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SARS-CoV-2 inactivated vaccine (Vero cells) shows good safety in repeated administration toxicity test of Sprague Dawley rats

The outbreak of COVID-19 has posed a serious threat to global public health. Vaccination may be the most effective way to prevent and control the spread of the virus. The safety of vaccines is the focus of preclinical research, and the repeated dose toxicity test is the key safety test to evaluate the vaccine before clinical trials. The purpose of this study was (i) to observe the toxicity and severity of an inactivated SARS-CoV-2 vaccine (Vero cells) in rodent Sprague Dawley rats after multiple intramuscular injections under the premise of Good Laboratory Practice principles and (ii) to provide a basis for the formulation of a clinical trial scheme. The results showed that all animals in the experimental group were in good condition, no regular changes related to the vaccine were found in the detection of various toxicological indexes, and no noticeable stimulating reaction related to the vaccine was found in the injected local tissues. The neutralizing antibodies in the low- and high-dose vaccine groups began to appear 14 days after the last administration. In the negative control group, no neutralizing antibodies were observed from the administration period to the recovery period. Therefore, the repeated administration toxicity test of the inactivated SARS-CoV-2 vaccine (Vero cells) in Sprague Dawley rats showed no obvious toxic reaction. It was preliminarily confirmed that the vaccine can stimulate production of neutralizing antibodies and is safe in Sprague Dawley rats.


Background
The rapid spread of COVID-19 since the outbreak has posed a serious threat to global public health. According to the World Health Organization (WHO) website, on November 17, 2020, the number of confirmed COVID-19 cases reached 39,023,292, and the number of deaths reached 1,099,586. The WHO has named it 2019 novel coronavirus (2019-nCoV) on January 12, 2020, and announced that the COVID-19 epidemic was listed as a "public health emergency of international concern" on January 30, 2020, and at the same time, the disease was officially named coronavirus disease 2019 (COVID-19) on February 11, 2020. 2019-nCoV is a novel coronavirus strain that has never been found in humans before. Middle East respiratory syndrome-related coronavirus (MERSr-CoV) and severe acute respiratory syndrome-related coronavirus (SARSr-CoV) belong to the β-coronavirus family , and have 88% nucleotide homology with SARS-CoV-2 .
In August 2020, the latest research results revealed that 2019-nCoV is highly infectious and its transmission is difficult to detect . Due to these features, cases of 2019-nCoV infection, especially asymptomatic ones, are not easy to find; hence, the virus easily spread globally and in medical institutions. Even if the virus can be completely eliminated from the population, the viral transmission mechanism from inactivated vaccine in animals and carried out necessary preclinical toxicological studies for clinical trials and marketing of the SARS-CoV-2 inactivated vaccine.

Vaccines
All SARS-CoV-2 virus strains used in this work were isolated from hospitalized patients including domestic and foreign patients with confirmed COVID-19 in Yunnan Hospital of Infectious Diseases from January to May 2020. A strain with a D614G mutation in the S protein was isolated from a pediatric patient who had returned from the U.S. to their hometown and was identified as being infected with SARS-CoV-2 through clinical diagnosis and q-RT-PCR in March 2020. The virus was proliferated in Vero cells (WHO) and was titrated with a microtitration assay. Vero cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Corning, NY, USA) containing 5% fetal bovine serum (FCS; HyClone, Logan, USA).
The SARS-CoV-2 inactivated vaccine was developed by the Institute of Medical Biology (IMB), Chinese Academy of Medical Sciences (CAMS). Briefly, the virus strain, named KMS-1 (GenBank No: MT226610.1), was inoculated into Vero cells for production in an environment that met the BSL requirements. The harvested viruses were inactivated by formaldehyde (v:v = 1:4000) for 48 h to lyse the viral membrane, purified via chromatography and concentrated. A second inactivation with beta-propiolactone (v:v = 1:2000) was performed to destroy the viral genomic structure, followed by a second purification and concentration using the same protocol. The vaccine stock was evaluated using various quality indexes, including antigen content, immunogenicity, sterility and residue testing. The viral antigen content was measured via ELISA. The vaccine contained 100 or 150 U of inactivated viral antigen adsorbed to 0.25 mg of Al(OH)3 adjuvant and suspended in 0.5 mL of buffered saline for each dose. The placebo contained only the same amount of Al(OH)3 in buffer.

