Publications by authors named "Parastoo Ehsani"

22 Publications

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In silico Analysis of Pasteurella multocida PlpE Protein Epitopes As Novel Subunit Vaccine Candidates.

Iran Biomed J 2021 01 4;25(1):41-6. Epub 2020 Jan 4.

Department of Microbiology and Immunology, University of Tehran, Iran.

Background: Pasteurella multocida is a Gram-negative, non-motile, non-spore forming, and aerobic/anaerobic cocobacillus known as the causative agent of human and animal diseases. Humans can often be affected by cat scratch or bite, which may lead to soft tissue infections and in rare cases to bacteremia and septicemia. Commercial vaccines against this agent include inactivated, live attenuated, and non-pathogenic bacteria. Current vaccines have certain disadvantages such as reactogenicity or reversion to virulence. Therefore, the aim of this study was to reach a multi-epitope vaccine candidate that could be serotype independent and covers most incident serotypes of P. multocida.

Methods: In this study, reverse vaccinology strategy was used to identify potentially immunogenic and protective epitopes. First, multiple alignments of different sequences of Pasteurella lipoprotein E (PlpE) from various serotypes of P. multocida were analyzed to identify the conserved regions. Bioinformatics tools were then applied to predict and select epitopes for further studies.

Results: Three different conserved immunogenic regions were selected according to the selected criteria, and their various sequential orders were evaluated structurally by in silico tools to find the best order.

Conclusion: In searching the epitopes of PlpE to design a new vaccine candidate against pasteurellosis, we found the region 1 + region 2 + region 3 (without any linker between regions) of epitope, including the regions of PlpE protein of P. multocida, as the appropriate serotype independent vaccine candidate against pasteurellosis.
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http://dx.doi.org/10.29252/ibj.25.1.41DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7748120PMC
January 2021

PlpE Epitopes of Fusion Protein as Novel Subunit Vaccine Candidates.

Adv Biomed Res 2020 28;9:43. Epub 2020 Aug 28.

Department of Microbiology and Immunology, University of Tehran, Tehran, Iran.

Background: is the causative agent of many diseases. Antimicrobial treatment disadvantages highlight the need to find other possible ways such as prophylaxis to manage infections. Current vaccines against this agent include inactivated bacteria, live-attenuated bacteria, and nonpathogenic bacteria, which have disadvantages such as lack of immunogenicity, reactogenicity, or reversion to virulence wild bacteria. Using bioinformatical approaches, potentially immunogenic and protective epitopes identified and merged to design the best epitope fusion form in case of immunogenicity as a vaccine candidate.

Materials And Methods: In this study, the fusion protein () and full genes () were cloned in pET28a in BL21 (DE3) firstly and later in pBAD/gIII A and expressed in Top10 coli. Overlap polymerase chain reaction (PCR) using different primers for 5' and 3' end of each segment produced fusion segment 1 + 2 and (1 + 2) +3 fragments and was used for cloning.

Results: Cloning of both and into the pET28a vector and their transform into the BL21 (DE3) host was successful, as the presence of the cassettes was proved by digestion and colony PCR, however, their expression faced some challenges independent of expression inducer (isopropyl β-d-1-thiogalactopyranoside) concentration.

Conclusion: Changing the vector to pBAD/gIII A and consequently changing the host to Top10 have resulted in sufficient expression, which shows that Top10 may be a good substitute for such cases. Furthermore, it is concluded that adding 8M urea results in sufficient purification, which hypothesizes that denature purification is better for such cases than native one. Purified proteins headed for further analysis as vaccine candidates.
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http://dx.doi.org/10.4103/abr.abr_245_19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7532835PMC
August 2020

Immunization with oral and parenteral subunit chimeric vaccine candidate confers protection against Necrotic Enteritis in chickens.

Vaccine 2020 10 2;38(46):7284-7291. Epub 2020 Oct 2.

Sari Agriculture Science and Natural Resource University (SANRU), Sari, Iran.

