ORIGINAL_ARTICLE Strategies of Hepatitis B vaccine export to the Middle East and Central Asian countries Introduction: This paper proposes an appropriate strategy for exporting Hepatitis B vaccine, manufactured by Pasteur Institute of Iran, to other countries. Methods: Eeffectiveness factors and their relative variables were found out by factor analysis method in SPSS software. Results: We could identify 6 main factors which can extremely affect the export of this product. We evaluated manufacturer’s strategic position among its competitors and the production market via SPACE model and put the obtained scores from the factor analysis method in this model matrix. Conclusion: We determined Iraq as the target market and identified suitable strategies for exporting the vaccine to this country. http://vacres.pasteur.ac.ir/article-1-63-en.pdf 2016-01-06 45 53 10.18869/acadpub.vacres.2.4.45 Strategy Hepatitis B Vaccine Factor analysis SPACE model. MR Maleki 1 Faculty member of Iran Medical and healthcare services University AUTHOR Y Diba ydreza1@gmail.com 2 Master of Business Administrative-Najafaabad Campus of Islamic Azad University AUTHOR
ORIGINAL_ARTICLE Different protein expression systems can influence the direction of the immune responses against HCV core protein in animal model Introduction: Hepatitis C virus (HCV) infection is a major public health problem which influences about 170 million people worldwide. Different types of vaccines have been designed using HCV structural proteins to control the viral infection. The core nucleocapsid protein is one of the most conserved proteins and a desirable target for HCV vaccines, especially the protein-based vaccines. In current study, we generated the core protein recombinantly in prokaryotic (Escherichia coli) and eukaryotic (Leishmania tarentolae) expression systems and compared the humoral immune responses stimulated by each protein in a BALB/c mice model. Methods: The expression of HCV core protein was performed using the prokaryotic pET-28a/ BL21 expression system and also the eukaryotic LEXSY expression system. The recombinant core proteins expressed in E. coli and Leishmania were purified using reverse staining method and affinity chromatography under native conditions, respectively. The purified core proteins were detected by SDS-PAGE and Western blotting using anti-His antibody and were assessed by NanoDrop spectrophotometer. Finally, the abilities of both recombinant core proteins to induce the effective humoral immune responses were evaluated using indirect ELISA. Results: Our data indicated a clear band of ~ 21 kDa for the purified HCV core proteins in both expression systems, confirmed by SDS-PAGE and Western blotting. The mice immunization with both recombinant core proteins was able to produce high levels of antibody isotypes (IgG1 and IgG2a) in comparison with the controls (p < 0.05). In addition, the level of IgG2a response was significantly higher in the group immunized with the core protein purified from leishmania compared to the protein generated from E. coli (p < 0.05). Conclusion: The recombinant core protein generated by the leishmania expression system could induce a Th1-biased immune response with respect to the increase of IgG2a to IgG1 ratio.  http://vacres.pasteur.ac.ir/article-1-65-en.pdf 2016-02-16 54 58 10.18869/acadpub.vacres.2.4.54 Hepatitis C virus LEXSY expression system E. coli expression system Core Humoral immune responses. SM Mirnurollahi mmirnorollahi@yahoo.com 1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. AUTHOR Sh Irani 2 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. AUTHOR N Davoudi 3 Department of Biotechnology, Pasteur Institute of Iran, Tehran, Iran. AUTHOR A Bolhassani azam.bolhassani@yahoo.com 4 Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran AUTHOR
ORIGINAL_ARTICLE Construction and evaluation of human papillomavirus genotype 18 pseudovirions Introduction: Cervical cancer is the second most common cancer in women worldwide and the role of human papillomavirus (HPV) has been proved in its etiology. The currentley available L1-capsid-protein-based vaccine is highly immunogenic and very high titers of serum antibodies can be obtained by its injection, but unfortunately it is restricted to only a few HPV genotypes and is relatively expensive. Therefore, development of the second-generation HPV vaccines has become the focus of the research and L2 capsid protein that is capable of producing broad spectrum antibodies has become one of the main candidates. Evaluation of the vaccine immunogenicity however, requires develoment of HPV pseudovirions (HPV PvSs) comprised of L1 and L2 virus protein and the present study was an attempt to produce PvSs of HPV18 genotype by an in-house method.  