The spike (S) gene of SARS-CoV-2 was designed according to the sequence of SARS-CoV-2/Wuhan-Hu-1 (GenBank ID: MN908947) (Yang et al., 2004) by adding the BamHI and XbaI at the 5’ and 3’ terminal of the gene, respectively. Then, a Syn21 sequence (AACTTAAAAAAAAAAATCAAA) (Liu et al., 2015) was also inserted before the start codon (ATG) of the spike gene to enhance transcription. Furthermore, the transmembrane and carboxyl terminus (TM/CT) of S was replaced by the corresponding transmembrane sequence of H5N1 hemagglutinin (HA) (aa 531-568, 38aa, A/Indonesia/5/2005 M1) (Liu et al., 2011). The resulting sequence was named the S gene ( Figure 1A ).

As reported, three sites of the Spike, including D614, RRAR (682-685), and KV986-987, are crucial for the virus infection. The mutation of D614 to A614 may avoid Antibody-dependent enhancement (ADE) (Wang et al., 2016). The mutation of RRAR (682-685) to GSAS can inhibit the recognition and cleavage by furin (Kalnin et al., 2021). Finally, the mutation of KV986-987 to PP986-987 can enhance immunogenicity (Kirchdoerfer et al., 2018; Dagotto et al., 2020; Wrapp et al., 2020). Therefore, these sites were replaced by the corresponding sequence, respectively, and the optimized sequence was designated the mS gene ( Figure 1A ). The S gene and mS gene were synthesized according to the codon usage preference of insect cells and linked downstream of promoter PH of pFastBac™ Dual donor plasmid (Invitrogen, USA) according to the protocol described previously (Xu et al., 2021). The resulting shuttle plasmids were designated as pFBD-SARS-CoV-2-S and pFBD-SARS-CoV-2-mS, respectively ( Figure 1B ).

Moreover, to improve the stability of the VLP (Liu et al., 2011), the expression frame of the H5N1 M1 gene (A/Indonesia/5/2005 M1, 252aa) was added to the downstream promoter P10 of pFastBac™ Dual donor plasmid (Invitrogen, USA), generating two shuttle plasmids pFBD-SARS-CoV-2-SM and pFBD-SARS-CoV-2-mSM, respectively ( Figure 1B ).

To generate recombinant bacmid, the donor plasmids pFBD-SARS-CoV-2-S, pFBD-SARS-CoV-2-mS, pFBD-SARS-CoV-2-SM, and pFBD-SARS-CoV-2-mSM were transformed into Competent DH10Bac™ E. coli cells, followed by the antibiotic selection. The positive E. coli colonies containing recombinant bacmids rBD-S, rBD-mS, rBD-SM, and rBD-mSM was identified by PCR. In addition, the recombinant bacmids were verified by PCR ( Table 1 ).

Recombinant baculovirus was rescued according to the previously described protocol (Xu et al., 2021). Briefly, sf9 cells in a 6-well plate (1×10⁶cells/well) were transfected with the recombinant bacmids using Cellfectin^® II Reagent (Gibco, USA) according to the manufacturer’s instructions and cultured in Grace’s medium (Gibco, USA) for 5-7 days at 27°C until apparent cytopathic effect (CPE) appeared. Then, cells and supernatant were collected, centrifuged at 3000 rpm, 4°C for 5 min, and filtered with a 0.22 μm filter. Next, the pellets were resuspended in PBS and evaluated using indirect fluorescence assay (IFA), Western blot, and transmission electron microscopy (TEM). The recombinant baculoviruses were named rBDV-S, rBDV-mS, rBDV-SM, and rBDV-mSM. Afterward, the baculoviruses were blind passaged in Sf9 cells and stored at -80°C. Finally, the viral titer of the third passage was examined according to the protocol described by BacPAK Baculovirus Rapid Titer Kit (Clontech, USA).

To generate VLPs, sf9 cells in a shake flask (1×10⁶cells/well) were infected with the third passage recombinant baculoviruses (multiplicity of infection, MOI=1) for 72 h. Medium supernatant was collected and ultracentrifuged through a 20% sucrose cushion at 30,000rpm, 4°C for 2 h. The pellets were resuspended in PBS and further purified using a stepwise sucrose density gradient consisting of 2 mL of 10%, 30%, and 60% sucrose in PBS and centrifuged at 30,000 rpm and 4°C for 2 h. The white circular target protein in 10%-30% was collected and transferred to the centrifuge tube containing 5mL PBS and centrifugated at 30,000 rpm, 4°C for 2 h. The pellets were resuspended in PBS and evaluated using SDS-PAGE and Western blot. At the same time, the target protein was also examined by SARS-CoV-2 (2019-nCoV) Spike ELISA Kit (Sino Biological, China) according to the manufacturer’s instructions. The standard curve was drawn according to the mean absorbance of optical density 450 (OD 450), and the concentration of the target antigen was calculated based on the standard curve.

