Comparison of numerical calculations and ALOHA modeling in consequence assessment of chlorine gas emissions from ethylene dichloride reactors.

Incidents involving chemical storage tanks in the petrochemical industry are significant events with severe consequences. Within the petrochemical industry, EDC is a sector that produces ethylene dichloride through the reaction of chlorine and ethylene. The present research was conducted to evaluate the consequences of chlorine gas released from the EDC reactor in a petrochemical industry in southern Iran. Data regarding reactor specifications were obtained from the factory's technical office, while climatic data was acquired from the Meteorological Organization. The consequences of chlorine gas release from the reactor were assessed in four predefined scenarios using numerical calculation methods and modeling with the ALOHA software. The numerical calculation method involved thermodynamic fluid path analysis, discharge coefficient calculations, and wind speed impact analysis. The hazard radius was determined based on the ERPG1-2-3 index. Results showed that in the scenario of chlorine gas release from EDC reactors, according to the ALOHA model, an increase in wind speed from 3 to 7 m/h led to an expanded dispersion radius. At a radius of 700 m from the reactor, the maximum outdoor concentration reached 3.12 ppm, decreasing to 2.27 ppm at 800 m and further to 1.53 ppm at 1000 m. The comparison of numerical calculations and modeling using the ALOHA software indicates the desirable conformity of the results with each other. The R2 coefficient for evaluating the conformity of the results was 0.9964, indicating the desired efficiency of the model in evaluating the consequences of the release of toxic gasses from the EDC tank. The results of this research can be useful in designing the site and emergency response plan.

Preclinical study of formulated recombinant nucleocapsid protein, the receptor binding domain of the spike protein, and truncated spike (S1) protein as vaccine candidates against COVID-19 in animal models.

BACKGROUND: In this pre-clinical study, we designed a candidate vaccine based on severe acute respiratory syndrome-related -coronavirus 2 (SARS-CoV-2) antigens and evaluated its safety and immunogenicity. METHODS: SARS-CoV-2 recombinant protein antigens, including truncated spike protein (SS1, lacking the N-terminal domain of S1), receptor-binding domain (RBD), and nucleoprotein (N) were used. Immunization program was performed via injection of RBD, SS1 +RBD, and SS1 +N along with different adjuvants, Alum, AS03, and Montanide at doses of 0, 40, 80, and 120 µg at three-time points in mice, rabbits, and primates. The humoral and cellular immunity were analyzed by ELISA, VNT, splenocyte cytokine assay, and flow cytometry. RESULTS: The candidate vaccine produced strong IgG antibody titers at doses of 80 and 120 µg on days 35 and 42. Even though AS03 and Montanide produced high-titer antibodies compared to Alum adjuvant, these sera did not neutralize the virus. Strong virus neutralization was recorded during immunization with SS1 +RBD and RBD with Alum. AS03 and Montanide showed a strong humoral and cellular immunity; however, Alum showed mild to moderate cellular responses. Ultimately, no cytotoxicity and pathologic change were observed. CONCLUSION: These findings strongly suggest that RBD with Alum adjuvant is highly immunogenic as a potential vaccine.

Designing and determining immunogenicity of a recombinant protein due to producing a new vaccine against Enterotoxigenic Escherichia coli containing CfaE and CotD subunits.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common bacterial causes of mortalities in developing countries due to diarrhea. Since mucosal immune responses to CFs can prevent the disease, a chimeric protein containing ETEC's CFA/I (CfaE) tip subunits and CS2 (CotD) sub-structural units is developed to produce effective vaccine. Using bioinformatics tools, the chimeric construct was analyzed and then the optimized gene was synthesized and expressed in E. coli. The recombinant protein was expressed and purified by the Ni-NTA chromatography column and confirmed by anti-his tag antibody by western blotting. Mice were immunized with recombinant protein, and the IgG and IgA antibodies' titrations of the sera were analyzed by ELISA. In addition, the immunogenicity and protective efficacy against the live ETEC bacteria in the challenge test were determined. Western blot analysis verified the chimeric protein expression of CotD-CfaE. The outcome of ELISA was a substantial improvement in the IgG antibody titer in immunized mice. In a live ETEC challenge, the survival percentage of 30% was shown for immunized mice. The developed recombinant chimeric protein could be suggested as an effective component in producing an efficient vaccine against Enterotoxigenic E. coli with other crucial subunits, different immunization route, and other factors.