Methods: The HPV18 L1/L2 coding plasmid and the reporter plasmid of pEGFP-N1 were amplified in E. coli DH5α and purified using silica oxide method. The plasmids were co-transfected into HEK 293FT cell line and the preliminary analysis of expression was performed using fluorescence microscopy. The PvSs were partially purified using gel filtration chromatography and were used to transduce the 293FT cells to evaluate the infectivity rate of the PvSs. The results were analyzed by fluorescent microscopy, flow cytometry and atomic force microscopy.  Results: The results showed that the HPV18 L1/L2 coding plasmid and the pEGFP-N1 reporter plasmid have been successfully co-transfected into HEK 293FT cells and the PvSs were constructed. The 293FT cells were successfully transduced by PvSs that had packaged reporter plasmid. These findings were confirmed by fluorescence microscopy and flow cytometry as well as AFM imaging. Conclusion: In this study, the cotransfection of HPV18 L1/L2 coding plasmid as well as pEGP-N1 reporter plasmid into the HEK 293FT cell led to the assembly of the pseudovirion  harboring the reporter gene. The protocols used in this study were easy to perform and relatively inexpensive and did not rely on the commercial kits. http://vacres.pasteur.ac.ir/article-1-67-en.pdf 2016-02-27 59 62 10.18869/acadpub.vacres.2.4.59 human papillomavirus cervical cancer pseudovirion. H Sharifi 1 Cellular and Molecular Biology Department, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran. AUTHOR H Barzegar 2 Cellular and Molecular Biology Department, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran. AUTHOR L Langroudi 3 Virology Department, Pasteur Institute of Iran, Tehran, Iran. AUTHOR K Azadmanesh 4 Virology Department, Pasteur Institute of Iran, Tehran, Iran. AUTHOR A Arashkia 5 Virology Department, Pasteur Institute of Iran, Tehran, Iran. AUTHOR
ORIGINAL_ARTICLE Construction of a recombinant bacmid DNA containing influenza A virus hemagglutinin gene using a site-specific transposition mechanism Introduction: In recent years, influenza viruses have caused moderate to severe infections all around the world while so far there is no influenza vaccine that can protect people with only one dose of injection. In this regard, producing a universal vaccine based on virus-like-particles (VLP) could be an ideal approach.  Methods: In this study, the full-length ORF of influenza hemagglutinin (HA) gene from Influenza A virus of H9N2 subtype was amplified by RT-PCR using specific primers to produce HA cDNA. The amplicon was cloned firstly into a T/A cloning vector and then was subcloned into a pFastBacDual donor plasmid through SalI/HindIII restriction sites.  The recombinant HA-pFastBacDual vector was transferred to Escherichia coli DH10Bac cells, to insert the HA gene into the bacmid DNA via a site-specific transposition process. The recombinant bacmid was then extracted and further analyzed by PCR.  Results: Our data indicated that the HA-containing recombinant bacmid was constructed successfully using the transposition mechanism between pFastBacDual-HA and the bacmid. Conclusion: The recombinant baculovirus construct in this work had proper characteristics to be used in production of H9N2 VLP in Sf9 insect cell line in the future studies. http://vacres.pasteur.ac.ir/article-1-68-en.pdf 2016-03-08 63 68 10.18869/acadpub.vacres.2.4.63 Influenza A virus Hemagglutinin protein Baculovirus. MR Shafaati shafaati@iauh.ac.ir 1 Department of Cellular & Molecular Biology IAU, Hamadan Branch, Hamadan, Iran. AUTHOR E Akhavan 2 Department of Microbiology IAU, Damaghan Branch, Damaghan, Semnan, Iran. AUTHOR SH Yazdani yazdani.vrs2@gmail.com 3 Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. AUTHOR M Shafaati 4 Department of Microbiology IAU, Jahrom Branch, Jahrom, Shiraz, Iran. AUTHOR
ORIGINAL_ARTICLE Comparison of Vero and MDCK cell lines transfected with human siat7e gene for conversion to suspension culture Introduction: Inactivated influenza vaccines are traditionally produced in chicken embryonated eggs but its limitations in producing the required doses in pandemic outbreaks quickly enough has made searching for alternative modes of production necessary. The use of cell culture-based vaccine production is one way of overcoming the limitations of the egg-based method and securing a more rapid response.  