Publications by authors named "Arash Arashkia"

37 Publications

The Inclusive Review on SARS-CoV-2 Biology, Epidemiology, Diagnosis, and Potential Management Options.

Curr Microbiol 2021 Feb 27. Epub 2021 Feb 27.

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

A novel coronavirus member was reported in Wuhan City, Hubei Province, China, at the end of the year 2019. Initially, the infection spread locally, affecting the Wuhan people, and then expanded rapidly throughout the world. On 11 March 2020, the World Health Organization (WHO) proclaimed it a global pandemic. The virus is a new strain most closely related to a bat coronavirus (RaTG13) which was not previously discovered in humans and is now formally known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronavirus disease 2019 (COVID-19) is the disease syndrome that the SARS-CoV-2 virus triggers. It is suggested that SARS-CoV-2 can be transmitted through aerosols, direct/indirect contact, and also during medical procedures and specimen handling. The infection is characterized by isolated flu-like symptoms, but there may be specific signs of fever, fatigue, cough, and shortness of breath, as well as the loss of smell and breathing difficulty. Within this report, we tried to review the most current scientific literature published by January 2021 on various aspects of the outbreak, including virus structure, pathogenesis, clinical presentation, epidemiology, diagnostic approaches, potential therapeutics and vaccines, and prospects. We hope this article makes a beneficial impact on public education to better deal with the SARS-CoV-2 crisis and push a step forward in the near term towards its prevention and control.
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http://dx.doi.org/10.1007/s00284-021-02396-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913045PMC
February 2021

Virus against virus: strategies for using adenovirus vectors in the treatment of HPV-induced cervical cancer.

Acta Pharmacol Sin 2021 Feb 25. Epub 2021 Feb 25.

Department of Microbiology, Ayatollah Amoli Branch, Islamic Azad University, Amol, Iran.

Although most human papillomavirus (HPV) infections are harmless, persistent infection with high-risk types of HPV is known to be the leading cause of cervical cancer. Following the infection of the epithelium and integration into the host genome, the oncogenic proteins E6 and E7 disrupt cell cycle control by inducing p53 and retinoblastoma (Rb) degradation. Despite the FDA approval of prophylactic vaccines, there are still issues with cervical cancer treatment; thus, many therapeutic approaches have been developed to date. Due to strong immunogenicity, a high capacity for packaging foreign DNA, safety, and the ability to infect a myriad of cells, adenoviruses have drawn attention of researchers. Adenovirus vectors have been used for different purposes, including as oncolytic agents to kill cancer cells, carrier for RNA interference to block oncoproteins expression, vaccines for eliciting immune responses, especially in cytotoxic T lymphocytes (CTLs), and gene therapy vehicles for restoring p53 and Rb function.
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http://dx.doi.org/10.1038/s41401-021-00616-5DOI Listing
February 2021

Severe acute respiratory syndrome-coronavirus-2 spike (S) protein based vaccine candidates: State of the art and future prospects.

Rev Med Virol 2020 Oct 15:e2183. Epub 2020 Oct 15.

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

Coronavirus disease 2019 (Covid-19) is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) which is responsible for a global pandemic that started in late 2019 in Wuhan, China. To prevent the worldwide spread of this highly pathogenic virus, development of an effective and safe vaccine is urgently needed. The SARS-CoV-2 and SARS-CoV share a high degree of genetic and pathologic identity and share safety and immune-enhancement concerns regarding vaccine development. Prior animal studies with first generation (whole virus-based) preparations of SARS-CoV vaccines (inactivated and attenuated vaccine modalities) indicated the possibility of increased infectivity or eosinophilic infiltration by immunization. Therefore, development of second and third generation safer vaccines (by using modern vaccine platforms) is actively sought for this viral infection. The spike (S) protein of SARS-CoVs is the main determinant of cell entry and tropism and is responsible for facilitating zoonosis into humans and sustained person-to-person transmission. Furthermore, 'S' protein contains multiple neutralizing epitopes that play an essential role in the induction of neutralizing antibodies (nAbs) and protective immunity. Moreover, T-cell responses against the SARS-CoV-2 'S' protein have also been characterized that correlate to the IgG and IgA antibody titres in Covid-19 patients. Thus, S protein is an obvious candidate antigen for inclusion into vaccine platforms against SARS-CoV-2 viral infection. This manuscript reviews different characteristics of S protein, its potency and 'state of the art' of the vaccine development strategies and platforms using this antigen, for construction of a safe and effective SARS-CoV-2 vaccine.
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http://dx.doi.org/10.1002/rmv.2183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7646037PMC
October 2020

Co-administration of 2'3'-cGAMP STING activator and CpG-C adjuvants with a mutated form of HPV 16 E7 protein leads to tumor growth inhibition in the mouse model.

Infect Agent Cancer 2021 Jan 26;16(1). Epub 2021 Jan 26.

Department of Virology, School of Public Health, Tehran University of Medical Sciences, 14155, Tehran, Iran.

Persistent infection with high-risk genotypes of human papillomavirus (HPV) is the leading cause of cervical cancer. The HPV oncoprotein E7 is constitutively expressed in cervical cancer and considered as an essential target for tumor-specific immunity. The goal of this study was to develop a candidate therapeutic vaccine based on the mutated E7 protein that had possibly reduced transformation capacity while was able to elicit a robust immune response. Therefore, the mutant type of HPV 16 E7 (E7GRG) protein was recombinantly expressed in E. coli. The protein was then purified and formulated with 2'-3'cGAMP CDN and/or CpG-C ODN adjuvants and subcutaneously injected to female C57BL/6 mice. To evaluate the immunogenic response, lymphocyte proliferation, secretion levels of IFN-γ and IL-4 cytokines, granzyme B level, and total IgG and subclasses of IgG antibody were measured. The anti-tumor activity was evaluated in tumor-harboring C57BL/6 mice. The highest rate of cell proliferation, IFN-γ and granzyme B levels, and amount of IgG antibody were found in mice group that were injected by E7GRG + 2'-3'cGAMP + CpG-C. Therapeutic immunization with E7GRG + 2'-3'cGAMP + CpG-C also significantly suppressed TC-1 tumor growth in mice. In conclusion, the results demonstrated that E7GRG + 2'-3'cGAMP + CpG-C induced strong cell-mediated and humoral immune responses that resulted in inhibition of tumor in mouse model.
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http://dx.doi.org/10.1186/s13027-021-00346-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836183PMC
January 2021

Association between circulating rotavirus genotypes and histo-blood group antigens in the children hospitalized with acute gastroenteritis in Iran.