Following the ban on the use of in-feed antimicrobials, necrotic enteritis (NE) NE is the most important clostridial disease. Vaccination has been considered as a possible approach to prevent NE. Our previous study showed that a chimeric protein product consisting of antigenic epitopes of NetB, Alpha-toxin and Zinc metallopeptidase (Zmp) triggered immune response against C. perfringens. In the current study we optimized the chimeric gene and constructed a fusion protein containing NetB, Alpha-toxin and Metallopeptidase (NAM) for expressing in tobacco plant to use as an edible vaccine for immunizing the chicken against NE. Simultaneously, we expressed and purified a His-tagged recombinant version of the NAM (rNAM) expressed in E. coli BL21 for subcutaneous immunization of chickens. Immunized birds produced strong humoral immune responses against both edible plant-based and parenteral purified rNAM. The responses were determined by the mean titer of antibody in blood samples to be around 9000 and 32,000, for edible and injected rNAM, respectively. Birds immunized subcutaneously showed the most striking responses. However the edible vaccine provided a more long lasting IgY response 14 days after the third vaccination compared to the injected birds. Chickens immunized with either lyophilized leaves expressing rNAM or purified rNAM, subsequently were subjected to the challenge with a virulent C. perfringens strain using an NE disease model. Our results showed that birds immunized both parenterally and orally with recombinant chimeric vaccine were significantly protected against the severity of lesion in the intestinal tract, but the protection provided with the injectable form of the antigen was greater than that of the oral form. Further analysis is needed to check whether these strategies can be used as the potential platform for developing an efficient vaccine against NE.
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http://dx.doi.org/10.1016/j.vaccine.2020.09.047DOI Listing
October 2020

Immobilization of recombinant lysostaphin on nanoparticle through biotin-streptavidin conjugation technology as a geometrical progressed confrontation against Staphylococcus aureus infection.

Biotechnol Appl Biochem 2020 Sep 12. Epub 2020 Sep 12.

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

Antibiotic resistance and the colonization of resistant bacteria such as Staphylococcus aureus on surfaces, often in the form of biofilms, prolong hospitalization periods and increase mortality, thus is a significant concern for healthcare providers. To prevent biofilm formation, the inadequate concentration of using nanoparticles as antibacterial coating agents is one of the major obstacles. This study aimed to design a hypervalency TiO nanocomposite as a reserved base to carry a high amount of active antibacterial agents such as lysostaphin via a biotin-streptavidin-biotin bridge. The utilization of the streptavidin-biotin system could increase the abundance of lysostaphin. Lysostaphin was expressed in Escherichia coli and purified. Both recombinant lysostaphin and titanium oxide nanocomposite were conjugated with biotin and linked to a streptavidin bridge. The kinetics and activity of the enzyme were examined after each step utilizing N-acetylhexaglycine as a substrate. Physical characteristics of nanoparticles containing lysostaphin were determined using AFM, SEM, FTIR, and zeta potential. The results showed changes in size, charge, and morphology of the nanoparticles following the lysostaphin attachment. Also, the stability and kinetics of the active biological enzymes on nanoparticles were reexamined following 8 months of storage. Exploiting this approach, various biotinylated antibacterial agents could be prepared and rapidly immobilized on a nanoparticle as an active net against related infectious agents.
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http://dx.doi.org/10.1002/bab.2025DOI Listing
September 2020

Canola oilseed- and Escherichia coli- derived hepatitis C virus (HCV) core proteins adjuvanted with oil bodies, induced robust Th1-oriented immune responses in immunized mice.

APMIS 2020 Nov 21;128(11):593-602. Epub 2020 Sep 21.

Department of Immunology, Pasteur Institute of Iran, Tehran, Iran.

Induction of broad Th1 cellular immune responses and cytokines is crucial characteristics for vaccines against intracellular infections such as hepatitis C virus (HCV). Plants (especially oilseed tissues) and plant-immunomodulators (like oil bodies) offer cost-effective and scalable possibilities for the production of immunologically relevant and safe vaccine antigens and adjuvants, respectively. Herein, we provide data of the murine immunization by transgenic canola oilseed-derived HCV core protein (HCVcp) soluble extract (TSE) and Escherichia coli- derived rHCVcp in combination with Canola oil bodies (oil) compared to that of the Freund's (FA) adjuvant. Mice immunized by TSE+ oil developed both strong humeral (IgG) and Th1-biased cellular responses, manifested by high levels of IFN-γ and lower IgG1/IgG2a ratio and IL-4 secretion. Results of the intracellular cytokine staining indicated that TSE+ oil immunization in mice triggered both CD4 and CD8 T cells to release IFN-γ, while CD4 cells were mostly triggered when FA was used. Analyses by qRT-PCR indicated that a combination of rHCVcp/TSE with oil body induced high levels of IL-10 cytokines compared to that of the FA adjuvant. These characteristics are important properties for the design of an HCV vaccine candidate and indicate the potential of Canola-derived antigen and oil bodies in addressing these concerns.
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http://dx.doi.org/10.1111/apm.13074DOI Listing
November 2020

Vaccine Candidates: A Systematic Review.