Sf9 cells (5×10⁵ cells/well) cultured in a 6-well plate were infected with the recombinant baculoviruses (MOI=1) for 48 h at 27°C. The IFA was performed as described by Xu et al. (Xu et al., 2021). Briefly, cells were fixed in 4% paraformaldehyde for 10 min at room temperature and washed three times with PBS for 5 min each. Next, the cells were blocked with 1% skim milk at 37°C for 30 min. After that, the cells were incubated with 500 μL AcMNPV GP64 antibody (1:1000, Sino Biological, China), SARS-CoV-2 (2019-nCoV) Spike RBD Antibody (1:1000, Sino Biological, China), or Influenza A M1 (1:1000, GeneTex, USA) at 37°C for 120 min. Then, the cells were incubated with 500 μL FITC-labeled Goat Anti-Rabbit IgG (H+L) (1:2,000, Beyotime, China) at 37°C for 40 min in the darkroom. Finally, the cells were examined using an Eclipse TE2000-V (Nikon, Japan).

Western blot was performed according to the protocol described by Xu et al. (Xu et al., 2021). Briefly, cell lysate or protein solution was loaded (1:5) and separated via 10% SDS-PAGE. Then, the protein was transferred onto the PVDF membrane, blocked with 5% skim milk for 1 h at room temperature, followed by incubating with the convalescent serum of the COVID-19 patient (1:1000) or Influenza A M1 (1:1000, GeneTex, USA) for 2 h at room temperature. After that, the membrane was incubated with HRP-labeled Goat Anti-Rabbit IgG (H+L) or HRP-labeled Goat Anti-Mouse IgG (H+L) (1:3000, Beyotime, China) for 1 h at room temperature. Subsequently, the band was developed using the GEGEGNOME XRQ enhanced chemiluminescence (ECL) (Thermo Fisher SCIENTIFIC, USA).

The sample was absorbed on the grid and stained with 1% Phosphotungstic Acid for 1-2 min. Then, TEM was performed to examine the VLPs using a JEM-1200EXII transmission electron microscope (JEOL USA, Peabody, MA, USA).

Specific pathogen-free (SPF, 6-8 weeks old) Balb/c mice and C57/BL/6N mice were purchased from Vital River Laboratory (Beijing, China). In addition, Humanized Balb/c mice containing human ACE2 (Balb/c-hACE2) were constructed by Cyagen (Suzhou, China). Mice were housed in SPF stainless steel with a constant atmosphere (22-25°C, 45-50% relative humidity), natural light cycle, and unlimited feeding and drinking. The virus infection experiment was conducted in the BSL-3 laboratory, Changchun Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences. Mice were monitored three times daily for changes in physical appearance and deaths (if any) and weighed every day during the experiment. In addition, the blood was collected weekly for specific antibody evaluation. At the end of the experiment, mice were anesthetized by carbon dioxide (CO₂), euthanized by cervical dislocation, and lung and other organs were collected for further evaluation.

Firstly, SPF Balb/c mice were randomly divided into five groups (6 mice in each group) and intramuscularly immunized with indicated VLPs and adjuvant ( Table 2 , group A). Then, SARS-CoV-2 specific antibody and T-cell subset distribution were evaluated at the indicated weeks via Enzyme-linked immunosorbent assay (ELISA) and Flow cytometry, respectively.

The effects of adjuvant and duration of immune interval on antibody production were evaluated to optimize the immunization strategy. For adjuvant ( Table 2 , group B), C57/BL/6N mice were divided into three groups (8 mice in each group), and intramuscular immunization was carried out on the hind legs with the mSM and candidate adjuvants, or PBS. For immune interval ( Table 2 , group C), C57/BL/6N mice were divided into five groups (6 mice in each group), and intramuscular immunization was carried out with the mSM and candidate adjuvants or PBS for indicated intervals. In addition, SARS-CoV-2 specific antibody was evaluated at the indicated weeks via ELISA.

