نیما خرم آبادی

Brucellosis is still an important zoonotic infection and evaluation of immunologic properties of various bacterial antigens along with different vaccination strategies helps in designing efficient vaccines against the disease. The aim of this study is to immunological evaluate the eukaryotic vector pVAX1, carrying the outer membrane protein gene of 31 kDa (Omp31) B.melitensis.
In this study which was carried out in 2014, whole sequence of omp31 of B. melitensis was inserted between BamHI and XhoI of pVAX1 plasmid vector. Female BALB/c mice aged 6-8 weeks (purchased from Pasteur Institute of Iran) were immunized intra-muscularly with 100μg of the construct, followed by either protein or plasmid boosters separately. The level of IL-4, IL-12, IFN-γ, total serum IgG, and specific IgG1 and IgG2a against recombinant Omp31 were evaluated. Finally the protective immune response following exposure to B. melitensis 16M was evaluated.
DNA-vaccine omp31 career with protein reminders Omp31, stimulate higher levels of IFN-γ, IL-12 and IgG2a compared to groups of DNA-vaccine or recombinant protein. Protective immunity was also significantly higher in mice which immunized with DNA vaccine– protein regimen.
Mice which immunized with DNA vaccine–protein regimen showed a significantly higher levels of IL-12 and IFN-γ along with serum IgG2a which together imply augmentation of T cell-mediated immune responses against Omp31. The latter was confirmed by significant protective response to B. melitensis 16M challenge.

Background and Methicillin resistant Staphylococcus aureus (MRSA) is a community-associated pathogen that is so common in hospitals. Antibiotic resistance and poor clinical outcome provide great reasons for using immunization strategies based on antibodies. The aim of this study was to investigate passive immunity using recombinant anti-MRSA ScaF antibody in a mouse model.
In this study, the recombinant protein, ScaF was purified through denaturation method. This protein combined with Freund’s adjuvant was injected into a rabbit to produce antibody. Then, the antibody produced against ScaF was injected to mice infected by S. aureus by two regimens of pre- and after infection treatment. Afterwards, the mortality rate in mice was compared between the control treatment group (vancomycin therapy) and negative control group (non-immune rabbit serum).
Out of 10 mice in two groups of pre- and after infection treatment that received antibody against ScaF antigen 20% and 10% were protected, respectively, that did not show a significant difference compared with non-immune rabbit serum group (negative control).
Passive immunization by antibody cannot play a considerable role in the control of infections caused by S. aureus. Therefore, other mechanisms of immune system for the protection against this bacterium including stimulating of cytokine-dependent cell-mediated immunity may be useful in prevention of infections caused by S. aureus.

Pathogenic Pseudomonas aeruginosa strains produce a polar flagellum that required for motility, adhesion, invasion and secretion of virulence factors. The aim of this study was to evaluate the effect of prevention and treatment with anti-recombinant type B flagellin antibody in a burned mouse model. One hundred twenty six mice were divided into 16 groups injected with different regimen of anti-recombinant type B flagellin antibody. Infections were caused by sub-dermal injection of P. aeruginosa PAO1 and PAK strains at the burn site. Groups were monitored for mortality for one week. Additionally, functional activity of this antibody was assessed by motility inhibition and opsonophagocytosis assays. Immunotherapy with rabbit antisera substantially increased (85.71%) survival rate of mice challenged with a homologous strain PAO1 compared with the control PBS. The mortality rate in mice infected by the heterologous strain PAK was only 28.57%. This antibody increased phagocytosis killing of the homologous strain but it only had a slight effect on the heterologous strain. Passive immunotherapy protected burned mice challenged with the homologous strain but showed lower efficacy against the heterologous strain.

Vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) are the leading nosocomial pathogens in Iran. We aimed at evaluating the resistance patterns of Staphylococcus aureus and enterococci to the antibiotics recommended by Clinical and Laboratory Standards Institute (CLSI).

Two-hundred forty Staphylococcus aureus and 203 Enterococcus isolated from urine, wound, blood, trachea, stool and other clinical specimens of inpatients and outpatients were obtain from some hospitals in Tehran. Sensitivity of S. aureus to oxacillin, vancomycin, chloramphenicol, nitrofurantoin, erythromycin, clindamycin and linezolid were determined by disk diffusion according to CLSI (M100-S22) guidelines. Likewise, sensitivity of enterococci to ampicillin, vancomycin, teicoplanin, tigecycline, linezolid, tetracycline, chloramphenicol and nitrofurantoin was invesditated by the same method.

