Pasteur Institute of Iran Vaccine Research 2383-2819 3 1 2016 5 1 New insight into the application of outer membrane vesicles of Gram-negative bacteria 1 4 FA A Fateh Mycobacteriology and Pulmonary Research Department, Pasteur Institute of Iran. Tehran, Iran, Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran F Vaziri Mycobacteriology and Pulmonary Research Department, Pasteur Institute of Iran. Tehran, Iran, Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran F Rahimi Janani Mycobacteriology and Pulmonary Research Department, Pasteur Institute of Iran. Tehran, Iran, Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran S Ahmadi Badi Mycobacteriology and Pulmonary Research Department, Pasteur Institute of Iran. Tehran, Iran M Ghazanfari Mycobacteriology and Pulmonary Research Department, Pasteur Institute of Iran. Tehran, Iran M Davari Mycobacteriology and Pulmonary Research Department, Pasteur Institute of Iran. Tehran, Iran A Arsang Mycobacteriology and Pulmonary Research Department, Pasteur Institute of Iran. Tehran, Iran SD Siadat Mycobacteriology and Pulmonary Research Department, Pasteur Institute of Iran. Tehran, Iran, Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran This review presents a brief outline of our current knowledge of the structure and chemical composition of the outer membrane vesicles (OMVs), originating from the surface of Gram negative bacteria including their outer membrane proteins and lipopolysaccharides. Moreover, the functional roles and applications of OMVs in medical research such as OMV-based vaccines, OMV adjuvants properties, OMV carriers in conjugated vaccines as well as in drug vehicles and delivery systems are discussed. Finally, new applications of these macromolecules as biotechnological tools are briefly presented.

Pasteur Institute of Iran Vaccine Research 2383-2819 3 1 2016 5 1 Synthesis and characterization of physicochemical and immunological properties of recombinant NS3-G2 dendrimer conjugate 5 8 FA F Javadi Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. P Rahimi Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran. MH Modaressi Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. M Shafiee Ardestani Department of Radiopharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. A Bolhassani Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran. MR Aghasadeghi Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran. SM Sadat Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran. Introduction: An effective vaccine against HCV infection is not available. The non-structural protein 3 (NS3) of the virus as an important immunogenic candidate has been utilized in various modules. Nanostructured polymers have been recently used for efficient vaccine and drug delivery. The aim of the current study was the synthesis of rNS3-G2 conjugate and preliminary evaluation of its immunogenicity. Methods: The dendrimer was synthesized and conjugated with purified recombinant NS3 (rNS3) protein. The physicochemical properties of the conjugate were evaluated by Zeta potential, FT-IR spectra and confirmed by atomic force microscopy (AFM). Immunogenicity of the conjugate was assessed in BALB/c mice. Results: Synthesis and conjugation of dendrimer G2 with the protein were confirmed and immunological assays showed that the conjugated form of the antigen induced higher titer of IgG compared to rNS3 antigen alone. Conclusion: The results showed that the antigenic structure of rNS3 was maintained when conjugated with the biodegradable and biocompatible G2 dendrimers and the immunogenic properties of the antigen were enhanced. Therefore the new formulation may have potential as a vaccine candidate.

Pasteur Institute of Iran Vaccine Research 2383-2819 3 1 2016 5 1 Establishment of NS3 tumor cell line expressing Hepatitis C virus Non-structural Protein 3 as valuable tool for HCV challenging in mice 9 14 FA MH Pouriayevali Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran . T Bamdad Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran . SM Sadat Department of Hepatitis and AIDS, Pasteur Institute of Iran. Tehran, Iran. F Sabahi Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran . MR Aghasadeghi Department of Hepatitis and AIDS, Pasteur Institute of Iran. Tehran, Iran. SH Yazdani Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. Introduction: Hepatitis C virus (HCV) is one of the major medical problems. Human and chimpanzees are the only specific hosts which are naturally susceptible to HCV infection. Mice and other common laboratory animals are resistant to the virus, hence HCV prophylactic and therapeutic researches are very difficult and challenging. HCV non-structural protein 3 (NS3) is one of the most attractive targets for developing novel anti-HCV therapies as it is essential for the viral replication. This study was designed to produce stable SP2/0 tumor cell lines expressing NS3 of HCV for future basic and vaccine studies. Methods: A lentivector expressing NS3, named PCDH-NS3, was constructed by cloning of NS3 cDNA into downstream of CMV promoter of pCDH-CMV-MCS-EF1-Puro-GFP. The constructed plasmid was co-transfected with pMD2.G plasmid which encodes envelope VSV G protein and psPAX2 packaging plasmid into HEK-293T cells. The lentivector-containing supernatant was collected every 12 h for 72 h and NS3-Lentivector was concentrated by ultracentrifugation. Titers of the NS3 lentivector were estimated using flow cytometry. The SP2/0 cells were then infected by NS3 lentivector. Puromycin as a selective antibiotic was added to the culture for 2 weeks to select NS3 positive cells. A single transfected clone was obtained using limiting dilution. The 1st and 6th passages of the cells cultured in vitro were harvested and NS3 mRNA was detected for by RT-PC. Results: The results showed that NS3 expressing lentivector plasmids and the two other helper plasmids could be transfected into HEK293T efficiently and packaged successfully as a pseudo-lentivector. Finally, the detection of NS3 mRNA in the 1st and 6th passages of SP2/NS3 cells was confirmed by establishment of a stable cell line. Conclusion: SP2/0 Cell line with stable expression of NS3 can be used as a suitable tumor model to facilitate research on HCV vaccine in vitro and in mice model and it could be served as a valuable tool for pharmaceutical HCV research to pave the way for further research on NS3 vaccine function.

