Engineering erucic acid biosynthesis in camelina (Camelina sativa) via FAE1 gene cloning and antisense technology.

Oil seeds now make up the world's second-largest food source after cereals. In recent years, the medicinal- oil plant Camelina sativa has attracted much attention for its high levels of unsaturated fatty acids and low levels of saturated fatty acids as well as its resistance to abiotic stresses. Improvement of oil quality is considered an important trait in this plant. Erucic acid is one of the fatty acids affecting the quality of camelina oil. Altering the fatty acid composition in camelina oil through genetic manipulation requires the identification, isolation, and cloning of genes involved in fatty acid biosynthesis. The Fatty Acid Elongase 1 (FAE1) gene encodes the enzyme ß-ketoacyl CoA synthase (KCS), a crucial enzyme in the biosynthesis of erucic acid. In this study, the isolation and cloning of the FAE1 gene from Camelina sativa were conducted to construct an antisense structure. The molecular homology modeling of DFAE1 proteins using the SWISS-MODEL server on ExPASy led to the generation of the 3D structures of FAE1 and DFAE1 proteins. The GMQE values of 0.44 for FAE1 and 0.08 for DFAE1 suggest high accuracy in the structural estimation of these genes. The fragments were isolated from the DNA source of the genomic Soheil cultivar with an erucic acid content of about 3% (in matured seeds) using PCR. After cloning the FAE1 gene into the Bluescript II SK+ vector and sequencing, the resulting fragments were utilized to construct the antisense structure in the pBI121 plant expression vector. The approved antisense structure was introduced into the Camelina plant using the Agrobacterium-mediated method, with optimization of tissue culture and gene transfer conditions. This approach holds potential to advance our knowledge of fat biosynthesis, leading to potential improvements in oil quality in Camelina sativa.

The heat-labile enterotoxin B subunit bio-adjuvant linked to Newcastle disease virus recombinant hemagglutinin neuraminidase elicited a humoral immune response in animal model.

Newcastle disease is a highly contagious viral infection primarily affecting poultry, leading to significant economic losses worldwide due to its high morbidity and mortality rates. Given the severity of the disease and its impact on the poultry industry, there is an urgent need for a preventative approach to tackle this issue. Developing an efficient and effective vaccine is a valuable step toward reducing the burden of this virus. Consequently, investing in preventive measures, such as vaccination programs, is a top priority to mitigate the economic losses associated with Newcastle disease and protect the livelihoods of those relying on the poultry industry. Despite many vaccines against this viral disease, it still infects many wild and domestic birds worldwide. In this work, chimeric proteins, composed of the recombinant B subunit of Enterotoxigenic E. coli with one or two HN (Hemagglutinin-neuraminidase) subunits of NDV (LHN and LHN2, respectively), expressed using E.coli host. In-silico, in-vitro, and In-vivo procedures were performed to evaluate the immunogenicity of these proteins. The sera from immunized mice were analyzed using Western Blotting and ELISA. The LHN2 protein with an extra HN subunit elicited a higher antibody titer than the LHN protein (P<0.05). Both products could effectively elicit an immune response against NDV and can be considered a component of Newcastle disease vaccine candidates.

Protective immunization against Enterohemorrhagic Escherichia coli and Shigella dysenteriae Type 1 by chitosan nanoparticle loaded with recombinant chimeric antigens comprising EIT and STX1B-IpaD.

