Protective efficacy of pDNA vaccine Candidates against lethal SARS-CoV-2 in Syrian Golden Hamsters

Protective efficacy of pDNA vaccine Candidates against lethal SARS-CoV-2 in Syrian Golden Hamsters | 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 Protective efficacy of pDNA vaccine Candidates against lethal SARS-CoV-2 in Syrian Golden Hamsters Iman Almansour Alzamil, Serguei Golovan, Jennifer Pickens, Krista Salley, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5664233/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 Seasonal Covid-19 vaccination is known as an efficient way to control Covid-19 pandemic. Despite these efforts, the currently approved mRNA and protein vaccines had been associated with risks of myocarditis and pericarditis. In addition, PEGylated nanoparticles have been associated with elevated risk of developing allergic reaction in people with high risk. To enhance safety of Covid-19 vaccines, alternative platform that can overcome this limitation are needed. Here, we developed pDNA based Covid-19 vaccine candidate administered by needle immunization. We assessed the immunogenicity of the vaccine candidate which predominantly produced high titer IgG2. In addition, the protective efficacy was evaluated. We determine that Th1 skewed immune response was important in conferring protection upon lethal virus challenge. These data shed new light on the importance of IgG isotype for the development of Covid-19 vaccine candidate. Furthermore, our finding is applicable for further testing on several vaccine candidates against other pathogens. Vaccine Development Vaccine pDNA SARS-CoC-2 Protective efficacy Spike Antibody Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Severe Acute Respiratory Syndrome Coronaviurs-2 (SARS-CoV-2) is a highly contagious virus that has been associated with Covid-19 pandemic ( 1 , 2 ). Since its discovery, substantial efforts have been made globally that have led to the development of the first generation Covid-19 vaccines. These vaccines have been instrumental in controlling the severity of Covid-19 pandemic. The currently FDA and EMA approved Covid-19 vaccines are based on either mRNA or protein platforms ( 2 , 3 , 4 , 5 , 6 , 7 , 8 ). Since then, the Covid-19 vaccine composition has been updated continuously and on a seasonal basis, when a newly circulating SARS-CoV-2 strain evades the host immunity generated from previous vaccination. While these vaccine platforms have been successful in containing the pandemic, side effects following the administration of these approved vaccines have been documented. For example, cases of myocarditis and pericarditis were observed in young adults and in adolescents following the administration of mRNA and protein vaccines ( 9 , 10 , 11 , 12 , 13 , 14 ). In addition, the PEG component included in composition of the lipid nanoparticles (LNP) in the approved mRNA vaccine has been associated with allergic reactions in individuals at an elevated risk of allergy ( 15 ). Importantly, some of major challenges of the mRNA vaccine platform is its structural instability and the requirements of ultra-cold chain shipment ( 16 ). Therefore, there is a demand to explore alternative vaccine platform that can overcome the limitations of the currently approved vaccines. pDNA vaccine is an attractive modern generation platform. One of major attributes of pDNA vaccine platform is its high thermal stability. This omits the requirements of further encapsulation and ultra-cold chain shipment ( 17 , 18 ). In addition, unlike viral vector-based vaccines, immunity can be boosted through multiple immunizations without the limitations of anti-vector responses ( 17 , 18 ). This makes the pDNA an appealing vaccine platform for the pathogen that requires frequent, seasonal updates. Over the past, several pDNA vaccine candidates have been explored in various preclinical and in clinical trials ( 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ). Most of these vaccine candidates relied on the electroporation method as delivery route for immunization. While several pDNA based platforms had been investigated as vaccine candidates against Covid-19, none of these vaccine candidates granted an approval by either EMA or FDA. In prior research, we developed pDNA based SARS-CoV-2 vaccine candidate encoding synthetic, codon optimized S gene (S.opt.FL) of SARS-CoV-2 and humoral and cellular immunogenicity were subsequently assesses in mice ( 25 ). In the present study, we further investigated pDNA based SARS-CoV-2 vaccine candidate in the pivotal protective efficacy study in Syrian golden hamsters. Results pDNA vaccine elicit potent antibody mediated immune response in Syrian Golden Hamsters Forty-five female Syrian golden hamsters, fifteen per group, were assigned to the following treatments: Group 1-PBS (0 mg/mL; Group 2-S.opt.FL pDNA 2 doses (0.2 mg/Animal); and Group 3-S.opt.FL 3 doses (0.2 mg/animal). Group 2 received two doses on day 1 and day 22. Group 3 received 3 doses of the vaccine candidate on day 1, 22, and 43. Group 1 (control group) received 3 doses of PBS on day 1, 22, and 43(Table 1 ). Key study procedures are summarized in (Table 2 ). Table 1 Group Assignment for the protective efficacy for the Covid-19 pDNA vaccine in Syrian Golden Hamsters. Group Test Article (TA) No. of Animals No. of TA Doses Dose Level per Animal per Dosing Day Challenge Dose (IN) 1 PBS Control 15F 3 0 2X10 5 TCID 50 /animal 2 S.opt.FL pDNA 15F 2 0.2 mg/0.2mL 3 S.opt.FL pDNA 15F 3 0.2 mg/0.2 mL Table 2 Key In-Life Study Procedures. Study Day 1 22 43 65 67 69 71 79 Day post infection (dpi) - - - 0 2 4 6 14 Vaccination X X X 1 IN Challenge X Mortality/Moribundity Check Performed twice daily (AM/PM) Clinical Observations Once weekly prior to challenge (post dose on dosing days) Daily post challenge Body Weight Once weekly prior to challenge Daily post challenge Food Consumption Twice weekly Total Blood Volume (mL) (pre-challenge, SST tubes) 2 (post challenge, EDTA tubes) 1.0 3 1.0 1.0 1.0 0.2 0.2 0.2 0.2 ELISA (spike full length) X X X X MN Assay X X X X Pseudovirus bases neutralization assay (future studies) X X X X sgRT-PCR (post challenge, EDTA tubes) X X X X sgRT-PCR: pharyngeal swabs X X X X Euthanasia and necropsy X X X X Microscopic pathology with H & E (nasal turbinates, lungs, GI tract, and kidney) X X X X We evaluated S-specific IgG for SARS-CoV-2 in sera collected on days 1, 22, 43, and 65. Three weeks after first dose of vaccine administration (Day 22), the mean ELISA titers for Group 2 and 3 were 4887 and 1580, respectively (Fig. 1 ). Three weeks after the second dose (Day 43), mean ELISA titers for Group 2 and 3 increased to 15160 and 5453, respectively. Three weeks after the 3rd dose of vaccination (Day 65), ELISA titer increased to 6187 for Group 3 that had received three doses of vaccination and decreased to 6567 for Group 2 that did not receive the third booster dose (Fig. 1 ). ELISA titer of vaccinated Groups 2 and 3 were significantly different (p < 0.01) compared to unvaccinated group (Group 1) at study Day 22, 43, and 65 (Fig. 1 ). Although slightly higher mean titer was detected in Group 2 compared to Group 3 on study Days 22 and 43, the difference was not statistically significant. Further, we evaluated IgG subclasses after eachvaccination. Overall, pDNA vaccine induced high titer of IgG2 specific antibodies responses following two and three doses (Fig. 2 ). These data confirms Th1 skewed immune responses induced by DNA vaccination. For neutralizing antibodies, the animals’ sera were assessed for 50% neutralizing antibody titer (MN 50 ) using microneutralization assay against “SARS-CoV-2 USA-WA-1/2020” strain. As expected, the mean MN50 the preimmunized sera collected form each animal groups (Day 1) were at basal levels for all animal groups. The highest MN50 titer was detected in Group 3, three weeks after receiving third dose (Day 65) (Fig. 3 ). The MN50 response was not detectable in Group 1 placebo group (Fig. 3 ). The MN50 titers for Group 2 at (Day 45) and (Day 65) were 69.91 and 31.69 at, respectively (Fig. 3 ). The MN50 titers for Group 4 at (Day 45) and (Day 65) were 69.91 and 31.69, respectively (Fig. 3 ). pDNA vaccine protects animals upon lethal challenge with SARS-CoV-2 virus To evaluate the protective efficacy of the SARS-CoV-2 pDNA vaccine candidates, animals were challenged intranasally at (Days 65) with 2X105 TCID50 of SARS-CoV-2 strain. Active virus replication as measured by the presence of (E sgRNA) in nasopharyngeal swab, trachea, and lungs were evaluated using sgRNA RT-qPCR. It is well established that at earlier days post challenge it is not possible to distinguish between live virus inoculum and active infection. Therefore, we took measurements at (Day 4) and (Day 6) post challenge to reflect active viral infection between vaccinated groups and control. For the nasopharyngeal swab, Group 2 showed 3-folds lower viral load and Group 3 showed 20 folds lower viral load on Day 69 (4 dpi) compared to Group 1 control group (Fig. 4 ). Likewise, substantial decrease was observed by Day 69 (4 dpi), with viral load 19,740-fold lower in Group 2 and 162-fold lower in Group 3 as compared to Group 1 (Fig. 4 ). Importantly, no active replication was detected in vaccinated Groups 2 or 3 by Study Day 71 (6 dpi) (Fig. 4 ). For trachea, peak viral load was 3-fold lower in Group 2 and 2-fold lower in Group 3 (Fig. 5 ). Even larger decrease was observed by at 4 dpi, with viral load 19,740-fold lower in Group 2 (34.35 copies/gram) and 162-fold lower in Group 3 (4.19E + 03 copies/gram) as compared to Group 1 (6.78E + 05copies/gram) (Fig. 5 ). For the lung tissue, active viral replication in peak value on Day 67 (2 dpi) in Group 1. On Day 69 (4 dpi), peak viral load was 60- fold lower in Group 2 and 14-fold lower in Group 3 compared to Group 1 (Fig. 6 ). Significant decrease in viral titer was observed by Day 71 (6 dpi), with viral load 2,062-fold lower in Group 2 and undetectable in Group 3 as compared to the placebo Group 1 (Fig. 6 ). No detectable active viral replication was observed in the brain, kidney, jejunum, illum, or whole blood. Clinical observations, body weight, and food consumptions Overall, the vaccine was well tolerated. Clinical observations and mortality data for