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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 to the use of peptides in the design of immunogenic vaccines. Therefore, finding more conserved sites on the surface glycoproteins (E1 and E2) of HCV, as major targets to design an effective vaccine against genetically different viruses in 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 NCBI website and analyzed in terms of mutations and critical sites by Bioedit 7.7.9, MEGA X software. Furthermore, HCV-1a samples 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 and E2 downloaded from the PDB database were analyzed by YASARA. In the next step, three interest areas of humoral immunity in the E2 glycoprotein were evaluated. OSPREY3.0 protein design software was performed to increase the affinity to neutralizing antibodies in these areas. Results: We found the effective in silico binding affinity of residues in three broadly neutralizing epitopes of E2 glycoprotein. First, positions that have substitution capacity were detected in these epitopes. Furthermore, residues that have high stability for substitution in these situations were indicated. Then, the mutants with the strongest affinity to neutralize antibodies were predicted. I414M, T416S, I422V, I414M-T416S, and Q412N-I414M-T416S substitutions theoretically 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 binding affinity to neutralizing antibodies were exhibited and a distinct peptide library platform was designed. [ABSTRACT FROM AUTHOR]

Vertical flow assays have been developed in recent years addressing limitations of the lateral flow assays, including limited multiplexing capability, quantitation, and hook effect. In the present study, the first passive paper-based vertical flow assay is developed for the detection of the nucleic acid target. Horseradish peroxidase was used as an enzymatic tracer with a high potential for signal amplification. In order to achieve the best signal-to-noise ratio, different parameters of paper-based assays were optimized. The sample is heat denatured and hybridized with a specific probe to form a dual-labeled hybridization complex. A small volume of diluted sample, 12 µl, can be analyzed within 6 min on the assay in a sandwich format. Assay specificity was evaluated by testing different unrelated samples, and also, 1.7 nM was obtained as the limit of detection (LOD) using the 0 + 3SD method, which is equivalent to 8.5 fmol of double-stranded DNA in the 12 µl sample volume. The linear range of 3–194 nM with a 0.978 correlation coefficient was obtained according to the calibration curve. The developed assay was evaluated with 45 hepatitis B virus clinical plasma samples, and the result showed 100% consistency of the assay with the real-time PCR benchmark. In the present study, we sought to develop a mere detection system for nucleic acid targets, and to investigate the possibility of using enzyme reporter in a passive vertical flow assay. [ABSTRACT FROM AUTHOR]

Introduction: Cancer immunotherapy is one of the effective treatment methods that provide a better quality of life with limited side effects for patients. Carcinoembryonic Antigen (CEA) can be an appropriate target for cancer immunotherapy. Methods: A lentivector expressing CEA antigen, pCDH-CEA, was constructed by cloning CEA cDNA downstream of the CMV promoter. The constructed plasmid was co-transfected with helper plasmids, into Lenti-X 293 T cells. The lentivector-containing supernatant was collected. Titers of the CEA- lentivector were estimated using the RT-PCR method. The CT26 cells were then infected by CEA- lentivector. Puromycin as a selective antibiotic was added to the culture for 2 weeks to select CEA-positive cells. The ability to produce tumors in BALB/c mice was investigated. Results: The results showed that CEA expressing lentivector plasmids and the two other helper plasmids could be transfected into Lenti-X 293T cells efficiently and packaged successfully as a pseudo-lentivector. The detection of CEA mRNA and protein expression in the 6th and 14th passages of CT26-CEA cells was confirmed in the engineered stable cell line. Tumor formation was confirmed in cell inoculated mice. Conclusion: CT26-CEA cell line with stable expression of CEA can be used as a suitable tumor model to facilitate research on colorectal cancer in vitro and in mice models; therefore, it could be served as a valuable tool for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Introduction: Cancer immunotherapy is one of the effective treatment methods that provide a better quality of life with limited side effects for patients. Carcinoembryonic Antigen (CEA) can be an appropriate target for cancer immunotherapy. Methods: A lentivector expressing CEA antigen, pCDH-CEA, was constructed by cloning CEA cDNA downstream of the CMV promoter. The constructed plasmid was co-transfected with helper plasmids, into Lenti-X 293 T cells. The lentivector-containing supernatant was collected. Titers of the CEA-lentivector were estimated using the RT-PCR method. The CT26 cells were then infected by CEA-lentivector. Puromycin as a selective antibiotic was added to the culture for 2 weeks to select CEA-positive cells. The ability to produce tumors in BALB/c mice was investigated. Results: The results showed that CEA expressing lentivector plasmids and the two other helper plasmids could be transfected into Lenti-X 293T cells efficiently and packaged successfully as a pseudo-lentivector. The detection of CEA mRNA and protein expression in the 6th and 14th passages of CT26-CEA cells was confirmed in the engineered stable cell line. Tumor formation was confirmed in cell inoculated mice. Conclusion: CT26-CEA cell line with stable expression of CEA can be used as a suitable tumor model to facilitate research on colorectal cancer in vitro and in mice models; therefore, it could be served as a valuable tool for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Alcohol consumption exacerbates the pathogenesis of hepatitis C virus (HCV) infection and aggravates disease consequences in alcohol‐abusing patients. Although the exact reasons by which alcohol consumption affects several cellular pathways in liver cells are not clear, they might be partially attributed to the ability of alcohol to further suppress the innate immunity, modulation of autophagy and also its relationship with reactive oxygen species (ROS) generation. To evaluate these issues, Huh7 cells harboring HCV replicon and Cytochrome p450 (CYP2E1) plasmid were exposed to ethanol and mRNA expression of Beclin-1, interferon-stimulated gene15 (ISG15) genes and HCV NS5B for two different times were relatively quantitated. ROS was determined by flow cytometry. The results showed that alcohol treatment in a short time caused an increase in HCV NS5B and Beclin-1 mRNA and decreased ISG 15 mRNA. Long-lasting alcohol treatment increased ROS production in Huh-7 cells and HCV replication was reduced. In conclusion, acute alcohol treatment might contribute to increase HCV replication by interference in innate immunity and induction of autophagy. Chronic alcohol treatment caused oxidative stress, which disrupts autophagy and thereby increased the rate of Huh7 cell injury. [ABSTRACT FROM AUTHOR]

