All animals were treated humanely and in accordance with institutional animal care guidelines. This study was approved by the Animal Care and Use Committee of Shihezi University.

Standard S. aureus [American Type Culture Collection (ATCC) 25923] and S. agalactiae (ATCC 13813) strains were provided by the ATCC and cultured in brain heart infusion (BHI) broth/agar (Hopebio, China) at 37°C. In addition, Escherichia coli strains DH5α (Sigma-Aldrich Corp., St. Louis, MO, USA) and C43 (DE3, Sigma, USA) were cultured in Luria–Bertani medium (Difco, Becton Dickinson, Franklin Lakes, New Jersey, USA). Five-week-old female BALB/c mice were purchased from the Experimental Animal Center of the Academy of Military Medical Science (Beijing, China). All experimental procedures and animal care protocols were performed in compliance with institutional animal care regulations.

FnBP (gene ID: DQ447162) and ClfA (gene ID: EF207779) gene sequences from S. aureus in addition to GapC (gene ID: af421899) and Sip (gene ID: fj808732) gene sequences of S. agalactiae were retrieved from the GenBank database. The Kolaskar and Tongaonkar methods were used to predict and analyze the B cell epitopes of FnBP, ClfA, GapC, and Sip gene sequences (http://imed.med.ucm.es/Tools/antigenic.pl) (23). The SignalP 5.0 Server (http://www.cbs.dtu.dk/services/SignalP/) (24, 25) was used to predict the presence of signal peptides in the proteins; and fragments with excellent immunogenicity were selected and divided into FC, GS, and FCGS combination groups by adding a linker sequence (-GGGGSGGGGSGGGGS-) to tightly combine FC, GS, and FCGS. After the target genes were optimized, we commissioned GENERAL BIOL (Anhui, China) to synthesize the sequences and constructed each tandem sequence (FC, GS, and FCGS) by ligation into the vector pET28a/pET32a (EMD Biosciences, Novagen, San Diego, CA, USA). The Phyre2 software program (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) was used to predict the tertiary structures of the recombinant fusion proteins (26).

Isopropyl β-d-1-thiogalactopyranoside (IPTG; Solarbio, Beijing, China) was used to induce recombinant expression in the strains pET32a-FC-DE3, pET28a-GS-DE3, and pET32a-FCGS-DE3. After cells were collected, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the soluble expression of the target proteins. Target protein purification was performed with a His-Tagged Protein Purification Kit using an inclusion body protein (CWBIO, Beijing, China).

Target protein concentrations were quantified with a Micro BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and Western blotting analysis was used to verify the reactogenicity of the purified proteins. The primary antibody used positive S. aureus/S. agalactiae antibody bovine serum (1:100) that was provided by International Joint Research Center for Animal Health Breeding (Shihezi University) (unpublished). The secondary antibody comprised rabbit anti-bovine IgG/horseradish peroxidase (HRP) (1:2,000; Solarbio, China). A SuperSignal West Femto Trial kit (Thermo, USA) was then used to develop color.

The total lethal levels of S. aureus and S. agalactiae toward BALB/c mice were first determined. S. aureus was cultivated to the order of 2.1 × 10¹⁰ CFU/ml. BALB/c mice were divided into six groups that were intraperitoneally inoculated with 5 μl (1.05 × 10⁸ CFU/mouse), 10 μl (2.1 × 10⁸ CFU/mouse), 15 μl (3.15 × 10⁸ CFU/mouse), 20 μl (4.2 × 10⁸ CFU/mouse), 200 μl (4.2 × 10⁹ CFU/mouse), and 400 μl (8.4 × 10⁹ CFU/mouse) of culture. S. agalactiae was also cultivated to 3.3 × 10¹⁰ CFU/ml, and BALB/c mice were divided into six groups that were intraperitoneally injected with 50 μl (1.65 × 10⁹ CFU/mouse), 100 μl (3.3 × 10⁹ CFU/mouse), 150 μl (4.95 × 10⁹ CFU/mouse), 200 μl (6.6 × 10⁹ CFU/mouse), and 400 μl (1.32 × 10¹⁰ CFU/mouse) of S. agalactiae culture. Six mice were immunized with each dose, and the clinical manifestations and time of death for mice were observed and recorded for seven consecutive days.

