Rational Combination of Dominant and Subdominant Antigens with Nanoadjuvant Elicits Durable Immunity Against Staphylococcus aureus

Abstract

Objectives: In response to the challenge that Staphylococcus aureus (S. aureus) vaccines fail to induce durable protective immunity, this study aims to develop a novel antigen-adjuvant co-design strategy. Specifically, we rationally combined the immunodominant toxin antigen LukS-PV with the immunologically subdominant adhesin antigen ClfA, co-delivered via the PLGA-PEG nanoadjuvant system, to elicit synergistic, durable, and balanced humoral and cellular immune responses. Methods: Firstly, recombinant antigens LukS-PV and ClfA were individually covalently conjugated to PLGA-PEG 25% nanoparticles (25% NPs) using EDC/NHS chemical coupling to construct a combined nanovaccine, followed by systemic safety verification in a mouse model. Subsequently, specific antibody titers were detected by ELISA, and the secretion levels of IL-4, IFN-γ, and IL-17A were measured by ELISPOT assay to comprehensively evaluate the humoral and cellular immune responses induced by the vaccine. Finally, the protective efficacy of the vaccine was assessed through acute and long-term (up to 180 days) lethal challenge experiments, thereby verifying the effectiveness of this co-design strategy based on rational antigen selection. Results: The combined vaccine group (25% NPs-rClfA + 25% NPs-rLukS-PV) not only elicited high levels of specific antibodies but, more importantly, induced robust cellular immune responses dominated by Th1 and Th17 cells. Challenge experiments confirmed that the protective efficacy of the combined vaccine was significantly superior to that of any single-antigen vaccine and provided complete protection for up to 180 days. Crucially, the same antigen combination formulated with a traditional aluminum adjuvant failed to confer this durable protection, underscoring the essential role of adjuvant synergy. Conclusions: This study demonstrates that rational combination of immunodominant and subdominant antigens with a compatible nanoadjuvant induces synergistic and durable immunity against S. aureus. This co-design strategy addresses key limitations of previous vaccines and provides a promising foundation for future clinical development.

1. Introduction

Staphylococcus aureus (S. aureus) represents one of the most significant challenges in contemporary global public health [1,2]. It colonizes asymptomatically in the nasal passages of approximately 30% of healthy adults, while also serving as the primary pathogen responsible for a spectrum of diseases ranging from mild skin and soft tissue infections to life-threatening conditions such as bacteraemia and septicaemic shock [2,3,4]. The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has further exacerbated this threat. In particular, community-acquired infections (CA-MRSA), due to carrying virulence factors such as Panton-Valentine leukocidin (PVL) which are related to virulence, are more invasive and have significantly increased the disease burden [5]. Therefore, the World Health Organization (WHO) has encouraged the development of new vaccines to address antimicrobial resistance (AMR) [6].

Despite high hopes for vaccines, multiple candidates entering clinical trials over recent decades have failed, revealing the limitations of traditional strategies [7]. For example, Nabi’s StaphVAX vaccine, targeting capsular polysaccharides (CPs) CP5 and CP8, failed to show significant protective efficacy in Phase III clinical trials among hemodialysis patients with S. aureus infections [8,9]. This failure indicates that humoral immune responses targeting CPs alone is insufficient for protection. Similarly, Pfizer’s SA4Ag vaccine, composed of CP5, CP8, recombinant clumping factor A (rClfA), and recombinant manganese transporter protein C (rMntC), demonstrated good safety, tolerability, and immunogenicity in preliminary clinical studies; however, it also ultimately failed to significantly reduce the rate of S. aureus infections in postoperative patients [9,10]. This suggests that relying solely on humoral immune responses that induce antibody production may be insufficient to combat S. aureus invasion. The reasons for the failures of these candidate vaccines reveal the limitations of traditional strategies. Firstly, antigen selection mostly targets a single virulence factor, while S. aureus has complex pathogenic mechanisms and potent immune evasion capabilities, resulting in a narrow protective range [11]. Secondly, the adjuvants employed (primarily aluminum-based) struggle to induce the requisite immune response type [12]. They predominantly drive Th2-mediated humoral immunity, whereas S. aureus possesses intracellular survival capabilities, its clearance relying on robust Th1/Th17-mediated cellular immunity [13]. Research indicates that the inability to establish effective T-cell responses is a key factor in recurrent S. aureus infections [14]. In infection models, IFN-γ-deficient mice exhibit high susceptibility, confirming the protective role of Th1 immunity [15]. Concurrently, the IL-17 signaling pathway has been identified as a critical component of defense; IL-17A/IL-17F double-deficient mice demonstrate markedly increased susceptibility to cutaneous and mucosal infections, indicating that Th17 immunity also plays a central role in host defense [16,17,18].

