, TMV-D and TMV-H showed neutralization higher than 50% at any dilution. Sera from mice vaccinated with the spike protein had a higher neutralizing antibody titer than the TMV-peptide fusions. IC\u003csub\u003e50\u003c/sub\u003e is a crucial parameter used to evaluate the efficacy of antibodies in preventing the replication or infectivity of a specific pathogen. It represents the concentration of antibody needed to inhibit the pathogen\u0026apos;s activity by 50%. Here Fig. 5E shows the IC50 for each of the constructs and controls. The constructs TMV-A (IC\u003csub\u003e50\u003c/sub\u003e \u0026ndash; 256.2), TMV-D (IC\u003csub\u003e50\u003c/sub\u003e \u0026ndash; 1270.0) and TMV-H (IC\u003csub\u003e50\u003c/sub\u003e \u0026ndash; 383.0) were neutralizing.\u003c/p\u003e\n\u003cp\u003eThermal stability\u003c/p\u003e\n\u003cp\u003eThe three TMV-epitope fusions that elicited neutralizing antibodies in mice were stored at various temperatures. After 28 days of storage, all the samples did not show any degradation when analyzed in an SDS PAGE using Coomassie staining.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the COVID-19 vaccine response, developing countries were among the last to receive vaccines, relying on other countries for vaccine development and high-technology production. Utilizing greenhouse-grown plants to produce vaccines provides several benefits that developing countries can use. Production can be rapid, with plants yielding large amounts of the vaccine in approximately a week. The cost of building large plant growth rooms is substantially lower than the expensive production facilities for the current FDA-approved COVID-19 vaccines. In addition, the cost of extracting the vaccines from the plants is also not overly expensive. The development and production of new vaccines for outbreaks and pandemics are within the capabilities of many developing countries. The thermal stability of the plant-produced vaccines will also be advantageous for new vaccines. Several studies have shown that TMV epitope display particles are stable for long periods at 4\u0026deg;C[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This is one of the limiting factors hindering the widespread use of the highly successful mRNA vaccines currently on the market[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. TMV epitope display vaccine technology is a step toward providing thermostable vaccines that cut down on the logistical problems posed by the need for cold-chain management.\u003c/p\u003e \u003cp\u003eHere, we report a platform capable of producing transiently expressed recombinant antigens as TMV epitope display particles in \u003cem\u003eN. benthamiana\u003c/em\u003e. We also show the simple purification of the recombinant TMV particles using PEG precipitation and sucrose gradient density centrifugation, which are both easily scalable. The yields obtained are relatively high and consistent over multiple extractions. This lends itself well to ease of production and scale-up. Through genetic fusion of the SARS-CoV2 antigens to the TMV particle, we augment stimulation of the immune system compared to what is achievable with peptides (SARS-CoV2 antigens) alone. Using short peptides allows for adapting a multivalent formulation in response to mutations to the SARS-CoV2 spike protein. An example is the emergence of the Brazil, South Africa, Delta, and Omicron variants that all have mutations in the spike protein RBD, an area covered by peptides D and H used in this study. Changing the sequence of just those two peptides would potentially elicit a variant-specific, protective immune response. The utility of immunity against circulating variants of SARS-CoV2, induced by the TMV-D and TMV-H constructs, can be investigated using the growing repertoire of SARS-CoV2 variant-specific pseudoviruses [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTMV epitope display vaccines have been shown to be stable at various temperatures[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Here we showed that the recombinant TMV particles we made were stable for four weeks at 0\u0026deg;C, 4\u0026deg;C, 25\u0026deg;C, and 37\u0026deg;C. One of the biggest challenges that hamstring vaccination campaigns in developing countries is maintaining the cold chain in remote areas. The areas that remain largely unvaccinated are potential reservoirs of pathogens that are the source of continued outbreaks. A vaccine that would remain viable and efficient over a long period would help resolve this issue. With COVID-19, most vaccines require stringent cold temperatures of at least 4\u0026deg;C while the mRNA (Moderna and Pfizer) have even more rigid temperature requirements to maintain viability[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe selected the S protein as it is the dominant target for neutralizing response in COVID-19 infection[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. We used short peptides because the full-length S protein of other coronaviruses has been implicated in the enhancement of viral infection or pulmonary toxicity in other studies [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile all the candidate vaccines injected into mice elicited a strong IgG response against SARS-CoV2 spike glycoprotein, not all candidate vaccines elicited a neutralizing antibody response. Three of the eight candidates in the mouse trials elicited high neutralizing antibody titers. Although \u003cem\u003ein vitro\u003c/em\u003e assays to detect neutralizing antibodies cannot completely predict \u003cem\u003ein vivo\u003c/em\u003e protection afforded by a vaccine, such assays are helpful for screening vaccine candidates. This suggests a multivalent formulation containing these three candidates would elicit a strong, protective immune reaction in humans[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. As with previous TMV epitope display vaccines and TMV-based VLPs, the presence of the TMV particle has been shown to elicit a potent cellular and humoral immune response [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. We suggest that this formulation would perform similarly, which is important for SARS-CoV2 as both arms of the immune system are essential for viral clearance and protection.