Increasing evidence demonstrated that Enterohemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae type 1 (S. dysenteriae1) are considered pathogens, that are connected with diarrhea and are still the greatest cause of death in children under the age of five years, worldwide. EHEC and S. dysenteriae 1 infections can be prevented and managed using a vaccination strategy against pathogen attachment stages. In this study, the chitosan nanostructures were loaded with recombinant EIT and STX1B-IpaD polypeptides. The immunogenic properties of this nano-vaccine candidate were investigated. The EIT and STX1B-IpaD recombinant proteins were heterologous expressed, purified, and confirmed by western blotting. The chitosan nanoparticles, were used to encapsulate the purified proteins. The immunogenicity of recombinant nano vaccine candidate, was examined in three groups of BalB/c mice by injection, oral delivery, and combination of oral-injection. ELISA and antibody titer, evaluated the humoral immune response. Finally, all three mice groups were challenged by two pathogens to test the ability of the nano-vaccine candidate to protect against bacterial infection. The Sereny test in guinea pigs was used to confirm the neutralizing effect of immune sera in controlling S. dysenteriae 1, infections. SDS-PAGE and western blotting, confirmed the presence and specificity of 63 and 27 kDa recombinant EIT and STX1B-IpaD, respectively. The results show that the nanoparticles containing recombinant proteins could stimulate the systemic and mucosal immune systems by producing IgG and IgA, respectively. The challenge test showed that, the candidate nano-vaccine could protect the animal model from bacterial infection. The combination of multiple recombinant proteins, carrying several epitopes and natural nanoparticles could evocate remarkable humoral and mucosal responses and improve the protection properties of synthetic antigens. Furthermore, compared with other available antigen delivery methods, using oral delivery as immune priming and injection as a booster method, could act as combinatorial methods to achieve a higher level of immunity. This approach could present an appropriate vaccine candidate against both EHEC and S. dysenteriae 1.

Control of erucic acid biosynthesis in Camelina (Camelina sativa) by antisense technology.

Oil seeds now make up the world's second-largest food source after cereals. In recent years, the medicinal- oil plant Camelina sativa has attracted much attention for its high levels of unsaturated fatty acids and low levels of saturated fatty acids as well as its resistance to abiotic stresses. Improvement of oil quality is considered an important trait in this plant. Erucic acid is one of the fatty acids affecting the quality of camelina oil. Altering the fatty acid composition in camelina oil through genetic manipulation requires the identification, isolation, and cloning of genes involved in fatty acid biosynthesis. The Fatty Acid Elangase 1 (FAE1) gene encoded the enzyme ß-ketoacyl COA synthase (KCS), which is a key factor in the biosynthesis of erucic acid. In this study, isolation and cloning of the FAE1 from the Camelina sativa were performed to prepare an antisense structure.  The fragments were isolated from the DNA source of the genomic Soheil cultivar with an erucic acid content of about 3% (in matured seeds) using PCR. After cloning FAE1 into the Bluescriprt II SK+ vector and sequencing, these fragments were used for the preparation of antisense structure in the pBI121 plant expression vector. The approved structure was transferred to the camelina plant via the Agrobacterium-mediated method. Also, the conditions of tissue culture and gene transfer were optimized. Moreover, the erucic acid content of the immature seeds of T0 transgenic plants was analyzed with gas chromatography (GC). Results showed significant changes in erucic acid levels of two control plants (0.88%), while two lines of the RFAE1 transgenic plants showed a decrease of approximately 0% in erucic acid level. It can be concluded that the antisense structure can be effective in reducing erucic acid.

Molecular Modeling and Optimization of Type II E.coli l-Asparginase Activity by in silico Design and in vitro Site-directed Mutagenesis.

INTRODUCTION: L-asparaginase (also known as L-ASNase) is a crucial therapeutic enzyme that is widely used in treatment of ALL (acute lymphoblastic leukemia) as a chemotherapeutic drug. Besides, this enzyme is used in the food industry as a food processing reagent to reduce the content of acrylamide in addition to the clinical industry. The improvement of activity and kinetic parameters of the L-ASNase enzyme may lead to higher efficiency resulting in practical achievement. In order to achieve this goal, we chosen glycine residue in position 88 as a potential mutation with advantageous outcomes. METHOD: In this study, firstly to find the appropriate mutation on glycine 88, various in silico analyses, such as MD simulation and molecular docking, were carried out. Then, the rational design was adopted as the best strategy for molecular modifications of the enzyme to improve its enzymatic properties. RESULT: Our in silico findings show that the four mutations G88Q, G88L, G88K, and G88A may be able to increase L-ASNase's asparaginase activity. The catalytic efficiency of each enzyme (kcat/Km) is the most important feature for comparing the catalytic activity of the mutants with the wild type form. The laboratory experiments showed that the kcat/Km for the G88Q mutant is 36.32% higher than the Escherichia coli K12 ASNase II (wild type), which suggests that L-ASNase activity is improved at lower concentration of L-ASN. Kinetic characterization of the mutants L-ASNase activity confirmed the high turnover rate (kcat) with ASN as substrate relative to the wild type enzyme. CONCLUSION: In silico analyses and laboratory experiments demonstrated that the G88Q mutation rather than other mutation (G88L, G88K, and G88A) could improve the kinetics of L-ASNase.

