Stable suppression of gene expression by short interfering RNAs targeted to promoter in a mouse embryonal carcinoma stem cell line

RNA interference (RNAi) can induce gene silencing via two pathways: post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). The mediators of gene inactivation in both pathways are 21-bp small interfering RNAs (siRNAs) generated from longer double-stranded RNA (dsRNA). PTGS involves siRNA-mediated targeting and degradation of mRNA. However, siRNAs induce TGS via DNA methylation at the targeted promoter. Synthetic siRNAs can induce loss of gene activity comparable to long dsRNA. The limitation of this method is that the transfected synthetic siRNA works for only a few days. In this study, we tested the RNAi response to siRNA (PTGS pathway) by using a plasmid containing an enhanced green fluorescent protein (eGFP) gene as a target as well as a plasmid creates siRNA transcript, in a form of a hairpin, against eGFP gene. To investigate TGS pathway via RNAi, we also used a plasmid creates hairpin siRNA transcript against pgk-1 promoter. The data presented here indicated long-lasting inhibition in expression of eGFP and puromycin genes, both under the control of the murine Pgk-1 promoter. However, Southern blot analysis showed no methylation in pgk-1 promoter. © 2010 The Society for In Vitro Biology

Introduction of RNA virus evolution

Lots of viruses, in particular RNA viruses, have high mutation rates and relatively short generation times. Particle stability during infection in nature or in laboratory triggers the evolutionary event toward different mechanisms such as genome segmentation, point mutation and  recombination. The frequency of mutant genomes increase and modify  the previous distribution, which, consequently, lead to emergence of a new infectious particle. Mutation and selection are the most fundamental processes in evolution. High mutation rate of RNA viruses has an important role in viral fitness. Therefore, it increase our understanding about molecular biology of viral infections and their evolution by selection, mutation could reliably  determine our ability to challenge destructive viruses. This review focuses on existing impressions of genetic organization and mechanisms of RNA viruses evolution

Autophagy knock down: as a booster for the replication of viruses in cell culture

Background: Autophagy suppression recently has been known to have a remarkable effect for cellular adjustment and viability in the final stages of cancer. On the other hand, autophagy has the potential effect in preventing many viruses from replication. Beclin1 is the most substantial constituent in autophagy apparatus regulation. This study was intended to investigate the beclin1 siRNA knockdown effect on the extent of activity of the oncolytic vesicular stomatitis virus (VSV) as a model in cell culture. Materials and Methods: In the current study, the cancer cell line , HeLa (cervical squamous cancer cell line ) was infected by VSV, followed by beclin1 siRNA vector transfection. The potential change in the expressions of gene beclin1 in transfected cells, as well as untransfected ones were examined by real time PCR, and also the titer of viruses was compared in cells with and without transfection.Results: The results revealed that the amount of putative gene beclin1 expression in HeLa cells decreased greatly due to siRNA suppressive impact, and also the sensitivity of the cells to VSV oncolytic effect increased upon decrease in beclin1gene expression.Conclusion: It seems that autophagy suppression by using siRNA with VSV is a substantial aid for increase in virus titer in cancer cell lines

Construction of pLLO vector encoding truncated form of Listeriolysin O as molecular adjuvant for DNA vaccine studies

Background: The major problem of DNA vaccine is less immunogenicity of them verses other killed or live whole organism vaccines therefore adjuvants for use in this kind vaccines is very necessary. Genetic adjuvants with bacterial sources are an appropriate approach to modulate immune responses to DNA vaccines. Listeria Monocytogenes proteins such as Listeriolysin O (LLO) with CD4 and CD8 epitopes can be as an adjuvant to initiate both innate and adaptive immune responses if the protein cytotoxicity can be eliminated. Herein we constructed a truncated LLO plasmid as genetic adjuvant and tested it in combination with a DNA construct as a model vaccine.Materials and Methods: About 1340bp of the 5' end of whole LLO gene was amplified by PCR on DNA purified from Listeria Monocytogenes. Sequential sub cloning of truncated LLO into the Xho I/EcoRV sites of pcDNA3.1 plasmid, downstream of CMV promoter was done. pLLO plasmid was transfected to HEK293T cell line by lipofection method. LLO protein expression from transiently transfected 293T cell lysates was confirmed by western blotting. Then the adjuvant activity of LLO in BALB/c mice model was analyzed using proliferation test.Results: Double digestion of pLLO plasmid with the enzymes that were applied for cloning led to the isolation of two fragments with expected sizes. The final plasmid was also confirmed following sequencing reactions. Moreover, expression of LLO was evidenced in transfected 293T cells, compared to non-transfected controls. In vivo study was shown, high significant proliferative responses in LLO co-immunization pattern.Conclusion: In the DNA vaccine study, LLO co-administration plasmid could be a suitable genetic adjuvant to enhance cellular immune response of vaccine