Although Vero cells are suitable for production of influenza vaccine, but their anchorage-dependency limits their production capability. In this study adherent Vero and MDCK cells were transfected with human siat7e gene in order to convert anchorage-dependent cells to those capable of growing in suspension. Methods: Human siat7e gene was amplified with primers containing restriction sites for Xho I and Hind III and the product was cloned into pEGFP-N1 vector upstream of GFP sequence. The cells were transfected with the construct containing the siat7e gene and a medium containing G418 was used to select stably transfected cells which were then evaluated using inverted immunofluorescence microscopy. Results: Anchorage-dependent cells exhibited changes in cell-cell adhesion and cell spreading behavior following transfection. Vero cells showed a higher longevity compared to MDCK cells as viability of the latter cells declined after 50 h. Conclusion: The data showed that adherent Vero cells can successfully be converted to anchorage- independent cells capable of growing in suspension through transfection with human siat7e gene. http://vacres.pasteur.ac.ir/article-1-69-en.pdf 2016-04-13 69 73 10.18869/acadpub.vacres.2.4.69 Human Siat7e influenza virus transfection Vero cell suspension culture. P Mehrbod mehrbode@yahoo.com 1 Influenza and Other Respiratory Viruses Department, Pasteur Institute of Iran, Tehran, Iran. AUTHOR F Fotouhi 2 Influenza and Other Respiratory Viruses Department, Pasteur Institute of Iran, Tehran, Iran. AUTHOR G Irani Mokhtari 3 Influenza and Other Respiratory Viruses Department, Pasteur Institute of Iran, Tehran, Iran. AUTHOR A Mohammadpour 4 Influenza and Other Respiratory Viruses Department, Pasteur Institute of Iran, Tehran, Iran. AUTHOR B Farahmand b_farahmand@pasteur.ac.ir 5 Influenza and Other Respiratory Viruses Department, Pasteur Institute of Iran, Tehran, Iran. AUTHOR
ORIGINAL_ARTICLE Evaluation of full length E1 and E2 glycoproteins of HCV expressed in P. pastoris as a protein-based vaccine candidate Introduction: The development of an effective vaccine against Hepatitis C virus (HCV) is still a target of intensive vaccine research. The HCV envelope proteins E1 and E2 which can induce broadly neutralizing antibodies are the major candidate for this purpose. Different types of expression systems have been used to express these glycoproteins. In this study, an expression system using Pichia pastoris was used to express E1 and E2 in full length. Methods: E1 and E2 regions containing the restriction sites from HCV 1b were separately amplified and cloned into a pPICZAa vector. The km71h strain of P. pastoris was transfected with the confirmed vectors separately using electroporation. The recombinant E1 and E2 proteins were evaluated for their antigenicity in an ELISA test and the induction of humoral immunity in mice. Results: The expression of full length HCV glycoproteins E1 and E2 in P. pastoris strain km71h was successfully achieved and their specific antibody was detected in serum samples from HCV infected patients. Furthermore, the recombinant glycoproteins could elicit a significant humoral immunity in mice as a vaccine candidate. Conclusion: P. pastoris is one of the best eukaryotic expression systems for the production of HCV glycoproteins in full length and the expressed proteins could be used in diagnostic tests such as ELISA. The induction of humoral immune responses in mice should lead to further studies on these glycoproteins for designing an effective vaccine. http://vacres.pasteur.ac.ir/article-1-70-en.pdf 2016-04-27 74 80 10.18869/acadpub.vacres.2.4.74 HCV E1 and E2 Pichia pastoris Yeast expression system Protein-based vaccine. P Rahimi pooneh5376@yahoo.com 1 AUTHOR R Solati 2 AUTHOR M Shokri 3 AUTHOR R Vahabpour 4 AUTHOR F Mahmoudizad 5 AUTHOR MR Aghasadeghi 6 AUTHOR F Motevalli 7 AUTHOR Sh Yazdani 8 AUTHOR M Shayestehpour 9 AUTHOR MR Amiran mohammadr.amiran@gmail.com 10 AUTHOR
ORIGINAL_ARTICLE Protein profiling and analysis of drug sensitive and multidrug resistant isolates of Mycobacterium tuberculosis by native polyacrylamide gel electrophoresis and mass spectrometry Introduction: Tuberculosis (TB) remains a deadly infectious disease despite all the efforts to reduce its incidence. Spread of multidrug resistant TB has seriously undermined the efforts to control the disease globally. In this study protein expression profile of MDR and sensitive isolates of MTB were analyzed and compared in order to identify proteins, which could be used in prevention, diagnosis and treatment. Methods: A sensitive and MDR isolate of Mycobacterium tuberculosis (MTB) were cultured on Middlebrook 7H9 medium and the whole cell lysates were subjected to native polyacrylamide gel electrophoresis (NPAGE) for protein expression profiling. Protein bands present in the MDR cell lysate that were not detected in the sensitive cell lysate were sent