Avicenna J Med Biotechnol 2020 Jul-Sep;12(3):140-147

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

() is the highly contagious causative agent of a broad range of diseases in animals as well as an occasional human pathogen. Economically significant infections caused by include avian fowl cholera, rabbit snuffles, and hemorrhagic septicemia in cattle, goats and pigs. Chemotherapy of pasteurellosis infections has some limitations, such as high cost of treatment, low efficacy, and the possibility of therapy failure due to antibiotic resistance. Prophylactic immunization offers a safe and effective preventive measure in case of zoonotic diseases. Bacterins, live attenuated and some old traditional vaccines against pasteurellosis remain in use today, beside their limitations. However, the past few years have seen significant progress in research to identify modern, effective vaccine candidates, but there is no new vaccine produced by new strategies. While scientists should struggle with a lot of aspects to design vaccine producing strategies, this review shows how pasteurellosis vaccine evolved and the limitations in its application which need to be overcome.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368114PMC
July 2020

Biomedical applications of yeasts - a patent view, part two: era of humanized yeasts and expanded applications.

Expert Opin Ther Pat 2020 Aug 30;30(8):609-631. Epub 2020 Jun 30.

; Universite de Versailles, Service de ne 'phrologie-transplantation re'nale, Hopital Foch, 40 rue Worth, Suresnes , Paris, France.

Introduction: Yeast humanization, ranging from a simple point mutation to substitution of yeast gene(s) or even a complete pathway by human counterparts has enormously expanded yeast biomedical applications.

Areas Covered: General and patent-oriented insights into the application of native and humanized yeasts for production of human glycoproteins (gps) and antibodies (Abs), toxicity/mutagenicity assays, treatments of gastrointestinal (GI) disorders and potential drug delivery as a probiotic (with emphasis on ) and studies on human diseases/cancers and screening effective drugs.

Expert Opinion: Humanized yeasts cover the classical advantageous features of a 'microbial eukaryote' together with advanced human cellular processes. These unique characteristics would permit their use in the production of functional and stable therapeutic gps and Abs in lower prices compared to mammalian (CHO) production-based systems. Availability of yeasts humanized for cytochrome P450 s will expand their application in metabolism-related chemical toxicity assays. Engineered for expression of human proteins might expand its application by synergistically combining the probiotic activity with the treatment of metabolic diseases such as phenylketonuria via GI-delivery. Yeast models of human diseases will facilitate rapid functional/phenotypic characterization of the disease-producing mutant genes and screening of the therapeutic compounds using yeast-based high-throughput research techniques (Yeast one/two hybrid systems) and viability assays.
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http://dx.doi.org/10.1080/13543776.2020.1781816DOI Listing
August 2020

A preliminary computational outputs versus experimental results: Application of sTRAP, a biophysical tool for the analysis of SNPs of transcription factor-binding sites.

Mol Genet Genomic Med 2020 05 10;8(5):e1219. Epub 2020 Mar 10.

Biochemistry Department, Pasteur Institute of Iran, Tehran, Iran.

Background: In the human genome, the transcription factors (TFs) and transcription factor-binding sites (TFBSs) network has a great regulatory function in the biological pathways. Such crosstalk might be affected by the single-nucleotide polymorphisms (SNPs), which could create or disrupt a TFBS, leading to either a disease or a phenotypic defect. Many computational resources have been introduced to predict the TFs binding variations due to SNPs inside TFBSs, sTRAP being one of them.

Methods: A literature review was performed and the experimental data for 18 TFBSs located in 12 genes was provided. The sequences of TFBS motifs were extracted using two different strategies; in the size similar with synthetic target sites used in the experimental techniques, and with 60 bp upstream and downstream of the SNPs. The sTRAP (http://trap.molgen.mpg.de/cgi-bin/trap_two_seq_form.cgi) was applied to compute the binding affinity scores of their cognate TFs in the context of reference and mutant sequences of TFBSs. The alternative bioinformatics model used in this study was regulatory analysis of variation in enhancers (RAVEN; http://www.cisreg.ca/cgi-bin/RAVEN/a). The bioinformatics outputs of our study were compared with experimental data, electrophoretic mobility shift assay (EMSA).