Moreover, humanized Balb/c mice were randomly divided into two groups (6 mice in each group), immunized with the mSM and Al/CpG adjuvant, or PBS, and boosted with the identical inocula at 3 weeks later ( Table 2 , Group D). SARS-CoV-2 specific antibody and neutralization activity of SARS-CoV-2 specific antibodies were determined by the pseudovirus-based neutralization assay at the indicated weeks via ELISA. Then, the mice were challenged with SARS-CoV-2/Wuhan-Hu-1 strain (Yan et al., 2022) (10^5.5 TCID₅₀/mL) by nasal drops (0.05 mL) 2 weeks post the boost immunization. The body weight was monitored at an interval of 2 days. Then, mice were euthanized, and viral loads in the lung were examined by real-time PCR on days 3, 5, and 7 post-infection (dpi).

To evaluate the protective efficiency of the candidate vaccine, SPF Balb/c mice were randomly divided into two groups (6 mice in each group), immunized with the mSM and Al/CpG adjuvant, or PBS, and boosted with the identical inocula at 3 weeks later ( Table 2 , group E). Then, the mice were infected with a mouse-adapted SARS-CoV-2/C57MA14 strain (Kindly provided by Prof. Yuwei Gao) (Yan et al., 2022) (10^5.5 TCID₅₀/mL) by nasal drops (0.05 mL) 10 days post the boost immunization. Mice were euthanized, and viral loads in the lung were examined by real-time PCR on 7 dpi.

SARS-CoV-2 specific antibody was examined using Mouse IgG Antibody Detection Kit for COVID-19 (ELISA) (Darui Technology, China) according to the manufacturer’s instruction. The coating antigen used in the kit was RBD protein. Briefly, serum was diluted with PBS (1:100) and added to the ELISA plate (100μL per well) at 37 °C for 40 min. After washing four times with PBS, 100μL HRP-labeled Goat pAb to Ms IgG (Darui Technology, China) or HRP-labeled Goat pAb to Ms IgG1, HRP-labeled Goat pAb to Ms IgG2a (Abcam, Britain) was added into the well and incubated at 37 °C for 20 min, followed by washing six times. Then, substrates A and B (50μL each) were added and reacted at 37 °C for 10 min in the dark. The reaction was stopped by adding 50μL stop buffer, and the absorbance values (optical density, OD) were examined at 450 nm or 630 nm using a microplate reader (TECAN SPARK, Switzerland). The serum of convalescent patients with COVID-19 and the normal person was used as the positive and negative control, respectively. Three replicates were used for each sample. When the OD value of the sample is greater than or equal to 2.1 times that of the negative control, the sample is defined as a positive. If the OD value of negative control is less than 0.1, it is calculated as 0.1.

The neutralization activity of SARS-CoV-2 specific antibodies was determined by the pseudovirus-based neutralization assay. Briefly, mice serum was inactivated at 56°C for 30 min, serially diluted with DMEM (3-fold dilutions), and added to a 96-well plate (50 μL/well). Then, SARS-CoV-2 pseudovirus (Du et al., 2022) was added to the well (100μL/well, MOI=100) and incubated at 37°C for 60 min. After that, Huh7 cells (2×10⁵ cells/mL) resuspended in DMEM (containing 10% FBS) were added to the well (100 μL/well) and incubated at 37°C. 48 h later, the cells were detected using One-Lumi™ Firefly Luciferase Reporter Gene Assay Kit (Beyotime, China) according to the manufacturer’s instruction and examined using a microplate reader (TECAN SPARK, Switzerland). The mock cell was used as negative control (CC), and SARS-CoV-2 pseudovirus was used as the positive control (VC). The Cut-Off Value is set as Value (sample)≤Value (VC)×0.5. The quality control is set as Value (VC)≥Value (CC)×3.

The splenocytes were isolated from immunized mice at the end of the experiment according to the previously described protocol (Liu et al., 2013; Xu et al., 2021). Briefly, the mouse spleen was homogenized and suspended in Roswell Park Memorial Institute (RPMI) 1640 (Hyclone, Beijing, China) to prepare cell suspension. Then, the splenic lymphocytes were isolated by centrifugation using a mouse lymphocyte isolation solution according to the manufacturer’s instructions (Hao Yang Biological Manufacture Co., Ltd., Tian Jin, China). Afterward, lymphocytes were resuspended in RPMI 1640 supplemented with 10% fetal bovine serum and counted.

