key: cord-0871555-a2g440z5 authors: Turan, Raife Dilek; Tastan, Cihan; Kancagi, Derya Dilek; Yurtsever, Bulut; Karakus, Gozde Sir; Ozer, Samed; Abanuz, Selen; Cakirsoy, Didem; Tumentemur, Gamze; Demir, Sevda; Seyis, Utku; Kuzay, Recai; Elek, Muhammer; Ertop, Gurcan; Arbak, Serap; Elmas, Merve Acikel; Hemsinlioglu, Cansu; Ng, Ozden Hatirnaz; Akyoney, Sezer; Sahin, Ilayda; Kayhan, Cavit Kerem; Tokat, Fatma; Akpinar, Gurler; Kasap, Murat; Kocagoz, Ayse Sesin; Ozbek, Ugur; Telci, Dilek; Sahin, Fikrettin; Yalcin, Koray; Ratip, Siret; Ince, Umit; Suyen, Guldal; Ovali, Ercument title: Gamma-irradiated SARS-CoV-2 vaccine candidate, OZG-38.61.3, confers protection from SARS-CoV-2 challenge in human ACEII-transgenic mice date: 2020-10-28 journal: bioRxiv DOI: 10.1101/2020.10.28.356667 sha: 56618e10b6d91487c830713e359fbc5b916c1ee9 doc_id: 871555 cord_uid: a2g440z5 The SARS-CoV-2 virus caused one of the severest pandemic around the world. The vaccine development for urgent use became more of an issue during the pandemic. An inactivated virus formulated vaccines such as Hepatitis A, inactivated polio, and influenza has been proven to be a reliable approach for immunization for long years. In this pandemic, we produced an inactivated SARS-CoV-2 vaccine candidate by modification of the oldest but the most experienced method that can be produced quickly and tested easily rather than the recombinant vaccines. Here, we optimized an inactivated virus vaccine which includes the gamma irradiation process for the inactivation as an alternative to classical chemical inactivation methods so that there is no extra purification required. Also, we applied the vaccine candidate (OZG-38.61.3) using the intradermal route in mice which decreased the requirement of a higher concentration of inactivated virus for proper immunization unlike most of the classical inactivated vaccine treatments. Thus, the novelty of our vaccine candidate (OZG-38.61.3) is a non-adjuvant added, gamma-irradiated, and intradermally applied inactive viral vaccine. We first determined the efficiency and safety dose (either 1013 or 1014 viral copy per dose) of the OZG-38.61.3 in Balb/c mice. Next, to test the immunogenicity and protective efficacy of the OZG-38.61.3, we immunized human ACE2-encoding transgenic mice and infected them with a dose of infective SARS-CoV-2 virus for the challenge test. We showed that the vaccinated mice showed lowered SARS-CoV-2 viral copy number in oropharyngeal specimens along with humoral and cellular immune responses against the SARS-CoV-2, including the neutralizing antibodies similar to those shown in Balb/c mice without substantial toxicity. This study encouraged us towards a new promising strategy for inactivated vaccine development (OZG-38.61.3) and the Phase 1 clinical trial for the COVID-19 pandemic. The development of a vaccine has a top biomedical priority due to the global COVID-19 pandemic caused by the SARS-CoV-2 virus. To end the global COVID-19 pandemic, a safe and effective SARS-CoV-2 vaccine may be needed. Several vaccine candidates have started clinical trials and several more are in preclinical research (B. L. Corey, Mascola, Fauci, & Collins, n.d.; Gao et al., 2020a; Yu et al., 2020) . Small animal model systems are critical for better understanding the COVID-19 disease pathways and to determine medical precautions for improved global health, considering that there are currently no approved vaccines and only one antiviral approved for emergency use for SARS-CoV-2 (Dinnon et al., 2020; Sheahan et al., 2017) . More significantly, several pioneering studies have shown that both SARS-CoV-2 and SARS-CoV use the same human angiotensin-converting enzyme 2 (hACE2) cellular receptor to enter cells (Li et al., 2003; Sun et al., 2020; Walls et al., 2020; Zhou et al., 2020b) . The crystal structure of the SARS-CoV-2 S protein receptor-binding domain (RBD) which binds to hACE2 has been described, with an approximately 10-to 20-fold greater affinity toward hACE2 than SARS-CoV binds. Unfortunately, standard laboratory mice cannot be infected with SARS-CoV-2 due to the discrepancy of the S protein to the murine orthologous (mACE2) of the human receptor, making model development complicated and difficult (Dinnon et al., 2020; Zhou et al., 2020a) . Thus, wild-type C57BL/6 mice cannot be infected efficiently with SARS-CoV-2 because there is no hACE2 protein expressed that supports SARS-CoV-2 binding and infection. On the other hand, both young and aged hACE2 positive mice showed high viral loads in the lung, trachea, and brain upon intranasal infection in the literature (Letko, Marzi, & Munster, 2020; Sun et al., 2020; Wan, Shang, Graham, Baric, & Li, 2020; Winkler et al., 2020) . For understanding viral pathogenesis, vaccine production, and drug screening, animal models are crucial. To assess preclinical efficacy, non-human primates (NHPs) are the best animal models. The implementation of NHPs, however, is limited by the high