J Med Virol 2021 Jan 19. Epub 2021 Jan 19.

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

Rotaviruses are the dominant cause of severe acute gastroenteritis in children under 5 years of age. Previous studies showed that some children are less susceptible to rotavirus gastroenteritis. It has been shown that this resistance depends on the rotavirus genotype and also human histo-blood group antigens (HBGAs), which works as a receptor for rotavirus surface protein (VP4). The present study aimed to evaluate the human genetic susceptibility to rotavirus gastroenteritis in Iran and to obtain a comparative analysis between rotavirus gastroenteritis and secretor or Lewis status in case and control groups in the Iranian population. The study was performed on fecal specimens from 108 children with acute rotavirus gastroenteritis from 2015 to 2017. A total of 50 fecal specimens from children with acute gastroenteritis of unknown etiology were also used as a control group. After the genotyping of positive rotavirus cases and human HBGAs by Sanger sequencing, the phylogenetic tree analysis showed that all rotavirus strains from Iran belonged to P[II]. The most common genotype was P[8] (n = 102; 94.4%), while the remaining belonged to P[4] (n = 3; 2.8%) and P[6] (n = 3; 2.8%) genotypes. The P[8] genotype was found to be associated with secretor and Lewis positive status (p < .05).
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http://dx.doi.org/10.1002/jmv.26808DOI Listing
January 2021

Estimation of genetic variation in the Secretor and Lewis genes in Iranian hospitalized children.

Transfus Clin Biol 2021 Feb 7;28(1):11-15. Epub 2020 Dec 7.

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

Background: The Secretor (FUT2) and lewis gene (FUT3) are in charge of the construction of histo-blood group antigens, which act as a receptor for some Pathogenes. This study aimed to estimate the prevalence of five significant single nucleotide polymorphisms (SNPs) in Iranian children.

Methods: In this cross-sectional study, 102 blood samples collected from hospitalized children. The FUT2 gene region was amplified and sequenced to explore rs1047781 and rs601338, and the FUT3 gene region was amplified to explore rs28362459, rs812936, rs778986 SNPs.

Results: In FUT2 gene, Se358,428 that produces Se phenotype with 63% (0.53 - 0.72) prevalence, was the most common genotype. For FUT3 gene Le59,202,314 with 80% prevalence was most common genotype (0.71 - 0.87).

Conclusion: This study genotyped Secretor and Lewis genes and designated SNPs' distinct distribution in Iran, and clarified at-risk groups for certain diseases.
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http://dx.doi.org/10.1016/j.tracli.2020.12.001DOI Listing
February 2021

Oncolytic virotherapy: Challenges and solutions.

Curr Probl Cancer 2021 Feb 15;45(1):100639. Epub 2020 Aug 15.

Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran. Electronic address:

Viruses as cancer therapies have attracted attention since the 19th century. Scientists observation that viruses can preferentially lyse cancer cells rather than healthy cells, created the field of oncolytic virology. Like other therapeutic strategies, oncolytic virotherapy has challenges, such as penetration into tumor bulk, anti-viral immune responses, off-target infection, adverse conditions in the tumor microenvironment, and the lack of specific predictive and therapeutic biomarkers. Whilst much progress has been made, as highlighted by the first Food and Drug Administration approval of an oncolytic virus talimogene laherparepvec (T-VEC) in 2015, addressing these issues remains a significant hurdle. Here we discuss different types of oncolytic viruses, their application in clinical trials, and finally challenges faced by the field of oncolytic virotherapy and strategies to overcome them.
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http://dx.doi.org/10.1016/j.currproblcancer.2020.100639DOI Listing
February 2021

Expression and Purification of a Bispecific Antibody against CD16 and Hemagglutinin Neuraminidase (HN) in for Cancer Immunotherapy.

Rep Biochem Mol Biol 2020 Apr;9(1):50-57

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

Background: : Immunotherapy of cancer by bispecific antibodies (bsAb) is an attractive approach for retargeting immune effector cells including natural killer (NK) cells to the tumor if the proper expression and purification of the bsAb for such applications could be addressed. Herein, we describe expression of a recombinant bsAb (bsHN-CD16) recognizing NK-CD16 and hemagglutinin neuraminidase (HN) of Newcastle Disease Virus (NDV). This bsAb might be efficient for stimulation of NK cells via coupling to HN on the surface of the NDV-infected tumor cells.

Methods: A bsAb-encoding pcDNA3.1 vector (anti-HN scFv-Fc-anti-CD16 scFv) was used as a template, and the scFv segments (after enzymatic digestion and cutting of the Fc part) were rejoined to construct the Fc-deprived bsAb (anti-HN scFv-anti-CD16 scFv; bsHN-CD16). The constructed bsHN-CD16 was inserted into the and site of the T7 promoter-based pET28a plasmid. Following restriction analyses and DNA sequencing to confirm the cloning steps, bsHN-CD16 encoding pET28a was transformed into the (Rosetta DE3 strain), induced for protein expression by IPTG, and the protein was purified under native condition by Ni/NTA column using imidazole.