for identification by Matrix-assisted laser desorption/ionization time-of-flightmass spectrometry (MALDI-TOF-MS). Results: Comparison of the protein expression profiles showed 6 bands that were not detected in the sensitive isolates. MTB Structural Annotation database search of the mass spectrometry results identified these bands as Rv3597c, Rv0379,Rv3614c, Rv0475, Rv0462, andRv0147and global transcriptional regulation, involvement in cell wall and cell processes and intermediary metabolism and respiration were the functions attributed to these proteins. Conclusion: Our results highlighted the complexities of linking protein expression to MDR phenotype as none of the proteins identified could be linked directly to drug resistance. The proteins identified in the present study were mostly those essential for survival or virulence of the bacteria, and could be used for diagnosis or as candidate vaccine, but with a better understanding of the function of these proteins their association with the MTB resistance to antibiotics might become clear. http://vacres.pasteur.ac.ir/article-1-74-en.pdf 2016-06-26 81 85 10.18869/acadpub.vacres.2.4.81 MALDI-TOF-mass spectrometry native PAGE multidrug resistant Mycobacterium tuberculosis. Sh Yari shami14479@yahoo.com 1 Tuberculosis Department, Pasteur Institute of Iran, Tehran, Iran, Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran. AUTHOR AR Hadizadeh Tasbiti hadi@pasteur.ac.ir 2 Tuberculosis Department, Pasteur Institute of Iran, Tehran, Iran, Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran. AUTHOR M Ghanei 3 Tuberculosis Department, Pasteur Institute of Iran, Tehran, Iran, Chemical Injury Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran. AUTHOR MA Shokrgozar 4 National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran. AUTHOR R Mahdian 5 Molecular Medicine, Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran. AUTHOR A Fateh afateh2@gmail.com 6 Tuberculosis Department, Pasteur Institute of Iran, Tehran, Iran, Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran. AUTHOR SD Siadat d.siadat@gmail.com 7 Tuberculosis Department, Pasteur Institute of Iran, Tehran, Iran, Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran. AUTHOR F Vaziri farzam_vaziri@yahoo.com 8 Tuberculosis Department, Pasteur Institute of Iran, Tehran, Iran, Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran. AUTHOR Sh Niknami 9 Department of Health Education, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran AUTHOR A Bahrmand padideh79@yahoo.com 10 Tuberculosis Department, Pasteur Institute of Iran, Tehran, Iran, AUTHOR
ORIGINAL_ARTICLE Sand fly saliva: toward a vaccine against leishmaniases Leishmaniases are a group of sand fly-borne diseases caused by protozoan parasites from species of Leishmania genus. These diseases are reported in about 100 countries with a prevalence of 12 million people infected and incidence of 2 million people per year, putting approximately 350 million people at risk of the infections. Leishmaniases are endemic and are considered as important public health problems in many provinces of Iran. The infection is transmitted through the bite of phlebotomine sand flies. The sand fly salivates while biting the vertebrate host. The saliva of phlebotomines consists of different molecules that are necessary for a sand fly to successfully take a blood meal. Additionally, previous exposures to sand fly saliva indirectly affect the establishment of Leishmania in the vertebrate host. Moreover, mice previously exposed to the saliva by injection or by uninfected sand fly bites have shown both humoral and cellular immune responses against the salivary antigens that protects them against Leishmania infection. Importantly, the immunization of mice with defined molecules from the saliva of the vector species has also conferred a strong protection against Leishmania infection. This suggests that such salivary components may be considered as candidates for a cocktail vaccine against leishmaniases. The current article briefly explains the potential of salivary components of sand fly vectors as immunological items to prevent leishmaniasis. So far, there is no efficient vaccine against these infections and efforts are required to be focused on developing effective and applicable vaccines against leishmaniases. http://vacres.pasteur.ac.ir/article-1-76-en.pdf 2016-07-09 86 92 10.18869/acadpub.vacres.2.4.86 Sand fly saliva vaccine leishmaniasis. N Hosseini- Vasoukolaei nasibeh.hoseini@gmail.com 1 Department of Medical Entomology and Vector Control, Health Sciences Research Center, Faculty of Health, Mazandaran University of Medical Sciences, Mazandaran, Sari, Iran. AUTHOR