Expression and purification of TolC as a recombinant protein vaccine against Shigella flexneri and evaluation of immunogenic response in mice.

BACKGROUND: Shigella is one of the major causes of dysenteric diarrhea, which is known shigelosis. Shigelosis causes 160,000 deaths annually of diarrheal disease in the global scale especially children less than 5 years old. No licensed vaccine is available against shigelosis, therefore, efforts for develop an effective and safe vaccine against Shigella as before needed. The reverse vaccinology (RV) is a novel strategy that evaluate genome or proteome of the organism to find a new promising vaccine candidate. In this study, immunogenicity of a designed-recombinant antigen is evaluated through the in silico studies and animal experiments to predict a new immunogenic candidate against Shigella. METHODS: In the first step, proteome of Shigella flexneri was obtained from UniProtKB and then the outer membrane and extracellular proteins were predicted. In this study TolC as an outer membrane protein was selected and confirmed among candidates. In next steps, pre-selected protein was evaluated for transmembrane domains, homology, conservation, antigenicity, solubility, and B- and T-cell prediction by different online servers. RESULT: TolC as a conserved outer membrane protein, using different immune-informatics tools had acceptable scores and was selected as the immunogenic antigen for animal experiment studies. Recombinant TolC protein after expression and purification, was administered to BALB/c mice over three intraperitoneal routes. The sera of mice was used to evaluate the IgG1 production assay by indirect-ELISA. The immunized mice depicted effective protection against 2LD50 of Shigella. Flexneri ATCC12022 (challenge study). CONCLUSION: Therefore, the reverse vaccinology approach and experimental test results demonstrated that TolC as a novel effective and immunogenic antigen is capable for protection against shigellosis.

Safety and immunogenicity of a recombinant receptor-binding domain-based protein subunit vaccine (Noora vaccine™) against COVID-19 in adults: A randomized, double-blind, placebo-controlled, Phase 1 trial.

The development of a safe and effective vaccine is essential to protect populations against coronavirus disease 2019 (COVID-19). There are several vaccine candidates under investigation with different mechanisms of action. In the present study, we have evaluated the safety and immunogenicity of a recombinant receptor-binding domain (RBD)-based protein subunit vaccine (Noora vaccine) against COVID-19 in adults. This Phase 1 trial is a randomized, double-blind, placebo-controlled study to evaluate the safety and immunogenicity of the recombinant RBD-based protein subunit vaccine (Noora vaccine) against COVID-19 in healthy adults volunteers. Eligible participants were included in this study after evaluating their health status and considering the exclusion criteria. They were then randomized into three groups and received three doses of vaccine (80 µg, 120 µg, and placebo) on Days 0, 21, and 35. Primary outcomes including solicited, unsolicited, and medically attended adverse events were recorded during this study. Secondary outcomes including the humoral and cellular immunity (including anti-RBD IgG antibody and neutralizing antibody) were measured on Days 0, 21, 28, 35, 42, and 49 by using the ELISA kit and the Virus Neutralization Test (VNT) was performed on day 49. Totally 70 cases were included in this Phase 1 trial and 60 of them completed the study. Safety assessments showed no severe adverse events. Local pain at the vaccine injection site occurred in 80% of the vaccinated volunteers. Induration and redness at the injection site were the other adverse reactions of this vaccine. There was no significant difference between the studied groups regarding adverse reactions. Anti-RBD IgG antibody and neutralizing antibody assessment showed significant seroconversion in comparison to the placebo group (80%, and 100% respectively, p < 0.001). The cellular immunity panel also showed mild to moderate induction of TH1 responses and the VNT showed 78% of seroprotection. The results of this Phase 1 trial showed acceptable safety without serious adverse events and significant seroconversions in the humoral and cellular immunity panel. The dose of 80 µg is an appropriate dose for injection in the next phases of the trial.

Bioinformatics design of recombinant chimeric protein containing SipD and LptD immunogens and evaluation of its immunogenicity against Salmonella Typhimurium.