Interferon lambda was discovered in recent years to be an antiviral agent, and research on different aspects of this antiviral factor in viral infection and investigations of its effectiveness are also progressing. The immunological effects of interferon lambda on different cell populations is not precisely known, which may be due to its use of a heterodimeric receptor consisting of IL-10R2 and IFN-λR1, which are not broadly expressed in all types of cells. In the present study, signaling by interferon lambda and its effect on the expression of hepatitis C virus (HCV) proteins were measured, and the expression pattern of some antiviral proteins and IL-10 levels were investigated in peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from 50 patients with chronic genotype 1a HCV infection and 10 healthy individuals as controls. The PBMCs were treated with various doses of interferon lambda at different times of cultivation. Real-time PCR was used for relative quantification of Mxa, PKR, OAS, ISG15 and HCV core mRNAs. Expression of the NS5A protein was measured by flow cytometry, and IL-10 production was assessed by ELISA. A significant increase in the expression of mRNA encoding antiviral proteins and a decrease in the expression of mRNAs encoding the HCV core protein were observed when cells were treated with interferon lambda in an intermittent manner. The expression of HCV NS5A protein and interleukin 10 levels were also lower than in the control group. It was shown that the maximum antiviral effect of interferon lambda in PBMCs is dependent on the dose and treatment time. [ABSTRACT FROM AUTHOR]

