Immune Responses Following Administration of Whole Gamma Irradiated SARS-CoV-2 Vaccine Stabilized With Disaccharide Trehalose on Syrian Hamster Model

Immune Responses Following Administration of Whole Gamma Irradiated SARS-CoV-2 Vaccine Stabilized With Disaccharide Trehalose on Syrian Hamster Model | 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 Immune Responses Following Administration of Whole Gamma Irradiated SARS-CoV-2 Vaccine Stabilized With Disaccharide Trehalose on Syrian Hamster Model Farahnaz Motamedi-Sedeh, Akbar Khorasani, Mohsen Lotfi, Seyed Morteza Moosavi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3405744/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 Background The SARS-CoV-2 virus is the causative agent of the emerging respiratory zoonosis disease. One of the most important requirements for the control of emerging diseases is the development of vaccines within a short period of time. Methods The use of ionizing radiation to inactivate pathogens has been developed for the rapid production of effective vaccines. In this study, the SARS-CoV-2 virus was isolated from tracheal swabs of an infected man, confirmed by RT-PCR, and propagated on Vero cells. The SARS-CoV-2 virus was irradiated with 14 kGy gamma radiation to completely inactivate it. Evaluation of the antigenic properties of the spike protein subunit S1 showed that the gamma-irradiated virus samples had intact antigens. The gamma-irradiated SARS-CoV-2 virus and formalin-treated virus were used to immunize Syrian hamsters in four vaccine formulations. Results The titer of neutralizing antibodies increased significantly in all vaccinated groups 3 weeks after the second and third vaccinations. Secretory IgA was examined in nasal lavage and NALT fluids and showed that the concentration of sIgA in irradiated vaccine plus trehalose increased significantly 3 weeks after the second and third vaccinations. The splenic lymphocyte proliferation assay showed a significant increase in all vaccinated hamsters, but the increase was greater in irradiated vaccine plus trehalose and irradiated vaccine plus alum. Conclusion In addition, we can introduce irradiated inactivated vaccine SARS-CoV-2 plus disaccharide trehalose via intranasal route of administration and another irradiated inactivated vaccine SARS-CoV-2 plus alum via subcutaneous route as safe and efficient vaccines against COVID-19 which can stimulate mucosal, humeral and cellular immunity. SARS-CoV-2 virus Inactivated Vaccine Gamma Irradiation Mucosal Immunity Immune Response Neutralizing Antibody Splenic Lymphocyte Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction Coronaviruses are enveloped RNA viruses that are widely distributed among humans, other mammals, and birds and cause acute and persistent infections. Members of this family were isolated as early as the 1930s as causative agents of infectious bronchitis in chickens, transmissible gastroenteritis in pigs, and severe hepatitis and neurologic disease in mice. Viruses of the family Coronaviridae are large, enveloped virions with positive RNA genomes (27–32 Kb) and diameters of 100–120 nm. The RNA genome of coronaviruses combines with the N-(nucleocapsid) phosphoprotein (50–60 kd) to form a long, flexible, helical nucleocapsid. Two types of human Coronavirus (HCV) have been identified: HCV-229E belongs to serotype I and causes respiratory tract infections, and HCV-OC43 belongs to serotype II and causes respiratory and intestinal infections. Severe acute respiratory syndrome ( SARS ) was spread worldwide in 2002 as a new human disease. Another zoonotic coronavirus, Middle East respiratory syndrome ( MERS ), was crossed species from camels to cause the often-fatal human disease in 2012 [ 1 , 2 ]. The SARS-CoV-2 virus is the causative agent of the emerging respiratory zoonosis known as COVID-19 from 2019. Neutralising antibodies (nAbs) against the disease agent, severe acute respiratory syndrome coronavirus 2 ( SARS-CoV-2 ), represent potential prophylactic and therapeutic options and could aid in vaccine development [ 3 ]. In general, nAbs with outstanding potency (so-called super-antibodies) [ 4 ] can be isolated by studying in detail the antibody responses of a sample of infected donors. The outstanding potency combined with the ability to extend the half-life of antibodies from weeks to many months lowers the effective cost of antibodies and provides more opportunities for prophylactic measures. As a cellular receptor, SARS-CoV-2 could use the human, bat, or civet ACE2 receptor, but no the mouse [ 5 , 6 , 7 ]. Therefore, it seems that mice expressing hACE2 would be a conceivable choice for vaccine challenge testing. Appropriate small animal models are critical for the study of viral pathogenesis, the production of vaccines, and the development of antiviral therapies. Hamster models are popular because of their affordability, availability, and simple genetic structure and have been widely used to study human coronavirus pathogenesis. The vaccinated hamsters against SARS-CoV-2 virus could effectively induce memory B and T cells and were able to protect reinfection of animals with the same strain [ 8 ]. One of the most important requirements for the control of emerging and re-emerging zoonosis is the development of vaccines within a short time. The most recent example is the inactivated whole virus vaccine against SARS-CoV-2 , which is still in use. The use of chemical substances such as formalin and beta-propiolactone is the common method for producing inactivated vaccines. Although inactivation with formalin is well established, the process has serious drawbacks, e.g., formalin is toxic and there are problems with residual traces in the final product. In addition, formalin leads to cross-linking and changes in structural components and damages antigenic structures, which affects the antigenicity of vaccines [ 9 ]. Other chemicals such as beta-propiolactone are also very dangerous. Therefore, an alternative to the chemical inactivation method must be found to produce an inactivated vaccine [ 9 ]. Recently, ionizing irradiation (gamma radiation by Co-60, X-rays, and electron beams) has been considered as an alternative for vaccine development. The irradiation inactivation method has some advantages over chemical inactivation for vaccine production. Recently, irradiation with low-energy electron beams has been shown to be much safer than gamma irradiation with a Co-60 source and can preserve viral and bacterial antigenicity [ 10 ]. The aim of this study was to investigate the efficacy of gamma irradiation in inactivating emerging/outbreaking pathogens. In this study, we tested our gamma irradiation and formalin-inactivated vaccine candidates for their immunogenicity and immune response against SARS-CoV-2 virus and demonstrated vaccine efficacy in Syrian hamsters. The vaccine candidates were administered intranasally and intradermally to the hamsters, requiring a higher amount of inactivated virus for proper immunization. 2 Materials and Methods 2.1 Virus (isolation, confirmation by RT-PCR, multiplication on Vero cell) The isolated SARS-CoV-2 virus from tracheal swabs of an infected man (43 years old) in Karaj, Iran, in May 2020 on Vero cells was used for this study. The Ct value of this sample in the RT-qP assay was 16. The tracheal swab samples were transferred to viral transport media (VTM) and transported to the cell culture laboratory under cold chain and inoculation on Vero cells with DMEM media plus 3% fetal calf serum and 1% penicillin-streptomycin solution (10000 units penicillin and 10 mg/ml streptomycin), at 37 ºC and 5% CO2. The cytopathic effect (CPE) was visible after 24–48 hours on Vero cells in the form of cell rounding and the development of syncytial cells. Virus samples were isolated and propagated in biosafety level facilities III at Razi Vaccine and Serum Research Institute, Karaj, Iran. The infected cells were harvested and confirmed using RT -qPCR kit (AccuPower® SARS-CoV-2 Real-Time RT-PCR Kit, Bioneer). The infected cells were used to infect four interval passages on Vero cells. After harvesting the fourth passage of infected cells, it was used for RT-PCR assay with specific primers for the spike (S) gene. The PCR product of the S gene was sequenced, and the nucleotide sequence was deposited in NCBI under accession number: MW709393 as the SARS-CoV-2/human/IRN/Alborz- IR /2020 surface glycoprotein (S) gene with 3822 bp length [ 11 ]. The isolated SARS CoV-2 virus was propagated on Vero cells, and the infected cell suspension was centrifuged at low speed for 15 min at 4 ºC to clear cell debris. The supernatant was taken as virus stock and divided into three parts. The 20% disaccharide trehalose (1 M) was added to the first part, and the second and third parts contained no trehalose. All virus strain stocks were aliquoted into 2-ml cryotubes and stored at -70 ºC. Two virus samples from two virus stocks (with trehalose and without trehalose) were used for virus titration by the TCID 50 method. The first and second virus stocks were used for irradiation, and the third was inactivated with formalin. 2.2 SARS CoV-2 Virus Inactivation using Gamma Irradiation and Formalin: The SARS-CoV-2 virus stocks were irradiated with a Cobalt-60 irradiator, Gamma cell 220 (MDS Nordion, Ottawa, Canada), at a dose rate of 0.93 Gy/s and an activity of 3985 Ci to inactivate viral infectivity and to inactivate SARS-CoV-2 virus genomic RNA in Nuclear Science and Technology Research Institute (NSTRI), Tehran, Iran. Gamma radiation doses of 2, 4, 5, 8, 10, and 15 kGy were administered to infected specimens frozen on dry ice. Each dose was repeated in three rounds [ 12 , 13 , and 14 ]. Viral titration of all irradiated samples was performed using the TCID 50 method. The third virus stock was inactivated with formalin 0.04% v/v at RT for 30 hours (final concentration 4:10000). The inactivated virus stocks (irradiated and treated with formalin) were concentrated by ultrafiltration and 8% PEG-6000. The concentrated and inactivated viruses were dialyzed against PBS and quantified for protein concentration using Nano-drop (Smart Nano-1000). 2.3 Safety Test and antigenicity evaluation by ELISA Test : The infectivity of the irradiated, inactivated SARS-CoV-2 virus was determined after inoculation of Vero cell monolayers at 37° for 48 hours, then sub-cultured in four blind cultures on fresh Vero cells, and viral titration was determined by TCID50 method [ 12 , 14 ]. In addition, the antigenic properties of irradiated and native virus samples were tested using the ELISA assay for S (spike) protein. The SARS-CoV-2 spike protein is one of the major surface glycoproteins. It consists of an S1 domain and S2 domain and binds to host cell receptors (ACE2). This protein plays a major role in viral infection and is involved in the fusion of the viral envelope with the cell membrane. To verify the antigenic properties of the irradiated virus samples in comparison with the control virus, the ELISA method was applied. SARS-CoV-2 anti-spike protein S1 monoclonal antibody (GT263) (1 mg/ml) 100 µl (Invitrogen) and goat anti-mouse IgG (H + L), HRP conjugate (Invitrogen) (REF: G21040) reconstituted in 1 ml PBS, pH 7.2, to obtain 1 mg/ml stock solution were used. The receptor-binding domain (RBD) of S1 can specifically bind to angiotensin-converting enzyme 2 (ACE2), the receptor on target cells. The diversity of SARS-CoV-2 is reflected in the variable spike proteins (S proteins) that have evolved in forms that differ in their receptor interactions and their response to various environmental triggers of virus-cell membrane fusion. Antigenicity evaluation of SARS-CoV-2 viruses was performed by ELISA assay, first preparing serial dilutions (1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256, and 1/512) of irradiated and unirradiated viral antigen in bicarbonate buffer as coating buffer (PH = 9.6). 