The growing emergence of resistant bacteria is the current global concern for the humans and animals. Vaccination could be the desirable method to preventing such infectious diseases. Safe and effective vaccines are urgently needed to manage and prevent Salmonella contamination. Subunit vaccines are safe approaches for the protection against Salmonella spp. The bioinformatics methods were performed to determine the gene structure. Gene cassette (rLPSI) was ordered in pET28a (+), and cloned into E.coli BL21 (DE3), and the recombinant protein was expressed using IPTG (1 mM). Mice were immunized by subcutaneous administration of recombinant protein. Then, the mice were challenged by oral administration of 100LD50 of live S. Typhimurium. The Codon adaptation index of the chimeric gene was multiplied by 0.92. Validation results showed that >90% of residues lie in the desired or extra allowed area of the Ramachandran plot. The recombinant protein (65.9 kDa) was expressed in E.coli. Antibody titers in vaccinated mice were significantly different from those in the control groups. Recombinant protein immunization of the mice provided 90% and 70% protection against 10LD50 and 100LD50 of S. Typhimurium, respectively. In general, the results showed the high efficiency of rLPSI chimeric protein as a protective antigen against S. Typhimurium infection. The rLPSI chimeric protein could be an effective recombinant vaccine candidate against S. Typhimurium infection, but more research is needed.

Investigate the immunogenic and protective effect of trivalent chimeric protein containing IpaD-StxB-TolC antigens as a vaccine candidate against S. dysenteri and S. flexneri.

BACKGROUNDS: Shigella spp. causes bloody diarrhea and leads to death, especially in children. Chimeric proteins containing virulence factors can prevent Shigella infection. The purpose of this study is to investigate the immunogenic and protective effect of trivalent chimeric protein containing IpaD-StxB-TolC antigens against shiga toxin, S. dysenteri and S. flexneri in vitro and in vivo conditions. METHODS: Recombinant vector was transferred to E. coli BL21. The expression of the chimeric protein was confirmed by SDS PAGE and purified using the Ni-NTA column. Mice were immunized with recombinant protein and antibody titer was evaluated by ELISA. 10, 25 and 50 LD50 of Shiga toxin neutralization was evaluated in vitro (Vero cell line) and in vivo conditions. Also, the challenge of immunized mice with 10, 25 and 50 LD50 of S. dysentery and S. flexneri was done. RESULTS: The expression and purification of the recombinant protein with 60.6 kDa was done. ELISA showed increased antibody titer against the chimeric protein. MTT assay indicated that 1/8000 dilution of the sera had a 51% of cell viability against the toxin in Vero cell line. The challenge of mice immunized with toxin showed that the mice had complete protection against 10 and 25 LD50 of toxin and had 40% survival against 50 LD50. Mice receiving 10 and 25 LD50 of S. dysenteri and S. flexneri had 100% protection and in 50 LD50 the survival rate was 60 and 50%, respectively. Organ burden showed that the amount of bacterial colonization in immunized mice was 1 × 104 CFU/mL, which was significantly different from the control group. CONCLUSION: This study showed that chimeric proteins can create favorable immunogenicity in the host as vaccine candidates.

Protective effects of anti-CfaB-EtpA-LTB IgY antibody against adherence and toxicity of enterotoxigenic Escherichia coli (ETEC).

AIM: Production of IgY antibodies against CfaB-EtpA-LTB (CEL) chimeric protein and evaluation of its protective effects against enterotoxigenic Escherichia coli (ETEC) by in vivo and in vitro investigation. METHODS AND RESULTS: Indirect ELISA and immunoblotting methods were applied to assess the immunogenicity and specificity of IgYs and also to evaluate the efficacy of IgYs in binding prevention and neutralizing the heat-labile (LT) toxin of ETEC bacteria. The results indicated that the anti-CEL IgY at a concentration of 2 mg ml-1 could decrease the bacterial adhesion to HT-29 cells by 74% compared to the control group.At a concentration of 750 µg ml-1, the IgY antibody managed to neutralize the disruptive LT toxin effect on the Y1 cell line. At a concentration of 2 mg ml-1, 81% reduction was observed in the fluid accumulation in the ileal loop assay. CONCLUSION: According to our findings, passive immunotherapy with anti-CEL IgY can prevent bacterial colonization and toxicity, thus facilitating in controlling the enteric diseases caused by ETEC infection.

