key: cord-0792175-tftrnxdo
authors: Sir Karakus, Gozde; Tastan, Cihan; Dilek Kancagi, Derya; Yurtsever, Bulut; Tumentemur, Gamze; Demir, Sevda; Turan, Raife Dilek; Abanuz, Selen; Cakirsoy, Didem; Seyis, Utku; Ozer, Samed; Elibol, Omer; Elek, Muhammer; Ertop, Gurcan; Arbak, Serap; Acikel Elmas, Merve; Hemsinlioglu, Cansu; Kocagoz, Ayse Sesin; Hatirnaz Ng, Ozden; Akyoney, Sezer; Sahin, Ilayda; Ozbek, Ugur; Telci, Dilek; Sahin, Fikrettin; Yalcin, Koray; Ratip, Siret; Ovali, Ercument
title: Preclinical efficacy and safety analysis of gamma-irradiated inactivated SARS-CoV-2 vaccine candidates
date: 2021-03-11
journal: Sci Rep
DOI: 10.1038/s41598-021-83930-6
sha: 728de86099189165053eebe4f8d859925ec7708e
doc_id: 792175
cord_uid: tftrnxdo

COVID-19 outbreak caused by SARS-CoV-2 created an unprecedented health crisis since there is no vaccine for this novel virus. Therefore, SARS-CoV-2 vaccines have become crucial for reducing morbidity and mortality. In this study, in vitro and in vivo safety and efficacy analyzes of lyophilized vaccine candidates inactivated by gamma-irradiation were performed. The candidate vaccines in this study were OZG-3861 version 1 (V1), an inactivated SARS-CoV-2 virus vaccine, and SK-01 version 1 (V1), a GM-CSF adjuvant added vaccine. The candidate vaccines were applied intradermally to BALB/c mice to assess toxicity and immunogenicity. Preliminary results in vaccinated mice are reported in this study. Especially, the vaccine models containing GM-CSF caused significant antibody production with neutralization capacity in absence of the antibody-dependent enhancement feature, when considered in terms of T and B cell responses. Another important finding was that the presence of adjuvant was more important in T cell in comparison with B cell response. Vaccinated mice showed T cell response upon restimulation with whole inactivated SARS-CoV-2 or peptide pool. This study shows that the vaccines are effective and leads us to start the challenge test to investigate the gamma-irradiated inactivated vaccine candidates for infective SARS-CoV-2 virus in humanized ACE2 + mice.

www.nature.com/scientificreports/ Inactivated SARS-CoV-2 virus imaging by transmission electron microscopy. 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.

LC-MSMS protein analysis. LC-MSMS protein analysis was performed at Acibadem Labmed Laboratory, Istanbul. ONAR data acquisition mode was applied by a WATERS XEVO G2-XS high-resolution mass spectrometer. Tryptic peptides were generated by overnight digestion with trypsin followed by reduction and alkylation steps with DTT and IAA, respectively, and fractionated by a 90 min reverse-phase gradient at 500 nL/ min flow rate on an HSS T3 (Waters-186008818) nano column. LC-MSMS data was searched against the NCBI RefSeq sequence database for protein identification. PROGENESIS QIP software was used for protein identification (Waters v4.1).

Replicative competent coronavirus test with gamma-irradiated inactivated SARS-CoV-2 vaccine candidates. 3 µg of lyophilized inactivated SARS-CoV-2 vaccine candidate in 100 µl apyrogenic 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 apyrogenic 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 10 × with 100 kDa 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.

For studies on BALB/c mice, we confirm that all methods were carried out in accordance with relevant guidelines and regulations. Furthermore, we confirmed that the study was carried out in compliance with the ARRIVE guidelines. Female or male 11-months-old BALB/c mice were housed in AAALAC International accredited Acıbadem Mehmet Ali Aydinlar University Laboratory Animal Application and Research Center (ACUDEHAM; Istanbul, Turkey) for 7-day toxicity and 21-day toxicity and efficacy tests. Light, temperature, humidity, and feeding conditions followed the ACUDEHAM accredited operating procedures. For 34-day efficacy tests, female or male 3-months-old BALB/c mice were housed in Yeditepe University Experimental Research Center. All animal studies received ethical approval by the Yeditepe University Animal Experiments Local Ethics Committee (Yeditepe-HADYEK). Mice in the intervention groups were identified as female and male plus a correlative number 1-10. Cages were identified with the study name and color codes. Each mouse was marked with its code including vaccine treatment with/without adjuvant in the base of the tail using a nontoxic permanent marker as single, double, or triple line and without a line.