individual animals are summarized in (Table 3 ). No abnormal clinical signs noted during the course of the study except for one animal (Animal from Group 2, which had a hunched posture and was hypoactive on study Days 71 (6 dpi) and 72 (7 dpi). Table 3 Summary of Clinical Observations and Mortality. Groups Observation type Days relative to start date 1 7 14 21 22 28 35 42 43 49 56 63 65 66 67 68 69 70 71 72 73 Group 1 Normal 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 9 9 6 6 Scheduled euthanasia - - - - - - - - - - - - - - 3 - 3 - 3 - - Group 2 Normal 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 12 12 9 8 5 5 Hunched posture - - - - - - - - - - - - - - - - - - 1 1 - Hypoactive - - - - - - - - - - - - - - - - - - 1 1 - Scheduled euthanasia - - - - - - - - - - - - - - 3 - 3 - 3 - - food spiller - - - - - - - - - - - - - - - - - - - - 1 Group 3 Normal 15 15 15 15 15 15 15 15 15 14 14 15 15 15 15 12 12 9 9 6 6 Scab, Face - - - - - - - - - 1 1 - - - - - - - - - - Scheduled euthanasia - - - - - - - - - - - - - - 3 - 3 - 3 - - Body weights were collected daily over the course of the study for all animal groups. Mean percent change in body weight was calculated for all groups with respect to their body weight on the day of SARS-CoV-2 infection (0 dpi). Mean percent change in body weights is shown in (Fig. 7 ). Hamsters infected with SARS-CoV-2 started to lose their body weight on Day 66 (1 dpi). Peak body weight loss was observed in both PBS and vaccine treated groups at Study Day 70 (6 dpi) for all animal groups. However, animals in Group 2 and Group 3 recovered their lost body weight faster after day 8 than the Placebo Group 1 (Fig. 7 ). Furthermore, Animals in Group 2 and Group 3 resume weight by day 9 and 10, respectively (Fig. 7 ). In contrast, the weight gain in Group 1 started slowly and resumed by day 14. These data demonstrate that intramuscular immunization with SARS-CoV-2 vaccine contribute to viral clearance. Percent change in mean food consumption was calculated for all groups with respect to -2 dpi (Fig. 8 ). The mean food consumption decreased to 54.6%, 53.9%, and 48.4% at 1 dpi. Mean food consumption was further decreased in Groups 1, 2, and 3 at 5 dpi to 28.6%, 31.9% and 36.6%, respectively (Fig. 8 ). Food intake increased in all three groups by 8 dpi and reached 73.6%, 104.3%, and 71.0% in Group 1, 2, and 3, respectively (Fig. 8 ). Of note, Group 2, which had received 2 doses of S.opt.FL pDNA vaccine, reached food intake level to pre-challenge level by 8 dpi (Fig. 8 ). Discussion Pivotal preclinical studies are crucial for evaluating vaccine candidates prior to entering clinical trials. For SARS-CoV-2 vaccine candidates, Golden Syrian Hamsters are prefect preclinical model as the angiotensin converting enzyme-2 (ACE-2) receptor is located at various cell tissue of the hamster which bind to the receptor binding sites (RBD) of the S protein of SARS-CoV-2 with high affinity. In addition, the infected Syrian hamsters are clinically characterized by viral load in the lung, viral shedding, and lung pathology. Therefore, it can give valuable indication of vaccine efficacy ( 31 ). Here, we report the results for the immunogenicity and protective efficacy studies for the Covid-19 pDNA vaccine candidates using intramuscular immunization. The S.opt.FL pDNA vaccine candidate was found to induce a potent immune response as well as protection in animal models upon lethal virus challenge with SARS-CoV-2 strain. S-specific IgG antibody responses were detected three weeks post immunization in the vaccinated animal groups. The highest increase of IgG titer was determined three weeks after second vaccination for Group 2 and three weeks after third vaccination for Group 3 weeks post immunization. Of note, vaccinated individual demonstrated high IgG2 response, which demonstrate skewed T helper 1 immune response. This is a unique characteristic of pDNA vaccine ( 32 , 33 , 34 ). In addition, serum samples were assessed for the 50% neutralizing antibody titer (MN 50 ) by microneutralization showed pre-immunization sera of all three groups (Day 1) and Group 1 (PBS control) at Day 22, 43, and 65 were at basal level of < 10 MN 50 . To evaluate the protective efficacy of S.opt.FL Covid-19 pDNA vaccine, all animals were challenged via intranasal route with an approximately 2X10 5 TCID 50 at study day 65 (3 weeks after third dose of vaccination). Intranasal challenge resulted in decreased food consumption at 1 dpi and further decrease was observed at 5 dpi. Hamsters infected with SARS-CoV-2 started to lose their body weight on 2 dpi and peak body weight loss was observed in both PBS and vaccine treated groups at 6 dpi. To measure the viral load in pharyngeal swabs, trachea, tissues including (lung, brain, kidney, jejunum, ilium) and whole blood, SARS-CoV-2 (E sgRNA) were measured. At earlier time of post challenge, it is not possible to distinguish between live virus inoculum and active infection ( 35 , 36 , 37 ). At 4 dpi, Two-dose vaccinated Group 2 (2.94E + 04 copies/swab) showed 3-fold lower and Group 3 (5.11E + 03 copies/swab) showed 20-fold lower of viral load when compared to Group 1. These results indicate decreased active replicating viral shedding in vaccinated animals. The peak viral load in trachea was 3-fold lower in Group 2 and 2-fold lower in Group 3. Even larger decrease was observed by at 4 dpi, with viral load 19,740-fold lower in Group 2 (34.35 copies/gram) and 162-fold lower in Group 3 (4.19E + 03 copies/gram) as compared to Group 1 (6.78E + 05copies/gram). Of note, no active replication was detected in vaccinated Group 2 and 3 by 6 dpi. In addition, SARS-CoV-2 active replication in lungs reached peak value of 8.78E + 07 copies/gram in Group 1 (PBS control) and 6.36E + 06 copies/gram in vaccinated Group 3 at 2 dpi, whereas vaccinated Group 2 reached peak value of 2.84E + 06 copies/gram at 4 dpi. An even larger decrease was observed at 6 dpi, with viral load 2,062-fold lower in Group 2 (1.42E + 02 copies/gram) and undetectable in Group 3 as compared to Group 1 (2.92E + 05 copies/gram), indicating virus clearance after vaccination. No deaths were noted in association with SARS-CoV-2 virus challenge. Taken together, these data of E sgRNA copies in lung, trachea and pharyngeal swabs demonstrate that both 2 doses and 3 doses of S.opt.FL pDNA plasmid vaccine are associated with protection against IN challenge of SARS-CoV-2 by reducing viral replication, viral shedding, and early clearance. As with other RNA viruses will be worthwhile to explore the effect of mutations on the cross-reactivities of the vaccine candidate against wide ranges of antigenically divergent strains ( 38 , 39 , 40 , 41 , 42 , 43 ) In conclusion, repeated-dose intramuscular administration of S.opt.FL pDNA vaccine showed protection from the lethal virus challenge. In addition, Th1 skewed immune response was observed in S.opt.FL Covid-19 vaccine candidate which is a unique attribute of pDNA vaccination. It has been demonstrated that IgG2 is more effective than IgG1 in the Fc mediated effector function. Changing the isotype of the S-specific antibody from IgG1 to IgG2 upon vaccination could be attributed to the immune protection. Previous reports have demonstrated that IgG2 might play role in virus protection. In this study, we have demonstrated the potent Th1 skewed IgG2 immune responses were likely associated with protection against intranasal challenge of SARS-CoV-2 USA-WA1-2020 with reduced viral load in lungs, trachea and nasopharyngeal swabs. This remarkable notification for pDNA vaccine. Methods Ethic statements Animal study was conducted in Southern Research, USA. Housing and animal care conformed to the guidelines of the U.S. Department of Agriculture (Animal Welfare Act; Public Law 99–198), the Guide for the Care and Use of Laboratory Animals. DNA vaccine The codon optimized, full length, spike gene insert S.opt.FL of (Wuhan/WIV04/2019) strain was previously synthesized ( 25 ). The gene insert was subcloned into pVAX1 plasmid. pDNA was transformed into E.Coli Dh5α. Restriction analysis and sequencing were preformed to confirm the accuracy of the construct. pDNA underwent expansion for culturing in LB broth with kanamycin. Plasmid was extracted and purified and diluted with TE buffer at 1mg/mL final concentration. Plasmid purity was measured by agarose gel electrophoresis. Study design Vaccine study was conducted to assess the immunogenicity and protective efficacy of Covid-19 pDNA vaccine candidate, against intranasal challenge of SARS-CoV-2 strain. A total of forty-five Female Syrian Golden Hamsters ( Mesocricetus auratus) were purchased from Charles River and received at southern research. Animals were housed at Southern Research, CRO (Alabama, USA). Animals were 8–9 weeks of age and weighed between 80–150 g at study day 1. Animals were randomized into three groups using the Provantis™ software. Animals were frequently monitored on daily basis for clinical observations such as food consumption, body weight, and temperature. Food consumption for each animal was measured quantitatively twice weekly throughout the study. For body weight, each animal was weighed prior to dosing (for randomization), once weekly prior to challenge, and then daily beginning on the day of challenge. Terminal weights were collected on the day of necropsy. Animals were anesthetized for viral challenge, collection of blood and swab samples, and euthanasia. Animals were anesthetized with either a solution of 1.0 mL of Ketamine HCl plus 0.1 mL Xylazine (100 mg/mL) dosed IP at 0.1 mL per grams of body weight or by inhalation of Isoflurane. Immunization The vaccine was administered intramuscularly by needle immunizations of 0.2 mL total volume (0.1 mL/hind leg) on each dosing day. Animals in Group 1 received 200 ul of PBS on days 1, 22, and 43. Animals in Group 2 received received two doses of 200 ug of pDNA vaccine candidate on study Days 1 and Day 22. Animals in group 3 received three doses of 200 ug pDNA vaccine candidate on study Days 1, 22, and 43. Blood was collected from each anesthetized animal via the retro-orbital or subclavian routes on Day 1 (prior to vaccination) and on Days 22, 43, and 65 (prior to challenge). Intranasal challenge Animals were transferred into the ABSL-3 