MOLECULAR MEDICINE REPORTS 15: 2369-2373, 2017 Protection against Staphylococcus aureus and tetanus infections by a combined vaccine containing SasA and TeNT‑Hc in mice YILONG YANG*, RUI YU*, XIUXU YANG, SHULING LIU, TING FANG, XIAOHONG SONG, LIHUA HOU, CHANGMING YU, JUNJIE XU, LING FU, SHAOQIONG YI and WEI CHEN Laboratory of Vaccine and Antibody Engineering, Beijing Institute of Biotechnology, Fengtai, Beijing 100071, P.R. China Received December 11, 2015; Accepted December 19, 2016 DOI: 10.3892/mmr.2017.6227 Abstract. In developing countries, trauma patients and Introduction neonates are vulnerable to Staphylococcus aureus (S. aureus) and Clostridium tetani infections. It has been suggested that a Clinical trauma patients are frequently diagnosed with combined vaccine against the two infections may be a reliable Staphylococcus aureus (S. aureus) and tetanus infections, and cost-effective strategy. Previous studies have indicated that particularly those in developing countries (1,2). These infec- the S. aureus surface protein A (SasA) and the C fragment of tions have contributed greatly to the morbidity and mortality tetanus neurotoxin (TeNT-Hc) may be suitable candidates for a rates of neonates in developing countries such as Nigeria (3). vaccine against S. aureus and tetanus infections, respectively. S. aureus colonizes in approximately one-third of the human In the present study, mice were immunized with a combined population (4), and is responsible for various diseases ranging vaccine containing SasA and TeNT-Hc, which induced a robust from skin and soft tissue infections to life-threatening septi- immune response to both antigens, and mutual interference cemia with metastatic complications (5). Tetanus is caused by between SasA and TeNT-Hc was not observed. In the S.aureus tetanospasmin, a neurotoxin produced by the obligate anaer- challenge model, the combined vaccine fully protected obic bacterium Clostridium tetani. Although the incidence of BALB/c mice against lethal intraperitoneal challenges with tetanus is low in developed countries, the worldwide mortality 3x109 colony-forming units of a methicillin-resistant S. aureus rates range from 6-72% (6), depending on the medical condi- USA300 strain. In the TeNT challenge model, the combined tion. In 2013, the World Health Organization estimated that vaccine conferred complete protection against a lethal dose of ~49,000 newborns died from neonatal tetanus (7). (2x103) xLD50 tetanus toxin. These results implied that SasA Combination vaccines are a reliable strategy to protect and TeNT-Hc promising components for a combined vaccine against two or more pathogens, and may reduce costs of against S. aureus and tetanus infections. mass vaccination and disposables (8). Those susceptible to trauma, including athletes, soldiers and the police, as well as women of childbearing age in developing countries, may benefit from the development of a combined vaccine against S. aureus and tetanus infections. S. aureus surface protein A (SasA), a cell wall-anchored protein of S. aureus, consisting Correspondence to: Professor Wei Chen or Dr Shaoquiong Yi, of 2,271 amino acid residues, is a potential vaccine candidate Laboratory of Vaccine and Antibody Engineering, Beijing Institute for S. aureus infections (9). SasA has been reported to be a of Biotechnology, 20 Dongdajie Street, Fengtai, Beijing 100071, virulence determinant in endovascular infections, as the SasA P. R. Ch i na mutant strain was observed to decrease the ability of catheter- E-mail: [email protected] ized rabbits to form vegetative plaques on heart valves (10). E-mail: [email protected] In addition, SasA is prevalent in S. aureus clinical isolates, and is expressed during in vivo growth of the bacteria (11). * Contributed equally Notably, immunization with SasA was able to protect mice against a lethal S. aureus challenge (9). The tetanus toxin is Abbreviations: S. aureus, Staphylococcus aureus; MRSA, a 150 kDa protein that has three domains (12), including the Methicillin-resistant S. aureus; IsdB, iron-responsive surface N-terminal zinc endopeptidase domain (13), the internal heavy determinant B; TeNT-Hc, C fragment of tetanus neurotoxin; SasA, Staphylococcus aureus surface protein A; ELISA, enzyme-linked chain translocation domain and the C-terminal heavy chain immunosorbent assay; TMB, 3,3',5,5'-tetramethylbenzidine dihydro- receptor-binding domain known as the C-fragment of tetanus chloride neurotoxin (TeNT-Hc) (14). The genetic recombinant TeNT-Hc, which is nontoxic but has ganglioside binding activities, has Key words: vaccine, Staphylococcus aureus, tetanus neurotoxin, been proposed as a possible replacement for the tetanus toxoid protective immunity trauma vaccine (15). TeNT-Hc is expected to demonstrate clear advan- tages over the existing tetanus toxoid vaccine with regard to ease of production, homogeneity and characterization. 