costs, availability, and complexity of the necessary husbandry settings. For research and antiviral therapeutic progress, suitable small animal models are therefore important. Mouse models are popular because of their affordability, availability, and simple genetic structure, and have been commonly used to research human coronavirus pathogenesis (Cockrell, Leist, Douglas, & Baric, 2018; Jiang et al., 2020) . As a cellular receptor, SARS-CoV-2 could use the ACE2 receptor of the human, bat, or civet but not the mouse (Jiang et al., 2020; Zhou et al., 2020a) . Therefore, it seems that mice expressing hACE2 would be a conceivable choice for the vaccine challenge tests. In this study, we tested our vaccine candidate .3) inactivated with gamma irradiation to assess their immunogenicity and protective efficacy against the SARS-CoV-2 viral challenge in K18-hACE2 mice and showed the efficacy of the vaccination in c57/Balb/C mice. K18-hACE2-transgenic mice, in which hACE2 expression is powered by the epithelial cell cytokeratin-18 (K18) promoter, were originally designed for the study of SARS-CoV pathogenesis and lead to a lethal infection model (McCray et al., 2007; Winkler et al., 2020; Yang, Pabon, & Murry, 2014) . We showed that the vaccinated transgenic mouse showed lowered SARS-CoV-2 viral copy number in nasal specimens along with humoral and cellular immune responses, including neutralizing antibodies similar to those shown in c57/Balb/C mouse design without substantial toxicity. In vitro isolation and propagation of SARS-CoV-2 from diagnosed COVID-19 patients were described in our previous study (Taştan et al., 2020) . The study for SARS-CoV-2 genome sequencing was approved by the Ethics Committee of Acıbadem Mehmet Ali Aydınlar University (ATADEK-2020/05/41) and informed consent from the patients was obtained to publish identifying information/images. These data do not contain any private information of the patients. All techniques had been executed according to the applicable guidelines. For the nasopharyngeal and oropharyngeal swab samples to have clinical significance, it is extremely important to comply with the rules regarding sample selection, taking into the appropriate transfer solution, transportation to the laboratory, and storage under appropriate conditions when necessary (Taştan et al., 2020) . The production of a candidate vaccine for gamma-irradiated inactivated SARS-CoV-2 was reported in our previous report (Sir Karakus et al., 2020) . In this study, the last version of our vaccine candidate, OZG-38.61.3 was constituted from 10 13 or 10 14 viral copy of SARS-CoV-2 in a dose without adjuvant. Viral RNA extractions were performed by Quick-RNA Viral Kit (Zymo Research, USA) in Acıbadem Labcell Cellular Therapy Laboratory BSL-3 Unit according to the manufacturer's protocols. Library preparation was performed by CleanPlex SARS-CoV-2 Research and Surveillance NGS Panel (Paragon Genomics, USA) according to the manufacturer's user guide. For the construction of the library, The CleanPlex® Dual-Indexed PCR Primers for Illumina® (Paragon Genomics, USA) were used by combining i5 and i7 primers. Samples were sequenced by Illumina MiSeq instrument with paired-end 131 bp long fragments. The data that passed the quality control were aligned to the reference genome (NC_045512.2) in Wuhan and a variant list was created with variant calling. The data analysis was described in detail in our previous study (Ozden Hatirnaz Ng et al., under revision) . Nanoparticle Tracking Analysis (NTA) measurements were carried out for SARS-CoV-2 titer in suspension by using The NanoSight NS300 (Amesbury, UK). Samples were diluted with distilled water 1:10 ratio and transferred to Nanosight cuvette as 1 ml. Measurements were performed at room temperature with 5 different 60-second video recording. Viruses were inactivated and fixed with 2.5% glutaraldehyde in PBS (0.1 M, pH 7.2) for 2.5 h. One drop of glutaraldehyde-treated virus suspension was placed on the carbon-coated grid for 10 min. The remaining solution was absorbed with a filter paper and the grid was stained by a negative staining procedure. Then, it was evaluated under a transmission electron microscope (Thermo Fisher Scientific-Talos L120C) and photographed. In-solution digestion was performed according to the manufacturer's instructions using 'insolution tryptic digestion and guanidination kit' (#89895, Thermo Fisher Scientific, USA). The protocol can be summarized as follow: 10 μg protein sample was added to 15 μL 50 mM Ambic containing 100 mM DTT solution. The volume was completed to 27 μL and incubated at 95ºC for 5 min. Iodoacetamide (IAA) was added to the heated sample to a 10 mM final concentration and incubated in the dark for 20 min. 1 μL of 100ng/μL trypsin was then added and incubated for 3 hours at 37ºC. 1 μL of 100ng/μL trypsin was added to the peptide mixture and incubated overnight at 30ºC. After incubation, the solution was vacuum concentrated to dryness and the peptides