facility three days prior to challenge for acclimation. On Day 65, All hamsters were anesthetized and were inoculated intranasally with a total of 200 µL of challenge inoculum (SARS-CoV-2). Euthanasia was scheduled on Days 67, 69, 71 (n = 3/group/timepoint) and 79 (n = 6/group). Swabs from animals were collected from all animals at Days 67, 69, 71, and 79. Immunogenicity Assessment ELISA 96-well clear polystyrene high binding antibody microtiter plates (Cat#9018, Corning) were coated with 1–2 ug/mL of S protein of SARS-CoV-2 (Cat#10549-CV, R&D System) and were incubated at 2–8 C for overnight. Plates were then washed four times with wash buffer (1X PBS, 0.05% tween). Afterwards, plates were blocked with SuperBlock buffer (37516, Thermo Scientific) for 1 h on an orbital shaker at 200 rpm at room temperature. Plates were washed five times with Serially diluted sera added in duplicate. After several washes, plates were coated with Mouse α-Hamster IgG-HRP or Mouse α-Hamster IgG2/IgG3-HRP (Cat#1935-05, Southern Biotech). After several washes, the plates were developed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Cat#34029, Thermo Scientific). The reaction was stopped with 1 N of H 2 SO 4 , and the absorbance measured at 450 nm using a SpectraMax M2 microplate reader. Antibody titer was calculated as reciprocal of the highest dilution. Microneutralization Vero E6 cell lines were seeded in 96 microtiter plates. Sera samples were serially diluted. Fixed concentration of SARS-CoV-2 (WA-1) virus strain were mixed with the serially diluted sera and incubated for 2-2.5 hours. Virus-serum mixture samples were added to 96-well plates previously seeded with Vero E6 cells and allowed to incubate for three days to allow infection of cells and replication of non-neutralized virus. Cytopathic effects were quantified by Cell-Titer Glo (Promega). Plate cutoffs were calculated using the following calculation [(CC average – VC average )/2] + VCaverage The highest serum dilution that was positive for neutralizing activity was used to calculate the titer for each serum sample. Statistical Analyses Statistical analyses were performed for body weight, food consumption, organ weights, and other parameters using Provantis™ software, GraphPad Prism, and Excel, including group means, standard deviations, and geometric mean titers. Declarations The animal study was approved by Southern Research Institute, Alabama, USA. The study was conducted in accordance with the local legislation and institutional requirements Funding This research was funded by Ministry of Education, Saudi Arabia. References Viner, R. M., Ward, J. L., Hudson, L. D., Ashe, M., Patel, S. V., Hargreaves, D., & Whittaker, E. (2021). Systematic review of reviews of symptoms and signs of COVID-19 in children and adolescents. Archives of disease in childhood , 106 (8), 802-807. Chen, X., Laurent, S., Onur, O. A., Kleineberg, N. N., Fink, G. R., Schweitzer, F., & Warnke, C. (2021). A systematic review of neurological symptoms and complications of COVID-19. Journal of neurology , 268 (2), 392-402. Oliver, S. E., Gargano, J. W., Marin, M., Wallace, M., Curran, K. G., Chamberland, M., ... & Dooling, K. (2020). The advisory committee on immunization practices’ interim recommendation for use of Pfizer-BioNTech COVID-19 vaccine—United States, December 2020. Morbidity and Mortality Weekly Report, 69(50), 1922. ‏ Roncati, L., & Roncati, M. (2021). Emergency use authorization (EUA), conditional marketing authorization (CMA), and the precautionary principle at the time of COVID-19 pandemic. Journal of Public Health Policy, 1-4. ‏ Krause, P. R., & Gruber, M. F. (2020). Emergency use authorization of Covid vaccines—safety and efficacy follow-up considerations. New England Journal of Medicine, 383(19), e107. Oliver, S. E. (2020). The advisory committee on immunization practices’ interim recommendation for use of moderna COVID-19 vaccine—United States, December 2020. MMWR. Morbidity and mortality weekly report, 69.‏ Tanne, J. H. (2022). Covid-19: CDC and FDA approve Pfizer and Moderna vaccines for under 5s. Parums, D. V. (2022). first approval of the protein-based adjuvanted nuvaxovid (NVX-CoV2373) novavax vaccine for SARS-CoV-2 could increase vaccine uptake and provide immune protection from viral variants. Medical science monitor: international medical journal of experimental and clinical research , 28 , e936523-1. Macías Saint-Gerons, D., Ibarz, M. T., Castro, J. L., Forés-Martos, J., & Tabarés-Seisdedos, R. (2023). Myopericarditis Associated with the Novavax COVID-19 Vaccine (NVX-CoV2373): A Retrospective Analysis of Individual Case Safety Reports from VigiBase. Drugs-Real World Outcomes , 1-8. Le Vu, S., Bertrand, M., Jabagi, M. J., Botton, J., Drouin, J., Baricault, B., ... & Zureik, M. (2022). Age and sex-specific risks of myocarditis and pericarditis following Covid-19 messenger RNA vaccines. Nature communications , 13 (1), 3633. Mevorach, D., Anis, E., Cedar, N., Bromberg, M., Haas, E. J., Nadir, E., ... & Alroy-Preis, S. (2021). Myocarditis after BNT162b2 mRNA vaccine against Covid-19 in Israel. New England Journal of Medicine , 385 (23), 2140-2149. Dionne, A., Sperotto, F., Chamberlain, S., Baker, A. L., Powell, A. J., Prakash, A., ... & Friedman, K. G. (2021). Association of myocarditis with BNT162b2 messenger RNA COVID-19 vaccine in a case series of children. JAMA cardiology , 6 (12), 1446-1450 Buoninfante, A., Andeweg, A., Genov, G., & Cavaleri, M. (2024). Myocarditis associated with COVID-19 vaccination. npj Vaccines , 9 (1), 122. Dong, Y. M., Liu, X., Yang, C. T., Qi, Q., Shi, W. B., Li, Y. M., ... & Cong, B. (2022). Case report: Myocarditis following COVID-19 protein subunit vaccination. Frontiers in Cardiovascular Medicine , 9 , 970045. Warren, C. M., Snow, T. T., Lee, A. S., Shah, M. M., Heider, A., Blomkalns, A., ... & Nadeau, K. C. (2021). Assessment of allergic and anaphylactic reactions to mRNA COVID-19 vaccines with confirmatory testing in a US regional health system. JAMA network open , 4 (9), e2125524-e2125524. Uddin, M. N., & Roni, M. A. (2021). Challenges of storage and stability of mRNA-based COVID-19 vaccines. Vaccines , 9 (9), 1033. Gary, E. N., & Weiner, D. B. (2020). DNA vaccines: prime time is now. Current Opinion in Immunology , 65 , 21-27. Chavda, V. P., Pandya, R., & Apostolopoulos, V. (2021). DNA vaccines for SARS-CoV-2: toward third-generation vaccination era. Expert Review of Vaccines , 20 (12), 1549-1560. Ljungman, P., Bermudez, A., Logan, A. C., Kharfan-Dabaja, M. A., Chevallier, P., Martino, R., ... & Maertens, J. (2021). A randomised, placebo-controlled phase 3 study to evaluate the efficacy and safety of ASP0113, a DNA-based CMV vaccine, in seropositive allogeneic haematopoietic cell transplant recipients. EClinicalMedicine , 33 . Youn, J. W., Hur, S. Y., Woo, J. W., Kim, Y. M., Lim, M. C., Park, S. Y., ... & Sung, Y. C. (2020). Pembrolizumab plus GX-188E therapeutic DNA vaccine in patients with HPV-16-positive or HPV-18-positive advanced cervical cancer: interim results of a single-arm, phase 2 trial. The Lancet Oncology , 21 (12), 1653-1660. Choi, Y. J., Hur, S. Y., Kim, T. J., Hong, S. R., Lee, J. K., Cho, C. H., ... & Park, J. S. (2020). A phase II, prospective, randomized, multicenter, open-label study of GX-188E, an HPV DNA vaccine, in patients with cervical intraepithelial neoplasia 3. Clinical Cancer Research , 26 (7), 1616-1623. Disc McNeel, D. G., Eickhoff, J. C., Johnson, L. E., Roth, A. R., Perk, T. G., Fong, L., ... & Liu, G. (2019). Phase II trial of a DNA vaccine encoding prostatic acid phosphatase (pTVG-HP [MVI-816]) in patients with progressive, nonmetastatic, castration-sensitive prostate cancer. Journal of Clinical Oncology , 37 (36), 3507. Ledwith, B. J., Manam, S., Troilo, P. J., Barnum, A. B., Pauley, C. J., Griffiths II, T. G., ... & Nichols, W. W. (2001). Plasmid DNA vaccines: investigation of integration into host cellular DNA following intramuscular injection in mice. Intervirology , 43 (4-6), 258-272. Sheets, R. L., Stein, J., Manetz, T. S., Duffy, C., Nason, M., Andrews, C., ... & Gomez, P. L. (2006). Biodistribution of DNA plasmid vaccines against HIV-1, Ebola, Severe Acute Respiratory Syndrome, or West Nile virus is similar, without integration, despite differing plasmid backbones or gene inserts. Toxicological Sciences , 91 (2), 610-619. Almansour, I., Macadato, N. C., & Alshammari, T. (2021). Immunogenicity of multiple doses of pDNA vaccines against SARS-CoV-2. Pharmaceuticals , 14 (1), 39. Modjarrad, K., Roberts, C. C., Mills, K. T., Castellano, A. R., Paolino, K., Muthumani, K., ... & Maslow, J. N. (2019). Safety and immunogenicity of an anti-Middle East respiratory syndrome coronavirus DNA vaccine: a phase 1, open-label, single-arm, dose-escalation trial. The Lancet Infectious Diseases , 19 (9), 1013-1022. Tebas, P., Roberts, C. C., Muthumani, K., Reuschel, E. L., Kudchodkar, S. B., Zaidi, F. I., ... & Maslow, J. N. (2017). Safety and immunogenicity of an anti–Zika virus DNA vaccine—preliminary report. New England Journal of Medicine . Tebas, P., Kraynyak, K. A., Patel, A., Maslow, J. N., Morrow, M. P., Sylvester, A. J., ... & White, S. M. (2019). Intradermal SynCon® Ebola GP DNA vaccine is temperature stable and safely demonstrates cellular and humoral immunogenicity advantages in healthy volunteers. The Journal of infectious diseases , 220 (3), 400-410. Muthumani, K., Falzarano, D., Reuschel, E. L., Tingey, C., Flingai, S., Villarreal, D. O., ... & Weiner, D. B. (2015). A synthetic consensus anti–spike protein DNA vaccine induces protective immunity against Middle East respiratory syndrome coronavirus in nonhuman primates. Science translational medicine , 7 (301), 301ra132-301ra132. Rosenke, K., Meade-White, K., Letko, M., Clancy, C., Hansen, F., Liu, Y., ... & Feldmann, H. (2020). Defining the Syrian hamster as a highly susceptible preclinical model for SARS-CoV-2 infection. Emerging Microbes & Infections, 9(1), 2673-2684. Prompetchara, E., Ketloy, C., Tharakhet, K., Kaewpang, P., Buranapraditkun, S., Techawiwattanaboon, T., ... & Ruxrungtham, K. (2021). DNA vaccine candidate encoding SARS-CoV-2 spike proteins elicited potent humoral and Th1 cell-mediated immune responses in mice. PLoS One , 16 (3), e0248007. Sankaradoss, A., Jagtap, S., Nazir, J., Moula, S. E., Modak, A., Fialho, J., ... & Sreekumar, E. (2022). Immune profile and responses of a novel dengue DNA vaccine encoding an EDIII-NS1 consensus design based on Indo-African sequences. Molecular Therapy , 30 (5), 2058-2077. Hayashi, H., Sun, J., Yanagida, Y., Otera, T., Kubota-Koketsu, R., Shioda, T., ... & Nakagami, H. (2022). Preclinical study of a DNA vaccine targeting SARS-CoV-2. Current research in translational medicine , 70 (4), 103348. Chi, X., Yan, R., Zhang, J., Zhang, G., Zhang, Y., Hao, M., Zhang, Z., Fan, P., Dong, Y., Yang, Y., et al. (2020). A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. Science 369 , 650–655. Chandrashekar, A., Liu, J., Martinot, A.J., McMahan, K., Mercado, N.B., Peter, L., Tostanoski, L.H., Yu, J., Maliga, Z., Nekorchuk, M., et al. (2020). SARS-CoV-2 infection protects against rechallenge in rhesus ma- caques. Science 369 , 812–817. Wo ̈lfel,R.,Corman,V.M.,Guggemos,W.,Seilmaier,M.,Zange,S.,Mu€ller, M.A., Niemeyer, D., Jones, T.C., Vollmar, P., Rothe, C., et al. (2020). Viro- logical assessment of hospitalized patients with COVID-2019. Nature 581 , 465–469. Almansour, I., & Jermy, B. R. (2024). Nucleic acid vaccine candidates encapsulated with mesoporous silica nanoparticles against MERS-CoV. Human Vaccines & Immunotherapeutics , 20 (1), 2346390. Almansour, I. (2020). Mumps vaccines: current challenges and future prospects. Frontiers in microbiology , 11 , 1999. Almansour, I., & Boudellioua, I. (2022). hCoronavirusesDB: an integrated bioinformatics resource for human coronaviruses. Database , 2022 , baac017. Almansour, I., & Alhagri, M. (2019). MMRdb: Measles, mumps, and rubella viruses database and analysis resource. Infection, Genetics and Evolution , 75 , 103982. Almansour, Iman, Mazen Alhagri, Rahaf Alfares, Manal Alshehri, Razan Bakhashwain, and Ahmed Maarouf. "IRAM: virus capsid database and analysis resource." Database 2019 (2019): baz079. Almansour, I., Alfares, R., & Aljofi, H. (2018). Large-scale analysis of B-cell epitopes of envelope: Implications for Zika vaccine and immunotherapeutic development. F1000Research , 7 . Additional Declarations The authors declare no competing interests. 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5664233","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":391560424,"identity":"dfbfb749-2a93-4b3d-9f9f-6e1fe961e24d","order_by":0,"name":"Iman Almansour Alzamil","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIie3OIW7DMBTG8WdFcogzU49sV3hoZCBXcVSpIY4UOBCckSh4vcV6g2pPCuoBDApSVQoaaEkUFC2ZNmnSVq9jA/4D2+QnfwA+3z+MC2Cbjzdr9futfyXwSQK8iMAXwhVcQq4EtS+22MVSZd1DWxDI0CAbCsewqEQyTZesntI7qxuC6+oVA9E4iISJcNJol9xqToDWYADcRcIjmZHimeR6JIjtPGx0DauQspLY80QgKadflEGISgcR25yympJV1QUqqVOhtl1OUX2e3Fbp+mB6imW4ZKehv7+Rj4v1fujPk2+J+dj8Afh8Pp/vh94AtytRL6cbCuQAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8193-9797","institution":"Imam Abdulrahman Bin Faisal University","correspondingAuthor":true,"prefix":"","firstName":"Iman","middleName":"Almansour","lastName":"Alzamil","suffix":""},{"id":394171058,"identity":"1565d040-e5b2-489a-8a83-c0081633e4da","order_by":1,"name":"Serguei Golovan","email":"","orcid":"","institution":"Southern Research, Birmingham, Alabama, United States","correspondingAuthor":false,"prefix":"","firstName":"Serguei","middleName":"","lastName":"Golovan","suffix":""},{"id":394171256,"identity":"b9efe0ce-a17c-4ed6-b066-b559b6383505","order_by":2,"name":"Jennifer Pickens","email":"","orcid":"","institution":"Southern Research, Birmingham, Alabama, United States","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"","lastName":"Pickens","suffix":""},{"id":394171257,"identity":"e458808d-c200-475f-8341-54a245216ac5","order_by":3,"name":"Krista Salley","email":"","orcid":"","institution":"Southern Research, Birmingham, Alabama, United States","correspondingAuthor":false,"prefix":"","firstName":"Krista","middleName":"","lastName":"Salley","suffix":""},{"id":394171258,"identity":"f8ab4f5a-9a83-42cd-b7a6-9ab6bc41908e","order_by":4,"name":"Michael Roberts","email":"","orcid":"","institution":"Southern Research, Birmingham, Alabama, United States","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Roberts","suffix":""}],"badges":[],"createdAt":"2024-12-17 18:55:58","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5664233/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5664233/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71992302,"identity":"27d2dbb6-f955-4f85-8da7-24acf4201a75","added_by":"auto","created_at":"2024-12-20 12:05:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":58611,"visible":true,"origin":"","legend":"\u003cp\u003eS antibodies endpoint titers in Syrian Golden Hamsters as measured by ELISA.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5664233/v1/9a3b5fc4e6d579822b43aae4.png"},{"id":71991979,"identity":"5bca5925-0b27-47bf-85fc-80a95faa31da","added_by":"auto","created_at":"2024-12-20 11:57:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":31470,"visible":true,"origin":"","legend":"\u003cp\u003eIgG2 specific antibody titer at day 43 in Syrian Golden Hamsters measured by ELISA.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5664233/v1/66e0c0f58776c4407d64b467.png"},{"id":71991943,"identity":"bcae81ae-4d31-46ca-b2c4-cb13075bea61","added_by":"auto","created_at":"2024-12-20 11:57:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":113587,"visible":true,"origin":"","legend":"\u003cp\u003eSARS-CoV-2 neutralizing antibody titers in Syrian Golden Hamsters as measured by neutralization test (NT) at day A). day 1, B). day 22, C). day 43, and D). day 65.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5664233/v1/891ca5d9435ad57bf16c2dc4.png"},{"id":71992316,"identity":"b6417506-b173-4c1d-8118-cc2121cde4b8","added_by":"auto","created_at":"2024-12-20 12:05:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":130252,"visible":true,"origin":"","legend":"\u003cp\u003eMean sgRNA copy number of SARS-CoV-2 in the lung as detected by sgRT-PCR.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5664233/v1/6788e3861549a9bb0cd673d5.png"},{"id":71991971,"identity":"993b4d15-619c-4b24-b600-79fc36d5b02b","added_by":"auto","created_at":"2024-12-20 11:57:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":54515,"visible":true,"origin":"","legend":"\u003cp\u003eMean sgRNA copy number of SARS-CoV-2 in the trachea as detected by sgRT-qPCR.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5664233/v1/31f907fc0ccb8c8e72c2430e.png"},{"id":71992311,"identity":"e3cc37ef-c286-4538-a769-569288296217","added_by":"auto","created_at":"2024-12-20 12:05:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":161855,"visible":true,"origin":"","legend":"\u003cp\u003eMean sgRNA copy number of SARS-CoV-2 in the lungs as detected by sgRT-qPCR.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5664233/v1/54f21f60ead4a60a3d471875.png"},{"id":71991942,"identity":"beb993a5-cc5b-4b68-a0ab-bb2dc51d8e32","added_by":"auto","created_at":"2024-12-20 11:57:20","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":87302,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of body Weight change after lethal virus challenges.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5664233/v1/66c42b2117bb6e979ea9f332.png"},{"id":71991982,"identity":"e705207f-1a39-42a5-86c0-39531bb88d6e","added_by":"auto","created_at":"2024-12-20 11:57:32","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":83110,"visible":true,"origin":"","legend":"\u003cp\u003eAverage food consumption for the protective efficacy study.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5664233/v1/3641a90a047e243397677698.png"},{"id":72288246,"identity":"a42671a4-c471-4a52-affc-d73c5f3b37a9","added_by":"auto","created_at":"2024-12-24 17:15:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1197195,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5664233/v1/455f2fb0-c124-440b-a439-d839794b4bf8.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eProtective efficacy of pDNA vaccine Candidates against lethal SARS-CoV-2 in Syrian Golden Hamsters\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSevere Acute Respiratory Syndrome Coronaviurs-2 (SARS-CoV-2) is a highly contagious virus that has been associated with Covid-19 pandemic (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Since its discovery, substantial efforts have been made globally that have led to the development of the first generation Covid-19 vaccines. These vaccines have been instrumental in controlling the severity of Covid-19 pandemic.\u003c/p\u003e \u003cp\u003eThe currently FDA and EMA approved Covid-19 vaccines are based on either mRNA or protein platforms (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Since then, the Covid-19 vaccine composition has been updated continuously and on a seasonal basis, when a newly circulating SARS-CoV-2 strain evades the host immunity generated from previous vaccination. While these vaccine platforms have been successful in containing the pandemic, side effects following the administration of these approved vaccines have been documented. For example, cases of myocarditis and pericarditis were observed in young adults and in adolescents following the administration of mRNA and protein vaccines (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). In addition, the PEG component included in composition of the lipid nanoparticles (LNP) in the approved mRNA vaccine has been associated with allergic reactions in individuals at an elevated risk of allergy (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eImportantly, some of major challenges of the mRNA vaccine platform is its structural instability and the requirements of ultra-cold chain shipment (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Therefore, there is a demand to explore alternative vaccine platform that can overcome the limitations of the currently approved vaccines.