The protection of CoronaVac against the infection of wild‐type SARS‐CoV‐2 (WH‐09) or Omicron variant in nude‐hACE2 mice

Abstract Background Immunocompromised individuals have an increased risk of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection and severe outcomes, but we pay less attention to these people. Athymic nude mice are a murine strain with a spontaneous deficiency of the Foxn1 gene, which can result in thymic degeneration or its absence, leading to immunosuppression and a decrease in the number of T cells, and are widely used in preclinical evaluations of disease in immunocompromised populations. Methods We investigated the protection of the coronavirus disease 2019 (COVID‐19) inactivated vaccine (CoronaVac) against the infection of wild‐type SARS‐CoV‐2 (WH‐09) or Omicron variant utilizing a hybrid‐type nude‐hACE2 mouse model. Results Compared with nude‐hACE2/W mice, the viral load in the brain and lung tissue of nude‐hACE2 mice (nude‐hACE2/WV) infected with WH‐09 after vaccination significantly decreased, and the histopathological changes were also reduced. The viral load in the brain and lung tissue of nude‐hACE2 mice (nude‐hACE2/OV) infected with the Omicron variant after vaccination was lower than that in nude‐hACE2/O, but histopathological symptoms did not improve significantly. Conclusion CoronaVac provides some protection against infection of both WH‐09 and the Omicron variant in the nude‐hACE2 mice. Our findings aimed to provide a reference for vaccination against SARS‐CoV‐2 in immunocompromised populations.

and respiratory symptoms, with most patients recovering within 1-2 weeks. However, some patients may progress to acute respiratory distress syndrome, multiple organ failure, and death. 3 The mass inoculation of a COVID-19 vaccine, inducing herd immunity, is considered a committed step toward ending the current global pandemic. However, several special populations, including those with weakened immunity and immune deficiency, were not included in sufficient numbers in the clinical trials of COVID-19 vaccines .4-6 One of the important reasons for excluding immune-deficient individuals is that immune deficiency can impair the humoral immune responses and cellular immune responses induced by vaccines, making it difficult to measure the effectiveness on the immune system. 7 The World Health Organization states that immunization plans should give preference to vulnerable groups, including the elderly, people with immune deficiencies, and those with underlying health. 8 It should be noted that there are many types of such diseases, and we have not yet fully understood the pathogenesis.
Athymic nude mice are a murine strain with a spontaneous deficiency of the Foxn1 gene, which can result in thymic degeneration or its absence, leading to immunosuppression and a decrease in the number of T cells. 9 The inherent characteristics of low immunity in nude mice was widely used to study animal immunodeficiency diseases and the abnormal response of pathogenic factors to the body.
Considering that nude mice cannot be infected with WH-09, we investigated the protective effect of CoronaVac against WH-09 or Omicron variant infection utilizing a hybrid-type nude-hACE2 mouse model. Our research aimed to provide basic research datas for vaccination against SARS-CoV-2 among immunodeficient individuals.
CoronaVac was produced by Sinovac Life Sciences with a labeled amount of 1200 SU/0.5 mL and a specification of 0.5 mL per dose. All experiments involving live SARS-CoV-2 were conducted in accordance with approved standard operating procedures in the Animal Biosafety Level 3 laboratory of ILAS and PUMC (CNAS BL0010).

| Animals
Nude-hACE2 (K18) mice were produced by ILAS and PUMC. F1 hybrid mice were obtained by hybridizing female hACE2 +/− (K18) mice with male BALB/c-nude mice in the ratio of 1:1. F1 female hACE2 +/− mice identified using PCR (polymerase chain reaction) amplification of DNA and agarose gel electrophoresis were backcrossed with male BALB/c-nude mice in the ratio of 1:1 to obtain F2 generation. The PCR primers were h-ACE2-MA-sl 5′-GGACA AGT TTA ACA CGA   AGCC-3′ and h-ACE2-MAas1 5′-CAGCT GAA GCT TAC ATG AGAT-3′. hACE2 +/− and hACE2 +/+ transgenic nude mice (nude-hACE2 mice) in F3 generation, obtained by crossing male hACE2 +/− nude mice with female hACE2 +/− hairy mice in F2 generation, were used in this study ( Figure S1). 10 Nude-hACE2 mice were kept in a 12-h light-dark cycle with free access to food and water. All the animal experiments were approved by the Institutional Animal Care and Use Committee of ILAS and PUMC (ILAS GH22001).

| Determination of viral gene copies in mouse tissues
Total RNA was extracted from homogenized lung samples. Tissue homogenates (1 g/mL) were prepared by homogenizing the perfused tissues using an electric homogenizer for 2 min 30 s in Dulbecco's modified Eagle's medium (DMEM). The homogenates were then centrifuged at 1509 g for 10 min at 4°C; the supernatant was collected and stored at −80°C for viral load detection. Quantitative real-time PCR (qRT-PCR) was performed using gene-specific primers to determine the copy number of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene. To determine the viral load, total RNA was extracted from 350 µl of clarified tissue homogenates using an RNA extraction kit. qRT-PCR was performed on a LightCycler 96 system using a one-step RT-PCR reaction kit with SARS-CoV-2 RdRp primers.