A novel EGFR-specific recombinant ricin-panitumumab (scFv) immunotoxin against breast and colorectal cancer cell lines; in silico and in vitro analyses.

The Epidermal Growth Factor Receptor (EGFR) has been of high importance as it is over expressed in a wide diversity of epithelial cancers, promoting cell proliferation and survival pathways. Recombinant immunotoxins (ITs) have emerged as a promising targeted therapy for cancer treatment. In this study, we aimed to investigate the antitumor activity of a novel recombinant immunotoxin designed against EGFR. Using an in silico approach, we confirmed the stability of the RTA-scFv fusion protein. The immunotoxin was successfully cloned and expressed in the pET32a vector, and the purified protein was analyzed by electrophoresis and western blotting. In vitro evaluations were conducted to assess the biological activities of the recombinant proteins (RTA-scFv, RTA, scFv). The novel immunotoxin demonstrated significant anti-proliferative and pro-apoptotic effects against cancer cell lines. The MTT cytotoxicity assay revealed a decrease in cell viability in the treated cancer cell lines. Additionally, Annexin V/Propidium iodide staining followed by flow cytometry analysis showed a significant induction of apoptosis in the cancer cell lines, with half maximal inhibitory concentration (IC50) values of 81.71 nM for MDA-MB-468 and 145.2 nM for HCT116 cells (P < 0.05). Furthermore, the EGFR-specific immunotoxin exhibited non-allergenic properties. The recombinant protein demonstrated high affinity binding to EGFR. Overall, this study presents a promising strategy for the development of recombinant immunotoxins as potential candidates for the treatment of EGFR-expressing cancers.

DNA barcoding and biomass accumulation rates of native Iranian duckweed species for biotechnological applications.

The Lemnaceae family (duckweed) consists of at least three recognized genera with six reported species in Iran that are distributed in wetlands. Duckweeds are the simplest and smallest flowering aquatic monocots with free-floating fronds that can reproduce asexually every 2-3 days. Duckweed could be a major source of balanced amino acids and high protein content, which is increasingly promising for biotechnological applications. For molecular classification and species identification of the collected samples, DNA barcoding was performed using two standard chloroplast markers, the spacer region between the ATP synthase subunits F and H (atpF-atpH) and the intron region of the ribosomal protein S16 (rps16). The results confirm the presence of four species belonging to the two genera Lemna and Spirodela. In addition, L. turionifera was detected for the first time in Iran. Due to the high growth rates of duckweed, measurement of biomass accumulation and doubling time are important factors in determining growth potential, especially for native species. The relative growth rates (RGR), doubling times (DT), biomass accumulation, and relative weekly yields (RY) of 40 distinct duckweed clones were determined under standard cultivation conditions. The dry weight-based RGR ranged from 0.149 to more than 0.600 per day, DT from 1.12 to 9 days, and RY from 7 to 108.9 per week. All values are comparable with previous studies. RGR and RY of selected clones are higher than the growth potential for a wide range of wild plants and common crops. These data support that native duckweed has high productivity value and should be further investigated as a potentially rich protein source for alternative human food, livestock feed, and recombinant protein production.

Immunological evaluation of recombinant chimeric construct from Enterotoxigenic E. coli expressed in hairy roots.

Enterotoxigenic Escherichia coli is the most important bacterial agent causing traveller's diarrhea in developing countries. Enterotoxins (LT & ST) and colonization factors (CFs) are two important factors in the ETEC pathogenesis. In the present study, a recombinant four-part fusion protein containing CFAB*ST, CFAE, and LTB (CCL) was expressed in hairy roots of Nicotiana tabacum. The synthetic gene sequence and gene order were designed based on bioinformatics analysis that predicted the best arrangement for antigenicity and stimulation of the immune response. Codon usage was optimized for expression in tobacco plant, under the control of promoter CaMV35S in plasmid pBI121. CCL was efficiently expressed in tobacco hairy roots to yield 1.11% soluble protein as determined by quantitative ELISA and Western blot. In this study, mice were immunized with purified CCL via the oral and subcutaneous route. Humoral immunity especially mucosal immunity with antigen specific IgG and IgA detected in serum and feces. The ability of CCL to elicit neutralizing antibodies was evaluated in the rabbit ileal loop model, using anti-CCL antibodies derived from immunized mice, and co-incubated with ETEC strains. A decrease in fluid accumulation in the intestinal lumen of rabbit ileal loops challenged with ETEC LT and ST positive strains, correlated with the presence of anti-CCL antibodies capable of toxin neutralization. The ability of these antibodies to neutralize toxin confirmed the recognition of epitopes, either linear or conformational, displayed by the recombinant chimeric protein expressed in transgenic tobacco hairy roots. Transgenic plants containing multivalent immunogenic vaccine candidates have the potential to be used for immunization with protection against gastrointestinal pathogens like ETEC.