The median lethal doses of S. aureus and S. agalactiae toward BALB/c mice were then determined. In these experiments, BALB/c mice were divided into six groups, and each group comprised six mice that were intraperitoneally injected with 16 μl (3.36 × 10⁸ CFU/mouse), 18 μl (3.9 × 10⁸ CFU/mouse), and 20 μl (4.2 × 10⁸ CFU/mouse) of S. aureus culture. In addition, BALB/c mice were divided into six groups, and six mice in each group were intraperitoneally injected with 160 μl (5.28 × 10⁹ CFU/mouse), 180 μl (6.0 × 10⁹ CFU/mouse), and 200 μl (6.6 × 10⁹ CFU/mouse) of S. agalactiae culture. The clinical manifestations and time of death for mice within each group were observed and recorded over 1 week.

The final concentrations of FC, GS, and FCGS proteins were 4.5, 0.13, and 0.3 mg/ml. The proteins were diluted in PBS and thoroughly mixed with a nano-adjuvant in a 1:1 ratio to obtain FC, GS, and FCGS nano-adjuvant vaccines. We previously screened nano-adjuvants to identify immunological effects of the nano-adjuvants compared with ordinary adjuvants (27). BALB/c mice were randomly divided into six groups, with six mice in each group. The FC, GS, and FCGS nano-adjuvant vaccines were subcutaneously injected (300-μl injections with 40 μg of immunogen per mouse) to immunize mice. PBS and an equal volume of nano-adjuvant were emulsified and injected into mice as a control (Figure 1).

Orbital blood sampling was used to collect serum from mice at 7, 14, and 21 days after immunization. Indirect ELISA was then used to detect IgG antibody levels in mouse sera. ELISA coating solution (Solarbio, China) was used to dilute the antigen to a working concentration; and FC (4.5 ng/ml), GS (2.6 ng/ml), and FCGS (3.0 ng/ml) were added to 96-well plates and kept overnight at 4°C. Each well was washed three times with PBST, 5% skim milk was added, and the plates incubated at 37°C for 2 h. The liquid in each well was then discarded, wells were washed with PBST, and mouse serum (1:2,000) was added and again incubated at 37°C for 1 h. After being washed with PBST, rabbit anti-mouse IgG H&L (HRP) (1:5,000) (Abcam, Cambridge, UK) was added to each well and incubated at 37°C for 1 h. After being washed with PBST, a one-component TMB substrate color developing solution (Solarbio, China) was added, and plates were maintained at room temperature in the dark for 15 min. An ELISA stop solution (Solarbio, China) was then added to each well, and the absorbance at 450 nm (OD value) was measured with a microplate reader within 5 min.

At 21-day post-immunization, S. aureus and S. agalactiae were cultured to appropriate concentrations, and the mice were intraperitoneally injected with the half-lethal dose. After the challenge, the appearance and mental condition of mice in each group were evaluated, and mouse deaths were recorded.

At 72 h post-bacterial challenge, mice were sacrificed using CO₂, immersed in 75% ethanol, and dissected under aseptic conditions. Livers and spleens were collected, and the organs were homogenized after adding PBS. Tissue homogenates were diluted to 10⁻¹, 10⁻², and 10⁻³. The homogenates were then spread on BHI Agar (Hopebio, China) and incubated at 37°C for 24–48 h, followed by colony enumeration. Tissue loads were calculated as CFU/g = average number of CFU in the plate × 5 × volume of homogenate (ml) × dilution factor/tissue weight (g). The design and schedule of the study are shown in Figure 1.

Statistical significance in differences and correlation coefficients were calculated with SPSS Statistics 23 program. Student's t-tests, Student–Newman–Keuls (SNK) tests, and one-way ANOVAs were used to compare measurements among groups. All data are presented as means ± SEM, and data represent the results of three independent experiments. The GraphPad Prism software was used to construct figures.

Epitope prediction was used as the basis for evaluating protein immunogenicity; and the epitopes of FnBP, ClfA, Sip, and GapC proteins served as the foundation for constructing and expression fusion proteins. The FnBP, ClfA, and GapC did not have signal peptide, while Sip did (20–40 amino acids in length) (Supplementary Figure 1). FnBP had three potential epitopes (propensity index = 0.9725), ClfA had 19 potential epitopes (propensity index = 0.9986), Sip had 19 potential epitopes (propensity index = 1.0315), and GapC had 17 potential epitopes (propensity index = 1.0260) (Supplementary Figure 2). Thus, the four proteins exhibited good immunogenicity. The tertiary structures of recombinant FC, GS, and FCGS were also predicting using Phyre2 (Supplementary Figure 3).

