Publications by authors named "Anis Jafari"

14 Publications

  • Page 1 of 1

Effect of nontypeable Haemophilus influenzae protein E (PE) as a microbial adjuvant on the amount of antibody against PRP of Haemophilus influenzae type b (Hib) in BALB/c mice.

Microb Pathog 2019 Apr 22;129:78-81. Epub 2019 Jan 22.

Mycobacteriology and Pulmonary Research Department, Microbiology Research Center, Pasteur Institute of Iran, Tehran, Iran.

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http://dx.doi.org/10.1016/j.micpath.2019.01.023DOI Listing
April 2019

Molecular Cloning, Expression and Purification of Truncated hpd Fragment of Haemophilus influenzae in Escherichia coli.

Jundishapur J Microbiol 2015 Aug 29;8(8):e23218. Epub 2015 Aug 29.

Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, IR Iran.

Background: Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in children, causing otitis media, sinusitis, conjunctivitis, pneumonia, and occasionally invasive infections. Protein D (PD) belongs to the minor outer-membrane proteins of H. influenza. Moreover, it has been shown that this protein is one of the most potent vaccine candidates against the NTHi strain.

Objectives: In the present study, a new truncated form of PD was designed based on conserved areas, and recombinant truncated PD was expressed.

Materials And Methods: Truncated PD was designed using bioinformatics tools, and a 345 bp fragment of the truncated hpd gene was amplified by polymerase chain reaction (PCR) from H. influenzae and subsequently cloned into the prokaryotic expression vector pBAD-gIIIA. In addition, for the expression of the recombinant protein, the pBAD-truncated PD plasmid was transformed into competent TOP10 cells. The recombinant protein was expressed with Arabinose. The expressed protein was purified by affinity chromatography using Ni-NTA resin.

Results: The cloning of PD was confirmed by colony-PCR and enzymatic digestion. Arabinose 0.2% was able to efficiently induce protein expression. The SDS-PAGE analysis showed that our constructed pBAD-PD-TOP10 efficiently produced a target recombinant protein with a molecular weight of 16 kDa. A high concentration of the recombinant protein was obtained via the purification process by affinity chromatography. The recombinant PD was reacted with peroxidase-conjugated rabbit anti-mouse immunoglobulins.

Conclusions: Our results showed that the recombinant protein produced by the pBAD vector in the Escherichia coli system was very efficient.
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http://dx.doi.org/10.5812/jjm.23218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4600343PMC
August 2015

Prokaryotic High-Level Expression System in Producing Adhesin Recombinant Protein E of Nontypeable Haemophilus influenzae.

Jundishapur J Microbiol 2015 Apr 18;8(4):e16377. Epub 2015 Apr 18.

Department of Molecular Biology, Pasteur Institute of Iran, Tehran, IR Iran.

Background: Adhesion protein E (PE) of Haemophilus influenzae is a 16 - 18 kDa protein with 160 amino acids which causes adhesion to epithelial cells and acts as a major factor in pathogenesis.

Objectives: In this study, we performed cloning, expression and purification of PE as a candidate antigen for vaccine design upon further study.

Materials And Methods: At first, the pe gene of NTHi ATCC 49766 strain (483 bp) was amplified by PCR. Then, to sequence the resulted amplicon, it was cloned into TA vector (pTZ57R/T). In the next step, the sequenced gene was sub-cloned in pBAD/gIII A vector and transformed into competent Escherichia coli TOP10. For overexpression, the recombinant bacteria were grown in broth medium containing arabinose and the recombinant protein was purified using metal affinity chromatography (Ni-nitrilotriacetic acid) (Ni-NTA agarose). Finally, the protein was detected using sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAG) and confirmed by western blotting.

Results: The cloned gene was confirmed by PCR, restriction digestion and sequencing. The sequenced gene was searched for homology in GenBank and 99% similarity was found to the already deposited genes in GenBank. Then we obtained PE using Ni-NTA agarose with up to 7 mg/mL concentration.

Conclusions: The pe gene was successfully cloned and confirmed by sequencing. Finally, PE was obtained with high concentration. Due to high homology and similarity among the pe gene from NTHi ATCC 49766 and other NTHi strains in GenBank, we believe that the protein is a universal antigen to be used as a vaccine design candidate and further studies to evaluate its immunogenicity is underway.
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http://dx.doi.org/10.5812/jjm.8(4)2015.16377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4449846PMC
April 2015

Co-expression of hepatitis C virus polytope-HBsAg and p19-silencing suppressor protein in tobacco leaves.