were resuspended in 0.1% FA for the nLC-MS/MS analysis. The peptides were analyzed by nLC-MS/MS using an Ultimate 3000 RSLC nanosystem (Dionex, Thermo Scientific, USA) coupled to a Q Exactive mass spectrometer (Thermo Scientific, USA). The entire system was controlled by Xcalibur 4.0 software (Thermo Fisher Scientific, USA). High-performance liquid chromatography(HPLC) separation was performed using mobiles phases of A (%0.1 Formic Acid) and B (%80 Acetonitril+%0.1 Formic Acid). Digested peptides were pre-concentrated and desalted on a trap column. Then the peptides were transferred to an Acclaim PepMap RSLC C18 analytical column (75 μmx15 cmx2 μm, 100 Å diameter, Thermo Scientific, USA). The gradient for separation was 6-32% B in 80 min, 32-50% B in 40 min, 50-90% B in 10 min, 90% in 15 min, 90-6% B in 10 min, and 6% B for 10 min with the flow rate of 300 nL/min. Full scan MS spectra were acquired with the following parameters: resolution 70.000, scan range 400-2000 m/z, target automatic gain control (AGC)3×106, maximum injection time 60 ms, spray voltage 2.3 kV. MS/MS analysis was performed by data-dependent acquisition selecting the top ten precursor ions. The instrument was calibrated using a standard positive calibrant (LTQ Velos ESI Positive Ion Calibration Solution 88323, Pierce, USA) before each analysis. Raw data were analyzed with Proteom Discoverer 2.2 (Thermo Scientific, USA) software for protein identification and the following parameters were used; peptide mass tolerance 10 ppm, MS/MS mass tolerance 0.2 Da, mass accuracy 2 ppm, tolerant miscarriage 1, minimum peptide length 6 amino acids, fixed changes cysteine carbamidomethylation, unstable changes methionine oxidation, and asparagine deamination. The minimum number of peptides identified for each protein was considered to be 1 and obtained data were searched in the Uniprot/Swissprot database. Residual Host Cell Protein (HCP) analysis in a viral product supernatant was performed with the manufacturer's protocol of the Cygnustechnologies-VERO Cell HCP ELISA kit (F500). The absorbance was read at 450/650nm with the microplate reader (Omega ELISA Reader). The vaccine candidate was solved in 100 cc pyrogen-free water. Firstly, pyrogen-free water was blanked and one drop sample was measured at the dsDNA program using Thermo Scientific NanoDrop™ One Spectrophotometers to determine Vero residual DNA and A260/A280 ratio for DNA/protein purity. 3µg of lyophilized inactivated SARS-CoV-2 vaccine candidate in 100 µl pyrogen-free water was inoculated into %90 confluent Vero cells at 37C. The supernatant of this culture was replenished with fresh Vero cell culture every 3-to-5 days up to 21 days of incubation. As a negative control, only 100 µl pyrogen-free water was inoculated into Vero cells and cultured for 21 days with the same treatments. At the end of the incubation, the final supernatant was collected, centrifuged at 2000G for 10 min to remove cell debris. Next, the supernatants were concentrated 10x with 100kDa Amplicon tubes. The concentrated samples were tested in the xCelligence RTCA system in a dose-dependent manner as 10-1 to 10-6 to determine the cytopathic effect. 5 μg / ml of SARS-COV-2 Spike S1 Monoclonal Antibody (ElabScience) antibodies were added in the gel at a concentration of 2%. Inactive SARS-CoV-2 was kept at room temperature for 15-30 minutes with 1% zwittergent detergent (mix 9 test antigens: 1 Zwittergent). Incubation was provided in a humid environment for 18 hours. The gel was washed with PBS, taken on the glass surface, and covered with blotter paper, and kept at 37 ° C until it dried. By staining the gel with Coomassie Brillant Blue, the presence of S antigen was determined according to the dark blue color (colorimetric). To analyze the efficiency and toxicology of the dose of inactive vaccine candidate parallel to challenge, 15 Female BALB/c mice were utilized from Acıbadem Mehmet Ali Aydinlar University Laboratory Animal Application and Research Center (ACUDEHAM; Istanbul, Turkey). All animal studies received ethical approval by the Acibadem Mehmet Ali Aydinlar University Animal Experiments Local Ethics Committee (ACU-HADYEK). BALB/c mice were randomly allocated into 3 groups, a negative control group (n=5) and 2 different dose groups (dose of 1x10 13 and 1x10 14 , n = 5 per group). To determine the immunogenicity with two different doses (dose 10 13 and dose 10 14 , n=5 per group) of inactive vaccine produced in Acibadem Labcell Cellular Therapy Laboratory, Istanbul, Turkey, on day 0 mice were vaccinated intradermally with the dose of 1x10 13 and 1x10 14 lyophilized vaccine candidate without adjuvant reconstituted in 100 cc pyrogen-free water and also control groups vaccinated with 100 cc pyrogen-free water. After 18 days booster dose was applied with the same vaccination strategies. Survival and weight change were evaluated daily and every week respectively. Blood samples were collected just before the sacrification on day 