Animals
Specific Pathogen Free Sprague Dawley rats, 6-8 weeks old; animal weight range when purchased: female, 150.8-192.7 g; male, 158.7-203.5 g; and the weight variance of the animals in the same batch did not exceed 20% of the average weight. Animals were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., production license No. SCXK (Jing) 2016-0006. During the test, rats were kept with two animals per cage, with free access to maintenance feed and sterilized water. This protocol has been approved by the Experimental Animal Ethics Committee of institute of Medical Biology, Chinese Academy of Medical Sciences before its implementation (approval No. DWSP202005  005). The use of animals conforms to the 3R (replace, reduce, refine) principle.

Animal grouping and administration
190 Sprague Dawley rats (half ♂ and half ♀) were randomly divided into four main test groups and three satellite groups. The main test groups included the following: a negative control group (normal saline), a solvent control group (SARS-CoV-2 inactivated vaccine blank control, 0 U in 0.5 mL/dose), a low-dose vaccine group (SARS-CoV-2 inactivated vaccine, 100 U in 0.5 mL/dose), and a high-dose vaccine group (SARS-CoV-2 inactivated vaccine, 150 U in 0.5 mL/dose), with 20 male and 20 female rats per group. The satellite groups, which were only prepared for blood collection for detection of neutralizing antibody, included a negative control satellite group, a low-dose vaccine satellite group, and a high-dose vaccine satellite group, with 5 male and 5 female rats per group. All satellite groups were administrated by vaccines or normal saline corresponding to the main test groups. A total volume of 0.5 mL (0.25 mL on both sides of the hind limb) was used per vaccination. The dosage of vaccine was selected according to the General Principles for the Technical Review of Pre-clinical Safety Evaluation of Biological Products for Prevention (CDE, 2008), which also conformed to WHO guidelines on the nonclinical evaluation of vaccine (WHO, 2014). All vaccines were administered intramuscularly by 1 mL aseptic injection syringe (Shaanxi Longkangxin Medical Instrument Co., Ltd). Referring to above guiding principles, animals received at least one more vaccination than clinically prescribed. Exposure intervals should generally be determined according to the immune response of animals. Because animals generally reach stable antibody formation after 2-3 weeks of inoculation, long-term toxicity tests generally use exposure intervals of 2-3 weeks. Referring to the above principles, the SARS-CoV-2 inactivated vaccine (Vero cells) was administered once each on days 0, 14, 28, and 42, for a total of four administrations, and the interval was 2 weeks, followed by a recovery period after the last administration of 2 weeks (14 days). Therefore, rats received four bilateral vaccinations Specific grouping information is shown in Table 1.

Clinical indicators
All indicators describe below was adopted to comply with recommendations contained in General Principles for the Technical Review of Pre-clinical Safety Evaluation of Biological Products for Prevention (CDE, 2008).

General clinical observations
Observations were carried out three times on days of administration: once in the morning before administration, once 30-60 min after administration, and once in the afternoon. Observation was conducted twice daily on the non-administration days: once in the morning and once in the afternoon.

wt and food intake test
During the experiment, body weight and food intake were measured once a week with a YP20001 balance (Sartorius Scientific Instruments (Beijing) Co., Ltd). Weight gain was calculated as follows: weightgainpercentage= currentweight− weightbeforeadministration weightbeforeadministration ×100% Each cage animal was given enough feed every week. The amount of food intake per rat per day was calculated as follows: amountgivenperweek − remainingamountperweek numberofanimals Note：Weight gain percentage = (Current weight -weight before administration)/weight before administration × 100%, ' n ' was the Sprague Dawley rats number of each group.

Eye and urine routine examination
At 2 and 13 days after the last administration, the animals were examined with a YZ11 ophthalmoscope (66 Vision Technology Co., Ltd., China) to check whether the optic nerve disc, macula, venules/arterioles on the macula, and retinal venules/arterioles were abnormal. At 2 and 13 days after the last administration, urine of animals was collected for analysis with a UA-66 urine automabic analyzer (Mindray Medical International Ltd., China) after 4 h of fasting.

