Immunogenicity Evaluation of Recombinant Staphylococcus aureus Enterotoxin B (rSEB) and rSEB-loaded Chitosan Nanoparticles Following Nasal Administration.

Staphylococcal enterotoxin B (SEB), apotent superantigen, is responsible for many disorders caused by Staphylococcus aureus. With regard to the appearance of multidrug-resistant strains of the bacteria, there is a great need to develop an efficient vaccine against this pathogen. In the present study, the immunogenicity of recombinant SEB was evaluated following nasal administration to BABLB/c mice. Indeed, the rSEB protein was entrapped into chitosan nanoparticles and the immunogenicity of nano-formulation was investigated. SEB protein was expressed in E. coli BL21 (DE3) and purified by using a nickel column. Chitosan nanoparticles were synthesized in the presence of rSEB; using the ionic gelation technique. Synthesized NPs containing rSEB and bare rSEB were administered to mice nasally. Serum and stool IgG and IgA antibody showed that both formulations were able to evoke the mice's immune responses and there was no significant difference between them. Results of the toxin neutralization test on Vero cells indicated that the sera of the immunized mice had an inhibitory effect on the growth of these cells (p<0.001). Nasal administration of bare rSEB could efficiently simulate the mice's immune system and nano-delivery of this protein via nasal route had not a significant impact on its immunogenicity improvement.

Production of egg yolk antibody (IgY) against shiga-like toxin (stx) and evaluation of its prophylaxis potency in mice.

OBJECTIVE: Enterohemorrhagic Escherichia coli (E. coli O157: H7) is an enteric pathogen, transmitted through contaminated water and food. Pathogenic factors include bacterial adhesion, invasion of intestinal epithelial and epithelium cells. The pathogenicity of EHEC is due to the production of Shiga-like toxin (Stx). This toxin binds to the ribosome and inhibits the synthesis of proteins. EHEC causes hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). The EHEC treatment with antibiotics leads to resistance. The best way to solve this problem is to use specific antibodies and prophylaxis. Egg yolk antibody (IgY) is a suitable method for prophylaxis. Hence, the aim of this study was to investigate the production of IgY against Stx toxin and its prophylaxis. RESULT: The produced antibodies were confirmed by SDS-PAGE and ELISA. IgY was obtained at a concentration of about 5 mg/ml (30 mg of each egg) and a purity of more than 90%. Toxin and antibody challenge was performed in mice. The obtained IgY was able to neutralize the effect of Stx at 2 mg/mice. CONCLUSION: This challenge showed that an antibody produced with an acceptable percentage was able to neutralize the effect of Stx.

Immunogenicity evaluation of rBoNT/E nanovaccine after mucosal administration.

OBJECTIVES: The Botulism syndrome is caused by types A to G of botulinum neurotoxins. The binding domains of these neurotoxins are immunogenic and considered as appropriate candidate vaccines. Due to the low immunogenicity of recombinant vaccines, there have been many studies on the use of biocompatible carriers such as chitosan nanoparticles for the delivery of these vaccines. The aim of this study was evaluating the efficiency of chitosan nanoparticles as carriers for a candidate vaccine, binding domain of BoNT/E, through oral and intranasal routes. MATERIALS AND METHODS: Chitosan nanoparticles containing rBoNT/E binding domain, were synthesized via ionic gelation. After administration of the nanoparticles to mice through oral and intranasal routes, antibody titers were assessed by ELISA and, finally, all groups were challenged by active botulinum neurotoxin type E. RESULTS: The groups that received nanoparticles containing the antigen, through oral and intranasal routes, and the group that received the bare antigen orally, were able to tolerate 5×102 folds of MLD. The intranasally immunized mice by the bare antigen were able to tolerate 2×103 folds of the toxin's MLD. CONCLUSION: It seems that the use of chitosan nanoparticles has no significant effect on the protective immunization of the mice against botulinum BoNT/E in either route (P>0.05), even intranasal administration of the bare antigen gives better mice immunization against the toxin.