Results: Analyses by SDS-PAGE and Western Blotting using Rabbit anti-human whole IgG-HRP conjugate, confirmed the expression and purification of the bsAb with the expected full size of 55 kDa and yields around 8% of the total protein.

Conclusion: Results showed efficient production of the bsAb in for future large-scale purification.
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http://dx.doi.org/10.29252/rbmb.9.1.50DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424422PMC
April 2020

Strategies for enhancing intratumoral spread of oncolytic adenoviruses.

Pharmacol Ther 2020 09 30;213:107586. Epub 2020 May 30.

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

Oncolytic viruses, effectively replicate viruses within malignant cells to lyse them without affecting normal ones, have recently shown great promise in developing therapeutic options for cancer. Adenoviruses (Ads) are one of the candidates in oncolytic virotheraoy due to its easily manipulated genomic DNA and expression of wide rane of its receptors on the various cancers. Although systematic delivery of oncolytic adenoviruses can target both primary and metastatic tumors, there are some drawbacks in the effective systematic delivery of oncolytic adenoviruses, including pre-existing antibodies and liver tropism. To overcome these limitations, intratumural (IT) administration of oncolytic viruses have been proposed. However, IT injection of Ads leaves much of the tumor mass unaffected and Ads are not able to disperse more in the tumor microenvironment (TME). To this end, various strategies have been developed to enhance the IT spread of oncolytic adenoviruses, such as using extracellular matrix degradation enzymes, junction opening peptides, and fusogenic proteins. In the present paper, we reviewed different oncolytic adenoviruses, their application in the clinical trials, and strategies for enhancing their IT spread.
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http://dx.doi.org/10.1016/j.pharmthera.2020.107586DOI Listing
September 2020

Expression analysis data of BCL11A and γ-globin genes in KU812 and KG-1 cell lines after CRISPR/Cas9-mediated BCL11A enhancer deletion.

Data Brief 2020 Feb 11;28:104974. Epub 2019 Dec 11.

Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

The data presented in this article are related to the research article entitled as "Targeted deletion of the BCL11A gene by CRISPR-Cas9 system for fetal hemoglobin reactivation: A promising approach for gene therapy of beta-thalassemia disease " [1]. BCL11A is a master regulator of γ-globin gene silencing, and suppresses fetal hemoglobin expression by association with other γ-globin suppressors, and also interacts with human beta-globin locus control region as well as intergenic region between the Aγ and δ-globin genes to reconfigure beta-globin cluster. Thus, HbF reactivation has been proposed to be an approach for the treatment of β-thalassemia through knockout of BCL11A. Accordingly, an erythroid enhancer sequence was identified that, when inactivated, led to repression of BCL11A and induction of γ-globin in the erythroid lineage [2-7]. This article describes data that obtained from BCL11A gene enhancer modification in KU812 and KG-1 cell lines using the CRISPR-Cas9 genome editing system in order to reactivate γ-globin gene expression.
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http://dx.doi.org/10.1016/j.dib.2019.104974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6933148PMC
February 2020

VP7 and VP4 genotypes of rotaviruses cocirculating in Iran, 2015 to 2017: Comparison with cogent sequences of Rotarix and RotaTeq vaccine strains before their use for universal mass vaccination.

J Med Virol 2020 08 9;92(8):1110-1123. Epub 2019 Dec 9.

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

The present study was conducted to analyze the genotypic diversity of circulating species A rotavirus (RVA) strains in Iran and also to investigate comparative analysis between the genotypes of VP4 and VP7 of cocirculating RVA and vaccine strains before the vaccine is introduced in the national immunization program. The G3-lineage I was found in this study as the most common G genotype which was followed by G9-lineage III, G1-lineages I, II, G12-lineage III, G2-lineage IV, and G4-lineage I. Also, P[8]-lineages III, IV was found as the predominant P genotype which was followed by P[4]-lineage V, and P[6]-lineage I. Overally, G3P[8] was determined as the most common combination. Moreover, the analysis of the VP7 antigenic epitopes showed that several amino acid differences existed between circulating Iranian and the vaccine strains. The comparison of genotype G1 of Iranian and vaccine strains (RotaTeq and Rotarix), and genotypes G2, G3, and G4 of Iranian and RotaTeq vaccine strains revealed three to five amino acids differences on the VP7 antigenic epitopes. Furthermore, analyzing of the VP8* epitopes of Iranian P[8] strains indicated that they contained up to 11 and 14 amino acid differences with Rotarix and RotaTeq, respectively. Based on different patterns of amino acid substitutions in circulating and vaccine strains, the emergence of antibody escaping mutants and potentially the decrease of immune protection might ensue in vaccinated children. However, considering the broad cross-protective activity of RVA vaccines, their efficacy should be monitored after the introduction in Iran.
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http://dx.doi.org/10.1002/jmv.25642DOI Listing
August 2020

Engineered Jurkat Cells for Targeting Prostate-Specific Membrane Antigen on Prostate Cancer Cells by Nanobody-Based Chimeric Antigen Receptor

Iran Biomed J 2020 03 23;24(2):81-8. Epub 2019 Oct 23.

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

Background: Recently, modification of T cells with chimeric antigen receptor (CAR) has been an attractive approach for adoptive immunotherapy of cancers. Typically, CARs contain a single-chain variable domain fragment (scFv). Most often, scfvs are derived from a monoclonal antibody of murine origin and may be a trigger for host immune system that leads to the T-cell clearance. Nanobody is a specific antigen-binding fragment derived from camelid that has great homology to human VH and low immunogenic potential. Therefore, in this study, nanobody was employed instead of scFv in CAR construct.

Methods: In this study, a CAR was constructed based on a nanobody against PSMA (NBPII-CAR). At first, Jurkat cells were electroporated with NBPII-CAR, and then flow cytometry was performed for NBPII-CAR expression. For functional analysis, CAR T cells were co-cultured with prostate cancer cells and analyzed for IL-2 secretion, CD25 expression, and cell proliferation.