Results: In 6 out of 18 TFBSs in the following genes COL1A1, Hb ḉᴪ, TF, FIX, MBL2, NOS2A, the outputs of sTRAP were inconsistent with the results of EMSA. Furthermore, no p value of the difference between the two scores of binding affinity under the wild and mutant conditions of TFBSs was presented. Nor, were any criteria for preference or selection of any of the measurements of different matrices used for the same analysis.

Conclusion: Our preliminary study indicated some paradoxical results between sTRAP and experimental data. However, to link the data of sTRAP to the biological functions, its optimization via experimental procedures with the integration of expanded data and applying several other bioinformatics tools might be required.
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http://dx.doi.org/10.1002/mgg3.1219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216802PMC
May 2020

Synergistic Anti-Staphylococcal Activity Of Niosomal Recombinant Lysostaphin-LL-37.

Int J Nanomedicine 2019 10;14:9777-9792. Epub 2019 Dec 10.

Department of Nano Biotechnology, New Technology Research Group, Pasteur Institute of Iran, Tehran, Iran.

Purpose: is the most common persistent pathogen in humans, so development of new formulations to combat pathogen invasion is quite necessary.

Methods: In the current study, for the first time, the synergistic activity of recombinant lysostaphin and LL-37 peptide was studied against . Moreover, different niosomal formulations of the peptide and protein were prepared and analyzed in terms of size, shape, zeta potential, and entrapment efficiency. Also, a long-term antibacterial activity of the best niosomal formulation and free forms was measured against in vitro.

Results: The optimal niosomal formulation was obtained by mixing the surfactants (span60 and tween60; 2:1 w/w), cholesterol, and dicetylphosphate at a ratio of 47:47:6, respectively. They showed uniform spherical shapes with the size of 565 and 325 nm for lysostaphin and LL-37, respectively. This formulation showed high entrapment efficiency for the peptide, protein, and a slow-release profile over time. Release kinetic was best fitted by Higuchi model indicating a diffusion-based release of the drugs. The lysostaphin/LL-37 niosomal formulation synergistically inhibited growth of for up to 72 hours. However, the same amounts of free forms of both anti-microbial agents could not hold the anti-microbial effect and growth was seen in the following 72 hours. Cytotoxicity assay specified that lysostaphin/LL-37 niosomal combination had no deleterious effect on normal fibroblast cells at effective antimicrobial concentrations.

Conclusion: This study indicated that the use of lysostaphin in combination with LL-37, either in niosomal or free forms, synergistically inhibited growth of in vitro. In addition, niosomal preparation of antimicrobial agents could provide a long-term protection against bacterial infections.
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http://dx.doi.org/10.2147/IJN.S230269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911324PMC
March 2020

In silico design and in vitro analysis of a recombinant trivalent fusion protein candidate vaccine targeting virulence factor of Clostridium perfringens.

Int J Biol Macromol 2020 Mar 11;146:1015-1023. Epub 2019 Nov 11.

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

Necrotic enteritis (NE) is a multifactorial disease in broiler that is caused by colonization of Clostridium perfringens in their gastrointestinal tract. Recently several immunogenic proteins from virulent C. perfringens have been considered as vaccines to provide protection against NE. In this study, a novel trivalent fusion protein including immunogenic epitopes of three virulence factors of, NetB, alpha toxin and a metallopeptidase protein (NAM) was designed using in silico studies. Circular dichroism spectra was applied for determination of secondary structure and folding properties of the purified recombinant NAM (rNAM) expressed in E. coli. The antigenicity of rNAM was confirmed by induction of immune response in rabbit and neutralization experiments of the toxins in cell culture studies. To this end, anti-rNAM antisera neutralized the crude toxins produced by a wild type virulent C. perfringens strain using chicken hepatocellular carcinoma (LMH) cell lines. The cells were exposed to a mixture of anti-rNAM antisera and 2 × LD50 doses of the toxins. The result showed 94% viability of the cells against the crude toxins, in the presence of anti-rNAM antisera. Our study suggests that combination of metallopeptidase protein along with alpha toxin and NetB toxins is a potent immunogen which is able to neutralize the toxicity of crude extracellular toxins. The recombinant chimeric NAM could be a suitable and effective subunit vaccine candidate to prevent NE disease caused by C. perfringens.
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http://dx.doi.org/10.1016/j.ijbiomac.2019.09.227DOI Listing
March 2020

Construction of bicistronic cassette for co-expressing hepatitis B surface antigen and mouse granulocyte-macrophage colony stimulating factor as adjuvant in tobacco plant.