100 µl of each antigen dilution was added to each well of the 96-well microplate in duplicate and incubated overnight at 4 ˚C. The suspension of Vero cells was used in duplicate as a negative control. Then all wells were washed 5 times with wash buffer (PBS + 0.05% Tween 20). 200 µl of blocking buffer (PBS + 5% skim milk) was added to each well and stored at 37 ºC for 2 hours to block the wells, after which they were washed three times. SARS-CoV-2 spike protein S1 monoclonal antibody (GT263) (1 mg/ml) (Invitrogen) was diluted 1/1000 and 100 µl of the dilution was added to each well for 1 hour at 37 ºC and then washed three times. Goat anti-mouse IgG (H + L), HRP conjugate (Invitrogen) (REF: G21040) reconstituted in 1 ml PBS, pH 7.2 to obtain 1 mg/ml stock solution, stored at -20 ºC, working solution 1/10000 was prepared and 100 µl of the working solution was added to each well 1 hour at 37 ºC. TMB (100 µl) was added to each well in the dark at room temperature for 30 minutes. Finally, a stop solution (sulfuric acid 1.25 mM) was added and the optical density was read at 450 nm using Hyperion MicroReader 4 Plus. 2.4 Vaccine Formulation The first vaccine formulation was irradiated SARS-CoV-2 virus, and the second vaccine formulation was irradiated SARS-CoV-2 virus plus 20% disaccharide trehalose (1 M). Trehalose can stabilize proteins as a cryoprotectant and as a free radical quencher and acts as a natural stabilizer of life processes [ 15 , 16 , 17 , and 18 ]. The third and fourth vaccine formulations were formalin-inactivated virus and irradiated virus mixed with alum. Alum compounds such as aluminum hydroxide (AH) or alhydrogel (chemically crystalline aluminum hydroxide) increase uptake and presentation of antigen by antigen-presenting cells (APCs) and enhance monocyte recruitment at the site of inoculation. 2.5 Syrian Hamster Immunization by Four Inactivated SARS CoV-2 vaccines Four regimens of inactivated SARS-CoV-2 vaccines were formulated and used to immunize male Syrian hamsters (weight 100 ± 10 g). Forty-two Syrian hamsters were purchased from Razi Vaccine and Serum Research Institute and divided into seven groups of 6 animals each (Table 1 ). The first group was used as preimmune animals and sampling was done before immunization. The other groups of animals were vaccinated with: irradiated inactivated SARS-CoV-2 antigen (intranasal), irradiated inactivated SARS-CoV-2 antigen + 20% trehalose (intranasal), formalin-inactivated SARS-CoV-2 antigen + alum (subcutaneous), irradiated inactivated SARS-CoV-2 antigen + alum (subcutaneous), negative control group inoculated with 100 µl sterile PBS (intranasal), negative control group injected with 100 µl sterile PBS (subcutaneous). Vaccination was administered via two routes of administration (intranasal and subcutaneous injection into the neck) as one prime and two booster doses three weeks apart. Blood samples were collected from the orbit of chloroform-anesthetized animals three weeks after each vaccination. Each hamster was anesthetized by inhalation, and then blood was collected from the inner corner of the eye using a sterile Pasteur pipette. Blood tubes were kept overnight at 4°C, and then sera were separated by centrifugation at 600g for 10 minutes at 4°C. The complement compounds in the isolated sera were inactivated at 56°C for 30 minutes. The sera were analyzed for the presence of antibodies to SARS-CoV-2 virus using the SARS-CoV-2 Neutralizing Antibody Detection Kit (Abeomics Inc.). Splenic lymphocytes from the hamster groups were cultured and stimulated by homologous-inactivated antigens to evaluate the splenic lymphocyte proliferation assay as cellular immunity three weeks after the second and third vaccinations (three animals in each group were used for each collection day). The schedule of animal vaccination was shown in Table 1 . Table 1 The schedule of the hamster vaccination against SARS-COV-2 virus and sampling No Vaccine Groups Route of adminstration Vaccine dose (µl) Vaccination day Number of animal Sampling day for Nt Ab Titration Sampling day for SLP assay Sampling day for IgA Titration 1 Pre-Immune 6 1 1 1 2 Irr.vac IN 100 1, 21, 42 6 21, 42, 63, 84 21, 42, 63, 84 21, 42, 63, 84 3 Irra.vac.T IN 100 1, 21, 42 6 21, 42, 63, 84 21, 42, 63, 84 21, 42, 63, 84 4 For.vac.alum SC 100 1, 21, 42 6 21, 42, 63, 84 21, 42, 63, 84 21, 42, 63, 84 5 Irr.vac.alum SC 100 1, 21, 42 6 21, 42, 63, 84 21, 42, 63, 84 21, 42, 63, 84 6 NC (PBS) IN 100 1, 21, 42 6 21, 42, 63, 84 21, 42, 63, 84 21, 42, 63, 84 7 NC (PBS) IN 100 1, 21, 42 6 21, 42, 63, 84 21, 42, 63, 84 21, 42, 63, 84 Irr.vac: Irradiated Vaccine, Irra.vac.T: Irradiated Vaccine plus trehalose, For.vac.alum: Formalin inactivated vaccine plus alum, Irr.vac.alum: Irradiated vaccine plus alum, IN: Intranasal, SC: subcoutanuse, NC: Negative Control 2.6 Neutralizing Antibody (NAb) Detection The SARS-CoV-2 Neutralizing Antibody Detection Kit (Abeomics Inc.) contains the key reagents required for functional neutralizing antibody testing against SARS-CoV-2 in sera. It is a colorimetric kit used to measure the neutralizing activity of antibodies present in sera upon binding of the SARS-CoV-2 spike (RBD domain) protein to the ACE2 receptor. The recombinant SARS-CoV-2 spike (RBD) protein was pre-coated onto the 96-well microtiter plate. The serum containing neutralizing antibodies against SARS-CoV-2 spike was pipetted into the wells to achieve binding to the coated viral protein and blocking of interaction with ACE2. After washing to remove unbound components, ACE2 protein coupled to HRP was added. After a final wash, peroxidase activity was quantified using the substrate TMB. The presence of neutralizing antibodies in the samples was detected by decreasing the optical density (OD), indicating inhibition of binding between spike (RBD)and ACE2, and was measured by calculating the percentage of inhibition of each sample using the following formula: the percent inhibition= (1-(OD of the sample)/ (OD of negative control)) ×100. 2.7 Mucosal Immunity Assay The Hamster Immunoglobulin A (IgA) ELISA Kit (MyBioSource, Cat. No.: MBS029668) as a quantitative sandwich ELISA kit was used to determine IgA content in original serum, tissue samples, or other fluids. Nasal cavity and nasopharyngeal-associated lymphoid tissue (NALT) fluids from the vaccinated hamsters were collected three weeks after the second and third vaccinations to determine IgA levels using the ELISA assay. Three hamsters per group were randomly scarified and then the nasal cavity was washed and the alveoli were rinsed with 1 ml of sterile PBS plus complete protease inhibitor from Roche. Then, the fluids from the nasal cavity and bronchoalveolar (BAL) were collected and used to detect mucosal antibodies (IgA) with the Hamster Immunoglobulin A (IgA) ELISA Kit. 2.8 Spleen lymphocyte Proliferation (SLP) Response Splenic lymphocytes from the vaccinated hamsters were collected aseptically three weeks after two booster vaccinations. The single splenic lymphocyte suspensions were prepared and incubated in 96-well plates containing 5 × 10 4 cells/well in RPMI 1640 plus 10% fetal calf serum at 37° in 5% CO 2. Cells were stimulated with 50 µl phytohemagglutinin (50 µg/ml; positive control), 2.5 µg/ml inactivated SARS-CoV-2 antigen (specific antigen stimulation), and no antigen (negative control) in triplicate. After 48 hours, the cell proliferation ELISA, BrdU assay (colorimetric kit, Roche), was performed according to the manufacturer’s instructions. The BrdU kit allows accurate assessment of cell proliferation by measuring BrdU incorporation into newly synthesized cellular DNA. Absorbance was measured at 450 nm, and the stimulation index (SI) was calculated as the mean OD of three wells corresponding to cells stimulated with an antigen divided by the mean OD of the triple-negative control wells [ 13 , 17 , 19 ]. 2.9 Statistical assay Comparisons of mean values for neutralizing antibody titration, lymphocyte proliferation, and mucosal immunity were performed using SPSS, version 16, by one-way test (ANOVA) and Duncan's multiple range tests in the hamster experiment with a sample size of forty-two Syrian hamsters. All values were expressed as mean ± standard deviation, and a significant P value was considered ≤ 0.05 statistically [ 17 ]. 3. Results 3.1 Inactivated SARS-CoV-2 Virus results To produce inactivated SARS-CoV-2 viruses, two frozen concentrated SARS-CoV-2 virus stocks, one with trehalose and the other without trehalose, were irradiated with increasing doses of gamma radiation (2–15 kGy) from a cobalt-60 gamma irradiator, and virus infectivity was assessed by a TCID 50 method. Using 50% endpoint titers of irradiated and unirradiated samples (Table 2 ), a dose-response curve (Fig. 1 ) was generated using OriginPro 6.1 software and used to calculate the D 10 value, i.e., the gamma radiation dose required to reduce viral infectivity by one log 10 the virus infectivity [ 20 , 21 ]. Based on the dose-survival curve (Fig. 1 and the log-linear regression equation (Y = 5.826–0.591X), the D 10 value was set at 1.7 kGy. Based on the D 10 value, the initial titer (10 5.93 /ml), and the 5.93 log 10 reductions in titer to achieve a complete loss of viral infectivity, the minimum dose required for complete inactivation (the optimal inactivation dose) was calculated using the formula "D 10 value × 5.93” and set at approximately 11 kGy. Table 2 The mean of virus titration in irradiated and un-irradiated virus samples Gamma Irradiation (kGy) Log of Virus (without Trehalose) Titration ± SD Log of Virus (plus Trehalose) Titration ± SD 0 5.93 ± 0.21 5.94 ± 0.24 2 4.56 ± 0.06 4.59 ± 0.15 4 3.31 ± 0.16 3.47 ± 0.15 5 2.75 ± 0.11 2.75 ± 0.07 6 1.93 ± 0.08 1.87 ± 0.06 8 1.5> 0.81 ± 0.03 10 1.5> 1.5> 15 1.5> 1.5> Table 3 Safety Test of Irradiated Inactivated SARS-CoV-2 Virus on Vero Cells Irradiated Virus Samples First Passage Second Passage Third Passage Fourth Passage 11 kGy + + + + 11 kGy + + + + 12 kGy + + 12 kGy + + 14 kGy 14 kGy 3.2 Evaluation of Irradiated SARS-Cov-2 Virus antigen by ELISA Test SARS CoV-2 virus samples irradiated with 12 and 14 kGy gamma rays were used to evaluate the antigenicity of the spike protein (S1 subunit). The optical densities of all samples at 450 nm are listed in Table 4 . Table 4 The mean of OD to evaluate Spike protein (S1 subunit) antigen Serial Dilution of Viral Antigen Samples 1 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 Irradiated SARS-Cov-2 Virus (12 kGy) 1.96 1.88 1.82 1.78 1.71 1.63 1.59 1.46 1.32 1.12 Irradiated SARS-Cov-2 Virus (14 kGy) 1.94 1.89 1.81 1.75 1.70 1.63 1.54 1.44 1.32 1.16 Un- Irradiated SARS-Cov-2 Virus 1.96 1.86 1.79 1.77 1.68 1.65 1.55 1.46 1.35 1.15 Irradiated SARS-Cov-2 Virus + Trehalose (12 kGy) 2.17 2.04 1.92 1.89 1.74 1.71 1.67 1.58 1.46 1.23 Irradiated SARS-Cov-2 Virus + Trehalose (14 kGy) 2.13 2.01 1.90 1.85 1.72 1.68 1.66 1.55 1.47 1.26 Un- Irradiated SARS-Cov-2 Virus + Trehalose 2.14 2.02 1.92 1.88 1.75 1.68 1.66 1.57 1.44 1.25 Negative Control 0.14 0.12 0.14 0.15 0.14 0.12 0.11 0.15 0.16 0.11 Blank 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.03 0.02 The results of the indirect ELISA assay to evaluate the antigenic properties of the spike protein subunit S1 of SARS-CoV-2 virus show that the gamma-irradiated virus samples (12 and 14 kGy) have no significant difference in antigenic properties compared with the control samples (non-irradiated virus) (P < 0.05). However, the optical density of the irradiated and non-irradiated virus samples plus 20% trehalose is higher than that of the samples without trehalose. It may be that the disaccharide plays an important role in maintaining the properties of proteins, which of course should be further investigated when evaluating the inactivated virus antigen in animal models. 