Production of Chicken Egg Yolk Antibody (IgY) Against Recombinant Cholera Toxin B Subunit and Evaluation of Its Prophylaxis Potency in Mice.

BACKGROUND: Cholera toxin (CT), responsible for the harmful effects of cholera infection, is made up of one A subunit (enzymatic), and five B subunits (cell binding). The release of cholera toxin is the main reason for the debilitating loss of intestinal fluid. Inhibition of the B subunit (CTB) may block CT activity. OBJECTIVE: To determine the effect of anti CTB-IgY against oral challenge with V. cholera in suckling infant mice. METHODS: The binding domain of cholera toxin was amplified and ligated into pET28a vector. The pET28a (+)/ctb expression vector was confirmed by endonuclease digestion and sequence analysis. The expression of recombinant CTB in E. coli was performed by induction with IPTG. After immunizing the chickens with recombinant CTB, IgY was purified by water dilution method and NaCl precipitation and analyzed by SDS-PAGE. Moreover, the activity and specificity of the IgY antibody were assessed by ELISA. RESULTS: The SDS-PAGE and western blot techniques showed that CTB protein was successfully expressed and specifically recognized by polyclonal antibodies against the cholera toxin. The oral administration of anti- (V. cholera+CTB) in infant mice in challenge with active V. cholera bacterium demonstrated high rate of survival. CONCLUSION: The increase in the number of antibiotic resistant bacteria implies the necessity of finding novel antibiotics. Our results suggest the possibility of passive protection from purified IgY, hence implying that anti CTB-IgY may be useful in the treatment of cholera infections.

EspA-loaded mesoporous silica nanoparticles can efficiently protect animal model against enterohaemorrhagic E. coli O157: H7.

In the present study, the application of mesoporous silica nanoparticles (MSNPs) loaded with recombinant EspA protein, an immunogen of enterohaemorrhagic E. coli, was investigated in the case of BALB/c mice immunization against the bacterium. MSNPs of 96.9 ± 15.9 nm in diameter were synthesized using template removing method. The immunization of mice was carried out orally and subcutaneously. Significant immune responses to the antigen were observed for the immunized mice when rEspA-loaded MSNPs were administered in both routes in comparison to that of the antigen formulated using a well-known adjuvant, i.e. Freund's. According to the titretitre of serum IL-4, the most potent humoral responses were observed when the mice were immunized subcutaneously with antigen-loaded MSNPs (244, 36 and 14 ng/dL of IL-4 in the serum of mice immunized subcutaneously or orally by antigen-loaded MSNPs, and subcutaneously by Freund's adjuvant formulated-antigen, respectively). However, the difference in serum IgG and serum IgA was not significant in mice subcutaneously immunized with antigen-loaded MSNPs and mice immunized with Freund's adjuvant formulated-antigen. Finally, the immunized mice were challenged orally by enterohaemorrhagic E. coli cells. The amount of bacterial shedding was significantly reduced in faecesfaeces of the animals immunized by antigen-loaded MSNPs in both subcutaneous and oral routes.

Nano-encapsulation of chicken immunoglobulin (IgY) in sodium alginate nanoparticles: In vitro characterization.

Controlled delivery of therapeutic agents by alginate nanoparticles became an attractive issue in the gastric organ. Some therapeutic agents such as proteins could not tolerate in severe condition in the gastrointestinal tract. In the present study, four concentrations of a specific IgY as a prophylactic agent against E. coli O157: H7 was entrapped in 0.2% w/v sodium alginate nanoparticles by ionic gelation method. Depending on the IgY concentration entrapment efficacy was 28.31-99.84%. The physicochemical and structural characteristics of free and IgY-loaded Alg NPs revealed that the individual particles exhibited a spherical shape with a diameter of 45-85 nm, and a negatively charged surface with a zeta potential value of 26-36 mV. In vitro release study showed a high significant difference of released amounts of IgY at 10% and 99.84% in simulated gastric fluid (pH 1.2) and simulated intestine fluid (pH 6.8), respectively. Also, the quality and activity of released IgY from Alg NPs not changed. The cytotoxicity of different concentrations of Alg NPs on the Vero cells was measured. Our results indicated that Alg NPs prepared from 0.2%w/v stock solution could be appropriate candidates for efficient and safe delivery of IgY through the gastrointestinal tract.