Root-preferential expression of Newcastle virus glycoproteins driven by NtREL1 promoter in tobacco hairy roots and evaluation of oral delivery in mice.

Newcastle disease virus (NDV) is a lethal virus in avian species with a disastrous effect on the poultry industry. NDV is enveloped by a host-derived membrane with two glycosylated haemagglutinin-neuraminidase (HN) and Fusion (F) proteins. NDV infection usually leads to death within 2-6 days, so the preexisting antibodies provide the most critical protection for this infection. The HN and F glycoproteins are considered the main targets of the immune system. In the present study, two constructs harboring the HN or F epitopes are sub-cloned separately under the control of a root-specific promoter NtREL1 or CaMV35S (35S Cauliflower Mosaic Virus promoter) as a constitutive promoter. The recombinant vectors were transformed into the Agrobacterium tumefaciens strain LBA4404 and then introduced to tobacco (Nicotiana tabacum L.) leaf disk explants. PCR with specific primers was performed to confirm the presence of the hn and f genes in the genome of the regenerated plants. Then, the positive lines were transformed via non-recombinant A. rhizogenes (strain ATCC15834) to develop hairy roots.HN and F were expressed at 0.37% and 0.33% of TSP using the CaMV35S promoter and at 0.75% and 0.54% of TSP using the NtREL1 promoter, respectively. Furthermore, the mice fed transgenic hairy roots showed a high level of antibody responses (IgG and IgA) against rHN and rF proteins.

In Silico designing and immunogenic production of the multimeric CfaB*ST, CfaE, LTB antigen as a peptide vaccine against Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the most frequent bacterial cause of diarrhea particularly reported in children of developing countries and also travelers. Enterotoxins and colonization factor antigens (CFAs) are two major virulence factors in ETEC pathogenesis. Colonization factor antigen I (CFA/I) includes major pilin subunit CfaB, and a minor adhesive subunit (CfaE), and enterotoxins consisting of heat-labile toxin subunit B (LTB) and heat-stable toxin (ST). Chimeric proteins (CCL) carrying epitopes and adjuvant sequences increase the possibility of eliciting a broad cellular or effective immune response. In the present study, a chimeric candidate vaccine containing CfaB*ST, CfaE, and LTB (CCL) was designed via in silico techniques. This chimeric gene was synthesized by using codon usage of E. coli for increasing the expression of the recombinant protein. After designing the chimeric construct, it showed a high antigenicity index estimated by the vaxiJen server. Linear and conformational B-cell epitopes were identified and indicated suitable immunogenicity of this multimeric recombinant protein. Thermodynamic analyses for mRNA structures revealed the appropriate folding of the RNA representative good stability of this molecule. In silico scanning was done to predict the 3D structure of the protein, and modeling was validated using the Ramachandran plot analysis. The chimeric protein (rCCL) was expressed in a prokaryotic expression system (E. coli), purified, and analyzed for their immunogenic properties. It was revealed that the production of a high titer of antibody produced in immunized mice could neutralize the ETEC using the rabbit ileal loop tests. The results indicated that the protein inferred from the recombinant protein (rCCL) construct could act as a proper vaccine candidate against three critical causative agents of diarrheal bacteria at the same time.

Efficacy assessment of a triple anthrax chimeric antigen as a vaccine candidate in guinea pigs: challenge test with Bacillus anthracis 17 JB strain spores.