The immunodominance of antigens determines vaccine efficacy. “Immunodominant” antigens refer to those that can continuously induce a high-titer antibody in healthy people but may not provide comprehensive protection. In contrast, “immunosubdominant” antigens are those that can only trigger low-titer immune responses under natural exposure, yet provide new targets for vaccine design. Population seroepidemiological data show that neutralizing antibodies against S. aureus toxins (such as alpha hemolysin, Hla) are ubiquitous and have high titers, while natural antibodies against the surface protein ClfA have low titers and lack protective functions [19]. Notably, ClfA is a highly conserved microbial surface component recognizing adhesive matrix molecules (MSCRAMM) across diverse S. aureus strains, particularly in its ligand-binding regions, making it a stable target for vaccine-induced antibodies [20,21]. This lays a theoretical foundation for the vaccine strategy using subdominant antigens represented by ClfA to avoid pre-existing immunity and induce new protective responses [19].

However, the flip side of “subdominance” is its relatively weak innate immunogenicity [19], potentially failing to rapidly elicit robust primary immune responses when used alone. Furthermore, the antibodies it induces are primarily opsonic, incapable of directly neutralizing bacterially secreted toxins. Complementary to this, PVL as a key immunodominant toxin antigen, possesses strong immunogenicity, rapidly eliciting high-titer neutralizing antibodies that provide a rapid protective barrier against toxin attack [22,23]. Furthermore, PVL genes are well conserved in different clones of S. aureus, leukocidins have two subunits that are classified as the host cell targeting S component (LukS-PV), and the polymerization F component (LukF–PV) [23,24]. This study uses an attenuated mutant of LukS-PV with natural immunogenicity and reduced toxicity [25]. More significantly, antibodies targeting its attenuated subunit (LukS-mut9) exhibit broad cross-neutralizing capacity against multiple homologous toxins including PVL, γ- hemolysin, and LukED, substantially broadening the scope of protection [25]. From an immunogenicity perspective, a vaccine based on the recombinant LukS-PV antigen primarily induces a specific immune response against the PVL toxin, and therefore offers little direct protective effect against PVL-negative strains. This further underscores the necessity of adopting a multivalent vaccine strategy, which can elicit a broad spectrum of immune responses, thereby effectively addressing the antigenic diversity and virulence complexity exhibited by S. aureus during infection.

Correspondingly, adjuvant selection should address limitations in conventional approaches to effectively stimulate comprehensive immune responses. We employed FDA-approved PLGA-PEG nanoparticles, which combine excellent biocompatibility with degradability [26,27]. These nanoparticles not only protect antigen stability and enable sustained release to mimic repeated immune stimulation, but can be efficiently taken up by antigen-presenting cells to promote cross-presentation and activate cellular immune responses [28,29,30]. For example, Thomas C et al. used PLGA nanoparticles for the pulmonary delivery of hepatitis B vaccines, and the results showed that this delivery system could more effectively promote the secretion of Th1-type cytokines (such as IFN-γ) [31]. Manish M et al. delivered the stable immunogenic domain 4 (PAD4) of Bacillus through the PLGA system, converting the vaccine response from a predominantly Th2-biased profile into a balanced Th1/Th2 mixed reaction, which is crucial for the defense against intracellular pathogens requiring cellular immunity [9,32].

Based on this background, this study proposes a synergistic “antigen-adjuvant” co-design strategy to construct a novel S. aureus vaccine (Figure 1). We selected the subdominant surface protein antigen ClfA as the foundation for sustained immunity, while incorporating the highly immunogenic dominant toxin antigen PVL to establish a rapid neutralization barrier. Both were covalently cross-linked with the PLGA-PEG nanoadjuvant, aiming to elicit a comprehensive, balanced, and enduring immune response, thereby offering a novel solution to this challenge.

2. Materials and Methods

2.1. Ethics Statement

All experimental procedures involving animals complied with China’s national regulations for laboratory animal administration. The female BALB/c mice used in this study were supplied by the Laboratory Animal Center of Air Force Medical University.