Results: Flow cytometry results confirmed the expression of NBPII-CAR on the transfected Jurkat cells. Our data showed the specificity of engineered Jurkat cells against prostate cancer cells by not only increasing the IL-2 cytokine (about 370 pg/ml) but also expressing the T-cell activation marker CD25 (about 30%). In addition, proliferation of engineered Jurkat cells increased nearly 60% when co-cultured with LNCaP (PSMA+), as compared with DU145 (PSMA-).

Conclusion: Here, we describe the ability of nanobody-based CAR to recognize PSMA that leads to the activation of Jurkat cells. This construct might be used as a promising candidate for clinical applications in prostate cancer therapy.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6984713PMC
March 2020

Contribution of Streptokinase-Domains from Groups G and A (SK2a) Streptococci in Amidolytic/Proteolytic Activities and Fibrin-Dependent Plasminogen activation: A Domain-Exchange Study

Iran Biomed J 2020 01 28;24(1):15-23. Epub 2019 Aug 28.

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

Background: Streptokinase (SK), a heterogeneous plasminogen activator (PA) protein from groups A, C, and G streptococci (GAS, GCS, GGS, respectively) contains three structural domains (SKα, SKβ, and SK). Based on the variable region of SKβ, GAS-SK (ska) are clustered as SK1 and SK2 (including cluster2-streptokinase (SK2a)/SK2b), which show low and high fibrinogen (FG)-dependent plasminogen (Plg) activation properties, respectively. Despite being co-clustered as SK2a, GCS/GGS-SK (skcg) variants display properties similar to SK1. Herein, by SKβ exchange between GGS (G88) and GAS-SK2a (STAB902) variants, the potential roles of SK domains in amidolytic/proteolytic activity and FG-bound-Plg activation are represented.

Methods: Two parental SKG88 and SKSTAB902 genes were cloned into the NdeI/XhoI site of pET26b expression vector. The two chimeric SKβ-exchanged constructs (SKC1: αG88-βSTAB-γG88 and SKC2; αSTAB-βG88-γSTAB) were constructed by BstEII/BsiWI digestion/cross-ligation in parental plasmids. SK were expressed in E. coli and purified by nickel-nitriloacetic acid chromatography. PA potencies of SKs were measured by colorimetric assay.

Results: SDS-PAGE and Western-blot analyses confirmed the proper expression of 47-kDa SK. Analyses indicated that the catalytic efficiency (Kcat/Km) for amidolytic and proteolytic activity were less and moderately dependent on SKβ, respectively. The increase of FG-bound-Plg activation for SKSTAB902/SKC1 containing SK2aβ was around six times, whereas for SKG88/SKC2 containing skcgβ, it was four times.

Conclusion: Although SKβ has noticeable contribution in FG-bound-Plg activation activity, it had minor contribution in fibrin-independent, amidolytic activity. These data might be of interest for engineering fibrin-specific versions of SK.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900475PMC
January 2020

Generation of recombinant measles virus containing the wild-type P gene to improve its oncolytic efficiency.

Microb Pathog 2019 Oct 2;135:103631. Epub 2019 Aug 2.

Department of Virology, Pasteur Institute of Iran, Tehran, Iran. Electronic address:

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http://dx.doi.org/10.1016/j.micpath.2019.103631DOI Listing
October 2019

Molecular analysis of human adenoviruses in hospitalized children <5 years old with acute gastroenteritis in Tehran, Iran.

J Med Virol 2019 11 18;91(11):1930-1936. Epub 2019 Jul 18.

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

Human adenoviruses (HAdVs), especially AdV-40 and 41, are common causes of nonbacterial sporadic and outbreak gastroenteritis in children. The present study aimed to describe the frequency and genetic analysis of HAdVs in hospitalized children <5 years old with acute gastroenteritis. A total of 376 stool samples obtained from June 2015 to December 2017 were investigated for the presence of HAdVs by polymerase chain reaction. The HAdV DNA was detected in 16 (4.3%) out of 376 stool samples. Based on the hexon hypervariable region (HVR), B, C, and F HADV species including five types HAdV-1, 2, 3, 6, and 41 were identified, among which enteric AdV species F (EAdV-41) was the most dominant. Moreover, our findings showed the presence of genomic type cluster 1 (GTC1) pattern in Iranian type 41 strains, which was closely similar to the D1 prototype strain (Tak) and D28. In this regard, a recombination was found in AdV-41 strains presenting the hexon sequence that belonged to GTC1, while fiber sequence clustered with GTC2.
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http://dx.doi.org/10.1002/jmv.25539DOI Listing
November 2019

The β-domain of streptokinase affects several functionalities, including specific/proteolytic activity kinetics.

FEBS Open Bio 2019 07 30;9(7):1259-1269. Epub 2019 May 30.

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

Streptokinase (SK) is a plasminogen activator which converts inactive plasminogen (Pg) to active plasmin (Pm), which cleaves fibrin clots. SK secreted by groups A, C, and G Streptococcus (SKA/SKC/SKG) is composed of three domains: SKα, SKβ and SKγ. Previous domain-swapping studies between SK1/SK2b-cluster variants revealed that SKβ plays a major role in the activation of human Pg. Here, we carried out domain-swapping between skcg-SK/SK2-cluster variants to determine the involvement of SKβ in several SK functionalities, including specific/proteolytic activity kinetics, fibrinogen-bound Pg activation and α -antiplasmin resistance. Our results indicate that SKβ has a minor to determining role in these diverse functionalities for skcg-SK and SK2b variants, which might potentially be accompanied by few critical residues acting as hot spots. Our findings enhance our understanding of the roles of SKβ and hot spots in different functional characteristics of SK clusters and may aid in the engineering of fibrin-specific variants of SK for breaking down blood clots with potentially higher efficacy and safety.
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http://dx.doi.org/10.1002/2211-5463.12657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609762PMC
July 2019

Targeted deletion of BCL11A gene by CRISPR-Cas9 system for fetal hemoglobin reactivation: A promising approach for gene therapy of beta thalassemia disease.