CONTEXT: Bacillus anthracis secretes a tripartite toxin comprising protective antigen (PA), edema factor (EF), and lethal factor (LF). The human anthrax vaccine is mainly composed of the anthrax protective antigen (PA). Considerable efforts are being directed towards improving the efficacy of vaccines because the use of commercial anthrax vaccines (human/veterinary) is associated with several limitations. OBJECTIVE: In this study, a triple chimeric antigen referred to as ELP (gene accession no: MT590758) comprising highly immunogenic domains of PA, LF, and EF was designed, constructed, and assessed for the immunization capacity against anthrax in a guinea pig model. MATERIALS AND METHODS: Immunization was carried out considering antigen titration and immunization protocol. The immunoprotective efficacy of the ELP was evaluated in guinea pigs and compared with the potency of veterinary anthrax vaccine using a challenge test with B. anthracis 17JB strain spores. RESULTS: The results demonstrated that the ELP antigen induced strong humoral responses. The T-cell response of the ELP was found to be similar to PA, and showed that the ELP could protect 100%, 100%, 100%, 80% and 60% of the animals from 50, 70, 90, 100 and 120 times the minimum lethal dose (MLD, equal 5 × 105 spore/ml), respectively, which killed control animals within 48 h. DISCUSSION AND CONCLUSIONS: It is concluded that the ELP antigen has the necessary requirement for proper immunization against anthrax and it can be used to develop an effective recombinant vaccine candidate against anthrax.

Efficient detection of eukaryotic calcium-sensing receptor (CaSR) by polyclonal antibody against prokaryotic expressed truncated CaSR.

Calcium-sensing receptor (CaSR), which is better known for its action as regulating calcium homeostasis, can bind various ligands. To facilitate research on CaSR and understand the receptor's function further, an in silico designed truncated protein was developed. The resulting protein folding indicated that 99% of predicted three dimensional (3D) structure residues are located in favored and allowed Ramachandran plots. However, it was found that such protein does not fold properly when expressed in prokaryotic host cells. Thioredoxin (Trx) tag was conjugated to increase the final protein's solubility, which could help obtain the soluble antigen with better immunogenic properties. The truncated recombinant proteins were expressed and purified in two forms (Trx-CaSR: RR19 and CaSR: RRJ19). The polyclonal antibody was induced by the rabbit immunization with the form of RR19. Western blot on mouse kidney lysates evidenced the proper immune recognition of the receptor by the produced antibody. The specificity and sensitivity of antibodies were also assayed by immunohistofluorescence. These experiments affirmed antibody's ability to indicate the receptor on the cell surface in native form and the possibility of applying such antibodies in further cellular and tissue assays.

Canola oilseed- and Escherichia coli- derived hepatitis C virus (HCV) core proteins adjuvanted with oil bodies, induced robust Th1-oriented immune responses in immunized mice.

Induction of broad Th1 cellular immune responses and cytokines is crucial characteristics for vaccines against intracellular infections such as hepatitis C virus (HCV). Plants (especially oilseed tissues) and plant-immunomodulators (like oil bodies) offer cost-effective and scalable possibilities for the production of immunologically relevant and safe vaccine antigens and adjuvants, respectively. Herein, we provide data of the murine immunization by transgenic canola oilseed-derived HCV core protein (HCVcp) soluble extract (TSE) and Escherichia coli- derived rHCVcp in combination with Canola oil bodies (oil) compared to that of the Freund's (FA) adjuvant. Mice immunized by TSE+ oil developed both strong humeral (IgG) and Th1-biased cellular responses, manifested by high levels of IFN-γ and lower IgG1/IgG2a ratio and IL-4 secretion. Results of the intracellular cytokine staining indicated that TSE+ oil immunization in mice triggered both CD4+ and CD8+ T cells to release IFN-γ, while CD4+ cells were mostly triggered when FA was used. Analyses by qRT-PCR indicated that a combination of rHCVcp/TSE with oil body induced high levels of IL-10 cytokines compared to that of the FA adjuvant. These characteristics are important properties for the design of an HCV vaccine candidate and indicate the potential of Canola-derived antigen and oil bodies in addressing these concerns.

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.

Immunogenic Evaluation of Bivalent Vaccine Candidate against Enterohemorrhagic and Enterotoxigenic Escherichia coli.