There is a relationship between the life cycle of the hepatitis C virus (HCV) and the synthesis and hemostasis of lipids as well as lipid metabolism and interferon (IFN) regulatory system. This study aimed to examine the effect of fluvastatin and IFN-γ in the expression of mediators involved in lipid metabolism and HCV proliferation in patients with rs12979860 CC polymorphism. Thirteen patients with HCV and five controls with rs1297986CC polymorphism were included in this study. Peripheral blood mononuclear cells (PBMCs) of patients and controls were treated by fluvastatin, IFN-γ or fluvastatin+IFN-γ. Assessment of IL-28B polymorphism, RNA extraction, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed. The mRNA expression of sterol regulatory element-binding protein 1 c (SREBP1c), ATP-binding cassette transporter A1 (ABCA1), diacylglycerol acyltransferase 1 (DGAT1), and HCV core as well as measurement of ABCA1 protein level were evaluated before and after treatment. The results indicated that IFN-γ +fluvastatinacted as an inhibitor in mRNA expression of SREBP1c; while acting as an inducer in the expression of ABCA-1. The results of ABCA1 assay showed a significant increase of this protein after treatment with fluvastatin and IFN-γ compared with untreated cells (p=0.02). Moreover, the mRNA expression of HCV core was suppressed in all experimental groups treated with fluvastatin, IFN-γ or their combination which was more significant after treatment with fluvastatin+IFN-γ (p<0.001). The results of this study demonstrated the significant effect of treatment with fluvastatin+IFN-γ in PBMCs of HCV patients with rs12979860 CC polymorphism. According to the drug resistance of viruses and prevention of virus-induced steatosis in patients with HCV, using regulatory agents of lipid mediators in parallel with current medications could be considered as an effective therapeutic strategy. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is a major health problem all over the world. Among HCV proteins, nonstructural protein 3 (NS3) is one of the most promising target for anti‐HCV therapy and a candidate for vaccine design. DNA vaccine is an efficient approach to stimulate antigen‐specific immunity but the main problem with that is less immunogenic efficiency in comparison with traditional vaccines. Several approaches have been applied to enhance the immunogenicity of DNA. Recently, bacteria‐derived substances are considered as one of the most attractive adjuvants for vaccines, which among them, Listeriolysin O (LLO) of Listeria monocytogenes is a toxin with an extremely immunogenic feature. We investigated detoxified form of LLO gene as genetic adjuvant to modulate NS3 DNA vaccine potency. Immunogenic truncated NS3 gene sequence of HCV (1095‐1380aa) and detoxified LLO gene region (5‐441aa) were amplified by PCR and cloned into the pcDNA3.1 plasmid separately. The expression of recombinant proteins (pc‐NS3, pLLO) was confirmed in HEK293T cell line by western blotting. BALB/c mice models received three doses of different formula of plasmids in two‐week intervals and two weeks after the final immunization, the immune responses were evaluated by specific total antibody level, lymphocyte proliferation, cytotoxicity, and cytokine levels assays. To evaluate in vivo cytotoxic activity, tumor challenge was performed. The recombinant plasmids were successfully expressed in mammalian cell line, and coadministration of pc‐NS3 with pLLO induced the highest titer of total IgG against NS3 antigen compared with other controls. Determination of IgG subclasses confirmed the efficient increase in mixed responses with Th1 dominancy. Furthermore, significant levels of cytokines (p < .05) and lymphocyte proliferation responses (p < .05) indicated the superiority of this regimen. The findings may have important implication for LLO gene application as genetic adjuvant in immune response against HCV. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is a blood-borne pathogen which has chronically infected people worldwide. Therefore, it is of utmost importance to design prophylactic and therapeutic vaccine in order to control HCV infection. To date, several researchers have attempted to improve the efficiency of HCV vaccine by using different adjuvants. However, a few studies have focused on the synthetic immunomodulatory drugs as adjuvants for HCV vaccine. Recently, researchers have shown that lenalidomide, which is used to treat the patients with multiple myeloma, is capable of improving the immune system factors. In this paper, two doses of lenalidomide along with pcDNA3.1 + NS3 as HCV DNA vaccine were administrated in mice models and the percentage of regulatory T cells (Treg cells) and the cells with PD-1 + expression in spleen of mice model were investigated by flow cytometry method. Additionally, activities of CTL cells and NK cells were evaluated in spleen of prophylactic and therapeutic mice models via LDH method. Results of the Treg and PD-1 analysis showed that low dose of lenalidomide along with pcDNA3.1 + NS3 can noticeably decrease the percentage of Treg cells and the cells with PD-1 + expression, while lenalidomide can significantly increase the CTL and NK activity in mice models. Also, results of the therapeutic mice model, in which SP2/0 cells- challenged mice were treated with 5 mg/kg lenalidomide in combination with pcDNA3.1 + NS3, reasonably agreed with those of the prophylactic model. Finally, it was found that lenalidomide can reduce the level of Treg cells which results in lower the cells with PD-1 + expression and subsequently higher CTL and NK cell activities. This study concluded that lenalidomide possess the characteristics of an ideal adjuvant candidate for use in combination with HCV DNA vaccine in order to promote the immune response and vaccine efficiency. [ABSTRACT FROM AUTHOR]

Introduction: Rabies is an acute viral disease that causes encephalomyelitis in mammals and human. The only way to prevent this disease is through vaccination before or after exposure. The aim of this study is to evaluate the efficiency of the Pasteur virus (PV) minigenome, using PV strain. Materials and Methods: Enhanced Green Fluorescent Protein (EGFP) sequence was placed between the designed necessary elements (Hammerhead, HDV ribozyme, 3' Leader, and 5' Trailer sequences), which resemble the rabies virus PV strain (PV2061) genome and anti-genome. These constructs were placed between T7 polymerase promoter and T7 polymerase terminator sequences. The accuracy of the minigenome was confirmed by the expression of EGFP using the helper virus in T7-BHK cell line. Results: The viral necessary elements of positive and negative sense strands were evaluated for the ability of EGFP expression in the presence of the helper virus. While the positive strand showed background results, no EGFP background was observed in the negative strand application. Discussion: Establishment of minigenome system does not require advanced biosafety levels. Furthermore, using minigenome system eliminates many potential confounding factors that may be present in coding regions of the genome. Use of the minigenome system is easier and more feasible than the full genome rescue of the virus. This study successfully shows the efficiency of the constructed rabies virus minigenome in expression of inserted gene. [ABSTRACT FROM AUTHOR]