Eur J Pharmacol 2019 Jul 27;854:398-405. Epub 2019 Apr 27.

Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran. Electronic address:

Hemoglobinopathies, such as β-thalassemia, and sickle cell disease (SCD) are caused by abnormal structure or reduced production of β-chains and affect millions of people worldwide. Hereditary persistence of fetal hemoglobin (HPFH) is a condition which is naturally occurring and characterized by a considerable elevation of fetal hemoglobin (HbF) in adult red blood cells. Individuals with compound heterozygous β-thalassemia or SCD and HPFH have milder clinical symptoms. So, HbF reactivation has long been sought as an approach to mitigate the clinical symptoms of β-thalassemia and SCD. Using CRISPR-Cas9 genome-editing strategy, we deleted a 200bp genomic region within the human erythroid-specific BCL11A (B-cell lymphoma/leukemia 11A) enhancer in KU-812, KG-1, and K562 cell lines. In our study, deletion of 200bp of BCL11A erythroid enhancer including GATAA motif leads to strong induction of γ-hemoglobin expression in K562 cells, but not in KU-812 and KG-1 cells. Altogether, our findings highlight the therapeutic potential of CRISPR-Cas9 as a precision genome editing tool for treating β-thalassemia. In addition, our data indicate that KU-812 and KG-1 cell lines are not good models for studying HbF reactivation through inactivation of BCL11A silencing pathway.
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http://dx.doi.org/10.1016/j.ejphar.2019.04.042DOI Listing
July 2019

T cell engineered with a novel nanobody-based chimeric antigen receptor against VEGFR2 as a candidate for tumor immunotherapy.

IUBMB Life 2019 09 6;71(9):1259-1267. Epub 2019 Feb 6.

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

Solid tumors that are responsible for more than 85% of cancer death cases need angiogenesis for their growth and metastasis. Among antiangiogenic therapies, targeting the vascular endothelial growth factor receptor 2 (VEGFR2) that is over-expressed on tumor vasculatures has been a promising strategy. In this study, we developed a second generation nanobody (VHH)-based CAR T cell targeting VEGFR2-expressing tumor cells. The CAR T cell was developed by linking the anti-VEGFR2 VHH to a spacer, and signaling domains of CD28 and CD3 ζ. The T cells were activated with anti-CD3 plus rIL-2 and electroporated with a plasmid encoding the CAR construct. The expression of activation markers, CD69 and CD25, on CAR T cells upon coculturing with VEGFR2-expressing cells were 41% and 48%, and the IL-2 and IFN-γ production were 470 pg/mL and 360 pg/mL, respectively. The expression of degranulation marker, CD107a, was 30% and the cytotoxic activity of the CAR T cells reached to more than 30% with E:T ratio of 9:1. The anti-VEGFR2 CAR but not mock T cells mediated specific lysis of 293-KDR cells expressing human VEGFR2 and might be considered as a candidate for adoptive T-cell immunotherapy of solid tumors. © 2019 IUBMB Life, 71(9):1259-1267, 2019.
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http://dx.doi.org/10.1002/iub.2019DOI Listing
September 2019

Construction of a chimeric antigen receptor bearing a nanobody against prostate a specific membrane antigen in prostate cancer.

J Cell Biochem 2019 06 22;120(6):10787-10795. Epub 2019 Jan 22.

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

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is considered to be a novel anticancer therapy. To date, in most cases, single-chain variable fragments (scFvs) of murine origin have been used in CARs. However, this structure has limitations relating to the potential immunogenicity of mouse antigens in humans and the relatively large size of scFvs. For the first time, we used camelid nanobody (VHH) to construct CAR T cells against prostate specific membrane antigen (PSMA). The nanobody against PSMA (NBP) was used to show the feasibility of CAR T cells against prostate cancer cells. T cells were transfected, and then the surface expression of the CAR T cells was confirmed. Then, the functions of VHH-CAR T cell were evaluated upon coculture with prostate cancer cells. At the end, the cytotoxicity potential of NBPII-CAR in T cells was approximated by determining the cell surface expression of CD107a after encountering PSMA. Our data show the specificity of VHH-CAR T cells against PSMA cells (LNCaP), not only by increasing the interleukin 2 (IL-2) cytokine (about 400 pg/mL), but also the expression of CD69 by almost 38%. In addition, VHH-CAR T cells were proliferated by nearly 60% when cocultured with LNCaP, as compared with PSMA negative prostate cancer cell (DU-145), which led to the upregulation of CD107a in T cells upto 31%. These results clearly show the possibility of using VHH-based CAR T cells for targeted immunotherapy, which may be developed to target virtually any tumor-associated antigen for adoptive T-cell immunotherapy of solid tumors.
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http://dx.doi.org/10.1002/jcb.28370DOI Listing
June 2019

Shedding light on the EpCAM: An overview.

J Cell Physiol 2019 08 9;234(8):12569-12580. Epub 2019 Jan 9.

Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran.