Cell death analysis of recombinant mature epsilon toxin on the kidney cell line.

BACKGROUND AND OBJECTIVES: Epsilon toxin is the third hazardous bacterial toxin causing ABS enterotoxaemia in domestic animal. In addition, epsilon toxin is known as a biological warfare agent. The aim of this study was to produce the recombinant mature epsilon toxin to evaluate cell death impact on the kidney cell line. MATERIALS AND METHODS: For this purpose, the sequence of mature epsilon toxin (46-328 aa) in pET28a was cloned and expressed in Escherichia coli BL21 (DE3) and purified by nickel-nitrilotriacetic acid (Ni-NTA) column and confirmed by western blot analysis using HRP conjugated anti-His antibody. Then, to assess the anti-proliferative effects of different concentrations of recombinant epsilon toxin, the MTT assay was done on the HEK293 cell line. The annexin V/PI staining was done to investigate the apoptotic and necrotic cell populations after exposure to epsilon toxin. RESULTS: Induction by 1 mM IPTG for 4 h at 37°C was an optimized condition for expressing mature epsilon toxin in E. coli strain BL21 (DE3). Electrophoresis on SDS-PAGE 12% gel showed the desired band approximately at 38 KDa. Our results showed that recombinant epsilon toxin is mainly expressed as an inclusion body. Furthermore, 100, 150, and 200 µg/mL of mature epsilon toxin are significantly reduced the cell viability (P≤0.05). The considerable increase of necrotic cell percentage was shown after exposing to 100, 150, and 200 µg/mL of mature epsilon toxin (P≤0.05). CONCLUSION: The recombinant mature epsilon toxin had cytotoxic effects and could induce necrosis.

The Immunogenicity of a Novel Chimeric Hemagglutinin-Neuraminidase-Fusion Antigen from Newcastle Disease Virus by Oral Delivery of Transgenic Canola Seeds to Chickens.

Newcastle disease (ND) is considered as one of the most devastating infectious diseases targeting domestic birds and has considerable threat to the commercial poultry production. Two surface glycoproteins, hemagglutinin-neuraminidase (HN) and fusion (F), act as antigens in the virus structure and also play important roles in infecting host cells. In the current study, the expression of the chimeric HN-F protein in canola seeds and its immunogenicity in chickens were investigated. The HN-F gene was cloned downstream of the fatty acid elongase 1 (FAE1) promoter in the binary expression vector, pBI1400-HN-F, and introduced into rapeseed (Brassica napus L.) using Agrobacterium-mediated transformation. The amount of the HN-F glycoprotein was estimated up to 0.18% and 0.11% of the total soluble protein (TSP) in transgenic seeds and leaves of canola, respectively. Confirmatory analyses of 36 transgenic lines revealed that the HN-F gene was integrated into the genome. Subsequently, HN-F protein could be expressed and accumulated in the seed tissue. Specific pathogen-free (SPF) chickens immunized orally with recombinant HN-F showed a significant rise in specific and hemagglutination inhibition (HI) antibodies 35 to 42 days post the first administration. The results implied the potential of transgenic canola seed-based expression for oral delivery of NDV immunogenic glycoproteins.

Immunization against Vibrio cholerae, ETEC, and EHEC with chitosan nanoparticle containing LSC chimeric protein.

INTRODUCTION: Severe intestinal infections caused by V. cholerae, ETEC and EHEC have contributed to the mortality rate in developing countries. Vibrio Cholera, ETEC and EHEC bacterium with the production of CT, LT and Stx2 toxins respectively lead to severe watery and bloody diarrhea. This study aimed to investigate a trimeric vaccine candidate containing recombinant chimeric protein, encapsulate the protein in chitosan nanoparticles and assess its immunogenicity. METHODS: The LSC recombinant gene was used. It is composed of LTB (L), STXB (S) and CTXB (C) subunits respectively. The LSC recombinant protein was expressed and purified and confirmed by western blotting. The purified protein was encapsulated in chitosan nanoparticles, and its size was measured. BalB/c mice were immunized in four groups through oral and injection methods by LSC protein. The antibody titer was then evaluated by ELISA, and finally, the challenge test of the toxins from all three bacteria was done on the immunized mouse. RESULTS: After expression and purification LSC protein size of nanoparticles containing protein was measured at 104.6 nm. Nanoparticles were able to induce systemic and mucosal immune responses by generating a useful titer of IgG and IgA. The challenge results with LT, CT and Stx toxins showed that the LSC protein might partially neutralize the effect of toxins. CONCLUSION: LSC chimeric protein with the simultaneous three essential antigens have a protective effect against the toxins produced by ETEC, EHEC and Vibrio cholera bacteria and it can be used in vaccines to prevent Diarrhea caused by these three bacteria.