HCV is a global health problem with an estimated 230 million chronically infected people worldwide. It has been reported that a 17-kd protein translated from core-encoding genomic region can contribute to immune-mediated mechanisms associated with the development of the chronic disease. Also, Treg cells can be contributed to an inadequate response against the viruses, leading to chronic infection. Here we evaluated the ability of protein F to modulate the frequency of CD4+CD25+FoxP3+T and IL-10+T cells in patients with chronic HCV infection. F gene was amplified and cloned in the expression vector. The protein was purified and used for stimulation of PBMCs in the HCV chronic patients and the control groups. The frequency of CD4+CD25+FoxP3+ T cell-like populations and IL-10-producing CD4+CD25+ T cells was assessed in the HCV-infected patients and in the healthy controls by flow cytometry, which showed an increase of both CD4+CD25+FoxP3+ T cell-like population and IL-10-producing CD4+CD25+ T cells in the HCV-infected patients positive for anti-F antibody. Our results suggest the potential involvement of F and core antigens in increasing the frequency of CD4+CD25+FoxP3+ T cell-like population and IL-10-producing CD4+CD25+ T cells which may be associated with HCV-persistent infection. [ABSTRACT FROM AUTHOR]

BACKGROUND: The role of different viral proteins in the progression of the disease to cirrhosis is not completely understood. The ARFP/F protein is a newly described protein synthesized from the +1 or -2 reading frames of the core protein gene, which its function remains unknown. The purpose of this study is to detect specific antibodies to HCV-ARF/Core+1 protein in cirrhotic and non-cirrhotic patients with HCV and investigate any possible association. METHODS: ARF/Core+1 recombinant proteins from HCV genotype 1a were expressed in Escherichia coli, and purified. Using an enzyme-linked immunosorbent assay, we assessed the prevalence of anti-ARF/Core+1 antibodies in 50 cirrhotic and 50 non-cirrhotic hepatitis C patients. RESULTS: All 50 cirrhotic patients were positive for anti-ARF/Core+1 antibody, while only 80% positive samples among non-cirrhotic patients were detected. The titer of anti-ARF/Core+1 antibody was also significantly higher in patients with cirrhosis than in non-cirrhotic patients. CONCLUSION: Compared to 80% positive samples among non-cirrhotic patients all 50 cirrhotic patients were positive for anti-ARF/Core+1 antibody and titer of anti-ARF/Core+1 antibody was significantly higher in patients with cirrhosis than in non-cirrhotic. These results suggest that ARF/Core+1 protein is associated with cirrhosis. A possible causative association between ARF/Core+1 and cirrhosis as well as the mechanism of this association needs to be further investigated. [ABSTRACT FROM AUTHOR]

BACKGROUND: The role of different viral proteins in the progression of the disease to cirrhosis is not completely understood. The ARFP/F protein is a newly described protein synthesized from the +1 or -2 reading frames of the core protein gene, which its function remains unknown. The purpose of this study is to detect specific antibodies to HCV-ARF/Core+1 protein in cirrhotic and non-cirrhotic patients with HCV and investigate any possible association. METHODS: ARF/Core+1 recombinant proteins from HCV genotype 1a were expressed in Escherichia coli, and purified. Using an enzyme-linked immunosorbent assay, we assessed the prevalence of anti-ARF/Core+1 antibodies in 50 cirrhotic and 50 non-cirrhotic hepatitis C patients. RESULTS: All 50 cirrhotic patients were positive for anti-ARF/Core+1 antibody, while only 80% positive samples among non-cirrhotic patients were detected. The titer of anti-ARF/Core+1 antibody was also significantly higher in patients with cirrhosis than in non-cirrhotic patients. CONCLUSION: Compared to 80% positive samples among non-cirrhotic patients all 50 cirrhotic patients were positive for anti-ARF/Core+1 antibody and titer of anti-ARF/Core+1 antibody was significantly higher in patients with cirrhosis than in non-cirrhotic. These results suggest that ARF/Core+1 protein is associated with cirrhosis. A possible causative association between ARF/Core+1 and cirrhosis as well as the mechanism of this association needs to be further investigated. [ABSTRACT FROM AUTHOR]

Background and Objectives: Various infectious and non-infectious factors can cause encephalitis in the central nervous system (CNS), the most important of which are viruses. Herpes viruses are one of the most important causes of encephalitis worldwide. PCR detected the virus on the cerebrospinal fluid (CSF) sample. The aim of this study was to set up an in-house PCR to identify herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) and determine the prevalence of these viruses in suspected children of encephalitis. Materials and Methods: This cross-sectional study was conducted on 160 suspected children with encephalitis cases referred to Dr. Kermanshahi Children Hospital, Kermanshah, Iran, from April to March 2021. CSF samples were extracted using a viral extraction kit, and a PCR was performed. The level of glucose and total protein of the samples was measured. Results: The total prevalence of HSV was 16.25%. 17 samples were positive for HSV-1 (10.6%), and 9 samples for HSV-2 (5.6%). There was a significant correlation between glucose, total protein, and HSV PCR positive, but no significant correlation between age and HSV PCR positive results. Conclusion: Rapid diagnosis of a virus may reduce the hospitalization rate and the use of unnecessary therapies and crease mortality, morbidity, and disability in children. Results in this study show that the distribution of HSV types in children with encephalitis predominantly was type 1 compared with type 2. [ABSTRACT FROM AUTHOR]