28 for serum preparation to be used for in vitro efficiency studies. Mice were sacrificed on day 28 post-immunization for analysis of B and T cell immune responses via SARS-Cov-2 specific IgG ELISA, IFN ELISPOT, and cytokine bead array analysis. Furthermore, dissected organs including the lungs, liver, kidneys of sacrificed mice were taken into 10% buffered formalin solution before they were got routine tissue processing for histopathological analysis. Also, the spleen tissues were taken into a normal saline solution including %2 Pen-Strep for T cell isolation following homogenization protocol. Transgenic mice were randomly allocated into 4 groups, negative control group (n=5), positive control group (n=6), and 2 different dose groups (dose of 1x10 13 and 1x10 14 , n = 7 per group). To determine the 21-day immunogenicity with two different doses (dose 10 13 and dose 10 14 , n=7 per group) of inactive vaccine produced in Acibadem Labcell Cellular Therapy Laboratory, Istanbul, Turkey, on day 0 mice were vaccinated intradermally with the dose of 1x10 13 and 1x10 14 SARS-CoV-2 viral copy per microliter lyophilized vaccine without adjuvant reconstituted in 100 cc pyrogen-free water and both negative and positive control groups vaccinated with 100 cc pyrogen-free water. In whole groups, a booster dose of 1x10 13 and 1x10 14 SARS-CoV-2 viral copy per microliter vaccine was administered intradermally on day 15 post-first vaccination. All hours. At 48 hours after the challenge, the nasopharyngeal swabs were collected from a dose of 1x10 13 or 1x10 14 and the positive control groups to analyze viral copy number. At 96 hours after the challenge, the nasopharyngeal swabs and sera were collected from whole groups including negative control groups to analyze immunological and virological assays. After serum collection, all mice were euthanized. Biopsy samples were collected including skin which was the vaccination part, brain, testis, ovarium, intestine, spleen, kidney, liver, lung, heart. Biopsy samples were collected and anatomically divided for qPCR analysis and histological and TEM examination. At 96 hours after the challenge, whole mice of each group imaged with the Siemens Arcadis Avantic C arms X-ray dark-field imaging system to evaluate the feasibility of early-stage imaging of acute lung inflammation in mice. 3 mice from each group were imaged and also all mice anesthetized once during imaging with Matrx VIP 3000 Isoflurane Vaporizer (MIDMARK) system. All images were acquired as the posterior prone position of mice. The X-ray ran at 48 kV, distance to source grating 70cm, 111°, and shooting with 0.2 and 0.3 mA. Transgenic mice and Balb/c mice were sacrificed on postimmunization for histopathology analysis. Dissected organs including the cerebellum, lungs, liver, kidneys, skin, intestine, and part of the spleen of sacrificed mice were taken into 10% buffered formalin solution before routine tissue processing for histopathological analysis after weighting. The histopathology analysis of the lung tissues of challenge mice groups was performed at the Department of Pathology at Acibadem Maslak Hospital. Before the sacrification, blood samples were collected from the whole group of mice. The serum was collected with centrifugation methods. Serum samples were stored at -40 C. To detect the SARS-COV-2 IgG antibody in mouse serum SARS-CoV-2 IgG ELISA Kit (Creative, DEIASL019) was used. Before starting the experiment with the whole sample, reagent and microplates pre-coated with whole SARS-CoV-2 lysate were brought to room temperature. As a positive control, 100 ng mouse SARS-CoV-2 Spike S1 monoclonal antibody was used which is commercially available (E-AB-V1005, Elabscience). Serum samples were diluted at 1:64, 1:128, and 1:256 in a sample diluent, provided in the kit. Anti-mouse IgG conjugated with Horseradish peroxidase enzyme (mHRP enzyme) was used as a detector. After incubation with the stopping solution, the color change was read at 450nm with the microplate reader (Omega ELISA Reader). instruments (ACEA, Roche) for 8 days (Sir Karakus et al., 2020) . Neutralization assay of sera from transgenic and balb/c mice groups was performed at 1:128, and 1:256 dilutions preincubated with a 10X TCID50 dose of SARS-CoV-2 at room temperature for 60 min. Next, the pre-incubated mixture was inoculated into the Vero-cell-coated flat-bottom 96-well plate which was analyzed at the end of 96 hr following standard MTT protocol. Viable cell analysis was determined by colorimetric change at the ELISA system. The neutralization ratio was determined by assessing percent neutralization by dividing the value of serum-virus treated condition wells by the value of untreated control Vero cells. 