Recombinant Bacillus subtilis spores expressing SARS-CoV-2 spike protein induced humoral, mucosal, and cellular immunity in mice

Introduction

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel member of the Coronaviridae family, has led to a global health emergency unparalleled in modern history¹. Since its first identification in December 2019, SARS-CoV-2 has caused the COVID-19 pandemic, resulting in over 777 million confirmed cases and more than 7 million deaths globally as of March 2025². In response to the severity of the pandemic, multiple vaccines, based on various platforms utilizing the spike proteins, were developed and deployed by the end of 2020. These first-generation vaccines were 70–95% effective in preventing symptomatic infection, severe disease, and hospitalizations^3,4,5,6.

Nevertheless, these vaccines have fallen short with several drawbacks. Despite their effectiveness in generating systemic immunity, the previously approved vaccines may not be as effective in inducing strong mucosal immunity, which is critical for preventing viral transmission at the entry sites⁷. Some studies reported that the vaccine efficacy against SARS-CoV-2 transmission ranged between 23 and 57% against the ancestral virus^8,9. The high transmission rate of the virus led to the rapid emergence of various variants of concern (VOCs), currently dominated by the Omicron variants (B.1.529) and subvariants such as BA.2, BA.5, XBB, JN.1, KP.2 and KP.3^10,11,12. This further compromised the efficacy of the previously approved vaccines¹¹. Many studies have also reported the rapid waning of vaccine-induced immunity, indicated by the loss of 20–50% humoral immunity 4 months after vaccination^13,14,15. These highlight the pressing need for a vaccination strategy that could elicit broad systemic and mucosal immune responses and the importance of administering booster doses to maintain and enhance protection^16,17.

Mucosal vaccination has emerged as a more promising approach to tackle the challenges of VOCs and the limited immune responses in the first-generation vaccines. Mucosal immunity, characterized by the production of secretory IgA (sIgA) antibodies and the activation of local immune cells such as the resident memory T (T_RM) cells and resident memory B cells, represents the first line of defense against respiratorypathogens^{18,19,20,21,22}. Following the natural infection, sIgA antibodies were highly stimulated in the mucosal secretions, including the bronchoalveolar lavage fluid (BALF) and saliva, and the sIgA was reported to be more neutralizing than the circulating IgG^19,23. sIgA antibodies exhibit broad cross-reactivity, as demonstrated by their ability to confer cross-protection against influenza infections of antigenically distinct lineages²⁴. This is relevant to SARS-CoV-2, which has acquired many mutations resulting in different variants and subvariants. Infection against the wild-type SARS-CoV-2 was also reported to confer sIgA neutralizing activity against the Omicron (B.1.1.529) variants, suggesting the potential of mucosal immunization to protect against infection with the VOCs^25,26. Since the infection of SARS-CoV-2 occurs through the respiratory route, mucosal delivery of vaccines, such as through oral, intranasal, or sublingual routes, is expected to stimulate both broadly protective mucosal and systemic immune responses against the wild-type and VOCs.

B. subtilis spore has attracted interest in vaccine research owing to its inherent stability, extreme resistance to harsh conditions, metabolically inactive, regarded as safe, and with natural adjuvant properties^27,28,29. These spores are resistant to gastrointestinal degradation and, hence, would be able to effectively deliver antigens of interest to the gut-associated lymphoid tissues (GALT), where they could stimulate potent mucosal and systemic immune responses³⁰. The immunostimulatory properties of B. subtilis spores are primarily driven by their interaction with innate immune receptors, mainly through the MyD88-dependent signaling pathway^31,32,33. This pathway is a central adaptor for most Toll-like receptors (TLRs) and facilitates immune recognition via receptors such as TLR2 and TLR4^31,32,33. The self-adjuvant properties of B. subtilis spores would eliminate the need for additional external adjuvants, primarily required in the recombinant protein vaccine platform, to mount sufficient immune responses^34,35. We had previously developed the B. subtilis spore-based vaccine platform and demonstrated the immunogenicity and protection against infection with the multidrug-resistant Acinetobacter baumanii^30,36. In the present study, we report the immunogenicity of the recombinant B. subtilis spore expressing the Wuhan-Hu-1 with D614G mutation SARS-CoV-2 spike proteins.

Results

Construction and expression of recombinant B. subtilis

The plasmid pHPS9-Prom-S1.1-Term, consisting of the SARS-CoV-2 spike protein fragment (S1.1) under the control of the cry1Aa promoter and cry1Ac terminator, was successfully constructed (Fig. 1A). DNA sequencing was performed to verify the integrity of the transformed plasmid, confirming the correct sequence of the inserted fragment (data not shown). A polypeptide band of around ~ 100 kDa, corresponding to the predicted size of the recombinant SARS-CoV-2 spike protein, was identified (Fig. 1B, Lane 3). In contrast, no band of similar size was detected in the protein lysate from the vector pHPS9. Flow cytometry analysis demonstrated that over 25% of the spores consisted of recombinant spores expressing the SARS-CoV-2 spike protein (Fig. 1C).

Results from IEM labeling further demonstrated the presence of 10 nm gold particles. These particles were specifically localized on the spore coat structures, corresponding to the binding of the SARS-CoV-2 spike-neutralizing antibody. The gold particles were distinctly observed on the surface of the spores, affirming the successful expression and localization of the SARS-CoV-2 spike proteins. This precise labeling pattern suggests that the SARS-CoV-2 spike protein was effectively expressed on the spore coat of the recombinant spores (Fig. 2).

Oral immunization of mice with the recombinant B. subtilis spores expressing SARS-CoV-2 spike protein

In the present study, mice were divided into several groups to evaluate the antibody responses following immunization with recombinant and non-recombinant B. subtilis spores. The mice were immunized with diluent as control group (group 1), low-dose non-recombinant B. subtilis spores (5 × 10⁸ CFU/ml) (group 2), medium-dose non-recombinant B. subtilis spores (1 × 10⁹ CFU/ml) (group 3), high-dose non-recombinant B. subtilis spores (5 × 10¹⁰ CFU/ml) (group 4), low-dose recombinant B. subtilis spores expressing SARS-CoV-2 spike proteins (5 × 10⁸ CFU/ml) (group 5), medium-dose recombinant B. subtilis spores expressing SARS-CoV-2 spike proteins (1 × 10⁹ CFU/ml) (group 6), and high-dose recombinant B. subtilis spores expressing SARS-CoV-2 spike proteins (5 × 10¹⁰ CFU/ml) (group 7).

At pre-immunization, the IgM levels of the different mice groups ranged at baseline levels between 0.617 ng/ml ± 0.617 to 16.416 ng/ml ± 9.87 (Fig. 3A). By day 16, mice of group 6 exhibited IgM levels of 21.655 ± 11.931 ng/ml, higher than the mice of group 5 (15.245 ± 5.318 ng/ml) and group 7 (9.783 ± 2.485 ng/ml). At day 32, two weeks post-second dose, the IgM levels in the mice of group 6 increased significantly to 46.015 ± 2.391 ng/ml (p = 0.001). Similarly, significant increases were observed in mice of group 7 (33.001 ± 6.252 ng/ml, p = 0.0421) and group 5 (37.303 ± 15.143 ng/ml, p = 0.0138). In contrast, mice immunized with the non-recombinant spores showed elevated IgM levels compared to controls, but these increases were not statistically significant (mice of group 2: 15.247 ± 9.521 ng/ml, group 3: 26.529 ± 12.546 ng/ml, and group 4: 14.911 ± 5.247 ng/ml; p > 0.05). By day 55, three weeks post-third dose, IgM levels remained elevated in most groups (Fig. 3A). The mice of group 6 maintained the highest IgM levels (37.089 ± 9.276 ng/ml, p = 0.0205), while the mice of group 7 showed elevated but non-significant levels (31.487 ± 11.956 ng/ml, p > 0.05). In contrast, the mice of group 5 experienced a decline in IgM levels to 15.239 ± 8.417 ng/ml. IgM levels in mice immunized with the non-recombinant B. subtilis spores remained detectable throughout the study period; however, no significant differences were observed compared to controls (mice of group 2: 3.807 ng/ml ± 2.479, group 3: 13.231 ng/ml ± 6.875, and group 4: 23.197 ng/ml ± 13.356; p > 0.05).

