Identification Of Active Components In Staphylococcin Injection And Biological Properties Of Recombinant Staphylococcal Enterotoxins

Staphylococcal enterotoxins (SE) belong to a representative group of superantigens secreted by Gram-positive bacteria Staphylococcus aureus. Currently more than seventeen SEs have been identified, including classical SEs and several newly described serological types. Unlike conventional protein antigens, SEs, when intact, could interact simultaneously with MHC-II molecules and T cell receptors. As the most powerful T cell mitogen, SEs can elicit massive T-cell proliferation and cytokine release both in vivo and in vitro even at a concentrations as low as pg-ng/mL, which suggests their use in immunotherapy of human malignant diseases. From the results of several preclinical studies and early-phase clinical trials, engineered antibody-targeted staphylococcal enterotoxins seemed to be desirable candidates for anti-tumor agents.In China, staphylococcal enterotoxins C2 (SEC2) is claimed as the main effective component in staphylococcin injection, which is prepared from the filtrate of fermentation broth of Staphylococcus aureus and commonly used as biological response modifier in cancer therapy. The immunomodulatory properties of staphylococcin injection were confirmed in many clinical reports. The injection could induce strong increase in CD4~+ cells, CD4~+/CD8~+ T cell ratio and percentage of NK cells in the treated patients. In addition the side effects correlated with chemotherapy or radiation therapy, such as gastrointestinal tract reaction and bone marrow suppression could be significantly reduced in the injection-treated patients. Short-term efficacy and long-term survival benefit provided by the staphylococcin injection are important for the patients with malignant disease. On the other hand, adverse events were encountered in approximately 20-40% of the patients treated with the injection. The most frequent side effect was mild to moderate fever. Local side effects at the injection site such as pain, swelling and redness also commonly occurred in the injection-treated patients.In this study, 1-D gel electrophoresis coupled with nano-LC-MS/MS was performed to identify the protein components in the staphylococcal injection products from different manufacturing companies ( Shenyang Xiehe Group Co., Ltd., Zhejiang provincial Yaojiang pharmaceutical Co., Ltd. and Hangzhou Guoguang Pharmaceutical Co., Ltd. The names of the manufacturing companies were replaced by A, B or C in the study on behalf of the companies, and there's no relationship between the order of the company names and A, B or C ) . The results showed more than seventy proteins from Staphylococcus aureus and other Gram-positive bacteria were confidently identified from the staphylococcin injection products from manufacturing company A; eighteen proteins from Staphylococcus aureus and other Gram-positive bacteria were confidently identified from the staphylococcin injection products from manufacturing company B and twelve proteins from Staphylococcus aureus and other Gram-positive bacteria were confidently identified from the staphylococcin injection products from manufacturing company C.In addition, the gene distribution of staphylococcal enterotoxins in one industrial strain of the injection from one manufacturing company (the name of the company was concealed on behalf of the company) was analyzed by PCR method. The results showed that seven enterotoxin genes (seg, sei, sek, sem, sen, seo and seq) were harbored by the genomic DNA of the industrial strain. Further, the genes of those staphylococcal enterotoxins along with sea and seb were cloned and corresponding recombinant staphylococcal enterotoxins were produced and purified. The in vitro superantigenicities of these recombinant staphylococcal enterotoxins were analyzed and the results showed that those recombinant enterotoxins could induce strong stimulatory effect on proliferation and inhibition effect on tumor cells of murine splenocytes.In this study, the murine monoclonal antibody and rabbit polyclonal antibody against recombinant staphylococcal enterotoxin I were produced by immunizing Balb/c mouse and New Zealand rabbit with recombinant staphylococcal enterotoxin I, respectively. The ELISA method for the detection and quantification for staphylococcal enterotoxin I was established using the newly-developed murine monoclonal antibody and rabbit polyclonal antibody. The operation conditions of the ELISA method were optimized. The detection linear range of the system was 0.2 - 7.8ng/mL. The intra-day precision and accuracy was 5.1-12.5% and 82.0-112.0%, respectively. The inter-day precision and accuracy was 5.7-13.6% and 88.0-96.5%, respectively. No cross reaction was observed when other rSEs were tested by the system.On the other hand, SEs are the only superantigens possessing emetic properties. Extensive epidemiological studies have demonstrated that SEA, SEB and SED are the most three important toxins responsible for staphylococcal food poisoning (SFP). However, little is known about the fate of these toxins in the digestive tract after ingestion and how SEs cause the symptoms of SFP. In addition, correlation between their emetic and superantigenic activities remains unclear. The potential transcytosis of the recombinant His-SEC2 was evaluated by using Caco-2 cell model and the established ELISA system for detection of SEC2. The results showed that the recombinant His-SEC2 could be transcytosed by Caco-2 cell monolayer in an intact form, and the amount of transcytosed His-SEC2 was significant higher than that of HRP both from apical side to basolateral side or from basolateral side to apical side in 24 hr.1. Identification of active components in the staphylococcin injection products1.1 SDS-PAGE analysis and in-gel digestion The staphylococcal injection solution from three manufacturing companies was concentrated approximately 40-fold by ultrafiltration and analyzed by SDS-PAGE. After visualization of the gel, the gel lane was divided into several sections based on band intensity and then cut into small pieces. All gel pieces were placed into small eppendorf tubes for in-gel digestion and manual extraction.1.2 Nano-LC-MS/MS analysisSamples were reconstituted in loading buffer and sonicated prior to nano-LC-MS/MS analysis. The MS/MS spectra generated by the hybrid triple quadrupole linear ion trap mass spectrometer were submitted to Mascot 1.9 for database searching