In Silico Analysis of Neutralizing Antibody Epitopes on The Hepatitis C Virus Surface Glycoproteins

Objective: Despite of antiviral drugs and successful treatment, an effective vaccine against hepatitis C virus (HCV)infection is still required. Recently, bioinformatic methods same as prediction algorithms, have greatly contributed tothe use of peptides in the design of immunogenic vaccines. Therefore, finding more conserved sites on the surfaceglycoproteins (E1 and E2) of HCV, as major targets to design an effective vaccine against genetically different virusesin each genotype was the goal of the study. Materials and Methods: In this experimental study, 100 entire sequences of E1 and E2 were retrieved from the NCBIwebsite and analyzed in terms of mutations and critical sites by Bioedit 7.7.9, MEGA X software. Furthermore, HCV-1asamples were obtained from some infected people in Iran, and reverse transcriptase-polymerase chain reaction (RTPCR)assay was optimized to amplify their E1 and E2 genes. Moreover, all three-dimensional structures of E1 andE2 downloaded from the PDB database were analyzed by YASARA. In the next step, three interest areas of humoralimmunity in the E2 glycoprotein were evaluated. OSPREY3.0 protein design software was performed to increase theaffinity to neutralizing antibodies in these areas. Results: We found the effective in silico binding affinity of residues in three broadly neutralizing epitopes of E2glycoprotein. First, positions that have substitution capacity were detected in these epitopes. Furthermore, residuesthat have high stability for substitution in these situations were indicated. Then, the mutants with the strongest affinityto neutralize antibodies were predicted. I414M, T416S, I422V, I414M-T416S, and Q412N-I414M-T416S substitutionstheoretically were exhibited as mutants with the best affinity binding. Conclusion: Using an innovative filtration strategy, the residues of E2 epitopes which have the best in silico bindingaffinity to neutralizing antibodies were exhibited and a distinct peptide library platform was designed

Autophagy induction regulates influenza virus replication in a time-dependent manner

Autophagy plays a key role in host defence responses against microbial infections by promoting degradation of pathogens and participating in acquired immunity. The interaction between autophagy and viruses is complex, and this pathway is hijacked by several viruses. Influenza virus (IV) interferes with autophagy through its replication and increases the accumulation of autophagosomes by blocking lysosome fusion. Thus, autophagy could be an effective area for antiviral research.Methodology. In this study, we evaluated the effect of autophagy on IV replication. Two cell lines were transfected with Beclin-1 expression plasmid before (prophylactic approach) and after (therapeutic approach) IV inoculation.Results/Key findings. Beclin-1 overexpression in the cells infected by virus induced autophagy to 26 %. The log10haemagglutinin titre and TCID50 (tissue culture infective dose giving 50 % infection) of replicating virus were measured at 24 and 48 h post-infection. In the prophylactic approach, the virus titre was enhanced significantly at 24 h post-infection (P≤0.01), but it was not significantly different from the control at 48 h post-infection. In contrast, the therapeutic approach of autophagy induction inhibited the virus replication at 24 and 48 h post-infection. Additionally, we showed that inhibition of autophagy using 3-methyladenine reduced viral replication. Conclusion. This study revealed that the virus (H1N1) titre was controlled in a time-dependent manner following autophagy induction in host cells. Manipulation of autophagy during the IV life cycle can be targeted both for antiviral aims and for increasing viral yield for virus production