Protective Immunity Against Enterotoxigenic Escherichia coli by Oral Vaccination of Engineered Lactococcus lactis.

Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of diarrhea in children globally, and thus suitable vaccines are desired. Antigen display on lactic acid bacteria is a reliable approach for efficient oral vaccination and preventing bowel diseases. To develop an oral vaccine against ETEC, the gene of the binding domain from heat-labile toxin (LTB), a key ETEC virulence factor, was codon-optimized and cloned into a construct containing a signal peptide and an anchor for display on L. lactis. Bioinformatics analysis showed a codon adaptation index of 0.95 for the codon-optimized gene. Cell surface expression of LTB was confirmed by transmission electron microscopy and blotting. White New Zealand rabbits were immunized per os (PO) with the recombinant L. lactis, and the antibody titers were assayed with ELISA. In vitro neutralization assay was performed using mouse adrenal tumor cells and rabbit ileal loop test was performed as the in vivo assay. ELISA results indicated that oral administration of the engineered L. lactis elicited a significant production of IgA in the intestine. In vitro neutralization assay showed that the effect of the toxin could be neutralized with 500 µg/ml of IgG isolated from the oral vaccine group. Furthermore, the dose of ETEC causing fluid accumulation in the ileal loop test showed a tenfold increase in rabbits immunized with either recombinant L. lactis or LTB protein compared to other groups. Our results imply that recombinant L. lactis could potentially be an effective live oral vaccine against ETEC toxicity.

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.

Recombinant HcpA-EspA-Tir-Stx2B chimeric protein induces immunity against attachment and toxicity of Escherichia coli O157:H7.

BACKGROUND: It is about 4 decades from the identification of Enterohemorrhagic Escherichia coli (EHEC) as a food-borne pathogen. There are many shreds of evidence that the bacteria are significant sources of intestinal infections and outbreaks even in developed countries. Developing an effective vaccine against O157 and non-O157 serotypes of EHEC is a good strategy to combat the bacteria. Raising antibody against main toxicity, adhering and colonizing apparatus of the bacteria using a multi-antigenic protein can be hopefully applicable. MATERIAL AND METHODS: A synthetic cassette consists of HcpA-EspA-Tir-Stx2B (HETS) subunit proteins were constructed and sub-cloned in pET32a (+). The protein was expressed and purified with Ni-NTA column and the BALB/c mice were immunized by the purified protein. HETS protein efficacy to elicit immune responses, O157 fecal shedding and immunity against Stx toxin were assessed. In addition, the cellular assays were performed to investigate the immune sera capability for neutralizing of Stx toxin and bacterial attachment apparatus. RESULTS: The HETS protein (611 amino acid length) was expressed and validated by Western blotting. Exerted EHEC bacteria ratio in the immunized mice was reduced close to 60% in shedding test. Cellular assays revealed that the sera of the immunized animals were able to neutralize Stx holotoxin in an extent of 70%; also, immunized mice were able to tolerate up to 200 LD50 of the active toxin. Moreover, toxin neutralization assay showed the capability of the immunized sera to block the cell adhesion. CONCLUSION: Regarding a lack of an efficient vaccine against EHEC, the proposed candidate immunogen, which consists of main adhesion and invasion factors, can overcome the lack of a vaccine against the bacteria.

Cloning and expression of a novel synthetic gene containing VP1 and 3A in Bacillus subtilis as a vaccine candidate against foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock animals and control of the disease based on vaccination against serotypes O, A and Asia 1 is important. VP1 (structural) protein and 3A (non-structural) protein is the important antigen in FMDV and they can be used to design recombinant vaccines. In this study the bioinformatics characteristics of VP1 [141-160 and 23-42] and 3A [21-35] of Iranian serotypes O, A and Asia 1 was obtained using on-line predicting software. Then the sequence VP1 [141-160]-GS-VP1 [23-42]-GS-3A [21-35]-GS were codon-optimized and cloned onpHT43shuttle vector and finally expressed in Bacillus subtilis WB600 strain. We could predict VP1 [141-160] as a B cell epitope, VP1 [23-42] as a CTL epitope and 3A [21-35] as a Th cell epitope. The 20KD recombinant protein expressed by Bacillus subtilis were detected by SDS-PAGE. The results showed that this recombinant protein had epitope characteristics and it could be useful as a vaccine candidate to control all serotypes of FMD in Iran.