The baseline pre-immunization IgG levels in mice across all groups ranged between − 2.018 ± 0.876 and 1.433 ng/ml ± 1.346 (Fig. 3B). Two weeks after the first dose, mice immunized with the recombinant spores exhibited an increase in anti-SARS-CoV-2 spike IgG levels compared to controls. The mice of group 6 demonstrated the highest IgG levels (3.326 ± 1.391 ng/ml), followed by the mice of group 7 (2.475 ± 1.215 ng/ml) and group 5 (2.138 ± 1.251 ng/ml). However, these differences were not statistically significant (p > 0.05). Mice immunized with the non-recombinant spores (groups 2, 3, and 4) showed IgG levels of 0.861 ng/ml ± 0.931, 0.589 ng/ml ± 1.008, and 0.566 ng/ml ± 1.064, respectively. By day 32, a significant increase in IgG levels was observed, particularly in mice of group 7 (7.129 ± 2.445 ng/ml, p = 0.0011). Similarly, mice of group 6 (6.042 ± 2.503 ng/ml, p = 0.009) and group 5 (5.296 ± 1.739 ng/ml, p = 0.0313) also demonstrated significant increases in IgG levels. The mice immunized with the non-recombinant spores also showed an increase in IgG levels; however, these differences remained statistically non-significant across all doses (0.847 ng/ml ± 1.523 for the mice in group 2, 0.625 ng/ml ± 1.452 for the mice in group 3, and 1.216 ng/ml ± 1.08 for the mice in group 4; p > 0.05). At day 55, IgG levels remained elevated in mice immunized with the recombinant spores. Mice in group 7 exhibited the highest levels (7.905 ± 1.279 ng/ml, p = 0.0001), while the mice in groups 6 and 5 showed elevated levels, however, these were not statistically significant compared to mice in the control group (p > 0.05). The IgG levels for the mice immunized with the non-recombinant spores remained low (mice in group 2 = − 0.046 ng/ml ± 1.093, group 3 = 0.122 ng/ml ± 1.515, and group 4 = 0.399 ng/ml ± 1.467). A dose-dependent effect was observed in the serum IgG level post-second and third doses of the recombinant spores. The mice immunized with high-dose recombinant spores exhibited significantly higher serum IgG levels than the mice immunized with medium- and low-dose recombinant spores (p = 0.0196 and p = 0.0019, respectively).

Overall, serum anti-SARS-CoV-2 spike IgM levels (Fig. 3A) and IgG levels (Fig. 3B) increased significantly following the second and third immunizations compared to baseline and control groups, with the most potent responses observed at medium and higher doses, or at 1 × 10⁹ CFU/ml and 5 × 10¹⁰ CFU/ml spores, respectively.

To assess the Th1/Th2 balance, serum IgG1 (indicative of Th2) and IgG2a (indicative of Th1) levels were measured in mice after immunization with three doses of the bacterial spores (Fig. 3C). The mice of group 7 exhibited an increase in IgG1 levels (OD 0.326 ± 0.107) compared to the mice of group 1 (OD 0.203 ± 0.022). The mice of groups 6 and 5 showed IgG1 levels of 0.184 ± 0.027 and 0.204 ± 0.039, respectively. Mice immunized with non-recombinant spores had IgG1 levels ranging from OD of 0.185 ± 0.027 to 0.208 ± 0.014.

IgG2a levels were higher in the mice of group 7 (OD 0.282 ± 0.081) compared to the mice of group 1 (OD 0.226 ± 0.023). Similarly, the mice of groups 5 and 6 exhibited high IgG2a levels (OD 0.269 ± 0.029 and 0.24 ± 0.033, respectively). The mice immunized with the non-recombinant spores exhibited low levels of IgG2a, ranging from 0.186 ± 0.014 to 0.223 ± 0.014.

The IgG2a/IgG1 ratio was highest in the mice of group 5 (1.514), followed by group 6 (1.201) and 7 (1.17). In comparison, the diluent control and the non-recombinant spore groups showed lower ratios, ranging from 0.9914 to 1.166. These data suggest that recombinant spores promoted a clearer skewing towards a Th1-biased humoral response relative to controls, although the overall magnitude of polarization remained moderate (Fig. 3D).

The neutralizing activity against SARS-CoV-2 in the sera obtained from immunized mice on Day 55 was measured using the sVNT assay. The assay detected the presence of neutralizing/blocking antibodies in the serum. The neutralizing/blocking antibodies bound to the HRP-conjugated RBD and blocked the binding to the pre-coated ACE2 receptor. The mean percentage inhibition for the control group mice was 20.53% ± 2.749 (Fig. 3E). Mice immunized with the non-recombinant spores at doses of 5 × 10⁸ CFU/ml, 1 × 10⁹ CFU/ml, and 5 × 10¹⁰ CFU/ml exhibited mean percentage inhibition values of 21.93% ± 3.137, 22.77% ± 5.097, and 22.66% ± 2.81, respectively. The mice of group 5 showed a mean of 29.87% ± 6.46 (p = 0.0414), while the mice of group 6 had a mean of 28.73% ± 5.154. Group 7 mice demonstrated the highest mean inhibition at 32.14% ± 10.83 (p = 0.0075), indicating significant neutralizing activity of the sera. Mice immunized with the recombinant spores, particularly at the highest dose, exhibited inhibition levels exceeding 30%, demonstrating substantial neutralizing activity against SARS-CoV-2 (Fig. 3E). A preliminary live-virus neutralization assay was conducted to assess the functional activity of antibodies elicited by the recombinant spores. Sera from mice receiving the highest dose of recombinant spores achieved 50% neutralization at a 1:100 dilution (PRNT₅₀ ≈ 100), confirming detectable neutralizing activity (Supplementary Fig. 2).

Fecal-specific anti-SARS-CoV-2 spike secretory IgA (sIgA) levels were determined at pre-immunization, day 16, day 32, and day 55 in mice receiving different doses of recombinant and non-recombinant B. subtilis spores (Fig. 4A). The sIgA levels at pre-immunization ranged from 0.006 ± 0.003 ng/ml to 0.035 ± 0.015 ng/ml across all groups. By day 16, the sIgA level in the mice of group 5 was at 0.052 ng/ml ± 0.008, the mice of group 6 demonstrated a sIgA level of 0.049 ng/ml ± 0.025, while the mice of group 7 showed a sIgA level of 0.043 ng/ml ± 0.016. These sIgA levels were elevated compared to the mice of the control group (0.007 ng/ml ± 0.004). The sIgA levels in the mice immunized with the non-recombinant spores were 0.011 ng/ml ± 0.006 in mice of group 2, 0.011 ng/ml ± 0.008 in mice of group 3, and 0.008 ng/ml ± 0.004 in mice of group 4. At day 32, the sIgA levels significantly increased in the mice immunized with the recombinant spores, with the high-dose immunization exhibiting the highest response (0.151 ± 0.036 ng/ml, p = 0.0006), followed by the medium-dose immunization (0.115 ng/ml ± 0.045, p = 0.035). The sIgA level in mice immunized with low-dose recombinant spores increased but did not reach statistical significance (0.093 ng/ml ± 0.044, p > 0.05). At day 55, sIgA levels remained elevated, with the mice of group 6 showing the highest level (0.108 ± 0.047 ng/ml), followed by the mice of groups 5 (0.083 ng/ml ± 0.043) and 7 (0.094 ng/ml ± 0.053), although a slight decline was observed compared to day 32 (Fig. 4A). Mice immunized with the non-recombinant spores showed low sIgA levels throughout, with 0.015 ng/ml ± 0.009 in mice of group 2, 0.013 ng/ml ± 0.009 in mice of group 3, and 0.047 ng/ml ± 0.014 in mice of group 4.