Designing Two Individual AcMNPV Polyhedrin-Plus Bac-to-Bac Expression System in order to Express GFP and CPV-VP2 in Insect Cells.

Background: The importance of viral protein-2 (VP2) of canine parvovirus (CPV) in binding to human cancer cells, production of veterinary vaccines and diagnostic kits has motivated several researches on producing this protein. Objectives: Our purpose was to construct recombinant bacmid shuttle vectors expressing VP2 of CPV using Bac-to-Bac baculoviral expression system. Materials and Methods: Mini-Tn7 transposones engineered in pFastBac1 donor vectors were used to construct expression cassettes of GFP and CPV-VP2. The plasmids were transferred into E. coli DH10Bac competent cells. Site-specific transposition of the genes into bacmid was accomplished using helper plasmid. Occurrence of Transposition was confirmed via PCR using specific primers and PUC/M13 universal primers. The recombinant bacmid DNAs were transfected into Sf9 cells using cationic lipids to generate new recombinant baculoviruses expressing GFP and CPV-VP2. GFP and VP2 expressions were evaluated by fluorescence microscopy and western analysis, respectively. Results: Cloning, subcloning and recombination processes of both GFP and VP2 were accomplished and verified. Accuracy of transfection process was confirmed by GFP fluorescence microscopy.VP2 expression was verified by SDS-PAGE and western analysis. Conclusions: Two Bac-to-Bac expression systems were designed to produce recombinant VP2 and GFP in insect cells.

Cloning, Expression, and Cost Effective Purification of Authentic Human Epidermal Growth Factor With High Activity.

BACKGROUND: Epidermal growth factor (EGF) plays a fundamental role in the healing of wounds relating to skin damage, the cornea, and the gastrointestinal tract. OBJECTIVES: The aim of this study is the cloning, expression, and purification of recombinant human EGF (rhEGF), and an assessment of its activity. MATERIALS AND METHODS: In the present experimental study, a synthetic pET28a (+) -hEGF construct was prepared. In order to ligate hEGF into pET24a (+), the PCR technique was performed, using special primers that possess restriction enzyme sites, which are also located in appropriate sites in pET24a (+). After transferring this construct into E. coli cells, protein expression was performed under standard conditions. Protein solubilization was done by urea. hEGF purification and refolding were carried out using gradient dialysis against the urea. We used RP-HPLC to compare between rhEGF and commercial rhEGF as a control. Finally, an MTT assay was performed to assess the viability of the NIH 3T3 cells treated with various concentrations of rhEGF. RESULTS: Dialysis after urea solubilization caused precipitation of unwanted proteins, resulting in achievement of purified EGF with > 90% purity, without the need for expensive and time-consuming process. The MTT assay results showed that our rhEGF activate significantly higher proliferation of NIH 3T3 cells in comparison to the control (P-values were < 0.0001), in total concentrations and times evaluated CONCLUSIONS: Via our purification protocol, a sufficient amount of bioactive recombinant human epidermal growth factor was obtained in just a few affordable steps, with superlative purity.

Expression and purification of neurotoxin-associated protein HA-33/A from Clostridium botulinum and evaluation of its antigenicity.

