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

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

Low prevalence of hepatitis B vaccine escape mutants among individuals born after the initiation of a nationwide vaccination program in Iran.

A nationwide hepatitis B virus (HBV) vaccination program for neonates was launched in Iran in 1993. Despite the success of this program, concern about its long-term success still remains, because breakthrough infections due to emergence of surface mutants have been reported in immunized children. We aimed to evaluate the seroprevalence of HBV and vaccine escape mutants among individuals born after the initiation of the nationwide vaccination program in Iran. This study included 1115 participants younger than 23 years old, with 223 in each age cohort. The presence of HBsAg, anti-HBs and anti-HBc was evaluated using an ELISA kit. HBV-DNA levels were measured in anti-HBc and/or HBsAg-positive subjects. PCR products were sequenced and mutations were identified. The overall HBsAg prevalence was 0.27 %. Anti-HBs and anti-HBc positive rates were 48 % and 0.18 %, respectively. Two individuals were positive for anti-HBc, one of whom was also positive for HBsAg, and the other was positive for anti-HBc only. HBV DNA was detected in three out of four anti-HBc-and /or HBsAg-positive subjects. An I195M mutation within the S gene was detected in two of the three HBV-DNA-positive cases. A very low prevalence of HBsAg and isolated anti-HBc were found in this study. The I195M mutation found in the surface gene could have been induced by immune pressure. Although the number of ''vaccine escape'' mutants found in this cohort was low, ongoing surveillance of breakthrough infections and escape mutants is still needed.

Expression of HCV Alternative Reading Frame Protein (Core+1/F) in Baculovirus Expression System and its Evaluation for Assessment of Specific Anti-core+1 Antibody in Iranian HCV Infected Patients.

BACKGROUND: Hepatitis C virus (HCV) genome contains an overlapping reading frame which results in alternative core protein (ARFP). Baculovirus expression system was used as a powerful eukaryotic vector system to express core+1/F protein for the first time. This recombinant core+1/F protein was used to assess the anti-core+1 antibody in anti-HCV drug resistant and sustained virologic response (SVR) patients. METHODS: The core+1 coding sequence from HCV genotype 1 was designed and synthesized in pUC57 vector. It was subcloned into baculovirus donor plasmid pFastBacTM HTA and transposed into baculovirus shuttle vector (bacmid) to transfect Sf9 cells. Recombinant core+1 protein was purified using Ni-NTA agarose under native condition and verified using SDS-PAGE electrophoresis and Western blotting. An enzyme-linked immunosorbent assay (ELISA) was developed using this purified protein to assess anti-core+1 antibody in 28 anti-HCV drug resistant patients and in 34 patients with sustained virologic response (SVR) in comparison with 31 healthy volunteers used as the negative control. RESULTS: Expression of HCV core+1 protein in Sf9 cells was confirmed by using SDS-PAGE and Western blotting. Antibody titer against core+1 protein in anti-HCV drug resistant patients was significantly higher than that in both the healthy volunteers and SVR patients (p < 0.0001). CONCLUSIONS: HCV core+1 protein was expressed successfully in a baculovirus expression system in high yield in order to develop an ELISA to assess the anti-core+1 antibody. Further studies are needed to reveal the potential application of core+1 protein in anti-HCV treatment prognosis.

Physicochemical properties of polymers: An important system to overcome the cell barriers in gene transfection.

Delivery of the macromolecules including DNA, miRNA, and antisense oligonucleotides is typically mediated by carriers due to the large size and negative charge. Different physical (e.g., gene gun or electroporation), and chemical (e.g., cationic polymer or lipid) vectors have been already used to improve the efficiency of gene transfer. Polymer-based DNA delivery systems have attracted special interest, in particular via intravenous injection with many intra- and extracellular barriers. The recent progress has shown that stimuli-responsive polymers entitled as multifunctional nucleic acid vehicles can act to target specific cells. These nonviral carriers are classified by the type of stimulus including reduction potential, pH, and temperature. Generally, the physicochemical characterization of DNA-polymer complexes is critical to enhance the transfection potency via protection of DNA from nuclease digestion, endosomal escape, and nuclear localization. The successful clinical applications will depend on an exact insight of barriers in gene delivery and development of carriers overcoming these barriers. Consequently, improvement of novel cationic polymers with low toxicity and effective for biomedical use has attracted a great attention in gene therapy. This article summarizes the main physicochemical and biological properties of polyplexes describing their gene transfection behavior, in vitro and in vivo. In this line, the relative efficiencies of various cationic polymers are compared.