Pharm Biol 2016 20;54(3):465-73. Epub 2015 May 20.

a Department of Molecular Biology .

Context: Plants transformed by virus-based vectors have emerged as promising tools to rapidly express large amounts and inexpensive antigens in transient condition.

Objective: We studied the possibility of transient-expression of an HBsAg-fused polytopic construct (HCVpc) [containing H-2d and HLA-A2-restricted CD8+CTL-epitopic peptides of C (Core; aa 132-142), E6 (Envelope2; aa 614-622), N (NS3; aa 1406-1415), and E4 (Envelope2; aa 405-414) in tandem of CE6NE4] in tobacco (Nicotiana tabacum) leaves for the development of a plant-based HCV vaccine.

Materials And Methods: A codon-optimized gene encoding the Kozak sequence, hexahistidine (6×His)-tag peptide, and HCVpc in tandem was designed, chemically synthesized, fused to HBsAg gene, and inserted into Potato virus X (PVX-GW) vector under the control of duplicated PVX coat protein promoter (CPP). The resulted recombinant plasmids (after confirmation by restriction and sequencing analyses) were transferred into Agrobacterium tumefaciens strain GV3101 and vacuum infiltrated into tobacco leaves. The effect of gene-silencing suppressor, p19 protein from tomato bushy stunt virus, on the expression yield of HCVpc-HBsAg was also evaluated by co-infiltration of a p19 expression vector.

Results: Codon-optimized gene increased adaptation index (CAI) value (from 0.61 to 0.92) in tobacco. The expression of the HCVpc-HBsAg was confirmed by western blot and HBsAg-based detection ELISA on total extractable proteins of tobacco leaves. The expression level of the fusion protein was significantly higher in p19 co-agroinfiltrated plants.

Discussion And Conclusion: The results indicated the possibility of expression of HCVpc-HBsAg constructs with proper protein conformations in tobacco for final application as a plant-derived HCV vaccine.
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http://dx.doi.org/10.3109/13880209.2015.1048371DOI Listing
October 2016

Vaccination with recombinant L7/L12-truncated Omp31 protein induces protection against Brucella infection in BALB/c mice.

Mol Immunol 2015 Jun 24;65(2):287-92. Epub 2015 Feb 24.

Department of Molecular Biology, Pasteur Institute of Iran, Pasteur Street, Tehran, Iran. Electronic address:

Brucellosis is the most common bacterial zoonotic disease worldwide and no vaccine is available for the prevention of human brucellosis. In humans, brucellosis is mostly caused by Brucella melitensis and Brucella abortus. The Outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved in human Brucella pathogens. In the present study, we evaluated the humoral and cellular immune responses induced by a fusion protein designed based on the Truncated form of Omp31 (TOmp31) and L7-L12 antigens. Vaccination of BALB/c mice with the recombinant fusion protein (rL7/L12-TOmp31) provided the significant protection level against B. melitensis and B. abortus challenge. Moreover, rL7/L12-TOmp31 elicited a strong specific IgG response (higher IgG2a titers) and significant IFN-γ/IL2 production and T-cell proliferation was also observed. The T helper1 (Th1) oriented response persisted for 12 weeks after the first immunization. The rL7/L12-TOmp31 could be a new potential antigen candidate for the development of a subunit vaccine against B. melitensis and B. abortus.
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http://dx.doi.org/10.1016/j.molimm.2015.01.009DOI Listing
June 2015

Intranasal immunization with fusion protein MrpH·FimH and MPL adjuvant confers protection against urinary tract infections caused by uropathogenic Escherichia coli and Proteus mirabilis.

Mol Immunol 2015 Apr 4;64(2):285-94. Epub 2015 Jan 4.