Hematology examination
At 3 days after the first administration, 3 and 14 days after the last administration, about 1.5 mL of blood was collected from the abdominal vein of rats in the experimental group (EDTA-2K anticoagulation). The samples were centrifuged at 3000 rpm for 10 min, and the plasma was collected and blood cells were counted by a 950-hematology analyzer (Drew Scientific Company, USA).

Serum biochemistry
At 3 days after the first administration, 3 and 14 days after the last administration, about 3 mL of blood was collected from the abdominal vein of rats in the experimental group. The blood samples were centrifuged at 3000 rpm for 10 min to separate the serum. About 100 μL of serum was taken 3 and 14 days after the last administration for antinuclear antibody (ANA) detection. The rest of the serum was detected by an Olympus AU400 automatic biochemical analyzer (Olympus Corporation, Japan).

Coagulation examination
At 3 and 14 days after the last administration, about 2 mL of blood was collected from the abdominal vein of rats in the main experimental group (9:1 sodium citrate anticoagulant) and centrifuged at 3000 rpm for 10 min, and plasma was detected by a CA500 automatic coagulation analyzer (Sysmex Co., Ltd, Japan).

CD4 + T cell and CD8 + T cell determination
At 3 and 14 days after the last administration, about 100 μL of blood added with EDTA-2K anticoagulant was taken as the sample for flow cytometry. Cells stained with CD3-PE (201412), CD4-FITC(201505), CD8a-PerCP(201712, all purchased from Biolegend Inc., USA) at room temperature for 15-30min followed by OpitiLyse C (A11895, Beckman Coulter Inc., USA) lysing solution at room temperature for 2-5min. Cells were then added with phosphate-buffered saline (PBS) and blended by vortex for testing by Cytoflex flow cytometry (Beckman Coulter Inc., USA). The proportions of CD3 + CD4 + cells and CD3 + CD8 + cells in T lymphocytes were analyzed by Cytexpert software, and the ratios of CD3 + CD4 + cells and CD3 + CD8 + cells were calculated.

Antinuclear antibody detection
About 100 μL of the remaining serum, which had been biochemically separated from the animals, was dissected 3 days after the last administration and tested by double-antigen enzyme-linked immunosorbent assay (ELISA) (Jiang Lai Bio Co., Ltd., China).

Neutralizing antibody detection
About 0.5 mL of venous blood was taken from the tail vein of animals in the satellite group before the first administration and 14 days after each administration. The blood was separated by centrifugation for 15 min at 3000 rpm without anticoagulant. The neutralization test was used to evaluate the efficacy of virus neutralization.

Gross anatomy
At 3 days after the first administration, autopsy was performed on the main experimental group by intraperitoneally injecting 2% pentobarbital sodium at a dose of 100 mg/kg and then dissecting the animals for visual observation. The specific number of dissected animals is shown in Table 2.

Pathological examination
At 3 and 14 days after the last administration, the animals in the main experimental group were anesthetized with 2% sodium pentobarbital at a dose of 100 mg/kg, sacrificed, and dissected. The wet weights of brain, heart, liver, thymus, spleen, lung, kidney, adrenal gland, testis, epididymis, ovary, and uterus were measured, and the organ coefficient was calculated as follows: organ coefficient = organ wet weight body weight × 100%. All organs were observed according to the guiding principles, including the sublingual gland, submandibular gland, parotid gland, spinal cord (neck, chest, and lumbar segment), pituitary gland, trachea, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, aorta, skin (abdomen), breast (female), vagina, fallopian tube, bladder, sciatic nerve, sternum, femur, thyroid, muscle (skeletal muscle), distal lymph nodes, eyes, Haugh's glands, adrenal glands, brain (brain, cerebellum, brainstem), prostate, seminal vesicle (male), thyroid (including parathyroid), the administration site and all abnormal tissues (CDE, 2008). The eyes were fixed with eye fixator, and other organs were fixed with neutral formalin solution. Paraffin-embedded sections were made and hematoxylin eosin staining was used for microscopic examination. The terms and criteria of pathological diagnosis were mainly based on the International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) (Berridge et al., 2016;Brändli-Baiocco et al., 2018;Mann et al., 2012;Renne et al., 2009;Thoolen et al., 2010).

Table 4
Food intake statistics of all animals (MEAN ± SD, g). Note: 'n' was Sprague Dawley rats number of each group.