Of 240 S. aureus isolates, 56%, 46.6% and 40.4%were resistant to oxacillin, erythromycin and clindamycin, respectively. We found one S. aureus isolate with low susceptibility to vancomycin (VISA; 0.5%). All S. aureus isolates were sensitive to chloramphenicol, nitrofurantoin and linezolid. Of 203 Enterococcus spp., 47.3%, 24.6%, 9.4%, 85.2%, 11.3% and 4.4% were resistant to ampicillin, vancomycin, teicoplanin, tetracycline, chloramphenicol and nitrofurantoin, respectively. All these isolates were sensitivity to tigecycline and linezolid.

Resistance of S. aureus isolates to oxacillin, erythromycin and clindamycin was significant. Enterococci isolates were remarkably resistant to ampicillin, vancomycin, teicoplanin, tetracycline, chloramphenicol and nitrofurantoin. Given our results, we suggest vancomycin, chloramphenicol, linezolid and nitrofurantoin for S. aureus infections and tigecycline and linezolid as drugs of choice for enterocoocal infections treatment.

One of the proteins shared in all strains of Brucella is 31 kDa surface protein (BCPS31) that could be an appropriate target for immunization and serological diagnosis. In the present study, BCSP31 produced as a recombinant protein in pET28a (+) expression system was utilized, using ELISA, to detect trace specific antibody (IgG) in brucellosis patient's serum that was confirmed by culture. We also evaluated cytokine response of peripheral blood lymphocytes to this protein in the cell culture. The results indicated a significant amount of surface protein antibodies (IgG) in the serum of patients with brucellosis. Evaluation of lymphocyte responses to rBCSP31 also showed a significant IL-12 and IFN-γ production in patients’ lymphocyte cultures. These results suggest that BCSP31 can elicit specific humoral and cellular responses during host infection and it can be used in designing immunization and serologic diagnosis systems.

One of the most common diseases between zoonosis - especially in developing countries – is brucellosis. Identification of Brucella cell antigen combinations in terms of the amount and type of immune response in infected hosts, are important in vaccine design. 31kDa Brucella cell surface protein (BCSP31) is shared among all Brucellae. We aimed to define specific immune responses to BCSP31 which are elicited during infection of murine host with B. abortus. Surface protein of 31 kDa (BCSP31) is present in all strains, and here, the host immune response during murine infection with Brucella abortus which are formed in proportion to the proteins are studied. Recombinant BCSP31 (rBCSP31) of B. abortus was produced and purified. One group of BALB/c mice were infected with pathogenic B. abortus 544 and maintained for 60 days; a no-infected group of mice also was considered. Specific serum antibodies directed to rBCSP31 was evaluated through ELISA. Splenocytes of mice were cultured and stimulated with rBCSP31; thereafter, IL-4, IL-12 and IFN-γ cytokine responses of spleen lymphocytes were assessed. We detected a remarkable IgG titer along with IgG1 and IgG2a specific to recombinant BCSP31 in serum samples from infected mice. Significant titers of IgG, IgG1 and IgG2a antibodies than BCSP31 in the the serum of mice infected with the virulent strain were observed. The Evaluation of Splenocytes responses to rBCSP31 also showed a significant IL-12 and IFN-γ production along with remarkable IL-4 production in infected mice. All responses were significantly different from that of non-infected mice (p<0.05). These findings suggest that specific humoral and cell-mediated responses to BCSP31 is formed during murine host infection with B. abortus. Based on these findings, rBCSP31 can be used in further design of immunogenic strategies for vaccination against brucellosis.

Haemophilus influenzae is a Gram-negative bacterium that is part of the normal nasopharyngeal flora of most humans. H. influenzae strains were defined in two categories: encapsulated (typeable) and non-capsulated (nontypeable) strains. Outer membrane protein P6 is a highly conserved and stable protein in the outer membrane of both encapsulated and non-capsulated strains of H. influenzae. As an immunogen, P6 protein is a potential candidate vaccine against H. influenzae strains. The aim of this study was to produce recombinant protein P6 as a carrier protein for production of conjugate vaccines. The sequence (324 bp) coding P647-153 protein of H. influenzae was successfully cloned in pJET1.2 and subsequently in pET28a (+). Expression of the recombinant protein was induced with 1mM IPTG. Recombinant P647-153 was purified through dissolving inclusions in 8M urea buffer, absorbing to Ni-NTA resins, washing by buffers with decreasing urea concentration and finally eluted in Imidazole solution. Imidazole was removed by dialysis against PBS (pH 7.4). The recombinant p647-153 was confirmed by western blot analysis using rabbit anti H. influenzae polyclonal antiserum. The recombinant P647-153 was successfully expressed in E. coli BL21 (DE3) and purified (4 mg /lit of broth culture). The immunoblotting showed that recombinant P647-153 conserved its native antigenic structure. Western blot results, along with that of sequencing, confirmed accurate production of recombinant P647-153 and partially retention of its conformational epitopes.