2370 YANG et al: COMBINED VACCINE AGAINST BOTH S. aureus AND TETANUS In the present study, mice were immunized with a combined Millipore) in PBS at 37˚C for 1 h. Duplicate two‑fold serial vaccine consisting of SasA (9) and TeNT-Hc (16) formulated dilutions of serum in an appropriate range (1:100~1:409,600) by adsorption to Alhydrogel. The antibody titers and protec- were incubated in the plates at 37˚C for 1 h followed by tive efficacy were measured for individual and combined washing with PBS with 3% Tween 20 (PBST). Then horse- vaccine administrations. The results demonstrated that the radish peroxidase-labelled goat anti-mouse total IgG, IgG1 combined vaccine was able to induce protective immunity (ab97240; 1:10,000) or IgG2a (ab97245; 1:10,000) antibodies against S. aureus and tetanus neurotoxin challenges. (Abcam, Cambridge, MA, USA) were applied for 1 h at 37˚C and then washed with PBST. The plates were incubated with Materials and methods 3,3',5,5'-tetramethylbenzidine dihydrochloride substrate (Sigma-Aldrich; Merck Millipore) at room temperature for Bacterial strains and growth conditions. The S. aureus strain 10 min in the dark. The colorimetric reaction was stopped with USA300 (BAA-1556™; American Type Culture Collection, 2 M sulfuric acid, and the optical density at 450 nm (OD450) Manassas, VA, USA) was cultivated in tryptic soy broth (TSB; was read in a microplate reader (Bio-Rad Laboratories, Inc., tryptone 15 g/l, soybean peptone 5 g/l, NaCl 5 g/l, pH 7.2, used Hercules, CA, USA). Each step was followed by washing after 121˚C, 20 min) at 37˚C. The Escherichia coli (E. coli) three times with PBST. The antibody-positive cut-off values strain BL21 (DE3; Merck Millipore, Darmstadt, Germany) were set as two times greater than the OD450 means of the were cultivated in Luria-Bertani broth (tryptone 10 g/l, yeast pre-immunized sera. The ELISA antibody titer was expressed extract 5 g/l, NaCl 10 g/l, pH 7.2, used after 121˚C, 20 min) as the highest serum dilution showing a positive reaction. at 37˚C and 100 µg/ml ampicillin (10 g ampicillin powder dissolved in 100 ml H2O, used after filtration and diluted S. aureus challenge model. Overnight cultures of S. aureus 1:1,000) was used for plasmid selection. strain USA300 were diluted 1:100 in fresh TSB and cultivated at 37˚C until reaching the mid‑late logarithmic phase. S. aureus Subunit antigens. The antigens used in the current study challenge was performed as described previously (17). were expressed in E. coli BL21 (DE3) and purified to >90%. Immunized mice were challenged by intraperitoneal injection The purification protocols were the same as described previ- with a lethal dose of S. aureus USA300 (3x109 colony-forming ously (9,16). Briefly, the polyhistidine-tagged recombinant units in 100 µl/mouse), at 6 weeks after the primary immuniza- SasA (rSasA) was purified by anion exchange, HisTrap and tion. Infected animals were monitored for survival for 5 days. gel filtered chromatography (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The non-tagged TeNT-Hc antigen was TeNT challenge model. At 6 weeks following primary purified by anion exchange, hydrophobic interactions and immunization, the mice were challenged with (2x103)xLD50s gel‑filtered chromatography (GE Healthcare Bio‑Sciences). (the LD50 in mice was determined by the improved Karber method (18). The LD50 of tetanus neurotoxin was ~15.8 ng/kg Active immunization. A total of 40 female BALB/c mice in mice) of tetanus neurotoxin [in 0.5 ml borate-buffered saline (age, 6-8 weeks; weight, 18-20 g) were purchased from The (0.5 g borax, 4.5 g boric acid and 8.5 g sodium chloride in Beijing Laboratory Animal Center (Beijing, China), and were 1 l distilled water)] by subcutaneous injection. Survival was divided into 4 groups of 10 mice each. All mice had free monitored for five days. access to food/water and were raised under standard condi- tions (temperature 25±2˚C, relative humidity 50±10%) with a Statistical analysis. Unpaired Student's two-tailed t-test was dark/light cycle (14/10 h). The mice were immunized intra- used to analyze the differences in ELISA titers between the peritoneally with 10 µg rSasA, 10 µg TeNT‑Hc, 10 µg rSasA groups. To compare the survival rates in the challenge models, + 10 µg TeNT‑Hc adsorbed to 0.75 mg aluminum hydroxide experiments were analyzed using the Gehan-Breslow-Wilcoxon adjuvant (Brenntag Biosector A/S, Frederikssund, Denmark). test with GraphPad Prism 5.01 (GraphPad Software, Inc., La The 0.75 mg aluminum hydroxide adjuvant was used as a Jolla, CA, USA). P≤0.05 was considered to indicate a statisti- negative control. Mice were injected at weeks 0, 2 and 4. Blood cally significant difference. samples were drawn at weeks 2, 4 and 6 and every 4 weeks thereafter until week 26 through the tail vein and screened for Results reactivity to rSasA or TeNT-Hc antigens. Subsequent to the experiments, all mice were sacrificed by CO2 asphyxiation. Antibody titer following immunization.