Statistical analysis
SPSS 21.0 was used for statistical analysis, and the t-test was used for comparison between the two groups. P < 0.05 was considered to indicate a statistical difference. Data, including body weight, food intake, organ weight and organ coefficients, hematological indexes, lymphocyte subsets, are expressed as mean ± SD. If the paired organs of an animal were missing on one side, the organ weight and organ coefficient of the animal were not used in the calculation of the average. Statistical and biological significance were considered in the analysis of results. The above data were analyzed according to the following process: firstly, Levene's test was conducted for the data homogeneity test. If the data are uniform (P > 0.05), then one-way ANOVA is conducted; if the ANOVA difference is significant (P < 0.05), Dunnett's multiple comparison (parametric method) is performed. If Levene's test results were significant (P < 0.05), the Kruskal-Wallis nonparametric test was performed. If the Kruskal-Wallis nonparametric test results were significant (P < 0.05), the Mann-Whitney U test was used for pairwise comparison. In addition, for the semiquantitative ordered data results of urine examination, the Ridit test of multiple groups was used. If the results were significant (P < 0.05), the Ridit test of two groups was used for further statistics. For the observation of clinical symptoms, no statistics were conducted because there were no abnormal symptoms related to the test product, and no statistical tests were performed in the pathological examination.

The clinical symptoms
The results of clinical symptoms showed that during the trial, the rats in each group were in good condition and exhibited normal activity. After receiving multiple repeated administrations, no obvious abnormalities were seen at the injection site, such as redness, swelling, bruising, induration, suppuration, fester, and other irritation related to the vaccine.

Body weight and food intake
During the administration and recovery period, the body weight of rats in all groups showed an increasing trend, and there were no statistically significant changes in body weight and body weight growth rates in the solvent control group, the low-dose vaccine group, and the high-dose vaccine group compared with the negative control group (P > 0.05), as shown in Table 3. During the whole experimental period, the food intake of male rats was significantly higher than that of female rats, but there was no difference between groups (P > 0.05), as shown in Table 4. It was concluded that the test product had no significant effect on the weight gain of the animals.

Urine routine examination
At 2 and 13 days after the last administration of the SARS-CoV-2 inactivated vaccine, compared with the negative control group, there was no statistically significant difference in urine biochemical indexes (P > 0.05) ( Table 5). At 2 days after the last administration, although occult blood (BLD) was not only found in an animal, there was no statistical difference between the groups, and it had no toxic significance.

Hematology and coagulation examination
At 3 days after the first administration of the SARS-CoV-2 inactivated vaccine, there were no statistically significant differences in various hematological indicators compared with the negative control group (P > 0.05). At 3 days after the last administration, white blood cell (WBC) and lymphocyte (LYM) were slightly increased in one rat in the negative control group, solvent control group, and low-dose vaccine group, but the difference was not statistically significant (P > 0.05). There were no statistically significant differences between the other dose groups and the negative control group (P > 0.05). At 14 days after administration withdrawal, there was no statistically significant difference between the other dose groups and the negative control group (P > 0.05), as shown in Table 6. There was no statistically significant change (P > 0.05) in coagulation indexes of rats in each group (Table 7). Thus, no toxicological changes were observed in the hematological and coagulation indexes of the main test group during the experiment.

Serum biochemistry and electrolyte examination
At 3 days after the first administration of the SARS-CoV-2 inactivated vaccine, the alkaline phosphatase (ALP) value of the solvent control group was increased compared with the negative control group, and the difference was statistically significant (P < 0.05). There were no statistically significant differences between the other dose groups and the negative control group (P > 0.05). At 3 and 14 days after administration withdrawal, there was no statistically significant difference in serum biochemical indexes between each group and the negative control group (P > 0.05), as shown in Table 8. During the study, the electrolyte including potassium (K+), sodium (Na+), chloride (Cl-), calcium (Ca+), had no significant difference between groups (Table 9).