The splenocytes (10⁶ cells) were incubated with 1 mL PE/Cyanine7 anti-mouse CD3ϵ Antibody, FITC anti-mouse CD4 Antibody, or APC anti-mouse CD8a Antibody (Biolegend, USA) at 4 °C for 30 min, centrifuged at 1500 rpm for 5 min. Next, the pellet was resuspended in PBS and centrifuged at 1500 rpm for 5 min, followed by examination using Flow cytometry (CytoFLEX, Beckman, USA).

Total RNA was extracted from the lung of the animals using QIAamp Viral RNA Mini Kit (QIAGEN, Germany) and examined using HiScript II U⁺ One Step qRT-PCR Probe Kit (Vazyme, China) with indicated primers ( Table 1 ).

Statistical analysis was performed using GraphPad 8.0 (GraphPad Software, SanDiego, CA) with the one-way analysis of variance (ANOVA; two-tailed, confidence intervals (CI) 95%), as indicated by the p-value. The results were statistically significant at p<0.05. At least three independent experiments were evaluated for each separate set of assays. The results are expressed as the mean ± standard deviation (SD).

SARS-CoV-2 S gene was optimized and synthesized ( Figure 1A ), followed by subcloning into shuttle plasmid pFastBac™ Dual according to the protocols described previously (Xu et al., 2021), resulting in four recombinant bacmids, including pFBD-SARS-CoV-2-S, pFBD-SARS-CoV-2-SM, pFBD-SARS-CoV-2-mS, and pFBD-SARS-CoV-2-mSM ( Figure 1B ). Then, the bacmids were identified using PCR ( Supplemental Figure S1 ), and the corrected bacmids were transfected into Sf9 cells to rescue recombinant baculoviruses ( Supplemental Figures S2 and S3 ), respectively. The recombinant baculoviruses are designated as rBDV-S, rBDV-SM, rBDV-mS, and rBDV-mSM. Thereafter, the expression of exogenous genes in the Sf9 cells was evaluated by IFA. As shown in Figure 1C , the cells infected by recombinant baculoviruses exhibited apparent fluorescence compared with the mock-infected group, suggesting exogenous genes were efficiently expressed in the cells. These results were further confirmed by Western blot. As expected, four kinds of S proteins can be detected in cells infected with the recombinant baculovirus ( Figure 1D ). There were several bands in the wild-type groups, while the optimized S protein (mS) had only one specific band at 180 kDa, indicating the optimized protein is more stable and not easily degraded by protease than the wild-type S. Moreover, the M1 protein of H5N1 can be detected between 22 and 25 kDa ( Figure 1D ), which further proves the successful expression of these proteins.

To prepare VLPs, sf9 cells were infected with recombinant baculoviruses, and the protein was purified by ultracentrifugation with sucrose cushions, followed by transmission electron microscope observation (TEM). As shown in Figure 1E , the VLPs showed spherical shapes as typical SARS-CoV-2 virions. Spike structure can be observed around VLP, similar to the characteristic spike structure of intact virus particles, indicating that the recombinant SARS-CoV-2 VLPs were generated successfully. The expression of mSM genes was more effective than in the other three groups, suggesting that codon optimization can enhance the expression of exogenous genes in the Sf9 cells.

Mice were immunized with the purified VLP or PBS, followed by an evaluation of the immune responses. Animals were monitored daily for adverse effects. No obvious adverse events were observed. Blood was collected at 0, 1, 2, 3, 4, 5, and 6 weeks after the first dose of the immunization, and the boost was conducted three weeks after the first vaccination ( Figure 2A and Table 2 , group A).