The fusion protein of S. aureus comprised residues 23–115 of FnBP and 159–282 of ClfA (Figure 2A), while the fusion protein of S. agalactiae comprised residues 183–312 of GapC and 162–338 of Sip (Figure 2B). The fusion protein of S. aureus and S. agalactiae comprised residues 23–115 of FnBP, 356–472 of ClfA, 88–162 of GapC, and 4–97 of Sip (Figure 2C). The amino acid sequences of FC, GS, and FCGS are shown in Supplementary Figure 4.

The recombinant plasmids of each expression vector were transferred into E. coli DE3 competent cells, resulting in the expression of the FC (pET32a-FC) (Figure 3A), GS (pET28a-GS) (Figure 3B), and FCGS (pET32a-FCGS) fusion proteins (Figure 3C). SDS-PAGE analysis indicated that the FCGS, FC, and GS recombinant proteins were all expressed in bacterial inclusion bodies (Figures 3A–C). Post-purification, the purity of FC, GS, and FCGS was >90%, >85%, and >85%, respectively (Supplementary Figure 5). Western blotting analysis confirmed that the target proteins reacted with the positive sera of S. aureus and S. agalactiae and had good reactogenicity (Figure 3D). A semi-quantitative analysis of Western blotting bands was also conducted (Figure 3E).

BALB/c mice displayed clinical symptoms within 12 h after intraperitoneal injection of S. aureus and S. agalactiae. Initial manifestations included a lack of energy, slower movements, reduced eating, reduced drinking, curling up in a corner, and rapid breathing. The mice did not die after 72 h (Figure 4). The total lethal doses were 4.2 × 10⁸ CFU/mouse for S. aureus and 6.6 × 10⁹ CFU/mouse for S. agalactiae (Figures 4A–D). For the LD50 tests, mice showed clinical manifestations similar to total lethal doses, but the symptoms were alleviated, and they were able to crawl slightly. After 72 h, LD50 values were 3.9 × 10⁸ CFU for S. aureus and 6.0 × 10⁹ CFU for S. agalactiae (Figures 4E,F).

The procedure shown in Figure 1 was followed for the sequential study. FC, GS, and FCGS fusion proteins were used to coat the ELISA plates to detect the production of antibodies in mice immunized with the three proteins. On the 14th and 21st days post-immunization, antibody levels in mice induced by the FC recombinant protein were significantly higher than in the control group (p < 0.001) (Figure 5A). It is worth noting that the FC fusion protein rapidly induced antibody production in mice on the seventh day (p < 0.05) (Figure 5A). The FCGS fusion protein-1 group produced significantly higher antibody levels, higher than those of the control group on the 14th day after immunization (p < 0.01), and the difference was extremely significant on the 21st day (Figure 5B). The GS fusion protein induced the mice to produce higher antibody levels than the control group on the 21st day after immunization, but the difference was not significant (Figure 5C). In addition, compared with the control group on the 14th and 21st days after immunization, the FCGS protein-2 group had significant difference (p < 0.01) (Figure 5D). Thus, the antibody levels induced by the FCGS fusion protein were higher than those due to the FC and GS recombinant proteins.

Standard S. aureus and S. agalactiae strains were used to challenge mice 21 days after immunization. The FC protein conferred good resistance to S. aureus, and the GS protein was effective against S. agalactiae. Significant resistance was also conferred by FCGS protein to S. agalactiae and S. aureus (Table 1).

At 72 h post-bacterial challenge, the bacterial loads in the spleens and livers were enumerated. Mice immunized with the FCGS and GS recombinant proteins withstood challenges with S. agalactiae, as indicated by fewer bacteria isolates compared with the control group (Figures 6A,B). The FCGS and FC recombinant proteins also conferred resistance to S. aureus after immunization compared with the control group, as evinced by significantly lower numbers of isolated colonies (Figures 6C,D). These results were consistent with FC, GS, and FCGS recombinant protein antibody levels (Figure 5) and challenge protection efficiency (Table 1), indicating that the FC, GS, and FCGS recombinant proteins can cause specific immune responses in mice. Thus, FC immunization was effective against S. aureus, and GS immunization was effective against S. agalactiae infection, while FCGS immunization was effective toward challenges of both S. aureus and S. agalactiae.