3.3 Neutralizing Antibody Response Following infection with SARS-CoV-2 , patients develop specific IgG and IgM antibodies. A subset of these antibodies to spike (RBD) can block viral infection and cell entry through a process called neutralization. A strong correlation has been observed between the levels of RBD-binding antibody and levels of SARS-CoV-2 blocked by neutralizing antibodies in patients [ 22 ]. Percent inhibition of spike (RBD)-ACE2 binding indicates the titration of neutralizing antibodies, as shown in Fig. 2 . Percent inhibition of spike (RBD)-ACE2 binding indicates a significant increase in neutralizing antibody titer three weeks after the third vaccination (P < 0.05). All vaccination groups showed a significant increase in neutralizing antibodies compared with the PBS groups (groups 6 and 7) three weeks after the first, second, and third vaccinations (P < 0.05). 3.4 IgA Concentration Nasal lavage and NALT fluid from vaccinated hamsters were used to evaluate mucosal immunity with the quantitative sandwich ELISA kit to determine IgA levels. The IgA concentration is shown in Fig. 3 . While circulating IgA is mostly monomeric and consists predominantly of the IgA1 subclass, secretory IgA (sIgA) is dimeric and consists of IgA1 and IgA2 [ 23 ]. SARS-CoV-2 primarily infects the upper respiratory tract (URT). Mucosal immune responses are expected to be initiated in the NALT (via the nasal epithelium and tonsils), which serve as sites of induction for the mucosal immune system [ 23 , 24 ]. An important effector molecule at mucosal sites is antibodies, which have two main sources: translocation of circulating IgG into the mucosa and local production of IgA [ 25 ]. In this study, secretory IgA was examined in nasal wash and NALT fluids, and the results showed that the concentration of sIgA in irradiated vaccine plus trehalose increased significantly 3 weeks after second and third vaccination ( P < 0.05 ). 4.5 Cellular Immunity T cells against coronaviruses persist in humans and can be detected months and/or years after infection; (2) memory Tc cells are directed against conserved regions of coronaviruses and are therefore largely cross-reactive. Therefore, T-cell-based vaccination approaches against COVID-19 are of realistic benefits for considering [ 26 ]. In this study, the splenic lymphocyte proliferation (SLP) assay was performed using a colorimetric BrdU kit, and the stimulation index of splenocytes was reported in Table 7. A significant increase in splenic lymphocyte proliferation was observed in all vaccinated hamsters (P < 0.05), but the increase was greater with irradiated vaccine plus trehalose and irradiated vaccine plus alum. 4. Discussion The SARS-CoV-2 virus has caused the most severe pandemic in the world. The development of a safe and effective vaccine is essential to manage the disease. We optimized an inactivated virus vaccine using the gamma irradiation method as an alternative to classical chemical inactivation methods. Previous studies have shown that gamma irradiation can induce immunogenicity more effectively than conventional inactivation methods [ 6 , 27 ]. The use of gamma radiation process (due to a cobalt-60 source) has been developed for the production of effective vaccines [ 28 , 29 ]. Gamma irradiation preserves antigen assembly, can reduce free radical damage due to water radiolysis, and can be used in a frozen state [ 20 , 30 ]. Gamma irradiation is a cost-effective and faster method of viral inactivation used to produce many inactivated viral vaccines, such as human and avian influenza vaccines. The current study reports on the efficacy of a gamma-irradiated virus vaccine that rather than formalin inactivated virus. The spike protein consists of two subunits, S1 and S2, which are cleaved by host cell furin-like proteases such as cathepsins during assembly and release of virus particles from infected cells. The S1 subunit has a variable sequence across coronavirus genera and species. The sequence of the spike protein in the genera of coronaviruses alpha and delta is very similar. The S1 subunit contains the receptor-binding domain (RBD) and plays an important role in binding to host receptors. Therefore, considering the important antigenic property of the spike protein in inducing immune responses in the host, the anti-S1 spike protein monoclonal antibody was used by ELISA method to compare the antigenic properties of the irradiated and un-irradiated virus samples. The spike (S) protein of the virus, which contains the major neutralizing epitopes in the receptor-binding domain (RBD) and the N-terminal domain (NTD), has been shown to be the most promising immunogen. Thus, most recently approved vaccines use full-length S (with or without modification) or whole virus (inactivated) as the target antigen [ 31 ]. Also in this study, evaluation of the antigenic properties of the spike protein using an ELISA kit showed no significant differences between irradiated and un-irradiated SARS-CoV- 2 virus samples with and without trehalose ( P > 0.05). However, the optical density of the irradiated and un-irradiated virus samples plus 20% trehalose is higher than that of the samples without trehalose. It may depend on the disaccharide playing an important role in maintaining the properties of proteins, which of course should be further investigated when evaluating the inactivated virus antigen in animal models. Neutralizing Antibody Offersgaard reported that increasing the dose of inactivated SARS-CoV-2 improved the induction of neutralizing antibodies when tested in mice. Although a second immunization in both mice and hamsters resulted in a large increase in nAbs titers compared with a single immunization, a third immunization in mice had little effect on nAb titers and S-specific antibody endpoint titers compared with two immunizations [ 32 ]. At times when the Delta or Omicron variant is prevalent, a third dose of vaccination has been shown to be highly effective in protecting individuals from severe COVID-19 -related sequelae and preventing COVID-19 -associated hospitalizations [ 33 , 34 ]. Furthermore, higher levels of neutralizing antibodies are associated with lower risk of symptomatic infection, and immune protection depends on levels of neutralizing antibodies [ 35 , 36 ]. Therefore, the vaccine-mediated antibody response against SARS-CoV-2 is required for higher efficacy of the SARS-CoV-2 vaccine [ 41 ]. Inactivated SARS-CoV − 2 vaccines based on a traditional platform show high safety and efficacy and prevent COVID-19 serum antibody response to currently commercially available inactivated vaccines has been studied in detail [ 38 , 39 , 40 , 41 ]. However, the profile of serum or plasma antibody response elicited by inactivated vaccines against all circulating variants of concern (VOCs) (alpha, beta, gamma, and delta) and circulating variants of interest (VOIs) (lambda, mu, kappa, eta, iota v1, iota v2, epsilon, and zeta) is less well defined. Also, the efficacy of vaccine-induced neutralizing antibodies is rarely reported. Therefore, a comprehensive analysis of the characteristics of antibody responses to inactivated vaccines and characterization of potent and broadly neutralizing antibodies is informative for optimizing and updating vaccine design and immunization strategies and therapeutics [ 37 ]. Passive antibodies administered are one of the most promising therapeutic and prophylactic anti- SARS-CoV- 2 agents. To date, the most potent monoclonal antibodies (mAbs) isolated from infected and vaccinated individuals were often dominant by those targeting RBD while many isolated NTD mAbs failed to reach 100% potency in neutralizing activity [ 39 ]. In this study, a gamma-irradiated vaccine against SARS-CoV-2 was used as an inactivated vaccine, and the neutralising antibody response showed a significant increase in four vaccinated hamster groups as early as three weeks after the first vaccination. However, it reached the peak antibody titer three weeks after the third vaccination. Therefore, we can suggest that the gamma-irradiated inactivated vaccine SARS-COV-2 can be vaccinated in three doses, one primer and two booster doses three weeks apart. Mucosal antibodies IgA can be enriched up to threefold in upper respiratory tract secretions compared to IgG, while IgG is the most common isotype in the blood and lower respiratory tract. The higher concentration of sIgA compared to IgG has been shown to result in greater avidity and higher neutralising capacity [ 25 , 42 , 43 , and 44 ]. Mucosal IgG is usually derived from plasma by transduction but can also be produced locally by mucosal B cells in the lamina propria. In addition to directly neutralising viruses, non-neutralising antibodies can also mediate the clearance of viruses and virus-infected cells via interactions of the Fc domain of the antibody with complement [ 45 ], thus enabling antibody-dependent cellular cytotoxicity (ADCC) [ 46 ]. Recently, survival after moderate-severe SARS-CoV-2 infection was associated with antibody responses with robust Fc effector activity, suggesting that such immunity may contribute to protection against respiratory disease. IgA can be expressed at mucosal surfaces in both monomeric and dimeric forms as secretory IgA (sIgA) and occurs in humans in two isotypes, with IgA1 present in both systemic and mucosal secretions and IgA2 predominantly in mucosa [ 25 ]. Although sIgA has been reported to be elicited at oral and nasal mucosal surfaces after intramuscular vaccination in both clinical and animal studies for influenza and SARS-CoV-2 , titers are generally modest and variable. In contrast, mucosal immunisation readily produces robust sIgA responses in upper and lower respiratory tract mucosa [ 25 ]. One of the most informative methods for evaluating effective immune responses against SARS-CoV-2 , whether triggered by natural infection or intranasal immunization, is the determination of mucosal sIgA in nasal secretions or saliva synthesized by IgA-secreting plasma cells. In this study, the concentration of sIgA in nasal washings and NALT was evaluated and it increased significantly when the irradiated vaccine plus trehalose was administered via the intranasal route three weeks after the second and third vaccinations ( P < 0.05 ). Spleen Lymphocyte proliferation as the T cell response (ref5) Previous experience with SARS-CoV-1 and MERS suggests that T cells may be the most important immune response for disease control [ 48 ]. However, T-cell immunity against coronaviruses is an important aspect of a successful vaccine, and it is a long-lived vaccine. In contrast to antibodies, cytotoxic T cells against coronaviruses persist and can be detected several months after an infection. Therefore, an ideal SARS-CoV-2 vaccine should stimulate both B and T cell immunity to provide optimal protection against COVID-19 [ 27 ]. Gamma radiation, as a superior inactivation method, can preserve T cell immunogenicity compared with other inactivation methods. Gamma rays can strongly penetrate the virus, resulting in direct damage to the genetic material without altering the structural proteins [ 49 ]. Mullbacher has previously shown that alphaviruses [ 50 ] and bunyaviruses [ 51 ] can be rendered noninfectious by gamma irradiation and yet have the ability to elicit cytotoxic T-cell responses. Gamma-irradiation can be used to produce an experimental influenza vaccine and reported that gamma-irradiated Influenza virus preparations promoted T-cell immunity [ 52 , 53 , 54 , 55 , and 56 ]. Viral replication can be eliminated during gamma irradiation, while immunogenicity and viral protein structure are preserved, so that viral proteins are naturally presented to the immune system, facilitating the induction of both T cells and humoral immunity. In the current study, splenic lymphocyte proliferation was increased in all vaccinated