To determine the 21-day immunogenicity (n = 3/ group) and 7-day (n = 4/group) or 21-day toxicity (n = 3/group) of inactive vaccine produced in Acibadem Labcell Cellular Therapy Laboratory, Istanbul, Turkey, on day 0 mice were inoculated with the dose of 3 µg/100 µl (4.2 × 10 6 SARS-CoV-2 viral copy per microgram) adjuvanted or nonadjuvanted vaccine intradermally and with apirogen water in the control group. In two other groups, a booster dose of 3 µg/100 µl adjuvanted or nonadjuvanted vaccine was administered on day 15 intradermally in addition to day 0. Survival and weight change were evaluated daily and every week respectively. To evaluate the fast response toxicity, on day 0 mouse was inoculated with the dose of 3 µg/100 µl adjuvanted or nonadjuvanted vaccine intradermally and with apirogen water in the control group (n = 4/group). Survival and weight change were evaluated on days 0, 3, and 7. Blood samples were collected just before the sacrification for hemogram and biochemical analysis on day 7. For long term toxicity and immunogenicity, blood samples were collected just before the sacrification on day 21 or day 34 for serum preparation to be used in preclinical in vitro studies. Mice treated with the vaccine candidates were sacrificed on day 21 or day 34 postimmunization for analysis of B and T cell immune responses via SARS-Cov-2 specific IgG ELISA, IFNγ ELISPOT, and cytokine bead array analysis.

Histopathological applications. Mice treated for both toxicity and efficacy tests were sacrificed on day 7 or day 21 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 prior to routine tissue processing for histopathological analysis. Tissue tracking was performed firstly in NBF 10% for 1 h and then in alcohol from 60% to absolute gradually for 1 h/each alcohol concentration. The tissue tracking was finalized in Xylene and Paraffin for 1 h/each. Blocking of tissues was performed by embedding them in paraffin and turned into blocks. Sections with 3-4 µm thickness were taken from paraffin blocks. Next, staining was performed following several procedures including deparaffinization, hydration, hematoxylin stage, acid alcohol phase, bluing, eosin phase, dehydration, transparency step, and closing with the non-aqueous closing agent.

Prior to 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 www.nature.com/scientificreports/ 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 stoping solution, the color change was read at 450 nm with the microplate reader (OMEGA ELISA Reader).

Neutralization assay using real-time cell analysis (RTCA), XCELLIGENCE. TCID50 (Median Tissue Culture Infectious Dose) of SARS-CoV-2 was determined by incubating the virus in a serial dilution manner with the Vero cell line (CCL81, ATCC) in gold microelectrodes embedded microtiter wells in XCELLIGENCE Real-Time Cell Analysis (RTCA) instruments (ACEA, Roche) for 8 days. Neutralization assay was performed with 1:64, 1:128, and 1:256 dilutions of mice serum pre-incubated with a 10X TCID50 dose of SARS-CoV-2 at room temperature for 60 min. Infective active SARS-CoV-2 virus to be used in neutralization tests was titrated in the RTCA system and the dose of TCID50 was determined. It was decided to use 10 times more than the dose of TCID50 in the following neutralization tests as 100X TCID50 dose. Next, the pre-incubated mixture was inoculated into the Vero-coated cells which were analyzed in real-time for 120 h (totally, 145 h). Cell analysis was normalized to the value at the 24th hour of culturing before culturing with serum-SARS-CoV-2 sample conditions. Normalized cell index shows the proliferation and viability of the adherent cells (the higher cell index means the higher viability and proliferation). The neutralization ratio was determined by assessing percent neutralization by dividing the index value of serum-virus treated condition wells by the cell index value of untreated control Vero cells (normalized to 100% Statistics. 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 non-normally distributed data. Statistical analysis of the presence or absence of toxicity including inflammation in the tissue sections was performed using the Chi-squared test. Statistical analyses were performed using SPSS Statistics software. No outliers were excluded in any of the statistical tests and 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.