Pasteur Institute of Iran Vaccine Research 2383-2819 3 1 2016 5 1 Immunization of Litopenaeus vannamei shrimp against white spot syndrome virus (WSSV) by gamma-irradiated WSSV plus Vibrio paraheomolyticus 15 20 FA F Motamedi-Sedeh Nuclear Science and Technology Research Institute, Tehran, Iran. M Afsharnasab Iranian Fisheries Science Research Institute, Tehran, Iran. M Heidarieh Nuclear Science and Technology Research Institute, Tehran, Iran. Introduction: White spot syndrome virus (WSSV) is one of the most deadly infectious pathogens of the shrimp culture industry. Neither effective vaccines nor efficient treatments are currently available for this disease. Vibrio species are well known dominant bacterial pathogens in the shrimp ponds. As facultative pathogenic bacteria, it is possible that Vibrio spp. along with WSSV to co-infect the shrimp species such as Litopenaeus vannamei. The aim of this study was to investigate the co-administration of gamma-irradiated Vibrio paraheomolyticus as a kind of probiotic and immune-stimulator with gamma-irradiated and inactivated WSSV as an intramuscular vaccine for protection of L. vannamei against WSSV infection. Methods: WSSV was isolated from the infected shrimp samples and multiplied in Astacus leptodactylus crayfish. Titration of WSSV was obtained in post-larvae as 105.4 LD50/ml. The virus was irradiated where D10 value and optimum dose of gamma ray were calculated to be 2.56 and 15 kGy, respectively. The gamma-irradiated WSSV samples, named GI-WSSV were used as a vaccine to immunize L. vannamei shrimps. The freeze-dried V. paraheomolyticus cultures, inactivated by gamma ray (8 kGy) were named GI-V.P and used as a probiotic. Results: Protective dose50 was calculated as 5.61 and 7.94 for the shrimp groups which were vaccinated by GI–WSSV vaccine and GI-WSSV vaccine + GI-V.P, respectively. Calculated RPS values were 73.3%, 86.66% and 26.66% for the GI-WSSV vaccine, GI-WSSV + GI-V.P and isolated probiotic groups, respectively. Significant differences in cumulative mortalities were observed between the vaccination groups and the positive control group    (P < 0.05). No significant difference was observed in cumulative mortalities between the two vaccination groups        (P > 0.05). Conclusion: GI-WSSV vaccine can induce immune responses in shrimps infected with WSSV and probiotic GI-V.P enhances these responses.

Pasteur Institute of Iran Vaccine Research 2383-2819 3 1 2016 5 1 Purification and evaluation of glycerol teichoic acid from biofilm forming Staphylococcus epidermidis as a putative vaccine candidate 21 25 FA B Mirzaei Department of Microbiology, Microbial research center, Pasteur Institute of Iran, Tehran, Iran, Microbiology Research Center (MRC) Pasteur Institute of Iran, Tehran, Iran. SF Moosavi Department of Microbiology, Microbial research center, Pasteur Institute of Iran, Tehran, Iran, Microbiology Research Center (MRC) Pasteur Institute of Iran, Tehran, Iran. R Babaei Microbiology Research Center (MRC) Pasteur Institute of Iran, Tehran, Iran, Department of Mycobacteriology and pulmonary research, Pasteur Institute of Iran, Tehran, Iran. SD Siadat Microbiology Research Center (MRC) Pasteur Institute of Iran, Tehran, Iran, Department of Mycobacteriology and pulmonary research, Pasteur Institute of Iran, Tehran, Iran. A Farjah Young Researchers and Elite Club, East Tehran Branch, Azad University, Tehran, Iran. M Shahrooei Laboratory of Medical Microbiology, Department of Medical Diagnostic Sciences, KU Leuven, U.Z. Gasthuisberg, Herestraat 49 CDG 8th floor, B-3000 Leuven, Belgium. Introduction: Biofilm forming Staphylococcus epidermidis is a main causative agent of infections related to medical devices. Purification and evaluation of Gly-TA polysaccharide from a biofilm-forming S. epidermidis as a putative vaccine candidate were the main goals of the current study. Methods: Taking advantage of size exclusion chromatography procedure, glycerol teichoic acid (Gly-TA) was purified from the above-mentioned strain and biochemical analyses including, Fourier Transform Infrared spectroscopy (FTIR) and Proton Nuclear Magnetic Resonance spectroscopy (H1-NMR) were conducted for the recovered polysaccharide. Results: Following PCR confirmation of a S. epidermidis strain, Gly-TA was extracted and its biochemical compositions (i.e. N-acetyl glucose amine residues) were obtained. Conclusion: It is envisaged that Gly-TA polysaccharide could be considered as a putative vaccine to inhibit formation of biofilm by S. epidermidis.