2.2. Materials

All commercial reagents and materials were sourced as follows: BL21(DE3) competent E. coli from TIANGENBIOTECH (Beijing, China); Ni-Sepharose from Cytiva (Shanghai, China); a High-Capacity Endotoxin Removal Column from Xiamen Bioendo Technology (Xiamen, China); PLGA_15,000-PEG₅₀₀₀-COOH from Xi’an Ruixi Biological Technology (Xi’an, China); Alhydrogel™ adjuvant from Croda (East Yorkshire, UK); Enzyme-linked immunosorbent assay (ELISA) plates from Corning (Corning, NY, USA); Enzyme-linked immunospot (ELISPOT) kits from Mabtech (Stockholm, Sweden); Bradford protein assay kits from Beijing Solarbio Science & Technology (Beijing, China). Additionally, Polyvinyl alcohol (PVA), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-Hydroxy succinimide (NHS), 2,2′-azino-bis (3-ethylbenzo thiazoline-6-sulfonic acid) (ABTS), horseradish peroxidase (HRP)-labeled goat anti-mouse IgG. Isopropylbeta-D-thiogalactopyranoside (IPTG), imidazole, dichloromethane (DCM), acetone, 2-(N-morpholino) ethanesulfonic acid hydrate (MES), Tween 20 were all obtained from Sinopharm Chemical Reagent (Shanghai, China) [33].

2.3. Synthesis of 25% NPs

The solvent emulsion volatilization method was employed to synthesize 25% NPs. Firstly, 100 mg of the material was dissolved in 2.5 mL of dichloromethane-acetone mixture (v/v = 3:2). After complete dissolution, it was dropped vertically into 10 mL of 0.5% PVA solution, and the solution was stirred at 600 rpm to mix well. The mixed liquid was sonicated with a cell breaker under ice bath conditions for 2 min (power 30 w, sonication 1 s, stop 2 s). The sonicated milky white liquid was solidified by dropping it vertically into 50 mL of pure water, stirred at 600 rpm, and the reaction was carried out overnight at room temperature. On the next day, the liquid was passed through a 0.45 μm filter, centrifuged 18,000× g force at 4 °C for 1 h to discard the supernatant, and the precipitate was resuspended and washed once with purified water, then the precipitate obtained by discarding the supernatant was the 25% NPs. Finally, it was resuspended with 16% alginate solution and lyophilized for storage.

2.4. Expression and Purification of ClfA and LukS-PV (Mutant LukST28F/K97A/S209A) Antigens

The base sequences were found on the NCBI website and the plasmids were synthesized by Beijing Tsingke Biotech. The synthesized plasmid was introduced into BL2 (DE3) receptor cells with subsequent expansion culture. For ClfA antigen, induce the bacteria with 1 mmol/L IPTG at 20 °C for 20 h. For LukS-PV mutant antigen, induce the bacteria with 0.001 mmol/L IPTG at 16 °C for 24 h. Bacteria were collected by centrifugation after the completion of induction at 4200 rpm at 4 °C for 10 min, resuspended in purified water and disrupted by sonication to release soluble proteins. Proteins were purified by affinity chromatography, eluting from Ni-Sepharose with 5 mmol/L, 100 mmol/L, 300 mmol/L, 500 mmol/L imidazole. The purity and specificity of the antigen was analyzed by SDS-PAGE, and the concentration was determined by BCA.

2.5. Preparation and Characterization of 25% NPs Conjugated with Antigen

Take 10 mg of lyophilized nanomaterials, dissolve with 10 mL of 25 mmol/L MES buffer, add 1 mol/L EDC 400 μL and 1 mol/L NHS 250 μL, aiming to activate the carboxyl terminus of the nanoparticles, at room temperature at 600 rpm for 3 h. Subsequently, centrifuge the nanoparticles at 18,000× g force at 4 °C for 60 min and the resulting precipitate is the activated nanoparticles. The activated NPs were resuspended with 1 mg/mL antigen and mixed by spinning at 4 °C overnight. On the following day, the vaccine was centrifuged at 14,000 rpm for 45 min, the supernatant was discarded, washed once with PBS and resuspended with PBS. The hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the NPs were measured via dynamic light scattering (DLS) (Delsa™ Nano, Beckman-Coulter, High Wycombe, UK). Successful conjugation with nanoparticles and antigen was verified by Fourier transform infrared spectroscopy (FTIR, Thermo Nicolet IS50, Thermo Fisher Scientific, Waltham, MA, USA). Bradford method was used to quantify the loading rate of antigen.