Manufacturing gamma-irradiated inactivated SARS-CoV-2 vaccine candidate. Most www.nature.com/scientificreports/ agents against these CoVs may be the relatively high cost and long production time 6 . Multiple strategies have been adopted in the development of CoV vaccines; most of these are recombinant adenovirus-based vaccines and immuno-informatics approaches used to identify cytotoxic T lymphocyte (CTL) and B cell epitopes 7, 8 .

Unlike the vaccine obtained with the recombinant protein cocktail of the virus, the whole of the virus in the vaccine candidates may enable to produce a vast amount of the immunoglobulin molecules that can recognize the virus antigens better and more specifically. With our straightforward manufacturing protocol of the whole inactivated lyophilized SARS-CoV-2 vaccine, two different formulations with or without GM-CSF as adjuvants were prepared (Fig. 1) . Furthermore, since the inactivated virus vaccine manufacturing process would require careful characterization of viral isolates as seeds, and demonstration of consistent in viral cultures, we have shown that our inactivated virus-based vaccine production procedure meets the criteria with the following three independent vaccine production (Supplementary Table 1 ). Thus, the end products were made available for preclinical in vitro and in vivo safety and efficacy analyzes.

Genome sequencing of the SARS-CoV-2 isolates. While evaluating the appropriate isolate for the inactive vaccine form, viral genome sequencing obtained from four patients was performed and a variant list was created (Table 1) . Representative IGV reads from each patient were depicted in Fig. 2 . The variants detected in patients were identified in previous sequencing results as well. Only one variant was novel according to the analysis in the GISAID database. The effect of the variants on the protein level and multiple alignment analysis results were presented in our viral genome sequencing study 5 .

Pooling was performed to obtain the final product with SARS-CoV-2 isolates which were sequenced genome. RT-PCR identification of the isolates was performed as stated in our previous publication 4 . The dry end product obtained after inactivation and lyophilization by gamma-irradiation was diluted to 3 ug / 200 µl and analyzed to measure particle count, size, and density. As a result of these analyzes, the average size of the particles in SK-01 V1 was 139.3 + / − 5.6 nm (Fig. 3A ,B) and the average size of the particles in OZG-3861 V1 was determined to be 144 nm + / − 51.8 nm (Fig. 3C,D) . However, the particle density in this size range was calculated to be 91.9% + / − 2.5% (Fig. 3D ). The number of viral particles per dose was found to be 2.6 × 10 8 + / − 2.61 × 10 7 . Results illustrate that the virus particles in the final product largely retain their compact structure. However, negative staining and transmission were analyzed with an electron microscope to display the compact structure of the virus particles in the final product (Fig. 4A ). In addition to RT-PCR analysis, the presence of SARS-CoV-2 specific protein sequences including Replicase polyprotein 1ab and Non-structural protein 3b) was confirmed by proteome analysis on the final product, Fig. 4B shows eluted peptides between m/z 50-2000 along a 90 min reverse-phase gradient elution. At the same time, the gamma-irradiated inactive virus strains lost their infective properties was confirmed by the replicative competitive coronavirus test using the RTCA analysis performed at the end of the 21-day repeated passage (Fig. 4C) . Also, to demonstrate the reliability and consistency of the inactivation assay, we repeated the test with colorimetric MTT assay post 21-day culturing of the inactivated virus samples. We showed inactivated virus samples in three representatives of inactivated vaccine samples ( Supplementary Fig. 1 ). As a result of these analyzes, it has been decided that vaccine candidates have been made final products for use in toxicity and efficacy studies.