100% of neutralization was normalized to only Vero condition while 0% of neutralization was normalized to the value of only 10x TCID50 dose of SARS-CoV-2 inoculated Vero cell condition. For example, for the sample of 1:128 serum sample, the value was 0,651 while the value for control Vero well as 0,715, and the value for control SARS-CoV-2 inoculated well was 0,2. The calculation is as %neutralization= ((0,651-0,2)*100)/(0,715-0,2). This gave 87,5% virus neutralization. This calculation was performed for each mouse in the group and the mean of the virus neutralization was determined. Mouse Spleen T cells were centrifuged with Phosphate Buffer Saline (PBS) at 300xg for 10 min. Pellet was resuspended in TexMACs (Miltenyi Biotech, GmbH, Bergisch Gladbach, Germany) cell culture media (%3 human AB serum and 1% Pen/Strep). 500,000 cells in 100 µl were added into microplate already coated with a monoclonal antibody specific for mouse IFN-γ. Normally distributed data in bar graphs was tested using student's t-tests for two independent means. The Mann-Whitney U test was employed for comparison between two groups of nonnormally distributed data. Statistical analyses were performed using the Graphpad Prism and SPSS Statistics software. Each data point represents an independent measurement. Bar plots report the mean and standard deviation of the mean. The threshold of significance for all tests was set at *p<0.05. ns is non-significant. In our first report, we determined that adjuvant positive vaccine administration should be removed in the newly designed version of the OZG-38.61 vaccine model due to the finding of inflammatory reaction in the skin, cerebellum, and kidney in toxicity analysis of vaccinated mice (Sir Karakus et al., 2020) . Therefore, it was decided to increase the SARS-CoV-2 effective viral copy dose (1x10 13 or 1x10 14 viral copies per dose) in the last version of vaccine candidates. In this study, we produced the third and final version of the OZG-38.61 without an adjuvant. First, we determined whether this vaccine product comprises all identified SARS-CoV-2 mutations. The obtained sequences from the propagated SARS-CoV-2 virus were compared with the GISAID database and the protein levels of the variant information were examined (Fig. 1A) . We determined that the SARS-CoV-2 strain forming OZG-38.61.3 vaccine covered previously identified mutation variants (red-colored) with new variants (blue colored) (Fig. 1B) . The data of all defined mutations were presented in detail in Supplementary Table 2 . Using Nanosight technology, we determined that the size of inactivated SARS-CoV-2 was 187.9 +/-10.0 nm (mode) with a concentration of 4.23x10 9 +/-1.88x10 8 particles/ ml (Fig. 1C) . As a result of the LC-MS-MS analysis, the presence of proteins belonging to the SARS-CoV-2 virus was detected in the analyzed sample (Fig. 1D) . Four of the defined proteins were Master Proteins and have been identified with high reliability (Supplementary Table 3 ). Other proteins were Master Candidate proteins and their identification confidence interval is medium. The data of all defined proteins were presented in detail in Supplementary Table 3 . Also, the transmission electron microscope was evaluated with the negative staining method and the main structures (envelope/spike) of the SARS-CoV-2 virus particles in the final product were well preserved (Fig. 1E) Acute toxicity and efficacy assays were studied in Balb/c mice. There were 3 groups in this study: control group (n=5), dose 10 13 viral particles per dose (n=5), and dose 10 14 viral particles per dose (n=5) (Fig. 2A) . There was no difference in nutrition and water consumption between the groups, as well as in total body weight and organ weight (Supplementary Table 4) . Version 3 of the OZG-38.61 vaccine did not differ in the histopathologic analysis from the control group, including the highest dose of 10 14 (Supplementary Table 4) . First, we collected blood to isolate serum from mice groups, we determined a significant increase in SARS-CoV-2 specific IgG at 1:128 dilution of serum of the highest dose (10 14 ) vaccinated group regarding control (Fig. 2B) . Next, to determine the neutralization capacity of serum collected from the immunized mice, 1:128 and 1:256 dilutions of sera were pre-inoculated with the SARS-CoV-2 virus and after 96hr incubation, MTT analysis was performed. We determined that dose 10 14 vaccinated mice achieved to neutralize the virus statistically significant (p<0.05) at both dilutions (Fig. 2C) . The Balb/c study shows that upon the restimulation, gamma interferon secretion of the T lymphocytes from both vaccination groups increased significantly rather than in the non-vaccinated mice group and PBS non-stimulated internal control groups (Fig. 2D) . Also, the spleen T cells stimulated with the peptides were analyzed through flow cytometry to determine proportions of activated (CD25+) CD4+ and CD8+ T cells; however, there was no significant change in other proportions of T cell (Fig. 2E) . Next, the supernatant of the incubated cells was analyzed using a cytokine bead array for a more detailed examination. Both doses of OZG-38.61.3 (especially dose 10 14 ) increased IL-2, GM-CSF, gamma-IFN levels and