\u003c/p\u003e \u003cp\u003eWe also observed high levels of the inflammatory marker IFNγ in mice immunized with TMV-A, TMV-D and TMV-H. These constructs also elicited the highest neutralization levels, suggesting that the selected peptides may contain both B and T-cell epitopes. The peptides A, B and H may elicit a robust T-cell response that leads to the activation of the cellular arm of the immune system[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The correlation between the levels of IFNγ observed in serum from mice immunized with TMV-A, TMV-D and TMV-H and the IC50s seen in the same sera suggest that the IFNγ response observed is SARS-CoV2 mainly specific.\u003c/p\u003e \u003cp\u003eSince the start of the COVID-19 pandemic, vaccine development has accelerated at a very high rate. Vaccines using different platforms have emerged, and even more technologies are being developed. These technologies include vaccines based on inactivated viruses, nucleic acid (mRNA and DNA), recombinant spike proteins (including those presented as VLPs), as well as replication-competent or incompetent virus vectors[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e][\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e][\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Recently, utilization of the 1018 CpG or AS02 adjuvant with a plant-based NP vaccine elicited high neutralizing antibody titers in humans [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. As such, we used Adavax, an adjuvant in the same class of adjuvants (squalene-based, oil-in-water nano-emulsion). The successful production of neutralizing antibodies provided here shows the utility of plant-based vaccine technology. When coupled with positive safety data, it suggests that NP vaccines could play a role in combating SARS-CoV2 by eliciting a robust immune response.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eSARS-CoV2: Severe acute respiratory syndrome coronavirus 2; COVID: coronaviral disease; TMV: Tobacco mosaic virus; NAb: Neutralizing antibody; S: spike; RBD: receptor binding domain; IC50: Half-maximal inhibitory concentration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eK.P. and \u0026nbsp;L.G. designed the research; K.P. performed the research; all analyzed data; and K.P. and L.G. wrote the paper, which was edited by all. The author (s) read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGift from Dr S Glanville\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsent for publication\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompeting interests\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthor details\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHenry E. Riggs School of Applied Life Sciences, Keck Graduate Institute,\u003c/p\u003e\n\u003cp\u003eClaremont, CA 91711, USA.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePhilip V. Coronavirus biology and replication : implications for SARS- . Nat Rev Microbiol [Internet]. Springer US; Available from: http://dx.doi.org/10.1038/s41579-020-00468-6\u003c/li\u003e\n\u003cli\u003ePetrosillo N, Viceconte G, Ergonul O, Ippolito G, Petersen E. COVID-19, SARS and MERS: are they closely related? Clin Microbiol Infect [Internet]. Elsevier Ltd; 2020;26:729\u0026ndash;34. Available from: https://doi.org/10.1016/j.cmi.2020.03.026\u003c/li\u003e\n\u003cli\u003eNo Title [Internet]. Available from: https://coronavirus.jhu.edu/map.html\u003c/li\u003e\n\u003cli\u003eAleem A, Akbar Samad AB, Vaqar S. Emerging Variants of SARS-CoV-2 And Novel Therapeutics Against Coronavirus (COVID-19). Treasure Island (FL); 2023. \u003c/li\u003e\n\u003cli\u003eYi C, Sun X, Ling Z, Sun B. 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Development of a Multivalent Subunit Vaccine against Tularemia Using Tobacco Mosaic Virus (TMV) Based Delivery System. PLoS One [Internet]. 2015;10:1\u0026ndash;22. Available from: http://dx.plos.org/10.1371/journal.pone.0130858\u003c/li\u003e\n\u003cli\u003eShivprasad S, Pogue GP, Lewandowski DJ, Hidalgo J, Donson J, Grill LK, et al. Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors. Virology. 1999;255:312\u0026ndash;23. \u003c/li\u003e\n\u003cli\u003eSukhikh GT, Priputnevich T V., Ogarkova DA, Pochtovyi AA, Kustova DD, Zlobin VI, et al. Sputnik Light and Sputnik V Vaccination Is Effective at Protecting Medical Personnel from COVID-19 during the Period of Delta Variant Dominance. Vaccines. 2022;10:1\u0026ndash;11. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3054976/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3054976/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEssential in halting the COVID-19 pandemic caused by SARS-CoV2, it is crucial to have stable, effective, and easy-to-manufacture vaccines. We developed a potential vaccine using a tobacco mosaic virus (TMV) epitope display model presenting peptides derived from the SARS-CoV2 spike protein. The TMV-epitope fusions in laboratory tests demonstrated binding to the SARS-CoV2 polyclonal antibodies. The fusion constructs maintained critical epitopes of the SARS-CoV-2 spike protein, and two in particular spanned regions of the receptor binding domain that have mutated in the more recent SARS-CoV2 variants. This would allow for rapid modification of vaccines in response to changes in circulating variants. The TMV-peptide fusion constructs also remained stable for over 28 days when stored at temperatures between \u0026minus;\u0026thinsp;20 and 37\u0026deg;C, an ideal property when targeting developing countries. Immunogenicity studies conducted on BALB/c mice elicited robust antibody responses against SARS-CoV2. A strong IFNγ response was also observed in immunized mice. Three of the six TMV-peptide fusion constructs produced virus-neutralizing titers, as measured by a pseudovirus neutralization assay. These TMV-peptide fusion constructs can be combined to make a multivalent vaccine that could be adapted to meet changing virus variants. These findings demonstrate the development of a stable COVID-19 vaccine candidate by combining SARS-CoV2 spike protein-derived peptides presented on the surface of a TMV nanoparticle.\u003c/p\u003e","manuscriptTitle":"Development of a candidate TMV epitope display vaccine against SARS-CoV2.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-06-26 18:02:59","doi":"10.21203/rs.3.rs-3054976/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dbb35391-6f22-4b62-8585-b4b781e1ad02","owner":[],"postedDate":"June 26th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-06-29T08:44:42+00:00","versionOfRecord":[],"versionCreatedAt":"2023-06-26 18:02:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3054976","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3054976","identity":"rs-3054976","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}