A Therapeutic Oral Vaccine Candidate against Propionibacterium acnes: Preparation and Immunological Evaluation of Nanoparticles Encapsulated Sialidase-CAMP Fusion Protein

Abstract Acne Vulgaris is a common skin disease caused by Propionibacterium acnes, an anaerobic microbiota of human skin that plays a vital role in the pathology of acne. The aim of this study was to prepare nanoparticles containing an acne recombinant protein and determine its ability as an oral acne vaccine in mice. The recombinant Sialidase-CAMP gene was expressed and purified in a prokaryotic host. The chitosan nanoparticles containing the recombinant protein were prepared, encapsulated, and administered by both oral and subcutaneous routes to Balb/c mice. Sera IgA and IgG and stool IgA titers were measured by ELISA, and the immunized mice were challenged against P. acnes. A 65 kDa recombinant protein was confirmed by SDS-PAGE and western blot. The size and zeta potential of nanoparticles were 80 nm and +18 mV, respectively. After oral immunization, the serum IgG and IgA titers were 1:3200 and 1:16, respectively, and the stool IgA titer was 1:8. In the subcutaneous route, the serum IgG titer was 1:51200. Immunized mice showed no inflammation in the ear of challenged mice. It is the first study that examines a chitosan-nanoparticulated acne fusion protein as an applicable acne vaccine candidate with appropriate immunogenicity potential. Further studies are required to validate the clinical usefulness of this vaccine candidate.

Immunogenicity of the nanovaccine containing intimin recombinant protein in the BALB/c mice

PURPOSE: Escherichia coli O157:H7 is one of the most important pathogens which create hemorrhagic colitis and hemolytic uremic syndrome in human. It is one of the most prevalent causes of diarrhea leading to death of many people every year. The first diagnosed gene in the locus of enterocyte effacement pathogenicity island is eae gene. The product of this gene is a binding protein called intimin belonging to the group of external membrane proteins regarded as a good stimulants of the immune system. Chitosan with its lipophilic property is an environmentally friendly agent able to return to the environment. MATERIALS AND METHODS: Intimin recombinant protein was expressed in pET28a vector with eae gene and purification was performed using Ni-NTA and finally the recombinant protein was approved through western blotting. This protein was encapsulated using chitosan nanoparticles and the size of nanoparticles was measured by Zetasizer. Intimin encapsulated was prescribed for three sessions among three groups of oral, injection, and oral-injection using Chitosan nanoparticles. Challenge was performed for all three groups with 108 E. coli O157:H7 bacteria. RESULTS: Intimin produced by chitosan nanoparticles improves immunological responses through the adjuvant nature of chitosan nanoparticles. Chitosan may be used as a carrier for transportation of the prescribed vaccine. Among the mice, encapsulated intimin could be able to provide suitable titers of IgG and IgA by the aid of chitosan nanoparticles. Results of mice challenge showed that decreased the bacterial shedding significantly. CONCLUSION: Results showed that the chitosan nanovaccine with intimin protein may be used as a suitable candidate vaccine against E. coli O157:H7.

Chitosan-based intranasal vaccine against Escherichia coli O157:H7

Background: Enterohemorrhagic Escherichia coli [EHEC] O157:H7 is an infectious zoonotic pathogen causing human infections. These infections, in some cases, can lead to hemolytic uremic syndrome and its life-threatening complications and even death worldwide. The first intimate bacterial adhesion, intimin [I], with its own receptor translocated intimin receptor [Tir] and E. coli secreted protein A, acting as Tir conduit, are highly immunogenic proteins for vaccine development against E. coli O157:H7

Methods: A chimeric trivalent recombinant protein was previously found to be a suitable strategy for developing vaccines against E. coli O157:H7. In this study, the recombinant EIT [rEIT] was used to design a protective EHEC nasal nanovaccine. Chitosan and its water-soluble derivative, trimethylated chitosan [TMC], as muco-adhesive biopolymers, are good candidates for preparation of nanovaccines. Using the electrospraying technique, as a novel method, we could obtain particles of rEIT loaded with chitosan and TMC on a nanometer scale. Mice were immunized with intranasal administration or intrapretoneal injection of rEIT