At baseline, saliva-specific sIgA levels ranged from OD 0 to 0.052 ± 0.01 (Fig. 4B). At day 16, the mice of group 6 showed significantly high saliva-specific sIgA levels (0.082 ± 0.018) (p < 0.0001), followed by the mice of group 5 (0.059 ± 0.014, p = 0.0046). In contrast, the mice of group 7 showed moderate sIgA levels (0.03 ± 0.011). Comparison against the group immunized with the non-recombinant spores (groups 2 and 3) of similar doses showed that the mice immunized with low- and medium-doses of recombinant spores exhibited significantly higher sIgA levels, p = 0.0046 and p < 0.0001, respectively. A comparison of the different dosages of the recombinant spores revealed that mice immunized with medium-dose showed a significantly high sIgA level compared to the high-dose immunization (p = 0.0168). By day 32, the mice of group 6 exhibited the highest sIgA level (0.095 ± 0.021, p < 0.0001), followed by the mice of groups 5 (0.063 ± 0.013, p = 0.0017) and 7 (0.054 ± 0.008, p = 0.0137). Medium-dose immunization with recombinant spores resulted in a significantly higher sIgA level than medium-dose immunization with non-recombinant spores (p < 0.0001). By day 55, sIgA levels in the mice of groups 6 and 5 were 0.117 ± 0.01 and 0.116 ± 0.015, respectively, significantly higher than in control groups (0, p < 0.0001) and the mice of groups 3 and 2 (0.013 ± 0.008, and 0, respectively, p < 0.0001). The high-dose immunized group showed a decline in sIgA level at 0.027 ± 0.026.

The sIgA level in BALF was analyzed on day 55 to assess mucosal immune responses in the respiratory tract. The mice of the control group (0.015 ± 0.007 ng/ml) and the mice immunized with the non-recombinant spores exhibited low sIgA levels (Group 2 and 3 = 0 ng/ml, Group 4 = 0.003 ± 0.003 ng/ml) (Fig. 4C). In contrast, mice immunized with the recombinant spores demonstrated high responses at 0.101 ± 0.035 ng/ml, p = 0.0125, 0.074 ± 0.024 ng/ml, p = 0.0104, and 0.028 ± 0.009 ng/ml for groups 7, 6, and 5, respectively.

The sIgA levels in intestinal wash samples, reflecting gut mucosal immunity, were higher in the mice of group 7 (0.296 ± 0.086 ng/ml), significantly surpassing the mice of groups 6 and 5 (p = 0.0005 and p = 0.0042, respectively) (Fig. 4D). Groups of mice immunized with the non-recombinant spores demonstrated undetectable or lower sIgA levels (mice of group 2 = 0 ng/ml, group 3 = 0.045 ± 0.02 ng/ml, and group 4 = 0.015 ± 0.015 ng/ml).

Cellular immune responses in mice immunized with the recombinant B. subtilis spores expressing SARS-CoV-2 spike protein

The cellular immune responses were analyzed in the mice immunized with diluents (group 1, control), medium-dose immunization with the non-recombinant spores, 1 × 10⁹ CFU/ml (group 3), and the mice immunized with low- (5 × 10⁸ CFU/ml), medium- (1 × 10⁹ CFU/ml) and high- (5 × 10¹⁰ CFU/ml) doses of the recombinant spores (groups 5, 6 and 7) using flow cytometry. The heatmap of the cellular immune responses revealed an elevated CD4⁺ population in the mice of groups 6 and 7, with median percentages of 49.45% and 43%, respectively. The mice of group 5 showed a lower CD4⁺ population (35.5%). The CD4⁺ population in the mice of the control group was 37.6%. The mice of group 3 exhibited a CD4⁺ population of 41.9%. The effector T cells (CD4⁺ CD44⁺) population was higher in the mice of groups 5 (53.6%) and 6 (41.3%), while mice of group 7 showed a reduction to 27.65%. The mice of the control group and mice of group 3 exhibited a CD4⁺CD44⁺ population of 32.55% and 28.6%, respectively (Fig. 5A). The median percentage of CD8a⁺ T cells was higher in the mice of group 6 (7%) compared to the mice of groups 7 (4.8%) and 5 (3.3%), with the control group showing the lowest level (1.8%). The CD8a⁺ T cell population in mice of group 3 (5.6%) was also elevated. Effector cytotoxic T cells (CD8a⁺CD44⁺) population were higher in the mice of group 6 (42.35%) compared to the mice of the control group (39.55%). The mice of groups 5 and 7 exhibited a CD8a⁺CD44⁺ population of 27.3% and 28%, respectively. The mice of group 3 exhibited a reduction of the CD8a⁺CD44⁺ population (34.55%). Overall, the mice of groups 6 and 7 showed elevated CD4⁺ and CD8a⁺ T cell populations, with the mice of group 6 also exhibiting higher effector T cells (CD4⁺CD44⁺) and cytotoxic T cells (CD8a⁺CD44⁺). The mice of group 5 had a lower CD4⁺ population but increased effector T cells, while mice of group 3 and the control group displayed moderate T cell levels. These findings suggest that medium-dose immunization with recombinant spores at 1 × 10⁹ CFU/ml resulted in enhanced immune activation.

The total B cell population (CD19⁺) was high in the mice of group 5 (38.5%) compared to the mice of the control group (30.8%). The activated B cell population (CD19⁺ CD44⁺) was lower at 55.7% in the mice of group 5 versus 84.55% in the mice of the control group (Fig. 5A). In contrast, mice of groups 6 and 7 showed a low B cell population (17.65% and 23.9%, respectively) but a higher activated B cell population, with median percentages of 90% and 89.7%, respectively. The mice of group 3 exhibited a lower B cell population (25%) and a reduced level of activated B cell population (66.45%). Overall, mice of group 5 exhibited a higher total B cell population, but a lower proportion of activated B cells compared to the control group. In contrast, mice of groups 6 and 7 showed reduced total B cell populations but substantially higher levels of activated B cells, suggesting enhanced B cell activation in these groups, thus indicating that the medium- and high-dose immunization with the recombinant spores at 1 × 10⁹ CFU/ml and 5 × 10¹⁰ CFU/ml may promote B cell activation despite lower overall B cell numbers.

Macrophage (CD11b⁺) populations were higher in the mice of group 5 (3.1%) compared to the mice of the control group (2.65%), whereas mice of groups 6 and 7 showed lower macrophage levels (1.85% and 1.6%, respectively) (Fig. 5A). However, the activated macrophage population (CD11b⁺ CD44⁺) was elevated in mice immunized with the recombinant spore groups, with high-dose immunization showing the highest percentage (85.4%), followed by low-dose (82.1%) and medium-dose (77.45%) immunization, compared to the mice of the control group (70.2%). The mice of group 3 had a lower activated macrophage population (58.9%). Overall, recombinant spore immunization enhanced macrophage activation, particularly at higher doses (5 × 10¹⁰ CFU/ml), despite varying total macrophage levels across groups.

Spike-specific T-cell responses were further evaluated by restimulating splenocytes with SARS-CoV-2 S1 peptide pools. Low-dose immunization with recombinant spores resulted in a mean CD4⁺ IFN-γ⁺ T cell frequency of 0.28% ± 0.17, while medium- and high-dose immunizations yielded mean frequencies of 0.17% ± 0.12 and 0.32% ± 0.09, respectively. In contrast, the control group exhibited a lower mean frequency of 0.02% ± 0.02 (Fig. 5B). However, these differences were not statistically significant. Similarly, CD8a⁺ IFN-γ⁺ T cell frequencies were significantly increased in mice from groups 5 (1.68% ± 0.62, p = 0.011) and 7 (1.1% ± 0.19, p = 0.0153) compared to the control group (0%) (Fig. 5C). Mice in group 6 also showed an increase (1.03% ± 0.49), though this did not reach statistical significance (p > 0.05). Oral immunization with recombinant B. subtilis spores expressing SARS-CoV-2 spike proteins induced cellular immune responses in the SPF Balb/c mice. CD8⁺ IFN-γ⁺ T cell frequencies were significantly increased in the low- and high-dose immunization groups, while CD4⁺ IFN-γ⁺ T cell responses were detected across all immunized groups but did not reach statistical significance (p > 0.05).