2.6. Animal Immunization

Female BALB/C mice, aged 6–8 weeks, are randomly divided into 10 groups (n = 10). The groups are as follows: PBS, rClfA, rLukS-PV, and rClfA + rLukS-PV serve as the negative control groups. Positive control groups are formed with alum-adjuvanted antigens: Alum-rClfA, Alum-rLukS-PV, and Alum-rClfA + Alum-rLukS-PV. The experimental groups receive 25% NPs-rClfA, 25% NPs-rLukS-PV, and 25% NPs-rClfA + 25% NPs-rLukS-PV. Mice are immunized subcutaneously with a single antigen dose of 25 μg per mouse, and for the mixed antigen groups, each antigen is administered at a dose of 25 μg per mouse. All groups are boosted with the same formulation 14 and 28 days after the initial immunization. Mouse serum samples are collected at specific time points.

2.7. Evaluation of In Vitro and In Vivo Biocompatibility of Vaccines

L929 cells were added to the 96-well plate with 100 μL and cultured in a CO₂ cell incubator at 37 °C for 24 h. Once the cells have completely adhered to the well surface, the culture medium is aspirated, and various materials, antigens, and vaccines at concentrations ranging from 7.8 μg/mL to 1 mg/mL are added to each well, with 100 μL per well. After 24 h, the liquid is aspirated again and the wells are rinsed 5 times with PBS. Subsequently, in a light-protected environment, 100 μL of CCK8 reagent is added to each well and incubated for 1 h. The optical density (OD) at 450 nm is then measured using a microplate reader, and the cell viability is calculated by comparing the OD values with those of the untreated control group.

Following the initial immunization, the weight fluctuation of the mice is monitored every 7 days to assess the in vivo toxicity of the nanovaccine. On day 35, the heart, liver, spleen, lung and kidney tissues of immunized mice were taken, and the toxicity of nanovaccine tissues was investigated by H&E staining.

2.8. ELISA

On day 35 and day 180 following the initial immunization, blood is collected from the immunized mice, and the serum titers are determined using an indirect ELISA. The antigen is diluted to a final concentration of 1~10 μg/mL with a carbonate-bicarbonate coating buffer at pH 9.6, and 100 μL per well is added to the ELISA plates. The plates are incubated overnight at 4 °C, after which the solution in the wells is discarded, and the wells are washed 3–5 times with PBST and dried. Mouse serum is then diluted in a gradient using a 0.1% casein sodium solution, starting with a dilution of 1:500 for the first well, and subsequent wells are diluted twofold, with 100 μL added to each well. The plates are incubated at 37 °C for 1 h, then washed and dried (as described above).

Next, horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibodies (at a dilution of 1:3300, 100 μL per well) are added and incubated at 37 °C for 45 min. The plates are again washed and dried (as described above). Color development is achieved using ABTS and H₂O₂ as substrate chromogens. The absorbance of each well is measured at 410 nm using a microplate reader, with the blank control well used to set the zero point. The highest dilution of the positive wells is taken as the titer of the immune mouse serum.

2.9. ELISPOT

The splenocytes of immunized animals were analyzed for IL-4, IFN-γ, and IL-17A production by ELISPOT assay on day 35 and day 180. Briefly, ELISPOT plates were washed four times with sterile PBS and incubated with complete medium (RPMI-1640, 10% FBS) for 30 min at room temperature. After removal of the complete medium, the immunized mouse splenocytes (1.0 × 10⁶ cells/well) were mixed with the stimulus (4 μg/mL), and medium alone were used as a negative control, and incubated for 24 h at 37 °C. The next day, the liquid in the wells was removed and the plates were washed 5 times with PBS; then, the detection antibody was added to PBS containing 0.5% FBS (PBS-0.5% FBS) and incubated for 2 h at room temperature. Wash the plate 5 times and add TMB substrate solution to the plate until visible spots appear then rinse the plate with plenty of purified water. Finally, spots were counted in an ELISPOT reader (Cellular Technology Limited, Cleveland, OH, USA).

2.10. Lethal Challenge

This study systematically evaluated the protective efficacy of the vaccine through a series of challenge experiments. On the 7th day after the third immunization, the mice were injected with 100 μL of S. aureus (ATCC25923) at a concentration of 2.56 × 10⁹ CFU/mL via the tail vein. This dose was determined as the lethal dose (1 × LD₁₀₀) through preliminary experiments. Thereafter, the survival status of the mice was recorded daily for 14 consecutive days to evaluate the protective effect after the initial immunization. To further distinguish the differences in protective efficacy among different antigen combinations, subsequent booster challenges were conducted on the mice using 2-fold (2 × LD₁₀₀) and 4-fold (4 × LD₁₀₀) lethal doses. To evaluate the long-term immune memory induced by the vaccine, on the 180th day after the initial immunization, the mice were challenged again with a 1 × LD₁₀₀ lethal dose, and observations were continued for 14 days to assess the long-lasting protective effect of the vaccine.