Results: The rEIT-specific immune responses [IgG and IgA] were measured by indirect ELISA. Only nasal administration of chitosan electrospray and TMC formulation produced significant secretion IgA. Intranasal administration of nanovaccine reduced the duration of bacterial fecal shedding on mice challenged with E. coli O157:H7

Conclusion: Since development of mucosal vaccines for the prevention of infectious diseases requires efficient antigen delivery; therefore, this research could be a new strategy for developing vaccine against E. coli O157:H7

[Cloning and expression of the binding subunit of shiga-like toxin type 2 gene and immunization study in an animal model]

Objective: Enterohemorrhagic Escherichia coli [EHEC] which produces shiga-like toxin type 2 [Stx2] is a major cause of bloody diarrhea. This pathogen can lead to hemolytic uremic syndrome [HUS] and renal failure with a high mortality rate. Stx2 is the major virulence factor of EHEC. Neutralization of toxin by specific antibodies is known to be the best way to prevent and cure HUS. In this study, we describe the cloning, expression, purification, and immunization of the Stx2B subunit which is responsible for toxin binding to the target cell surface

Methods: The Stx2B gene was amplified by PCR and subcloned into a pET28a expression vector and transformed into E. coli BL21-DE3. We evaluated recombinant protein expression and rSTX2B was purified by the Ni-NTA column. The purified rSTX2B was administered subcutaneously to BALB/c mice in three separate doses as an immunogenic candidate. The raising of anti-rSTX2B antibodies in immunized mice sera was evaluated by Elisa assay. The neutralizing immune response was verified by an in vitro assay on HeLa cells and an in vivo assay on mice by challenging them with a lethal dose of Stx2

Results: The IgG titration verified the induction of a humeral response in immunized mice. The HeLa cell assay indicated that the Stx2 toxin was neutralized by immune mice sera. In the challenge assay, 70% of immunized mice survived

Conclusion: Recombinant rSTX2B can induce a neutralizing immune response in mice. It can be used as a major component in development of EHEC vaccines

A potent multivalent vaccine for modulation of immune system in atherosclerosis: an in silico approach

PURPOSE: Atherosclerosis is classically defined as an immune-mediated disease characterized by accumulation of low-density lipoprotein cholesterol over intima in medium sized and large arteries. Recent studies have demonstrated that both innate and adaptive immune responses are involved in atherosclerosis. In addition, experimental and human models have recognized many autoantigens in pathophysiology of this disease. Oxidized low-density lipoproteins, beta2 glycoprotein I (beta-2-GPI), and heat shock protein 60 (HSP60) are the best studied of them which can represent promising approach to design worthwhile vaccines for modulation of atherosclerosis. MATERIALS AND METHODS: In silico approaches are the best tools for design and evaluation of the vaccines before initiating the experimental study. In this study, we identified immunogenic epitopes of HSP60, ApoB-100, and beta-2-GPI as major antigens to construct a chimeric protein through bioinformatics tools. Additionally, we have evaluated physico-chemical properties, structures, stability, MHC binding properties, humoral and cellular immune responses, and allergenicity of this chimeric protein by means of bioinformatics tools and servers. RESULTS: Validation results indicated that 89.1% residues locate in favorite or additional allowed region of Ramachandran plot. Also, based on Ramachandran plot analysis this protein could be classified as a stable fusion protein. In addition, the epitopes in the chimeric protein had strong potential to induce both the B-cell and T-cell mediated immune responses. CONCLUSION: Our results supported that this chimeric vaccine could be effectively utilized as a multivalent vaccine for prevention and modulation of atherosclerosis.

[Design and expression of recombinant HER-2 antigen as a marker for detection of breast cancer]

Objective: The incidence of breast cancer is approximately one million which makes this cancer one of the most common among women worldwide. Breast cancer comprises 7% of the total death rate caused by cancers. Several strategies that use tumor-associated antigen [TAA] vaccination and early detection of breast cancer are clinically being developed. Breast cancer is caused by increased over expression of certain genes. HER-2 is a tyrosine kinase receptor in the epidermal growth factor family. The role of HER-2 in breast cancer has been extensively studied. HER-2 is found in 25%-30% of breast cancer patients. Herceptin, a human antibody, is used as a therapeutic target for HER-2. The purpose of this study is to produce recombinant protein HER-2 for early detection of breast cancer cells

Methods: We used specific primers to amplify the HER-2 gene. The amplified gene was cloned into pET28a as an expression vector. Cloning was confirmed by restriction analysis and sequencing. Expression was induced using IPTG and the recombinant protein was analyzed by SDS-PAGE

Results: Cloning of the HER-2 gene was confirmed by enzyme digestion and sequencing. The gene was expressed in E.coli BL21 DE3. The pET-28a vector which contained the HER-2 gene showed a high level of expression. The recombinant protein was confirmed by Western blot analysis

Conclusions: A portion of the HER-2 gene was expressed as a recombinant in E.coli. This could be a good diagnostic test for breast cancer

in silico chimeric vaccine targeting breast cancer containing inherent adjuvant

Today, Lack of efficient therapeutic strategy for breast cancer [the most common cause of death in women] is one of the momentous problematic topics for all health care committees. Designing new specific vaccine, based on antigens located on the surface of cancer cells can be useful. Over expression of ROR1, lacked of HER2/neu, and hormone receptors on cell surface in the breast cancer, introduce this protein as an appropriate candidate for designing cancer vaccine. We hypothesized the extracellular domain of receptor tyrosine kinase like orphan receptor 1 [ROR-1] along with a super antigen such as staphylococcal enterotoxin B could be a potent vaccine for drug resistant breast cancer. Here, we assessed the findings of bioinformatics analysis to identify the antitumor immune properties of this chimeric construct. In addition, the stability, physic-chemical properties and allergic potency of designed fusion protein were investigated by valid bioinformatics software. Our result suggested that chimeric model is capable to be a stimulant of both T-cell and B- cell mediated immune responses with an acceptable accessibility and solubility but without any allergenicity. The ROR-1 with an enterotoxin B could be a potent vaccine for breast cancer

[Cloning and expression of muc-1 gene in prokaryotic host for early detection of breast cancer]

Background and Aim: Breast cancer is the second most common cause of death among women in the world. Normal mammary gland and breast carcinomas are under the control of regulatory factors, activators and inhibitors inside the breast tissue, as well as a number of growth factors, receptors and proteins outside the breast tissue. Levels of tumor-associated antigens can be used as a predictor in the treatment of this disease. Use of antibodies against MUC1 antigen which is over expressed in 90% of breast cancers is a modern method of treatment. MUC1 tumor antigen disturbs the function of E-cadherin as a cell adhesion molecule. The purpose of this study was to produce MUC-1 recombinant protein for early diagnosis of breast cancer

Material and Methods: A part of MUC-1 gene was amplified by PCR. Then it was cloned into plasmid pET28a in order to be expressed in prokaryotic system. Plasmid pET28a was entered into E.coli BL21DE3 using heat shock method. Cloning process by digestive enzymes and sequence determination were confirmed. Bacteria containing recombinant plasmids were induced by using IPTG and the protein expression was investigated by SDS-PAGE gel

Results: The gene was cloned in the plasmid and the method was confirmed by enzyme digestion and sequencing. Gene expression was confirmed by western blotting

Conclusion: A part of human recombinant MUC-1 gene was produced in E.coli bacteria which can be used as a suitable diagnostic candidate for breast cancer

In silico analysis for identifying potential vaccine candidates against Staphylococcus aureus

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

In silico design and analysis of TGF alpha L3-SEB fusion protein as "a new antitumor agent" candidate by ligand-targeted superantigens technique

