Preparation and In Vitro Evaluation of Antitumor Activity of TGFαL3-SEB as a Ligand-Targeted Superantigen.

Tumor-targeted superantigens (TTSs) have been used to treat a variety of tumors in preclinical studies. The TTS utilizes the powerful T-cell activation strategy by means of staphylococcal enterotoxins (SEs) as superantigens (Sags) to target tumor cells. Monoclonal antibodies and tumor-related ligands have been used as targeting molecules of Sag. In this study, we assessed the antitumor potency of tumor-targeted superantigen (TTS) strategy to design and produce fusion protein as a new antitumor candidate. The third loop (L3) of transforming growth factor α (TGF-α) was genetically conjugated to staphylococcal enterotoxin type B (TGFαL3-SEB), and its in vitro antitumor activity against murine breast cancer cells (A431 cell line) was evaluated. We designed and prepared TGFαL3-SEB chimeric protein and evaluated superantigenic activity, binding property to cancer cells, overexpression of epidermal growth factor receptor (EGFR), and in vitro antitumor activities. Cloning of tgfαl3-seb was confirmed by colony-polymerase chain reaction, enzymatic digestion, and sequencing. The recombinant TGFαL3-SEB fusion protein with molecular weight of 31 kDa was expressed and confirmed by anti-His Western-blot analysis. The TGFαL3-SEB fusion protein attached to A431 cell line with proper affinity and induced dose-dependent cytotoxicity against EGFR-expressing cancer cells in vitro. The TGFαL3-SEB chimeric protein exhibited potent in vitro antitumor activity. Our findings indicated that TGFαL3-SEB may be a promising anticancer candidate in cancer immunotherapy, and further studies are required to explore its potential in vivo therapeutic applications.

The distribution of pathogenic and toxigenic genes among MRSA and MSSA clinical isolates.

Staphylococcus aureus (S. aureus) is considered as a notorious nosocomial pathogen among hospitalized patients and community-dwelling subjects. Its increasing morbidity and mortality is believed to be due to antibiotic resistance. However, the data concerning molecular properties of infecting strains are few. In this study, a total of 192 S. aureus strains, including 88 (45.8%) meticillin-sensitive S. aureus (MSSA) and 104 (54.2%) meticillin-resistant S. aureus (MRSA) were recovered from clinical samples. The prevalence of subtypes containing staphylococcal cassette chromosome mec (SSCmec), staphylococcal enterotoxins (SEs), toxic shock syndrome toxin (TSST) and exfoliative toxin was assessed by PCR. Antibiotic susceptibility pattern and vancomycin resistance of each isolate were evaluated by disk diffusion method and micro-dilution method, respectively. 9 (2.3%) strains required MIC > 2 mg/l of vancomycin, which significantly increased among multi drug resistant (MDR), MRSA and SCCmec type III strains (p < 0.05). 171 (89%), 140 (72.91%), 7 (3.6), 78 (48.6%), 5 (2.6%), 151 (78.64%), 129 (67.18%), 178 (92.7%) and 15 (7.8%) of 192 isolates harbored mecA, entA, entB, entC, entD, entE, eta, etb and tsst-1 genes, respectively. 31 (16.14%), 5 (2.6%), 95 (49.48%) and 7 (3.64%) of 192 isolates carried SCCmec type I, II, III and IV, respectively. We found a significantly higher rate of MRSA and resistance to all tested antibiotics, except to penicillin G, kanamycin and linezolide among the SCCmec type III class (p < 0.05). According to our findings, MSSA isolates should be taken as seriously as MRSA strains due to the potential presence of broad spectrum virulence factor genes.

In silico analysis for identifying potential vaccine candidates against Staphylococcus aureus

PURPOSE: Staphylococcus aureus is one of the most important causes of nosocomial and community-acquired infections. The increasing incidence of multiple antibiotic-resistant S. aureus strains and the emergence of vancomycin resistant S. aureus strains have placed renewed interest on alternative means of prevention and control of infection. S. aureus produces a variety of virulence factors, so a multi-subunit vaccine will be more successful for preventing S. aureus infections than a mono-subunit vaccine. MATERIALS AND METHODS: We selected three important virulence factors of S. aureus, clumping factor A (ClfA), iron-regulated surface determinant (IsdB), and gamma hemolysin (Hlg) that are potential candidates for vaccine development. We designed synthetic genes encoding the clfA, isdB, and hlg and used bioinformatics tools to predict structure of the synthetic construct and its stabilities. VaxiJen analysis of the protein showed a high antigenicity. Linear and conformational B-cell epitopes were identified. RESULTS: The proteins encoded by these genes were useful as vaccine candidates against S. aureus infections. CONCLUSION: In silico tools are highly suited to study, design, and evaluate vaccine strategies.

EspA-Intimin chimeric protein, a candidate vaccine against Escherichia coli O157:H7.

BACKGROUND AND OBJECTIVE: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important enteric pathogen in human causing bloody or nonbloody diarrhea, which may be complicated by hemolytic uremic syndrome (HUS). Cattle are an important reservoir of EHEC. This research aims at vaccination with a divalent chimer protein composed of EspA120 and Intimin 282 and its preventive effect of EHEC O157 colonization in mice rectal epithelium. MATERIALS AND METHODS: A divalent recombinant EspA-Intimin (EI) protein containing EspA120 and Intimin280 attached with a linker was amplified from a trivalent construct and cloned in pET-28a (+) vector. The immunization was conducted in mice after expression and purification of the recombinant EI (rEI). RESULTS: Mice subcutaneously immunized with rEI, elicited significant rEI specific serum IgG antibodies and showed significantly decreased E.coli O157:H7 shedding compared to the control group. CONCLUSION: The chimeric recombinant protein induced strong humoral response as well as protection against oral challenges with live E.coli O157:H7.