Comparison of Immune Response in Mice Immunized with Recombinant PreS2/S-C18-27 Protein Derived from Hepatitis B Virus with Commercial Vaccine.

Background & Objective: The vaccine available to prevent Hepatitis B virus disease is ineffective in 5% of people due to the use of HBsAg as a weak immunogenic factor. In the present study, PreS2/S fused to C18-27 peptide fragment as an effective antigen and is proposed as a promising vaccine candidate compared with the conventional vaccine prescribed in the vaccination program. Methods: After the synthesis of PreS2/S genes and C18-27 peptide fragment in pET28a, the recombinant protein was confirmed by Western blotting. The efficacy of the PreS2/S-C18-27 protein was compared with the conventional vaccine injected into five groups of rats. Finally, the cytokine level of IF-r, IL-2, IL-4, IL-10, TNF-a, IgG1, and IgG2a were measured using the ELISA method. Results: This study showed no significant difference between the recombinant vaccine group and PBS control group in the IF-r test, but there was a significant difference between groups testing IL-2 and IL-10. In addition, the group receiving the recombinant vaccine with CPG adjuvant at a dilution of 1/10 in the IgG total test on days 14 and 45 after the first injection showed a significant difference in comparison with other groups. Conclusion: This study showed no statistically significant difference between the recombinant protein vaccine group and the conventional vaccine group. The Th1- mediated immune responses obtained from recombinant proteins with and without CPG performed better than conventional vaccines, possibly due to the functional deficiency of the available vaccines.

Evaluation of Triple Fragment Vaccine HSPX (Rv2031c) + PPE44 (Rv2770c) + Mouse IgG1 (Fcγ2a) with Auxiliary Adjuncts IL-22 in Comparison with BCG Vaccine.

Background & Objective: Despite the vaccination with the BCG vaccine, tuberculosis (TB) remains one of the major health problems in the world. The aim of this study was to evaluate our newly designed vaccine using IL-22 as an adjuvant in comparison with the common BCG vaccine. Methods: The gene constructs were cloned into the expression vector of pET28a and then into the recombinant vector of PET28a - HSPX, and PPE44 was transformed into Escherichia coli BL21 (DE3). Finally, the immunogenicity of recombinant proteins with and without BCG and IL-22 in BALB/c mice was investigated. Results: The key cytokines INF-γ and TNF-α were elevated more greatly in BCG immunized group than in PHF immunized group. Immunization with PHF showed a significant increase in IL-4 levels versus the BCG group. Adding IL-22 to the vaccine formulations indicated a tiny increase in IL-4 levels compared to their related vaccine groups.Specific total IgG1 in the experimental groups showed an increase in comparison with control groups, but in the vaccinated groups, no significant differences were observed, and the presence of IL-22 in the vaccine formulations indicated a slight decrease compared with the related mere vaccine groups. Results of specific total IgG2a in the experimental groups revealed that only in the PHF group formulated with IL-22 a significant increase occurs compared with all other experimental groups. Conclusion: It seems that BCG, as the only licensed vaccine for TB infection, could be more potent than a recombinant vaccine in the induction of cellular and humoral immune responses.

Evaluating the Performance of PPE44, HSPX, ESAT-6 and CFP-10 Factors in Tuberculosis Subunit Vaccines.

Mycobacterium tuberculosis (M. tuberculosis) is an intracellular pathogen causing long-term infection in humans that mainly attacks macrophages and can escape from the immune system with the various mechanisms. The only FDA-approved vaccine against M. tuberculosis (MTB) is Mycobacterium bovis bacillus Calmette-Guérin (BCG). The protection of this vaccine typically lasts 10-15 years. Due to the increasing number of people becoming ill with MTB each year worldwide, the need to develop a new effective treatment against the disease has been increased. During the past two decades, the research budget for TB vaccine has quadrupled to over half a billion dollars. Most of these research projects were based on amplifying and stimulating the response of T-cells and developing the subunit vaccines. Additionally, these studies have demonstrated that secretory and immunogenic proteins of MTB play a key role in the pathogenesis of the bacteria. Therefore, these proteins were used to develop the new subunit vaccines. In this review, based on the use of these proteins in the successful new subunit vaccines, the PPE44, HSPX, CFP-10 and ESAT-6 antigens were selected and the role of these antigens in designing and developing new subunit vaccines against TB and for the prevention of TB were investigated.

Comparing HBV Viral Load in Serum, Cerumen, and Saliva and Correlation With HBeAg Serum Status in Patients With Chronic Hepatitis B Infection.