Bacterial superantigen Staphylococcal Enterotoxins [SEs], has stimulated polyclonal T cells irrespective of their antigen specificity, resulted a massive release of cytokines, and suggested that they could be assigned as a candidate of new antitumor agents. Recent attempts have done to specifically target superantigens towards tumors, subsequently Monoclonal antibodies and tumor-related ligands have employed as targeting molecules of superantigen for the preclinical treatment of different tumors. Here, we have evaluated TGF alpha L3-SEB fusion protein as a new antitumor candidate by genetically fusing the third loop of transforming growth factor alpha [TGF alpha L3] to Staphylococcal Enterotoxin type B. An in silico techniques have launched to characterize the properties and structure of the protein, before initiating the experimental study, we have predicted physicochemical properties, structures, stability, MHC binding properties and ligand-receptor interaction of this chimeric protein by means of computational bioinformatics tools and servers. Our results have indicated codon adaptation index of tgf alpha l3-seb fusion gene has increased from 0.5 in the wild type sequences to 0.85 in the chimeric optimized gene. The mfold data has shown the tgf alpha l3-seb mRNA was stable enough for efficient translation in the new host. Based on Ramachandran plot TGF alpha L3-SEB has classified as a stable fusion protein. Our result has shown fusing of TGFalphaL3 in N-terminal of the TGF alpha L3-SEB construct, had no effects on MHC binding and subsequently superantigenic activity of SEB. Finally based on ligand-receptor docking the binding ability of TGFalphaL3 was strong enough to its receptor, so TGF alpha L3-SEB could be assigned as a new antitumor candidate in cancer immunotherapy. Our results have proposed that TGF alpha L3-SEB was a stable fusion protein with proper affinity to its receptor that overexpressed in various human carcinomas, so it could generate potent immune response towards tumors

[Production of chimeric tir-intimin protein from E. coli O157:H7 in the tobacco [Nicotiana tobbacum] plant and its immunological evaluation in an animal model]

Escherichia coli [E.coli] O157:H7 is one of the most important pathogenic causes of hemorrhagic colitis in humans. Cattle are the main reservoirs of this bacteria and vaccination is a key mechanism for its control. The intimin, translocated intimin receptor [tir], and EspA proteins are virulence factors expressed by the LEE locus of enterohemorrhagic E. coli. EspA protein is a member of the type III secretion system [TTSS] needle complexes that delivers the tir protein into the host cell. Surface arrayed intimin docks the bacterium to the translocated intimin receptor [Tir]. This intimate linkage is the starting point for attachment and effacing lesions. We hypothesize that the chimeric recombinant forms of two of these three effectors, as edible-based immunogens, would reduce colonization of E. coli O157:H7 in the mice model. We constructed a synthetic gene [it] composed of eae [i] and tir [t] attached together by a peptide linker. The synthetic gene [it] was codon optimized based on the tobacco [Nicotiana tobbacum] plant and cloned into plant expression vectors adjacent to CaMV35S promoters for expression in transgenic tobacco plants. The antigen produced in this plant was orally fed to mice. Immunization of the mice model by the transgenic plant that contained the divalent immunogen showed the presence of IgG antibodies against E. coli O157:H7. This method could be an effective tool for protecting against E. coli O157:H7 hemorrhagic colitis

DNA vaccine against escherichia coli o157:H7

Infection with Escherichia coli O157:H7 rarely leads to bloody diarrhea and causes hemolytic uremic syndrome with renal failure that can be deadly dangerous. Intimin, translocated Intimin receptor [Tir], and enterohemorrhagic E. coli [EHEC] secreted protein A [EspA] proteins are the virulence factors expressed by locus of enterocyte effacement locus of EHEC. This bacterium needs EspA as a conduit for Tir delivery into the host cell and the surface arrayed Intimin, which docks the bacterium to the translocated Tir. Here we used triplet synthetic gene [eit] which was designed from three genes: espA coding EspA 120 lacking 36 amino acids from the N-terminal of the protein, eae coding Intimin constructed of 282 amino acids from the C-terminal and tir coding Tir 103, residues 258-361 which interacts with Intimin. The multimeric gene was cloned in two eukaryotic vectors pAAV-multiple cloning site-green fluorescent protein and pCI-neo. The pAAV was used for gene expression assay in cell line 293T and pCI-neo-EIT [EspA, Intimin, Tir] was used as DNA vaccine in mice. Test groups were injected intramuscularly with pCI-neo-EIT four times and mice control group was injected under the same conditions with PBS or pCI-neo vector. The titration of serums showed that BALB/c mice were successfully immunized with DNA vaccine compared to control groups and also they were protected against challenges of live oral using E. coli O157:H7. Conclusion: The results suggest that the DNA vaccine could induce protective immunity either alone or in combination with purified antigens to reduce EHEC infection

[Expression of optimized gene of Enterotoxigenic Escherichia coli CFA/I major subunit]