| Live virus neutralization assays
Cytopathic effect (CPE) was used to detect neutralizing antibody (NAb) titers in serum. Serum samples were heat inactivated at 56°C for 30 min and diluted twofold using cell culture medium. Heatinactivated serum was mixed with a viral suspension containing 100 TCID 50 in the ratio of 1:1 in 96-well plates and incubated for 1 h at 36.5°C in a 5% CO 2 incubator. Then, 1 × 10 4 Vero E6 cells were added to the serum-virus mixture and incubated for 72 h after washing away the inoculum with PBS. The CPE was observed, and the NAb titer was calculated using the dilution number of 50% protective condition.

| Histopathology and immunohistochemistry studies of mouse tissue sections
Formalin-fixed and paraffin-embedded tissues were cut into 4μm slices and stained with hematoxylin and eosin (H&E) for histopathological examination. The expression of the virus antigen in tissue was detected using mouse anti-SARS-CoV-2 S protein antibodies, followed by staining with goat anti-mouse IgG secondary antibody. The expression of hACE2 antigen in tissues was detected using staining with an rabbit anti-hACE2 antibody, followed by staining with goat anti-rabbit IgG secondary antibody.

| Statistical analysis
All data were analyzed using GraphPad Prism 8.0 software. Twotailed unpaired Student's t-test was used to compare the differences between the two groups, whereas one-way analysis of variance (ANOVA) was used to compare data among the three groups. Statistical significant difference was defined as *p < 0.05 and **p < 0.01.

| CoronaVac did not improve clinical symptoms in nude-hACE2 mice infected with WH-09
To evaluate the protective effect of CoronaVac on nude-hACE2 mice infected with WH-09, 24 mice were used in this study. The experimental design and longitudinal sampling plan are shown in Figure 1A.
Twelve nude-hACE2 mice (nude-hACE2/WV group) were inoculated with 0.1 mL of CoronaVac two times (on days 0 and 28) and intranasally infected with WH-09 at 100 TCID 50 at 14 days after the final immunization. The control group (nude-hACE2/W group, n = 12) was  Figure 1C). All mice died on 5 dpi ( Figure 1D).
To further evaluate the virus replication and histopathological changes in nude-hACE2 mice, we harvested samples from the infected mice at 3-4 dpi for PCR, H&E staining, and immunohistochemistry staining. The mean viral RNA loads in the lungs and brain of nude-hACE2/W mice were 10 7.83 and 10 9.75 copies/g ( Figure 1E). The copy number of SARS-CoV-2 S protein in the brain of nude-hACE2/W mice was significantly higher than that in the lungs (p < 0.01; Figure 1E), which was consistent with the results of nude-hACE2 mice. 11 Compared with nude-hACE2/W mice, the viral load in the brain tissue of nude-hACE2/WV mice significantly decreased (p < 0.01) but not eliminated.
Significantly, the viral load in the lung tissue of five of six nude-hACE2/ WV mice was reduced to an undetectable level ( Figure 1E). The NAb titers of the nude-hACE2/WV mice were 16-32 arbitrary unit (AU) at 0 dpi ( Figure 1F), which was 16.5-fold lower than that in hACE2. 6 The WH-09 can infect the central nervous system of hACE2 mice, 12 in which we can detect a high level of viral load. The brain tissue was collected for H&E staining and immunohistochemical study to further study the pathological changes and virus infection in nude-hACE2 mice infected with WH-09. Consistent with virus load results, the immunohistochemical staining results of hACE2 and SARS-CoV-2 S protein showed that the brain tissues of nude-hACE2 mice expressed abundant hACE2 protein, and SARS-CoV-2 S protein was significantly distributed in the brain tissues in both groups ( Figure 1G).

| CoronaVac slightly improved the histopathological characteristics in nude-hACE2 mice infected with WH-09
The main complications caused by COVID-19 are pneumonia and acute respiratory distress syndrome, which may also cause other To further evaluate the virus replication of nude-hACE2/O and nude-hACE2/OV, we harvested samples for PCR at 4 dpi. The mean viral RNA loads were 10 6.63 and 10 5.2 copies/g in the lungs and brain of nude-hACE2/O mice and were 10 6.06 and 10 4.48 copies/g in the lungs and brain of nude-hACE2/OV mice. Compared with nude-hACE2/O, the viral load in the lung tissue of nude-hACE2/OV mice was significantly reduced (p < 0.01) but not eliminated ( Figure 3E).