Department of Molecular Biology, Pasteur Institute of Iran, Pasteur Ave., Tehran 13164, Iran. Electronic address:

Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) and Proteus mirabilis are among the most common infections in the world. Currently there are no vaccines available to confer protection against UTI in humans. In this study, the immune responses and protection of FimH of UPEC with MrpH antigen of P. mirabilis in different vaccine formulations with and without MPL adjuvant were assessed. Mice intranasally immunized with the novel fusion protein MrpH·FimH induced a significant increase in IgG and IgA in serum, nasal wash, vaginal wash, and urine samples. Mice immunized with fusion MrpH·FimH also showed a significant boost in cellular immunity. Addition of MPL as the adjuvant enhanced FimH and MrpH specific humoral and cellular responses in both systemic and mucosal samples. Vaccination with MrpH·FimH alone or in combination with MPL showed the highest efficiency in clearing bladder and kidney infections in mice challenged with UPEC and P. mirabilis. These findings may indicate that the protection observed correlates with the systemic, mucosal and cellular immune responses induced by vaccination with these preparations. Our data suggest MrpH·FimH fusion protein with or without MPL as adjuvant could be potential vaccine candidates for elimination of UPEC and P. mirabilis. These data altogether are promising and these formulations are good candidates for elimination of UPEC and P. mirabilis.
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http://dx.doi.org/10.1016/j.molimm.2014.12.008DOI Listing
April 2015

Antigenicity and immunogenicity of fused B-subunit of heat labile toxin of Escherichia coli and colonization factor antigen I polyepitopes.

J Microbiol Methods 2014 Nov 7;106:40-46. Epub 2014 Aug 7.

Department of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran. Electronic address:

Linear B-cell epitopes ((93)AKEFEAAAL(101) and (66)PQLTDVLN(73)) of CfaB were genetically fused to ltb-(gly)5-cfaB(1-25). Sera of rabbits immunized with fusion proteins reacted strongly with solid-phase bound ETEC bacteria bearing CFA/I fimbriae. Sera failed to agglutinate or inhibit hemagglutination promoted by CFA/I-positive strain which may be due to solvent inaccessibility of epitope residues on intact fimbriae.
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http://dx.doi.org/10.1016/j.mimet.2014.07.035DOI Listing
November 2014

Enteroaggregative Escherichia coli, a heterogenous, underestimated and under-diagnosed E. coli pathotype in Iran.

Gastroenterol Hepatol Bed Bench 2013 ;6(2):71-9

Molecular Biology Unit, Pasteur Institute of Iran, Tehran, Iran ; Escherichia coli Reference Laboratory, Molecular Biology Unit, Pasteur Institute of Iran, Tehran, Iran.

The main features of enteroaggregative Escherichia coli (EAEC) pathogenesis include attachment of bacteria to the intestinal mucosa, production of various toxins and cytotoxins, and stimulation of mucosal inflammation. 'Virulence' genes encode these features. Comparison of different EAEC isolates has shown that the virulence gene content of these isolates varies considerably. The heterogeneity of EAEC strains was concluded from the results obtained from the volunteer as well as other studies. Although the underlying mechanism behind the apparent increase in O104:H4 virulence is not known, several bacterial factors have been implicated. In this review, the known virulence factors involved in pathogenesis of EAEC pathotype are summarized.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4017499PMC
June 2014

In silico and in vivo studies of truncated forms of flagellin (FliC) of enteroaggregative Escherichia coli fused to FimH from uropathogenic Escherichia coli as a vaccine candidate against urinary tract infections.

J Biotechnol 2014 Apr 12;175:31-7. Epub 2014 Feb 12.

Department of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran. Electronic address:

The new generation of vaccines against infectious diseases is based on recombinant fusion proteins. Flagellin (FliC) of enteroaggregative Escherichia coli (EAEC) could be considered as a potent adjuvant in designing new vaccines. However, because of its large size, incorporation of this protein with a vaccine antigen might negatively influence recognition of the vaccine epitopes by the immune system. Designing the truncated forms of FliC, capable of inducing innate immune response, enhances the immune responses to the target antigen. We have previously shown that two truncated forms of FliC are able to induce Interleukine-8 production in HT-29 epithelial cell line. In this study we designed recombinant vaccine against urinary tract infections (UTIs) using truncated forms of FliC and type 1 fimbrial FimH adhesin from uropathogenic Escherichia coli (UPEC) and studied their in silico interactions with Toll-like receptor 5 (TLR-5) via docking protocols. The best fusion protein was subjected to cloning and expression. The ability of the recombinant vaccine and the truncated forms in inducing immune responses was investigated. Our results showed that truncated forms are capable of inducing Th1 (forms A and B) and Th2 (form A) responses and fusion vaccine induced strong cellular and humoral immune responses.
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http://dx.doi.org/10.1016/j.jbiotec.2014.01.037DOI Listing
April 2014

Genotypic and phenotypic comparison of enteroaggregative Escherichia coli isolates from HIV-positive and non-HIV diarrheal samples.

