The S. aureus strain USA300 (BAA-1556™; American Type Culture Collection, Manassas, VA, USA) was cultivated in tryptic soy broth (TSB; tryptone 15 g/l, soybean peptone 5 g/l, NaCl 5 g/l, pH 7.2, used after 121°C, 20 min) at 37°C. The Escherichia coli (E. coli) strain BL21 (DE3; Merck Millipore, Darmstadt, Germany) were cultivated in Luria-Bertani broth (tryptone 10 g/l, yeast extract 5 g/l, NaCl 10 g/l, pH 7.2, used after 121°C, 20 min) at 37°C and 100 µg/ml ampicillin (10 g ampicillin powder dissolved in 100 ml H₂O, used after filtration and diluted 1:1,000) was used for plasmid selection.

The antigens used in the current study were expressed in E. coli BL21 (DE3) and purified to >90%. The purification protocols were the same as described previously (9,16). Briefly, the polyhistidine-tagged recombinant SasA (rSasA) was purified by anion exchange, HisTrap and gel filtered chromatography (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The non-tagged TeNT-Hc antigen was purified by anion exchange, hydrophobic interactions and gel-filtered chromatography (GE Healthcare Bio-Sciences).

A total of 40 female BALB/c mice (age, 6–8 weeks; weight, 18–20 g) were purchased from The Beijing Laboratory Animal Center (Beijing, China), and were divided into 4 groups of 10 mice each. All mice had free access to food/water and were raised under standard conditions (temperature 25±2°C, relative humidity 50±10%) with a dark/light cycle (14/10 h). The mice were immunized intraperitoneally with 10 µg rSasA, 10 µg TeNT-Hc, 10 µg rSasA + 10 µg TeNT-Hc adsorbed to 0.75 mg aluminum hydroxide adjuvant (Brenntag Biosector A/S, Frederikssund, Denmark). The 0.75 mg aluminum hydroxide adjuvant was used as a negative control. Mice were injected at weeks 0, 2 and 4. Blood samples were drawn at weeks 2, 4 and 6 and every 4 weeks thereafter until week 26 through the tail vein and screened for reactivity to rSasA or TeNT-Hc antigens. Subsequent to the experiments, all mice were sacrificed by CO₂ asphyxiation. All experiments were performed in agreement with the institutional guidelines approved by the Laboratory Animal Care and Use Committee of the Beijing Institute of Biotechnology (IACUC of AMMS-08-2014-006).

Serum antibody titers of the antigen-specific total IgG, IgG1 and IgG2a were determined by enzyme-linked immunosorbent assay (ELISA). Microplates (96-well) were coated overnight with rSasA or TeNT-Hc (2 µg/ml) in coating buffer (50 mM carbonate buffer, pH 9.6) at 4°C. The plates were blocked with 2% (w/v) bovine serum albumin (2 g dissolved in 100 ml H₂O; Sigma-Aldrich; Merck Millipore) in PBS at 37°C for 1 h. Duplicate two-fold serial dilutions of serum in an appropriate range (1:100~1:409,600) were incubated in the plates at 37°C for 1 h followed by washing with PBS with 3% Tween 20 (PBST). Then horseradish peroxidase-labelled goat anti-mouse total IgG, IgG1 (ab97240; 1:10,000) or IgG2a (ab97245; 1:10,000) antibodies (Abcam, Cambridge, MA, USA) were applied for 1 h at 37°C and then washed with PBST. The plates were incubated with 3,3′,5,5′-tetramethylbenzidine dihydrochloride substrate (Sigma-Aldrich; Merck Millipore) at room temperature for 10 min in the dark. The colorimetric reaction was stopped with 2 M sulfuric acid, and the optical density at 450 nm (OD₄₅₀) was read in a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Each step was followed by washing three times with PBST. The antibody-positive cut-off values were set as two times greater than the OD₄₅₀ means of the pre-immunized sera. The ELISA antibody titer was expressed as the highest serum dilution showing a positive reaction.

Overnight cultures of S. aureus strain USA300 were diluted 1:100 in fresh TSB and cultivated at 37°C until reaching the mid-late logarithmic phase. S. aureus challenge was performed as described previously (17). Immunized mice were challenged by intraperitoneal injection with a lethal dose of S. aureus USA300 (3×109 colony-forming units in 100 µl/mouse), at 6 weeks after the primary immunization. Infected animals were monitored for survival for 5 days.