Safety analysis of the vaccine candidates, SK-01 V1 and OZG-3861 V1. In order to test the reliability including the 7-day and 21-day toxicity of the vaccine candidates, intradermal administration was performed to the mouse groups as a single dose with adjuvant (SK-01 V1) and single-dose without adjuvant (OZG-3861 V1) www.nature.com/scientificreports/ ( Fig. 5A) . At a one-week follow-up, no significant weight change was detected in groups compared to the control mouse group (Fig. 5B) . There was also no significant difference in CBC analysis (red blood cell, RBC; white blood cell, WBC; hemoglobin; HGB and platelet rates) (Fig. 5C,D) . However, when the study groups were compared with the control, there was a significant increase in gammaglobulin and related protein increase in the vaccine group containing adjuvant (Fig. 5E ). In toxicity analyzes, Ca, ALT, and LDH values did not differ significantly between the groups (Fig. 5F-H) . In the histopathological analysis on day 7, no difference was observed in the samples of spleen, liver, lung, intestine, hippocampus, kidney, and skin among the groups (Fig. 5I ). In the examination of cerebellum tissues, no statistically significant pathology in comparison with the control group was observed (Fig. 5I ). In the adjuvant negative single dose (OZG-3861 V1) group, dense Purkinje cells were observed. However, this density did not appear to be significant (p > 0.05) in comparison with the control group www.nature.com/scientificreports/ (Fig. 5I) . These toxicity analyzes encouraged us to start an in vivo efficacy and dose study with both adjuvant SK-01 V1 and OZG-3861 V1 vaccine candidates without adjuvant in mice. Subsequently, long-term toxicity analysis at day 21 was determined. Histopathology analysis showed no significant pathological finding in the lung, liver, jejunum of intestine, spleen, cerebellum, hippocampus, kidney, and skin tissues (Fig. 6A,B) . Numerous foci of megakaryocytes (marked by a star) and trabeculae (marked by arrow) were determined in the histological sections of the spleen in all groups (Fig. 6A) . Cerebellum sections were studied in brain tissues obtained from mouse groups. In particular, there was no statistically significant difference in the shape and staining properties of Purkinje neurons in the cerebellum cortex of all groups. Interestingly, the cells in the adjuvant-negative single-dose group had better shapes in comparison with the other groups (Fig. 6B) . No pathological finding was observed in the dentate gyrus and the pyramidal layer of the hippocampus in all groups (Fig. 6B) . Distal and proximal tubules in the kidney were observed similarly in all groups (Fig. 6B) . On the other hand, a statistically significant (p < 0.05) inflammatory reaction was observed in the analyzed skin and kidney tissues in the adjuvant positive double dose vaccine administration group (Fig. 6B) . Furthermore, glomerulus structures in all vaccinated groups were normal. In toxicity analysis of skin tissue, inflammatory cells infiltration, and eosinophils in some dermis area of the vaccination points of the skin were detected in the double-dose groups (Fig. 6B ). The vaccine candidates had no significant toxicity on the tissues. The analysis was also performed to investigate whether there was an increase in Th1, Th2, and Th17 dependent cytokine releases in the blood sera collected from the mouse groups that received the vaccine either at day 21. Compared to the control groups, no statistically significant cytokine increase was observed in any application group (Fig. 6C ). Findings show that there was no toxic side effect of SK-01 V1 and OZG-3861 V1 in mouse groups.