2.11. Statistical Analysis

All data are expressed as mean ± S.D. and were analyzed using GraphPad Prism 9.0. Normality was confirmed via the Shapiro–Wilk test, followed by one-way ANOVA for group comparisons. Survival data were analyzed using the Log-rank test, with Bonferroni correction for multiple comparisons. Statistical significance was defined as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; p ≥ 0.05 was considered not significant (ns).

3. Results

3.1. Antigen Selection and Purification

ClfA as S. aureus cell wall anchoring protein can bind to host extracellular matrix proteins, PVL leucocytocidal toxin is an extracellular two-component perforated cytolytic toxin produced by S. aureus, and they are all important virulence factors involved in a variety of infections. In this experiment, the two antigens were successfully prepared by biotechnological synthesis methods, and by comparing the synthesized proteins with standard proteins through SDS-PAGE. The synthesized proteins were verified and characterized, and the results showed that the relative molecular weight of rClfA was about 34 KD, and the relative molecular weight of rPVL (Mutant LukS-PV) was about 32 KD in agreement with the position of the standard protein bands (Figure S1a,b).

3.2. Characterizations of the PLGA-PEG 25% NPs Conjugated with Antigen

Nanoparticles were prepared by the emulsification-solvent evaporation method and conjugated through EDC/NHS with the rClfA and rLukS-PV. SEM results showed that 25% NPs were spherical with a smooth surface (Figure 2d). The particle size of the nanoparticles was determined by DLS to be around 170 nm with good dispersion. The particle size of nanoparticles increased to about 190 nm after conjugation with antigen (Figure 2a and Figure S2a,c,e). The absolute value of the zeta potential increased by 2 mV~5 mV (Figure 2b and Figure S2b,d,f). BCA assay revealed that the loading efficiency of both 25% NPs-rClfA and 25% NPs-rLukS-PV were 1%~1.5% (mass ratio) (Figure 2c). The FTIR demonstrated that the bimodal peaks induced by the amino group of the pure antigen at 3500 cm⁻¹ phenomenon disappeared after cross-linking to form amide bonds, indicating that the antigen was successfully conjugated with the nanoparticles (Figure 2e,f).

3.3. Biocompatibility of the Antigen Conjugated 25% NPs

To evaluate the biocompatibility of antigen-conjugated 25% NPs, we conducted a series of in vitro and in vivo experiments. In vitro, the CCK-8 assay using human skin fibroblasts showed no significant cytotoxicity at concentrations ranging from 7.8 μg/mL to 1 mg/mL, with cell viability remaining above 90% (Figure 3a). After subcutaneous inoculation in mice, there was no significant weight loss (Figure 3b). In addition, H&E staining analysis of major organs (heart, liver, spleen, lung, and kidney) did not observe obvious pathological damage (Figure 3c). In conclusion, the vaccine system exhibited good biocompatibility under the experimental conditions.

3.4. Determination of Serum Antibody Titers in Immunized Mice

Following the confirmation of vaccine safety, we assessed the humoral immunogenicity of the 25% NPs nanovaccine system by measuring antigen-specific IgG antibody titers in mouse serum via ELISA. As shown in Figure 4a, mice were immunized subcutaneously three times on days 0, 14, and 28. The experimental groups included PBS, free antigen, alum-adjuvanted antigen, and 25% NPs-adjuvanted antigen groups. Serum was collected on days 35 and 180 after the final immunization to systematically evaluate the magnitude and durability of the humoral immune response.

The results demonstrated that at both day 35 and day 180, antibody titers induced by both the alum and 25% NPs adjuvant groups were significantly higher than those in the PBS and free antigen groups (p < 0.0001), confirming the efficacy of the immunization protocol. On the 35th day after the first vaccination, in-depth analysis revealed that the synergistic effect of different adjuvants on the immune response to antigens is specific. The 25% NPs adjuvant system showed a more potent effect for the single antigen rLukS-PV and the combined antigens (rClfA + rLukS-PV), with antibody levels approximately 3.5-fold and 2.5-fold higher, respectively, than those in their alum-adjuvanted counterparts (Figure 4b). In contrast, the traditional alum adjuvant exhibited a stronger enhancing effect for the single antigen rClfA, inducing antibody titers about 2-fold higher than those in the 25% NPs-rClfA group.