At 6 weeks following primary immunization, the mice were challenged with (2×103)xLD50s (the LD50 in mice was determined by the improved Karber method (18). The LD50 of tetanus neurotoxin was ~15.8 ng/kg in mice) of tetanus neurotoxin [in 0.5 ml borate-buffered saline (0.5 g borax, 4.5 g boric acid and 8.5 g sodium chloride in 1 l distilled water)] by subcutaneous injection. Survival was monitored for five days.

Unpaired Student's two-tailed t-test was used to analyze the differences in ELISA titers between the groups. To compare the survival rates in the challenge models, experiments were analyzed using the Gehan-Breslow-Wilcoxon test with GraphPad Prism 5.01 (GraphPad Software, Inc., La Jolla, CA, USA). P≤0.05 was considered to indicate a statistically significant difference.

A total of 3 groups of 10 mice were intraperitoneally immunized three times (at weeks 0, 2 and 4) with 10 µg rSasA, 10 µg TeNT-Hc or 10 µg rSasA + 10 µg TeNT-Hc. Blood samples were collected periodically through the tail vein to assay antigen-specific antibody responses by ELISA. The responses were compared between the groups over 26 weeks (Fig. 1). Detectable levels of IgG against rSasA and TeNT-Hc antigens were observed at 2 weeks following primary immunization, and the titers were maintained over 6 months.

No significant differences in TeNT-Hc specific IgG titers were observed between the TeNT-Hc immunized group and TeNT-Hc + SasA immunized group over the 26 weeks (Fig. 1A), except for the week 6, when the combined vaccine induced anti-TeNT-Hc IgG titers 1.683-fold higher than TeNT-Hc (P=0.0313). Significant differences in rSasA specific IgG titers were observed between the rSasA immunized group and the TeNT-Hc + SasA immunized group in weeks 2, 6 and 10 (Fig. 1B). The combined vaccine induced rSasA specific IgG titers 3.083-fold higher than rSasA alone at week 2 (P=0.0210). However, at weeks 6 and 10, the combined vaccine induced rSasA specific IgG titers 3.999 times (P=0.0032) and 2.377 times (P=0.0323) lower than rSasA alone, respectively.

Serum samples from immunized mice at week 6 were assayed for the presence of antigen-specific IgG1 and IgG2a antibodies by ELISA. rSasA and the combined vaccine induced the robust production of IgG1 and IgG2a antibodies specific to rSasA (Fig. 2). No significant differences in rSasA IgG1 and IgG2a titers were observed between the rSasA and rSasA + TeNT-Hc groups. Similar results were observed from TeNT-Hc-immunized mice and the combined vaccine-immunized mice (Fig. 2). The combined vaccine induced specific IgG1 and IgG2a titers comparable to rSasA or TeNT-Hc vaccines. Additionally, the antigen-specific IgG1/IgG2a ratio was not influenced by co-administration of the two antigens (data not shown).

Immunized mice were challenged by intraperitoneal inoculation with 3×109 colony-forming units of S. aureus USA300, 6 weeks following primary immunization. Approximately 90% of mice in the control group died within 24 h (Fig. 3A). By contrast, immunized mice survived for 120 h post challenge. All mice immunized with 10 µg rSasA or 10 µg rSasA+10 µg TeNT-HC survived the challenge with symptoms of infection such as temporary leg paralysis.

The mice vaccinated three times with 10 µg TeNT-Hc or 10 µg rSasA + 10 µg TeNT-Hc were challenged with tetanus neurotoxin, and its protective abilities were evaluated. When challenged with 2×103 LD50s of tetanus neurotoxin, TeNT-Hc and the combined vaccine provided excellent protection; 9 mice vaccinated by TeNT-Hc survived and 1 mouse died from tetanus poisoning. All mice vaccinated by 10 µg rSasA + 10 µg TeNT-Hc survived without any observed symptoms (Fig. 3B). However, all mice immunized with aluminum hydroxide adjuvant alone did not survive at 24 h post-challenge.