Pre-clinical efficacy and dose study of vaccine candidates, SK-01 V1 and OZG-3861 V1. In order to perform in vivo efficacy and dose studies of vaccine candidates, OZG-3861 V1 and SK-01 V1 were administered intradermally to the mouse groups as single or 15-day booster doses (Fig. 5A) . SARS-CoV-2 specific IgG antibody analysis was performed in three different titrations (1:64, 1: 128, and 1: 256) in serum isolated from blood. According to the IgG ELISA result, a significantly increased SARS-CoV-2 IgG antibody www.nature.com/scientificreports/ was detected in all groups in comparison with the control (non-vaccinated) mouse group (*p < 0.05) (Fig. 7A) . Mouse SARS-CoV-2 Spike S1 monoclonal antibody was used in the same test as a positive control for the accuracy of the analysis (Fig. 7A) . The proof-of-concept has been optimized with the real-time cell analyzing (RTCA) www.nature.com/scientificreports/ system to determine the neutralization efficiency of SARS-CoV-2 specific antibodies in serum content. As a representative data, with double dose SK-01 V1, control group serums were pre-incubated with SARS-CoV-2 virus at 100 × TCID50 dose (Fig. 7B ) in 1: 128 and 1: 256 ratios, followed by Vero cell viability for approximately 6 days according to normalized cell index value in the RTCA system (Fig. 7C) . The results showed that double dose SK-01 V1 can neutralize the infective virus significantly in comparison with the control serum group even at 1: 256 dilutions (Fig. 7C) . Conduction of the same study for a single dose of SK-01 V1 and a single and double dose of OZG-3861 V1 showed that double dose OZG-3861 V1 at 1: 256 dilution also had virus neutralization capacity (Fig. 7D) . Although single-dose SK-01 V1 or OZG-3861 V1 did not show a significant neutralization efficacy at 1: 256 dilution (p > 0.05), it was evaluated that it had neutralization capacity at 1: 128 dilution (Fig. 7D) . The high rate of neutralizing antibodies detected in control mice (in 2 out of 3 mice, %66) in this study suggests that the mice may have previously had a viral infection like the mouse hepatitis virus (MHV), a member of the coronavirus family, related to SARS-CoV-2. Therefore, the findings of this study show the need to repeat the assay with mice that were considered to be negative for spontaneous neutralizing antibodies. However, the ADE (antibody-dependent enhancement) test worked almost like a confirmation of the neutralizing antibody test, showing that the antibodies formed neutralized the virus without causing ADE (Fig. 7E ). This in vitro analysis with mice serum showed that the SARS-CoV-2 specific neutralizing antibody was produced with the help of immunization of mice with the first versions of SK-01 and OZG-3861 vaccine candidates without an ADE effect. In this study, following the finding of the presence of antibodies due to B cell activity, T cell response was tested upon re-stimulation either with whole inactivated SARS-CoV-2 virus or SARS-CoV-2 specific S, N, and M-protein peptide pool. T cells were isolated from the spleen tissue of mice dissected either on day 21 or day 34. As T cells were incubated with the SARS-CoV-2 antigens, the cytokine secretion profile was evaluated for 72 h (Fig. 8A) . Subsequently, the balance of Th1 and Th2 cell responses was determined and showed an increase in the ratio of IL-12 to IL-4 and IFNγ to IL-4 (Fig. 8B ). This illustrated that our vaccine candidates were predominantly biased towards Th1 CD4 T cell response regarding control T cells isolated from untreated mice spleens. Furthermore, a significant increase in the proportion of cytotoxic CD8 T cells from an adjuvant negative single dose (OZG-3861 V1) and adjuvant positive double dose (SK-01 V1) immunized mice upon re-stimulation with www.nature.com/scientificreports/ the peptide pool was detected (Fig. 8C ). This showed that viral antigens caused CD8 T cell proliferation 34 days after vaccination. However, there was no increase in the T cell activation marker, CD25 on both T cell subtypes (Fig. 8C) . In order to evaluate the SARS-CoV-2 specific T cell response, stimulated T cells providing specific IFNg secretion that were counted as spots in the IFNγ ELISPOT plate were analyzed (Fig. 8D,E) . Findings especially showed that IFNg increase in T cells isolated from day21-dissected spleens was in the single or double dose of SK-01 V1 vaccine candidate containing adjuvant as opposed to the control mouse group (Fig. 8D) . Although there was no significant difference in the double dose of the OZG-3861 V1 vaccine candidate without adjuvant, an increase in IFNg was detected in single-dose administration (Fig. 8D) . On the other hand, a significant IFNg secretion from the T cells isolated from day34-dissected spleens of the single or double dose of OZG-3861 V1 and a single dose of SK-01 V1 was detected (Fig. 8E ). This analysis illustrated that SK-01 V1 and OZG-3861 V1 vaccine candidates can achieve not only B cell response but also T cell response. These encouraging pre-clinical in vivo efficacy studies will lead us to challenge humanized ACE2 + mice with SK-01 V1 and OZG-3861 V1.