An Endogenous Immune Adjuvant Released by Necrotic Cells for Enhancement of DNA Vaccine Potency

ABSTRACT Background: Improving vaccine potency in the induction of a strong cell-mediated cytotoxicity can enhance the efficacy of vaccines. Necrotic cells and the supernatant of necrotic tumor cells are attractive adjuvants, on account of their ability to recruit antigen-presenting cells to the site of antigen synthesis as well as its ability to stimulate the maturation of dendritic cells. Objective: To evaluate the utility of supernatant of necrotic tumor cells as a DNA vaccine adjuvant in a murine model. Method: The supernatant of EL4 necrotic cells was co-administered with a DNA vaccine expressing the glycoprotein B of Herpes simplex virus-1 as an antigen model under the control of Cytomegalovirus promoter. C57BL/6 mice were vaccinated three times at two weeks intervals with glycoprotein B DNA vaccine and supernatant of necrotic EL4 cells. Five days after the last immunization, cell cytotoxicity, IFN-γ and IL-4 were evaluated. Results: The obtained data showed that the production of IFN-γ from the splenocytes after antigenic stimulation in the presence of the supernatant of necrotic EL4 cells was significantly higher than the other groups (p<0.002). The flow cytometry results showed a significant increase in the apoptosis/necrosis of EL4 cells in the mice immunized with DNA vaccine and supernatant of necrotic EL4 cells comparing to the other groups (p<0.001). Conclusion: The supernatant of necrotic cells contains adjuvant properties that can be considered as a candidate for tumor vaccination

Induction of protective anti-CTL epitope responses against HER-2-positive breast cancer based on multivalent T7 phage nanoparticles.

We report here the development of multivalent T7 bacteriophage nanoparticles displaying an immunodominant H-2k(d)-restricted CTL epitope derived from the rat HER2/neu oncoprotein. The immunotherapeutic potential of the chimeric T7 nanoparticles as anti-cancer vaccine was investigated in BALB/c mice in an implantable breast tumor model. The results showed that T7 phage nanoparticles confer a high immunogenicity to the HER-2-derived minimal CTL epitope, as shown by inducing robust CTL responses. Furthermore, the chimeric nanoparticles protected mice against HER-2-positive tumor challenge in both prophylactic and therapeutic setting. In conclusion, these results suggest that CTL epitope-carrying T7 phage nanoparticles might be a promising approach for development of T cell epitope-based cancer vaccines

Investigation In Vitro Expression of CatSper Sub Fragment followed by Production of Polyclonal Antibody: Potential Candidate for The Next Generation of Non Hormonal Contraceptive

Objective: CatSper is a voltage-sensitive calcium channel that is specifically expressed in the testis and it has a significant role in sperm performance. CatSper (1-4) ion channel subunit genes, causes sperm cell hyperactivation and male fertility. In this study, we have explored targeting of the extracellular loop as an approach for the generation of antibodies with the potential ability to block the ion channel and applicable method to the next generation of non-hormonal contraceptive.Materials and Methods: In this experimental study, a small extracellular fragment of CatSper1 channel was cloned in pET-32a and pEGFP-N1 plasmids. Then, subsequent methods were performed to evaluate production of antibody: 1) pEGFP-N1/CatSper was used as a DNA vaccine to immunize Balb/c mice, 2) The purified protein of pET-32a/CatSper was used as an antigen in an enzyme-linked immunosorbent assay (ELISA) and western- blot, and 3) The serum of Balb/-c mice was used as an antibody in ELISA and western-blot. The statistical analysis was performed using the Mann Whitney test.Results: The results showed that vaccination of the experimental group with DNA vaccine caused to produce antibody with (p< 0.05) unlike the control group. This antibody extracted from Balb/c serum could recognize the antigen, and it may be used potentially as a male contraception to prevent sperm motility.Conclusion: CatSpers are the promising targets to develop male contraceptive because they are designed highly specific for sperm; although, no antagonists of these channels have been reported in the literature to date. As results showed, this antibody can be used in male for blocking CatSper channel and it has the potential ability to use as a contraceptive