\u003c/p\u003e \u003cp\u003epDNA vaccine is an attractive modern generation platform. One of major attributes of pDNA vaccine platform is its high thermal stability. This omits the requirements of further encapsulation and ultra-cold chain shipment (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). In addition, unlike viral vector-based vaccines, immunity can be boosted through multiple immunizations without the limitations of anti-vector responses (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). This makes the pDNA an appealing vaccine platform for the pathogen that requires frequent, seasonal updates. Over the past, several pDNA vaccine candidates have been explored in various preclinical and in clinical trials (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Most of these vaccine candidates relied on the electroporation method as delivery route for immunization. While several pDNA based platforms had been investigated as vaccine candidates against Covid-19, none of these vaccine candidates granted an approval by either EMA or FDA.\u003c/p\u003e \u003cp\u003eIn prior research, we developed pDNA based SARS-CoV-2 vaccine candidate encoding synthetic, codon optimized S gene (S.opt.FL) of SARS-CoV-2 and humoral and cellular immunogenicity were subsequently assesses in mice (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In the present study, we further investigated pDNA based SARS-CoV-2 vaccine candidate in the pivotal protective efficacy study in Syrian golden hamsters.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003epDNA vaccine elicit potent antibody mediated immune response in Syrian Golden Hamsters\u003c/h2\u003e \u003cp\u003eForty-five female Syrian golden hamsters, fifteen per group, were assigned to the following treatments: Group 1-PBS (0 mg/mL; Group 2-S.opt.FL pDNA 2 doses (0.2 mg/Animal); and Group 3-S.opt.FL 3 doses (0.2 mg/animal). Group 2 received two doses on day 1 and day 22. Group 3 received 3 doses of the vaccine candidate on day 1, 22, and 43. Group 1 (control group) received 3 doses of PBS on day 1, 22, and 43(Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Key study procedures are summarized in (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGroup Assignment for the protective efficacy for the Covid-19 pDNA vaccine in Syrian Golden Hamsters.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTest Article (TA)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. of Animals\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo. of TA Doses\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDose Level per Animal per Dosing Day\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChallenge Dose (IN)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePBS Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2X10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTCID\u003csub\u003e50\u003c/sub\u003e/animal\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS.opt.FL pDNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.2 mg/0.2mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS.opt.FL pDNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.2 mg/0.2\u0026nbsp;mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKey In-Life Study Procedures.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStudy Day\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay post infection (dpi)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVaccination\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIN Challenge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMortality/Moribundity Check\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e \u003cp\u003ePerformed twice daily (AM/PM)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical Observations\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eOnce weekly prior to challenge (post dose on dosing days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e \u003cp\u003eDaily post challenge\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBody Weight\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eOnce weekly prior to challenge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e \u003cp\u003eDaily post challenge\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFood Consumption\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e \u003cp\u003eTwice weekly\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Blood Volume (mL) (pre-challenge, SST tubes)\u003csup\u003e2\u003c/sup\u003e (post challenge, EDTA tubes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eELISA (spike full length)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMN Assay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePseudovirus bases neutralization assay\u003c/p\u003e \u003cp\u003e(future studies)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esgRT-PCR (post challenge, EDTA tubes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esgRT-PCR: pharyngeal swabs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEuthanasia and necropsy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMicroscopic pathology with H \u0026amp; E (nasal turbinates, lungs, GI tract, and kidney)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWe evaluated S-specific IgG for SARS-CoV-2 in sera collected on days 1, 22, 43, and 65. Three weeks after first dose of vaccine administration (Day 22), the mean ELISA titers for Group 2 and 3 were 4887 and 1580, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Three weeks after the second dose (Day 43), mean ELISA titers for Group 2 and 3 increased to 15160 and 5453, respectively. Three weeks after the 3rd dose of vaccination (Day 65), ELISA titer increased to 6187 for Group 3 that had received three doses of vaccination and decreased to 6567 for Group 2 that did not receive the third booster dose (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003e). ELISA titer of vaccinated Groups 2 and 3 were significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to unvaccinated group (Group 1) at study Day 22, 43, and 65 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although slightly higher mean titer was detected in Group 2 compared to Group 3 on study Days 22 and 43, the difference was not statistically significant. Further, we evaluated IgG subclasses after eachvaccination. Overall, pDNA vaccine induced high titer of IgG2 specific antibodies responses following two and three doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These data confirms Th1 skewed immune responses induced by DNA vaccination.\u003c/p\u003e \u003cp\u003eFor neutralizing antibodies, the animals\u0026rsquo; sera were assessed for 50% neutralizing antibody titer (MN\u003csub\u003e50\u003c/sub\u003e) using microneutralization assay against \u0026ldquo;SARS-CoV-2 USA-WA-1/2020\u0026rdquo; strain. As expected, the mean MN50 the preimmunized sera collected form each animal groups (Day 1) were at basal levels for all animal groups. The highest MN50 titer was detected in Group 3, three weeks after receiving third dose (Day 65) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The MN50 response was not detectable in Group 1 placebo group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The MN50 titers for Group 2 at (Day 45) and (Day 65) were 69.91 and 31.69 at, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The MN50 titers for Group 4 at (Day 45) and (Day 65) were 69.91 and 31.69, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003epDNA vaccine protects animals upon lethal challenge with SARS-CoV-2 virus\u003c/h3\u003e\n\u003cp\u003eTo evaluate the protective efficacy of the SARS-CoV-2 pDNA vaccine candidates, animals were challenged intranasally at (Days 65) with 2X105 TCID50 of SARS-CoV-2 strain. Active virus replication as measured by the presence of (E sgRNA) in nasopharyngeal swab, trachea, and lungs were evaluated using sgRNA RT-qPCR.\u003c/p\u003e \u003cp\u003eIt is well established that at earlier days post challenge it is not possible to distinguish between live virus inoculum and active infection. Therefore, we took measurements at (Day 4) and (Day 6) post challenge to reflect active viral infection between vaccinated groups and control.\u003c/p\u003e \u003cp\u003eFor the nasopharyngeal swab, Group 2 showed 3-folds lower viral load and Group 3 showed 20 folds lower viral load on Day 69 (4 dpi) compared to Group 1 control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Likewise, substantial decrease was observed by Day 69 (4 dpi), with viral load 19,740-fold lower in Group 2 and 162-fold lower in Group 3 as compared to Group 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Importantly, no active replication was detected in vaccinated Groups 2 or 3 by Study Day 71 (6 dpi) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor trachea, peak viral load was 3-fold lower in Group 2 and 2-fold lower in Group 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Even larger decrease was observed by at 4 dpi, with viral load 19,740-fold lower in Group 2 (34.35 copies/gram) and 162-fold lower in Group 3 (4.19E\u0026thinsp;+\u0026thinsp;03 copies/gram) as compared to Group 1 (6.78E\u0026thinsp;+\u0026thinsp;05copies/gram) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor the lung tissue, active viral replication in peak value on Day 67 (2 dpi) in Group 1. On Day 69 (4 dpi), peak viral load was 60- fold lower in Group 2 and 14-fold lower in Group 3 compared to Group 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Significant decrease in viral titer was observed by Day 71 (6 dpi), with viral load 2,062-fold lower in Group 2 and undetectable in Group 3 as compared to the placebo Group 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNo detectable active viral replication was observed in the brain, kidney, jejunum, illum, or whole blood.\u003c/p\u003e\n\u003ch3\u003eClinical observations, body weight, and food consumptions\u003c/h3\u003e\n\u003cp\u003eOverall, the vaccine was well tolerated. Clinical observations and mortality data for individual animals are summarized in (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). No abnormal clinical signs noted during the course of the study except for one animal (Animal from Group 2, which had a hunched posture and was hypoactive on study Days 71 (6 dpi) and 72 (7 dpi).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of Clinical Observations and Mortality.