Cytokine responses in mice immunized with the recombinant B. subtilis spores expressing SARS-CoV-2 spike protein

Key pro-inflammatory cytokines, including IFN-γ, IL-1α, IL-1β, IL-6, IL-12p70, IL-2, IL-17A, and TNF-α, were measured to evaluate the cytokine responses in mice immunized with the recombinant B. subtilis spores (Fig. 6A). The mice immunized with the recombinant spores exhibited elevated IFN-γ levels, with median concentrations of 2.226 pg/ml and 2.572 pg/ml in low- and high-dose immunization, respectively, compared to the mice of the control group and group 3 (undetected level). The medium-dose immunization showed a higher IFN-γ level of 12.822 pg/ml. IL-1α levels were low in the control group (0.211 pg/ml) but increased in mice immunized with the recombinant spores: group 5 (2.669 pg/ml), group 6 (1.09 pg/ml), and group 7 (1.389 pg/ml). IL-1α was undetectable in the mice immunized with the non-recombinant spores. Similarly, IL-1β levels were elevated in recombinant spore-immunized mice: group 5 (1.779 pg/ml), group 6 (1.523 pg/ml), and group 7 (1.97 pg/ml), compared to the control group (− 0.126 pg/ml) and group 3 (− 2.897 pg/ml). IL-6 levels also increased in recombinant spore-immunized mice: group 5 (18.765 pg/ml), group 6 (0.736 pg/ml), and group 7 (1.99 pg/ml), whereas IL-6 was undetectable in the control group. For IL-12p70, the control group exhibited a level of 3.347 pg/ml, while low- and high-dose recombinant spore immunization resulted in elevated levels of 12.962 pg/ml and 10.343 pg/ml, respectively. IL-12p70 was undetectable in the mice of groups 6 and 3. IL-2 levels were highest in the mice of group 7 (15.693 pg/ml) compared to the control group (0.782 pg/ml). IL-2 remained undetectable in the mice of groups 3, 5, and 6. IL-17A levels were higher in the mice of groups 7 (1.473 pg/ml) and 5 (0.869 pg/ml) but undetectable in the mice of groups 5 and 3. The mice of group 3 showed a higher TNF-α level (47.305 pg/ml), while recombinant spores immunized mice exhibited variable TNF-α levels: 10.656 pg/ml (group 5), − 1.212 pg/ml (group 6), and 4.068 pg/ml (group 7). These findings suggested that immunization with the recombinant spores expressing SARS-CoV-2 spike proteins stimulated pro-inflammatory cytokine responses, with low- and high-dose immunization exhibiting higher response levels.

The anti-inflammatory cytokines were also assessed by determining the levels of IL-10 and IL-13 (Fig. 6A). The heatmap showed that immunization with the low-dose recombinant spores induced the highest IL-10 level (34.731 pg/ml), followed by medium-dose (10.376 pg/ml) and high-dose (10.638 pg/ml), compared to the control group (3.144 pg/ml). Non-recombinant spores immunization resulted in low IL-10 level (0.617 pg/ml). The level of IL-13 was undetectable across all samples. The findings suggested that the recombinant spores expressing SARS-CoV-2 spike proteins immunization, especially at low-dose, stimulated high anti-inflammatory cytokine responses.

Other cytokines such IL-12p40, IL-3, IL-4, IL-5 and IL-9 were also evaluated (Fig. 6A). The heatmap revealed that IL-12p40 concentrations were higher in the mice immunized with the recombinant spores (group 5, 24.449 pg/ml, and group 7, 22.807 pg/ml) compared to controls (8.978 pg/ml). The IL-3 levels were high in the mice of groups 6 (0.162 pg/ml) and 7 (0.172 pg/ml). The mice of group 5 exhibited an increase in the IL-4 level (0.743 pg/ml), followed by the mice of groups 7 (0.643 pg/ml) and 6 (0.115 pg/ml). Recombinant spores immunization increased IL-5 production in the mice of groups 6 (0.528 pg/ml) and 7 (0.558 pg/ml), while the mice of group 5 showed suppression (− 0.522 pg/ml). Low-dose immunization with recombinant spores resulted in elevated IL-9 levels (1.157 pg/ml), while medium- and high-doses immunization resulted in IL-9 levels of 0.649 pg/ml and 0.84 pg/ml, respectively. These findings suggested that the recombinant spore immunization modulated cytokine responses in a dose-dependent manner, with low-dose immunization particularly enhancing IL-12p40, IL-9 and IL-4 levels, while medium-dose immunization stimulated higher IL-3 and IL-5 production.

Chemokines such as Eotaxin, MCP-1, MIP-1α, MIP-1β, RANTES, and KC were also determined using the multiplex assay (Fig. 6B). Elevated eotaxin levels were detected in the recombinant spores immunized groups, with mice of group 5 exhibiting the highest increase (41.86 pg/ml), followed by the mice of group 7 (39.95 pg/ml), compared to the control group (10.34 pg/ml). The immunization with non-recombinant spores also showed an elevation (16.88 pg/ml). Recombinant spores immunization showed an increase in the MCP-1 level, with the highest observed in the high-dose immunized mice (57.53 pg/ml), compared to the control group (− 5.84 pg/ml). The immunization with non-recombinant spores resulted in a strong suppression (− 22.33 pg/ml). High expressions of MIP-1α and MIP-1β were observed in mice immunized with the recombinant spores, with low-dose immunization showing the highest level (513.173 pg/ml and 540.224 pg/ml, respectively). High-dose recombinant spores immunization caused the highest increase in RANTES expression (459.10 pg/ml), followed by low-dose immunization (354.30 pg/ml), compared to the control group (− 1.185 pg/ml). The expression of KC was also increased in the mice of groups 5 (14.802 pg/ml) and 6 (7.908 pg/ml), compared to the control group (− 0.895 pg/ml). Growth factors, including G-CSF and GM-CSF, were increased, particularly in the mice of groups 5 (G-CSF levels, 5.31 pg/ml, GM-CSF, 30.42 pg/ml) and 7 (G-CSF, 1.37 pg/ml, GM-CSF, 17.63 pg/ml) (Fig. 6B). These findings suggested that oral immunization with recombinant spores expressing SARS-CoV-2 spike proteins stimulated chemokines and growth factors.

To assess the Th1/Th2 immune responses, the level of key cytokines associated with cell-mediated (Th1; IFN-γ, TNF-α, IL-2, and IL-12p70) (Fig. 6C–F) and humoral-mediated (Th2; IL-4, IL-5, IL-6 and IL-10) (Fig. 6G–J) immunity were measured. The cytokine levels were measured in the supernatant of SARS-CoV-2 spike protein peptide pool-restimulated immunized mice splenocytes.

The mice of group 1 and group 3 showed lower IFN-γ levels of − 0.165 pg/ml and − 0.505 pg/ml, respectively. The immunization with recombinant spores resulted in an increase in the IFN-γ levels with the highest in the low-dose immunization (4.929 pg/ml). Medium and high-dose immunization resulted in IFN-γ levels of 0.492 pg/ml and 2.402 pg/ml, respectively (Fig. 6C). TNF-α levels were reduced in the mice of group 1, group 3, group 5, and group 6 (0.782 pg/ml, − 0.231 pg/ml, 0.815 pg/ml, and 0.312 pg/ml, respectively). The mice in group 7, which were immunized with a high dose of recombinant spores showed the highest TNF-α level (15.69 pg/ml) (Fig. 6D). IL-2 levels were highest in the mice of group 5 and group 7 (11.22 pg/ml and 2.959 pg/ml, respectively). The mice of group 1, group 3 and group 5 showed a reduction in the IL-2 levels (− 0.979 pg/ml, − 25.69 pg/ml, and − 1.212 pg/ml, respectively) (Fig. 6E). IL-12p70 levels were significantly highest in the mice of group 5 (12.96 pg/ml, p = 0.024) and group 7 (10.55 pg/ml, p = 0.035) compared to the mice of group 3 (− 9.78 pg/ml). The IL-12p70 levels in the mice of group 6, however, showed a reduction to − 3.415 pg/ml. (Fig. 6F). Overall, these data suggested that the recombinant spores expressing SARS-CoV-2 spike proteins stimulated Th1 cytokine production, particularly at low-dose immunization of 5 × 10⁸ CFU/ml spore.