Enterotoxigenic Escherichia coli is considered as the most important agent of children diarrhea and mortality in developing countries. This bacterium causes 300-600 thousands of deaths in the children under 5 years of age per year. With difficulties in treatment as well as its wide prevalence, designing an effective vaccine against this microorganism is the objective of world Health Organization [WHO]. The CfaB protein as immunogen and major subunit of fimberia has a critical role in the bacterial attachment to small intestine epithelium and the produced antibody against this protein can prevent attachment of bacterium to epithelial surface. Hence, this molecule alone or with other virulent factors has been considered by many researchers in vaccine designing. In this study, expression of colonization factor B with the aim of studying the immunogenesity of this protein as a component of vaccine candidate was performed. cfaB gene was amplified by PCR and cloned into pET28a and its expression was evaluated. Since there was no expression, which was due to presence of rare codon, the cfaB gene was again cloned into pET28a using codon bias in E. coli and subsequently expressed. Presence of a 20KD band on SDS-PAGE gel indicated the expression of CfaB protein, which was later confirmed by immunoblotting with anti-His tag and anti CfaB antibodies and purified on Ni-NTA column. Codon optimization and expression in heterologous hosts is a useful approach for obtaining large quantities of recombinant protein

Immunogenic and protective potentials of recombinant receptor binding domain and a C-terminal fragment of Clostridium botulinum neurotoxin type E

Clostridium Botulinum Type E neurotoxin heavy chain consists of two domains: the translocation domain as the N-terminal half and the binding domain as the Cterminal half [Hc]. One effective way to neutralize botulinum neurotoxin is to inhibit binding of this toxin to neuromuscular synapses with antibodies against binding domain. Two synthetic genes, coding for Hc [the full length binding domain] and the c-terminal quarter of binding domain [HcQ], were cloned in pET-28a vector and over-expressed in E. coli BL21 [DE3] cells. These recombinant proteins were purified by affinity Ni-NTA column [under native condition]. Mice were vaccinated with 2 microg of purified proteins, respectively; at step one with complete adjuvant, steps two and three with incomplete adjuvant and step four only with phosphate buffered saline [PBS]. Enzyme-linked immunosorbent assay [ELISA] has been performed with mice serum samples 14 days following their third and final vaccination. Binding activity of the purified proteins to ganglioside and synaptotagmin II was analyzed by ELISA. The results showed that HcQ and Hc could bind with ganglioside. Based on challenge experiments it was revealed that HcQ, Hc and BoNT/E toxoid could give protections in mice challenged with 10[2], 10[4] and 10[5] minimum lethal dose [MLD] dose of BoNT/E

simple and rapid leaf genomic DNA extraction method for polymerase chain reaction analysis

In plants, secondary metabolites and polysaccharides interfere with genomic isolation procedures and downstream reactions such as restriction enzyme analysis and gene amplification. The removal of such contaminants needs complicated and time-consuming protocols. In this study, a simple, rapid and efficient method for leaf DNA extraction was optimized. This method use small amount of plant material to reduce inhibitory agents [alkaloids, phenolic]. The procedure involves homogenization of the plant leaf in extraction buffer, incubation at 60°C, extraction by chloroform: iso-amyl alcohol and finally DNA precipitation by cold isopropanol. The results showed that the extracted DNA could be used directly for PCR

Simultaneous detection of Escherichia coli O157:H7, toxigenic Vibrio cholera, and Salmonella typhimurium by multiplex PCR

Escherichia coli O157:H7, Vibrio cholerae, and Salmonella typhimurium are pathogenic bacteria found inn contaminated water and food. No assay method is currently available on simultaneous detection or identification of all the three pathogens. Our aim was to develop a rapid and reliable method for this purpose. A protocol for sample collection, and a PCR procedure was designed specifically for the assay. Selected fragments of 239 bp, 432 bp, and 360 bp for E. coli O157 lipopolysaccharide [LPS] gene [rfbE], V. cholerae toxin gene [ctx], and Salmonella typhimurium putative cytoplasmic protein gene [STM4497], respectively, were amplified from the extracted bacterial DNA samples in a single tube by multiplex PCR. The multiplex PCR products were analyzed by gel electrophoresis. All unknown samples were verifiably identified. The assay was sensitive enough to detect and identify as few as 100 cells of E. coli O157:H7, V. cholerae and Salmonella typhimurium. The presence of other bacteria did not interfere with the analysis. This assay is a specific and reliable tool which allows cost-effective detection o all three bacterial pathogens in one reaction tube