Various methods are available in obtaining an effective and safe immunization in inactive vaccine production.

Besides chemical modifications such as formaldehyde or β-propiolactone, physical manipulations with ultraviolet radiation or gamma radiation are also available 9 . Chemical modifications in the vaccine inactivation process are time-consuming methods due to the need for purification. They also have disadvantages associated with toxicity with changes in viral structure and product loss due to the necessity to purify the final product. The physical www.nature.com/scientificreports/ inactivation process in the three separate animal experiments in this study showed that single or double dose administrations with or without adjuvant were non-toxic and effective. All viral vaccines contain virus-like materials that they try to protect. This directs the immune system to generate a response and to produce antibodies ready for use if it encounters a true viral infection. However, it is worrying that the virus mutates to form "escape mutants". These are mutated versions of the virus that vaccine-induced antibodies do not recognize. For a significant immunization, it is necessary to create a vaccine profile that covers the genetic variation of the virus within the community. If the genetic variation represented by the vaccine is small, triggering social immunity will not be at the desired rate. Therefore, the production and selection of more than one inactive viral strain remain an important mechanism for producing successful viral vaccines. Different variations may occur when producing large quantities (bulk) in the laboratory. Due to the low sensitivity of RNA-bound RNA polymerase, RNA viruses always produce a pool of variants during replication 10 . This phenomenon provides a potential for the rapid evolution of the virus, but it also makes up the majority of mutations that have detrimental effects on virus stability. Increased virus complexity can cause a weakening of the population's virulence degree; therefore, the characterization of individual variants can provide useful information for the design of a new generation of more effective and safer vaccines. Lyophilization (freeze-drying) is expressed as a process that combines the benefits of freezing and drying to obtain a dry, active, long shelf life, and easily soluble product 11 , and it is an important process for the preservation of heat-sensitive biological materials 12 .

In the production of inactive vaccines in this study, both inactivation and sterilization steps were carried out with a double dose (fractionated) gamma irradiation (25 kGy / single dose). With gamma irradiation, the frozen product can be irradiated, thus reducing the risk of toxicity as a free radical formation is prevented, and the risk of possible changes in the viral protein structure is reduced. Since functional viral structures are preserved, both B cell and T cell responses are triggered. With the first dose of irradiation, the raw product containing the live and infective virus is transformed into an intermediate product containing an inactive virus. Thus, prior to bottling, both environment and personnel are protected. A radiation dose over 25 kGy single dose was sufficient for inactivation is reported 13 . Both RTCA assay and colorimetric MTT assay for cell viability and proliferation confirmed the inactivation of the virus propagated in this study following 21-day of incubation with the three independent inactivated virus samples, and no suspicious situation was observed. In this study protocol, following the conversion of the frozen raw product into an inactive form by gamma irradiation, it is melted, bottled, and lyophilized. Lyophilized formulations, together with the advantage of better stability, provide easy handling during transportation and storage. The second dose of irradiation, performed after bottling (vialing) and lyophilization, functions to eliminate the presence of the replicant virus and end product sterilization. Also, unlike chemical inactivation methods, isolation and purification processes are not required. As a result, while achieving inactivation and sterility, fractionated (2-stage) gamma irradiation leads to less damage to the final product virus structure and allows the maximization of the preserved products. Besides, in our recently published pre-print 14 , we optimized an inactivated virus vaccine that 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 third version of our 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 [14] [15] [16] . In this study, 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 14 .

In toxicity analysis of vaccinated mice in this study, it was decided that adjuvant positive double dose administration should be removed in the newly designed version 2 vaccine model due to the finding of inflammatory reaction in the skin and kidney. Furthermore, for the overall picture immunization in the presence or absence of GM-CSF adjuvant did not yield significant differences in antibody and T cell responses. With this study, no significant difference was observed on immunization when GM-CSF was used as an adjuvant. Studies also showed that injection with intradermal GM-CSF leads to significant increases in grafting power in intradermally applied areas compared to distant areas 17 . This may explain the inability of GM-CSF in intradermally administered inactive virus vaccines. Therefore, it was decided to increase the SARS-CoV-2 effective viral copy dose (1 × 10 13 or 1 × 10 14 viral copies per dose) in version 2 of vaccine candidates. In terms of T and B cell responses, it was observed that especially the vaccine models containing GM-CSF as an adjuvant lead to significant antibody production with neutralization capacity in the absence of ADE feature.