Curr HIV Res 2013 Dec;11(8):635-41

Molecular Biology Department, Pasteur Institute of Iran, Tehran, Iran.

Enteroaggregative Escherichia coli (EAEC) have been isolated from both HIV-positive and non-HIV diarrheal samples. In this study a collection of 18 isolates from these two groups were compared for biofilm formation and antibiotic resistance and for the presence of 14 virulence-related genes. All the HIV-positive and over 66% of the non- HIV strains were PCR-negative for adhesion-related sequences indicating that as yet unknown adhesins may play a role. However, despite some variations, the prevalence rate of the virulence-related genes was not significantly different in the two groups. HIV-positive isolates were biofilm producer but only a single weak biofilm former was observed among the non-HIV strains. The rate of resistance to most of the antibiotics used was higher among the HIV-positive group than the non-HIV isolates, but was significantly higher for amoxicillin-calvulanic acid (100%) and nalidixic acid (55.5%). Pulse field gel electrophoresis of the isolates produced 17 unique profiles reflecting the exiting heterogeneity of the isolates.
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http://dx.doi.org/10.2174/1570162x12666140207161000DOI Listing
December 2013

Expression of Shigella flexneri ipaB Gene in Tobacco.

Avicenna J Med Biotechnol 2013 Apr;5(2):118-24

Molecular Biology Unit, Pasteur Institute of Iran, Tehran, Iran.

Background: Shigellosis is a leading cause of diarrhea in many developing countries and although the disease can be controlled and managed with antibiotics, the constant emergence of resistant species requiring ever newer antibacterial drugs make development of an effective vaccine necessary. The bacteria are highly contagious and since immunity to Shigella is serotype-specific a multi-serotype vaccine is required for adequate protection. Proteins encoded by Shigella invasion plasmid, which are part of the Type Three Secretion System (TTSS) of this bacteria, are good candidate as vaccine targets since they are both immunogenic and conserved between different Shigella species. The advent of molecular farming, which is a low cost system, has opened up new venues for production of recombinant proteins. In view of the difficulties encountered in expressing IpaB in Escherichia coli (E. coli), the feasibility of the expression of this protein in tobacco has been investigated.

Methods: The ipaB gene was cloned in place of the Hygromycin gene in pCambia1304 containing GFP as a reporter gene. The vector was then transferred into competent Agrobacterium tumefaciens (A. tumefaciens) strain LBA4404 which was used for agro-infiltration of Nicotiana tobaccum (N. tobaccum) leaves. Transformation was confirmed by expression of GFP. The gene was also cloned in pBAD/geneIII A and transformed E. coli host containing the construct was induced using different amounts of L-arabinose as inducer. Expression of IpaB gene by both hosts was determined by Western blotting using anti-IpaB monoclonal antibody.

Results: The data obtained showed that IpaB was expressed in plant leaves but expression in E. coli was not detectable.

Conclusion: This study showed that N. tobaccum is capable of expressing this protein without its specific chaperon and in levels detectable by Western blotting.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3689555PMC
April 2013

Comparison of multiplex PCR with serogrouping and PCR-RFLP of fliC gene for the detection of enteropathogenic Escherichia coli (EPEC).

Braz J Infect Dis 2011 Jul-Aug;15(4):365-9

Molecular Biology Unit, Pasteur Institute of Iran, Tehran, Iran.