The epithelial cell adhesion molecule (EpCAM) is a Type I transmembrane superficial glycoprotein antigen that is expressed on the surface of basolateral membrane of multiple epithelial cells with some exceptions such as epidermal keratinocytes, hepatocytes, thymic cortical epithelial cells, squamous stratified epithelial cells, and myoepithelial cells that do not express the molecule. The molecule plays a pivotal role in the structural integrity, adhesion of the epithelial tissues and their interaction with the underlying layers. EpCAM prevents claudin-7 and claudin-1 molecules from degradation, thereby, decreasing the number of tight junctions and cellular interconnections, and promoting the cells toward carcinogenic transformation. Moreover, the mutations in the EpCAM gene lead to congenital tufting enteropathy, severe intestinal epithelium homeostasis disorders, and Lynch and Lynch syndrome. Overexpression of EpCAM on stem cells of some cancers and the presence of this molecule on circulating tumor cells (CTCs) makes it a promising candidate for cancer diagnosis as well as tracing and isolation of CTCs.
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http://dx.doi.org/10.1002/jcp.28132DOI Listing
August 2019

Oncolytic adenovirus: A tool for cancer therapy in combination with other therapeutic approaches.

J Cell Physiol 2019 06 4;234(6):8636-8646. Epub 2018 Dec 4.

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

Cancer therapy using oncolytic viruses is an emerging area, in which viruses are engineered to selectively propagate in tumor tissues without affecting healthy cells. Because of the advantages that adenoviruses (Ads) have over other viruses, they are more considered. To achieve tumor selectivity, two main modifications on Ads genome have been applied: small deletions and insertion of tissue- or tumor-specific promoters. Despite oncolytic adenoviruses ability in tumor cell lysis and immune responses stimulation, to further increase their antitumor effects, genomic modifications have been carried out including insertion of checkpoint inhibitors and antigenic or immunostimulatory molecules into the adenovirus genome and combination with dendritic cells and chemotherapeutic agents. This study reviews oncolytic adenoviruses structures, their antitumor efficacy in combination with other therapeutic strategies, and finally challenges around this treatment approach.
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http://dx.doi.org/10.1002/jcp.27850DOI Listing
June 2019

A Dual-Type L2 11-88 Peptide from HPV Types 16/18 Formulated in Montanide ISA 720 Induced Strong and Balanced Th1/Th2 Immune Responses, Associated with High Titers of Broad Spectrum Cross-Reactive Antibodies in Vaccinated Mice.

J Immunol Res 2018 3;2018:9464186. Epub 2018 May 3.

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

-derived concatenated, multitype L2-conserved epitopes of human papillomavirus (HPV) L2 protein might represent a less expensive and pan-type vaccine alternative (compared to type-specific HPV L1 virus-like particles), if stable protein expression and strong immunogenicity features could be met. Herein, three dual-type- (DT-) HPV L2 fusion peptides comprising the three head-to-tail tandem repeats (multimers) of either HPV 16 epitope "17-36" or "69-81" or one copy (monomer) of 11-88 fused to the same residues of HPV 18 were constructed and expressed in . SDS-PAGE and Western blot analyses indicated the proper expression and stability of the -derived DT peptides. Mice immunized by formulation of the purified DT peptides and Freund's adjuvant (CFA/IFA) raised neutralizing antibodies (NAbs; the highest for DT: 11-88 peptide) which showed proper cross-reactivity to HPV types: 18, 16, 31, and 45 and efficiently neutralized HPV 18/16 pseudoviruses . Immunization studies in mice by formulation of the DT: 11-88 × 1 peptide with various adjuvants (alum, MF59, and Montanides ISA 720 and 50) indicated that Montanide adjuvants elicited the highest cross-reactive titers of NAbs and similar levels of IgG1 and IgG2a (switching towards balanced Th1/Th2 responses). The results implied development of low-cost -derived DT: 11-88 peptide formulated in human compatible ISA 720 adjuvant as a HPV vaccine.
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http://dx.doi.org/10.1155/2018/9464186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5960516PMC
October 2018

Prevalence and genetic diversity of norovirus genogroup II in children less than 5 years of age with acute gastroenteritis in Tehran, Iran.

Med Microbiol Immunol 2018 Aug 4;207(3-4):201-210. Epub 2018 Apr 4.

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

Viral gastroenteritis is a major public health problem worldwide. In Iran, very limited studies have been performed with regard to the epidemiology of noroviruses. This study aimed to evaluate the prevalence and molecular epidemiology of GII noroviruses in hospitalized children less than 5 years of age with acute gastroenteritis (AGE). A total of 210 stool specimens were collected from Ali Asghar Children's Hospital and Bahrami Children's Hospital in Tehran, from June 2015 to June 2016. The samples were screened by real-time RT-PCR for genogroup II (GII). Positive samples were genotyped by semi-nested PCR followed by Sanger sequencing and phylogenetic analysis. Norovirus was identified in 36 (17.1%) of 210 specimens. Based on genetic analysis of RdRp and capsid sequences, the strains were clustered into eight RdRp-capsid genotypes: GII.P4-GII.4 Sydney_2012 (41.7%), GII.Pe-GII.4 Sydney_2012 (30.6%), GII.P21-GII.3 (13.9%), GII.P16-GII.4 Sydney_2012 (2.8%), GII.P16-GII.12 (2.8%), GII.P2-GII.4 Sydney_2012 (2.8%), GII.P7-GII.7 (2.8%) and GII.P2-GII.2 (2.8%). We determined several different co-circulating norovirus genotypes in children < 5 years of age with AGE in our hospital in Tehran, Iran. Continued molecular surveillance of noroviruses, including typing of both RdRp and capsid genes, is important for monitoring emerging strains in our continued efforts to reduce the overall burden of norovirus disease.
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http://dx.doi.org/10.1007/s00430-018-0541-6DOI Listing
August 2018

Evaluation of autophagy induction on HEV 239 vaccine immune response in a mouse model.

IUBMB Life 2018 03 25;70(3):207-214. Epub 2018 Jan 25.

Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran.