BACKGROUND: Hepatitis B is a disease that is prevalent worldwide and is responsible for 10% of the deaths that occur every year. The virus persists in 5% of infected adults and 90% of infected children and can cause chronic hepatitis. In addition to blood, the virus may also be present in other secretions. Transmission through saliva, sexual fluids, and urine has also been confirmed. OBJECTIVES: The main aim of this study was to compare viral DNA copies in the serum, cerumen, and saliva of patients with HBeAg levels in their sera. PATIENTS AND METHODS: This was a cross-sectional study and subjects were selected by non-randomized methods. Serum, cerumen, and saliva samples were collected from 50 patients who were diagnosed with chronic hepatitis B about a year prior to the study. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the presence of HBsAg and HBeAg in the gathered specimens. Viral DNA was extracted from specimens by using a Qiagen kit. The number of viral DNA copies was determined using a real-time polymerase chain reaction (PCR) assay. The study was performed in Ilam province in western Iran. RESULTS: Twenty-eight percent of the patients were HBeAg positive. The average number of viral copies in serum, cerumen, and saliva was higher in women than in men, and a significant correlation was observed between the gender and average viral copies. However, no significant correlation was observed between viral copies present in the serum and cerumen with the age and gender of patients. In addition, no correlation was observed between serum HBeAg and viral copies present in serum, cerumen, and saliva. The correlation analysis confirmed a direct and definite correlation between viral DNA loads in the patients' serum and cerumen. CONCLUSIONS: A significant direct correlation was observed between the viral DNA copies present in patients' cerumen and serum. However, the correlation between saliva viral load with serum and cerumen viral load was very low and inverse. These findings suggest that the presence of the hepatitis B virus (HBV) in non-invasive specimens (such as cerumen and saliva) should also be evaluated when monitoring patients to determine the course of infection and disease.

The Application of Pulsed Field Gel Electrophoresis in Clinical Studies.

Pulsed-field gel electrophoresis is a method applied in separating large segments of deoxyribonucleotide using an alternating and cross field. In a uniform magnetic field, components larger than 50kb pass a route through the gel and since the movement of DNA (Deoxyribonucleic acid) molecules are in a Zigzag form, separation of DNAs as bands carried out better via gel. PFGE in microbiology is a standard method which is used for typing of bacteria. It is also a very useful tool in epidemiological studies and gene mapping in microbes and mammalian cell, also motivated development of large-insert cloning system such as bacterial and yeast artifical chromosomes. In this method, close and similar species in terms of genetic patterns show alike profiles regarding DNA separation, and those ones which don't have similarity or are less similar, reveal different separation profiles. So this feature can be used to determine the common species as the prevalence agent of a disease. PFGE can be utilized for monitoring and evaluating different micro-organisms in clinical samples and existing ones in soil and water. This method can also be a reliable and standard method in vaccine preparation. In recent decades, PFGE is highly regarded as a powerful tool in control, prevention and monitoring diseases in different populations.

Role of IL-25 in Immunity.

IL-25 a 2o KDa protein mostly known as IL-17E, encoded by chromosome 14, and containing 117 amino acids. Cytokine IL-17 family consists of 6 members; IL-17A to IL-17F, among which IL-25 has a unique structure and function. The receptor of IL-25 (IL-17BR) is highly expressed in the main Th2 cells. IL-25 regulates the internal safety of adaptive immune responses which leads to begin allergic diseases and plays a role in stimulation of pulmonary mucosal cells and fibroblasts. IL-25 can also have some effects on production of other cytokines. For instance, production of IL-25 in human and mice or injection of IL-25 to animals has resulted in production of high concentrations of Th2 cytokines, including IL-4, IL-5, and IL-13. Pilot studies have shown that mRNA of IL-25 has a high expression in Th2 cells. However, the mechanism through which IL-25 leads to Th2 immune response is still unknown. Reaction between IL-25 and IL-17BR leads to activation of transcription factors, such as NF-KB, STAT6, GATA3, NF-ATC1, JUNNB, MAPK, and JNK. IL-25 has been used against the kidney damage in mice. A large number of researchers in various countries, including the U.S. and Taiwan, have stated that IL-25 is a strong inflammatory cytokine protein which is involved in allergic inflammations.

Relationship between the Presence of the nalC Mutation and Multidrug Resistance in Pseudomonas aeruginosa.

Objectives. The current study was conducted to determine the relationship between the presences of significant multidrug resistance in Pseudomonas aeruginosa (P. aeruginosa) having intact mexR genes (nalC) to different antibiotics. Methods. In order to identify nalC, fifty strains of P. aeruginosa were obtained. All isolates were found in urinary tract infections. They were evaluated against different antibiotics. The nalC mutant was identified by PCR. Results. The 50 clinical isolates of P. aeruginosa originated from two hospitals in Iran, in which 32 isolates were found in Milad hospital, and 18 isolates were collected in the Ilam Hospital. The results in Milad hospital of nalC revealed that all P. aeruginosa resistant to oxacillin showed the presence of nalC. In Ilam hospital only three (16.6%) isolates were resistant to oxacilin and aztreonam, and among these three isolates only one isolate revealed resistance to ceftazidime and amikacin. The resistant isolates showed the presence of both OXA-10 and nalC. Conclusion. Our results showed that the presence of nalC was observed among P. aeruginosa resistance to oxacilin. Thus, the finding suggested relationship between oxacilin resistance and presence of nalC and consequently overproduction of the MexABOprM efflux system.