Objective: Infectious microorganisms are major sources of illness and death worldwide, and the leading cause of death in neonates. Effective vaccination of this age group is of particular importance. The lack of a response and greater susceptibility to tolerance are two major features that limit the effectiveness of vaccines in neonates. In this study we compare the cellular immune response generated following antigen injections at different times of life in newborn mice to that of adult mice. Methods: Adult and different age neonate mice were vaccinated with vesicular stomatitis virus (VSV). One week after the last injection, cellular immunity was assayed on spleen cells that targeted EL4 infected cells using lactate dehydrogenase cytotoxicity assay. Results: Antigen injection induced a decreased immune response in newborn mice compared with mice that had been immunized with subsequent injections. In the adult group, due to the evolution of the immune system, we observed a stronger immune response. Conclusion: Immunization of newborn mice may induce a reduced response when compared to adult vaccinations. However this can be corrected by the administration of additional booster doses. [ABSTRACT FROM AUTHOR]

We report here the development of multivalent T7 bacteriophage nanoparticles displaying an immunodominant H-2kd- 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. [ABSTRACT FROM AUTHOR]

Abstract: Although CD8+ cytotoxic T lymphocyte (CTL) epitope-based DNA vaccination is valuable experience on vaccine research but many attempts are still continued to achieve acceptable protective response. To study the role of full length antigen in CTL epitope immunization, we evaluated cellular immunity of diverse patterns of complete Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) and the immunodominant CTL epitope (498–505) DNA injection in C57BL/6 mice. Optimal immune response was observed in the group immunized with the full length of gB in the first injection and CTL epitope in the second and third vaccination as assessed by lymphocyte proliferation assay (MTT), cytokine assay (ELISA) and CTL assay. B cell and spatially CD4+ T cell epitopes in full length protein might be important for appropriate priming of CTL immune response. These findings may have important implication for the improvement of CTL epitope based DNA vaccine against HSV and other pathogens. [Copyright &y& Elsevier]

Abstract: Although the role of various cytokines on stimulating the immune responses is characterized well, the importance of LIGHT, a member of TNF superfamily, is less clear. In the current study, we administrated LIGHT expression plasmid as an adjuvant to HSV-1 gB DNA vaccine. HSV-1 gB DNA can elicit vigorous humoral and cell mediated immunity in BALB/c mice. LIGHT could potentiate the proliferation of T lymphocytes and induction of T CD8+ cells performing by measuring Granzyme B, a specific marker of CMI immunity and virus neutralization antibody titer. In this study, timing effect of cytokine administration on the resultant immune pattern was evaluated in three different timing groups. The group received LIGHT 3days before DNA vaccine, demonstrated significant increase in cell mediated immunity. So, utilization of an adjuvant to DNA vaccine can significantly influences the induced immune response consequently and this phenomenon could be important to obtain the optimal response in DNA vaccine strategy. Given the growing use of plasmid-based immune adjuvants to improve the immunogenicity and efficacy of DNA vaccines, these findings support the need for further detailed study of this class of agent. [Copyright &y& Elsevier]

Abstract: Many approaches have so far been tried to enhance the immunogenicity of DNA vaccine. These include the use of various factors that induce apoptosis or anti-apoptosis effects when co-delivered with DNA vaccine. In the present study, the effects of pro-apoptotic Bax encoding plasmid (pBax) and anti-apoptotic Bcl-XL encoding plasmid (pBcl-xl), intradermally co-injected with glycoprotein B (gB) of Herpes Simplex Virus (HSV)-1 encoding plasmid (pgB) into the C57BL/6 mice were evaluated. Immune responses of the mice to the antigen were assessed by antibody assay, lymphoproliferative responses as well as cytokine and cytotoxic T-lymphocyte (CTL) assay. Analysis of the humoral and cellular responses showed that the mice immunized with pBax and pgB induced higher levels of antibody and Interleukin-4 as well as stronger lymphocyte proliferative responses and cytotoxic activity compared to those mice received pgB alone. pBcl-xl when intradermally co-injected with pgB showed no significant enhancement in immune responses comparing to pgB. [Copyright &y& Elsevier]

Abstract: Phage display is based on expressing peptides as a fusion to one of the phage coat proteins. To date, many vaccine researches have been conducted to display immunogenic peptides or mimotopes of various pathogens and tumors on the surface of filamentous bacteriophages. In recent years as a new approach to application of phages, recombinant bacteriophage lambda particles were used as DNA delivery vehicles to mammalian cells. In this study, recombinant filamentous phage whole particles were used for vaccination of mice. BALB/c mice were inoculated with filamentous phage particles containing expression cassette of Herpes simplex virus 1 (HSV-1) glycoprotein D that has essential roles in the virus attachment and entry. Both humoral and cellular immune responses were measured in the immunized mice and compared to conventional DNA vaccination. A dose–response relationship was observed in both arms of immune responses induced by recombinant filamentous phage inoculation. The results were similar to those from DNA vaccination. Filamentous phages can be considered as suitable alternative candidate vaccines because of easier and more cost-effective production and purification over plasmid DNA or bacteriophage lambda particles. [Copyright &y& Elsevier]