Review on pathogenicity mechanism of enterotoxigenic Escherichia coli and vaccines against it.

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of diarrhea in children. Colonization factors (CFs) and LT enterotoxin are the major ETEC candidate vaccines. To cause disease, ETEC must adhere to the epithelium of the small intestine by means of CFs. Watery diarrhea is produced due to the effects of the enterotoxins. Vaccine development against ETEC has been identified as an important primary prevention strategy in developing countries and for travelers to these regions. Mucosal immunization can cause secretory IgA antibody (sIgA) responses that prevents the attachment of bacteria to the intestine and are of particular importance for provide protection against ETEC infection. The design of multivalent ETEC vaccine containing various colonization factors and ETEC toxin may provide protection against a wide range of bacterial strains. In this review, the importance and pathogenesis of ETEC, and the latest ETEC vaccine research results are discussed.

Preparation and evaluation of chitosan nanoparticles containing CtxB antigen against Vibrio cholera.

Vibrio cholera is a Gram-negative pathogen that causes diarrheal disease. The B subunit of Chlora toxin (CtxB) is one of the most important antigens of Vibrio cholera in which mediates the attachment of the bacteria to target cells. The aim of this study was to prepare chitosan nanoparticles containing CtxB and evaluate the effect of the antigen entrapment on the immunogenicity of this antigen. For this, the pET28a vector was induced using IPTG. Recombinant CtxB protein was expressed and purified using Ni-NTA column and finally was confirmed by western blotting. Following the confirmation of the protein entrapment onto the chitosan nanoparticles, the formulation was prescribed to BALB/c mice in three groups, including oral, oral-injection and injection groups. Serum and fecal IgA and IgG were evaluated by ELISA test. Finally, challenge of immunized mice was performed using Ctx toxin and rabbit ileal loop test. Using SDS-PAGE and western blotting, the 17.5 kDa recombinant CtxB was confirmed. Size electrical charge and of nanoparticles were determined and approved by Zetasizer. Nanoparticles prescription showed 1/102400 IgG endpoint titers for injection groups and 1/1600, 1/6400 for oral, oral-injection groups respectively and Serum and fecal IgA endpoint titers showed above 1/160 in all groups. Furthermore, immunized mice were able to neutralize Ctx toxin by ileal loop test. The CtxB is a suitable immunogen of V. cholera to be incorporated in both protective and preventive vaccines. Chitosan nanoparticles improve the immune responses and it may be used as a carrier for vaccine delivery.

A review on strategies for decreasing E. coli O157:H7 risk in animals.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a food-borne pathogen that younger children are most prone to this microorganism. Hemolytic Uremic Syndrome (HUS) caused by EHEC, leads to the destruction of red blood cells and kidney failure. The virulence of E.coli O157:H7 is attributed to fimbriae, that facilitate colonization of bacteria within the colon and verotoxins (VT) or Shiga toxins (Stx) that are released into the blood. Although, in most cases, the infection is self-limitedin young children and aged population, it may cause HUS. Therefore, several investigations are performed in order to offer effective therapies and vaccines, which can prevent and treat the infection in appropriate time. As the pathogenesis of this infection is complicated, a multi-targeted strategy is required. Since cattle are the most important reservoir of EHEC and the root of contamination, reducing E. coli O157:H7 at the farm level should decrease the risk of human illness. Several vaccine approaches have been employed with different proper outcomes in animal models, including recombinant proteins (virulence factors such as; Stx1/2, intimin, EspA, fusion proteins of A and B Stx subunits), avirulent ghost cells of EHEC O157:H7, live attenuated bacteria expressing recombinant proteins, recombinant fimbrial proteins. In addition to protein-based vaccines, DNA vaccines have provided proper prevention in the laboratory animal model. This review paper summarizes the previous studies, current status and future perspective of different immunization strategies for eradicating Enterohemorrhagic Escherichia coli O157:H7.

