Pasteur Institute of Iran

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Introduction: Most B-cell malignancies are diagnosed based on morphologic and immunohistochemical criteria. Some cases, however, still present a challenge for the pathologist to discriminate between reactive hyperplasia and neoplastic disorders. Molecular techniques can be used as a helpful diagnostic tool in these cases. In this study, we assessed the value of polymerase chain reaction (PCR) technique in determination of monoclonality of immunoglobulin heavy chain gene rearrangements for the diagnosis of large B-cell non-Hodgkin's lymphoma (NHL) in paraffin embedded tissue samples. Methods: DNA was extracted from paraffin embedded tissues of 44 diffuse large B-cell lymphoma (DLBCL) cases and 20 samples of reactive lymphoid tissues from appendix and tonsils as control. Framework 3 and the joining region (FR3/JH) of the variable segment of the immunoglobulin heavy chain gene were amplified using degenerated primers. PCR products from each sample were analyzed on 8% polyacrylamide gels following AgNO3 staining. Results: Monoclonal rearrangements were identified in 33 of 44 cases (75%) of DLBCL using FR3/JH primers. Monoclonal IgH gene rearrangements were not detected in any of the reactive lymphoid hyperplasic samples. All these control cases showed polyclonal pattern. Conclusion: Through PCR analysis, using degenerated primers, monoclonality was demonstrated in 75% of DLBCL cases. This technique can thus be used as a sensitive, reliable and valuable diagnostic supplement to conventional morphologic examination and immunohistocytochemical evaluation of lymphoproliferative disorders, particularly in cases with restrictions in amount or type of analytic material.

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Diagnosis of infectious diseases remains an important issue in medical science. Identification of biomarkers can be used to predict early infections. Recently, heat shock proteins (HSPs) have been known as the conserved compounds expressed under stress conditions in both prokaryotic and eukaryotic systems. These proteins act as molecular chaperones. Several studies showed the increased levels of HSPs in patients suffering from infectious diseases suggesting the role of HSPs as promising biomarkers. Also, Hsps possess significant roles in antigen presentation, the maturation of dendritic cells and the activation of lymphocytes. Thus, these proteins can be utilized to develop vaccines in bacterial and viral infections. In this mini-review, we will briefly describe the important roles of HSPs in diagnosis and immunity in bacterial and viral infections.

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Neutrophils are part of the first line of immune response and are essential for resistance against a variety of pathogens. They professionally mediate direct killing of pathogens, recruit other phagocytes by specific chemokines, produce cytokines and interact with different immune cells to shape the adaptive response. Leishmania as an obligatory intracellular parasite has evolved to benefit this early innate response to find its way into macrophages, the final host cells. Therefore it is important to reconsider the role of neutrophils for further improvement of the current vaccine status.

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Introduction: Hepatitis A virus (HAV) infection poses a significant public health problem worldwide, especially in developing countries. This study investigated the effect of vaccination policies on the HAV seropositivity of Syrian immigrants and local Turkish people. Methods: The anti-HAV antibodies of 6007 patients, including 5613 (93.4%) Turks and 394 (6.6%) Syrian suspected of HAV infection, were analyzed by the chemiluminescent microparticle immunoassay (CMIA) method. Results: In our study, total anti-HAV positivity was higher in Turkish patients than in Syrian patients in the 0-9 age group, while in the 10-19, 20-29, and 30-39 age groups, the rate was higher in Syrians. Anti-HAV seropositivity was significantly higher in Turkish male patients than female patients. The young adult and adult age groups of Turks were more at risk of HAV infection, i.e., when the disease is symptomatic. Conclusion: Vaccination of young and young adult seronegative Turks and ensuring Syrian children's participation in the routine vaccination program implemented in our country is a requirement for preventing HAV infection.

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Introduction: This study aimed to evaluate rSAG1-PLGA efficacy as a particulate vaccine in conferring protection against Toxoplasma gondii infection in C57BL/6 mice. In light of our previous studies, we studied mice genotype role in eliciting immune responses by rSAG1-PLGA nanoparticles in this study. Methods: Poly (DL-lactide-co-glycolide) (PLGA) nanoparticles loaded by rSAG1 as a subunit vaccine were prepared, and C57BL/6 mice were subcutaneously immunized twice at a 3-week interval by rSAG1-PLGA, soluble rSAG1, blank PLGA, and one group kept unvaccinated. The characteristics of PLGA nanoparticles, the amounts of produced IFN-γ, IL-10, specific anti-ToxoplasmaIgGs, and the conferred protection against infection by T. gondii RH tachyzoite were assessed. Results: rSAG1-PLGA nanoparticles shared a z-average of about 450nm with negative Zeta potential. Compared with the negative control group, the mice vaccinated with rSAG1-PLGA nanoparticles produced significantly higher amounts of IFN-γ, specific anti-T. gondii IgG antibodies and higher titer of IgG2a, which resulted in longer survival times. Conclusion: The efficiency of rSAG1-PLGA nanoparticles in inducing humoral and cellular responses and consequently partial protection against acute toxoplasmosis in C57BL/6 was confirmed.