Pharm Biol 2019 Dec;57(1):669-675

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

The co-delivery of adjuvant and antigen has shown to be more effective for targeting the immune response than antigen alone. Therefore, designing an efficient bicistronic system is more assuring for production of both elements in the same tobacco cells as a plant model system. Comparing the efficient transient co-expression of hepatitis B surface antigen (HBsAg) and mouse granulocyte macrophage colony stimulating factor (mGM-CSF) in tobacco leaves by designing either mono or bicistronic cassettes. Four expression cassettes containing tobacco etch virus (TEV) leader sequence were constructed with and without above genes in different orders. The cassettes were transferred into tobacco, L. (Solanaceae), leaves by agroinfiltration technique. The expression levels were compared using ELISA and western blotting and bioactivity of cytokine was assessed by proliferation of mouse GM-CSF-responsive progenitor cells. Agroinfiltrated leaves contained recombinant HBsAg protein at 20-50 ng/mg and mGM-CSF at 0.2-4 ng/mg in both nonglycosylated and glycosylated forms. The highest expression obtained in HBsAg and mGM-CSF monocistronic co-agroinfiltrated leaves. The expression of mGM-CSF was 1.1 and 0.2 ng/mg in two different orders of bicistronic cassettes. The growth frequency of GM progenitors was approximately 1/187 cells for standard rGM-CSF and 3.2 times less activity for the plant produced. The recombinant mGM-CSF was produced less in bicistronic cassette than other forms; however, co-presenting of both vaccine candidate and adjuvant is confirmed and could be promising for amelioration of plant expression system as a means for vaccine production.
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http://dx.doi.org/10.1080/13880209.2019.1662458DOI Listing
December 2019

Salmonella Typhimurium in Iran: Contribution of molecular and IS200 PCR methods in variants detection.

PLoS One 2019 13;14(3):e0213726. Epub 2019 Mar 13.

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

Salmonella Typhimurium, a zoonotic pathogen, is regarded as a major health and economic concern worldwide. Recently, monophasic variants of this serovar have been significantly associated with human gastroenteritis outbreaks globally, making its accurate identification essential for epidemiological and control purposes. We have identified and analyzed 150 S. Typhimurium from 884 Salmonella genus isolated from humans, domestic animals, poultry, food items and abattoirs origins. The Salmonella isolates were obtained from Iranian National Veterinary Reference Laboratories of 9 provinces during 2007-2016, and from five hospitals in Tehran in 2015. The isolates were evaluated biochemically, serologically, and by PCR amplification of invA, mdh, STM4492, fliC, fljA, fljB, hin genes, IS200 and DT104. invA and mdh genes were used to confirm the S. Typhimurium serotype, fliC and fljB genes for determination of monophasic variants and amplification of IS200 to discriminate the monophasic variants from the closely related serotypes. We identified 78.6% (118/150) as classical S. Typhimurium (fliC, fljB and IS200 positive), 12.6% (19/150) were IS200 negative from all isolates. DT104 is another marker for S.Typhimurium serovar typing. Contrary to EFSA guidelines 20.6% (19/29) of human isolates that lacked IS200 insertion sequence, were confirmed as S.Typhimurium. Compared to the North American/European isolates the low prevalence of fljB negative 6% (9/150) and the high abundance of fliC negative 23.3% (35/150) isolates also were indicative of a different regional atypical population. Studies have shown that the prevalence of monophasic (fljB-) S. Typhimurium worldwide is promoted by the Swine industry. Thus, one reason for this high number of different atypical strains could be inhibition of swine breeding system (house hold and industry) in Iran. These results demonstrate a need for a modified identifying protocol to overcome the regional differences.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0213726PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6415898PMC
December 2019

Transient Expression of Biologically Active Anti-rabies Virus Monoclonal Antibody in Tobacco Leaves.

Iran J Biotechnol 2018 Apr 18;16(1):e1774. Epub 2018 Apr 18.

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

Background: Rabies virus is a neurotropic virus that causes fatal, but, a preventable disease in mammals. Administration of rabies immunoglobulin (RIG) is essential for the post-exposure of the prophylaxis to prevent the disease. However, replacement of polyclonal RIGs with alternative monoclonal antibodies (MAbs) that are capable of neutralizing rabies virus has been recommended.