against the NCBI firmicutes database.2. Detection of staphylococcal enterotoxins genes in the genomic DNA of industrial strain of staphylococcin injection and production and superantigenicity of several recombinant staphylococcal enterotoxins2.1 Detection of staphylococcal enterotoxins genes and production of recombinant staphylococcal enterotoxinsThe gene distribution of staphylococcal enterotoxins in the genomic DNA of one industrial strain from one manufacturing company was analyzed by PCR method. The expression vectors for recombinant staphylococcal enterotoxins were constructed by cloning the gene fragments amplified by PCR reactions into pGEX-4T-1 vectors. The recombinant GST-tagged staphylococcal enterotoxins were produced by Escherichia coli BL21 and purified by affinity chromatography. The GST-tag was then released by thrombin digestion from the fusion protein and the recombinant staphylococcal enterotoxin was purified by anion ion-exchange chromatography.2.2 The superantigenicities of recombinant staphylococcal enterotoxinsStaphylococcal enterotoxin-induced proliferation of murine lymphocytes was measured by MTT assay. The degree of proliferation rates of the lymphocytes induced by the recombinant proteins were presented as stimulation index. Staphylococcal enterotoxin-induced cellular cytotoxicity of murine lymphocytes was also assessed by MTT method. K562-ADM cells were served as target cells and murine splenic lymphocytes were used as effector cells. In addition, rSEI-mediated celluar cytotoxicity of murine lymphocytes was also measured by the RT-CES system. A549 cells were served as target cells and murine splenic lymphocytes were used as effector cells.3. Establishment of immunoassay for SEI3.1 Production of rabbit polyclonal antibodies and murine monoclonal antibodies against SEINew Zealand rabbits were immunized subcutaneously on the back with purified recombinant SEI over a period of approximate 8 weeks and the anti-sera was collected. Balb/c mice were injected subcutaneously on the back with purified recombinant SEI and the animals were boosted with recombinant SEI three days before cell fusion. The splenic lymphocytes obtained from the immunized mice were fused with murine SP2/0 myeloma cells using PEG 4000. The hybridoma cells capable of producing antibodies against recombinant SEI were screened by indirect ELISA and cloned by limiting dilution. Positive hybridoma cells were further cultured injected intraperitoneally to Balb/c mice pretreated with paraffin oil. Ascite fluid was collected from the mice 10-14 days later and the monoclonal antibodies against recombinant SEI were purified with caprylic acid - ammonium sulfate method.3.2 Establishment of ELISA systemTwo procedures were investigated: (1) use of plate coated with rabbit polyclonal antibodies and murine monoclonal antibodies as secondary antibody and (2) use of plate coated with murine monoclonal antibodies and rabbit polyclonal antibodies as secondary antibody. The ELISA assay was optimized by choosing various dilutions of coating antibody, secondary antibody and other operation conditions.4. Transcytosis of His-tagged SEC2 The ability of Caco-2 cells to transcytose recombinant His-tagged SEC2 was tested using confluent monolayers grown on Transwell filters. His-SEC2 was added to either the apical or basal chamber, horseradish peroxidase was added as an internal control for nonspecific transcytosis. After incubation, apical or basal media were collected depending on whether His-SEC2 was added to the basal or apical media. The established ABS-ELISA system was used to determine amounts of His-SEC2 present in sample solution and the protein was also detected using western-blotting method.Conclusion:In this study, LC-MS coupled with SDS-PAGE was performed to identify protein components in three staphylococcin injection products. The results showed that many types of proteins from S.aureus and other Gram-positive bacteria were confidently identified. Investigation of the relationship between those identified proteins and the clinical efficacy or toxicities of the staphylococcin injection is required in future. In addition, the manufacturing process as well as the standard quality control of injection preparation should be improved to avoid bacteria contamination, reduce impurities and increase clinical efficacies. In this study, the gene distribution of staphylococcal enterotoxins of one industrial strain from one manufacturing company was analyzed, and the results demonstrated that seven se genes were harbored by the strain, indicating that one or a plurality of them may be present in the related staphylococcin injection products. Several recombinant staphylococcal enterotoxins, including those seven staphylococcal enterotoxins, were produced using gene engineering approaches. Results showed that the superantigenicity of those recombinant staphylococcal enterotoxins were close to that of native staphylococcal enterotoxin C2. Thus, recombinant staphylococcal enterotoxins provide a new approach for the production of next-generation staphylococcin injection. In this study, murine monoclonal and rabbit polyclonal antibodies against SEI were produced. Using the newly-developed antibodies, sandwich ELISA system for the detection of SEI at concentration of ng/mL was established, which could be used in the clinical diagnosis of SEI-related diseases and development of SEI-related drugs in future.The fate and mechanism of enterotoxic activities of SEs in human gastrointestinal tract remains to be determined. In this study, the transcytosis of recombinant His-SEC2 was preliminarily investigated. The results indicated that the recombinant His-SEC2 could specifically cross the Caco-2 cell monolayer in an intact form, indicating that SEC2 and other serological types of staphylococcal enterotoxins could cross the intestinal epithelial cells and then cause local or systemic symptoms. In addition, the results also have implications for the development of oral drugs in which staphylococcal enterotoxins are used as the main effective components.In conclusion, in this study, the protein components in the staphylococcin injection products and the superantigenic activities of recombinant staphylococcal enterotoxins were preliminarily investigated. In addition, the immunoassay for SEI was also established and the transcytosis of recombinant His-SEC2 was preliminarily investigated. The study would shed light into the future research on the staphylococcal enterotoxins...