IL-4 levels in the mice of groups 1 and 3 were determined at 0.09 pg/ml and 0.091 pg/ml, respectively. The immunization with the recombinant spores increased the IL-4 levels to 1.045 pg/ml, 0.442 pg/ml, and 0.643 pg/ml in groups 5, 6, and 7, respectively (Fig. 6G). Similarly, IL-5 levels were − 0.285 pg/ml in the mice of group 1, increasing to 0.204 pg/ml in the mice of group 3, and further rising to 1.331 pg/ml, 1.092 pg/ml, and 0.558 pg/ml in the mice of groups 5, 6 and 7 at the respective concentrations (Fig. 6H). For IL-6, the mice of group 1 showed a baseline level of − 0.635 pg/ml, which increased to 0.204 pg/ml in the mice of group 3. An increase to 19.66 pg/ml was observed in the mice of group 5, while IL-6 levels in the mice of groups 6 and 7 were 0.97 pg/ml and 1.99 pg/ml, respectively (Fig. 6I). IL-10 levels were − 2.126 pg/ml in the mice of group 1 and decreased to − 10.82 pg/ml in the mice of group 3. Significant elevations were observed in the mice of group 5, with IL-10 levels reaching 39.2 pg/ml. Increased IL-10 levels were also observed in the mice of groups 6 and 7, with IL-10 levels of 10.38 pg/ml and 10.64 pg/ml, respectively, however, the value did not reach statistical significance (p > 0.05) (Fig. 6J). These results suggested that immunization with the recombinant spores expressing SARS-CoV-2 spike proteins, particularly at lower spore concentrations of 5 × 10⁸ CFU/ml, modulated the Th2-associated cytokine production compared to those obtained from mice treated with just the diluent or the non-recombinant spores.

In line with the observed Th1 versus Th2 bias in the IgG2a and IgG1 levels, the cytokine ratios derived from SARS-CoV-2 spike proteins peptides-stimulated splenocytes supernatant at day 55 revealed significant shifts in Th1/Th2 balance. The IFN-γ/IL-4 ratio, a key indicator of Th1 bias, was − 1.07 in the mice of group 1 and decreased further to − 2.06 in the mice of group 3. However, in mice immunized with the recombinant spores, this ratio shifted to positive values, reaching 2.3, 2.68, and 3.53 at low-, medium-, and high-immunization doses, respectively, with significant Th1-biased in mice immunized with high-dose recombinant spores (p = 0.0317) (Fig. 6K). Similarly, the IL-2/IL-4 ratio was 20.87 in the mice of group 1 but decreased to 0.67 in the mice of group 3. In contrast, the mice of groups 5, 6, and 7 exhibited higher ratios of 18.64, 32.69, and 17.01 at the respective doses, further supporting a shift toward Th1 immunity in group 7 (Fig. 6L). The IFN-γ/IL-10 ratio, which reflects the balance between Th1 and regulatory responses, was − 0.09 in the mice of group 1 and increased slightly to 0.07 in the mice of group 3. In the mice immunized with the recombinant spores, this ratio showed increases, with values of 0.43, 0.30, and 0.22 in groups 5, 6, and 7, respectively. While these changes were not as pronounced as the IFN-γ/IL-4 ratio, they still suggest a trend toward Th1 dominance (Fig. 6M). The IL-2/IL-10 ratios were − 2.36 in the mice of group 1 and − 0.02 in the mice of group 3. In the mice immunized with the recombinant spores, this ratio further increased to 3.52, 3.73, and 1.05 in the mice of groups 5, 6, and 7, respectively, reinforcing the observed Th1 bias (Fig. 6N). Overall, these findings suggested that the immunization with recombinant spores expressing SARS-CoV-2 spike proteins stimulated Th-1-biased immune responses.

Th17-mediated immunity, crucial for mucosal defense, was evaluated by measuring IL-17A levels. The IL-17A levels in the mice of groups 1 and 3 were reduced to − 0.139 pg/ml and − 0.127 pg/ml, respectively. Increased IL-17A levels were observed in the mice immunized with the recombinant spores with 1.202 pg/ml, 0.264 pg/ml, and 1.473 pg/ml for low-, medium-, and high-dose immunizations, respectively (Fig. 6O). These data suggested that the high-dose immunization of 5 × 10¹⁰ CFU/ml of spores was most effective in inducing the Th17 responses, which are crucial for enhancing mucosal immunity.

Discussion

SARS-CoV-2 infections primarily occur through the mucosal tissues of the upper respiratory tract, highlighting the pivotal role of mucosal immunity as the primary barrier against viral entry³⁷. Secretory IgA (sIgA) antibodies, a key component of mucosal immunity, play a critical role in neutralizing viral pathogens at mucosal interfaces, including the respiratory and gastrointestinal tracts³⁸. Although current intramuscular SARS-CoV-2 vaccines have demonstrated significant efficacy in reducing severe disease and mortality, their capacity to elicit robust mucosal immune responses remains suboptimal^{7,39,40,41,42}. This limitation compromises their ability to prevent initial viral colonization and interrupt transmission chains. Additionally, the reactogenicity associated with intramuscular vaccination often results in mild to moderate adverse effects, such as localized inflammation and systemic symptoms^43,44. These observations highlight the necessity for novel immunization strategies that concurrently enhance systemic and mucosal immunity while minimizing reactogenicity, thereby improving overall vaccine efficacy and safety.

The present study explored Bacillus subtilis spores as a novel vaccine delivery platform. The spores were engineered to express the SARS-CoV-2 spike protein, leveraging their inherent stability, safety profile, and suitability for oral administration^27,28. Given the well-documented challenges posed by the acidic gastrointestinal environment to the efficacy of oral vaccines, this approach was designed to overcome the limitations of traditional injectable vaccines. By leveraging the robust nature of spores, the strategy aims to enhance both mucosal and systemic immune responses while ensuring the preservation of antigen integrity throughout the harsh gastrointestinal tract^45,46. In the present study, the expression of the SARS-CoV-2 spike protein on recombinant B. subtilis spores was confirmed using immunoblotting and flow cytometry, demonstrating successful surface display. Approximately 27% of the spore preparation displayed positive fluorescence, confirming successful spike protein expression. This aligns with earlier findings that reported recombinant protein display on bacterial spores in the range of 24–30%³⁰. Immunoelectron microscopy (IEM) further revealed that the expressed proteins were localized on the spore coat, supporting the assertion that B. subtilis spores can serve as a viable antigen presentation system without the need to attach the heterologous protein to the spore coat protein³⁰. The ability to display antigens on the spore surface is particularly advantageous for oral vaccines, as it facilitates direct interaction with antigen-presenting cells in the gut-associated lymphoid tissue (GALT), which is critical for initiating mucosal immune responses.

Results from this study demonstrated that oral immunization with recombinant B. subtilis spores induced systemic and mucosal immune responses. A significant increase in serum SARS-CoV-2 spike-specific IgM and IgG was observed after the second and third immunization doses. Although the absolute antibody concentrations and sVNT inhibition appeared modest, these responses were statistically significant and supported by live-virus neutralization assays, which confirmed functional protection (PRNT₅₀ ≈ 100 in the high-dose group). The discrepancy between assays likely reflects methodological differences, as sVNT measures only ACE2–RBD inhibition, whereas live-virus assays capture broader neutralizing mechanisms. The comparatively lower neutralization compared to conventional vaccines may be explained by limited spike incorporation into the spore coat⁴⁷ Nevertheless, spore-based vaccines are primarily intended to elicit mucosal and cellular immunity, which play critical roles in blocking infection at entry sites and providing cross-variant protection.

In this study, recombinant spores induced strong mucosal immune responses, with elevated spike-specific sIgA detected in feces, saliva, BALF, and intestinal washes. Such responses suggest effective priming of GALT and BALT, leading to localized protection at both the gastrointestinal and respiratory mucosa^7,23,48. Moreover, T cell responses, which are broadly conserved across SARS-CoV-2 variants^49,50,51, further support the potential for durable protection, even when neutralizing antibody levels are moderate.