hamsters and was higher in the irradiated vaccine groups. In addition, we may introduce an irradiated inactivated vaccine SARS-CoV-2 plus disaccharide trehalose via the intranasal route of administration and another irradiated inactivated vaccine SARS-CoV-2 plus alum via the subcutaneous route of administration as safe and efficient vaccines against COVID-19 . Furthermore, cytokine assay will be done in the future study to evaluate exact immune cells. Declarations Acknowledgments The authors would like to gratitude Dr. Viskam Vijewardana in the Department of Nuclear Sciences and Applications, Animal Production and Health Section, International Atomic Energy Agency (IAEA), VIC, Vienna, Austria to purchase SARS-CoV-2 anti-spike protein S1 monoclonal antibody (GT263-Invitrogen) and goat anti-mouse IgG (H+L), HRP conjugate (Invitrogen). Also, we really appreciate Dr. Ivancho Naletoski in the Department of Nuclear Sciences and Applications, Animal Production and Health Section, IAEA, Vienna, Austria for ordering to purchase the SARS-CoV-2 Neutralizing Antibody Detection Kit (Abeomics Inc.) and Hamster Immunoglobulin A (IgA) ELISA Kit (MyBioSource, Cat. No.: MBS029668) and sent for my institute. Funding This study was supported by Iran National Science Foundation (INSF) (Project No.99024693). The paid fund by INSF was used just for buying hamsters and some feed for animals (400 $), but it was not enough for article processing charge. My institution and my research grant provide no fund for Open Access fee support. My country is low in-coming; also sanction makes an inhibition for money transfer from Iran. Also, this research was supported by IAEA to purchase some necessary reagents and kits. Conflict of Interest The authors declare that they have no conflict of interest. The funders had no role in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Author contribution statement These authors have contributed in different parts of the manuscript such as; Farahnaz Motamedi Sedeh is the corresponding author and senior authorship. Akbar Khorasani is the senior authorship. Mohsen Lotfi contributed in methodology. Seyed Morteza Moosavi has contributed in writing and editing. Arash Arbabi and Seyedeh Maede Hosseini have contributed in methodology and writing. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval The animal study was reviewed and approved by Tehran University of Medical Science. For this research, all institutional and national guidelines adopted by the horizontal legislation on the protection of animals used for scientific purposes (Directive 2010/63/EU, amended by Regulation (EU) 2019/1010) were approved for implementation. Consent to participate The authors acknowledge affirming that freely given informed consent was obtained from the infected man (43 years old) in the study for the collection of tracheal swabs in Karaj, Iran, in May 2020. References Cui J, Li F, Shi Z-L (2019) Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol 17(3):181–192 Santos IdeA, Grosche VR, Bergamini FRG, Sabino-Silva R, Jardim ACG (2020) Antivirals Against Coronaviruses: Candidate Drugs for SARS-CoV-2 Treatment? 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J Vet Med Sci 64(4):377–379 Ong WT, Omar AR, Ideris A, Hassan SS (2007) Development of a multiplex real-time PCR assay using SYBR Green 1 chemistry for simultaneous detection and subtyping of H9N2 influenza virus type A. J Virol Methods 144(1–2):57–64 Sandbulte MR, Roth JA (2004) Methods for analysis of cell-mediated immunity in domestic animal species. J Am Vet Med Assoc 225(4):522–530 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3405744","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":238380412,"identity":"caf23049-4797-44be-ade6-b98c02284f0b","order_by":0,"name":"Farahnaz Motamedi-Sedeh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYBAC9gYGBmY4j8fABkgyNh7Ap4XnAFQLD0RLGkhLAylaGA6DRfFrYT/78HMBwzY5e/bTiQ/eFJy3W9t+GGhLjU00Ti086cbSMxhuG/Pw5G42nGNwO3nbmUSglmNpuQ04tNgzpDFI8zDcTuxhyN0mzQPUYnYAqIWx4TBOLTz8z5h/g7Xwv93+m8fgXLLZ+YcEtEiksUFskcjdxsxjcMDO7AYhWySesVkD3WPMc+PtZsk5BskJZjeAtiTg8QsPfxrzbZ6K23Ls/bkbP7z5Y2dvdj794YMPNTY4tUCAAYKZCFaZgFc5GrAnRfEoGAWjYBSMDAAAPklcfM6uLkMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-3388-9670","institution":"Nuclear Science and Technology Research Institute","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Farahnaz","middleName":"","lastName":"Motamedi-Sedeh","suffix":""},{"id":238380413,"identity":"1ea0f7ea-01d5-45eb-ba2c-db7beade15e4","order_by":1,"name":"Akbar Khorasani","email":"","orcid":"","institution":"Razi Vaccine and Serum Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Akbar","middleName":"","lastName":"Khorasani","suffix":""},{"id":238380414,"identity":"f8db9cdc-9007-4f6d-95d2-f2280b7cac77","order_by":2,"name":"Mohsen Lotfi","email":"","orcid":"","institution":"Razi Vaccine and Serum Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mohsen","middleName":"","lastName":"Lotfi","suffix":""},{"id":238380415,"identity":"a56e2964-c1d1-4132-b3d3-b7175fcb9eb9","order_by":3,"name":"Seyed Morteza Moosavi","email":"","orcid":"","institution":"Nuclear Science and Technology Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seyed","middleName":"Morteza","lastName":"Moosavi","suffix":""},{"id":238380416,"identity":"781209be-99ed-4323-8d6e-933c5dc11ef9","order_by":4,"name":"Arash Arbabi","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Arash","middleName":"","lastName":"Arbabi","suffix":""},{"id":238380417,"identity":"a7d8313c-142d-485a-b2fd-a811eacd710a","order_by":5,"name":"Seyed Maede Hosseini","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seyed","middleName":"Maede","lastName":"Hosseini","suffix":""}],"badges":[],"createdAt":"2023-10-02 19:07:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3405744/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3405744/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":44451691,"identity":"c7df4b5b-6173-4f2e-b528-2dea98449a87","added_by":"auto","created_at":"2023-10-11 16:46:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":318317,"visible":true,"origin":"","legend":"\u003cp\u003eDose/Response Curve of Gamma Irradiated SARS CoV-2 virus samples\u003c/p\u003e","description":"","filename":"Fig1.D10.png","url":"https://assets-eu.researchsquare.com/files/rs-3405744/v1/8cfed2d52a50e7d110c75646.png"},{"id":44451850,"identity":"ff48b12e-965e-443b-b113-1cf0c8251c1e","added_by":"auto","created_at":"2023-10-11 16:54:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3038797,"visible":true,"origin":"","legend":"\u003cp\u003eThe inhibition percent of the Spike (RBD)-ACE2 binding indicate the neutralizing antibody titration; (Wafv: week after first vaccination, wasv: week after second vaccination, watv: week after third vaccination, IN: intranasal, SC: subcutaneous).\u003c/p\u003e","description":"","filename":"Fig2.nAb.png","url":"https://assets-eu.researchsquare.com/files/rs-3405744/v1/f063886fd75713b82a786c7e.png"},{"id":44450584,"identity":"400d201e-4fe4-4880-9127-74202580163e","added_by":"auto","created_at":"2023-10-11 16:38:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1579130,"visible":true,"origin":"","legend":"\u003cp\u003eIgA concentration (µg/ml); (Wafv: week after first vaccination, wasv: week after second vaccination, watv: week after third vaccination, IN: intranasal, SC: subcutaneous).\u003c/p\u003e","description":"","filename":"Fig3.IgA.png","url":"https://assets-eu.researchsquare.com/files/rs-3405744/v1/10b6936f317c7dc5c250ed80.png"},{"id":44450586,"identity":"b4b6d570-3d14-41a5-9877-8c87adcfcb9e","added_by":"auto","created_at":"2023-10-11 16:38:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1869057,"visible":true,"origin":"","legend":"\u003cp\u003eThe spleen lymphocyte Proliferation (SLP) results showed as Stimulation Index (SI); (Wafv: week after first vaccination, wasv: week after second vaccination, watv: week after third vaccination, IN: intranasal, SC: subcutaneous).\u003c/p\u003e","description":"","filename":"Fig4.SI.png","url":"https://assets-eu.researchsquare.com/files/rs-3405744/v1/3cae750b65421261317e0e60.png"},{"id":45998710,"identity":"4220c00e-16f0-4edc-a3f7-5b010b65bd4b","added_by":"auto","created_at":"2023-11-07 10:59:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1948035,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3405744/v1/dd1244e2-59fb-4b69-a664-5b62154cb39e.pdf"}],"financialInterests":"","formattedTitle":"Immune Responses Following Administration of Whole Gamma Irradiated SARS-CoV-2 Vaccine Stabilized With Disaccharide Trehalose on Syrian Hamster Model","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003e \u003cem\u003eCoronaviruses\u003c/em\u003e are enveloped RNA viruses that are widely distributed among humans, other mammals, and birds and cause acute and persistent infections. Members of this family were isolated as early as the 1930s as causative agents of infectious bronchitis in chickens, transmissible gastroenteritis in pigs, and severe hepatitis and neurologic disease in mice. Viruses of the family \u003cem\u003eCoronaviridae\u003c/em\u003e are large, enveloped virions with positive RNA genomes (27\u0026ndash;32 Kb) and diameters of 100\u0026ndash;120 nm. The RNA genome of coronaviruses combines with the N-(nucleocapsid) phosphoprotein (50\u0026ndash;60 kd) to form a long, flexible, helical nucleocapsid. Two types of human \u003cem\u003eCoronavirus\u003c/em\u003e (HCV) have been identified: HCV-229E belongs to serotype I and causes respiratory tract infections, and HCV-OC43 belongs to serotype II and causes respiratory and intestinal infections. Severe acute respiratory syndrome (\u003cem\u003eSARS\u003c/em\u003e) was spread worldwide in 2002 as a new human disease. Another zoonotic coronavirus, Middle East respiratory syndrome (\u003cem\u003eMERS\u003c/em\u003e), was crossed species from camels to cause the often-fatal human disease in 2012 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The \u003cem\u003eSARS-CoV-2\u003c/em\u003e virus is the causative agent of the emerging respiratory zoonosis known as \u003cem\u003eCOVID-19\u003c/em\u003e from 2019. Neutralising antibodies (nAbs) against the disease agent, severe acute respiratory syndrome coronavirus 2 (\u003cem\u003eSARS-CoV-2\u003c/em\u003e), represent potential prophylactic and therapeutic options and could aid in vaccine development [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In general, nAbs with outstanding potency (so-called super-antibodies) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] can be isolated by studying in detail the antibody responses of a sample of infected donors. The outstanding potency combined with the ability to extend the half-life of antibodies from weeks to many months lowers the effective cost of antibodies and provides more opportunities for prophylactic measures. As a cellular receptor, \u003cem\u003eSARS-CoV-2\u003c/em\u003e could use the human, bat, or civet ACE2 receptor, but no the mouse [\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]. Therefore, it seems that mice expressing hACE2 would be a conceivable choice for vaccine challenge testing. Appropriate small animal models are critical for the study of viral pathogenesis, the production of vaccines, and the development of antiviral therapies. Hamster models are popular because of their affordability, availability, and simple genetic structure and have been widely used to study human coronavirus pathogenesis. The vaccinated hamsters against \u003cem\u003eSARS-CoV-2\u003c/em\u003e virus could effectively induce memory B and T cells and were able to protect reinfection of animals with the same strain [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. One of the most important requirements for the control of emerging and re-emerging zoonosis is the development of vaccines within a short