Hepatitis E virus (HEV) infection remains a serious threat to life and productivity in developing world. Vaccine seems to be an effective, safe, and affordable approach to address HEV disease burden. The HEV genome consists of three open reading frames (ORFs). Of these, ORF2 encodes a single structural protein (pORF2) for the HEV capsid which has been studied extensively as vaccine candidates. Recently, it has been recognized that autophagy plays an important role in innate and adaptive immunity defense against intracellular pathogens. This mechanism could therefore promote a protective immune response by inducing CD4 and CD8 T cells. In this study, HEV 239 and Beclin1 proteins were expressed in prokaryotic host cell [Escherichia coli (BL21)]. HEV 239 protein with different formulations (+Alum, +Beclin1, and +Alum-Beclin1) were used as candidate vaccines and administrated subcutaneously in BALB/c mice on 0, 14, and 28 days. Finally, elicited cellular and humoral immunity were evaluated. Taken together, although our results indicated that mice immunized with HEV 239 protein formulated with Alum, Beclin1, and Alum + Beclin1 displayed humoral and cellular response that was not significant in comparison with each other (P > 0.05); whereas they were significant while compared with control groups (P < 0.05). A comprehensive understanding of the intricate interplay between autophagy and immune response remains to be unraveled. Further study will clear the detailed impact of autophagy manipulation to enhance vaccine efficacy and boost the immune responses against the disease. © 2018 IUBMB Life, 70(3):207-214, 2018.
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http://dx.doi.org/10.1002/iub.1719DOI Listing
March 2018

Generation of the Fluorescent HPV16 E7 Protein for Detection of Delivery In vitro.

Protein Pept Lett 2018 ;25(3):244-252

Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.

Background: Immunotherapies targeting the human papillomavirus (HPV) oncogenic proteins, E6 and E7, are effective to treat HPV-associated cervical malignancies.

Objective: The main objective of this study was to generate the fluorescent HPV16 E7 protein for detection of delivery in vitro.

Methods: Two types of the fusion E7-GFP proteins (i.e., with or without linker) were expressed in different E. coli strains. Then, the efficiency of GFP and E7-GFP transfection was compared with FITC-antibody protein control using TurboFect reagent in the HEK-293T cell line.

Results: Our data indicated that both E7-GFP fusion proteins were efficiently produced in M15 E. coli strain, but not in BL21 or Rosetta strains. The E7-GFP fusion showed a clear band of ~ 50 kDa in SDS-PAGE. Moreover, the E7-GFP protein maintained the fluorescent properties only when there was a distance between E7 and GFP genes, suggesting a promising potential to use GFP fusion protein in generating soluble form of protein. This fluorescent property was stable and could be detected in vitro. Moreover, the HEK-293T cells transfected by GFP/TurboFect and E7- GFP/TurboFect complexes demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the GFP fluorescence was stable even at 24 h post-transfection.

Conclusion: Briefly, the E7-GFP fusion protein with linker can be useful for the development of protein vaccines against HPV16 infections and detection in vivo.
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http://dx.doi.org/10.2174/0929866525666180115123620DOI Listing
March 2019

Immunization of Mice by Rotavirus NSP4-VP6 Fusion Protein Elicited Stronger Responses Compared to VP6 Alone.

Viral Immunol 2018 04 29;31(3):233-241. Epub 2017 Nov 29.

2 Virology Department, Pasteur Institute of Iran , Tehran, Iran .

Due to the limitations and safety issues of the two currently approved live attenuated rotavirus (RV) vaccines "RotaTeq and Rotarix," studies on nonreplicating sources of RV vaccines and search for proper RV antigens are actively carried out. The adjuvant activity of NSP4 and highly immunogenic properties of RV VP6 protein prompted us to consider the construction of a NSP4-VP6 fusion protein and to assess the anti-VP6 IgG, IgA, and IgG subclass responses induced by Escherichia coli-derived NSP4-VP6 fusion protein compared to that of VP6 protein with/without formulation in Montanide ISA 50V2 (M50) in BALB/c mice. Results indicated to the proper expression of the fused NSP4-VP6 and VP6 proteins in E. coli. Intraperitoneal immunization by M50 formulated NSP4-VP6 fusion protein (M5+NSP4-VP6) induced the highest titration of VP6-specific IgG and IgA responses compared to the other groups. Indeed, the presence of NSP4 resulted to the induction of stronger humoral immune responses against the fused protein compared to that elicited by administration of VP6 protein alone (with/without M50 formulation), implying the adjuvant properties of NSP4 for the fused protein. Moreover, the "M50+NSP4-VP6" formulation induced higher serum IgG2a titers than IgG1 and increased Interferon-γ levels, despite unchanged interleukin-4 amounts compared to other groups, indicating Th1-oriented responses with a possible role of NSP4. In conclusion, this study further highlights the potentiality of NSP4-VP6 fusion protein as an efficient and cost-effective immunogen in the field of RV vaccine development.
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http://dx.doi.org/10.1089/vim.2017.0075DOI Listing
April 2018

Immunization of mice by a multimeric L2-based linear epitope (17-36) from HPV type 16/18 induced cross reactive neutralizing antibodies.

Res Pharm Sci 2017 Aug;12(4):265-273

Department of Virology, Pasteur Institute of Iran, Tehran, I.R. Iran.