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"23\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c17\" colnum=\"17\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c18\" colnum=\"18\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c19\" colnum=\"19\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c20\" colnum=\"20\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c21\" colnum=\"21\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c22\" colnum=\"22\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c23\" colnum=\"23\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eObservation type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"21\" nameend=\"c23\" namest=\"c3\"\u003e \u003cp\u003eDays relative to start date\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eGroup 1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eScheduled euthanasia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cb\u003eGroup 2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHunched posture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHypoactive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eScheduled euthanasia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003efood spiller\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eGroup 3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eScab, Face\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eScheduled euthanasia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBody weights were collected daily over the course of the study for all animal groups. Mean percent change in body weight was calculated for all groups with respect to their body weight on the day of SARS-CoV-2 infection (0 dpi). Mean percent change in body weights is shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Hamsters infected with SARS-CoV-2 started to lose their body weight on Day 66 (1 dpi). Peak body weight loss was observed in both PBS and vaccine treated groups at Study Day 70 (6 dpi) for all animal groups. However, animals in Group 2 and Group 3 recovered their lost body weight faster after day 8 than the Placebo Group 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Furthermore, Animals in Group 2 and Group 3 resume weight by day 9 and 10, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In contrast, the weight gain in Group 1 started slowly and resumed by day 14. These data demonstrate that intramuscular immunization with SARS-CoV-2 vaccine contribute to viral clearance.\u003c/p\u003e \u003cp\u003ePercent change in mean food consumption was calculated for all groups with respect to -2 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The mean food consumption decreased to 54.6%, 53.9%, and 48.4% at 1 dpi. Mean food consumption was further decreased in Groups 1, 2, and 3 at 5 dpi to 28.6%, 31.9% and 36.6%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Food intake increased in all three groups by 8 dpi and reached 73.6%, 104.3%, and 71.0% in Group 1, 2, and 3, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Of note, Group 2, which had received 2 doses of S.opt.FL pDNA vaccine, reached food intake level to pre-challenge level by 8 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePivotal preclinical studies are crucial for evaluating vaccine candidates prior to entering clinical trials. For SARS-CoV-2 vaccine candidates, Golden Syrian Hamsters are prefect preclinical model as the angiotensin converting enzyme-2 (ACE-2) receptor is located at various cell tissue of the hamster which bind to the receptor binding sites (RBD) of the S protein of SARS-CoV-2 with high affinity. In addition, the infected Syrian hamsters are clinically characterized by viral load in the lung, viral shedding, and lung pathology. Therefore, it can give valuable indication of vaccine efficacy (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Here, we report the results for the immunogenicity and protective efficacy studies for the Covid-19 pDNA vaccine candidates using intramuscular immunization. The S.opt.FL pDNA vaccine candidate was found to induce a potent immune response as well as protection in animal models upon lethal virus challenge with SARS-CoV-2 strain.\u003c/p\u003e \u003cp\u003eS-specific IgG antibody responses were detected three weeks post immunization in the vaccinated animal groups. The highest increase of IgG titer was determined three weeks after second vaccination for Group 2 and three weeks after third vaccination for Group 3 weeks post immunization. Of note, vaccinated individual demonstrated high IgG2 response, which demonstrate skewed T helper 1 immune response. This is a unique characteristic of pDNA vaccine (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). In addition, serum samples were assessed for the 50% neutralizing antibody titer (MN\u003csub\u003e50\u003c/sub\u003e) by microneutralization showed pre-immunization sera of all three groups (Day 1) and Group 1 (PBS control) at Day 22, 43, and 65 were at basal level of \u0026lt;\u0026thinsp;10 MN\u003csub\u003e50\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eTo evaluate the protective efficacy of S.opt.FL Covid-19 pDNA vaccine, all animals were challenged via intranasal route with an approximately 2X10\u003csup\u003e5\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e at study day 65 (3 weeks after third dose of vaccination). Intranasal challenge resulted in decreased food consumption at 1 dpi and further decrease was observed at 5 dpi. Hamsters infected with SARS-CoV-2 started to lose their body weight on 2 dpi and peak body weight loss was observed in both PBS and vaccine treated groups at 6 dpi.\u003c/p\u003e \u003cp\u003eTo measure the viral load in pharyngeal swabs, trachea, tissues including (lung, brain, kidney, jejunum, ilium) and whole blood, SARS-CoV-2 (E sgRNA) were measured. At earlier time of post challenge, it is not possible to distinguish between live virus inoculum and active infection (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). At 4 dpi, Two-dose vaccinated Group 2 (2.94E\u0026thinsp;+\u0026thinsp;04 copies/swab) showed 3-fold lower and Group 3 (5.11E\u0026thinsp;+\u0026thinsp;03 copies/swab) showed 20-fold lower of viral load when compared to Group 1. These results indicate decreased active replicating viral shedding in vaccinated animals. The peak viral load in trachea was 3-fold lower in Group 2 and 2-fold lower in Group 3. Even larger decrease was observed by at 4 dpi, with viral load 19,740-fold lower in Group 2 (34.35 copies/gram) and 162-fold lower in Group 3 (4.19E\u0026thinsp;+\u0026thinsp;03 copies/gram) as compared to Group 1 (6.78E\u0026thinsp;+\u0026thinsp;05copies/gram). Of note, no active replication was detected in vaccinated Group 2 and 3 by 6 dpi. In addition, SARS-CoV-2 active replication in lungs reached peak value of 8.78E\u0026thinsp;+\u0026thinsp;07 copies/gram in Group 1 (PBS control) and 6.36E\u0026thinsp;+\u0026thinsp;06 copies/gram in vaccinated Group 3 at 2 dpi, whereas vaccinated Group 2 reached peak value of 2.84E\u0026thinsp;+\u0026thinsp;06 copies/gram at 4 dpi. An even larger decrease was observed at 6 dpi, with viral load 2,062-fold lower in Group 2 (1.42E\u0026thinsp;+\u0026thinsp;02 copies/gram) and undetectable in Group 3 as compared to Group 1 (2.92E\u0026thinsp;+\u0026thinsp;05 copies/gram), indicating virus clearance after vaccination. No deaths were noted in association with SARS-CoV-2 virus challenge. Taken together, these data of E sgRNA copies in lung, trachea and pharyngeal swabs demonstrate that both 2 doses and 3 doses of S.opt.FL pDNA plasmid vaccine are associated with protection against IN challenge of SARS-CoV-2 by reducing viral replication, viral shedding, and early clearance. As with other RNA viruses will be worthwhile to explore the effect of mutations on the cross-reactivities of the vaccine candidate against wide ranges of antigenically divergent strains (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eIn conclusion, repeated-dose intramuscular administration of S.opt.FL pDNA vaccine showed protection from the lethal virus challenge. In addition, Th1 skewed immune response was observed in S.opt.FL Covid-19 vaccine candidate which is a unique attribute of pDNA vaccination. It has been demonstrated that IgG2 is more effective than IgG1 in the Fc mediated effector function. Changing the isotype of the S-specific antibody from IgG1 to IgG2 upon vaccination could be attributed to the immune protection. Previous reports have demonstrated that IgG2 might play role in virus protection. In this study, we have demonstrated the potent Th1 skewed IgG2 immune responses were likely associated with protection against intranasal challenge of SARS-CoV-2 USA-WA1-2020 with reduced viral load in lungs, trachea and nasopharyngeal swabs. This remarkable notification for pDNA vaccine.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEthic statements\u003c/h2\u003e \u003cp\u003eAnimal study was conducted in Southern Research, USA. Housing and animal care conformed to the guidelines of the U.S. Department of Agriculture (Animal Welfare Act; Public Law 99\u0026ndash;198), the \u003cem\u003eGuide for the Care and Use of Laboratory Animals.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA vaccine\u003c/h3\u003e\n\u003cp\u003eThe codon optimized, full length, spike gene insert S.opt.FL of (Wuhan/WIV04/2019) strain was previously synthesized (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The gene insert was subcloned into pVAX1 plasmid. pDNA was transformed into E.Coli Dh5α. Restriction analysis and sequencing were preformed to confirm the accuracy of the construct. pDNA underwent expansion for culturing in LB broth with kanamycin. Plasmid was extracted and purified and diluted with TE buffer at 1mg/mL final concentration. Plasmid purity was measured by agarose gel electrophoresis.\u003c/p\u003e\n\u003ch3\u003eStudy design\u003c/h3\u003e\n\u003cp\u003eVaccine study was conducted to assess the immunogenicity and protective efficacy of Covid-19 pDNA vaccine candidate, against intranasal challenge of SARS-CoV-2 strain. A total of forty-five Female Syrian Golden Hamsters (\u003cem\u003eMesocricetus auratus)\u003c/em\u003e were purchased from Charles River and received at southern research. Animals were housed at Southern Research, CRO (Alabama, USA). Animals were 8\u0026ndash;9 weeks of age and weighed between 80\u0026ndash;150 g at study day 1. Animals were randomized into three groups using the Provantis\u0026trade; software.