Objectives: Here, we have investigated the transient expression of the full-size human MAb against rabies virus glycoprotein; the MAb SO57 in the tobacco plants using vacuum agro-infiltration. Previously, stably transformed plants expressing the MAb have been reported.

Materials And Methods: In this study three vectors carrying the codon-optimized genes for the heavy or light chain and p19 silencing-suppressor were constructed. These vectors were co-infiltrated into leaves and the transgenes were expressed.

Results: Dot blot, Western blotting, ELISA, and in vitro neutralization assays of the plant extracts showed that the human MAb could assemble in tobacco leaves and was able to neutralize rabies virus.

Conclusions: This study is the first report of transient expression of human MAb SO57 gene in tobacco plant within a few days after vacuum agro-infiltration.
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http://dx.doi.org/10.21859/ijb.1774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6217261PMC
April 2018

In silico and in vivo analyses of the mutated human tissue plasminogen activator (mtPA) and the antithetical effects of P19 silencing suppressor on its expression in two Nicotiana species.

Sci Rep 2018 09 19;8(1):14079. Epub 2018 Sep 19.

Department of Molecular Biology, Pasteur Institute of Iran (IPI), Tehran, Iran.

Human tissue-type plasminogen activator is one of the most important therapeutic proteins involved in the breakdown of blood clots following the stroke. A mutation was found at position 1541 bp (G514E) and the mutated form was cloned into the binary vector pTRAc-ERH. In silico analysis showed that this mutation might have no significant effect on the active site of the tissue plasminogen activator enzyme. Accordingly, zymography assay confirmed the serine protease activity of the mutated form and its derivatives. The expression of the mutated form was verified with/without co-agroinjection of the P19 gene silencing suppressor in both Nicotiana tabacum and N. benthamiana. The ELISA results showed that the concentration of the mutated form in the absence of P19 was 0.65% and 0.74% of total soluble protein versus 0.141% and 1.36% in the presence of P19 in N. benthamiana and N. tabacum, respectively. In N. tabacum, co-agroinjection of P19 had the synergistic effect and increased the mutated tissue plasminogen activator production two-fold higher. However, in N. benthamiana, the presence of P19 had the adverse effect of five-fold reduction in the concentration. Moreover, results showed that the activity of the mutated form and its derivatives was more than that of the purified commercial tissue plasminogen activator.
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http://dx.doi.org/10.1038/s41598-018-32099-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6145930PMC
September 2018

Determination immunogenic property of truncated MrpH.FliC as a vaccine candidate against urinary tract infections caused by Proteus mirabilis.

Microb Pathog 2018 Jan 11;114:99-106. Epub 2017 Nov 11.

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

Proteus mirabilis is common cause of urinary tract infections (UTIs) especially in complicated UTIs which are resistant to antibiotic therapy, Consequently, an ideal vaccine is inevitably required. The N-terminal domain of MrpH (Truncated form of MrpH) lies between the most critical antigens of P. mirabilis to consider as vaccine candidate. FliC of Salmonella typhimurium induces several pathways of immunity system, which leads to produce antibody and cytokines. In this study, adjuvant properties of FliC and efficacy of truncated MrpH as important antigen, in tMrpH.FliC were determined in in vitro and in vivo circumstances. Three proteins including: FliC, MrpH and tMrpH.FliC were injected to mice and subsequently sera and supernatant of cell culture were collected to evaluate different immune responses. According to our findings, tMrpH.FliC could stimulate both humoral and cellular immune responses, so that serum IgG, urine IgA, IL.4, IFN-γ and IL.17 were increased significantly in comparison to MrpH and FliC alone, this augmentation was considerable. Results showed significant decrease of bacterial load in all of the challenged groups compared to the control group, although this protective effect was the highest in mice vaccinated with tMrpH.FliC. Our results showed truncated MrpH, without an unwanted domain is an ideal vaccine target and FliC, as adjuvant, increases its immunogenic property. Thus, fusion protein tMrpH.FliC can be considered as promising vaccine against P. mirabilis.
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http://dx.doi.org/10.1016/j.micpath.2017.11.015DOI Listing
January 2018

Biomedical applications of yeast- a patent view, part one: yeasts as workhorses for the production of therapeutics and vaccines.