caused Th-1 response (Fig. 2F) . At the same time, IL-10 was increased in both dose groups, suggesting that the Tr1 (Regulatory T lymphocyte type 1 response) response was stimulated (Fig. 2F ) (Andolfi et al., 2012) . Moreover, we performed histopathology analysis of the lung, liver, and kidney to determine inflammation, hemorrhage, and eosinophil infiltration ( Fig. 2G and Supplementary Table 4) . There was no significant toxicity including hemorrhage and eosinophil infiltration in overall organs ( Fig. 2G and Supplementary Table 4 ). Therefore, 1x10 13 and 1x10 14 doses of OZG-38.61.3 caused the effective neutralizing SARS-CoV-2 specific IgG antibody production and cytokine secretion of T so that both vaccine doses caused Th1 response without significant toxicity. Following efficacy and safety analysis of OZG-38.61.3 in Balb/c mice, we wanted to determine protection from SARS-CoV-2 infection in human ACE2 expressing transgenic mice postimmunization. Viral challenge analyzes were performed in K18-hACE2 (Jackson Lab). Two mice groups were vaccinated with the 10 13 and 10 14 doses of the vaccine. On day 4 postchallenge, mice were euthanized for in vitro efficacy tests and histopathology analysis (Fig. 3A) . During the challenge, there was no significant change in food and water consumption along with temperature ( Supplementary Figure 1) . Also, although it is not statistically significant, weight distribution is better in vaccine groups (Supplementary Figure 1) . Next, viral load analyzes were performed on oropharyngeal swab samples on the 2nd and 4th days of the challenge test. While there was no significant change in the virus load in the positive control group, it was observed that the virus load decreased in the vaccine groups except for one mouse and completely disappeared in one (Fig. 3B) . It was observed that the mean virus load decreased statistically significantly over time, especially at the highest dose (10 14 ), between 48-96 hours. There was no change in the positive control group (Fig. 3C) . When compared with the positive control group at 96th hour, a 3-log decrease in viral copy number was determined especially in the highest dose vaccine group (Fig. 3D) . No difference was observed between the groups in the lung X-ray imaging analysis of the mice groups taken in our study (Fig. 4A) . Also, it is observed that both vaccine doses do not cause antibody-dependent enhancement (ADE) side effects in the lung in histopathology analysis (Fig. 4B) . No histologically significant change was observed in the positive control and vaccine groups, although positive control has signs of partial alveolar fusion and inflammation in 1 mouse (Fig. 4B) . This finding is similar to chest radiographs. The absence of an additional pathology in the lung, especially in vaccine groups, was another additional finding confirming that ADE does not occur and the inactivity of our vaccine. Thus, viral load analyses in the oropharyngeal specimens showed that the SARS-CoV-2 infection was significantly disseminated in the vaccinated groups. SARS-CoV-2 specific IgG antibody analysis was performed in 1: 128 and 1: 256 titrations of serum isolated from blood. In SARS-CoV-2 antibody measurements, antibody development was observed in the vaccine groups, including the virus-administered group (Fig. 5A) . According to the IgG ELISA result, the SARS-CoV-2 IgG antibody increase was significantly detected at 1:256 dilution in the dose 10 14 vaccinated group compared to the positive control group (nonvaccinated) (p=0.001) (Fig. 5A) . The neutralizing antibody study also showed a significant increase in both vaccine groups compared to the positive control at 1: 256, similar to the antibody levels ( Fig. 5B) . However, there was no significant change in gamma interferon responses from mouse spleen T cells without re-stimulation (Fig. 5C) . Next, we wanted to determine the cytokine secretion profile and T cell frequencies between groups without a re-stimulation. Although it was not statistically significant, TNFsecretion was also seen to increase in the dose 10 14 group (Fig. 5D) . The increase of IL-2 in the highest-dose (10 14 ) vaccine group indicates that the mice vaccinated after viral challenge show a Th1 type response (Fig. 5D) . Also, when we compare SARS-CoV-2 infected non-vaccinated positive control with non-vaccinated and uninfected negative control, we determined that IL-10 cytokine, known as cytokine synthesis inhibitory factor, was significantly increased (Fig. 5D) , suggesting downregulation of the expression of cytokines (Eskdale, Kube, Tesch, & Gallagher, 1997) . On the other hand, we wanted to determine a change in the proportion of spleen T cell subsets upon re-stimulation with SARS-CoV-2 peptides (Fig. 5E) . Although total CD3+ T and CD4+ T cell populations did not increase in the vaccinated groups regarding control groups, CD25+ CD4+ T cell population was determined to increase in dose groups (Fig. 5E) . Depending on the