Importantly, although IgM, IgG, and fecal sIgA levels declined after the third dose, the concentration remained significantly above baseline. This pattern is consistent with the well-described kinetics of vaccine-induced immunity, in which peak responses are followed by contraction to a stable plateau maintained by long-lived plasma cells⁵². In addition, prior studies have suggested that once anti-spore antibodies are generated, subsequent spores may be cleared more rapidly, potentially limiting the extent of further boosting⁵³. However, the persistence of elevated IgG and mucosal sIgA responses in our study, together with evidence of functional neutralization, suggests that protective immunity can still be maintained despite this effect. Moreover, previous work has shown that repeated exposures or very high oral doses do not necessarily amplify mucosal IgA responses and may instead lead to a plateau or tolerogenic effects^54,55. Overall, these findings extend prior reports on spore-based vaccines^31,56,57 and highlight their potential as a dual mucosal–systemic platform, in which durable mucosal protection can be maintained even as antibody levels naturally wane.

Future optimization strategies such as enhancing antigen display, incorporating mucosal adjuvants, or adopting heterologous prime–boost regimens may further strengthen systemic antibody responses while preserving efficacious mucosal immunity.

Aside from the humoral immunity, vaccine-induced cellular immune responses are also crucial to directly attack and kill virus-infected cells. In the present study, we demonstrated that oral immunization with recombinant B. subtilis spores expressing the SARS-CoV-2 spike protein elicited robust cellular immune responses with the elevation of CD4⁺ and CD8⁺ T cells, particularly in the medium- and high-dose immunization. Ex vivo restimulation of the splenocytes with SARS-CoV-2 spike protein peptide pools also revealed SARS-CoV-2 spike-specific CD4⁺ and CD8⁺ T cells recall responses in the mice immunized with the recombinant spores with significantly elevated CD8⁺ T cells producing IFN-γ, particularly in low- and high-dose immunization. These findings suggest a balanced helper and cytotoxic T-cell response critical for viral clearance and limiting disease severity^58,59,60. CD4⁺ T cells may be more involved in aiding immune memory and have been linked to the control of primary infection of SARS-CoV-2⁶⁰, while the functional CD8⁺ T cells are likely essential for viral clearance and reducing viral replication⁶¹. This could be particularly important in limiting disease severity and preventing the further spread of multiple SARS-CoV-2 variants. Prior studies have reported that T-cell responses are highly cross-reactive against emerging variants^{62,63,64,65,66,67}. This cross-reactivity persists even as neutralizing activities decline. T cell epitopes on spike proteins are more conserved across different variants and show high cross-recognition capacity⁴⁹. Furthermore, more than 90% of the native CD4⁺ and CD8⁺ T cell epitopes were conserved, which further supports the assertion that the cellular immune responses will likely remain uncompromised against emerging SARS-CoV-2 variants, even in the case of reduced neutralizing antibodies^49,50,51.

Multiplex analysis of the supernatant from splenocytes stimulated with SARS-CoV-2 S1 peptide pools revealed a well-coordinated immune response in mice immunized with the recombinant spores. The low-dose immunization with the recombinant spores, in particular, resulted in the elevation of pro-inflammatory cytokines such as IFN-γ, IL-1α, IL-1β, IL-6, IL-12p70, IL-17A, and TNF-α, as it has been reported in earlier studies^31,56. Interestingly, alongside the increase in pro-inflammatory cytokines, the anti-inflammatory cytokine IL-10 was also elevated, particularly in the low-dose immunization group. This concurrent rise in IL-10 suggests a regulatory mechanism that prevents excessive inflammation, hence reducing the risk of adverse immune responses^68,69,70. Such a balanced immune response potentially enhances the recombinant spore vaccine’s safety while maintaining its efficacy.

In this study, restimulation with S1 peptide pools induced elevated levels of key Th1 cytokines, such as IFN-γ, TNF-α, IL-2, and IL-12p70, particularly in the low- and high-dose immunized groups. While Th2 cytokines, including IL-4, IL-5, IL-6, and IL-10, were also detected, the overall immune response was strongly Th1-biased, especially in the high-dose immunized group. This Th1 bias was further supported by the increased IFN-γ/IL-4 ratio, a key indicator of Th1 dominance, which is critical for effective antiviral immunity against intracellular pathogens like SARS-CoV-2. Additionally, the IFN-γ/IL-10 and IL-2/IL-10 ratios provided insights into the balance between Th1 and regulatory responses. Although changes in these ratios were less pronounced, they indicated a trend toward Th1 dominance, particularly in the high-dose group. The increase in these ratios suggests a reduction in IL-10-mediated regulatory suppression of Th1 responses, potentially enhancing the vaccine’s efficacy.

The Th1 cellular responses are essential in the defense against intracellular pathogens such as viruses, while Th2 responses are most commonly associated with the protection against parasites and allergens^71,72,73. It has been reported that Th1 dominance is linked to protective T-cell-mediated immunity, while Th2 dominance can lead to pathogenic effects^74,75,76, including vaccine-associated enhanced respiratory disease (VAERD), a significant concern in vaccine development^77,78,79,80.

In this study, the IgG subclass profile further corroborated the Th1-biased immune response. The IgG2a/IgG1 ratio, a well-established marker for Th1/Th2 polarization, was higher in the recombinant spore immunized mice groups, particularly in high-dose immunization. This aligns with the elevated levels of Th1-associated cytokines (IFN-γ, TNF-α, IL-2, and IL-12p70) and the increased IFN-γ/IL-4 ratio, confirming a robust Th1-biased immunity. The concurrent increase in IL-10 alongside pro-inflammatory cytokines suggests a regulatory mechanism that maintains a strong Th1 response while preventing excessive inflammation. This balance is reflected in the IgG2a/IgG1 ratio, which suggests dominant Th1 immunity without complete suppression of Th2-associated humoral immunity. Dose-dependent effects were also observed, in that the low- and medium-dose immunization groups showed moderate Th1 bias, while the high-dose group exhibited the most pronounced Th1 response. This suggests that higher doses of recombinant spores may enhance Th1 immunity, potentially due to increased antigen availability or stronger activation of antigen-presenting cells. Furthermore, in the low-dose and high-dose immunization groups, concurrent activation of the Th17 pathways, marked by increased IL-17A levels, underscores the potential of recombinant spores to fortify mucosal barriers, particularly in the respiratory tract^81,82,83. This response could provide better protection against SARS-CoV-2 at mucosal surfaces where the SARS-CoV-2 virus first enters.

In conclusion, the present study demonstrates the potential of recombinant B. subtilis spores expressing the SARS-CoV-2 spike protein to stimulate efficacious immune responses. The B. subtilis spores antigen delivery platform generated strong systemic and mucosal immune responses, including significant IgM and IgG production, neutralizing activity, enhanced mucosal immunity, and balanced cellular immune responses as indicated by high T cell, B cell, and macrophage, as well as cytokines and chemokines responses. These results showed the recombinant spores ability to provide broad protection against SARS-CoV-2.

A key limitation of this study is the lack of evaluation against emerging SARS-CoV-2 variants. Although the present work focused on the wild-type virus, the spore-based platform can be rapidly adapted to incorporate updated antigens, if required. Importantly, many conserved regions of the spike protein remain targets for cross-reactive antibodies and T cells, suggesting that some protection against variants of concern may still be achieved. This raises an important question of whether it is more feasible to continue updating the vaccine to match emerging variants, or if vaccination with the ancestral variant is sufficient to confer broad protection^84,85. Nonetheless, future studies should directly assess variant-specific immune responses to determine whether updating the vaccine antigen or relying on ancestral antigen-induced cross-reactivity provides broader and more durable protection.