Immune function-related indicators
ANA levels were determined by double antigen ELISA after repeated administration of the SARS-CoV-2 inactivated vaccine. The results showed that the serum ANA was negative in the negative control group, solvent control group, and low-and high-dose groups during the experiment, as shown in Table 10. CD4 + and CD8 + T cell counts, as determined by flow cytometry, at different time points (3 and 14 days after the last administration) are shown in Table 10 and Fig. 1. Compared with the negative control group, there was no significant difference in the proportion of peripheral blood CD4 + and CD8 + T cells in the solvent control group and the low-and high-dose groups. Furthermore, the CD4+/CD8+ cell ratio of each group showed no statistical difference with the negative control group. The neutralizing antibodies in all groups were negative before grouping. At 14 days after the first administration, the neutralizing antibodies in the low-dose vaccine satellite group and the high-dose vaccine satellite group began to appear, with average titers of 1:5.2 and 1:17.6, respectively; at 14 days after the second administration, the levels of neutralizing antibodies of the two groups gradually increased, with average titers of 1:80 and 1:108.8, respectively; and at 14 days after the third administration, the levels of neutralizing antibodies of the two groups continued to rise, with average titers of 1:128 and 1:128, respectively. The neutralizing antibody levels of the two groups remained at 1:128 until 14 days after the fourth administration, as shown in Table 11 and Fig. 2.

Eye exam
During the trial, no abnormal changes were found in the eyelid, conjunctiva, sclera, cornea, iris, pupil, lens, optic disc, macula, arterioles, retinal arterioles, vitreous body, and fundus of the rats in the test group.

Gross necropsy
Rats were dissected 3 days after the first administration and 3 and 14 days after the last administration. There were no obvious abnormalities in the apparent pathology of each group of animals. The skin, subcutaneous tissue, and muscle of all animals in all autopsies were examined at the original injection site, and no irritant reaction related to the SARS-CoV-2 inactivated vaccine was found.

Organ weight
During the test period, compared with the negative control group in the same period, there was no statistically significant difference in the weight and relative weight of all organs in the test group (P > 0. 05), as shown in Table 12, indicating that this vaccine does not affect the weight of organs.

Histopathological examination
At 3 days after the last dose, a small number of inflammatory cells were found in the myocardium of two animals in the low-dose vaccine group, 14 days after the last dose, and one animal in the solvent control group, one in the low-dose group, and one in the high-dose vaccine group had the same pathological symptoms (Fig. 3A). At 3 days after the last dose, there were two small foci of inflammatory necrosis in the liver parenchyma and sink area, mainly including lymphocytes and emptying hepatocytes. This pathological symptom also appeared 14 days after the last dose in two animals in the negative control group and the solvent control group (Fig. 3B); these two animals also developed glomerular atrophy or increased glomerular capillaries (Fig. 3C), but its structure was clear and complete, and there was no obvious structural cell necrosis. At 3 days after the last dose, pulmonary pathology showed that one or two animals in each group showed local pulmonary interstitial thickening, alveolar epithelial cell proliferation, shedding and varying degrees of vascular and alveolar septal capillary congestion, a small amount of inflammatory cell infiltration, or thickened alveolar septa in the vascular lumen and perivascular and interstitial lung ( Fig. 3D and E). In the negative control group, two animals showed a large amount of exudate and a small amount of monocytes and local glomerular atrophy with increased glomerular capillaries (Fig. 3C).
Even though some minor lesion was observed in each group, there no significant group correlation for injuries. Most pathological results of rats in each group, no matter at 3 or 14 days after the last administration, were normal. Fig. 4 showed that there were no obvious histopathological changes in main organs including lung, liver, kidney and heart in each group. Meanwhile, muscles at injection site were also pathologically examined. No obvious pathological changes were found in each group as well (Fig. 5).