viral challenge, frequencies of CD3+ and CD4+ T lymphocytes significantly increased in the positive control group (nonvaccinated viral challenge group), while this increase was not observed in the vaccinated group ( Fig. 5E) . Only an increase in the amount of activated (CD25+) CD4+ T cell was observed in the vaccinated groups (Fig. 5E) . This data showing that SARS-CoV-2 viral infection was caused to stimulate T cell response along with increase of Th1 inhibitory Tr1 (T cell regulatory)-related IL-10 cytokine secretion and with the absence of Th2-related cytokine response. To sum up, the in vitro efficacy analysis of the challenge test showed that the presence of active T lymphocytes significantly increased in the highest dose (10 14 ) vaccine group. The study indicated that viral dissemination was blocked by SARS-CoV-2 specific antibodies and neutralizing antibodies. It was also determined that the ADE effect was not observed, and also confirming that OZG-38.61.3 was non-replicative. As the cellular immune response, CD4+ T cell activation was present, especially at the highest dose, and T cell response was biased to the Th1 response type as desired in the immunization. The SARS-CoV-2 virus caused one of the severest pandemic around the world. The safe and effective vaccine development for urgent use became more of an issue to end the global COVID-19 pandemic. Several vaccine candidates have recently begun clinical phase studies, and many others are in preclinical development (L. Corey, Mascola, Fauci, & Collins, 2020; Gao et al., 2020b) . Here, we optimized an inactivated virus vaccine which includes the gamma irradiation process for the inactivation as an alternative to classical chemical inactivation methods so that there is no extra purification required. Previous studies showed that gamma-radiation can induce immunogenicity more effectively rather than conventional inactivation procedures (Seo, 2015) . Also, we applied the vaccine candidate (OZG-38.61.3) using the intradermal route in mice which decreased the requirement of a higher concentration of inactivated virus for proper immunization unlike most of the classical inactivated vaccine treatments (Hickling et al., 2011; Lambert & Laurent, 2008) . Different variations may occur when producing large quantities (bulk) of the virus in a laboratory (Zhang et al., 2019) . For this reason, 50% of the unit volume of virus isolates cultured in multilayered flasks was frozen in each passage. While preparing the final product (OZG-38.61.3), frozen raw intermediate products were pooled. Thus, pre-pooling genomic characterization of individual variants between passages was made and the final product was created for a more effective and safer vaccine design. When we look at the variety of mutations that occur in our SARS-CoV-2 strain at the end of the production, it was found that it contains most of the defined mutations. In addition, the SARS-CoV-2 virus was passaged 3 times for the isolation from the first donor and 6 times for the final production of OZG-38.61.3. We determined that genome analysis of the OZG-38.61.3 vaccine retained >%99.5 homology with the starting virus stock isolated from the COVID-19 patient. This may enable our inactive virus vaccine to be effective in a large population. Zeta-sizer along with Nanosight size analysis, proteome, and electron microscopic data showed that the OZG-38.61.3 vaccine preserved its compact structure despite gamma irradiation and lyophilization. However, we also detected aggregate formation, especially in electron microscope images. We added human serum albumin (<0.02%) to the final product to increase the stability, to prevent viral particles from adhering to the injection vial walls, and efficacy of the vaccine candidate (Prymula, Simko, Povey, & Kulcsar, 2016) . Besides, when we assess residual Vero host cell protein and DNA level in each vaccine dose, it was found that the protein level was <4ng and DNA was absent in the dose. This showed us that the vaccine production process is efficiently pure from the residual products. In this study, we generated a prototype gamma-irradiated inactive SARS-CoV-2 vaccine (OZG-38.61.3) and assessed protective efficacy against the intranasal SARS-CoV-2 challenge in transgenic human ACE2 encoding mice. We demonstrate vaccine protection with substantial ~3 log10 reductions in mean viral loads in dose 10 14 immunized mice compared with non-vaccinated infected positive control mice. We showed humoral and cellular immune responses against the SARS-CoV-2, including the neutralizing antibodies similar to those shown in Balb/c mice, without substantial toxicity. This study encouraged us towards initiating Phase 1 clinical trial for the COVID-19 pandemic. When we performed the efficacy and safety test of the final product, OZG-38.61.3, vaccine candidate on Balb/ c mice at two different doses (10 13 and 10 14 ), the presence of SARS-CoV-2 specific neutralizing antibodies was significantly