Notably, when evaluating the durability of the immune response, the combined antigen group (25% NPs-rClfA + 25% NPs-rLukS-PV) demonstrated superior long-term immunogenicity. This group maintained the highest antibody levels at day 180, with the smallest decline in titer compared to day 35 (Figure 4b,c). This performance was significantly better than that of the single-antigen groups (25% NPs-rClfA, 25% NPs-rLukS-PV). These results indicate that the multivalent vaccine composed of 25% NPs with rClfA and rLukS-PV not only elicits a robust primary immune response but also induces stable and long-lasting immunological memory, establishing a critical foundation for long-term anti-infective protection.

3.5. ELISPOT Analysis of Splenic Cytokine Secretion Profiles

To systematically evaluate the cellular immune response induced by the nanoadjuvant vaccine, we measured the secretion of IFN-γ (Th1 type), IL-4 (Th2 type), and IL-17A (Th17 type) cytokines in splenocytes of mice at 35 days and 180 days post-immunization. The experimental procedure is shown in Figure 5a.

Regarding IL-4, a core cytokine of the Th2 response, results on day 35 showed that for the antigen rClfA, the number of IL-4 spot-forming units induced by the 25% NPs-rClfA group (291) was significantly lower than that induced by the Alum-rClfA group (321). Conversely, for the antigen rLukS-PV and the combined antigens, the IL-4 secretion levels in the 25% NPs-adjuvanted groups were significantly higher than those in the corresponding alum-adjuvanted groups (Figure 5b), and this result is consistent with the ELISA antibody titer data.

IFN-γ, secreted by Th1 cells, is a key effector molecule that activates phagocytic functions of macrophages and promotes cellular immunity. The experimental results confirmed that the 25% NPs adjuvant efficiently elicits a Th1-type immune response. At 35 days after immunization, the IFN-γ response intensity in each group showed obvious antigen dependence. The 25% NPs-rClfA group exhibited the highest IFN-γ secretion level, followed by the combined antigen group (25% NPs-rClfA + 25% NPs-rLukS-PV), and then the 25% NPs-rLukS-PV group (Figure 5d). It is noteworthy that although the prior antibody titer experiment indicated relatively lower antibody levels in the 25% NPs-rClfA group, subsequent challenge experiments demonstrated that this did not compromise its protective efficacy. This result suggests that the Th1-type cellular immunity driven by high levels of IFN-γ plays an important role in protecting against S. aureus infection.

IL-17A is a key cytokine mediating protection against extracellular S. aureus infection, primarily involved in the recruitment and activation of neutrophils. The results indicated that all 25% NPs-adjuvanted groups induced a significantly higher number of IL-17A spots compared to the alum-adjuvanted groups. Furthermore, the combined antigen group (25% NPs-rClfA + 25% NPs-rLukS-PV) showed the highest level of IL-17A secretion (Figure 5f), highlighting the potential of this adjuvant system to potently activate inflammation and innate immunity related to anti-S. aureus defense.

Long-term monitoring on day 180 post-immunization revealed a decline in cytokine levels across all groups compared to day 35. However, the combined antigen group (25% NPs-rClfA + 25% NPs-rLukS-PV) maintained comparatively higher levels of cytokine secretion (Figure 5c,e,g). Particularly, the IL-17A secretion in this group remained significantly higher than that of other cytokines, and its decrease was the most moderate compared with other groups (Figure 5f,g). These results collectively indicate the sustained and potent activation of the Th17 pathway, which may contribute to enhanced long-term immune memory and neutrophil-mediated protective responses.

3.6. Lethal Challenge Assay

To systematically evaluate the protective efficacy of the vaccines, a series of lethal challenge experiments using S. aureus (ATCC25923) were conducted on days 35 and 180 post-immunization.

On day 35 after the primary immunization, mice were first challenged intravenously with a 1× LD₁₀₀ dose. The results showed that all mice in the PBS and free antigen groups died within 72 h (Figure S3). In contrast, all groups receiving 25% NPs-adjuvanted vaccines exhibited significant protection, with a 100% survival rate, which was significantly superior to the survival rate in the alum-adjuvanted group (Figure 6a–d). This confirmed the protective advantage of the nanoadjuvant system.

To further differentiate the protective efficacy of different antigen combinations, a subsequent challenge was performed using a 2 × LD₁₀₀ dose. Under this condition, all mice in the PBS and free antigen groups died within 48 h (Figure S4), the survival rate of the 25% NPs-rLukS-PV group dropped sharply to 10% (Figure 7c), which was significantly lower than that of the 25% NPs-rClfA group (50% survival, Figure 7b) and the combined antigen group (25% NPs-rClfA + 25% NPs-rLukS-PV, 80% survival, Figure 7d). Notably, despite the ELISA data showing that the antibody titer of the 25% NPs-rClfA group was only 38% of that in the 25% NPs-rLukS-PV group (p < 0.0001, Figure 4b), its protective efficacy was stronger, suggesting a compensatory role for cellular immunity.