BACKGROUND: Caused by bacterial, viral, and parasitic pathogens, diarrhea is the second leading cause of death among children under five. Two strains of E. coli, namely Enterotoxigenic, ETEC and Enterohemorrhagic EHEC are the most important causes of this disease in developing countries. EHEC is a major causative agent of bloody diarrhea and hemorrhagic uremic syndrome, while ETEC is the most important cause of diarrhea in neonates and travelers. OBJECTIVES: To evaluate the immunologic properties of a subunit vaccine candidate comprising the main immunogenic epitopes from these two bacterial strains. METHODS: The construct comprised of LTB and CfaB antigens from ETEC, and Intimin and Stx2B antigens from EHEC, was designed, analyzed and synthesized using bioinformatics methods. The chimeric gene was sub-cloned in the expression vector and expressed in E. coli host. The purified chimera protein was injected subcutaneously into the experimental animals. The production of specific antibodies was confirmed by immunological methods, and the protection capacity was evaluated by the challenge of immunized mice with the pathogenic bacteria. RESULTS: Chimeric recombinant protein was able to increase IgG titer. Neutralization assay indicated that the antibodies generated against LtB moiety were able to neutralize ETEC toxin. In animal challenge study, all non-immune mice died within 3 days after the injection of toxin, but all immunized mice survived from Stx toxin. CONCLUSIONS: The immunity to both ETEC and EHEC bacteria is significant, and this structure can be considered as a candidate for vaccine production against these bacterial strains.

Chitosan nano-structure loaded with recombinant E. coli O157:H7 antigens as a vaccine candidate can effectively increase immunization capacity.

Escherichia coli O157:H7 is considered as emerging foodborne pathogens that occur globally. Three major virulence protein factors; EspA(E), intimin(I), Tir(T) and Stx2 toxin have been found to be highly associated with bloody diarrhoea or, Haemolytic Uremic Syndrome. In this study, a trivalent recombinant EIT in combination with the binding domain of STX toxin were encapsulated with chitosan nanoparticles as a combination vaccine candidate. Mice were immunized either subcutaneously or orally with these antigens and challenged with E. coli O157:H7. Results of the binding inhibition assay with caco2 cell monolayer show a significant reduction in the adhesion percentage of pre-treated E. coli O157:H7 with immunized mice sera. Evaluation of neutralizing abilities of immune sera pre-incubated with CD50 dose of STX2 by Vero cells cytotoxicity neutralization assay shows less morphological reforms in comparison with the control groups. Results of mice mortality challenge with STX2 demonstrate around 66% of survived in immunized mice. In a challenge experiment with E. coli O157:H7, all the immunized mice showed a significant decrease in bacterial colonization and shedding. The results indicate that the use of multiple recombinant proteins in combination with natural nanostructure effectively evocated strong humoral and mucosal response, increasing the protection capacity of the synthetic antigen.

Prevention of EHEC infection by chitosan nano-structure coupled with synthetic recombinant antigen.

One of highly effective methods for prevention and control of Entrohemorragic Esherichia coli (EHEC) infections is to use vaccination against extremely immunogenic part of attachment factors. In this study rEIT (EspA, Intimin, Tir) was produced in bacteria and then encapsulated with chitosan nanoparticle as a candidate nanovaccine. A chimeric trivalent recombinant protein which was previously found to provide reasonable immunogenicity against E.coli O157:H7 was used as a base. Mice immunized orally with chitosan based nanoparticle containing rEIT antigen. The rEIT-specific immune responses (IgG and IgA) were measured by indirect ELISA. In challenging tests different groups of immunized mice were infected orally with E.coli O157:H7. The results showed that the recombinant nanovaccine candidate could induce the strong humoral and mucosal immune responses and protect the mice from live EHEC O157:H7 challenge. Higher titers of serum anti rEIT IgG were achieved after the last immunization in all of the groups. Comparison of the amount of IgA titers in serum and feces showed higher values for the latter. In vitro study of binding inhibition assay on Caco-2 cell monolayers by pre-incubated antisera with EHEC bacteria, showed that immunized mice antibody could reduce adhesion properties of E. coli O157:H7. In a challenging study with EHEC bacteria, reduction in number of colonies was observed in all of the immunized groups for over two weeks. Results from the present study prove that nanovaccine candidate with rEIT can reduce signs and symptoms of EHEC infections. This novel approach can be a new strategy for inducing immunity against E. coli O157:H7. This study suggests the use of oral -injection combined vaccination routes comparing to other methods available in order to achieve higher humoral and mucosal immunogenicity levels.

