All experiments and methods were performed in accordance with relevant guidelines and regulations. Mice experiments were reviewed and approved by the Research Ethics Review Committee of Shanghai Public Health Clinical Center.

The full-length s genes of the SARS-CoV-2 Wuhan and Omicron strain were optimized according to the preference of human codon usage and synthesized by Genewiz (Genewiz Biotech Co., Ltd., Suchow, China). The codon optimized spike genes were subcloned into the pJW4303 eukaryotic expression vector (Kindly gifted by Dr. shan Lu’s laboratory at the University of Massachusetts). The sequence of insertion was confirmed by Sanger sequencing (Sangon Biotech Co., Ltd., Shanghai, China). The recombinant plasmids for mouse vaccination were prepared using an EndoFree Plasmid Purification Kit (Cat# 12391, Qiagen, Hilden, USA).

Female C57BL/6J mice, 6-8-week-old, were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed in the SPF animal facility of Shanghai Public Health Clinical Center. The schedule of vaccination is shown in Figure 1A . Briefly, 20μg of the S_Wuhan DNA vaccine was injected intramuscularly into each mouse at Week 0 and Week 2. Subsequently, the mice were boosted with 50μg of either the S_Omicron or the S_Wuhan DNA vaccine at Week 6. The regimen is designed based on our previous findings, which show that increasing DNA dosage for the 3^rd vaccination can ensure the efficiency of in vivo antigen expression (30). The control group was boosted with PBS. Four weeks after the final vaccination, the mice were euthanized. Peripheral blood and splenocytes were collected for assays of S protein specific immune responses.

An in-house enzyme-linked immunosorbent assays (ELISA) were developed to measure RBD of Wuhan or Omicron strain of SARS-CoV-2 specific binding antibodies. High-binding 96-well EIA plates (Cat# 9018, Corning, USA) were coated with purified SARS-CoV-2 RBD protein of Wuhan strain (Cat# 40592-V08B, Sino Biological, China) or RBD protein of Omicron strain (Cat# 40592-V08H122, Sino Biological, China) at a final concentration of 1µg/ml in carbonate/bi-carbonate coating buffer (30mM NaHCO₃,10mM Na₂CO₃, pH 9.6). After coating overnight at 4°C, the plates were blocked with 1×PBS containing 5% milk for 1 hour at 37°C. Subsequently, 100μl of serial dilutions of mouse serum was added to each well. After 1 h incubation at 37°C, the plates were washed with 1×PBS containing 0.05% Tween 20 for 5 times. Then, 100μl of an HRP labeled goat anti-mouse IgG antibody (Cat# 115-035-003, Jackson Immuno Research, USA) diluted in 1×PBS containing 5% milk were added to each well and incubated for 1 hour at 37°C. After a second round of wash, 100μl of TMB substrate reagent (Cat# MG882, MESGEN, China) was added to each well. 6 minutes later, the color development was stopped by adding 100μl of 1M H₂SO₄ to each well and the values of optical density at OD450nm and OD630nm were measured using 800 TS microplate reader (Cat# 800TS, Biotek, USA).

S specific T cell responses in splenocytes were detected by flowcytometry assay. Briefly, fresh splenocytes were prepared and stimulated with R10 containing synthesized peptides covering the full-length spike protein of Wuhan strain (152 peptides in total, 0.66 μg/ml per peptide) or peptides covering the fragments of spike protein containing mutations specific to Omicron variant (36 peptides in total, 0.66 μg/ml per peptide) in round-bottom 96-well plates. The detailed information about the synthesized peptides is shown in Supplementary Table 1 . 2 hours later, brefeldin A were added to each well at final concentrations of 1 μg/ml. After 12 hours, the cells were collected and stained sequentially with Live/Dead dye (Fixable Viability Stain 510, Cat# 564406, BD Pharmingen) for 15 min at room temperature, surface markers (PE/Cyanine7-labeled anti-mouse CD3, Cat# 100220, BioLegend; APC-labeled anti-mouse CD4, Cat# 100412, BioLegend; PE-labeled anti-mouse CD8, Cat# 100708, BioLegend) for 30min at 4 ˚C and intracellular markers (BV421-labeled anti-mouse IFN-γ, Cat# 505830, BioLegend; FITC-labeled anti-mouse IL-2, Cat# 503806, BioLegend; BV711-labeled anti-mouse TNF-α, Cat# 506349, BioLegend) for 30min at 4 °C. After washing, the stained cells were resuspended in 200 μl 1×PBS and analyzed using a BD LSRFortessa™ Flow Cytometer. The data were analyzed using the FlowJo 10 software (BD Biosciences, USA). The gating strategy was shown in Supplementary Figure 1 . The percentages for specific T-cell populations were defined as the percentages of cytokines producing CD4⁺ or CD8⁺ cells in all CD4⁺ or CD8⁺ T cells.