As shown in Figure 2B , levels of SARS-CoV-2 specific antibody increased gradually in the VLP groups after the first dose of the immunization, which was significantly higher than that of the PBS group. The highest titer of specific antibodies in serum reached the peak four weeks post the first immunization in the VLP groups, with the highest level in the mSM group, and then decreased slightly in each group. After the boost immunization, the titers of the mSM and mS groups were higher than those of other groups, up to 1: 102,400 ( Figure 2C ). Compared with the control group, IgG, IgG1, and IgG2a at six weeks post initial immunization in the immunized groups increased to different degrees ( Figures 2D–F ), and the levels of specific IgG2a antibodies were higher than that of IgG1 ( Figures 2E, F ). In addition, the levels of specific IgG2a in the mSM and mS groups were higher than in other infected groups ( Figure 2F ). Three weeks post the boost immunization, the splenocytes were isolated from immunized mice according to a previously described protocol (Liu et al., 2013), followed by an evaluation of the T-cell subset distribution. As shown in Figure 2G , the levels of CD3⁺CD4⁺T and CD3⁺CD8⁺T lymphocytes in the mSM group were highest than in the other groups. These results suggest that the optimized S proteins, especially the mSM protein, had more robust immunogenicity than the wild-type S. Therefore, recombinant baculovirus rBDV-mSM and the optimized mSM were further evaluated in the subsequent studies. Then, the mSM protein in the supernatant was further purified using a stepwise sucrose density gradient and evaluated using SDS-PAGE, Western blot, and ELISA. Figure The purified mSM protein can be efficiently recognized by the convalescent serum of a COVID-19 patient (primary antibody), while the unpurified protein in the supernatant has no obvious band ( Supplemental Figure S4 ). The concentration of the purified mSM protein was quantified via standard curve ( Supplemental Figure S5 ), and it was 0.2 μg/μL.

To optimize the immunization strategy of the mSM group, we evaluated the effects of adjuvant and duration of immune interval on antibody production and compared it with that of the PBS group ( Table 2 , group B-C). As shown in Figure 3A , the effect of the Al/CpG adjuvant on antibody production was better than MF59/CpG adjuvant. However, the duration of the immune interval has no significant effect on antibody production ( Figures 3B , C ). Meanwhile, the optimized immunization with mSM and Al/CpG adjuvant can stimulate animals to produce sustained high-level antibodies, which can still be detected after 53 weeks of vaccination ( Figure 3D ). The proportion of positive serum of immunized mice in Figure 3D (positive rate) was about 33.33% ( Figure 3E ).

To clarify the immunogenicity and protective efficiency of the VLP-based vaccine, humanized ACE2 mice (Balb/c-hACE2) were immunized with the VLP or PBS, followed by an evaluation of the immune responses and virus challenge ( Figure 4A and Table 2 , group D). Figures 4B , C shows that the optimized vaccinaion with mSM and Al/CpG adjuvant can stimulate humanized mice to produce sustained high-level antibodies at 14, 21, 28, and 35 days post initial vaccination, with the highest titer of about 1:204,800 at 35 days post initial immunization. Furthermore, the titers of neutralizing antibodies reached 1:1,620 on the 35^th day of the initial vaccination ( Figure 4D ).

Furthermore, the immunized mice were infected with SARS-CoV-2/Wuhan-Hu-1 14 days post the boost immunization, and animals were monitored at an interval of 2 days for adverse effects. As a result, no apparent adverse events were observed, and the body weights of the vaccine-immunized group were similar to that of the PBS group ( Figure 4E ). Moreover, the viral loads of the vaccine-immunized group were significantly decreased compared with that of the PBS group 3 days post-challenge ( Figure 4F ). These results indicate that the optimized immunization with SARS-CoV-2 VLP (mSM) and Al/CpG adjuvant can induce effective immune responses.

To further clarify the protective efficiency of the mSM-based vaccine, BABL/c mice were immunized with the VLP or PBS, followed by an evaluation of the immune responses and challenge with a mouse-adapted SARS-CoV-2 strain ( Figure 5A and Table 2 , group E). As shown in Figure 5B , the SARS-CoV-2 specific antibody increased gradually in the mSM group, but there was no significant increase in the PBS group. There was a significant difference between the two groups 28 dpi. Furthermore, the body weights of the two groups decreased gradually after being challenged with the virus at 10 days post the boost immunization, but the weight-loss trend of the mSM-immunized mice was significantly lower than that of the control group ( Figure 5C ). On the fifth day of the virus challenge, one mouse in the PBS group died, while the others survived. At the end of the experiment, the survival rate of the mSM group was 100%, and that of the PBS group was 83.3% ( Figure 5D ). Then, the viral loads were examined on the seven^th dpi. As shown in Figure 5E and 5F, the copy numbers of viral genomic RNA (N gRNA, Figure 5E ) and viral subgenomic RNA (sgERNA, Figure 5F ) in the lung of the mSM group decreased significantly from that of the PBS group. These results indicate that the candidate vaccine based on the mSM VLP and Al/CpG adjuvant can establish an effective protective barrier to protect mice from virus attacks.