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The production of recombinant vaccines in green plants is an attractive and promising topic in genetic engineering. However, the stable transformation of green plants is a time-consuming, costly, and labor-intensive practice. Moreover, public concerns about genetically modified plants put another limitation on the development and release of transgenic plant-based recombinant vaccines. These shortcomings were addressed by developing transient gene expression systems that allow researchers to investigate candidate recombinant vaccines quickly without tedious work and high costs. A comprehensive literature review was used to gather relevant information. This approach has received much attention in various recombinant vaccine production platforms, including mammalian cell culture, insect cell culture, yeast expression systems, and, more importantly, in plant hosts. Due to their simplicity and efficiency, transient gene expression systems are now widely used to validate gene constructs and transgene expression within plant tissues. This paper describes the concept of transient gene expression and discusses the significant advantages of this approach for producing recombinant vaccines. Notably, the major types of transient gene expression viz. agroinfiltration, viral-based systems, and application of naked plasmid in plant cell culture are introduced, and some examples illustrate the pros and cons of each system. Our literature review also discusses some practical notes on the successful application of this system to provide a more comprehensive image of transient gene expression applicability in green plants. As a whole, this review contributes to the existing literature by shedding more light on various aspects of transient gene expression that have not been addressed thoroughly yet.

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Introduction: The angiotensin-converting enzyme 2 (ACE2) is the effective primary receptor for SARS-CoV-2. The interaction between ACE2 and the spike protein of the virus is the crucial step for virus entry into the target cells. ACE2 receptor can be blocked by neutralizing antibodies (nAbs) such as CR3022 which targets the virus receptor-binding site. Enhancing the binding affinity between CR3022 and ACE2 would lead to a more efficient blockade of virus entry. Methods:  In this regard, the amino acids with central roles in the binding affinity of CR3022 antibody to spike protein were substituted. The best mutations to increase the affinity of antibodies were also selected based on protein-protein docking and molecular dynamics simulations. Result: The variants 45 (H:30I/G, H:55D/F, H: 103S/Y, L:59T/F, L:98Y/A), 60(H:31T/D, H:55D/E,  H:103S/Y, L:59T/D, L:98Y/F), 67(H:30I/G, H:55D/F, H:103S/Y, L:56 W/L, L:59T/Y, L:61E/G), 69(H:31T/D,  H:55D/F,   H:103S/Y, L:59T/F, L:98Y/A), and 71(H: 31T/D, H:55D/F, H:103S/Y) with respective binding affinities of -167.3, -167.5, -161.6, -173.0, and -169.8 Kcal/mol had higher binding affinities against the RBD of the SARS-CoV2 spike protein compared to the wild-type Ab. Conclusion: The engineered antibodies with higher binding affinities against the target protein can improve specificity and sensitivity. Thus, a more successful blockade of the ACE2 is achieved, resulting in a better therapeutic outcome. In silico studies can pave the way for designing these engineered molecules avoiding the economic and ethical challenges.
 

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Introduction: HIV-1 gp120 V3 GPGR motif has an essential role in viral invasion, cell fusion, and pathogenesis but presents a significant variability that can implicate neutralizing antibodies and antiretroviral drug resistance. Methods: We performed a comprehensive analysis based on 259,288 HIV-1 gp120 amino acid sequences obtained from the Los Alamos National Laboratory (LANL) HIV Sequence Database to infer the global distribution of V3 tetrapeptide motifs. We calculated the frequencies and presented the main variants according to continents and countries. Furthermore, the clinical importance of the most distributed V3 motifs was detailed. Results: Our results showed GPGR and GPGQ as the most commonly found V3 motifs among more than five hundred V3 variant motifs. Motifs with clinical implications are widely distributed around the world. Within the most frequent V3 tetrapeptide motifs set, some variants enable the escape from fusion inhibitor drugs and neutralizing antibodies. Conclusion: Considering that an effective vaccine candidate should elicit broadly neutralizing antibodies while fusion inhibitor drug interaction requires conserved amino acids, the diversity of V3 motifs implicates a great challenge in developing an effective HIV-1 vaccine.