Enteropathogenic Escherichia coli (EPEC) comprise one of the six categories of diarrhoeagenic E. coli (DEC). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC). The identification of DEC cannot be based only on cultural and biochemical criteria, since they are indistinguishable from the non-pathogenic E. coli commonly found in human feces. Several PCR methods, with both single and multiple target genes, have been reported for detecting the different DEC pathotypes. In the present study five hundred E. coli isolates from children with diarrhea were subjected into multiplex PCR. Furthermore the strains were typed serologically with O antisera and their fliC gene was characterized by PCR-RFLP. The results obtained revealed that overall 41 (8.2%) isolates could be detected as EPEC by this multiplex PCR assay. Of these isolates; 27 (66%) were typical (escv+, bfp+) and 14 (34%) atypical EPEC (escv+, bfp-). None of these 41 isolates contained the Stx1 and Stx2 genes. Among 37 (90%) typeable strains, nine different serogroups were present. The most common serogroups were O111, followed by O86, O55 and O119 and 10 different H types were found among these isolates. The multiplex PCR assay was found to be rapid and reliable in comparison to serological test; especially when screening the large number of isolates.
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http://dx.doi.org/10.1016/s1413-8670(11)70206-9DOI Listing
February 2012

Comparison of virulence markers and antibiotic resistance in enterotoxigenic Escherichia coli isolated ten years apart in Tehran.

J Infect Dev Ctries 2011 Apr 26;5(4):248-54. Epub 2011 Apr 26.

Molecular Biology Unit, Pasteur Institute of Iran, Tehran 13164, Iran.

Introduction: Enterotoxigenic Escherichia coli (ETEC) causes diarrhoea by producing heat-labile (LT) or heat-stable (ST) enterotoxins after colonizing the small intestine by means of colonization factors (CFs). Although detection of the toxins is sufficient for verification of ETEC isolates, toxin-positive strains may be further analyzed for the presence of CFs. Antibiotics may shorten the duration of diarrhoea caused by ETEC, but the rapid emergence of resistant strains limits their usefulness.

Methodology: ETEC isolates collected 10 years apart were compared for the prevalence of toxin types, CFs and antibiotic resistance. DNA/DNA hybridization with digoxigenin (DIG)-labeled probes was used for the detection of toxin types, and CF-typing was performed by DNA hybridization using DIG-labeled probes for cfaD and CS6 with slide agglutination. Disk diffusion was used to determine antibiotic resistance. The presence of class 1 integrons was detected by PCR.

Results: ST-positive isolates were the most prevalent among the isolates from 1988, but a significant shift towards LT-gene carriage was observed in the 1998 group. CFA/I and CFA/IV were the most common CF types within both groups. The most prevalent resistance patterns among these isolates were ACSTSXT followed by ASTSXT and ASSXT.

Conclusion: Our study of the two groups of isolates showed that the rate of LT and ST gene carriage, as well as antibiotic resistance markers, has changed in the ten years separating the two bacterial populations. These variations show the importance of monitoring pathogenic bacteria to obtain a near realistic picture of the circulating bacterial pathogens.
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http://dx.doi.org/10.3855/jidc.1206DOI Listing
April 2011

Immune response against adhesins of enteroaggregative Escherichia coli immunized by three different vaccination strategies (DNA/DNA, Protein/Protein, and DNA/Protein) in mice.

Comp Immunol Microbiol Infect Dis 2010 May 3;33(3):215-25. Epub 2008 Dec 3.

Molecular Biology Unit, Pasteur Institute of Iran, Tehran, Iran.

Enteroaggregative Escherichia coli (EAEC) are an increasingly recognized enteric pathogen. It is a cause of both acute and persistent diarrhoea among children, adults and HIV-infected persons, in both developing and developed countries. The aggregative adherence of EAEC is due to the presence of aggregative adherence fimbriaes (AAFs). To elucidate the possible protective role of these adhesins in diarrheagenic E. coli with DNA immunization approach, Balb/c mice were immunized with three different modes of vaccination, i.e. DNA/DNA, DNA/Protein, or Protein/Protein of Aggregative Adherence Factors, AAF/I or AAF/II of enteroaggregative Escherichia coli (EAEC), respectively. Overall, AAF/I and AAF/II in DNA/DNA mode could not induce the immune response. However, the DNA/Protein immunization of AAF/I significantly (P<0.05) induced total IgG level, and in the case of Protein/Protein approach, the induction of immune system was more significant (P<0.02). The DNA/Protein regimen of AAF/II induced total IgG significantly (P<0.03). But in the case of Protein/Protein immunization, the induced response was not significant. These preliminary data revealed that as an antigen, these two adhesins behave in a different manner, although AAF/I and AAF/II are known adhesins of EAEC with putatively similar functions.
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http://dx.doi.org/10.1016/j.cimid.2008.10.002DOI Listing
May 2010