Antigen 85 complex of Mycobacterium tuberculosis includes three immunogenic proteins which are important TB vaccine candidates. The use of these protein as a component of subunit vaccines, as a part of DNA vaccines or recombinant BCG boosters, enhances their recombinant production. Recombinant production of these mycobacterial proteins located in the cell wall somehow differs from the other proteins, as they are partially apolar. Therefore, this study aims to produce recombinant Ag85C as a vaccine candidate. Ag85C gene was cloned in pJET1.2 and subsequently in pET32a(+). Both recombinant plasmids were sequenced. Expression of the recombinant protein was induced with 1mM IPTG. Recombinant Ag85C was purified through dissolving the inclusions in 8M urea buffer, absorbed to Ni-NTA resins, washed by buffers with decreasing urea concentrations, and finally eluted in aqueous solution. Western blot analysis was performed using anti-6His tag antibody, rabbit anti-M. tuberculosis polyclonal antibody, and the serum from hospitalized TB patients. Ag85C successfully cloned in both plasmid vectors. The recombinant Ag85C expressed in the E. coli host and was purified with significant yield. Western blot results along with that of sequencing ensure accurate production of recombinant Ag85C, retaining its partial epitopes.

The identification of Brucella spp. antigens with the capacity to elicit a protective immune response is of the great interest for the researchers. So, characterization and assessment of diverse antigens of Brucella need to be evaluated. In this study, we report the cloning and expression of the gene coding for 31 KDa OMP (OMP31) of Brucella melitensis 16M. Brucella melitensis Omp31 gene was amplified with specific primers, cloned into pJET1/2 and subsequently subcloned in pET28a (+) vector. Both these recombinant plasmids were sequenced and then after, expression of recombinant protein was induced by 1mM IPTG. Western blot analysis was also performed by polyclonal rabbit antiserum. Omp31 successfully was cloned in both plasmid vectors. The recombinant Omp31 was expressed in E.coli host and purified with significant yield. Western blot results along with those of sequencing ensured accurate production of recombinant omp31 and retaining of its partial epitopes. Our results show that, an expression host such as E. coli is suitable for omp31 production.

Haemophilus influenzae type b (Hib) is a gram negative bacterium and one of the most common causative agents of acute meningitis in infants and less than 5 years old children worldwide. The production of Hib capsular polysaccharide; polyribosyl ribitolphosphate (PRP) is important for the production of conjugate vaccines against Hib infections. The aim of this study is the improvement of Large-scale PRP production by Hib. Haemophilus influenzae type b standard strain ATCC10211 was cultivated in 2L fermentors contain 1.5L CY (casaminoacid yeast extract) medium with normal or modified concentrations of glucose, yeast extract, hemin and NAD (nicotinamide adenine dinucleotide). Seed culture of two fermentors was inoculated to 50 L fermentor, separately and range of PRP production and Dry cell weight (DCW) were studied. Cultivation of Hib in 50L fermentor contained modified CY medium with 6gl-1 Glucose, 2.5 gl-1 Yeast extract, 0.03 gl-1 Hemin and 0.015 gl-1 NAD, with controlled pH at 7.3 and 30% Dissolved oxygen tension (DOT) resulted to about 5.1 gl-1 DCW and 1.16 gl-1 PRP, that was significantly higher than normal CY medium. In conclusion, by modification in some medium components of CY medium, control of Dissolved oxygen tension and pH, the Large-scale production of PRP is improved. Improvement of PRP production leads to reduce the final cost of Hib conjugate vaccines.

A quantitative TaqMn Real-Time PCR assay was developed and its diagnostic value on human serum and livestock samples were evaluated. Brucella species could be distributed through communities as a biological agent. Rapid detection of biological threat agents is critical for timely therapeutic administration. Quantitative real-time PCR pro vides a rapid, sensitive and specific tool for molecular identification of this agent. We evaluated a new real-time PCR set by numerous human and livestock samples. For primers and probe designation BCSP31 of B. melitensis 16 M was used. Different sero-positive human samples and tissue samples livestock with suspected brucellosis were then assayed after primary adaptation of Q-PCR in the laboratory. The detection limit of the assay was 10 fg (equivalent to 2 genome). Totally 50 samples (25 sero-positive, 25 sero-negative) of patients and 75 samples from slaughtered livestock (due to brucellosis) were collected and assayed by Q-PCR, along with control samples. Brucella genome were detected by this real-time set from nearly 42% (5 out of 12) of negative samples by conventional PCR. This system also detected brucella contamination in 46 out of 75 animal samples.