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.

In vivo validation of the immunogenicity of recombinant Baumannii Acinetobactin Utilization A protein (rBauA).

Acinetobacter baumannii has become a tremendous challenge to modern healthcare as an antimicrobial resistant. Replication and persistence of A. baumannii within eukaryotes is based on iron acquisition functions including siderophore biosynthesis. Iron transport into the cytosol is mediated by specific membrane receptors which recognize the iron-siderophore complexes. Expression of this acinetobactin mediated Iron uptake system is vital for intracellular growth of A. baumannii. Baumannii acinetobactin utilization (BauA), is an outer membrane protein, acting out the siderophore-ferric complex receptor. This study was aimed at analysis of immunogenicity and specificity of BauA. The genomic bauA was amplified via PCR method and after digestion, bauA was ligated into pET28a. The recombinant gene was expressed in Escherichia coli BL21(DE3) and the product was analyzed by SDS-PAGE and purified by Ni-NTA affinity chromatography method. The recombinant BauA (rBauA) was confirmed by western blot analysis using anti-His antibodies and its immunogenicity was assessed by injecting the rBauA to BALB/c mice. Antibodies produced therein could effectively recognize and bind rBauA. The immunized mice challenged with bacterial doses higher than LD50 survived. The antibodies were highly specific to A. baumannii and its clinical isolates. Passive immunization using serum raised against BauA protected mice from infection. BauA can be nominated as an immunogen against A. baumannii.

Camelid-derived heavy-chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris.

Botulinum neurotoxins (BoNTs) result in severe and often fatal disease, botulism. Common remedial measures such as equine antitoxin and human botulism immunoglobulin in turn are problematic and time-consuming. Therefore, diagnosis and therapy of BoNTs are vital. The variable domain of heavy-chain antibodies (VHH) has unique features, such as the ability to identify and bind specifically to target epitopes and ease of production in bacteria and yeast. The Pichia pastoris is suitable for expression of recombinant antibody fragments. Disulfide bond formation and correct folds of protein with a high yield are some of the advantages of this eukaryotic host. In this study, we have expressed and purified the camelid VHH against BoNT/E in P. pastoris. The final yield of P. pastoris-expressed antibody was estimated to be 16 mg/l, which is higher than that expressed by Escherichia coli. The nanobody expressed in P. pastoris neutralized 4LD50 of the BoNT/E upon i.p. injection in 25% of mice. The nanobody expressed in E. coli extended the mice's survival to 1.5-fold compared to the control. This experiment indicated that the quality of expressed protein in the yeast is superior to that of the bacterial expression. Favorable protein folding by P. pastoris seems to play a role in its better toxin-binding property.

Construction and immunogenic properties of a chimeric protein comprising CfaE, CfaB and LTB against Enterotoxigenic Escherichia coli.

ETEC (Enterotoxigenic Escherichia coli) is a major cause of diarrhea in developing countries and children. ETEC has two virulence factors including colonization factors antigen (CFA) and labile enterotoxins (LTs). CFA/I consists the major pilin subunit CfaB and a minor adhesive subunit, CfaE. In this study a tripartite fusion protein containing CfaB, CfaE and LTB was designed. In silico analysis of the tertiary structure of the chimeric protein showed a protein with three main domains linked together with linkers. Linear and conformational B-cell epitopes were identified. A chimera consisting cfaB, cfaE and ltB(BET)was then synthesized with E. coli codon bias in pUC57 and sub cloned into pET32 vector. Recombinant protein was expressed and purified by affinity chromatography and confirmed by western blotting. Mice were immunized with recombinant protein and the antibody titer and specificity of the sera were analyzed by ELISA. The efficiency of the immune sera against ETEC was evaluated by binding assay and GM1-ELISA. VaxiJen analysis of the protein showed high antigenicity. Post-immune sera contained high titers of anti-BET IgG. Pretreatment of ETEC cells with sera from immunized mice decreased their ability to adhere to cells of the human colon adenocarcinoma cell line HT29.

