Cellular immunity survey against urinary tract infection using pVAX/fimH cassette with mammalian and wild type codon usage as a DNA vaccine

PURPOSE: FimH (the adhesion fragment of type 1 fimbriae) is implicated in uropathogenic Escherichia coli (UPEC) attachment to epithelial cells through interaction with mannose. Recently, some studies have found that UPEC can thrive intracellularly causing recurrent urinary tract infection (UTI). Almost all vaccines have been designed to induce antibodies against UPEC. Yet, the humoral immune response is not potent enough to overcome neither the primary UTI nor recurrent infections. However, DNA vaccines offer the possibility of inducing cell mediated immune responses and may be a promising preventive tool. MATERIALS AND METHODS: In this study, we employed two different open reading frames within mammalian (mam) and wild type (wt) codons of fimH gene. Optimized fragments were cloned in pVAX-1. Expression of the protein in COS-7 was confirmed by western blot analysis after assessing pVAX/fimH(mam) and pVAX/fimH(wt). The constructs were injected to BALB/c mice at plantar surface of feet followed by electroporation. RESULTS: The mice immunized with both constructs following booster injection with recombinant FimH showed increased interferon-gamma and interleukin-12 responses significantly higher than non-immunized ones (p<0.05). The immunized mice were challenged with UPEC and then the number of bacteria recovered from the immunized mice was compared with the non-immunized ones. Decreased colony count in immunized mice along with cytokine responses confirmed the promising immune response by the DNA vaccines developed in this study. CONCLUSION: In conclusion, DNA vaccines of UPEC proteins may confer some levels of protection which can be improved by multiple constructs or boosters.

[Designing and optimizing of multiplex polymerase chain reaction for amplification of human anti-tetanus toxoid antibody genes]

Antibodies are used in many areas of diagnosis and treatment. Fully human monoclonal antibodies [mAb] have attracted high attention due to not stimulating immune response in the body. This study was carried out with the purpose of designing and optimizing multiplex polymerase chain reaction for amplification human anti-tetanus toxoid antibody genes. After collecting blood samples from a human immunized with tetanus toxoid and lymphocyte separation, total RNA was extracted and cDNA was synthesized. all varieties of light [Kapa] and heavy chains of antibody were amplified by two separated multiplex PCR reactions using 14 pair primers. PCR was performed to link VH and VL together and make a ScFv segment, and semi nested PCR was performed to add cutting sites of restriction enzymes at the two ends of the segment. In two multiplex PCR reactions, VH and VL segments were amplified with the length of 410 and 680 bp, respectively. After gel extraction, VH and VL were linked together as a 1070 bp ScFv segment. Restriction sites were added to the two ends of ScFv. By selection of an appropriate primer set and optimization of effective factors of multiplex PCR, all varieties of antibody genes derived from total human lymphocyte cDNA could be amplified and extracted using two multiplex PCR reactions instead of several uniplex ones. Therefore, using only two Multiplex PCR reactions, all genetic varieties of human antibody could be amplified as VH and VL segments from the blood of a subject immunized with tetanus toxoid

new approach for designing a potentially vaccine candidate against urinary tract infection by using protein display on lactobacillus surface

The prevalence of Urinary Tract Infection [UTI] is really high in the world. Escherichia coli is a major agent of UTI. One of the strategies for decreasing UTI infections is vaccine development. As the attachment is a really important stage in colonization and infection, attachment inhibition has an applied strategy. FimH protein is a major factor during bacterial colonization in urinary tract and could be used as a vaccine. Thus, it was considered in this research as a candidate antigen. The sequences of fimH and acmA genes were used for designing a synthetic gene. It was cloned to pET23a expression vector and transformed to E. coli [DE3] Origami. To confirm the expression of recombinant protein, SDS-PAGE and western blotting methods were used. Subsequently, recombinant protein was purified. On the other hand, Lactobacillus reuteri was cultured and mixed with FimH / AcmA recombinant protein. The rate of protein localization on lactobacillus surface was assessed using ELISA method. It was showed that the recombinant protein was expressed in E. coli [DE3] Origami and purified by affinity chromatography. Moreover, this protein could be localized on lactobacillus surface by 5 days. In current study, a fusion recombinant protein was prepared and displayed on L. reuteri surface. This strain could be used for animal experiment as a competitor against Uropathogenic E. coli [UPEC]. Using manipulated probiotics strains instead of antibiotic therapy could decrease the antibiotic consumption and reduce multi-drug resistant strains

Prevalence and evaluation of toxin genes among uropathogenic escherichia coli clinical isolates by duplex PCR

One of the most common infections in human is urinary tract infection [UTI] and Uropathogenic Escherichia coli is one of its major causative agents. UTI is extremely common among young women. Children under age 5 are also highly at risk. Considering the prevalence of this disease, it is necessary to design an appropriate diagnostic method for its effective diagnosis. The aim of present study was to identify the prevalence of two virulence genes [sat and vat] among Uropathogenic E. coli isolates. Urine samples were taken from 350 patients with urinary tract infection. The samples were cultured on EMB agar and Blood agar. The suspected E. coli colonies were isolated and confirmed by biochemical tests. The genomic DNA was extracted from 297 isolated E. coli and target genes were amplified by PCR. The amplicons were sequenced and analyzed with ClustalW software. Moreover, data analysis was performed by using SPSS software. Subsequently, Duplex PCR was optimized for simultaneous detection of two genes. The prevalence of sat and vat genes were 75 [n: 225] and 36 [n: 106] percent, respectively. In addition, less than 4% [n: 11] of clinical isolates comprised two genes. According to the conducted research, molecular identification of Uropathogenic E .coli strains according to detection of sat gene is potentially an appropriate method and could be noted for diagnosis.

[ prevalence of extended-spectrum beta-lactamases and CTX-M-1 producing escherichia coli in urine samples collected at Tabriz city hospitals]

Numerous use of Beta Lactame in treatment of bacterial infections resulted in increments of drug resistance of such bacteria. One of difficulties in treatment of hospital infections is Extended Spectrum Beta Lactamase [ESBL] among isolated clinical strains of E.coli. Since some of ESBL strains shows double reaction in drug sensitivity test at in vitro and in vivo condition, therefore it makes difficulties in selection of right treatment. In the last years, CTX-M enzymes have become the most prevalent ESBLs in worldwide. The prevalence of ESBL types largely remains unknown in many parts of the Iran. This study was made to find the prevalence of ESBLproducing E.coli and molecular detection of CTX-M-1 in Tabriz. In the present study, 400 urine samples collected between November 2009 and April 2010, from Tabriz Hospitals were studied. Out of the 400 samples, 188 E.coli isolates were detected by standard biochemical tests. Susceptibility to antimicrobial agents was tested to 10 antibiotics by the disk agar diffusion [DAD] method. ESBL production was screened by phenotypic test that included both separate and combined disk agar diffusion techniques. The screened isolates were investigated by PCR assay to detect CTX-M-1 gene. From the total 188 E.coli isolates, 82 [43.6%] were shown to produce ESBLs by phenotypic test. During the PCR method on the 82 isolates, 69 [84.1%] were confirmed as CTX-M-1 producing group. The present study showed that CTX-M-producing isolates were increasing among E.coli strains and indicated the need for adequate susceptibility tests in laboratories for choosing the appropriate antibiotics for treatment