time. The most recent example is the inactivated whole virus vaccine against \u003cem\u003eSARS-CoV-2\u003c/em\u003e, which is still in use. The use of chemical substances such as formalin and beta-propiolactone is the common method for producing inactivated vaccines. Although inactivation with formalin is well established, the process has serious drawbacks, e.g., formalin is toxic and there are problems with residual traces in the final product. In addition, formalin leads to cross-linking and changes in structural components and damages antigenic structures, which affects the antigenicity of vaccines [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Other chemicals such as beta-propiolactone are also very dangerous. Therefore, an alternative to the chemical inactivation method must be found to produce an inactivated vaccine [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Recently, ionizing irradiation (gamma radiation by Co-60, X-rays, and electron beams) has been considered as an alternative for vaccine development. The irradiation inactivation method has some advantages over chemical inactivation for vaccine production. Recently, irradiation with low-energy electron beams has been shown to be much safer than gamma irradiation with a Co-60 source and can preserve viral and bacterial antigenicity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The aim of this study was to investigate the efficacy of gamma irradiation in inactivating emerging/outbreaking pathogens. In this study, we tested our gamma irradiation and formalin-inactivated vaccine candidates for their immunogenicity and immune response against \u003cem\u003eSARS-CoV-2\u003c/em\u003e virus and demonstrated vaccine efficacy in Syrian hamsters. The vaccine candidates were administered intranasally and intradermally to the hamsters, requiring a higher amount of inactivated virus for proper immunization.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Virus (isolation, confirmation by RT-PCR, multiplication on Vero cell)\u003c/h2\u003e \u003cp\u003eThe isolated \u003cem\u003eSARS-CoV-2\u003c/em\u003e virus from tracheal swabs of an infected man (43 years old) in Karaj, Iran, in May 2020 on Vero cells was used for this study. The Ct value of this sample in the RT-qP assay was 16. The tracheal swab samples were transferred to viral transport media (VTM) and transported to the cell culture laboratory under cold chain and inoculation on Vero cells with DMEM media plus 3% fetal calf serum and 1% penicillin-streptomycin solution (10000 units penicillin and 10 mg/ml streptomycin), at 37 \u0026ordm;C and 5% CO2. The cytopathic effect (CPE) was visible after 24\u0026ndash;48 hours on Vero cells in the form of cell rounding and the development of syncytial cells. Virus samples were isolated and propagated in biosafety level facilities III at Razi Vaccine and Serum Research Institute, Karaj, Iran. The infected cells were harvested and confirmed using RT -qPCR kit (AccuPower\u0026reg; SARS-CoV-2 Real-Time RT-PCR Kit, Bioneer). The infected cells were used to infect four interval passages on Vero cells. After harvesting the fourth passage of infected cells, it was used for RT-PCR assay with specific primers for the spike (S) gene. The PCR product of the S gene was sequenced, and the nucleotide sequence was deposited in NCBI under accession number: MW709393 as the \u003cem\u003eSARS-CoV-2/human/IRN/Alborz- IR /2020\u003c/em\u003e surface glycoprotein (S) gene with 3822 bp length [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe isolated \u003cem\u003eSARS CoV-2\u003c/em\u003e virus was propagated on Vero cells, and the infected cell suspension was centrifuged at low speed for 15 min at 4 \u0026ordm;C to clear cell debris. The supernatant was taken as virus stock and divided into three parts. The 20% disaccharide trehalose (1 M) was added to the first part, and the second and third parts contained no trehalose. All virus strain stocks were aliquoted into 2-ml cryotubes and stored at -70 \u0026ordm;C. Two virus samples from two virus stocks (with trehalose and without trehalose) were used for virus titration by the TCID\u003csub\u003e50\u003c/sub\u003e method. The first and second virus stocks were used for irradiation, and the third was inactivated with formalin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 \u003cem\u003eSARS CoV-2\u003c/em\u003e Virus Inactivation using Gamma Irradiation and Formalin:\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eSARS-CoV-2\u003c/em\u003e virus stocks were irradiated with a Cobalt-60 irradiator, Gamma cell 220 (MDS Nordion, Ottawa, Canada), at a dose rate of 0.93 Gy/s and an activity of 3985 Ci to inactivate viral infectivity and to inactivate \u003cem\u003eSARS-CoV-2\u003c/em\u003e virus genomic RNA in Nuclear Science and Technology Research Institute (NSTRI), Tehran, Iran. Gamma radiation doses of 2, 4, 5, 8, 10, and 15 kGy were administered to infected specimens frozen on dry ice. Each dose was repeated in three rounds [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, and \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Viral titration of all irradiated samples was performed using the TCID\u003csub\u003e50\u003c/sub\u003e method. The third virus stock was inactivated with formalin 0.04% v/v at RT for 30 hours (final concentration 4:10000). The inactivated virus stocks (irradiated and treated with formalin) were concentrated by ultrafiltration and 8% PEG-6000. The concentrated and inactivated viruses were dialyzed against PBS and quantified for protein concentration using Nano-drop (Smart Nano-1000).\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.3 Safety Test and antigenicity evaluation by ELISA Test\u003c/b\u003e: The infectivity of the irradiated, inactivated \u003cem\u003eSARS-CoV-2\u003c/em\u003e virus was determined after inoculation of Vero cell monolayers at 37\u0026deg; for 48 hours, then sub-cultured in four blind cultures on fresh Vero cells, and viral titration was determined by TCID50 method [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In addition, the antigenic properties of irradiated and native virus samples were tested using the ELISA assay for S (spike) protein. The \u003cem\u003eSARS-CoV-2\u003c/em\u003e spike protein is one of the major surface glycoproteins. It consists of an S1 domain and S2 domain and binds to host cell receptors (ACE2). This protein plays a major role in viral infection and is involved in the fusion of the viral envelope with the cell membrane. To verify the antigenic properties of the irradiated virus samples in comparison with the control virus, the ELISA method was applied. \u003cem\u003eSARS-CoV-2\u003c/em\u003e anti-spike protein S1 monoclonal antibody (GT263) (1 mg/ml) 100 \u0026micro;l (Invitrogen) and goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L), HRP conjugate (Invitrogen) (REF: G21040) reconstituted in 1 ml PBS, pH 7.2, to obtain 1 mg/ml stock solution were used. The receptor-binding domain (RBD) of S1 can specifically bind to angiotensin-converting enzyme 2 (ACE2), the receptor on target cells. The diversity of \u003cem\u003eSARS-CoV-2\u003c/em\u003e is reflected in the variable spike proteins (S proteins) that have evolved in forms that differ in their receptor interactions and their response to various environmental triggers of virus-cell membrane fusion. Antigenicity evaluation of \u003cem\u003eSARS-CoV-2\u003c/em\u003e viruses was performed by ELISA assay, first preparing serial dilutions (1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256, and 1/512) of irradiated and unirradiated viral antigen in bicarbonate buffer as coating buffer (PH\u0026thinsp;=\u0026thinsp;9.6). 100 \u0026micro;l of each antigen dilution was added to each well of the 96-well microplate in duplicate and incubated overnight at 4 ˚C. The suspension of Vero cells was used in duplicate as a negative control. Then all wells were washed 5 times with wash buffer (PBS\u0026thinsp;+\u0026thinsp;0.05% Tween 20). 200 \u0026micro;l of blocking buffer (PBS\u0026thinsp;+\u0026thinsp;5% skim milk) was added to each well and stored at 37 \u0026ordm;C for 2 hours to block the wells, after which they were washed three times. \u003cem\u003eSARS-CoV-2\u003c/em\u003e spike protein S1 monoclonal antibody (GT263) (1 mg/ml) (Invitrogen) was diluted 1/1000 and 100 \u0026micro;l of the dilution was added to each well for 1 hour at 37 \u0026ordm;C and then washed three times. Goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L), HRP conjugate (Invitrogen) (REF: G21040) reconstituted in 1 ml PBS, pH 7.2 to obtain 1 mg/ml stock solution, stored at -20 \u0026ordm;C, working solution 1/10000 was prepared and 100 \u0026micro;l of the working solution was added to each well 1 hour at 37 \u0026ordm;C. TMB (100 \u0026micro;l) was added to each well in the dark at room temperature for 30 minutes. Finally, a stop solution (sulfuric acid 1.25 mM) was added and the optical density was read at 450 nm using Hyperion MicroReader 4 Plus.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Vaccine Formulation\u003c/h2\u003e \u003cp\u003eThe first vaccine formulation was irradiated \u003cem\u003eSARS-CoV-2\u003c/em\u003e virus, and the second vaccine formulation was irradiated \u003cem\u003eSARS-CoV-2\u003c/em\u003e virus plus 20% disaccharide trehalose (1 M). Trehalose can stabilize proteins as a cryoprotectant and as a free radical quencher and acts as a natural stabilizer of life processes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, and \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The third and fourth vaccine formulations were formalin-inactivated virus and irradiated virus mixed with alum. Alum compounds such as aluminum hydroxide (AH) or alhydrogel (chemically crystalline aluminum hydroxide) increase uptake and presentation of antigen by antigen-presenting cells (APCs) and enhance monocyte recruitment at the site of inoculation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Syrian Hamster Immunization by Four Inactivated \u003cem\u003eSARS CoV-2\u003c/em\u003e vaccines\u003c/h2\u003e \u003cp\u003eFour regimens of inactivated \u003cem\u003eSARS-CoV-2\u003c/em\u003e vaccines were formulated and used to immunize male Syrian hamsters (weight 100\u0026thinsp;\u0026plusmn;\u0026thinsp;10 g). Forty-two Syrian hamsters were purchased from Razi Vaccine and Serum Research Institute and divided into seven groups of 6 animals each (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The first group was used as preimmune animals and sampling was done before immunization. The other groups of animals were vaccinated with: irradiated inactivated SARS-CoV-2 antigen (intranasal), irradiated inactivated \u003cem\u003eSARS-CoV-2\u003c/em\u003e antigen\u0026thinsp;+\u0026thinsp;20% trehalose (intranasal), formalin-inactivated \u003cem\u003eSARS-CoV-2\u003c/em\u003e antigen\u0026thinsp;+\u0026thinsp;alum (subcutaneous), irradiated inactivated \u003cem\u003eSARS-CoV-2\u003c/em\u003e antigen\u0026thinsp;+\u0026thinsp;alum (subcutaneous), negative control group inoculated with 100 \u0026micro;l sterile PBS (intranasal), negative control group injected with 100 \u0026micro;l sterile PBS (subcutaneous). Vaccination was administered via two routes of administration (intranasal and subcutaneous injection into the neck) as one prime and two booster doses three weeks apart. Blood samples were collected from the orbit of chloroform-anesthetized animals three weeks after each vaccination. Each hamster was anesthetized by inhalation, and then blood was collected from the inner corner of the eye using a sterile Pasteur pipette. Blood tubes were kept overnight at 4\u0026deg;C, and then sera were separated by centrifugation at 600g for 10 minutes at 4\u0026deg;C. The complement compounds in the isolated sera were inactivated at 56\u0026deg;C for 30 minutes. The sera were analyzed for the presence of antibodies to \u003cem\u003eSARS-CoV-2\u003c/em\u003e virus using the \u003cem\u003eSARS-CoV-2\u003c/em\u003e Neutralizing Antibody Detection Kit (Abeomics Inc.). Splenic lymphocytes from the hamster groups were cultured and stimulated by homologous-inactivated antigens to evaluate the splenic lymphocyte proliferation assay as cellular immunity three weeks after the second and third vaccinations (three animals in each group were used for each collection day). The schedule of animal vaccination was shown in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\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\u003eThe schedule of the hamster vaccination against SARS-COV-2 virus and sampling\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=\"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=\"char\" char=\".