Discussions
Vaccination is the most effective and economical means to prevent and control many infectious diseases (Iwasaki and Omer, 2020;Leidner et al., 2019). Because vaccines are mainly used for immunization of healthy people, the safety requirements of vaccines should be much higher than those of therapeutic drugs. Pre-clinical safety evaluation is one of the important phases in the development of new vaccines. However, due to the sudden outbreak, the pre-clinical safety evaluation of the SARS-CoV-2 inactivated vaccine remains to be conducted. How to evaluate the safety of the vaccine is also under continuous exploration.
It is generally considered that the vaccination dose is small and the adverse reactions are mild. According to the WHO guidelines on vaccine toxicology studies, no matter what animal model is used, it is recommended to give at least the dose that one person would receive (WHO, 2005(WHO, , 2014. The General Principles for the Technical Review of Pre-clinical Safety Evaluation of Biological Products for Prevention also indicate that the vaccine dose should, in general, enable the vaccine to achieve the best immune response in animals, and that long-term toxicity tests can be conducted directly using the high dose (per person) proposed in clinical trials (CDE, 2008). In the current study, the candidate dose of the SARS-CoV-2 inactivated vaccine for clinical trial, 100 U/person and 150 U/person, proposed by the results of previous immunogenicity and protection studies was respectively used as low dose and high dose.
In the pre-clinical safety evaluation of vaccine products, a single animal species should be used to study the toxicity of repeated administration (CDE, 2008). The Sprague Dawley rats used in this study are the most commonly used rodents in vaccines general toxicity studies (Mancebo et al., 2012;Sifontes-Rodríguez et al., 2009;Stokes et al., 2020). The background information of this family of animals is abundant, and the supply of animals is sufficient. The candidate vaccine was planned to apply for adults over 18 years old. Therefore, the choice of Sprague Dawley rats on 5-6 weeks, which reached sexual maturity, were more reasonable in repeated dose toxicity study. In neutralizing Table 7 Statistical data of coagulation examination in all animals (MEAN ± SD).  antibody testing experiment, both low-dose vaccine group and the high-dose vaccine group could be detected at 14 days after the first administration. By 14 days after the second vaccination, the neutralizing antibodies were positive in all vaccinated groups. From 14 days after the second dose to 14 days after the third dose, the levels of neutralizing antibodies of these two groups continued to rise, and this trend remained until 14 days after the fourth administration, showing that the SARS-CoV-2 inactivated vaccine (Vero cells) could stimulate Sprague Dawley rats to produce a humoral immune response, and the antibody is maintained at a high level for at least 2 weeks. Therefore, the dose set in the repeated administration toxicity study produced a good immune response in Sprague Dawley rats. These results conformed to further support the applicability of the animal species in the repeated administration toxicity study. For the purpose that providing sufficient evidence of toxicity might induced by proposed dose of SARS-Cov-2 inactivated vaccine, as many indicators as possible were tested in the repeated dose toxicity study. One of the potential toxicity of vaccines comes from immune systemrelated toxicity, including hypersensitivity reactions and autoimmunity. Autoimmunity is the main research content of immunotoxicology, especially for adjuvanted vaccines (Agmon-Levin et al., 2009;Batista--Duharte et al., 2014). So far, only a few autoimmune diseases caused by vaccine immunization have been reported, such as Guillain-Barré syndrome, caused by influenza vaccination in the United States (Schonberger et al., 1979). However, the detailed mechanism is still not fully understood. From the perspective of strict regulation, product-related immunotoxicological evaluation should be carried out during the safety evaluation of new vaccines before clinical trials. Serum autoantibodies have been widely used for the detection of specific autoimmune diseases (Descotes, 2000;Verdier et al., 1997), although the association between autoantibody levels and the development and severity of autoimmune diseases is not fully established. In this study, no  Note:CD4+,CD4+ positive T cell; CD8 + ,CD8+ positive T cell; CD4+/CD8+,CD4+ positive T cell/CD8+ positive T cell; ANA,Antinuclear antibody. Fig. 1. The results of the CD4 + and CD8 + T cell counts. Compared with the negative control group, there was no significant difference in the proportion of peripheral blood CD4 + and CD8 + T cells in the solvent control group and the low-and high-dose groups. Furthermore, the CD4 + /CD8 + cell ratio of each group showed no statistical difference with the negative control group. Note：if the neutralizing antibody titer is lower than 1:4, it is judged as "negative", and if it is higher than or equal to 1:4, it is judged as positive. Neg,Negative.