Current licensed and commercially available prophylactic human papillomavirus (HPV) vaccines (Cervarix and quadrivalent/nine valents Gardasil) are based on major capsid protein L1 virus-like particles (VLPs) production which are expensive and type specific. Minor capsid L2-RG1 linear epitope (17-36) is a known candidate for induction of cross-neutralizing antibodies to develop low-cost pan-HPV vaccines. Herein, we report construction and expression of a three tandem repeats of L2-RG1 derived from HPV16 and 18 fused with the same head to tail pattern (HPV16:17-36×3+ HPV18:17-36×3; hereafter termed dual-type fusion L2 peptide) in and provide the results of its immunogenicity in mice. SDS-PAGE and western blot analyses indicated proper expression of the peptide that could be further purified by Ni-NTA affinity chromatography via the located C-terminal 6xHis-tag. Mice immunized by formulation of the purified peptide and Freund adjuvant raised neutralizing antibodies which showed proper cross reactivity to HPV L2 (11-200) of types: 18, 16, 31 and 45 (which totally are responsible for 90% of cervical cancers) and efficiently neutralized HPV18/16 pseudoviruses . Our results imply the possibility of development of a simple, low-cost preventive HPV vaccine based on this dual-type fusion L2 peptide in bacterial expression system with broad spectrum.
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http://dx.doi.org/10.4103/1735-5362.212043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566000PMC
August 2017

Differential Maturation of miR-17 ~ 92 Cluster Members in Human Cancer Cell Lines.

Appl Biochem Biotechnol 2017 Aug 28;182(4):1540-1547. Epub 2017 Feb 28.

Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

While some microRNAs are transcribed from a specific promoter, at least one third of human miRNA genes are clustered, wherein multiple miRNA genes are generated from a single primary transcript such as miR-17 ~ 92 cluster. Although six members of the cluster are generated from a single transcript, the mature level of each member may be diverse in various cell types. Here, we attempt to monitor the mature level of miR-17, miR-92a, and miR-20a from miR-17 ~ 92 cluster in blood (HL60 (human promyelocytic leukemia cells) and Jurkat) and breast (MDA-MB-231 and MCF-7) cancer cell lines. Interestingly, different mature levels of the miRNAs were observed in each cell line. While miR-20 was highly matured in HL60 and MDA-MB-231 cell lines, higher mature level of miR-92a was observed in Jurkat cell line compared to that of miR-20 and miR-17. Further, the mature level of miRNAs was also measured in normal and cancer cell lines. Although the mature level of miR-17 and miR-92a increased in HL60 and Jurkat cell lines, miR-20 expression showed an almost identical level in blood cancer cell lines compared to controls. Conversely, miR-20 mature level significantly increased in breast cancer cell lines whereas the expression level of miR-92a was comparable in MDA-MB-231, MCF-7, and MCF-10A cell lines.
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http://dx.doi.org/10.1007/s12010-017-2416-5DOI Listing
August 2017

Immuno-Stimulating Peptide Derived from HMGB1 is More Effective Than the N-Terminal Domain of Gp96 as an Endogenous Adjuvant for Improvement of Protein Vaccines.

Protein Pept Lett 2017 ;24(3):190-196

Department of Hepatitis and AIDs, Pasteur Institute of Iran, Tehran, Iran.

Up to now, different protein vaccine modalities against human papillomavirus (HPV) have been designed to control cervical cancer. The important issue is to increase their immunogenicity using appropriate adjuvants. Among heat shock proteins (HSPs), glycoprotein 96 (Gp96) and its Nterminal region (NT-gp96) have attracted a specific interest in stimulation of antigen-specific immune responses in vivo. Furthermore, the potency of high mobility group box 1 (HMGB1) protein and its fragment (Hp91) was reported to enhance the desired immune responses against various disorders. In this study, the recombinant (r) HPV16 E7 and rNT-gp96 proteins were generated in bacterial expression system. Mice were vaccinated three times with E7 antigen mixed with Montanide, Hp91, and NT-gp96 as the adjuvant and their preventive and therapeutic efficiencies were evaluated in a murine tumor model. Mice vaccinated with E7 co-delivered by Hp91 peptide induced higher IgG2a and IFN-γ responses in comparison with E7 co-injected with Montanide and NT-gp96 protein suggesting a strong Th1 cellular immune response. The data showed that vaccination with noncovalent rE7 + rNT-gp96 complex delayed the tumor growth as compared to control groups. Mice immunized with rE7 + Montanide and rE7 + Hp91 protected 100% of mice versus 75% survival in groups vaccinated with rE7 + rNT-gp96 after TC-1 tumor challenge. The percentage of tumor free mice was decreased in group immunized with rE7 + rNT-gp96 in therapeutic experiments (~ 50%). These results demonstrated that Hp91 peptide is a safe and strong adjuvant against rNT-gp96 with the potent anti-tumor effects similar to Montanide adjuvant.
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http://dx.doi.org/10.2174/0929866523666161220111833DOI Listing
July 2017

A novel dendritic cell-targeted lentiviral vector, encoding Ag85A-ESAT6 fusion gene of Mycobacterium tuberculosis, could elicit potent cell-mediated immune responses in mice.

Mol Immunol 2016 07 3;75:101-11. Epub 2016 Jun 3.

Department of Virology, Pasteur Institute of Iran, Tehran, Iran. Electronic address:

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), leading to high mortality worldwide. It is well-established that cellular immunity plays a critical role to control Mtb infection. Dendritic Cells (DCs) are potent antigen presenting cells, which play an important role to prime cell-mediated immune responses. In vivo targeting of DCs has been shown to induce both strong cellular immunity and protection against tumor challenges. The aim of the present study was not only to assess the immunizing potential of a novel DC-targeted recombinant lentivirus expressing fusion antigen Ag85A-ESAT6 of Mtb, but also to compare it with a recombinant lentivirus with broad cellular tropism expressing the same antigen in mice. The findings demonstrated that our novel recombinant DC-targeted lentivector was able to successfully transduce and express the fusion antigen Ag85A-E6 in vitro and in vivo. Moreover, a single footpad injection of targeted lentivectors could elicit strong T-helper 1 (Th1) immunity against the above mentioned antigen, as indicated by the specific high-level production of IFN-γ and IL-2 using spleen lymphocytes and lymphoproliferative responses. Despite of these promising results, more attempts are required to elucidate the protective and therapeutic efficacy of this approach in future.
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http://dx.doi.org/10.1016/j.molimm.2016.04.014DOI Listing
July 2016