Activation of the immune system to fight cancer is a major goal in immunology and oncology. Although cancer treatment using oncolytic viruses shows promising results, virus mediated oncolysis induces a weak anti-tumor immune response. Upon application of viruses, immune responses against the virus play a significant role in limiting tumor virotherapy. Although suppression of host immunity increases the efficacy of virotherapy against the tumor, but inhibits anti-tumor immune responses. Induction of viral specific tolerance before viral replication may cause the virus to efficiently replicate in tumor cells without affecting the immune responses against tumor antigens. Investigation of the combined strategy of virotherapy and immunotherapy using irradiated tumor cells along with IL-2 and interferon-alpha in virus specific tolerant mice was the goal of this study. For tolerance induction, the newborn mice were injected with vesicular stomatitis virus (VSV) subcutaneously. After injection of TC-1 tumor cells to adult tolerant mice and formation of a tumor, irradiated TC-1 cells along with IL-2 and Interferon-alpha expression plasmid were injected twice in mice and followed by virotherapy. Size of tumors and CTL activity against the virus and tumor cells were measured. The results showed increased efficacy of virotherapy in combination with immune-stimulators and tumor cells injection in tolerant mice compared to normal mice. Specific tolerance against the oncolytic virus enhances the efficacy of virotherapy both in monotherapy and in combination with immunotherapy. • The combination of virotherapy and immunotherapy synergistically, Enhanced the reduction of tumor size. • Combination of tumor vaccines and virotherapy effectively enhanced CTL activity against the virus and tumor cells. • The reduction of tumor size was associated with invivo specific Tcell activation against the virus and tumor antigens. • The induction of specific viral immune tolerance increases the efficacy of virotherapy against the tumor. [ABSTRACT FROM AUTHOR]

Objective: Hepatitis C virus is a main cause of progressive and chronic liver disease such as hepatitis, cirrhosis and hepatocellular carcinoma which resulted in global health concern. There are 170 million people infecting HCV in the world. Autophagy is a catabolic process 'degrading damaged organelles and cellular components for recycling. There are conflict reports about the role of autophagy in production of cytokines in viral infection. It may depend on the type of virus and the type of infected cells. However, it has been reported that in HCV infected cells, dysfunction of autophagy led to enhancement in some products of native immunity factors such as interferon-α. HCV has been shown to induce autophagy to increase viral growth in vitro. Pharmacological inhibition of autophagic process remarkably inhibited expression level of HCV replicon. In this study we examined the impact of autophagy inhibition by chloroquine'a lysosomal acidification inhibitor'on IFNα mRNA expression and HCV replication in PBMCs. Material and Methods: Immediately after blood collection from HCV positive patients who have chronic HCV genotype1a before treatment the PBMCs were separated from whole blood by centrifugation on a Ficoll-Hypaque density gradient. The same number of normal samples were tested as controls. The cells were cultured in RPMI medium and incubated with 50μgr chloroquine. MTT assay was applied to indicate the dose of chloroquine that has minimum cytotoxicity for cells. After 72h, viral RNA was extracted. The real-time PCR was carried out after a reverse transcription step. The result had been analyzed by REST software. Results: The use of CQ for inhibition of autophagy in infected PBMCs with HCV resulted in a decrease amount HCV mRNA with an increase in IFNα mRNA expression. These results indicated that chloroquine may consider as a complementary new anti-HCV agent that targets the autophagic proteolysis. Conclusion: Inhibition of autophagy may be a new therapeutic strategy for HCV treatment. CQ (chloroquine), inhibits autophagy in infected PBMCs and led to increase of IFNα mRNA expression. This increase has an inhibitory effect on HCV replication. These findings imply that chloroquine effectively impairs the function of autophagy in our experiment. [ABSTRACT FROM AUTHOR]

Background and Objectives: Coronavirus disease 2019 (COVID-19) pandemic has affected most countries in the world. Monitoring the humoral immune responses during the natural course of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection and the duration of them provide useful information for the development of vaccination strategies against this virus and its emerging variants. The importance of the antibody response especially neutralizing antibodies in long-term immunity to SARS-CoV-2 is significant. Materials and Methods: The present study is a cross-sectional study of sero-epidemiological type that has been proposed to compare the persistence of Immunoglobulin G (IgG) against N (nucleocapsid), S (spike) and RBD (receptor-binding domain) proteins in the community after the time of primary disease. A total of 652 serum samples were collected from hospital staff working in COVID wards, as well as a number of community members with different occupations, among those with positive antibody titers, 86 participated in the resampling test before vaccination. Results: There was no association between antibody titer and disease severity (p>0.05). A significant decrease in Ab levels was observed in the paired second samples. The highest rate of decrease was related to anti-N, then anti-RBD and anti-S IgG levels, respectively. There is a significant relationship between the initial antibody titer and its reduction over time (p-value <0.05). Conclusion: Our data revealed that humoral immunity following natural infection of SARS-CoV-2 is detectable for at least 4 months, regardless of disease severity. The most decrease in antibody titer over time was related to anti-N IgG levels. [ABSTRACT FROM AUTHOR]