Expert Opin Ther Pat 2017 Aug 22;27(8):929-951. Epub 2017 Jun 22.

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

Introduction: Yeasts, as Eukaryotes, offer unique features for ease of growth and genetic manipulation possibilities, making it an exceptional microbial host. Areas covered: This review provides general and patent-oriented insights into production of biopharmaceuticals by yeasts. Patents, wherever possible, were correlated to the original or review articles. The review describes applications of major GRAS (generally regarded as safe) yeasts for the production of therapeutic proteins and subunit vaccines; additionally, immunomodulatory properties of yeast cell wall components were reviewed for use of whole yeast cells as a new vaccine platform. The second part of the review will discuss yeast- humanization strategies and innovative applications. Expert opinion: Biomedical applications of yeasts were initiated by utilization of Saccharomyces cerevisiae, for production of leavened (fermented) products, and advanced to serve to produce biopharmaceuticals. Higher biomass production and expression/secretion yields, more similarity of glycosylation patterns to mammals and possibility of host-improvement strategies through application of synthetic biology might enhance selection of Pichia pastoris (instead of S. cerevisiae) as a host for production of biopharmaceutical in future. Immunomodulatory properties of yeast cell wall β-glucans and possibility of intracellular expression of heterologous pathogen/tumor antigens in yeast cells have expanded their application as a new platform, 'Whole Yeast Vaccines'.
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http://dx.doi.org/10.1080/13543776.2017.1339789DOI Listing
August 2017

Heterologous Expression of Hepatitis C Virus Core Protein in Oil Seeds of Brassica napus L.

Jundishapur J Microbiol 2015 Nov 7;8(11):e25462. Epub 2015 Nov 7.

Agricultural Biotechnology Department, National Institute of Genetic Engineering and Biotechnology, Tehran, IR Iran.

Background: Hepatitis c virus (HCV), prevalent among 3% of the world population, is a major worldwide public health concern and an effective vaccination could help to overcome this problem. Plant seeds as low-cost vaccine expression platforms are highly desirable to produce antigens.

Objectives: The present study was aimed at investigating the possible expression of recombinant HCV core protein, as a leading HCV vaccine candidate, in canola (Brassica napus) plant seeds in order to be used as an effective immunogen for vaccine researches.

Materials And Methods: A codon-optimized gene harboring the Kozak sequence, 6 × His-tag, HCVcp (1 - 122 residues) and KDEL (Lys-Asp-Glu-Leu) peptide in tandem was designed and expressed under the control of the seed specific promoter, fatty acid elongase 1 (FAE1), to accumulate the recombinant protein in canola (B. napus L.) seeds. Transgenic lines were screened and the presence of the transgene was confirmed in the T0 plants by polymerase chain reaction (PCR). The quantity and quality of the HCV core protein (HCVcp) in transgenic seeds were evaluated by enzyme-linked immunosorbent assay (ELISA) and western blot, respectively.

Results: Western blot analysis using anti-His antibody confirmed the presence of a 15 kDa protein in the seeds of T1 transgenic lines. The amount of antigenic protein accumulated in the seeds of these transgenic lines was up to 0.05% of the total soluble protein (TSP).

Conclusions: The canola oilseeds could provide a useful expression system to produce HCV core protein as a vaccine candidate.
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http://dx.doi.org/10.5812/jjm.25462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4735835PMC
November 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

Enhanced-Transient Expression of Hepatitis C Virus Core Protein in Nicotiana tabacum, a Protein With Potential Clinical Applications.

Hepat Mon 2014 Nov 24;14(11):e20524. Epub 2014 Nov 24.

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

Background: Hepatitis C virus (HCV) is major cause of liver cirrhosis in humans. HCV capsid (core) protein (HCVcp) is a highly demanded antigen for various diagnostic, immunization and pathogenesis studies. Plants are considered as an expression system for producing safe and inexpensive biopharmaceutical proteins. Although invention of transgenic (stable) tobacco plants expressing HCVcp with proper antigenic properties was recently reported, no data for "transient-expression" that is currently the method of choice for rapid, simple and lower-priced protein expression in plants is available for HCVcp.

Objectives: The purpose of this study was to design a highly codon-optimized HCVcp gene for construction of an efficient transient-plant expression system for production of HCVcp with proper antigenic properties in a regional tobacco plant (Iranian Jafarabadi-cultivar) by evaluation of different classes of vectors and suppression of gene-silencing in tobacco.