| CoronaVac did not improve the histopathological characteristics in nude-hACE2 mice infected with the Omicron variant
To further study the histopathological changes in nude-hACE2 mice infected or postimmune infected with the Omicron variant, we collected the brain and lung tissues for H&E staining. The lungs of nude-hACE2/O mice showed mild interstitial pneumonia, including mild and focal thickening of alveolar septa, peribronchiolar and perivascular mild inflammatory infiltration, and mild collagen fiber hyperplasia ( Figure 4A). The brain tissue of nude-hACE2/O mice showed local vacuolar degeneration, neuronal degeneration and necrosis, and dissolved nucleus. Nude-hACE2/OV mice showed the same histopathological features in the brain and lungs as nude-hACE2/OV mice ( Figure 4B).
CoronaVac did not improve the histopathological changes in nude-hACE2/O and nude-hACE2/OV mice. There were no obvious pathological changes in the kidney, liver, or spleen of both groups ( Figure 4C).

| DISCUSS ION
Immunocompromised individuals often experience inflammation and are at an increased risk of contracting SARS-CoV-2. They are more likely to develop severe illness as a result of infection. 8,[14][15][16] Rodents have been widely used to study the pathogenesis and vaccination response of SARS-CoV-2 due to their wide availability and short growth cycle. Athymic nude mice are a murine strain with a spontaneous deficiency of the Foxn1 gene, which can result in thymic deterioration or its absence, leading to immunosuppression and a decrease in the number of T cells. This model is widely used in preclinical evaluations of immunocompromised populations and in the study of infectious diseases. However, the WH-09 of SARS-CoV-2 F I G U R E 4 Histopathological characteristics of brain, lungs, renal interstitium, renal tubules, liver, and spleen in nude-hACE2/O and nude-hACE2/OV groups infected with the Omicron variant. The tissue samples of mice were collected, fixed in formalin, and embedded in paraffin for histopathological examination. (A) Histopathological characteristics and immunohistochemistry of lungs in nude-hACE2 mice at 4 dpi were analyzed. Histopathological characteristics of (B) brain and (C) kidney, liver, and spleen. that was prevalent in the early stages of COVID-19 had a low binding efficiency with mACE2, and infected athymic nude mice showed almost no symptoms of the disease. 17 In this study, we used 3-to 4-month-old nude-hACE2 mice as research objects to investigate the protection of CoronaVac against WH-09 infection. First, we demonstrated that nude-hACE2 mice infected with WH-09 showed obvious clinical symptoms such as weight loss, hunched back, movement retardation, and respiratory distress. The replication of the virus in lung and brain tissues was obvious and showed corresponding histopathological changes, which was consistent with the hACE2 mice model. Second, CoronaVac reduced viral load replication and alleviated the histopathological changes in nude-hACE2/WV mice. Compared with nude-hACE2/W, the viral load was significantly reduced in brain tissue and was reduced to an undetectable level in the lungs of nude-hACE2/WV mice. The histopathological changes in the brain, lungs, kidneys, and liver in nude-hACE2/WV mice subsided. Third, the NAb titers in the serum of nude-hACE2 mice with two doses of CoronaVac were significantly lower than those in wide-type hACE2 mice. Overall, our research showed that CoronaVac inoculation can partly alleviate the symptoms in nude-hACE2 mice infected with WH-09, suggesting that CoronaVac can play a certain role in the prevention of critically ill patients.
During COVID-19 transmission, emerging SARS-CoV-2 variants, including the Delta and Omicron variants, reduced their fusogenicity and pathogenicity. 18 The titers of NAbs in the serum of nude-hACE2 mice immunized with two doses of CoronaVac were far lower than those in normal hACE2 mice and showed limited protection against WH-09. Therefore, we further stud- showed only mild histopathological changes in lung and brain tissues, including mild interstitial pneumonia and local vacuoles in brain tissue. However, the replication of the virus in lung and brain tissues was obvious. Second, compared with nude-hACE2/O mice, the viral load in the lung and brain tissue of nude-hACE2/O mice decreased, and mild histopathological features were still observed in the lungs and brain, indicating that vaccination reduced virus replication in mice. Third, the NAb titers of the Omicron variant of nude-hACE2/OV mice with three doses of vaccination were lower than those of nude-hACE2/WV mice. Lv et al. found that in inactivated vaccine sera, at 1-3 months post three doses, geometric mean titers of NAb against the Omicron variant were 4.9/5.2-fold lower than those of the WH-09, which was consistent with the results of this study. 19 The infection symptoms of the Omicron variant were mild, and no obvious histopathological changes in nude-hACE2 mice were observed after vaccination.
In general, our research shows that CoronaVac conferred some protection among the nude-hACE2 mice infected with the WH-09 or Omicron variant. However, in this experiment, we chose the antibody peak time to carry out the study, as the time point of infection postimmunization in the population is uncertain. Therefore, it is still necessary to further carry out long-term follow-up research in animals.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no competing financial interests or other associations that may pose a conflict of interest (e.g., pharmaceutical stock ownership, consultancy).

E TH I C S S TATEM ENT
All the animal experiments were approved by the Institutional Animal Care and Use Committee of ILAS & PUMC (ILAS GH22001).