Background: Suppression of p53 is an important mechanism in Epstein-Barr virus associate-tumors and described as EBNA1-USP7 which is a key axis in p53 suppression. Thus, in this study, we aimed to evaluate the function of EBNA1 on the expression of p53-inhibiting genes including HDAC-1, MDM2, MDM4, Sirt-3, and PSMD10 and the influence of USP7 inhibition using GNE-6776 on p53 at protein/mRNA level. Methods: The electroporation method was used to transfect the BL28 cell line with EBNA1. Cells with stable EBNA1 expression were selected by Hygromycin B treatment. The expression of seven genes, including PSMD10, HDAC-1, USP7, MDM2, P53, Sirt-3, and MDM4, was evaluated using a real-time PCR assay. For evaluating the effects of USP7 inhibition, the cells were treated with GNE-6776; after 24 hours and 4 days, the cells were collected and again expression of interest genes was evaluated. Results: MDM2 (P=0.028), MDM4 (P=0.028), USP7 (P=0.028), and HDAC1 (P=0.015) all showed significantly higher expression in EBNA1-harboring cells compared to control plasmid transfected cells, while p53 mRNA expression was only marginally downregulated in EBNA1 harboring cells (P=0.685). Four-day after treatment, none of the studied genes was significantly changed. Also, in the first 24-hour after treatment, mRNA expression of p53 was downregulated (P=0.685), but after 4 days it was upregulated (P=0.7) insignificantly. Conclusions: It seems that EBNA1 could strongly upregulate p53-inhibiting genes including HDAC1, MDM2, MDM4, and USP7. Moreover, it appears that the effects of USP7 suppression on p53 at protein/mRNA level depend on the cell nature; however, further research is needed. [ABSTRACT FROM AUTHOR]

Background and aim: struct in a prime-boost vaccination regimen, compared to commercial vaccine. Methods: Cloning to pcDNA3.1(+) and expression of rabies virus glycoprotein gene in BSR cell line were performed followed by SDSPAGE and Western blot analysis of the expressed glycoprotein. The resulting genetic construct was used as a DNA vaccine by injecting 80 μg of the plasmid to MNRI mice twice. Prime-Boost vaccination strategy was performed using 80 μg plasmid construct as prime dose and the second dose of an inactivated rabies virus vaccine. Production of rabies virus neutralizing antibody (RVNA) titers of the serum samples were determined by RFFIT. Results: In comparisons between heterologous prime-boost vaccination strategy and DNA vaccinations, the potency of group D that received Prime-Boost vaccine with the second dose of pcDNA3.1(+)-Gp was enhanced significantly compared to the group C which had received pcDNA3.1(+)-Gp as first injection. Conclusion: In this study, RVGP expressing construct was used in a comparative approach between Prime-Boost vaccination strategy and DNA vaccination and compared with the standard method of rabies vaccination. It was concluded that this strategy could lead to induction of acceptable humoral immunity. [ABSTRACT FROM AUTHOR]

Abstract: Previously, we have reported that the injection of an expression vector containing Herpes simplex virus (HSV) Glycoprotein D-1 (gD-1) generated a significant antibody response in mice and protected them against HSV lethal challenge. We tested its potential to induce antibody and cell mediated immune responses in latently infected mice. Positive control group (KOS) and HSV gD-1 vaccinated mice demonstrated protection against a lethal ocularly challenge of 105.5 plaque-forming units (pfu)/eye of wild HSV-1 versus negative control groups. For neutralizing antibody titers, delayed-type hypersensitivity (DTH), lymphocyte proliferation responses, clinical evaluation and survival following lethal challenge, no considerable difference was observed between mice vaccinated with DNA plasmid and those vaccinated with KOS. KOS-vaccinated mice demonstrated the ability to completely prevent latency whereas DNA vaccinated group showed some degree of protection and displayed less latency than negative control groups and had considerably high levels of IFN-γ and strong CTL responses versus negative control groups. It can be concluded that although immunization with the DNA vaccine is more effective in both protecting mice and induction of immune response, however it could not completely block the latent infection in sensory nerves. [Copyright &y& Elsevier]