Materials And Methods: A codon-optimized gene encoding the Kozak sequence, 6xHis-tag, HCVcp (1-122) and KDEL peptide in tandem (from N- to C-terminal) was designed and inserted into potato virus-X (PVX) and classic pBI121 binary vectors in separate cloning reactions. The resulted recombinant plasmids were transferred into Agrobacterium tumefaciens and vacuum infiltrated into tobacco leaves. The effect of gene silencing suppressor P19 protein derived from tomato bushy stunt virus on the expression yield of HCVcp by each construct was also evaluated by co-infiltration in separate groups. The expressed HCVcp was evaluated by dot and western blotting and ELISA assays.

Results: The codon-optimized gene had an increased adaptation index value (from 0.65 to 0.85) and reduced GC content (from 62.62 to 51.05) in tobacco and removed the possible deleterious effect of "GGTAAG" splice site in native HCVcp. Blotting assays via specific antibodies confirmed the expression of the 15 kDa HCVcp. The expression level of HCVcp was enhanced by 4-5 times in P19 co-agroinfiltrated plants with better outcomes for PVX, compared to pBI121 vector (0.022% versus 0.019% of the total soluble protein). The plant-derived HCVcp (pHCVcp) could properly identify the HCVcp antibody in HCV-infected human sera compared to Escherichia coli-derived HCVcp (eHCVcp), indicating its potential for diagnostic/immunization applications.

Conclusions: By employment of gene optimization strategies, use of viral-based vectors and suppression of plant-derived gene silencing effect, efficient transient expression of HCVcp in tobacco with proper antigenic properties could be possible.
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http://dx.doi.org/10.5812/hepatmon.20524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4286711PMC
November 2014

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

Prognosis for splicing factor PRPF8 retinitis pigmentosa, novel mutations and correlation between human and yeast phenotypes.

Hum Mutat 2010 May;31(5):E1361-76

Section of Ophthalmology and Neuroscience, Leeds Institute of Molecular Medicine, St James's University Hospital, Leeds, UK.

PRPF8-retinitis pigmentosa is said to be severe but there has been no overview of phenotype across different mutations. We screened RP patients for PRPF8 mutations and identified three new missense mutations, including the first documented mutation outside exon 42 and the first de novo mutation. This brings the known RP-causing mutations in PRPF8 to nineteen. We then collated clinical data from new and published cases to determine an accurate prognosis for PRPF8-RP. Clinical data for 75 PRPF8-RP patients were compared, revealing that while the effect on peripheral retinal function is severe, patients generally retain good visual acuity in at least one eye until the fifth or sixth decade. We also noted that prognosis for PRPF8-RP differs with different mutations, with p.H2309P or p.H2309R having a worse prognosis than p.R2310K. This correlates with the observed difference in growth defect severity in yeast lines carrying the equivalent mutations, though such correlation remains tentative given the limited number of mutations for which information is available. The yeast phenotype is caused by lack of mature spliceosomes in the nucleus, leading to reduced RNA splicing function. Correlation between yeast and human phenotypes suggests that splicing factor RP may also result from an underlying splicing deficit.
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http://dx.doi.org/10.1002/humu.21236DOI Listing
May 2010

prp8 mutations that cause human retinitis pigmentosa lead to a U5 snRNP maturation defect in yeast.

Nat Struct Mol Biol 2007 Nov 14;14(11):1077-83. Epub 2007 Oct 14.

Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK.

Prp8 protein (Prp8p) is a highly conserved pre-mRNA splicing factor and a component of spliceosomal U5 small nuclear ribonucleoproteins (snRNPs). Although it is ubiquitously expressed, mutations in the C terminus of human Prp8p cause the retina-specific disease retinitis pigmentosa (RP). The biogenesis of U5 snRNPs is poorly characterized. We present evidence for a cytoplasmic precursor U5 snRNP in yeast that lacks the mature U5 snRNP component Brr2p and depends on a nuclear localization signal in Prp8p for its efficient nuclear import. The association of Brr2p with the U5 snRNP occurs within the nucleus. RP mutations in Prp8p in yeast result in nuclear accumulation of the precursor U5 snRNP, apparently as a consequence of disrupting the interaction of Prp8p with Brr2p. We therefore propose a novel assembly pathway for U5 snRNP complexes that is disrupted by mutations that cause human RP.
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http://dx.doi.org/10.1038/nsmb1303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2584834PMC
November 2007