On the other hand, ACE2 is ubiquitously expressed in various types of cells in humans 18 . Interaction between SARS-CoV-2 and ACE2 stimulates various pathways which some of which are known to determine the pathogenesis of SARS-CoV2 infection 19 . It was reported that ACE2-mediated cardiovascular protection was lost, multiorgan failure and gut dysbiosis were taken place following endocytosis of the enzyme following interaction with SARS-CoV-2 20 . However, we applied the vaccine candidate (OZG-38.61.1) 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 15, 16 . Therefore, we expected that only specialized dendritic cells named Langerhans cells (LCs) that populate the epidermal layer of the skin would be primed with the inactivated SARS-CoV-2 particles in the site of injection for the proper immunization 21 . Furthermore, in the histopathological analysis that was performed in this study and our recent preprint 14 , we did not determine any signature of multiorgan failure or cardiovascular impairment, supporting the safety of our vaccination procedure.

In the formation of the SARS-CoV-2 specific antibody, the antibody was detected up to 1: 256 titration in all doses and formulations of vaccine candidates administered to mice. On the other hand, in the control mice, www.nature.com/scientificreports/ Figure 6 . Day-21 safety analysis of the vaccine candidates, SK-01 V1, and OZG-3861 V1. Histopathologic analysis on day 21 of (A) lung (the arrow; chronic inflammation X100), jejunum of the intestine, liver, spleen, and (B) cerebellum of brain, hippocampus, kidney, and skin. In the spleen, stars show foci of megakaryocytes and arrows were trabeculae (X400 H + E stain). In the cerebellum, the thin arrow shows dendrites, the thick arrow shows picnotic cell, the thin double-headed arrow shows space. In the hippocampus, P is the pyramidal cell layer and D is the dentate gyrus (X100 H + E stain). In the kidney, the arrow shows interstitial chronic inflammation (X400 H + E stain www.nature.com/scientificreports/ we determined a low level of spontaneous neutralizing antibodies, which may be because the control mice may meet coronavirus like infections such as mouse hepatitis virus (MHV) 22, 23 . Also, no traces of MHV was detected when stool samples from 5 mice were tested for MHV copy using qRT-PCR. This neutralizing antibody ratio was seen in the first version of vaccine candidates with a viral copy number of approximately 10 7 /dose is predicted to achieve higher and longer-term antibody concentration in the second version which will have 9 × 10 11 or 1 × 10 13 viral copies per dose of SK-01 V2 and OZG-3861 V2 vaccine candidates. To assess the sensitivity or specificity of the XCELLIGENCE assay, we tested the OZG-38.61.3 vaccine candidate in our recent challenge study with ACE2 transgenic mice. No significant neutralizing capacity was observed in the neutralizing test using SARS-CoV2 at the same proportions with convalescent plasma or standard control sera 14 .

We also determined the balance of Th1 and Th2 cell responses, because Vaccine-Associated Enhanced Respiratory Disease (VAERD) was reported to be associated with Th2-biased cell responses, both in some animal models and children vaccinated with whole-inactivated RSV and measles virus vaccines [24] [25] [26] [27] . In this study, Th1 to Th2 was balanced towards Th1 response suggesting that VAERD risk is low. Another finding showed that the presence of adjuvant is more important in T cell response in comparison with the B cell response, which may lead to long-term immunization. In addition, here, the ADE test was the in vitro equivalent of VAERD. It has been reported indirectly that there is no vaccine-related ADE effect within the macrophage. The absence of this effect has been confirmed in Version 3 of our vaccine OZG-38.61 in challenge tests with ACE2 transgenic mice 14 . VAERD evolves the ADE in in-vitro conditions. It has been reported in our latest report indirectly that there is no vaccine-related ADE effect within the macrophage. The absence of this effect has been confirmed in the Version 3 challenge tests 14 . In the viral challenge study, viral dissemination was blocked by SARS-CoV-2 specific antibodies and neutralizing antibodies in both vaccine groups, unlike other groups, especially at high doses, CD4 activation is also present in the immune response that occurs. the resulting T cell response is in the Th1 response type as desired; the resulting T cell response is in the Th1 response type as desired. It has been determined that the ADE effect is not observed. These findings also confirm that our vaccine is non-replicative 14 .