\u003c/p\u003e \u003cp\u003eAnimals were frequently monitored on daily basis for clinical observations such as food consumption, body weight, and temperature. Food consumption for each animal was measured quantitatively twice weekly throughout the study. For body weight, each animal was weighed prior to dosing (for randomization), once weekly prior to challenge, and then daily beginning on the day of challenge. Terminal weights were collected on the day of necropsy. Animals were anesthetized for viral challenge, collection of blood and swab samples, and euthanasia. Animals were anesthetized with either a solution of 1.0 mL of Ketamine HCl plus 0.1 mL Xylazine (100 mg/mL) dosed IP at 0.1 mL per grams of body weight or by inhalation of Isoflurane.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunization\u003c/h2\u003e \u003cp\u003eThe vaccine was administered intramuscularly by needle immunizations of 0.2 mL total volume (0.1 mL/hind leg) on each dosing day. Animals in Group 1 received 200 ul of PBS on days 1, 22, and 43. Animals in Group 2 received received two doses of 200 ug of pDNA vaccine candidate on study Days 1 and Day 22. Animals in group 3 received three doses of 200 ug pDNA vaccine candidate on study Days 1, 22, and 43. Blood was collected from each anesthetized animal via the retro-orbital or subclavian routes on Day 1 (prior to vaccination) and on Days 22, 43, and 65 (prior to challenge).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIntranasal challenge\u003c/h2\u003e \u003cp\u003eAnimals were transferred into the ABSL-3 facility three days prior to challenge for acclimation. On Day 65, All hamsters were anesthetized and were inoculated intranasally with a total of 200 \u0026micro;L of challenge inoculum (SARS-CoV-2). Euthanasia was scheduled on Days 67, 69, 71 (n\u0026thinsp;=\u0026thinsp;3/group/timepoint) and 79 (n\u0026thinsp;=\u0026thinsp;6/group). Swabs from animals were collected from all animals at Days 67, 69, 71, and 79.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunogenicity Assessment\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003e96-well clear polystyrene high binding antibody microtiter plates (Cat#9018, Corning) were coated with 1\u0026ndash;2 ug/mL of S protein of SARS-CoV-2 (Cat#10549-CV, R\u0026amp;D System) and were incubated at 2\u0026ndash;8 C for overnight. Plates were then washed four times with wash buffer (1X PBS, 0.05% tween). Afterwards, plates were blocked with SuperBlock buffer (37516, Thermo Scientific) for 1 h on an orbital shaker at 200 rpm at room temperature. Plates were washed five times with Serially diluted sera added in duplicate. After several washes, plates were coated with Mouse α-Hamster IgG-HRP or Mouse α-Hamster IgG2/IgG3-HRP (Cat#1935-05, Southern Biotech). After several washes, the plates were developed with 3,3\u0026prime;,5,5\u0026prime;-tetramethylbenzidine (TMB) substrate (Cat#34029, Thermo Scientific). The reaction was stopped with 1 N of H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, and the absorbance measured at 450 nm using a SpectraMax M2 microplate reader. Antibody titer was calculated as reciprocal of the highest dilution.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMicroneutralization\u003c/h2\u003e \u003cp\u003eVero E6 cell lines were seeded in 96 microtiter plates. Sera samples were serially diluted. Fixed concentration of SARS-CoV-2 (WA-1) virus strain were mixed with the serially diluted sera and incubated for 2-2.5 hours. Virus-serum mixture samples were added to 96-well plates previously seeded with Vero E6 cells and allowed to incubate for three days to allow infection of cells and replication of non-neutralized virus. Cytopathic effects were quantified by Cell-Titer Glo (Promega). Plate cutoffs were calculated using the following calculation\u003c/p\u003e \u003cp\u003e[(CC\u003csub\u003eaverage\u003c/sub\u003e \u0026ndash; VC\u003csub\u003eaverage\u003c/sub\u003e)/2]\u0026thinsp;+\u0026thinsp;VCaverage\u003c/p\u003e \u003cp\u003eThe highest serum dilution that was positive for neutralizing activity was used to calculate the titer for each serum sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed for body weight, food consumption, organ weights, and other parameters using Provantis\u0026trade; software, GraphPad Prism, and Excel, including group means, standard deviations, and geometric mean titers.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u0026nbsp;The animal study was approved by Southern Research Institute, Alabama, USA. The study was conducted in accordance with the local legislation and institutional requirements\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by Ministry of Education, Saudi Arabia.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eViner, R. M., Ward, J. L., Hudson, L. D., Ashe, M., Patel, S. V., Hargreaves, D., \u0026amp; Whittaker, E. (2021). Systematic review of reviews of symptoms and signs of COVID-19 in children and adolescents. \u003cem\u003eArchives of disease in childhood\u003c/em\u003e, \u003cem\u003e106\u003c/em\u003e(8), 802-807.\u003c/li\u003e\n \u003cli\u003eChen, X., Laurent, S., Onur, O. A., Kleineberg, N. N., Fink, G. R., Schweitzer, F., \u0026amp; Warnke, C. (2021).\u0026nbsp;A systematic review of neurological symptoms and complications of COVID-19. \u003cem\u003eJournal of neurology\u003c/em\u003e, \u003cem\u003e268\u003c/em\u003e(2), 392-402.\u003c/li\u003e\n \u003cli\u003eOliver, S. E., Gargano, J. W., Marin, M., Wallace, M., Curran, K. G., Chamberland, M., ... \u0026amp; Dooling, K. (2020). The advisory committee on immunization practices\u0026rsquo; interim recommendation for use of Pfizer-BioNTech COVID-19 vaccine\u0026mdash;United States, December 2020. Morbidity and Mortality Weekly Report, 69(50), 1922.\u003cspan dir=\"RTL\"\u003e\u0026rlm;\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eRoncati, L., \u0026amp; Roncati, M. (2021). Emergency use authorization (EUA), conditional marketing authorization (CMA), and the precautionary principle at the time of COVID-19 pandemic. Journal of Public Health Policy, 1-4.\u003cspan dir=\"RTL\"\u003e\u0026rlm;\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eKrause, P. R., \u0026amp; Gruber, M. F. (2020). Emergency use authorization of Covid vaccines\u0026mdash;safety and efficacy follow-up considerations. New England Journal of Medicine, 383(19), e107.\u003c/li\u003e\n \u003cli\u003eOliver, S. E. (2020). The advisory committee on immunization practices\u0026rsquo; interim recommendation for use of moderna COVID-19 vaccine\u0026mdash;United States, December 2020. MMWR. Morbidity and mortality weekly report, 69.\u0026rlm;\u003c/li\u003e\n \u003cli\u003eTanne, J. H. (2022). Covid-19: CDC and FDA approve Pfizer and Moderna vaccines for under 5s.\u003c/li\u003e\n \u003cli\u003eParums, D. V. (2022). first approval of the protein-based adjuvanted nuvaxovid (NVX-CoV2373) novavax vaccine for SARS-CoV-2 could increase vaccine uptake and provide immune protection from viral variants. \u003cem\u003eMedical science monitor: international medical journal of experimental and clinical research\u003c/em\u003e, \u003cem\u003e28\u003c/em\u003e, e936523-1.\u003c/li\u003e\n \u003cli\u003eMac\u0026iacute;as Saint-Gerons, D., Ibarz, M. T., Castro, J. L., For\u0026eacute;s-Martos, J., \u0026amp; Tabar\u0026eacute;s-Seisdedos, R. (2023). Myopericarditis Associated with the Novavax COVID-19 Vaccine (NVX-CoV2373): A Retrospective Analysis of Individual Case Safety Reports from VigiBase. \u003cem\u003eDrugs-Real World Outcomes\u003c/em\u003e, 1-8.\u003c/li\u003e\n \u003cli\u003eLe Vu, S., Bertrand, M., Jabagi, M. J., Botton, J., Drouin, J., Baricault, B., ... \u0026amp; Zureik, M. (2022). Age and sex-specific risks of myocarditis and pericarditis following Covid-19 messenger RNA vaccines. \u003cem\u003eNature communications\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(1), 3633.\u003c/li\u003e\n \u003cli\u003eMevorach, D., Anis, E., Cedar, N., Bromberg, M., Haas, E. J., Nadir, E., ... \u0026amp; Alroy-Preis, S. (2021). Myocarditis after BNT162b2 mRNA vaccine against Covid-19 in Israel. \u003cem\u003eNew England Journal of Medicine\u003c/em\u003e, \u003cem\u003e385\u003c/em\u003e(23), 2140-2149.\u003c/li\u003e\n \u003cli\u003eDionne, A., Sperotto, F., Chamberlain, S., Baker, A. L., Powell, A. J., Prakash, A., ... \u0026amp; Friedman, K. G. (2021). Association of myocarditis with BNT162b2 messenger RNA COVID-19 vaccine in a case series of children. \u003cem\u003eJAMA cardiology\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e(12), 1446-1450\u003c/li\u003e\n \u003cli\u003eBuoninfante, A., Andeweg, A., Genov, G., \u0026amp; Cavaleri, M. (2024). Myocarditis associated with COVID-19 vaccination. \u003cem\u003enpj Vaccines\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(1), 122.\u003c/li\u003e\n \u003cli\u003eDong, Y. M., Liu, X., Yang, C. T., Qi, Q., Shi, W. B., Li, Y. M., ... \u0026amp; Cong, B. (2022). Case report: Myocarditis following COVID-19 protein subunit vaccination. \u003cem\u003eFrontiers in Cardiovascular Medicine\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e, 970045.\u003c/li\u003e\n \u003cli\u003eWarren, C. M., Snow, T. T., Lee, A. S., Shah, M. M., Heider, A., Blomkalns, A., ... \u0026amp; Nadeau, K. C. (2021). Assessment of allergic and anaphylactic reactions to mRNA COVID-19 vaccines with confirmatory testing in a US regional health system. \u003cem\u003eJAMA network open\u003c/em\u003e, \u003cem\u003e4\u003c/em\u003e(9), e2125524-e2125524.\u003c/li\u003e\n \u003cli\u003eUddin, M. N., \u0026amp; Roni, M. A. (2021). Challenges of storage and stability of mRNA-based COVID-19 vaccines.\u0026nbsp;\u003cem\u003eVaccines\u003c/em\u003e,\u0026nbsp;\u003cem\u003e9\u003c/em\u003e(9), 1033.\u003c/li\u003e\n \u003cli\u003eGary, E. N., \u0026amp; Weiner, D. B. (2020). DNA vaccines: prime time is now.\u0026nbsp;\u003cem\u003eCurrent Opinion in Immunology\u003c/em\u003e,\u0026nbsp;\u003cem\u003e65\u003c/em\u003e, 21-27.\u003c/li\u003e\n \u003cli\u003eChavda, V. P., Pandya, R., \u0026amp; Apostolopoulos, V. (2021). DNA vaccines for SARS-CoV-2: toward third-generation vaccination era. \u003cem\u003eExpert Review of Vaccines\u003c/em\u003e, \u003cem\u003e20\u003c/em\u003e(12), 1549-1560.