\" 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\u003eNo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVaccine Groups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoute of adminstration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVaccine dose (\u0026micro;l)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eVaccination day\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNumber of animal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSampling day for Nt Ab Titration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSampling day for SLP assay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eSampling day for IgA Titration\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\u003ePre-Immune\u003c/p\u003e \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=\"char\" char=\".\" colname=\"c6\"\u003e 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84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e21, 42, 63, 84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e21, 42, 63, 84\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\u003eIrra.vac.T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1, 21, 42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21, 42, 63, 84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e21, 42, 63, 84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e21, 42, 63, 84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFor.vac.alum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1, 21, 42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21, 42, 63, 84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e21, 42, 63, 84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e21, 42, 63, 84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIrr.vac.alum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1, 21, 42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21, 42, 63, 84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e21, 42, 63, 84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e21, 42, 63, 84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC (PBS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1, 21, 42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21, 42, 63, 84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e21, 42, 63, 84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e21, 42, 63, 84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC (PBS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1, 21, 42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21, 42, 63, 84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e21, 42, 63, 84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e21, 42, 63, 84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eIrr.vac: Irradiated Vaccine, Irra.vac.T: Irradiated Vaccine plus trehalose, For.vac.alum: Formalin inactivated vaccine plus alum, Irr.vac.alum: Irradiated vaccine plus alum, IN: Intranasal, SC: subcoutanuse, NC: Negative Control\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Neutralizing Antibody (NAb) Detection\u003c/h2\u003e \u003cp\u003eThe SARS-CoV-2 Neutralizing Antibody Detection Kit (Abeomics Inc.) contains the key reagents required for functional neutralizing antibody testing against SARS-CoV-2 in sera. It is a colorimetric kit used to measure the neutralizing activity of antibodies present in sera upon binding of the \u003cem\u003eSARS-CoV-2\u003c/em\u003e spike (RBD domain) protein to the ACE2 receptor. The recombinant \u003cem\u003eSARS-CoV-2\u003c/em\u003e spike (RBD) protein was pre-coated onto the 96-well microtiter plate. The serum containing neutralizing antibodies against SARS-CoV-2 spike was pipetted into the wells to achieve binding to the coated viral protein and blocking of interaction with ACE2. After washing to remove unbound components, ACE2 protein coupled to HRP was added. After a final wash, peroxidase activity was quantified using the substrate TMB. The presence of neutralizing antibodies in the samples was detected by decreasing the optical density (OD), indicating inhibition of binding between spike (RBD)and ACE2, and was measured by calculating the percentage of inhibition of each sample using the following formula: the percent inhibition= (1-(OD of the sample)/ (OD of negative control)) \u0026times;100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Mucosal Immunity Assay\u003c/h2\u003e \u003cp\u003eThe Hamster Immunoglobulin A (IgA) ELISA Kit (MyBioSource, Cat. No.: MBS029668) as a quantitative sandwich ELISA kit was used to determine IgA content in original serum, tissue samples, or other fluids. Nasal cavity and nasopharyngeal-associated lymphoid tissue (NALT) fluids from the vaccinated hamsters were collected three weeks after the second and third vaccinations to determine IgA levels using the ELISA assay. Three hamsters per group were randomly scarified and then the nasal cavity was washed and the alveoli were rinsed with 1 ml of sterile PBS plus complete protease inhibitor from Roche. Then, the fluids from the nasal cavity and bronchoalveolar (BAL) were collected and used to detect mucosal antibodies (IgA) with the Hamster Immunoglobulin A (IgA) ELISA Kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Spleen lymphocyte Proliferation (SLP) Response\u003c/h2\u003e \u003cp\u003eSplenic lymphocytes from the vaccinated hamsters were collected aseptically three weeks after two booster vaccinations. The single splenic lymphocyte suspensions were prepared and incubated in 96-well plates containing 5 \u0026times; 10 4 cells/well in RPMI 1640 plus 10% fetal calf serum at 37\u0026deg; in 5% CO 2. Cells were stimulated with 50 \u0026micro;l phytohemagglutinin (50 \u0026micro;g/ml; positive control), 2.5 \u0026micro;g/ml inactivated SARS-CoV-2 antigen (specific antigen stimulation), and no antigen (negative control) in triplicate. After 48 hours, the cell proliferation ELISA, BrdU assay (colorimetric kit, Roche), was performed according to the manufacturer\u0026rsquo;s instructions. The BrdU kit allows accurate assessment of cell proliferation by measuring BrdU incorporation into newly synthesized cellular DNA. Absorbance was measured at 450 nm, and the stimulation index (SI) was calculated as the mean OD of three wells corresponding to cells stimulated with an antigen divided by the mean OD of the triple-negative control wells [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical assay\u003c/h2\u003e \u003cp\u003eComparisons of mean values for neutralizing antibody titration, lymphocyte proliferation, and mucosal immunity were performed using SPSS, version 16, by one-way test (ANOVA) and Duncan's multiple range tests in the hamster experiment with a sample size of forty-two Syrian hamsters. All values were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, and a significant P value was considered\u0026thinsp;\u0026le;\u0026thinsp;0.05 statistically [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Inactivated SARS-CoV-2 Virus results\u003c/h2\u003e \u003cp\u003eTo produce inactivated \u003cem\u003eSARS-CoV-2\u003c/em\u003e viruses, two frozen concentrated \u003cem\u003eSARS-CoV-2\u003c/em\u003e virus stocks, one with trehalose and the other without trehalose, were irradiated with increasing doses of gamma radiation (2\u0026ndash;15 kGy) from a cobalt-60 gamma irradiator, and virus infectivity was assessed by a TCID\u003csub\u003e50\u003c/sub\u003e method. Using 50% endpoint titers of irradiated and unirradiated samples (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), a dose-response curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) was generated using OriginPro 6.1 software and used to calculate the D\u003csub\u003e10\u003c/sub\u003e value, i.e., the gamma radiation dose required to reduce viral infectivity by one log\u003csub\u003e10\u003c/sub\u003e the virus infectivity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Based on the dose-survival curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and the log-linear regression equation (Y\u0026thinsp;=\u0026thinsp;5.826\u0026ndash;0.591X), the D\u003csub\u003e10\u003c/sub\u003e value was set at 1.7 kGy. Based on the D\u003csub\u003e10\u003c/sub\u003e value, the initial titer (10\u003csup\u003e5.93\u003c/sup\u003e/ml), and the 5.93 log\u003csub\u003e10\u003c/sub\u003e reductions in titer to achieve a complete loss of viral infectivity, the minimum dose required for complete inactivation (the optimal inactivation dose) was calculated using the formula \"D\u003csub\u003e10\u003c/sub\u003e value \u0026times; 5.93\u0026rdquo; and set at approximately 11 kGy.\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\u003eThe mean of virus titration in irradiated and un-irradiated virus samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGamma Irradiation (kGy)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLog of Virus (without Trehalose) Titration\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLog of Virus (plus Trehalose) Titration\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\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\u003e4.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5\u0026gt;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5\u0026gt;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5\u0026gt;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5\u0026gt;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5\u0026gt;\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 \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\u003eSafety Test of Irradiated Inactivated SARS-CoV-2 Virus on Vero Cells\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIrradiated Virus Samples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFirst Passage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSecond Passage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThird Passage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFourth Passage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11 kGy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\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 \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11 kGy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\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 \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12 kGy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\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\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12 kGy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\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\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14 kGy\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 \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14 kGy\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 \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Evaluation of Irradiated SARS-Cov-2 Virus antigen by ELISA Test\u003c/h2\u003e \u003cp\u003e \u003cem\u003eSARS CoV-2\u003c/em\u003e virus samples irradiated with 12 and 14 kGy gamma rays were used to evaluate the antigenicity of the spike protein (S1 subunit). The optical densities of all samples at 450 nm are listed in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe mean of OD to evaluate Spike protein (S1 subunit) antigen\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e \u003cp\u003eSerial Dilution of Viral Antigen\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSamples\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\u003e1/2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1/4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1/8\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1/16\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1/32\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1/64\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1/128\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1/256\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1/512\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIrradiated SARS-Cov-2 Virus (12 kGy)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e1.