VSV-backboned SARS-CoV-2 pseudo-viruses were prepared according to a reported method (31). The neutralization assay was conducted by following the previously described procedure (31, 32). Briefly, 100μl of serially diluted mice sera were added into 96-well cell culture plates. Then, 50μl of pseudo-viruses with a titer of 13000 TCID₅₀/ml were added into each well and the plates were incubated at 37°C for 1 hour. Next, Vero cells were added into each well (2×10⁴ cells/well) and the plates were incubated at 37°C in a humidified incubator with 5% CO₂. 24 hours later, luminescence detection reagent (Bright-Glo™ Luciferase Assay System, Promega, USA) was added to each well following the manufacturer`s instruction. The luminescence was measured using a luminescence microplate reader (GloMax^® Navigator Microplate Luminometer, Promega, USA) within 5 minutes. The Reed-Muench method was used to calculate the virus neutralization titer. Antibody neutralization titers were presented as 50% maximal inhibitory concentration (IC₅₀).

All statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, Inc., La Jolla, CA, USA). Comparisons among three groups were conducted by the method of one-way ANOVA. P<0.05 was considered as statistically significant.

As afore mentioned, female C57BL/6J mice were immunized with 20μg of the S_Wuhan DNA vaccine at Week 0 and Week 2. Subsequently, the mice were boosted with 50μg of DNA vaccines expressing the S proteins of either the Omicron variant or the Wuhan strain ( Figure 1A ) at Week 6. Sera were collected at the 4^th week post the last vaccination to measure RBD specific binding antibodies by a method of ELISA ( Figures 1B, C ). Our data showed that booster vaccination with S_Wuhan DNA vaccine significantly enhanced the binding antibody response to RBD protein of ancestral Wuhan strain compared with that of PBS group. A booster shot of the S_Omicron DNA vaccine significantly increased RBD binding antibody responses against both the ancestral Wuhan and the Omicron variant compared to that of the PBS control. Additionally, the mean titer of RBD binding antibodies against the Wuhan and the Omicron variant tended to be higher in the group boosted with the S_Omicron DNA vaccine than the group boosted with the S_Wuhan DNA vaccine, although the difference is not statistically significant.

Then the neutralizing antibody titers were assessed using a VSV-backboned SARS-CoV-2 pseudo-virus assay. Similar to the results of binding antibody assays, we found that a booster shot of the S_Omicron DNA vaccine elicited significantly stronger neutralizing antibody responses against the Omicron variant compared with booster doses of the S_Wuhan DNA vaccine and the PBS control ( Figure 1D ). To further investigate the impact of S_Omicron DNA vaccine against SARS-CoV-2 variants, neutralizing antibodies were measured for against Delta variant, which was an important variant of concern ( Figure 1E ). The results showed that a booster with the S_Omicron DNA vaccine significantly enhanced the neutralizing antibody responses against the Delta variant compared with the PBS control. In addition, the mean titer of neutralizing antibodies against the Delta variant also tended to be higher in the group boosted with the S_Omicron DNA vaccine than the group boosted with the S_Wuhan DNA vaccine, although the difference is not statistically significant.

At the 4^th week post the booster vaccinations, mouse splenocytes were freshly isolated and stimulated with peptides covering the full-length spike protein of Wuhan strain or the fragments of Omicron spike protein containing mutations. Specific T cell responses were assessed by intracellular cytokine staining assays ( Figure 2 and Figure 3 ). Our data showed that a booster vaccination of the S_Wuhan and the S_Omicron DNA vaccine failed to improve IL-2⁺, TNF-α⁺ or IFN-γ⁺ CD4⁺ T cell responses against the spikes protein of both the Wuhan and the Omicron variant as compared to those of the PBS control ( Figure 2 ). Multifunction analyses showed that the booster vaccination of the S_Omicron DNA vaccine significantly enhanced IL-2⁺TNF-α⁺IFN-γ⁺ polyfunctional CD4⁺ T cell responses against the spike protein of Wuhan strain ( Figure 4A ), but it failed to improve the polyfunctional CD4⁺ T cell responses against the Omicron variant ( Figure 4B ).

In comparison with CD4⁺ T cell responses, a booster with the S_Wuhan DNA vaccine significantly augmented the IFN-γ⁺ CD8⁺ T cell responses against the spike protein of Wuhan strain as compared to the PBS control ( Figure 3A ). While, a booster with the S_Omicron DNA vaccine significantly enhanced IFN-γ⁺CD8⁺ ( Figures 3A, D ) and TNF-α⁺CD8⁺ T responses ( Figures 3B, E ) against both the Wuhan and the Omicron variant. The IL-2⁺CD8⁺ T cell responses against the Wuhan and the Omicron variant were comparable among all three groups ( Figures 3C, F ). The polyfunctional (IL-2^-TNF-α⁺IFN-γ⁺) CD8⁺ T cell responses against the spike protein of both the Wuhan and the Omicron variant were significantly improved in mice boosted with the S_Omicron DNA vaccine as compared to those boosted with PBS control ( Figures 4C, D ). However, no significant difference was observed between the polyfunctional CD8⁺ T cell responses of mice boosted with the S_Omicron and the S_Wuhan DNA vaccines.