Immunogenicity Evaluation of Chimeric Subunit Vaccine Comprising Adhesion Coli Surface Antigens from Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the most common agent of diarrhea morbidity in developing countries. ETEC adheres to host intestinal epithelial cells via various colonization factors. The CooD and CotD proteins play a significant role in bacteria binding to the intestinal epithelial cells as adhesin tip subunits of CS1 and CS2 pili. The purpose here was to design a new construction containing cooD and cotD genes and use several types of bioinformatics software to predict the structural and immunological properties of the designed antigen. The fusion gene was synthesized with codon bias of E. coli in order to increase the expression level of the protein. The amino acid sequences, protein structure, and immunogenicity properties of potential antigens were analyzed in silico. The chimeric protein was expressed in E. coliBL21 (DE3). The antigenicity of the recombinant proteins was verified by Western blotting and ELISA. In order to assess the induced immunity, the immunized mice were challenged with wild-type ETEC by an intraperitoneal route. Immunological analyses showed the production of a high titer of IgG serum with no sign of serum-mucosal IgA antibody response. The result of the challenge assay showed that 30% of immunized mice survived. The results of this study showed that CooD-CotD recombinant protein can stimulate immunity against ETEC. The designed chimera could be a prototype for the subunit vaccine, which is worthy of further consideration.

Oral immunization with a plant-derived chimeric protein in mice: Toward the development of a multipotent edible vaccine against E. coli O157: H7 and ETEC.

The most bacterial cause of infectious diseases associated with diarrhea are enterotoxigenic and enterohemorrhagic Escherichia coli (ETEC and EHEC, respectively). These strains use colonization factors for the attachment to the human intestinal mucosa, followed by enterotoxins production that could induce more host damage. The Heat-labile enterotoxin (LT) and colonization factors (CFs) are momentous factors for the pathogenesis of ETEC. Also, Intimin and Shiga like toxin (STX) are the main pathogenic factors expressed by EHEC. Because of mucosal surfaces are the major entry site for these pathogens, oral immunization with providing the protective secretary IgA antibody (sIgA) responses in the mucosa, could prevent the bacterial adherence to the intestine. In this study oral immunogenicity of a synthetic recombinant protein containing StxB, Intimin, CfaB and LtB (SICL) was investigated. For specific expression in canola seeds, the optimized gene was cloned in to plant expression vector containing the Fatty Acid Elongase (FAE) promoter. The evaluation of the expression level in canola seeds was approximately 0.4% of total soluble protein (TSP). Following to oral immunization of mice, serum IgG and fecal IgA antibody responses induced. Caco-2 cell binding assay with ETEC shows that the sera from immunized mice could neutralize the attachment properties of toxigenic E. coli. The reduction of bacterial shedding after the challenge of immunized mice with E. coli O157:H7 was significant. The sera from immunized mice in the rabbit ileal loop experiment exhibited a significant decrease in the fluid accumulation compared to the control. The results indicate efficacy of the recombinant chimeric protein SICL in transgenic canola seed as an effective immunogen, which elicits both systemic and mucosal immune responses as well as protection against EHEC and ETEC adherence and toxicity.

Immunogenicity of the Multi-Epitopic Recombinant Glycoproteins of Newcastle Disease Virus: Implications for the Serodiagnosis Applications.