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Introduction: Developing potent therapeutic vaccines against human papillomaviruses (HPVs) is crucial for the effective management of various HPV-associated cancers. DNA-based vaccines are attractive due to their safety, stability, and capacity to elicit a targeted immune response against specific antigens. Heat shock proteins (HSPs) can enhance the efficacy of DNA vaccines when used as adjuvants. In this study, we created a recombinant DNA molecule by fusing the HPV16 e7 gene with either the hspB1 or hsp27 gene and assessed its expression in a eukaryotic cell line. Methods: Initially, we constructed a recombinant eukaryotic expression vector by inserting the hsp27-e7 fusion gene into the pcDNA3.1 (-) vector. The concentration and purity of the sample were evaluated using NanoDrop spectrophotometry. We cultured human embryonic kidney 293T (HEK-293T) cells in RPMI 1640 medium and transfected them with the pcDNA3.1-hsp27-e7 construct using Lipofectamine 2000 transfection reagent. After 48 hours, we assessed the expression of the Hsp27-E7 fusion protein by western blotting using an anti-E7 monoclonal antibody. Results: We successfully subcloned the hsp27-e7 fusion gene into the pcDNA3.1 (-) vector, and enzymatic digestion confirmed a distinct ~975 bp band on an agarose gel. The concentration and purity of the recombinant DNA vector in a 10 mL culture were measured to be 210 ng/µL and 1.86, respectively. Furthermore, the expression of the Hsp27-E7 fusion protein in HEK-293T cells was confirmed by Western blot analysis, which detected a distinct band of approximately 38 kDa. Conclusion: Our in vitro findings demonstrate successful expression of the DNA construct encoding the hsp27-e7 gene, which can be utilized as a DNA vaccine for future in vivo investigations.
 

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Introduction: Antibiotic-resistant Pseudomonas aeruginosa has been designated by the World Health Organization (WHO) as a critical priority pathogen, highlighting the critical need for developing new strategies, particularly prophylactic measures. This research focuses on incorporating highly antigenic elements from essential, surface-exposed outer membrane proteins of P. aeruginosa to design a polypeptide-based subunit vaccine capable of inducing a strong immune response, using immunoinformatics approaches. Methods: Ten essential outer membrane proteins of P. aeruginosa were analyzed using three online servers (ABCpred, BCPREDS, and LBtope) to predict B-cell epitopes and the IEDB server to predict CD8+ and CD4+ T-cell epitopes. The predicted epitopes were then assessed for physicochemical properties, allergenicity, and toxicity using relevant web servers. A vaccine construct incorporating the selected epitopes and an adjuvant was designed, and its 3D structure was modeled to study its interaction with Toll-like receptor-4 (TLR-4). Results: The final vaccine construct consisted of a 478-amino acid polypeptide incorporating 5 CD8+ T-cell, 5 CD4+ T-cell, and 15 B-cell epitopes. In silico analysis predicted the vaccine construct to be immunogenic, non-toxic, non-allergenic, and possess favorable physicochemical properties. Molecular docking simulations predicted strong binding affinity between the vaccine construct and TLR-4, suggesting its potential to elicit a robust immune response. Conclusion: These in silico analyses suggest that the designed subunit vaccine is potentially safe and effective against P. aeruginosa. However, experimental validation is necessary to confirm these predictions.
 

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Introduction: Green fluorescent protein (GFP) and its variants are pivotal in tracking gene expression across various gene delivery systems. While GFP is typically employed for intracellular reporting, it can be modified to display on cell surfaces for labeling. Previous research indicates GFP might have immunogenic effects, notably enhancing tumor-specific T cell responses. This study explores the immunostimulatory differences between enhanced GFP (EGFP) and the supercharged variant, +36 GFP. Methods: Recombinant EGFP and +36 GFP proteins were generated using an Escherichia coli expression system. Murine bone marrow-derived dendritic cells (BMDCs) were generated using established protocols. Splenocytes were isolated from murine spleens via mechanical disruption and red blood cell lysis. The RAW 264.7 macrophage cell line was cultured in complete DMEM medium. Immune cells were then incubated with varying concentrations of EGFP and +36 GFP, separately, for 48 h. Cytokine levels (IFN-γ, TNF-α, IL-10) were quantified using sandwich ELISA.