Fig. 2.
The results of the neutralizing antibodies. The neutralizing antibodies in all groups were negative before grouping. At 14 days after the first administration, the neutralizing antibodies in the low-dose vaccine satellite group and the high-dose vaccine satellite group began to appear, with average titers of 1:5.2 and 1:17.6, respectively; at 14 days after the second administration, the levels of neutralizing antibodies of the two groups gradually increased, with average titers of 1:80 and 1:108.8, respectively; and at 14 days after the third administration, the levels of neutralizing antibodies of the two groups continued to rise, with average titers of 1:128 and 1:128, respectively. The neutralizing antibody levels of the two groups remained at 1:128 until 14 days after the fourth administration. antinuclear antibodies were detected in rats after repeated immunization with the SARS-CoV-2 inactivated vaccine. These results indicate that the experimental vaccine might have a low risk of causing an autoimmune response. Meanwhile, the results of the present study showed that after repeated inoculations with SARS-CoV-2 inactivated vaccine (Vero cells) in rats, no specific affected organs were found. In addition, histopathological examination of the animals' immune organs including bone marrow, spleen, thymus, inguinal lymph nodes, mesenteric lymph nodes and tonsils, showed no significant changes, and repeated administration did not cause a local reflux lymph node stress response at the injection site, which further indicated that the SARS-CoV-2 inactivated vaccine have no obvious immunotoxicity in Sprague Dawley rats (Fig. S2).
CD4 + and CD8 + T cells respectively represent helper T cells and cytotoxic T cells, play a key role in antiviral immunity (Yang et al., 2018), the level of cellular immunity is another important consideration. In this study, the detection of rat CD4 + and CD8 + T cells was carried out, and the ratio of both kinds of cells had not changed significantly (Fig. S1). At the same time, immune cells (including lymphocytes, mononuclear phagocytes, neutrophils, basophils, eosinophils, and platelets) in the blood did not increase or decrease after the first dose or the last dose or during the recovery period. It indicated that the vaccine had no obvious effect on lymphocytes and there was no infectious antigen in inactivated vaccines, which would attack antigen presenting cells to produce cytotoxic T cells (CD8 + T cells) (Yang et al., 2018).
Although there was a difference in serum ALP levels between the negative control group and the solvent control group 3 days after the first administration of the vaccine (P < 0.05), it returned to the normal value 3 and 14 days after the last administration, and there was no dose-response relationship. ALP was one of indicators related with liver function, especially serum ALP which was hepatic origin. The increase of ALP usually associated with liver lesions. However, other indexes related to liver function, including γ-glutamyl transpeptidase (GGT), total bilirubin (TBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (ALB), were normal at 3 days after first administration. Only one indicator change was not clinically significant. Meanwhile, the level of ALP in this study was normal comparing with other Sprague Dawley rats in toxic studies (Mancebo et al., 2012;Mary L.A. Giknis, 2006;Sifontes-Rodríguez et al., 2009). No abnormality was found in pathological examination of solvent control group at 3 days after first administration as well. Therefore, it suggested that the inactivated SARS-CoV-2 vaccine has no obvious hepatotoxicity in Sprague Dawley rats. At 3 and 14 days after the last administration, the results of urine routine examination showed that occult blood appeared in all groups, and the index was on the high side. In the comparative analysis with the negative control group, considering that the change of the index was not related to the vaccine, the 4-h urine content is the most ideal in the determination of various indicators of urine. Because the amount of urine is greatly affected by the amount of drinking water, especially for experimental animals, the retention process of collecting 4-h urine is relatively complex. Single measurements of the concentrations of substances in random urine samples give an incomplete view of the real situation. There was no statistically significant difference in other urine indicators between the two groups. In addition, urea nitrogen (BUN) and creatinine (CREA) levels were normal. Although a small number of animals (5/120) showed glomerular atrophy, only a small number of local changes were not obvious, and the incidence rate was low. In this study, the occurrence of renal lesions was not associated with certain doses or groups. We speculate that the lesions were spontaneous changes in the kidney and were due to accidental events unrelated to the vaccine. We conclude that the inactivated SARS-CoV-2 vaccine has no nephrotoxic effects in Sprague Dawley rats.
Histopathology examination of heart, liver, lung, and kidney at 3 and 14 days after the last administration showed some mild inflammatory symptoms, but its tissue structure was complete, clear, and no large area of cell injury was observed. These lesions did not have significant temporal differences and group distribution specificity, and there was no significant gender difference, nor was it a specific change of a certain type of disease. In compilation of histopathological findings in 4-26week old Sprague Dawley rats compiled by Charles River Laboratory, the incidence of pathological changes in heart, liver, kidney and lung was higher than other organs no matter in male or female rats. All rats reported in this compilation were from 34 toxicity studies conducted in different laboratories (Mary L.A. Giknis, 2012). Also, it has been shown that Sprague Dawley rats often develop spontaneous diseases with chronic inflammation as the main lesion, including chronic interstitial pneumonia or liver and cardiac focal necrosis (Noto et al., 1998;Weber et al., 2011). Spontaneous renal lesions in rats are dominated by tubular lesions, including dilation of renal tubules, protein cast, tubular atrophy, pigmentation, and calcium deposition. The incidence increases with age and may be associated with many factors such as nutrition (Chandra and Frith, 1994;Hannerz et al., 1989). The indicators related to relevant organs function in the same batch were normal. Therefore, the above lesions were probably spontaneous or occasional lesions in Sprague Dawley rats, and some of them may be physical injuries caused by the operation processes. They have no toxic pathological significance, and no dose-effect-related lesions and target organs have been found. The development of SARS-CoV-2 vaccine was still ongoing. A SARS-CoV-2 inactivated vaccine developed by Wuhan Institute of Biological Products Co. Ltd evaluated safety and immunogenicity of vaccine on seven different species of animals. Sprague Dawley rats were used in this study for repeated dose study as well. After different doses administration and 2-week recovery, there was no obvious systemic toxicity observed in rats . Similar to the study above, repeated administration of high-dose (1.5 person dosages) SARS-CoV-2 inactivated vaccine did not cause damage to the animals' immune system, and no obvious dose-response histopathological changes were seen in various tissues and organs. Most importantly, in both the low-dose vaccine group (1 person dosage) and the high-dose vaccine group (1.5person dosages), no serious pathological changes and abnormal clinical indicators, predicting systemic toxicity, were found. There was no obvious dose-response change where found in SARS-CoV-2 viral target organ: the lungs and bronchi. The vaccine was preliminarily confirmed to have good safety, providing an experimental basis for further clinical trials and a reference framework for related follow-up research.