BACKGROUND: Newcastle disease virus (NDV) is a dangerous viral disease, infecting a broad range of birds, and has a fatal effect on the poultry industries. The attachment and consequently fusion of the virus to the host cell membrane is directed by the two superficial glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion (F) which is considered as the important targets for the poultry immune response. OBJECTIVES: The principal goal of this investigation was to realize the potential efficacy of the E. coli expression system for the production of the multi-epitopic HN, and F proteins with respect to the ability for the stimulation of the immune system and production of the cross-reactive antibodies in mice. MATERIALS AND METHODS: The recombinant HN and F (rHN, rF) have accumulated almost 40% of the total bacterial proteins. The presence of rHN and rF proteins recognized by the Western blotting with specific anti-HN, anti-F, anti-Newcastle B1, and anti-poly 6x His-tag antibodies. Furthermore, both rHN and rF have shown the specific reactivity against the Newcastle B1 antiserum as a standard strain. RESULTS: The ELISA analysis showed that the higher dilutions of the antibody against Newcastle B1 could react with the as least quantity as 100 ng of the purified rHN, and rF. Cross-reactivity analysis of the sera from the mice immunized with Newcastle B1 in two time points indicated that the raise of anti-Newcastle B1, anti-HN and anti-F antibodies peaked at 28 days post immunization (dpi). Moreover, temporal variation in IgG titration between both time points was significant at 5% probability level. CONCLUSION: The results provided valuable information about the cross-reactivity patterns and biological activity of the multi-epitopic proteins compared to the NDV standard strain which was determined by the Western blotting and ELISA.

Preparation of chitosan nanoparticle containing recombinant StxB antigen of EHEC and evaluation its immunogenicity in BALB/c mice.

BACKGROUND AND OBJECTIVES: Escherichia coli O157:H7 is one of the most important food pathogens that produces colitis and bloody urine in humans. The Stx2B subunit is considered as one of the candidates for vaccine due to its immunogenic and adjuvant properties. Designing a mucosal vaccine using nanoparticles for protecting the antigen against degradation and controlling the release of antigen are important. The objective of the current study was to prepare nanoparticles containing the Stx2B subunit of E. coli O157:H7 and evaluation of its immunogenicity in the mouse model. MATERIALS AND METHODS: E. coli BL21 DE3 and pET28a-stxB were used for expression of the stx2b gene. After inducing gene expression, purification of the Stx2b protein was performed. Then, chitosan nanoparticle containing recombinant Stx2B was prepared and administered to BALB/c mice. IgA and IgG titers in serum and IgA titers in feces of immunized and control mice were evaluated by the ELISA method. RESULTS: After expression and purification of the Stx2B recombinant protein, an expected band of 13 kDa was observed on the SDS-PAGE gel and confirmed by Western Blot analysis. The size of the nanoparticle containing Stx2B was 290 nm. In the immunized mice, IgG and IgA titers were significantly increased. The immunized mice were challenged against E. coli O157:H7 Stx+ and the shedding analysis showed that colonization of bacteria in the intestinal tract decreased. CONCLUSION: Oral administration of nanoparticles containing Stx2B as a candidate for the vaccine can induce a systemic and mucosal immune response against Stx2 toxin and can provide acceptable protection.

Immunogenic properties of trivalent recombinant protein composed of B-subunits of LT, STX-2, and CT toxins.

Infectious diarrhoea remains an emerging problem in the world health program. Among diarrheagenic agents, Vibrio cholerae and enterotoxigenic and enterohemorrhagic Escherichia coli are critical enteropathogens. AB5 toxin produced by these bacteria, heat-labile enterotoxin (LT), cholera enterotoxin (CT), and shiga-like cytotoxin (STX) can target the immune system and are subunit vaccine candidates. A chemically-synthesized chimeric construct composed of the binding subunits of these toxins (LTB, STXB, and CTXB) was developed based on bioinformatics studies. The whole chimeric protein (rLSC) and each of the segments (rLTB, rSTXB, and rCTXB) were expressed in a prokaryotic expression system (E. coli), purified, and analysed for their immunogenic properties. The results indicate that these recombinant proteins were effectively able to present appropriate epitopes to an animal model of the immune system which could result in and increase IgG in serum and immune responses that protect against the binding activity of these toxins. The immunological assays revealed that the sera of immunized mice prevented toxins from binding to their specific receptors and neutralized their toxic effects. The proposed construct should be considered as a potent immunogen to prevent toxicity and diarrhoea.