In a subsequent dose challenge of 4 × LD₁₀₀, the survival rate was 0% for the 25% NPs-rLukS-PV group and 20% for the 25% NPs-rClfA group, while the combined antigen group (25% NPs-rClfA + 25% NPs-rLukS-PV) maintained a survival rate as high as 60% (Figure 8b–e). This further indicates that the combined antigen has a significant advantage in resisting high-dose infections.

To monitor its long-term immune efficacy, in the lethal challenge experiment with 1 × LD₁₀₀ conducted on day 180 after immunization, mice in the PBS and free antigen groups succumbed rapidly, whereas all 25% NPs-adjuvanted groups maintained a significant survival advantage (Figure 9b–e). Most notably, the survival rate in the combined antigen group (25% NPs-rClfA + 25% NPs-rLukS-PV) remained at 100% (Figure 9d). Combined with the long-term ELISPOT data, we speculate that the protective mechanism may be related to enhanced neutrophil recruitment and bactericidal function due to the sustained activation of the Th17 pathway, indicating that this combined antigen nanovaccine can establish a durable anti-infective immune barrier.

4. Discussion

Conventional S. aureus vaccine design has predominantly focused on immunodominant antigens [12,19]. Although this approach can rapidly elicit strong responses, its protective efficacy is often limited by pre-existing, non-protective immune imprinting in the host. In contrast, the multivalent antigen combination strategy developed in this study plays distinct yet complementary roles [34]. We combined the immunologically subdominant adhesin ClfA with the immunodominant toxin antigen LukS-PV and delivered this combination via a PLGA-PEG nanoadjuvant platform. This formulation induced synergistic protective immunity in a mouse model and provided complete protection for up to 180 days.

First, our antigen combination strategy is based on a rational design of immunogenicity complementarity, rather than simple additive mixing. The work of Cui et al. revealed a significant “antigen dependency” of nanoadjuvant efficacy. Specifically, the same PLGA-PEG nanoparticle carrying either rHlaH35L or rSpam elicited vastly different immune strengths and protective outcomes, and their simple combination showed no additive advantage, indicating that random antigen addition cannot ensure the success of the vaccine [35]. In comparison, our combination of ClfA and LukS-PV is rationally designed for “immunogenicity complementarity.” Here, LukS-PV, as a strongly immunogenic antigen, aims to rapidly generate high-titer neutralizing antibodies against toxin attack. In contrast, ClfA is oriented towards mobilizing cellular immunity, predominantly of the Th1/Th17 type, targeting the clearance of the bacteria themselves. Our challenge experiment data confirmed that this design produced a significant synergistic effect. The combined vaccine group showed markedly superior protective efficacy against high-dose challenges compared to any single-component vaccine. Furthermore, the ClfA antigen selected in our study, similar to the metabolic antigen PDHC investigated by Huang et al., belongs to the category of “immunologically subdominant” antigens [36]. Their work similarly suggests that targeting such antigens may effectively circumvent the “non-protective immune imprinting” resulting from widespread prior exposure to S. aureus in the human population, a finding that provides a crucial theoretical foundation for our vaccine design.

Our results further indicate that protective efficacy depends on the “quality” of the immune response rather than merely the antibody “quantity.” Although the serum antibody titer in the 25% NPs-rClfA group was relatively low, it induced robust IFN-γ and IL-17A responses and demonstrated excellent protection in serial challenge experiments. Interestingly, we find differences in the protective efficacy of ClfA with different adjuvants. Although the Alum-rClfA group induced a much higher antibody titer, only 60% of mice survived from S. aureus lethal challenge, while the survival rate of the 25% NPs-rClfA group was 100% with a lower antibody titer. Since the secretion of IL-17A in the 25% NPs-ClfA group was approximately three times that of the Alum-ClfA group, the fundamental reason might lie in the strong Th17 immune response driven by 25% NPs. This phenomenon strongly suggests that for the ClfA antigen, antibody may not be the main mechanism of protective immunity against S. aureus; instead, the recruitment and activation of neutrophils, which are dominated by effector factors such as IL-17A, might play a decisive role in clearing S. aureus. Indeed, studies have shown that Th17 cells and their signature cytokine IL-17A play a supportive role in anti-S. aureus defense, as evidenced by multiple clinical and basic studies [37,38]. For instance, patients with STAT3 mutations leading to impaired Th17 cell differentiation often present with recurrent, severe S. aureus skin and mucosal infections [39]. Similarly, early depletion of Th17 cells in HIV infection is closely associated with poor control of skin and soft tissue infections [18]. This collective evidence underscores that maintaining intact and durable Th17 immunity is a key mechanism for establishing effective defense against S. aureus.