A new triple chimeric protein as a high immunogenic antigen against anthrax toxins: theoretical and experimental analyses.

Background: Anthrax is a zoonotic disease caused by Bacillus anthracis and it can be deadly in 6 days. Considerable efforts have been conducted toward developing more effective veterinary and human anthrax vaccines because these common vaccines have several limitations. B. anthracis secretes a tripartite toxin, comprising protective antigen (PA), edema factor (EF), and lethal factor (LF). Several studies have shown important role of PA in protection of anthrax. LF and EF induce production of toxin neutralizing antibodies too. PA in fusion form with LF/EF has synergistic effects as a potential subunit vaccine. Methods: In this study, for the first time, a triple chimeric protein called ELP was modeled by fusing three different domains of anthrax toxic antigens, the N-terminal domains of EF and LF, and the C-terminal domain of PA as a high immunogenic antigen using Modeller 9.19 software. Immunogenicity of the ELP was assessed in guinea pigs using enzyme-linked immunosorbent assay (ELISA) test and MTT assay. Results: Theoretical studies and molecular dynamics (MD) simulation results suggest that the ELP model had acceptable quality and stability. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified ELP, its domains, and PA were matched with their molecular size and confirmed by western blotting analysis. In the immune guinea pigs, antibody was produced against all of the ELP domains. It was observed that ELP induced strong humoral response and could protect murine macrophage cell line (RAW 264.7 cells) against anthrax lethal toxin (LeTx). Conclusions: ELP chimeric antigen could be considered as a high immunogenic antigen.

Immunogenicity Evaluation of Recombinant Edible Vaccine Candidate Containing HER2-MUC1 against Breast Cancer.

Human epithelial growth factor receptor2 (Her2) and polymorphic epithelial mucin (MUC1) are tumor-associated antigens that have been extensively investigated in adenocarcinomas. Generally, each of these molecules was used separately for diagnosis of adenocarcinomas and as an injective vaccines in cancer therapy researches, but not in the chimeric form as an edible immunogen. In this study, Her2, MUC1, and a novel fusion structure were expressed in the seeds and hairy roots of transgenic plants appropriately. The mice groups were immunized either by feeding of transgenic seeds or hairy roots. All immunized groups showed a considerable rise in anti-glycoprotein serum IgG and IgA, and IFNÉ£ cytokine. However, the animals received chimeric protein showed significant higher immune responses in comparison to ones received one of these immunogen. The results indicated that the oral immunization of an animal model with transgenic plants could effectively elicit immune responses against two major tumor-associated antigens.

Induction of protective immunity by recombinant peptidoglycan associated lipoprotein (rPAL) protein of Legionella pneumophila in a BALB/c mouse model.

Legionella pneumophila causes a severe form of pneumonia known as Legionnaires' disease especially in patients with impaired cellular immune response. In order to prevent the disease, immunogenicity and the level of the induction of protective immunity from the recombinant peptidoglycan-associated lipoprotein (rPAL) against Legionella pneumophila in BALB/c mice was examined. Mice immunized with (rPAL) rapidly increased an antibody response in serum and also displayed a strong activation of both innate and adaptive cell-mediated immunity as determined by antigen-specific splenocyte proliferation, an early production of pro-inflammatory cytokines in the serum and in the splenocyte cultures. Infection with a primary sublethal does of Legionella pneumophila serogroup 1, strain paris, caused resistance to a lethal challenge infection in the animals with 100% survival rate. However, mice treated with rPAL survived with 60% rate in 10 days after a lethal i.v challenge with L. pneumophila. All of the control animals receiving PBS died within 24 h. The present study indicates that recombinant protein PAL of Legionella pneumophila is strongly immunogenic and capable to elicit early innate and adaptive immune responses and lasting immunity against a lethal dose of Legionella pneumophila challenge. Antigenic characterization and immune protection of recombinant protein PAL would be of considerable value in comprehension the immune-pathogenesis of the disease and in development possible vaccine against the Legionella.