While the present study has demonstrated the effectiveness of the B. subtilis spore antigen delivery platform against SARS-CoV-2, one major limitation is the absence of animal challenge studies. Such experiments are essential to directly assess whether immunization with the recombinant spores can prevent SARS-CoV-2 infection and block viral transmission. This limitation primarily reflects the lack of access to an Animal Biosafety Level 3 (ABSL-3) facility, which is required to safely perform live virus challenge studies. Future work incorporating these experiments will be critical to confirm the protective efficacy and translational potential of this vaccine approach.

The present study focused on early and intermediate immune responses (baseline, days 16, 32, and 55) and did not include extended follow-up to determine the long-term durability of anti-SARS-CoV-2 IgG. This design was intended to prioritize demonstration of mucosal induction and cellular activation following immunization. We acknowledge that evaluating antibody waning and long-term memory is critical for vaccine assessment; therefore, future longitudinal studies will be required to investigate IgG and sIgA decay kinetics, memory B-cell frequencies, and persistence of tissue-resident T cells in order to define the recombinant spore’s potential for durable immunity and sustained protection.

While this study focused on oral immunization to investigate mucosal and systemic immune responses, future work could explore alternative routes such as intranasal, intramuscular, or subcutaneous delivery. Comparative or combined approaches, including prime-boost strategies, may further enhance the overall efficacy and durability of vaccines. Another limitation of this study is the absence of a comparator group immunized with an established vaccine, such as mRNA or inactivated SARS-CoV-2 vaccines. Such comparisons would provide important context for interpreting the relative immunogenicity of the spore-based platform. Due to the limited availability of licensed vaccines for animal studies at the time, this was not feasible. Future work will address this by including benchmark vaccine controls to directly compare the magnitude and durability of immune responses and protection under the same experimental conditions.

Findings from the present study, nonetheless, support the use of B. subtilis spores as an effective oral vaccine delivery platform against SARS-CoV-2 and potentially other infections transmitted through mucosal routes, offering a practical and scalable approach for global vaccination efforts. Although B. subtilis is GRAS, the safety of recombinant spores was also specifically evaluated. In a separate acute toxicity study, oral administration of recombinant spores in mice did not result in weight loss, clinical abnormalities, or histopathological changes in the major organs, confirming the absence of detectable adverse effects^36,86. These findings provide additional assurance of the platform’s safety for vaccine development.

Methods

Construction, expression, and purification of recombinant B. subtilis expressing spike protein

The target gene sequence encoding the spike protein, which contains a D614G mutation (S1.1) (GenBank Accession number: UNY50970.1), was submitted for codon optimization for expression within the Bacillus subtilis. The gene of interest was commercially synthesized into the cloning vector pUC57 by incorporating the cry1Aa promoter sequence, which was derived from B. thuringiensis, and the cry1Ac terminator sequence (GenScript, USA). It was further cloned into the shuttle vector pHPS9 for transformation into B. subtilis strain WB800N (MobiTec, Germany) for protein expression. The recombinant construct employed the cry1Aa promoter (pCry1Aa) and cry1Ac terminator (tCry1Ac) to achieve sporulation-specific expression in B. subtilis. The pCry1Aa promoter, originally derived from B. thuringiensis, exhibits strong homology with B. subtilis sporulation sigma factors, allowing autoinduced expression during sporulation. The tCry1Ac terminator was incorporated to enhance transcript stability and prevent transcriptional read-through. In addition, a leader sequence together with a Shine–Dalgarno (L + SD) element was included to improve translational efficiency. The transformation was verified by polymerase chain reaction (PCR) amplification and sequencing.

The recombinant B. subtilis was subjected to several treatments and cultivated in a GYS medium to induce sporulation as previously described³⁰. After the sporulation, the harvested spores were heated at 90 °C for 30 min to kill the remaining non-sporulating and vegetative cells. Following centrifugation, the spores were resuspended in a two-phase separation system consisting of 11.18% w/v PEG 4000, 34% v/v 3 M potassium phosphate buffer (1.76 M K2HPO4, 1.24 M KH2PO4), and 50% v/v nucleus-free water. The spore-containing fractions at the interphase were collected and washed five times to remove the residual extraction buffer.

Immunoblot and flow cytometry analyses were performed to validate the spores’ expression of spike proteins. For immunoblotting, after separation by SDS-PAGE, the recombinant proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA), blocked in 10% BSA Diluent/Blocking Solution (Seracare, MA, USA) followed by overnight incubation with mouse monoclonal anti-SARS-CoV-2 Spike glycoprotein antibody (40,591-MM42, Sino Biological Inc., China) at 1:1000 dilution and then stained with goat anti-mouse IgG H&L (HRP) (ab6721, AbCam, Cambridge, UK) at 1:4000 dilution.

Recombinant spores expressing the SARS-CoV-2 spike protein were analyzed using flow cytometry as described earlier⁸⁷. In brief, 1 × 10⁶ CFU/ml of recombinant spores were incubated at room temperature for 30 min in 1 × PBS containing 3% fetal bovine serum (FBS). The spores were then incubated with mouse monoclonal anti-SARS-CoV-2 spike glycoprotein antibody (Sino Biological Inc., China) diluted 1:20, followed by incubation with goat anti-mouse IgG H&L (Alexa Fluor® 488) (Abcam, Fremont, CA, USA) at a 1:2000 dilution. After four additional washes, the spores were resuspended in 1 mL PBS. Negative controls were included to assess nonspecific fluorescence, including non-recombinant B. subtilis spores and samples without the secondary antibody. Positive events were defined as those with a fluorescence intensity threshold of 1 × 10³. The stained spores were analyzed using a fluorescence-activated cell sorting (FACS) CANTO™ II Flow Cytometer (BD Biosciences, Bredford, MA, USA).

Immunogold electron microscopy (IEM)

The immunogold labeling of bacterial spores was performed following a previously established protocol⁸⁸ with minor adjustments. In brief, spore pellets were fixed overnight using 4% glutaraldehyde, followed by post-fixation in 1% buffered osmium tetroxide and subsequent storage in cacodylate buffer overnight. The fixed pellets were dehydrated through a graded ethanol series. The pellets were then washed with a mixture of propylene oxide and epoxy resin [1 mL Agar-100, 0.6 mL dodecenylsuccinic anhydride (DDSA)] and then embedded in 100% epoxy resin. Thin sections were mounted on 200-mesh copper grids (Ted Pella Inc., USA) blocked with 10% BSA (in PBS, pH 7.2) and incubated with a 1:100 dilution of SARS-CoV-2 (2019-nCoV) spike neutralizing mouse monoclonal antibody (40,591-MM43, Sino Biological Inc., China). After five washes, the grids were incubated with goat anti-mouse IgG H&L (10 nm Gold) at a 1:50 dilution and following another five washes the grids were stained with 4% uranyl acetate and Reynold’s lead citrate. After several rinses with deionized water, the grids were dried on clean filter paper and examined using a ZEISS LIBRA transmission electron microscope.

Immunization of mice

All procedures involving the use of laboratory animals were conducted in accordance with relevant guidelines and regulations and were reviewed and approved by the Faculty of Medicine Institutional Animal Care and Use Committee (FOM-IACUC), ethics reference no: 2022-240,110/TIDREC/R/SAB (Fig. 7). All methods are reported in accordance with the ARRIVE guidelines. Specific-pathogen-free female BALB/c mice, 5–6 weeks old, were sourced from the Malaysian Institute of Pharmaceuticals and Nutraceuticals (IPharm, Penang, Malaysia). Mice were housed in the Association for Assessment and Accreditation of Laboratory Animal Care International-certified Animal Experimental Unit at the Faculty of Medicine, Universiti Malaya, under specific pathogen-free conditions. They were kept in individually ventilated cages (IVC) with ad libitum access to water and commercial pellets on a 12-h light–dark cycle. Clinical signs, food consumption, and body weight were monitored daily from Day 0 to Day 55.

Fifty-six mice (n = 8/group) were randomly assigned to seven groups for oral vaccination. Mice were administered either the blank control, diluent, 5 × 10⁸ CFU/ml (low dose), 1 × 10⁹ CFU/ml (medium dose), or 5 × 10¹⁰ CFU/ml (high dose) of negative control, the empty vector (B. sub/pHPS9) or the same doses of recombinant B. subtilis spores expressing the SARS-CoV-2 spike protein (B. sub/pHPS9/S1.1) via intra-gastric lavage for three consecutive days (Table 1). Immunizations were repeated three times at 2-week intervals.