\u003c/li\u003e\n \u003cli\u003eLjungman, P., Bermudez, A., Logan, A. C., Kharfan-Dabaja, M. A., Chevallier, P., Martino, R., ... \u0026amp; Maertens, J. (2021). A randomised, placebo-controlled phase 3 study to evaluate the efficacy and safety of ASP0113, a DNA-based CMV vaccine, in seropositive allogeneic haematopoietic cell transplant recipients. \u003cem\u003eEClinicalMedicine\u003c/em\u003e, \u003cem\u003e33\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eYoun, J. W., Hur, S. Y., Woo, J. W., Kim, Y. M., Lim, M. C., Park, S. Y., ... \u0026amp; Sung, Y. C. (2020). Pembrolizumab plus GX-188E therapeutic DNA vaccine in patients with HPV-16-positive or HPV-18-positive advanced cervical cancer: interim results of a single-arm, phase 2 trial. \u003cem\u003eThe Lancet Oncology\u003c/em\u003e, \u003cem\u003e21\u003c/em\u003e(12), 1653-1660.\u003c/li\u003e\n \u003cli\u003eChoi, Y. J., Hur, S. Y., Kim, T. J., Hong, S. R., Lee, J. K., Cho, C. H., ... \u0026amp; Park, J. S. (2020). A phase II, prospective, randomized, multicenter, open-label study of GX-188E, an HPV DNA vaccine, in patients with cervical intraepithelial neoplasia 3. \u003cem\u003eClinical Cancer Research\u003c/em\u003e, \u003cem\u003e26\u003c/em\u003e(7), 1616-1623.\u003c/li\u003e\n \u003cli\u003eDisc\u003c/li\u003e\n \u003cli\u003eMcNeel, D. G., Eickhoff, J. C., Johnson, L. E., Roth, A. R., Perk, T. G., Fong, L., ... \u0026amp; Liu, G. (2019). Phase II trial of a DNA vaccine encoding prostatic acid phosphatase (pTVG-HP [MVI-816]) in patients with progressive, nonmetastatic, castration-sensitive prostate cancer. \u003cem\u003eJournal of Clinical Oncology\u003c/em\u003e, \u003cem\u003e37\u003c/em\u003e(36), 3507.\u003c/li\u003e\n \u003cli\u003eLedwith, B. J., Manam, S., Troilo, P. J., Barnum, A. B., Pauley, C. J., Griffiths II, T. G., ... \u0026amp; Nichols, W. W. (2001). Plasmid DNA vaccines: investigation of integration into host cellular DNA following intramuscular injection in mice. \u003cem\u003eIntervirology\u003c/em\u003e, \u003cem\u003e43\u003c/em\u003e(4-6), 258-272.\u003c/li\u003e\n \u003cli\u003eSheets, R. L., Stein, J., Manetz, T. S., Duffy, C., Nason, M., Andrews, C., ... \u0026amp; Gomez, P. L. (2006). Biodistribution of DNA plasmid vaccines against HIV-1, Ebola, Severe Acute Respiratory Syndrome, or West Nile virus is similar, without integration, despite differing plasmid backbones or gene inserts. \u003cem\u003eToxicological Sciences\u003c/em\u003e, \u003cem\u003e91\u003c/em\u003e(2), 610-619.\u003c/li\u003e\n \u003cli\u003eAlmansour, I., Macadato, N. C., \u0026amp; Alshammari, T. (2021). Immunogenicity of multiple doses of pDNA vaccines against SARS-CoV-2. \u003cem\u003ePharmaceuticals\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(1), 39.\u003c/li\u003e\n \u003cli\u003eModjarrad, K., Roberts, C. C., Mills, K. T., Castellano, A. R., Paolino, K., Muthumani, K., ... \u0026amp; Maslow, J. N. (2019). Safety and immunogenicity of an anti-Middle East respiratory syndrome coronavirus DNA vaccine: a phase 1, open-label, single-arm, dose-escalation trial. \u003cem\u003eThe Lancet Infectious Diseases\u003c/em\u003e, \u003cem\u003e19\u003c/em\u003e(9), 1013-1022.\u003c/li\u003e\n \u003cli\u003eTebas, P., Roberts, C. C., Muthumani, K., Reuschel, E. L., Kudchodkar, S. B., Zaidi, F. I., ... \u0026amp; Maslow, J. N. (2017). Safety and immunogenicity of an anti\u0026ndash;Zika virus DNA vaccine\u0026mdash;preliminary report. \u003cem\u003eNew England Journal of Medicine\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eTebas, P., Kraynyak, K. A., Patel, A., Maslow, J. N., Morrow, M. P., Sylvester, A. J., ... \u0026amp; White, S. M. (2019). Intradermal SynCon\u0026reg; Ebola GP DNA vaccine is temperature stable and safely demonstrates cellular and humoral immunogenicity advantages in healthy volunteers. \u003cem\u003eThe Journal of infectious diseases\u003c/em\u003e, \u003cem\u003e220\u003c/em\u003e(3), 400-410.\u003c/li\u003e\n \u003cli\u003eMuthumani, K., Falzarano, D., Reuschel, E. L., Tingey, C., Flingai, S., Villarreal, D. O., ... \u0026amp; Weiner, D. B. (2015). A synthetic consensus anti\u0026ndash;spike protein DNA vaccine induces protective immunity against Middle East respiratory syndrome coronavirus in nonhuman primates. \u003cem\u003eScience translational medicine\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e(301), 301ra132-301ra132.\u003c/li\u003e\n \u003cli\u003eRosenke, K., Meade-White, K., Letko, M., Clancy, C., Hansen, F., Liu, Y., ... \u0026amp; Feldmann, H. (2020). Defining the Syrian hamster as a highly susceptible preclinical model for SARS-CoV-2 infection.\u0026nbsp;Emerging Microbes \u0026amp; Infections,\u0026nbsp;9(1), 2673-2684.\u003c/li\u003e\n \u003cli\u003ePrompetchara, E., Ketloy, C., Tharakhet, K., Kaewpang, P., Buranapraditkun, S., Techawiwattanaboon, T., ... \u0026amp; Ruxrungtham, K. (2021). DNA vaccine candidate encoding SARS-CoV-2 spike proteins elicited potent humoral and Th1 cell-mediated immune responses in mice.\u0026nbsp;\u003cem\u003ePLoS One\u003c/em\u003e,\u0026nbsp;\u003cem\u003e16\u003c/em\u003e(3), e0248007.\u003c/li\u003e\n \u003cli\u003eSankaradoss, A., Jagtap, S., Nazir, J., Moula, S. E., Modak, A., Fialho, J., ... \u0026amp; Sreekumar, E. (2022). Immune profile and responses of a novel dengue DNA vaccine encoding an EDIII-NS1 consensus design based on Indo-African sequences.\u0026nbsp;\u003cem\u003eMolecular Therapy\u003c/em\u003e,\u0026nbsp;\u003cem\u003e30\u003c/em\u003e(5), 2058-2077.\u003c/li\u003e\n \u003cli\u003eHayashi, H., Sun, J., Yanagida, Y., Otera, T., Kubota-Koketsu, R., Shioda, T., ... \u0026amp; Nakagami, H. (2022). Preclinical study of a DNA vaccine targeting SARS-CoV-2.\u0026nbsp;\u003cem\u003eCurrent research in translational medicine\u003c/em\u003e,\u0026nbsp;\u003cem\u003e70\u003c/em\u003e(4), 103348.\u003c/li\u003e\n \u003cli\u003eChi, X., Yan, R., Zhang, J., Zhang, G., Zhang, Y., Hao, M., Zhang, Z., Fan, P., Dong, Y., Yang, Y., et al. (2020). A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. Science \u003cem\u003e369\u003c/em\u003e, 650\u0026ndash;655.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eChandrashekar, A., Liu, J., Martinot, A.J., McMahan, K., Mercado, N.B., Peter, L., Tostanoski, L.H., Yu, J., Maliga, Z., Nekorchuk, M., et al. (2020). SARS-CoV-2 infection protects against rechallenge in rhesus ma- caques. Science \u003cem\u003e369\u003c/em\u003e, 812\u0026ndash;817.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWo ̈lfel,R.,Corman,V.M.,Guggemos,W.,Seilmaier,M.,Zange,S.,Mu\u0026euro;ller, M.A., Niemeyer, D., Jones, T.C., Vollmar, P., Rothe, C., et al. (2020). Viro- logical assessment of hospitalized patients with COVID-2019. Nature \u003cem\u003e581\u003c/em\u003e, 465\u0026ndash;469. \u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAlmansour, I., \u0026amp; Jermy, B. R. (2024). Nucleic acid vaccine candidates encapsulated with mesoporous silica nanoparticles against MERS-CoV. \u003cem\u003eHuman Vaccines \u0026amp; Immunotherapeutics\u003c/em\u003e, \u003cem\u003e20\u003c/em\u003e(1), 2346390.\u003c/li\u003e\n \u003cli\u003eAlmansour, I. (2020). Mumps vaccines: current challenges and future prospects. \u003cem\u003eFrontiers in microbiology\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e, 1999.\u003c/li\u003e\n \u003cli\u003eAlmansour, I., \u0026amp; Boudellioua, I. (2022). hCoronavirusesDB: an integrated bioinformatics resource for human coronaviruses. \u003cem\u003eDatabase\u003c/em\u003e, \u003cem\u003e2022\u003c/em\u003e, baac017.\u003c/li\u003e\n \u003cli\u003eAlmansour, I., \u0026amp; Alhagri, M. (2019). MMRdb: Measles, mumps, and rubella viruses database and analysis resource. \u003cem\u003eInfection, Genetics and Evolution\u003c/em\u003e, \u003cem\u003e75\u003c/em\u003e, 103982.\u003c/li\u003e\n \u003cli\u003eAlmansour, Iman, Mazen Alhagri, Rahaf Alfares, Manal Alshehri, Razan Bakhashwain, and Ahmed Maarouf. \u0026quot;IRAM: virus capsid database and analysis resource.\u0026quot; \u003cem\u003eDatabase\u003c/em\u003e 2019 (2019): baz079.\u003c/li\u003e\n \u003cli\u003eAlmansour, I., Alfares, R., \u0026amp; Aljofi, H. (2018). Large-scale analysis of B-cell epitopes of envelope: Implications for Zika vaccine and immunotherapeutic development. \u003cem\u003eF1000Research\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Vaccine, pDNA, SARS-CoC-2, Protective efficacy, Spike, Antibody","lastPublishedDoi":"10.21203/rs.3.rs-5664233/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5664233/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSeasonal Covid-19 vaccination is known as an efficient way to control Covid-19 pandemic. Despite these efforts, the currently approved mRNA and protein vaccines had been associated with risks of myocarditis and pericarditis. In addition, PEGylated nanoparticles have been associated with elevated risk of developing allergic reaction in people with high risk. To enhance safety of Covid-19 vaccines, alternative platform that can overcome this limitation are needed. Here, we developed pDNA based Covid-19 vaccine candidate administered by needle immunization. We assessed the immunogenicity of the vaccine candidate which predominantly produced high titer IgG2. \u0026nbsp;In addition, the protective efficacy was evaluated. We determine that Th1 skewed immune response was important in conferring protection upon lethal virus challenge. These data shed new light on the importance of IgG isotype for the development of Covid-19 vaccine candidate. Furthermore, our finding is applicable for further testing on several vaccine candidates against other pathogens.\u003c/p\u003e","manuscriptTitle":"Protective efficacy of pDNA vaccine Candidates against lethal SARS-CoV-2 in Syrian Golden Hamsters","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-20 11:57:12","doi":"10.21203/rs.3.rs-5664233/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":"3cccaecc-93ef-4ceb-8e62-631590f850be","owner":[],"postedDate":"December 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":41736830,"name":"Vaccine Development"}],"tags":[],"updatedAt":"2024-12-24T17:09:24+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-20 11:57:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5664233","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5664233","identity":"rs-5664233","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}