Pasteur Institute of Iran Vaccine Research 2383-2819 3 1 2016 5 1 Differential protein expression in Mycobacterium tuberculosis susceptible and multidrug resistant isolates 26 30 FA AR Hadizadeh Tasbiti Sh Yari M Ghanei SD Siadat S Niknami A Bahrmand Introduction: Infections by multidrug resistant Mycobacterium tuberculosis (MDR-TB) is a major public health challenge. Secretion of proteins by M. tuberculosis plays an important role in the pathogenesis of the bacterium. We compared the protein profiles of susceptible M. tuberculosis and MDR-TB isolates using proteomic analyses, namely two dimensional gel electrophoresis (2DE) and mass spectrometry (MS). Methods: The bacilli were cultured on Middlebrook 7H9 medium and bacterial colonies were mechanically disrupted and proteins were extracted by ammonium sulfate. The 2DE and MS analyses were performed using Ettan IP Gphor 3 isoelecteric system and Autoflex II TOF/TOF, respectively. Results: Our study showed that in comparison to the sensitive strains, 27 proteins were over-expressed in the MDR isolates and these proteins were mainly involved in the cellular metabolism, cell wall and membrane structures and bacterial respiration. Bactoferritin (Rv1876) has been shown to play a role in antibacterial resistance. Increased intensity of Rv2031c, a heat shock protein (Alpha-crystallin/HspX), was also observed in the whole cell lysate of the MDR-TB. This protein is a marker of the latent TB and has been proposed as a target for vaccine development. Conclusion: Our results identified proteins that are overexpressed in the resistant M. tuberculosis which could be used as antibacterial targets or vaccine candidates.  

Pasteur Institute of Iran Vaccine Research 2383-2819 3 1 2016 5 1 Bioinformatics study of complete amino acid sequences of neuraminidase (NA) antigen of H1N1 influenza viruses from 2006 to 2013 in Iran 31 37 FA A Farhangi Institute of Biochemistry and Biophysics, University of Tehran. Tehran. Iran. B Goliaei Institute of Biochemistry and Biophysics, University of Tehran. Tehran. Iran. K Kavousi Institute of Biochemistry and Biophysics, University of Tehran. Tehran. Iran. A Ashtari Razi Vaccine and Serum Research Institute, Karaj, Alborz, Iran. MA Bayatzadeh Razi Vaccine and Serum Research Institute, Karaj, Alborz, Iran. A Pourbakhsh Razi Vaccine and Serum Research Institute, Karaj, Alborz, Iran. Introduction: Influenza is a contagious acute viral disease of the respiratory tract that causes fever, headache, muscle aches and cough. One of the unique features of influenza virus is antigenic variation in viral protein neuraminidase (NA) which causes emergence of new virus variants. NA is responsible for the release and spread of progeny virions. Due to the continuous changes of NA genes, vaccine strains must be re-selected annually. Methods: Complete NA amino acid sequences of 97 strains circulating from 2006 to 2013 in Iran were downloaded from NCBI. The sequences were edited and classified by the year of isolation and their diversity and important changes as well as changes in the predicted ligand binding sites and their resistance to anti-NA drugs, were analyzed. Bioinformatics software such as MEGA6.0, BioEdit, DNAsisMAX and DNAstar were used for the sequence alignments and phylogenetic analyses. Web-based analysis such as SWISS-MODEL, Phyre2 and 3DLigandSite were used for evaluation of the second and third protein structures and prediction of the ligand binding sites. Results: The results showed that 2009 could be considered as an important transition year which caused to classify the isolates into two different distinct groups. This shows the importance of changes made during possible mutations in the genomic structure of the virus which have made it antigenically different from the previous years. Anti-NA drug resistance was observed in 2009. This pandemic strain has become dominant in the following years and is used as a standard vaccine strain from 2010 onwards. Conclusion: The results obtained in this study can aid in better understanding of the antigenic evolution of H1N1 influenza viruses and can potentially accelerate the selection of the vaccine strains.  

Pasteur Institute of Iran Vaccine Research 2383-2819 3 1 2016 5 1 The Shift of HAV Infection in to adulthood in Iran: Consideration for HAV Vaccination 38 38 FA MR Aghasadeghi Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran