Piroxicam Analogs: Design, Synthesis, Docking Study and Biological Evaluation as Promising Anti-HIV-1 Agents.

OBJECTIVE: In this study, we describe the synthesis, docking study and biological evaluation of 1,2-benzothiazines 1,1-dioxide derivatives. METHODS: Taking the well-known drug, Piroxicam as a lead compound, we designed and synthesized two series of 1,2-benzothiazines 1,1-dioxide derivatives to assay their ability in inhibition of HIV-1 replication in cell culture. RESULTS: Most of the new compounds were active in the cell-based anti-HIV-1 assay with EC50 < 50 µM. Among them, compound 7g was found to be the most active molecule.Docking study using 3OYA pdb code on the most active molecule 7g with EC50 values of 10 µM showed a similar binding mode to the HIV integrase inhibitors. CONCLUSION: Since all the compounds showed no remarkable cytotoxicity (CC50> 500 µM), the designed scaffold is promising structure for the development of new anti-HIV-1 agents.

Design, Synthesis, Docking Study and Biological Evaluation of 4-Hydroxy-2H-benzo[e][1,2]thiazine-3-carboxamide 1,1-dioxide Derivatives as Anti-HIV Agents.

A novel series of benzothiazine-3-carboxamide 1,1-dioxide derivatives by modifying the piroxicam scaffold was designed, synthesized, and evaluated as anti-HIV agents. The 1,2-benzothiazine-3-carboxamide 1,1-dioxide scaffold consists of hydroxy and carboxamide groups as a chelating motif to form an interaction with Mg2+ ions within the integrase active site as a target. Most of the compounds displayed encouraging anti-HIV activity in a cell-based assay. Among them, compounds 13d, 13l and 13m were the most potent with EC50 values ranging from 20-25 µM and SI > 26. Docking study of compounds in integrase active site proposed that the mechanism of action of compounds might be through Mg2+ chelation within integrase active site. The lack of severe cytotoxicity and favorable anti-HIV activity of benzothiazine-3-carboxamide 1,1-dioxide derivatives support further modifications to improve the potency.

Parvovirus 4 in Individuals with Severe Hemophilia A and Matched Control Group.

Background: Hemophilia is a well-known bleeding disorder with worldwide distribution. Replacement therapy, using plasma-derived or recombinant coagulation factors, comprises a gold standard regimen for the treatment. Regardless of the advancements made in viral inactivation methods in the production of plasma-derived coagulation factors, the possibility of transmission of new viral infections remained as a noticeable concern yet. The aim of the current study was to investigate the status of parvovirus 4 (PARV4) in severe hemophilia A, von Willebrand disease (vWD), and healthy control. Materials and Methods: In the current case-control study, 76 patients with hemophilia and vWD and 60 individuals from their family members entered the study. Nested PCR used to determine the presence of PARV4 in study subjects (76 cases). To characterize the PARV4 genotype, positive samples subjected to sequencing and phylogenetic analysis. Results: PARV4 genome detected in 11 (14.47%) patients with bleeding disorders. Among whom, nine patients (14.75%) were with severe hemophilia A and two (13.33%) patients with vWD. Only five healthy controls (8.33%) were positive for PARV4. All PARV4 sequences were found to be genotype 1. Conclusion: PARV4 infection in patients with hemophilia and vWD was higher than the control group. While detection of PARV4 DNA in patients with bleeding disorders may not necessarily reflect a clinical urgency, future investigations are needed to define the clinical significance of PARV4. It seems the detection of the virus immune signature of PARV4 infection, particularly in the context of acute and persistent infections, needs to focus on cellular and tissue targets.

In vitro Anti-HIV-1 Activity of the Recombinant HIV-1 TAT Protein Along With Tenofovir Drug.

BACKGROUND: HIV-1 TAT protein is essential for the regulation of viral genome transcription. The first exon of TAT protein has a fundamental role in the stimulation of the extrinsic and intrinsic apoptosis pathways, but its anti-HIV activity is not clear yet. METHODS: In the current study, we firstly cloned the first exon of the TAT coding sequence in the pET-24a expression vector and then protein expression was done in the Rosetta expression host. Next, the expressed TAT protein was purified by Ni-NTA column under native conditions. After that, the protein yield was determined by Bradford kit and NanoDrop spectrophotometry. Finally, the cytotoxicity effect and anti-Scr-HIV-1 activity of the recombinant TAT protein alone and along with Tenofovir drug were assessed by MTT and ELISA, respectively. RESULTS: The recombinant TAT protein was successfully generated in E. coli, as confirmed by 13.5% SDS-PAGE and western blotting. The protein yield was ~150-200 µg/ml. In addition, the recombinant TAT protein at a certain dose with low toxicity could suppress Scr-HIV replication in the infected HeLa cells (~30%) that was comparable with a low toxic dose of Tenofovir drug (~40%). It was interesting that the recombinant TAT protein could enhance anti-HIV potency of Tenofovir drug up to 66%. CONCLUSION: Generally, a combination of TAT protein and Tenofovir drug could significantly inhibit HIV-1 replication. It will be required to determine their mechanism of action in the next studies.

Novel 4-Oxo-4,10-dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine-3-carboxylic Acid Derivatives as HIV-1 Integrase Inhibitors: Synthesis, Docking Studies, Molecular Dynamics Simulation and Biological Activities.

BACKGROUND: HIV-1 integrase (IN) has been considered as an important target for the development of novel anti-HIV-1 drugs. OBJECTIVE: The aim of this study was to design novel groups of HIV IN inhibitors. METHODS: In this study, we presented a novel series of 4-oxo-4,10-dihydrobenzo[4,5]imidazo[1,2- a]pyrimidine-3-carboxylic acid derivatives by structural modification of N-arylindole ß-diketoacids as a well-known group of IN inhibitors. RESULTS: Based on in-vitro anti-HIV-1 activity in a cell-based assay, compounds 5, 6a and 6k displayed moderate to good inhibitory activity with EC50 values of 4.14, 1.68 and 0.8 µM, respectively. However, integrase inhibition assay showed that most of the analogues did not have significant effects against integrase enzyme except compound 5 with an IC50 value of 45 µM. Our results indicated that compound 6k was the best one among synthesized compounds with an EC50 of 0.8 µM and SI of 175. Docking and molecular dynamics simulation studies were also performed to provide some insights into the probable mechanism of tested compounds. CONCLUSION: These findings suggest that 4-oxo-4,10-dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine-3- carboxylic acid derivatives may consider as promising lead compounds for the development of new anti-HIV-1 drugs.

Design, Synthesis, Molecular Modeling and Anti-HIV Assay of Novel Quinazolinone Incorporated Coumarin Derivatives.

BACKGROUND: The emergence of drug-resistant viral strains has created the need for the development of novel anti-HIV agents with a diverse structure that targets key enzymes in the HIV lifecycle. OBJECTIVE: Considering the pharmacophore of integrase inhibitors, one of the validated targets for anti-HIV therapy, we designed a quinazolinone incorporated coumarin scaffold to affect HIV. METHODS: Coumarin is a beta enol ester and also a well-known drug scaffold. Designed structures were prepared using a one-pot three-component reaction from 3-amino-4-hydroxycoumarin, isatoic anhydride and benzaldehyde derivatives. RESULTS: In vitro anti-HIV and cytotoxicity assay indicated that more than half of the compounds had EC50 values lower than 50 µM. Unsubstituted phenyl derivative showed the highest activity and selectivity with an EC50 value of 5 µM and a therapeutic index of 7. Compounds were docked into the integrase active site to investigate the probable mechanism of action. Accordingly, the hydroxyl moiety of coumarin along with the carbonyl of the quinazolinone ring could function as the metal chelating group. Quinazolinone and phenyl groups interact with side chains of IN residues, as well. CONCLUSION: Here, a novel anti-HIV scaffold is represented for further modification and in-vivo studies.

Novel Benzoxazin-3-one Derivatives: Design, Synthesis, Molecular Modeling, Anti-HIV-1 and Integrase Inhibitory Assay.

INTRODUCTION: Integrase is a validated drug target for anti-HIV-1 therapy. The second generation integrase inhibitors display π-stacking interaction ability with 3'-end nucleotide as a streamlined metal chelating pharmacophore. METHODS: In this study, we introduced benzoxazin-3-one scaffold for integrase inhibitory potential as bioisostere replacement strategy of 2-benzoxazolinone. RESULTS: Molecular modeling studies revealed that amide functionality alongside oxadiazole heteroatoms and sulfur in the second position of oxadiazole ring could mimic the metal chelating pharmacophore. The halobenzyl ring occupies hydrophobic site created by the cytidylate nucleotide (DC-16). CONCLUSION: The most potent and selective compound displayed 110 µM IC50 with a selectivity index of more than 2.

Design, synthesis and evaluation of cytotoxic, antimicrobial, and anti-HIV-1 activities of new 1,2,3,4-tetrahydropyrimidine derivatives.

A series of new 1,2,3,4-tetrahydropyrimidine (THPM) derivatives were designed and synthesized within a one-pot three component Biginelli reaction. The structures of compounds were characterized by FT-IR, 1HNMR, mass spectroscopy, and elemental analysis. All synthesized derivatives were screened for their cytotoxic, antimicrobial, and anti-HIV activities. Due to significant cytotoxic and antimicrobial effects of 1,2,3,4-THPM scaffold, in this study, cytotoxic and antimicrobial activities of synthesized derivatives were evaluated on two cell lines and four bacterial strains. Compounds 4e and 4k showed highest cytotoxic activity against HeLa and MCF-7 cell lines. In addition, 4c and 4d were most active against MCF-7 and HeLa cell lines, respectively. Among the compounds, 4e revealed high antimicrobial activity against four strains. According to the results, 4e possessing m-bromophenyl group at C-4 position of THPM exhibited the highest cytotoxic and antimicrobial effects. Also, all the newly synthesized compounds were evaluated for their anti-HIV-1 assay. Compounds 4l and 4a indicated remarkable anti-HIV-1 activity. It is concluded from cytotoxic, antimicrobial, and anti-HIV-1 activities that the 1,2,3,4-tertahydropyrimidines may serve as hit compounds for development of new anticancer small-molecules.

Anti-viral Effects of Superpositively Charged Mutant of Green Fluorescent Protein.

BACKGROUND: Supercharged GFP proteins were known as effective carriers for delivery of macromolecules into eukaryotic cells as well as fluorescent fusion tags for in vitro and in vivo detection. OBJECTIVE: Herein, anti-viral effects of +36 GFP and its anti-tumor effects were studied in vitro and in vivo, respectively. METHODS: We evaluated anti-HIV, anti-HSV, and anti-HCV effects of +36 GFP in vitro using ELISA, and real time PCR as common techniques for their detection, respectively. Moreover, we assessed the role of +36 GFP for eliciting HPV-related anti-tumor effects in mice due to the lack of HPV replication in vitro. RESULTS: Our data showed that +36 GFP efficiently enter the cells and augment the transfection rate of HPV16E7 antigen, as well. Furthermore, +36 GFP significantly reduced HCV, HIV and HSV replication up to 75%, 49% and 43% in HCV-infected Huh7.5 cells, HIV-infected Hela cells and HSV-infected Vero cells, respectively. On the other hand, mice immunization with +36 GFP complexed with HPV16 E7 antigen (+36GFP + E7) or fused to HPV16 E7 antigen (+36GFP-E7) elicited a higher Th1 cellular immune response with the predominant IgG2a, IgG2b, IFN-γ and Granzyme B levels than those induced by other groups. These regimens protected mice against TC- 1 tumor challenge (~ 67%) compared to E7 protein alone (~ 33%). These data suggested that +36 GFP can act as an anti-viral agent at certain dose due to its high efficiency in cell penetration in vitro and in vivo. CONCLUSION: Generally, +36 GFP targets viral replication in vitro as well as helps to suppress the growth of HPV-related tumors in vivo.

In Vitro Anti-Viral Effects of Small Heat Shock Proteins 20 and 27: A Novel Therapeutic Approach.

BACKGROUND: The protective effects of heat shock proteins (Hsps) were studied in some infectious and non-infectious diseases, but their specificity was slightly known in various disorders. Among Hsps, small Hsps (e.g. Hsp27 and Hsp20) have important roles in protein folding and translocation, and also in immunity. METHODS: In this study, overexpression of Hsp20 and Hsp27 was performed by transfection of the plasmids encoding Hsp20 and Hsp27 (pEGFP-Hsp20 and pEGFP-Hsp27) into Huh7.5, Hela and Vero cells using Lipofectamine along with heat shock. Then, their anti-herpes simplex virus-1 (HSV-1), anti- human immunodeficiency virus-1 (HIV-1) and anti-hepatitis C virus (HCV) effects, as well as cytotoxicity, were evaluated in vitro, for the first time. RESULTS: Our data showed that simultaneous treatment with Lipofectamine and heat shock augmented the rate of transfection and subsequently the expression of Hsps in these cells. Moreover, overexpression of Hsp20 in HCV-infected Huh7.5 cells, HIV-infected Hela cells and HSV-infected Vero cells reduced the replication of HCV, HIV and HSV, respectively. In contrast, overexpression of Hsp27 significantly decreased HSV replication similar to Hsp20, but it did not affect the replication of HIV and HCV. CONCLUSION: Generally, Hsp20 was identified as a novel anti-HCV, anti-HSV and anti-HIV agent, but Hsp27 was efficient in the suppression of HSV infection. These Hsps may act through suppression of virus entry and/ or through interaction with viral proteins. Thus, it is necessary to determine their exact mechanisms in the near future.

Rabies Prophylaxis Strategy in Iran, a Need for an Alternative Strategy: Would the Essen Regimen be Replaced by Zagreb Protocol?

BACKGROUND: Rabies is a fatal zoonotic infectious disease, which can be prevented by prompt post-exposure prophylaxis that could be expensive in countries with a large population. The Essen protocol with injection of 5 single doses of human rabies vaccine on separate days is a well-known rabies prophylaxis schedule. Decreasing the number of vaccine doses and the number of clinical visits due to an effective alternative schedule is strongly needed. The 2-1-1 regimen, known as Zagreb, is one of the best candidates to succeed Essen. METHODS: To evaluate the effectiveness of Zagreb regimen in the Iranian population by using the Purified Vero cell Rabies Vaccine (PVRV), anti-rabies antibody titer was measured in volunteers with second and third exposure through Rapid Fluorescent Focus Inhibition Test (RFFIT) and Enzyme Linked Immunosorbent Assay (ELISA) test and compared with patients, who were treated according to the Essen protocol. RESULTS: In all participants, anti-rabies antibody titer reached the protective level with no suppressive effect of rabies immunoglobulin in patients with third exposure in Zagreb regimen. CONCLUSIONS: Zagreb regimen could be considered a suitable alternative for the Essen protocol.

Optimization of chitosan nanoparticles as an anti-HIV siRNA delivery vehicle.

Chitosan has emerged as a promising polysaccharide for gene/siRNA delivery. However, additional works will be required to modify chitosan nanoparticles. In the present study, chitosan nanoparticles were well modified to introduce anti-HIV siRNA into two mammalian cell lines, macrophage RAW 264.7 and HEK293. We first generated two stable cell lines expressing HIV-1 Tat, and then designed and generated an efficient anti-tat siRNA. The nanoparticles were prepared by using different concentrations of chitosan, polyethylenimine (PEI) and carboxymethyl dextran (CMD) in various formulations and then their physicochemical and biological properties were investigated. The results demonstrated that the combination of chitosan with both CMD and PEI significantly improved both cell viability and siRNA delivery. The modified chitosan nanoparticles (ChNPs) at the N:P ratio of 50 were approximately uniform spheres with sizes ranging from 100 to 150 nm and a positive zeta potential of about +22 mV. In both cell types, the nanoparticles noticeably increased siRNA delivery efficiency with no significant cytotoxicity or apoptosis-inducing effects compared to the control cells. In addition, the nanoparticles significantly reduced the RNA and protein expression of HIV-1 tat in both stable cells. These data show that the nanoparticle formulation could potentially be used in gene therapy, especially against HIV infection.

Design, Synthesis, Molecular Modeling Study and Biological Evaluation of New N'-Arylidene-pyrido [2,3-d]pyrimidine-5-carbohydrazide Derivatives as Anti-HIV-1 Agents.

In an attempt to identify potential new agents that are active against HIV-1, a series of novel pyridopyrimidine-5-carbohydrazide derivatives featuring a substituted benzylidene fragment were designed and synthesized based on the general pharmacophore of HIV-1 integrase inhibitors. The cytotoxicity profiles of these compounds showed no significant toxicity to human cells and they exhibited anti-HIV-1 activity with EC50 values ranging from 90 to 155 µM. Compound 5j bearing 4-methylbenzylidene group was found to be the most active compound with EC50 = 90 µM and selectivity index, CC50/EC50 = 6.4. Molecular modeling studies indicated the capacity of compound 5j to interact with two Mg2+ cations and several residues that are important in HIV-1 integrase inhibition. These findings suggested that pyridopyrimidine-5-carbohydrazide scaffold might become a promising template for development of novel anti-HIV-1 agents.

Design, Synthesis, Molecular Modeling Studies and Biological Evaluation of N'-Arylidene-6-(benzyloxy)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide Derivatives as Novel Anti-HCV Agents.

HCV-induced hepatitis is one of the most debilitating diseases. The limited number of anti-HCV drugs and drug-resistance necessitate developing of new scaffolds with different mode of actions. HCV non-structural protein 5B (NS5B) is an attractive target for development of novel inhibitors of HCV replication. In this paper, new N'-arylidene-6-(benzyloxy)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide derivatives were designed based on the pharmacophores of HCV NS5B active site binding inhibitors. Designed compounds were synthesized and evaluated for their inhibitory activities in a cell-based HCV replicon system assay. Among tested compounds, compounds 18 and 20 were found to be the most active (EC50 = 35 and 70 µM, respectively) with good selectivity index (SI > 2) in the corresponding series. Molecular modeling studies showed that the designed compounds are capable of forming key coordination with the two magnesium ions as well as interactions with other key residues at the active site of HCV NS5B.

Long non-coding RNA LY86-AS1 and HCG27_201 expression in type 2 diabetes mellitus.

Long non-coding RNAs (LncRNAs) are non-coding RNAs. The potential roles of lncRNAs in type 2 diabetes mellitus (T2DM) are not well-known. In this study, we aim to assess the expression levels of LY86-AS1 and HCG27_201 in T2DM patients and a healthy control group. We obtained whole blood and serum samples from 100 T2DM and 100 non-diabetic subjects. Peripheral blood mononuclear cells (PBMCs) were extracted from whole blood samples using Ficoll. Total RNA was isolated from PBMCs obtained from patients with type 2 diabetes mellitus and healthy control individuals using TRIzol LS reagent (GeneAll Biotechnology Co., LTD.). Extracted RNA was used to synthesize complementary DNA (cDNA) with a Reverse Transcription Kit (Takara). Real-time was performed with SYBR Green (Takara) and monitored by a Rotor-Gene (Qiagen) system. We performed quantitative PCR analysis of the LY86-AS1 and HCG27_201 lncRNA expression levels in the 200 samples. Here we found that the expression of LY86-AS1 and HCG27_201 were down regulated in the T2DM group compared with the control group. We further identify that the expression of both lncRNAs was negatively correlated with fasting blood sugar (FBS) levels. Receiver operating characteristic (ROC) analysis was used to assess the diagnostic value of LY86-AS1 and HCG27_201 as biomarkers for T2DM. ROC analysis demonstrated that LY86-AS1 with an area under the ROC curve (AUC) of 0.747 (P < 0.0001, sensitivity: 64.6, and specificity: 79.8) might be the potential novel diagnostic biomarkers for T2DM. Lower expression of our two studied long non-coding RNAs LY86-AS1 and HCG27_201 in type 2 diabetes mellitus patients indicates their role in the pathogenesis of T2DM. Furthermore, LY86-AS1 could possibly be used as a diagnostic marker for T2DM.

Downregulation of long non-coding RNAs LINC00523 and LINC00994 in type 2 diabetes in an Iranian cohort.

Long non-coding RNAs (lncRNAs) are a subclass within the non-coding RNA repertoire that have potential roles in type 2 diabetes mellitus (T2DM). However, the biological function and molecular mechanisms of lncRNAs in T2DM remain largely unknown. The purpose of this study is to investigate the association between LINC00523 and LINC00994 expressions and T2DM susceptibility in an Iranian cohort. In this case-control study, we obtained whole blood and serum samples from 100 T2DM patients and 100 healthy subjects. We extracted peripheral blood mononuclear cells (PBMCs) from whole blood samples using Ficoll-Hypaque density-gradient centrifugation. Total RNA was extracted from the PBMC lysates by using the TRIzol-LS reagent (GeneAll). Finally, a quantitative real-time PCR (qPCR) assay was used to detect LINC00523 and LINC00994 lncRNA expression levels in the 200 samples. LINC00523 and LINC00994 expressions significantly decreased in patients with T2DM compared to the healthy participants, with a fold change for LINC00523 of 0.157 and 0.159 for LINC00994. We observed a significant inverse correlation between the expressions of these lncRNAs with FBS. Receiver operating characteristic (ROC) curve analysis revealed that LINC00523 has a higher area under the ROC curve (AUC) of 0.7430 and a lower P-value (P < 0.0001), in addition to a sensitivity of 81.44% and specificity of 61.11%. Therefore, LINC00523 could be considered a potential diagnostic biomarker for T2DM. Decreased expressions of LncRNAs LINC00523 and LINC00994 in T2DM is possibly associated with pathogenicity of T2DM in Iranian population. Moreover, LINC00523 can perhaps be considered as effective diagnostic biomarkers for T2DM.

ZnO Q-dots as a potent therapeutic nanomedicine for in vitro cytotoxicity evaluation of mouth KB44, breast MCF7, colon HT29 and HeLa cancer cell lines, mouse ear swelling tests in vivo and its side effects using the animal model.

Nanoformulations derived from fine porous ZnO quantum dot nanoparticles (QD NPs) can offer strong potential medical applications; especially in cancer therapy. ZnO QD NPs was synthesized by sol-gel hydrothermal process, fast cold quenching and further smart surface functionalization methods to obtain ultrasmall size (1-4 nm) NPs. ZnO nanopolymer, a wetting agent, PEG co-solvent and water/oil emulsion stabilizer were considered in our nanofluid formulation. The resulting nanofluid was characterized by SEM, FTIR, photoluminescence, band gap energy, zeta potential and UV-Vis spectroscopy. The cytotoxic effects on the growth of four cancer cell lines were evaluated by MTT assay. The IC50 (µg/ml) values of 30, 41, 40 and 35 for KB44, MCF-7, HT29 and HeLa cells, respectively, after 48 h of nanoformulation treatment suggested the cytotoxic effect of this nanoformulation on these cell lines in a concentration-dependent manner (p < .05). ZnO nanofluid destroyed cancer cell lines more efficiently than the normal HFF-2 (IC50 = 105 µg/ml). The reduction in cell viability in response to ZnO nanofluid treatment induced apoptosis in the cultured cells. Skin sensitization test plus antibacterial activity were also measured. Side effect tests on 70 white mice in vivo resulted in only 3-4 abnormal situations in hepatic tissue section possibly due to the idiosyncratic drug reactions.

Truncated JFH1 E2 Protein: Is it Reliable to be Used in Diagnostic ELISA Test and as a Protein-Based Vaccine Candidate?

BACKGROUND: HCV E2 glycoprotein is one of the most attractive proteins for designing an effective vaccine. Deletion of hydrophobic carboxyl-terminal region of this protein is necessary for its secretion, especially when it is expressed in E-coli. In this study we expressed this protein in truncated form and evaluated its application in developing an ELISA test and induction of humoral response in immunized mice. OBJECTIVES: The purpose of this study was expression of HCV truncated E2 protein from JFH1 strain in E-coli BL21(DE3) and evaluation of its antigenicity. METHODS: Truncated E2 region from HCV genotype 2a (JFH1) was amplified by PCR and cloned into a pET28a (+) vector and was used to transform the E-coli DH5α strain. The recombinant E2 protein was evaluated both in an ELISA test and induction of humoral immunity in mice. RESULTS: Truncated E2 protein was expressed in BL21(DE3). Its specific antibody was detected in serum samples from HCV infected patients. Also, it could elicit a significant humoral immunity in mice. CONCLUSION: Truncated form of E2 protein which has been expressed in E-coli could be used as an effective antigen both in diagnostic tests such as ELISA and also, as a protein-based vaccine candidate.

Generation of the Fluorescent HMGB1-GFP Fusion Protein in Insect Cells and Evaluation of its Immunogenicity in Two Mice Models.

BACKGROUND: High mobility group box 1 (HMGB1) is a highly conserved protein present in the nuclei and cytoplasm of cells which has an important role as a mediator of inflammation in the extracellular environment. HMGB1 was identified as an innate adjuvant that induces immune responses against soluble antigens in vivo. OBJECTIVE: Our goal is the generation of recombinant HMGB1-GFP fusion protein in insect cells for evaluation of immune responses in mouse model. METHOD: In the current study, we used a baculovirus expression system for insect cells that was based on expression of HMGB1 with target gene (GFP), and purified the recombinant HMGB1- GFP fusion protein. We then demonstrated whether immunogenicity of GFP changes in the presence or absence of recombinant HMGB1 acting as an adjuvant in C57BL/6 and BALB/c mice. RESULTS: Our data showed that HMGB1 had a major influence on antibody immune responses induced by GFP in both animal models. The groups receiving HMGB1-GFP fusion protein showed total IgG and IgG2a responses significantly higher than IgG1 in BALB/c mice. Indeed, a mixed IgG1/IgG2a response was observed with high intensity toward IgG2a. In contrast, C57BL/6 mice immunized by HMGB1-GFP protein elicited the same levels of IgG1 and IgG2a. However, the levels of IgG2a and total IgG against the recombinant GFP (rGFP) in C57BL/6 mice were lower than those in BALB/c mice. CONCLUSION: We concluded that fusion of HMGB1 with GFP was immunologically more effective than GFP alone.

HIV-1 genetic diversity and transmitted drug resistance frequency among Iranian treatment-naive, sexually infected individuals.

In recent years, the patterns of human immunodeficiency virus 1 (HIV-1) transmission in Iran have been changing gradually from drug injection to unprotected sexual contact. This study sought to investigate the phylogenetic trends and characteristics of transmitted drug resistance (TDR) mutations of HIV-1 in a population that is mainly infected through homo/heterosexual contacts. Sixty newly diagnosed antiretroviral-naive individuals with HIV infection living in Tehran were recruited to this survey, and among them, 42 subjects were established to be infected through sexual intercourse. Following amplification and sequencing of the main part of the HIV-1 pol region, phylogenetic and drug-resistance mutation (DRM) analysis was successfully performed on these 42 patients. Phylogenetic analysis showed that the majority of the subjects were infected with subtype CRF35_AD (88%), followed by subtype B, with 7.1%, and subtype CRF01_AE, with 4.7%. A total of 7.1% of the subjects were found to be infected with HIV-1 variants with surveillance drug-resistant mutations (SDRMs) according to the last world health organisation (WHO) algorithm. All of the identified SDRMs belonged to the non-nucleoside reverse transcriptase inhibitors (NNRTIs) class, including K103 N and V106A, which were found in three patients. Two minor HIV protease-inhibitor-related mutations (L10I and G73S) were detected in two patients, but these mutations are not included in the WHO SDRMs list. The dominance of HIV-1 subtype CRF35_AD was observed among subjects of this study who were infected through sexual contact. The moderate prevalence of SDRMs (7.1%) in this population emphasises the fact that the risk of treatment failure in HIV-infected individuals might increase in the future, and preventive measures should be considered by health authorities.