High level expression, purification and immunogenicity analysis of a protective recombinant protein against botulinum neurotoxin type E.

Botulinum neurotoxin type E heavy chain consists of two domains: N-terminal half as a translocation domain and C-terminal half (Hcc) as a binding domain. In this research a synthetic gene fragment encoding the binding domain of botulinum neurotoxin type E (BoNT/E-Hcc) was highly expressed in Escherichia coli by pGEX4T-1 vector. After purification, the recombinant BoNT/E-Hcc was evaluated by SDS-PAGE and western blot (immunoblot) analysis. Average yields obtained in this research were 3.7 mg recombinant BoNT/E-Hcc per liter of bacterial culture. The recombinant protein was injected in mice for study of its protection ability against botulinum neurotoxin type E challenges. The challenge studies showed that, vaccinated mice were fully protected against 104 × minimum lethal dose of botulinum neurotoxin type E.

Production of egg yolk antibody (IgY) against a chimeric protein containing IpaD, StxB, and TolC antigens from Shigella: An investigation of its prophylactic effects against Shiga toxin (Stx) and Shigella dysenteriae in vitro and in vivo.

Shigella is a major problem in developing countries. Immunoglobulin Y (IgY) can be used for prophylaxis and neutralize bacteria. The aim of this study was to produce IgY against the chimeric protein containing IpaD, StxB, and TolC antigens from Shigella, investigate its prophylactic and neutralizing effects against Stx and Shigella dysenteriae. The nucleotide sequence corresponding to the chimeric protein was cloned into pET28a plasmid and expressed in E. coli BL21 (DE3). Protein expression was confirmed by SDS-PAGE and the recombinant protein was purified by Ni-NTA affinity chromatography. The 150 µg of chimeric protein was mixed with Freund's adjutant and injected into laying hens (Leghorn). IgY was purified using PEG6000 precipitation. Antibody titer in the serum and egg yolk was evaluated by ELISA. IgY challenge against 1,10 and 50 LD50 of Stx and S. dysenteriae was investigated. A 60.6 kDa recombinant protein was confirmed by SDS-PAGE. ELISA showed that the antibody titer was significantly increased. MTT assay [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] showed that at 16 µmol/L, IgY protected HeLa cells against Stx. Treatment of mice with 1000 and 1500 µg IgY leads to complete survival of the mice against 1LD50 toxin and 4000 µg of IgY led to complete survival against 1LD50, also 70% and 30% survival against 10 and 50 LD50S. dysenteriae. This study showed that IgY produced against Stx and Shigella virulence factors could cause high protective effects against bacteria and toxins.

Expression of Recombinant Stonustoxin Alpha Subunit and Preparation of polyclonal antiserum for Stonustoxin Neutralization Studies.

Stonustoxin (SNTX) is a lethal protein found in stonefish venom, responsible for many of the symptoms associated with stonefish envenomation. To counter stonefish venom challenges, antivenom is a well-established and effective solution. In this study, we aimed to produce the recombinant alpha subunit protein of Stonustoxin from Synanceia horrida and prepare antibodies against it The SNTXα gene sequence was optimized for E. coli BL21 (DE3) expression and cloned into the pET17b vector. Following purification, the recombinant protein was subcutaneously injected into rabbits, and antibodies were extracted from rabbit´s serum using a G protein column As a result of codon optimization, the codon adaptation index for the SNTXα cassette increased to 0.94. SDS-PAGE analysis validated the expression of SNTXα, with a band observed at 73.5 kDa with a yield of 60 mg/l. ELISA results demonstrated rabbits antibody titers were detectable up to a 1:256,000 dilution. The isolated antibody from rabbit´s serum exhibited a concentration of 1.5 mg/ml, and its sensitivity allowed the detection of a minimum protein concentration of 9.7 ng. In the neutralization assay the purified antibody against SNTXα protected mice challenged with 2 LD50. In conclusion, our study successfully expressed the alpha subunit of Stonustoxin in a prokaryotic host, enabling the production of antibodies for potential use in developing stonefish antivenom.