With this study, it was seen that our gamma-irradiated inactivated vaccine candidates can effectively trigger the production of SARS-CoV-2 specific antibodies along with long-term T cell response. Hence, the findings of this study prompted us to plan a new in vivo experiment with the second version of SK-01 and OZG-3861 (1 × 10 13 or 1 × 10 14 viral copies per dose) in humanized ACE2 + mice 14 . In this report, we determined that GMCSF 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. Therefore, it was decided to increase the SARS-CoV-2 effective viral copy dose (1 × 10 13 or 1 × 10 14 viral copies per dose) in the last version of vaccine candidates. In the challenge study, we produced the third and final version of the OZG-38.61 without an adjuvant 14 . In this study, it was demonstrated 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 14 . This preclinical study has encouraged us to try phase 1 vaccine clinical trials to avoid the COVID-19 pandemic. www.nature.com/scientificreports/ 

World Health Organization. Draft Landscape of COVID-19 Candidate Vaccines-14th

An mRNA vaccine against SARS-CoV-2-preliminary report

Development of an inactivated vaccine candidate for SARS-CoV-2

SARS-CoV-2 isolation and propagation from Turkish COVID-19 patients

Genome analysis of SARS-CoV-2 and effects of spike glycoprotein variants on protein structure

This scientist hopes to test coronavirus drugs on animals in locked-down Wuhan

Proposal for vaccination against SARS coronavirus using avian infectious bronchitis virus strain H from The Netherlands

Potential interventions for novel coronavirus in China: a systemic review

Inactivation methods for whole influenza vaccine production

Long-term passage of duck Tembusu virus in BHK-21 cells generates a completely attenuated and immunogenic population with increased genetic diversity

Genel Proses Değerlendirmesi. Etlik Vet. Mikrobiyoloji Derg

A bread-based lyophilized C-strain CSF virus vaccine as an oral vaccine in pigs

The effect of gamma-irradiation conditions on the immunogenicity of whole-inactivated Influenza A virus vaccine

Gamma-irradiated SARS-CoV-2 vaccine candidate, OZG-38.61.3, confers protection from SARS-CoV-2 challenge in human ACEII-transgenic mice

Intradermal delivery of vaccines: potential benefits and current challenges

Intradermal vaccine delivery: will new delivery systems transform vaccine administration

Intradermal granulocyte-macrophage colony-stimulating factor alters cutaneous antigen-presenting cells and differentially affects local versus distant immunization in humans

The pivotal link between ACE2 deficiency and SARS-CoV-2 infection

Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response

Angiotensin-converting enzyme 2: SARS-CoV-2 receptor and regulator of the renin-angiotensin system

Origin, homeostasis and function of Langerhans cells and other langerin-expressing dendritic cells

Lessons for COVID-19 immunity from other coronavirus infections

Coronaviruses and sars-cov-2. Turk

SARS-CoV-2 mRNA vaccine development enabled by prototype pathogen preparedness

Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine

Altered reactivity to measles virus: atypical measles in children previously immunized with inactivated measles virus vaccines

A Double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge

Sevda Demir and Sezer Akyoney are funded by TÜBİTAK 2247-C Trainee Researcher Scholarship Program (STAR). Selen Abanuz and Ilayda Sahin are supported by the TUBITAK-BIDEB 2244-University-Industry Ph.D. program, Project number 118C082. Figure 1 and 5A of the study was drawn by G.S.K. and C.T. with BIOREN-DER.com.

The online version contains supplementary material available at https ://doi. org/10.1038/s4159 8-021-83930 -6.Correspondence and requests for materials should be addressed to E.O.Reprints and permissions information is available at www.nature.com/reprints.Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.