Critically, durable protection relies on the precise synergy between antigens and adjuvant. Our control experiments showed that the same antigen combination failed to induce similarly durable (180 day) complete protection when formulated with the traditional aluminum adjuvant. This contrast clearly reveals the necessity of antigen-adjuvant synergy in shaping the immune response. The aluminum adjuvant tends to induce an antibody-focused immune response (Th2-type) but is ineffective at activating the Th1/Th17 cellular immune pathways essential for clearing S. aureus. Consequently, even with the correct antigen combination, the aluminum adjuvant cannot generate the crucial cellular immune memory required for long-term protection. Conversely, the PLGA-PEG nanoadjuvant more effectively delivers antigens to antigen-presenting cells and provides sustained immune stimulation through its controlled-release properties [40]. This activates potent Th1/Th17 responses and durable immune memory, thereby translating the theoretical protective potential of the antigen combination into practical, long-lasting protection. Specifically, the 25% NPs can significantly enhance the uptake of dendritic cells (DCs), resulting in the upregulation of MHC class II and CD80/CD86 molecules, promoting antigen presentation by DCs and then the activation of T cells (unpublished data). In addition, PLGA can be recognized as an endogenous danger signal, activating the NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome and triggering the release of IL-1β, thereby amplifying the Th17 response [41]. In addition, 25% NPs could also induce Th1-type immune responses by promoting the secretion of IFN-γ [33]. Meanwhile, PEGylation further prolongs the systemic circulation time of nanoparticles, thereby providing more sustained and concentrated antigen stimulation at key sites for immune induction [42,43]. These mechanisms work synergistically to collectively drive Th1 and Th17 cellular immune responses.

The current conclusions are drawn solely from a prophylactic systemic infection model in mice and do not encompass other important clinical scenarios of S. aureus infection. Furthermore, the vaccine’s protection has been primarily validated against a standard laboratory strain. Future work is needed to test its broad-spectrum efficacy against a wider range of clinical isolates, especially multidrug-resistant strains. The intravenous systemic challenge model used in this study (ATCC 25923 strain) is primarily designed to simulate the most critical systemic dissemination stages of S. aureus infection, such as bacteremia and sepsis. Although this model does not cover more common local initial infection scenarios like skin and soft tissue infections, it can directly and effectively evaluate the host immune system’s defense capability against circulating bacteria and the toxins they release (such as PVL, which was focused on in this study). The relevant conclusions provide a specific perspective for understanding the immune defense against systemic infections, and their significance for local infections needs to be further verified in combination with other models [44].

Additionally, this study raises several mechanistic questions requiring further investigation. These include elucidating how the low-titer antibodies induced by ClfA confer superior protection, which necessitates analysis from qualitative perspectives such as antibody affinity and Fc-mediated effector functions; the hypothesis that the combination of dominant and subdominant antigens may achieve long-lasting immunity needs to be verified using immune repertoire sequencing technology, the specific mechanism by which PLGA-PEG nanoadjuvants initiate Th1/Th17 immune responses and their essential differences from aluminum adjuvants will also be the core direction of future research. While this study demonstrates that sustained Th17/IL-17A responses play key role in durable protection of S. aureus, the direct causal role of IL-17A requires validation through functional experiments. Future studies should further verify the role of Th17/IL-17A responses, such as blocking IL-17A function with neutralizing antibodies before or after challenge, adoptively transferring antigen-specific T cells from vaccinated mice into normal recipient mice, or evaluating vaccine efficacy in IL-17A gene-deficient mouse models. These experiments would directly test whether the Th17 response is essential for the protection of S. aureus.

5. Conclusions

In conclusion, this study constructed a novel combined vaccine by rationally combining the immunologically subdominant antigen ClfA with the immunodominant toxin antigen LukS-PV, delivered via a PLGA-PEG nanoadjuvant. This strategy successfully induced Th1/Th17-dominated cellular immunity and an efficient antibody response, achieving durable complete protection superior to single-antigen or conventional adjuvant formulations. These findings establish that effective vaccine design depends not only on complementary antigen selection but also on precise adjuvant compatibility to drive balanced and sustained immune responses, offering a generalizable paradigm for combating complex pathogens.