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIrradiated SARS-Cov-2 Virus (14 kGy)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e1.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUn- Irradiated SARS-Cov-2 Virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e1.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIrradiated SARS-Cov-2 Virus\u0026thinsp;+\u0026thinsp;Trehalose (12 kGy)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e1.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIrradiated SARS-Cov-2 Virus\u0026thinsp;+\u0026thinsp;Trehalose (14 kGy)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e1.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUn- Irradiated SARS-Cov-2 Virus\u0026thinsp;+\u0026thinsp;Trehalose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.02\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 \u003c/p\u003e \u003cp\u003eThe results of the indirect ELISA assay to evaluate the antigenic properties of the spike protein subunit S1 of \u003cem\u003eSARS-CoV-2\u003c/em\u003e virus show that the gamma-irradiated virus samples (12 and 14 kGy) have no significant difference in antigenic properties compared with the control samples (non-irradiated virus) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the optical density of the irradiated and non-irradiated virus samples plus 20% trehalose is higher than that of the samples without trehalose. It may be that the disaccharide plays an important role in maintaining the properties of proteins, which of course should be further investigated when evaluating the inactivated virus antigen in animal models.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Neutralizing Antibody Response\u003c/h2\u003e \u003cp\u003eFollowing infection with \u003cem\u003eSARS-CoV-2\u003c/em\u003e, patients develop specific IgG and IgM antibodies. A subset of these antibodies to spike (RBD) can block viral infection and cell entry through a process called neutralization. A strong correlation has been observed between the levels of RBD-binding antibody and levels of \u003cem\u003eSARS-CoV-2\u003c/em\u003e blocked by neutralizing antibodies in patients [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Percent inhibition of spike (RBD)-ACE2 binding indicates the titration of neutralizing antibodies, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePercent inhibition of spike (RBD)-ACE2 binding indicates a significant increase in neutralizing antibody titer three weeks after the third vaccination (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). All vaccination groups showed a significant increase in neutralizing antibodies compared with the PBS groups (groups 6 and 7) three weeks after the first, second, and third vaccinations (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 IgA Concentration\u003c/h2\u003e \u003cp\u003eNasal lavage and NALT fluid from vaccinated hamsters were used to evaluate mucosal immunity with the quantitative sandwich ELISA kit to determine IgA levels. The IgA concentration is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhile circulating IgA is mostly monomeric and consists predominantly of the IgA1 subclass, secretory IgA (sIgA) is dimeric and consists of IgA1 and IgA2 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. \u003cem\u003eSARS-CoV-2\u003c/em\u003e primarily infects the upper respiratory tract (URT). Mucosal immune responses are expected to be initiated in the NALT (via the nasal epithelium and tonsils), which serve as sites of induction for the mucosal immune system [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. An important effector molecule at mucosal sites is antibodies, which have two main sources: translocation of circulating IgG into the mucosa and local production of IgA [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this study, secretory IgA was examined in nasal wash and NALT fluids, and the results showed that the concentration of sIgA in irradiated vaccine plus trehalose increased significantly 3 weeks after second and third vaccination (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Cellular Immunity\u003c/h2\u003e \u003cp\u003eT cells against coronaviruses persist in humans and can be detected months and/or years after infection; (2) memory Tc cells are directed against conserved regions of coronaviruses and are therefore largely cross-reactive. Therefore, T-cell-based vaccination approaches against \u003cem\u003eCOVID-19\u003c/em\u003e are of realistic benefits for considering [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this study, the splenic lymphocyte proliferation (SLP) assay was performed using a colorimetric BrdU kit, and the stimulation index of splenocytes was reported in Table\u0026nbsp;7. A significant increase in splenic lymphocyte proliferation was observed in all vaccinated hamsters (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but the increase was greater with irradiated vaccine plus trehalose and irradiated vaccine plus alum.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe SARS-CoV-2 virus has caused the most severe pandemic in the world. The development of a safe and effective vaccine is essential to manage the disease. We optimized an inactivated virus vaccine using the gamma irradiation method as an alternative to classical chemical inactivation methods. Previous studies have shown that gamma irradiation can induce immunogenicity more effectively than conventional inactivation methods [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The use of gamma radiation process (due to a cobalt-60 source) has been developed for the production of effective vaccines [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Gamma irradiation preserves antigen assembly, can reduce free radical damage due to water radiolysis, and can be used in a frozen state [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Gamma irradiation is a cost-effective and faster method of viral inactivation used to produce many inactivated viral vaccines, such as human and avian influenza vaccines. The current study reports on the efficacy of a gamma-irradiated virus vaccine that rather than formalin inactivated virus.\u003c/p\u003e \u003cp\u003eThe spike protein consists of two subunits, S1 and S2, which are cleaved by host cell furin-like proteases such as cathepsins during assembly and release of virus particles from infected cells. The S1 subunit has a variable sequence across coronavirus genera and species. The sequence of the spike protein in the genera of coronaviruses alpha and delta is very similar. The S1 subunit contains the receptor-binding domain (RBD) and plays an important role in binding to host receptors. Therefore, considering the important antigenic property of the spike protein in inducing immune responses in the host, the anti-S1 spike protein monoclonal antibody was used by ELISA method to compare the antigenic properties of the irradiated and un-irradiated virus samples. The spike (S) protein of the virus, which contains the major neutralizing epitopes in the receptor-binding domain (RBD) and the N-terminal domain (NTD), has been shown to be the most promising immunogen. Thus, most recently approved vaccines use full-length S (with or without modification) or whole virus (inactivated) as the target antigen [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Also in this study, evaluation of the antigenic properties of the spike protein using an ELISA kit showed no significant differences between irradiated and un-irradiated \u003cem\u003eSARS-CoV-\u003c/em\u003e2 virus samples with and without trehalose (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/em\u003e However, the optical density of the irradiated and un-irradiated virus samples plus 20% trehalose is higher than that of the samples without trehalose. It may depend on the disaccharide playing an important role in maintaining the properties of proteins, which of course should be further investigated when evaluating the inactivated virus antigen in animal models.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNeutralizing Antibody\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOffersgaard reported that increasing the dose of inactivated \u003cem\u003eSARS-CoV-2\u003c/em\u003e improved the induction of neutralizing antibodies when tested in mice. Although a second immunization in both mice and hamsters resulted in a large increase in nAbs titers compared with a single immunization, a third immunization in mice had little effect on nAb titers and S-specific antibody endpoint titers compared with two immunizations [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. At times when the Delta or Omicron variant is prevalent, a third dose of vaccination has been shown to be highly effective in protecting individuals from severe \u003cem\u003eCOVID-19\u003c/em\u003e-related sequelae and preventing \u003cem\u003eCOVID-19\u003c/em\u003e-associated hospitalizations [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Furthermore, higher levels of neutralizing antibodies are associated with lower risk of symptomatic infection, and immune protection depends on levels of neutralizing antibodies [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Therefore, the vaccine-mediated antibody response against \u003cem\u003eSARS-CoV-2\u003c/em\u003e is required for higher efficacy of the \u003cem\u003eSARS-CoV-2\u003c/em\u003e vaccine [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInactivated \u003cem\u003eSARS-CoV \u0026minus;\u0026thinsp;2\u003c/em\u003e vaccines based on a traditional platform show high safety and efficacy and prevent \u003cem\u003eCOVID-19\u003c/em\u003e serum antibody response to currently commercially available inactivated vaccines has been studied in detail [\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]. However, the profile of serum or plasma antibody response elicited by inactivated vaccines against all circulating variants of concern (VOCs) (alpha, beta, gamma, and delta) and circulating variants of interest (VOIs) (lambda, mu, kappa, eta, iota v1, iota v2, epsilon, and zeta) is less well defined. Also, the efficacy of vaccine-induced neutralizing antibodies is rarely reported. Therefore, a comprehensive analysis of the characteristics of antibody responses to inactivated vaccines and characterization of potent and broadly neutralizing antibodies is informative for optimizing and updating vaccine design and immunization strategies and therapeutics [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Passive antibodies administered are one of the most promising therapeutic and prophylactic anti-\u003cem\u003eSARS-CoV-\u003c/em\u003e2 agents. To date, the most potent monoclonal antibodies (mAbs) isolated from infected and vaccinated individuals were often dominant by those targeting RBD while many isolated NTD mAbs failed to reach 100% potency in neutralizing activity [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this study, a gamma-irradiated vaccine against \u003cem\u003eSARS-CoV-2\u003c/em\u003e was used as an inactivated vaccine, and the neutralising antibody response showed a significant increase in four vaccinated hamster groups as early as three weeks after the first vaccination. However, it reached the peak antibody titer three weeks after the third vaccination. Therefore, we can suggest that the gamma-irradiated inactivated vaccine SARS-COV-2 can be vaccinated in three doses, one primer and two booster doses three weeks apart.