Authors' contributions
Kaili Ma and Qihan Li conceived the idea and designed the experiment. Zhangqiong Huang, Qinfang Jiang, Yixuan Wang, Jinling Yang and Tingfu Du performed the main experiments, Zhangqiong Huang analyzed the data and wrote the main manuscript. Hongkun Yi, Cong Li, Yun Li, Zhengcun Wu, Shengtao Fan, Yun Liao, Ying Zhang, Lichun Wang, Guorun Jiang, Donghong Tang, Yousong Ye, Chenyun Wang, Zheli Li, Zhisai Li and Caixing Zhang participated in this work. Kaili Ma and Qihan Li revised the manuscript. We also thank all participants involved in this work. All authors reviewed the manuscript.  The results of the histopathological examination. A: At 3 days after the last dose, a small number of inflammatory cells were found in the myocardium of two animals in the low-dose vaccine group, 14 days after the last dose, and one animal in the solvent control group, one in the low-dose group, and one in the high-dose vaccine group had the same pathological symptoms; B: At 3 days after the last dose, there were two small foci of inflammatory necrosis in the liver parenchyma and sink area, mainly including lymphocytes and emptying hepatocytes. This pathological symptom also appeared 14 days after the last dose in two animals in the negative control group and the solvent control group; C: Two animals in the negative control group also developed glomerular atrophy or increased glomerular capillaries, but its structure was clear and complete, and there was no obvious structural cell necrosis. Also in the negative control group, two animals showed a large amount of exudate and a small amount of monocytes and local glomerular atrophy with increased glomerular capillaries; D,E: At 3 days after the last dose, pulmonary pathology showed that one or two animals in each group showed local pulmonary interstitial thickening, alveolar epithelial cell proliferation, shedding and varying degrees of vascular and alveolar septal capillary congestion, a small amount of inflammatory cell infiltration, or thickened alveolar septa in the vascular lumen and perivascular and interstitial lung.  Pathological results of injection sites muscle from each group. A. The injection sites muscle from negative control group administrated with saline. B. The injection sites muscle from solvent control group administrated with SARS-CoV-2 inactivated vaccine blank control. C. The injection sites muscle from low-dose group administrated with 100U/dose SARS-CoV-2 inactivated vaccine. D. The injection sites muscle from high-dose group administrated with 150U/dose SARS-CoV-2 inactivated vaccine. There was no significant lesion in each group at the injection sites.