BACKGROUND: Botulinum neurotoxin (BoNT) complexes consist of neurotoxin and neurotoxin-associated proteins. Hemagglutinin-33 (HA-33) is a member of BoNT type A (BoNT/A) complex. Considering the protective role of HA-33 in preservation of BoNT/A in gastrointestinal harsh conditions and also its adjuvant role, recombinant production of this protein is favorable. Thus in this study, HA-33 was expressed and purified, and subsequently its antigenicity in mice was studied. METHODS: Initially, ha-33 gene sequence of Clostridium botulinum serotype A was adopted from GenBank. The gene sequence was optimized and synthesized in pET28a (+) vector. E. coli BL21 (DE3) strain was transformed by the recombinant vector and the expression of HA-33 was optimized at 37°C and 5 h induction time. RESULTS: The recombinant protein was purified by nickel nitrilotriacetic acid agarose affinity chromatography and confirmed by immunoblotting. Enzyme Linked Immunoassay showed a high titer antibody production in mice. CONCLUSION: The results indicated a highly expressed and purified recombinant protein, which is able to evoke high antibody titers in mice.

Comparative study of immunological and structural properties of two recombinant vaccine candidates against botulinum neurotoxin type E.

BACKGROUND: Recently, botulinum neurotoxin (BoNT)-derived recombinant proteins have been suggested as potential botulism vaccines. Here, with concentrating on BoNT type E (BoNT/E), we studied two of these binding domain-based recombinant proteins: a multivalent chimer protein, which is composed of BoNT serotypes A, B and E binding subdomains, and a monovalent recombinant protein, which contains 93 amino acid residues from recombinant C-terminal heavy chain of BoNT/E (rBoNT/E-HCC). Both proteins have an identical region (48 aa) that contains one of the most important BoNT/E epitopes (YLTHMRD sequence). METHODS: The recombinant protein efficiency in antibody production, their structural differences, and their BoNT/E-epitope location were compared by using ELISA, circular dichroism, computational modeling, and hydrophobicity predictions. RESULTS: Immunological studies indicated that the antibody yield against rBoNT/E-HCC was higher than chimer protein. Cross ELISA confirmed that the antibodies against the chimer protein recognized rBoNT/E-HCC more efficiently. However, both antibody groups (anti-chimer and anti-rBoNT/E-HCC antibodies) were able to recognize other proteins. Structural studies with circular dichroism showed that chimer proteins have slightly more secondary structures than rBoNT/E-HCC. CONCLUSION: The immunological results suggested that the above-mentioned identical region in rBoNT/E-HCC is more exposed. Circular dichroism, computational protein modeling and hydrophobicity predictions indicated a more exposed location for the identical region in rBoNT/E-HCC than the chimer protein, which is strongly in agreement with immunological results.

Production and characterization of a recombinant chimeric antigen consisting botulinum neurotoxin serotypes A, B and E binding subdomains.

Botulinum neurotoxins (BoNTs) are potent toxicant proteins composed of a heavy chain (100 kDa) and a light chain (50 kDa) of seven (A-G) serotypes that is responsible for botulism syndrome. In this study, polypeptides from C-terminal heavy chain of BoNTs serotypes A, B and E to the length of 54, 45 and 48 amino acid respectively were selected, linked together using a hydrophobic linker and expressed in E. coli. The expression efficiency of the chimeric protein was found to be 51%. The chimeric protein was produced in the form of inclusion body (IB) both at two studied temperatures, 30 degrees C and 37 degrees C. This IB was extracted by ultracentrifugation and followed for chimeric protein solubilization and purification using of ultrafiltration and preparative electrophoresis. The purified chimeric protein was characterized using blotting and ELISA. To evaluate the protection ability of this chimeric antigen against their active toxins, it was injected to mice and the antibody titer as well as the extent of protectivity were determined. Mice given three injections (10 microg/mice) of the antigen were protected against an intra-peritoneal administration of 10 LD(50 )of serotypes A and E, but 100 LD(50) of serotype B. We conclude that a significant correlation exists between the antigenic characteristics and protection capability of the chimeric protein prepared in this study.