Table 1 Details of the immunogen, volume of immunogen and dose inoculated, and the immunization regimen.

Full size table

Prior to all procedures involving blood collection, mice were anesthetized via intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg) to minimize movement and prevent discomfort or injury. Blood was collected via retro-orbital puncture before each immunization and three weeks after the final immunization, and serum was harvested for specific antibody IgM and IgG level determination and neutralization assays. For each serum collection, saliva and fecal pellets were also collected to evaluate secretory IgA antibody levels. Four weeks after the last immunization, mice were euthanized by an intraperitoneal overdose of ketamine (240–360 mg/kg) and xylazine (30–48 mg/kg), followed by cervical dislocation to confirm death, in accordance with approved institutional animal care guidelines. After euthanasia, the heart, lungs, spleen, liver, kidneys, and small intestines were harvested for macroscopic and histopathological examinations. BALF was collected via the trachea through two rounds of injection and aspiration using 1 × PBS. Small intestinal washes were also collected to evaluate specific secretory IgA antibody levels.

Detection of antigen-specific antibody by indirect ELISA

Specific anti-SARS-CoV-2 IgG and IgM were performed on the sera samples collected from the experimental mice using the Mouse Anti-2019 nCoV(S)IgG ELISA Kit and Mouse Anti-2019 nCoV(S)IgM ELISA Kit (Fine Biotech Co, Wuhan, China), respectively, according to the manufacturer’s instructions. Fecal secretory IgA (sIgA) specific to SARS-CoV-2 was determined using the Mouse Anti-2019 nCoV(S) IgA ELISA Kit (Fine Biotech Co., Wuhan, China), also following the manufacturer’s instructions.

Specific anti-SARS-CoV-2 serum IgG isotypes and saliva, intestinal washes, and BALF sIgA were detected by indirect ELISA as described by ³⁰. Briefly, 96-well microtiter plates (Nest Scientific, USA) were coated with SARS-CoV-2 Spike S1 (D614G) recombinant protein. Diluted serum, saliva, intestinal washes, or BALF were added and developed with horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse IgG1 HRP (Abcam, USA) at 1:10,000 and goat anti-mouse IgG2a HRP (Abcam, USA) at 1:1,000 for the serum IgG isotypes detection, or goat anti-mouse IgA HRP (Abcam, USA) at 1:10,000 for the detection of sIgA in saliva, intestinal washes, and BALF).

Virus neutralization assay

The surrogate viral neutralization test (sVNT) was performed strictly following the manufacturer’s instructions (GenScript, Piscataway, NJ, USA). In brief, mouse sera (diluted 1:100) were pre-incubated with horseradish peroxidase (HRP)-conjugated receptor binding domain (RBD), and the mixtures were then added to the angiotensin-converting enzyme 2 (ACE2)-coated plates (GenScript, Piscataway, NJ, USA) and developed with TMB substrate. Percent inhibition was calculated by comparing the OD values of the samples to those of the negative control.

Flow cytometry

The proportions of T cells, B cells, and macrophage populations in the lymphocytes were determined by flow cytometry. Briefly, spleens isolated from each group were mechanically disrupted and cultured in RPMI1640 followed by stimulation in-vitro under three conditions: (i) stimulation with a 1:1 mix with peptide pools containing 166 peptides covering the S1 domain of the spike protein (amino acid 13-685) (ii) unstimulated (negative control) (iii) stimulation with PMA + Ionomycin (positive control) for 16 h. 12 h before the incubation time ended, Monensin (BD Biosciences, Franklin Lakes, NJ, USA) was added to inhibit protein transport. After the stimulation, the cells were stained with BD Horizon™ FVS 780 Staining Solution for dead cell exclusion. The cell suspension was further preincubated with FC blocker and stained with surface staining reagents (BD Pharmingen), followed by resuspension in BD Perm/Wash™ buffer containing anti-cytokine antibody. Following after, cells were washed, resuspended in BD Pharmingen™ Stain Buffer (FBS) (BD Biosciences, Franklin Lakes, NJ, USA), and analyzed by flow cytometric analysis using FACS Canto (BD Biosciences, Franklin Lakes, NJ, USA).

Multiplex analysis

Cytokines, chemokines, and growth factors secretion from mouse splenocyte supernatants was measured using the Bio-Plex Pro Mouse Cytokine 23-plex (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions. The panel was designed to measure the following analytes: Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-10, IL-12p40, IL-12p70, IL-13, IL-17A, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, KC, MCP-1, MIP-1α, MIP-1β, RANTES, and TNF-α. Data were collected using the Luminex 200™ System (Luminex Corporation, Austin, TX, USA), and analysis was performed using xPONENT for LX100/LX200 v3.1 software, based on standard curve fitting, as per the manufacturer’s protocol.

Statistical analysis

Statistical analysis and graph generation were conducted using GraphPad Prism 9 (San Diego, CA, USA), as described in the figure legends. The Shapiro–Wilk test was used to assess the normality of the sample distribution. Statistical significance was considered when the p-value was less than 0.05.

Data availability

The data used and/or analyzed during the current study are available from the corresponding author on reasonable request.

References

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Acknowledgements

We thank the Animal Experimental Unit (AEU), Universiti Malaya for their invaluable assistance and supports during the animal studies. We also thank the Central Unit of Advanced Research Imaging (CENTUARI), Universiti Malaya for the assistance with the TEM imaging. This work was supported by the Ministry of Science, Technology and Innovation (MOSTI), Malaysia under the MOSTI Combating Covid-19 (MCCOF) Grant (GA002-2021) and the Ministry of Higher Education (MOHE), Malaysia under the Fund for the SUKUK under the Prihatin People’s Economic Stimulus Package (SUKUK PRIHATIN) – Phase 2 (MO002i-2021).

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Tropical Infectious Diseases Research & Education Centre (TIDREC), Universiti Malaya, 50603, Kuala Lumpur, Malaysia

Atiqah Hazan, Amalia A. Saperi, Nurfatihah Zulkifli, Vunjia Tiong, Hai-Yen Lee & Sazaly AbuBakar
Institute for Advanced Studies (IAS), Universiti Malaya, 50603, Kuala Lumpur, Malaysia

Atiqah Hazan
Virology Unit, Infectious Diseases Research Centre, Institute for Medical Research, National Institutes of Health, 40170, Shah Alam, Malaysia

Nor-Aziyah MatRahim

Authors

Atiqah Hazan
Amalia A. Saperi
Nurfatihah Zulkifli
Nor-Aziyah MatRahim
Vunjia Tiong
Hai-Yen Lee
Sazaly AbuBakar

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A.H. has made substantial contributions to the conception AND design of the work; AND the acquisition, analysis, AND interpretation of data; AND has drafted the work and the main manuscript; AND has approved the submitted version; AND has agreed both to be personally accountable for the author’s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature. A.A.S. and N.Z. have made substantial contributions to the acquisition of data; AND have approved the submitted version; AND have agreed both to be personally accountable for the author’s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature. N.M., V.T. and L.H.Y. have made substantial contributions to the conception AND design of the work; AND have approved the submitted version; AND have agreed both to be personally accountable for the author’s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature. S.A. has made substantial contributions to the conception AND design of the work; AND interpretation of data; AND has substantively revised the work and the main manuscript; AND has approved the submitted version; AND has agreed both to be personally accountable for the author’s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.

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Correspondence to Sazaly AbuBakar.

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Hazan, A., Saperi, A.A., Zulkifli, N. et al. Recombinant Bacillus subtilis spores expressing SARS-CoV-2 spike protein induced humoral, mucosal, and cellular immunity in mice. Sci Rep 15, 44552 (2025). https://doi.org/10.1038/s41598-025-28250-9

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Received: 21 April 2025
Accepted: 10 November 2025
Published: 24 December 2025
Version of record: 24 December 2025
DOI: https://doi.org/10.1038/s41598-025-28250-9