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMucosal antibodies\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIgA can be enriched up to threefold in upper respiratory tract secretions compared to IgG, while IgG is the most common isotype in the blood and lower respiratory tract. The higher concentration of sIgA compared to IgG has been shown to result in greater avidity and higher neutralising capacity [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, and \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Mucosal IgG is usually derived from plasma by transduction but can also be produced locally by mucosal B cells in the lamina propria. In addition to directly neutralising viruses, non-neutralising antibodies can also mediate the clearance of viruses and virus-infected cells via interactions of the Fc domain of the antibody with complement [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], thus enabling antibody-dependent cellular cytotoxicity (ADCC) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Recently, survival after moderate-severe \u003cem\u003eSARS-CoV-2\u003c/em\u003e infection was associated with antibody responses with robust Fc effector activity, suggesting that such immunity may contribute to protection against respiratory disease. IgA can be expressed at mucosal surfaces in both monomeric and dimeric forms as secretory IgA (sIgA) and occurs in humans in two isotypes, with IgA1 present in both systemic and mucosal secretions and IgA2 predominantly in mucosa [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Although sIgA has been reported to be elicited at oral and nasal mucosal surfaces after intramuscular vaccination in both clinical and animal studies for influenza and \u003cem\u003eSARS-CoV-2\u003c/em\u003e, titers are generally modest and variable. In contrast, mucosal immunisation readily produces robust sIgA responses in upper and lower respiratory tract mucosa [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. One of the most informative methods for evaluating effective immune responses against \u003cem\u003eSARS-CoV-2\u003c/em\u003e, whether triggered by natural infection or intranasal immunization, is the determination of mucosal sIgA in nasal secretions or saliva synthesized by IgA-secreting plasma cells. In this study, the concentration of sIgA in nasal washings and NALT was evaluated and it increased significantly when the irradiated vaccine plus trehalose was administered via the intranasal route three weeks after the second and third vaccinations (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSpleen Lymphocyte proliferation as the T cell response (ref5)\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious experience with \u003cem\u003eSARS-CoV-1\u003c/em\u003e and \u003cem\u003eMERS\u003c/em\u003e suggests that T cells may be the most important immune response for disease control [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. However, T-cell immunity against coronaviruses is an important aspect of a successful vaccine, and it is a long-lived vaccine. In contrast to antibodies, cytotoxic T cells against coronaviruses persist and can be detected several months after an infection. Therefore, an ideal \u003cem\u003eSARS-CoV-2\u003c/em\u003e vaccine should stimulate both B and T cell immunity to provide optimal protection against \u003cem\u003eCOVID-19\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGamma radiation, as a superior inactivation method, can preserve T cell immunogenicity compared with other inactivation methods. Gamma rays can strongly penetrate the virus, resulting in direct damage to the genetic material without altering the structural proteins [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Mullbacher has previously shown that alphaviruses [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] and bunyaviruses [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] can be rendered noninfectious by gamma irradiation and yet have the ability to elicit cytotoxic T-cell responses. Gamma-irradiation can be used to produce an experimental influenza vaccine and reported that gamma-irradiated Influenza virus preparations promoted T-cell immunity [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, and \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Viral replication can be eliminated during gamma irradiation, while immunogenicity and viral protein structure are preserved, so that viral proteins are naturally presented to the immune system, facilitating the induction of both T cells and humoral immunity. In the current study, splenic lymphocyte proliferation was increased in all vaccinated hamsters and was higher in the irradiated vaccine groups. In addition, we may introduce an irradiated inactivated vaccine \u003cem\u003eSARS-CoV-2\u003c/em\u003e plus disaccharide trehalose via the intranasal route of administration and another irradiated inactivated vaccine SARS-CoV-2 plus alum via the subcutaneous route of administration as safe and efficient vaccines against \u003cem\u003eCOVID-19\u003c/em\u003e. Furthermore, cytokine assay will be done in the future study to evaluate exact immune cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to gratitude Dr. Viskam Vijewardana in the Department of Nuclear Sciences and Applications, Animal Production and Health Section, International Atomic Energy Agency (IAEA), VIC, Vienna, Austria to purchase SARS-CoV-2 anti-spike protein S1 monoclonal antibody (GT263-Invitrogen) and goat anti-mouse IgG (H+L), HRP conjugate (Invitrogen). Also, we really appreciate Dr. Ivancho Naletoski in the Department of Nuclear Sciences and Applications, Animal Production and Health Section, IAEA, Vienna, Austria for ordering to purchase the SARS-CoV-2 Neutralizing Antibody Detection Kit (Abeomics Inc.) and Hamster Immunoglobulin A (IgA) ELISA Kit (MyBioSource, Cat. No.: MBS029668) and sent for my institute.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Iran National Science Foundation (INSF) (Project No.99024693). The paid fund by INSF was used just for buying hamsters and some feed for animals (400 $), but it was not enough for article processing charge. My institution and my research grant provide no fund for Open Access fee support. My country is low in-coming; also sanction makes an inhibition for money transfer from Iran. Also, this research was supported by IAEA to purchase some necessary reagents and kits.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest. The funders had no role in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese authors have contributed in different parts of the manuscript such as; Farahnaz Motamedi Sedeh is the corresponding author and senior authorship. Akbar Khorasani is the senior authorship. Mohsen Lotfi contributed in methodology. Seyed Morteza Moosavi has contributed in writing and editing. Arash Arbabi\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand Seyedeh Maede Hosseini\u003csup\u003e\u0026nbsp;\u003c/sup\u003ehave contributed in methodology and writing.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eData Availability\u003c/h4\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study was reviewed and approved by Tehran University of Medical Science. For this research, all institutional and national guidelines adopted by the horizontal legislation on the protection of animals used for scientific purposes (Directive 2010/63/EU, amended by Regulation (EU) 2019/1010) were approved for implementation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge affirming that freely given informed consent was obtained from the infected man (43 years old) in the study for the collection of tracheal swabs in Karaj, Iran, in May 2020.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCui J, Li F, Shi Z-L (2019) Origin and evolution of pathogenic coronaviruses. 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J Am Vet Med Assoc 225(4):522\u0026ndash;530\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SARS-CoV-2 virus, Inactivated Vaccine, Gamma Irradiation, Mucosal Immunity, Immune Response, Neutralizing Antibody, Splenic Lymphocyte","lastPublishedDoi":"10.21203/rs.3.rs-3405744/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3405744/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eSARS-CoV-2\u003c/em\u003e virus is the causative agent of the emerging respiratory zoonosis disease. One of the most important requirements for the control of emerging diseases is the development of vaccines within a short period of time.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe use of ionizing radiation to inactivate pathogens has been developed for the rapid production of effective vaccines. In this study, the \u003cem\u003eSARS-CoV-2\u003c/em\u003e virus was isolated from tracheal swabs of an infected man, confirmed by RT-PCR, and propagated on Vero cells. The \u003cem\u003eSARS-CoV-2\u003c/em\u003e virus was irradiated with 14 kGy gamma radiation to completely inactivate it. Evaluation of the antigenic properties of the spike protein subunit S1 showed that the gamma-irradiated virus samples had intact antigens. The gamma-irradiated \u003cem\u003eSARS-CoV-2\u003c/em\u003e virus and formalin-treated virus were used to immunize Syrian hamsters in four vaccine formulations.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe titer of neutralizing antibodies increased significantly in all vaccinated groups 3 weeks after the second and third vaccinations. Secretory IgA was examined in nasal lavage and NALT fluids and showed that the concentration of sIgA in irradiated vaccine plus trehalose increased significantly 3 weeks after the second and third vaccinations. The splenic lymphocyte proliferation assay showed a significant increase in all vaccinated hamsters, but the increase was greater in irradiated vaccine plus trehalose and irradiated vaccine plus alum.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn addition, we can introduce irradiated inactivated vaccine \u003cem\u003eSARS-CoV-2\u003c/em\u003e plus disaccharide trehalose via intranasal route of administration and another irradiated inactivated vaccine \u003cem\u003eSARS-CoV-2\u003c/em\u003e plus alum via subcutaneous route as safe and efficient vaccines against \u003cem\u003eCOVID-19\u003c/em\u003e which can stimulate mucosal, humeral and cellular immunity.\u003c/p\u003e","manuscriptTitle":"Immune Responses Following Administration of Whole Gamma Irradiated SARS-CoV-2 Vaccine Stabilized With Disaccharide Trehalose on Syrian Hamster Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-11 16:38:54","doi":"10.21203/rs.3.rs-3405744/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":"54123694-57c9-4743-b8d2-d3799a026e63","owner":[],"postedDate":"October 11th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-11-07T10:59:16+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-11 16:38:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3405744","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3405744","identity":"rs-3405744","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}