Objective(s): The rotavirus nonstructural protein 4 (NSP4) is responsible for the increase in cytoplasmic calcium concentration through a phospholipase C-dependent and phospholipase Cindependent pathways in infected cells. It is shown that increasing of intracellular calcium concentration in rotavirus infected cells is associated with the activation of some members of protein kinases family such as calcium/calmodulin-dependent kinase II, which plays a crucial role in replication and pathogenesis of the virus. The aim of this study was to expression bovine rotavirus NSP4 gene in HEK293 cell and evaluation of its biological effect related to activation of calcium/calmodulin-dependent kinase II in cell culture. Materials and Methods: MA104 cells was used as a sensitive cell for propagation of virus and defined as a positive control. The NSP4 gene was amplified and inserted into an expression vector, and introduced as a recombinant plasmid into HEK293T cells. Western blot analysis was performed as a confirmation test for both expression of NSP4 protein and activation of calcium/calmodulin-dependent kinase II. Results: Expression of NSP4 and activated form of calcium/calmodulin-dependent kinase II were demonstrated by western blotting. Conclusion: It was shown that the expression of biologically active full- length NSP4 protein in HEK293T cells may be associated with some biological properties such as calcium calmodulin kinase II activation, which was indicator of rotaviruses replication and pathogenesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: It has been demonstrated that the detection of enteroviruses and mumps virus nucleic acid in cerebrospinal fluid (CSF) specimens improves the management of the patients with aseptic meningitis. To determine the effect of overnight enrichment of mumps and enteroviruses in CSF samples on cell culture for increasing the sensitivity of viral detection, we developed a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR). CSFs were collected from 100 patients younger than 7 years. The samples were tested for the presence of enteroviruses and mumps virus by multiplex RT-PCR method. Negative samples in Multiplex RT-PCR were enriched for viral template RNAs by overnight culture of CSF samples on cells and followed by the optimized Multiplex RT-PCR. Overall, 35% of the CSF samples were positive for enteroviruses, whereas only 1% of CSF samples were positive for both viruses. After enriching in cell culture, 34% of the negative samples showed a positive polymerase chain reaction band for enteroviruses, and a 3% increase was observed for both viruses. The results showed that enrichment of viral template RNAs in cell culture can increase the sensitivity of the multiplex RT-PCR assay and provide a rapid and sensitive method for detection of viral infections. [Copyright &y& Elsevier]

Abstract: The immunomodulatory effects of exogenous opioids on induction of acquired immunity during microbial infection are now well known; however, our knowledge about the relationship between endogenous opioid response and microbial infections is rudimentary. Here, we report the effect of administration of Naloxone (NLX), an opioid receptor antagonist, on induction of acquired immunity during primary herpes simplex virus type 1 (HSV-1) infection. BALB/c mice received NLX, twice daily, 2h before infection with HSV-1 until 7 days after infection. Cell-mediated immunity was assessed by evaluating lymphocyte proliferation, interferon-γ (IFN-γ) production, delayed type hypersensitivity (DTH) and mortality rate after acute HSV-1 challenge. The findings showed that a higher level of cell-mediated immunity was induced in the NLX-treated animals compared to the control group after induction of HSV-1 infection. However, the data indicate similar neutralizing antibody production in NLX-treated animals and control animals. This observation and further studies in this field may lead to the use of NLX as an adjuvant for designing microbial vaccines and adjunctive therapy of viral infections. [Copyright &y& Elsevier]

Abstract: The use of morphine has been demonstrated to increase susceptibility to infections. Herpes simplex virus type 1 (HSV-1) is a highly successful pathogen among immunocompromised individuals. In the present study, due to the importance of HSV vaccination in morphine abusers, the effects of chronic morphine exposure on the host response to a HSV-1 gB DNA-based vaccine have been investigated. The study is addressing an important aspect of vaccine development among the susceptible (immunocompromised) hosts. BALB/c mice were exposed to morphine over 11 days. They were then vaccinated with DNA vaccine or KOS strain as a live vaccine. The findings showed that the morphine-treated animals failed to respond to DNA vaccination evaluated by the anti-HSV gB antibody titer, delayed type hypersensitivity (DTH) and lethal HSV-1 challenge. Under the same conditions, the KOS vaccine showed a reduced Ab titer and DTH response in morphine-treated mice, but could protect mice against the lethal challenge and was safe for vaccination of morphine-treated animals. [Copyright &y& Elsevier]

Herpes simplex virus produces primary and latent infections with periodic recurrency. The prime-boost immunization strategies were studied using a DNA vaccine carrying the full-length glycoprotein D-1 gene and a baculovirus-derived recombinant glycoprotein D, both expressing herpes simplex virus glycoprotein D-1 protein. Immunization with recombinant DNAs encoding antigenic proteins could induce cellular and humoral responses by providing antigen expression in vivo. Higher immune response, however, occurred when the recombinant proteins followed DNA inoculation. While all groups of the immunized mice and positive control group could resist virus challenge, a higher virus neutralizing antibody level was detected in the animals receiving recombinant protein following DNA vaccination. [ABSTRACT FROM AUTHOR]