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.

[Production of antibody against CtxB recombinant protein based on nanofibers and nanocapsules]

Objectives: CtxB [Cholera toxin B subunit] contributes to a vaccine's efficacy by stimulating production of the anti-CtxB antibody. Various attempts have been made to increase production of this antibody. Chitosan is a mucoadhesive polysaccharide that has tremendous potential for oral vaccine delivery in terms of its exclusive features that include biocompatibility, biodegradability, high charge density and non-toxicity. We investigated the potential for chitosan nanofibers and nanocapsules as novel carrier systems for the oral delivery of CtxB

Methods: Antigen-containing chitosan nanofibers were prepared by electrospinning a chitosan/AcOH solution. Encapsulation of the antigen inside the chitosan nanofibers was confirmed through infrared spectroscopy analysis [FTIR]. Guinea pigs were immunized with free antigen and CtxB antigen or antigen alone by direct administration of antigencontaining chitosan nanofibers into the buccal cavity. Serum immunoglobulin G [IgG] and intestinal immunoglobulin A [IgA] antibody responses were determined

Results: The results indicated that antigen in the chitosan nanofibers or nanocapsules elicited very high IgA and IgG responses. No detectable IgA and IgG responses were obtained after oral immunization with CtxB. The results of the antibody titer were analyzed using the ANOVA and LSD tests

Conclusion: CtxB inside the nanofiber increased antibody production when administered orally. This system might be used for delivery of other antigens

Immunogenicity of a fusion protein comprising coli surface antigen 3 and labile b subunit of enterotoxigenic escherichia coll

Enterotoxigenic Escherichia coli [ETEC] strains are the major causes of diarrheal disease in humans and animals. Colonization factors and enterotoxins are the major virulence factors in ETEC pathogenesis. For the broad-spectrum protection against ETEC, one could focus on colonization factors and non-toxic heat labile as a vaccine candidate. A fusion protein is composed of a major fimbrial subunit of coli surface antigen 3, and the heat-labile B subunit [LTB] was constructed as a chimeric immunogen. For optimum level expression of protein, the gene was synthesized with codon bias of E. coli. Also, recombinant protein was expressed in E. coli BL21DE3. ELISA and Western tests were carried out for determination of antigen and specificity of antibody raised against recombinant protein in animals. The anti-toxicity and anti-adherence properties of the immune sera against ETEC were also evaluated. Immunological analyses showed the production of high titer of specific antibody in immunized mice. The built-in LTB retains native toxin properties which were approved by GMi binding assay. Pre-treatment of the ETEC cells with anti-sera significantly decreased their adhesion to Caco-2 cells. The results indicated the efficacy of the recombinant chimeric protein as an effective immunogen inducing strong humoral response. The designated chimer would be an interesting prototype for a vaccine and worthy of further investigation

Immunogenicity of a new recombinant IpaC from shigella dysenteriae type I in guinea pig as a vaccine candidate

Recombinant vaccine technology is one of the most developed means in controlling infectious diseases. However, an effective vaccine against Shigella is still missing. To evaluate recombinant IpaC protein of Shigella as a vaccine candidate. In this study we cloned IpaC gene into an expression vector in prokaryotic system. The protein expression was evaluated by SDS-PAGE and Western-Blotting analysis. The recombinant protein was purified using Ni-NTA affinity chromatography. Guinea pigs were immunized with the recombinant protein and the level of immunogenicity was examined by ELISA and Western blotting of IpaC. Challenge test was done through the intraoculary injection of Shigella dysenteriae [6×108 CFU/eye] and after 48 hours was scored for keratoconjunctivitis. The results showed a remarkable level of immunogenicity in terms of antibody response and protection against keratoconjunctivitis in tested animals. The recombinant IpaC protein provided a protective system against Shigella dysenteriae type I during the challenge test. The results showed the potential of using recombinant IpaC in preparation of vaccine in perspective studies

[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

Cloning and Expression of N-terminal Region of IpaD from Shigella dysenteriae in E. coli

Genus Shigella is one of the important members of the family Enterobacteriaceae. There are numerous antigens in Shigella carrying by a 220 kb plasmid. Among them, IpaD is the key virulence factor of S. flexneri. Apart from having effectors function that is essential for host cell invasion and intracellular survival, this protein also controls the secretion and translocation of other effector proteins into eukaryotic host cells. In the present study, we have cloned and expressed the ipaD in E. coli. The ipaD gene was amplified by PCR. Prokaryote expression vector pET-28a [+] - ipaD was constructed, and used to transform E. coli BL21DE3 plySs. The expression of recombinant protein induced by IPTG was examined by SDS-PAGE. Western blot were used to determine immunoreactivity of IpaD-His by a rabbit monoclonal antibodies against his-tag. SDS-PAGE demonstrated that the constructed prokaryotic expression efficiently produced IpaD at the 1 mmol/L of IPTG. IpaD protein was able to react with the rabbit monoclonal antibody against His-tag. IpaD is essential for Shigella spp invasion. N-terminal region is most significant functional fragment of IpaD. Purification of IpaD from the wild type of Shigella is difficult furthermore profound study on a specific domain on the N-terminal of IpaD by using the wild type of purified IpaD is not feasible

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

Simultaneous molecular detection of Neisseria Meningitidis and Haemophilus Influenzae

Bacterial meningitis is a dangerous and sometimes fatal infection that affects the central nervous system. Because some antibiotics can prevent some types of these Bacteria and suppress them from spreading and infecting, therefore it is important to know what type of virus or bacterium is causing meningitis. Haemophilus influenzae and Neisseria meningitides are the two main pathogens causing acute bacterial meningitis. Different methods are used for the detection of H. influenzae and N. meningitidis but they are of low sensitivity, taking long time and difficult to perform . Therefore, complementary methods are necessary for more sensitive detection of these agents. In this study, a muliplex polymerase chain reation [mPCR] assay was developed for detection of H. influenzae and N. meningitidis. These strains were confirmed by biochemical methods. Two specific primer pairs were designed for lic-1 and opa genes of H. influenzae and N. meningitidis respectively. DNA amplification product fragments were 150 bp and 320 bp for H. influenzae and N. meningitidis, respectively. Streptococcus pneumoniae used as a negative control and did not yield a PCR product. The result of this study indicated that PCR is a useful complementary diagnostic technique, especially when Gram stain, culture, or antigenic detection is negative or inconclusive