detected in the highest-dose vaccination (10 14 ). However, at both dose groups, significant IFN secretion from the spleen T cells detected concerning the controls, showing that cellular immune response developed earlier than the humoral immune response. The fact that the neutralizing test was more accurate than the IgG ELISA analysis, which may be due to the increased levels of SARS-CoV-2 specific IgA and IgM antibodies (Demers-Mathieu et al., 2020; Poland, Ovsyannikova, & Kennedy, 2020; Woo et al., 2004) . Moreover, the fact that mice vaccinated with both doses showed a significant increase in T cell IFN responses and Th1 dominant cytokine release encouraged us at the point of vaccine efficacy. At the same time, since we did not encounter any significant toxicity in the histopathological analysis of Balb/c mice vaccinated with both doses, we decided to start testing with these two doses for the challenge test. On the other hand, we faced difficulties in the intradermal vaccination of mice. We saw that some of our mice had skin injury due to the vaccination. Hence, it may be a factor reducing the efficacy of intradermal vaccine tests during studying with mice. This may be the reason why have a high standard deviation and also we cannot see a parallel neutralization capacity in each mouse. In the Challenge test, we collected oropharyngeal samples to determine the viral copy number following the administration of intranasal infective SARS-CoV-2 virus. When we compared the viral copy numbers at 48 and 96 hours, we observed that the copy numbers in our unvaccinated but virus-infected positive control mice did not change or even increased. However, in our groups of mice vaccinated with both doses, we observed that copy numbers effectively decreased around 3 log10, and even a few mice were completely lost viral load. We performed X-rays to see a similar effect in the lung lobes, but we did not encounter the icy structure, which is the classic COVID-19 infection image. It was probably due to the short 96-hour infection. Besides, the low amount of virus (30,000 TCID50) used in the 96-hour challenge test might not be sufficient to descend into the lungs in this period. We performed qRT-PCR studies from tissue samples to detect the change in viral copy number in the lung lobes, but we did not encounter any viral copy number in any group. We confirmed this finding in histopathological analyzes as there was no inflammation in the lung tissues. Furthermore, studies show that the reason why no viral load could be observed in the lung tissue was the amount of infected dose or the virus could be detected in the specific locations of the lung (Gao et al., 2020b; Subbarao et al., 2004) . As a result, we showed the anti-viral effect of the vaccination with the viral load determined on mouth swabs. When we looked at the neutralizing antibody capacity of mice vaccinated within the scope of the Challenge test, we observed that both doses of vaccination could significantly neutralize SARS- CoV-2. This shows us that the reduction in viral copy rates is consistent. However, when we looked at the T cell response, we could not see any difference in IFN release. Presumably, because groups of mice are infected with the virus, T cells may already be stimulated and this may not make a difference in IFN release. A significant decrease in CD3+ and CD4+ T cell ratios and an increase in CD25+ CD4+ T cell ratio show that these cells have already been activated. On the other hand, the fact that the virus was neutralized here prevented the increase in CD3+ T cell proportion, therefore viral challenge resulted in only the increase of active T cell. When we looked at spleen T cells that were not re-stimulated, we detected Th1-type cytokine release, as we expected, especially in the 10 14 dose vaccine group. On the other hand, the significant increase in the ratios of total CD3+ and CD4+ T cells and the ratios of activated (CD25+) CD8+ T cells and the level of the Th1 cytokine and inhibitor IL-10 between the negative control and positive control mice that had an only viral infection. It shows that in a short time such as 96 hours, it started to generate T cell response more effectively than antibody response. This has shown that T cell response occurs in individuals exposed to the virus without sufficient time for neutralizing antibody formation. In summary, this study demonstrates that the OZG-38.61.3 vaccine candidates that we created with gamma-irradiated inactivated SARS-CoV-2 viruses produced neutralizing antibodies, especially effective in 10 14 viral copy formulation, and this was effective in transgenic human ACE2 expressing mice. We showed that it can protect against infection. We observed that vaccine candidates were safe to the tissues with our study in Balb/ c and transgenic mice. This preclinical study has encouraged us to try phase 1 vaccine clinical trials to avoid the COVID-19 pandemic. 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