Brucellosis is an important zoonotic disease of economic significance. Brucella species are gram-negative, facultative intracellular bacteria, and are capable of replicating in the phagosomes of macrophages. They cause infection in several animal species and humans. Prevention of new diseases and diagnosis of cases infected with the organism are both essential for eradication of the disease. Characterization and evaluation of different antigens of Brucella cells has a key role in progression of prevention and diagnosis programs. Here, we report the production and purification of recombinant 3 kDa outer membrane protein Brucella abortus (Omp2b). Brucella abortus 36kDa outer membrane protein gene was amplified with PrimeSTAR® HS DNA polymerase, cloned in pJET1.2. The target gene was subcloned in pET28a (+). Recombinant pET28a vectors were transformed into E coli BL21 (DE3). Expression of recombinant protein was induced with 1mM IPTG. Proteins were absorbed to Ni-NTA agarose resins and Recombinant proteins were eluted with 250mM imidazol. Imidazol removed by dialysis. Proteins were assayed by Western-blotting and rOmp2b was probed by Brucella rabbit anti serum. Appearance of a golden brown band at the site of reaction, in Western blotting confirmed successfully clone and expression. We purified Omp2b by affinity chromatography and this method prepared refolds proteins on the column. Omp2b were successfully cloned, expressed and purified. The recombinant proteins were recognized by polyclonal antiserum which suggests the accuracy of procedure.

Molecular detection techniques are believed to be key tools for both prevention and treatment follow up of brucellosis within live stock and human beings. Consequently rapid، reliable، easy to perform and automated systems for Brucella detection are urgently needed to allow early diagnosis and adequate antibiotic therapy in time. Brucellosis is a worldwide re-emerging zoonosis causing high economic losses and severe human disease. In attempt to improve current molecular detection of Brucella melitensis، we compared a conventional PCR with PCR- ELISA، to detect brucella genome within standard strains and clinical isolates. Primer sets based on “omp-31” sequence of B. melitensis 16M were designed. The primer specificity was checked with appropriate online bioinformatics softwares. The primer specificity was also confirmed by testing the reaction with non-Brucella strains. A biotinylated probe complementary to an internal sequence of the PCR products was designed. The labeled non-specific fragments bound to streptavidin-coated wells، saturating the solid phase streptavidin by biotin-streptavidin interaction. Compared with conventional PCR، the PCR- ELISA proved to have more sensitivity for Brucella genome after appropriate optimization. Few human serum، whole blood and also different affected tissue samples from slaughtered livestock with brucellosis were used for protocol evaluation. Further samples should be tested before final conclusion about the results.

The most important cause of urinary tract infection is Uropathogenic Escherichia coli (UPEC) which produces type I pili. UPEC has intracellular duplication after invasion to bladder epithelial cells; therefore, cellular immunity is important in this case. This study was performed in order to design a gene cassette inducing cellular immune system against Uropathogenic Escherichia coli.

At first, fimH gene was amplified by PCR. PCR product was inserted to pcDNA.1 eukaryotic expression vector and the recombinant vector was confirmed by sequencing. The clone was inserted into COS7 cell and then assessed by RT-PCR in order to evaluate gene expression and the correct function of the gene cassette. Gene expression at mRNA level was evaluated by florescence microscope.

fimH gene was well amplified by PCR. PCR product was inserted to pcDNA.1 eukaryotic expression vector and pcDNA/fimH gene cassette was constructed. COS7 cells were transfected in order to evaluate the correct function of this cassette. Expression of fimH gene in COS7 was confirmed by RT-PCR.

pcDNA/fimH cassette could express inserted fimH gene in eukaryotic cells and is a valuable DNA cassette for urinary tract infection vaccination.

Exposure of T cells with staphylococcal enterotoxns invitro condition is known to activate T cells in a subset restricted manner based on β-chain variable region (Vβ) gene expression. In particular, staphylococcal enterotoxin B (SEB) activates T cells bearing V β8. We examined the ability of SEB to activate T cells in vitro. Exposure of lymphocyte cells origin of lymph node Balb/c mice (auxiliary, Inguinal and Mesenteric node) with doses of SEB ranging from 1, 10, 100, 1000 and 10000 ng/µl resulted in a dose dependent activation of T cells as reflected by increased proliferation that was detected by MTT assay. Our results showed that in order increase of proliferation T cells, 1000 ng/µl of SEB was the best concentration. In additional, we studied the ability of SEB in create of apoptosis with Hoechst staining. Our result demonstrate that in a statistically there are not significant difference between above SEB concentrations and negative control. Therefore 1000 ng/µl of SEB don’t create apoptosis but it is just inducible lymphocyte cell. Staphylococcal Enterotoxins is the best super antigen, and induce T-Cell suppressor, that it increase tumor cell death. Therefore, SEB is a very excellent candidate for tumor therapy.