Pitfalls in screening streptococci for retrieving superior streptokinase (SK) genes: no activity correlation for streptococcal culture supernatant and recombinant SK.

Streptokinase (SK), the heterogeneous protein family secreted by some groups of ß-hemolytic streptococci (ßHS), is a plasminogen activator and well-known drug for thrombolytic therapy. Differences in plasminogen activation property of streptococcal culture supernatants (SCS) have been traditionally used to identify superior producer strains and SK genes (skc) for recombinant SK (rSK) production. However, the role of SK heterogeneity and whether SK activities in SCS correlate with that of their corresponding rSK is a matter of debate. To address these concerns, SCS of nine group C streptococci (GCS) screened among 252 ßHS clinical isolates were compared for plasminogen activation using S-2251 chromogenic assay. The GCS (Streptococcus equisimilis) showing the highest (GCS-S87) and lowest (GCS-S131) activities were selected for PCR-based isolation of skc, cloning and rSK production in Escherichia coli. The 6×His-tagged rSK proteins were purified by NI-NTA chromatography, analyzed by SDS-PAGE and Western blotting and their activities were determined. While SCS of GCS-S87 and GCS-S131 showed different plasminogen activations (95 and 35 %, respectively) compared to that of the reference strain (GCS-9542), but interestingly rSK of all three strains showed close specific activities (1.33, 1.70, and 1.55 × 10(4) IU mg(-1)). Accordingly, SKS87 and SKS131 had more than 90 % sequence identity at the amino acids level compared to SK9542. Therefore, SK heterogeneity by itself may not contribute to the differences in plasminogen activation properties of SCS and evaluation of this activity in SCS might not be a proper assay for screening superior skc.

Reverse Vaccinology and Immunoinformatic Approach for Designing a Bivalent Vaccine Candidate Against Hepatitis A and Hepatitis B Viruses.

Hepatitis A and B are two crucial viral infections that still dramatically affect public health worldwide. Hepatitis A Virus (HAV) is the main cause of acute hepatitis, whereas Hepatitis B Virus (HBV) leads to the chronic form of the disease, possibly cirrhosis or liver failure. Therefore, vaccination has always been considered the most effective preventive method against pathogens. At this moment, we aimed at the immunoinformatic analysis of HAV-Viral Protein 1 (VP1) as the major capsid protein to come up with the most conserved immunogenic truncated protein to be fused by HBV surface antigen (HBs Ag) to achieve a bivalent vaccine against HAV and HBV using an AAY linker. Various computational approaches were employed to predict highly conserved regions and the most immunogenic B-cell and T-cell epitopes of HAV-VP1 capsid protein in both humans and BALB/c. Moreover, the predicted fusion protein was analyzed regarding primary and secondary structures and also homology validation. Afterward, the three-dimensional structure of vaccine constructs docked with various toll-like receptors (TLR) 2, 4 and 7. According to the bioinformatics tools, the region of 99-259 amino acids of VP1 was selected with high immunogenicity and conserved epitopes. T-cell epitope prediction showed that this region contains 32 antigenic peptides for Human leukocyte antigen (HLA) class I and 20 antigenic peptides in terms of HLA class II which are almost fully conserved in the Iranian population. The vaccine design includes 5 linear and 4 conformational B-cell lymphocyte (BCL) epitopes to induce humoral immune responses. The designed VP1-AAY-HBsAg fusion protein has the potency to be constructed and expressed to achieve a bivalent vaccine candidate, especially in the Iranian population. These findings led us to claim that the designed vaccine candidate provides potential pathways for creating an exploratory vaccine against Hepatitis A and Hepatitis B Viruses with high confidence for the identified strains.

Different dendritic cells-based vaccine constructs influence HIV-1 antigen-specific immunological responses and cytokine generation in virion-exposed splenocytes.

In recent years, dendritic cells (DCs)-based vaccines have been developed to combat HIV-1 infection in preclinical and clinical trials. In this study, mice bone marrow cells-derived DCs were pulsed with the recombinant Nef, heat shock protein 27 (Hsp27) and Hsp27-Nef proteins, and also green fluorescent protein (GFP) as a positive control. Then, new platforms of DCs loaded with HIV-1 Nef and Hsp27-Nef proteins (i.e., DC prime/DC boost, DNA prime/DC boost, and DC prime/protein boost) were used to evaluate immune responses in BALB/c mice. Finally, the potency of splenocytes exposed to single-cycle replicable (SCR) HIV-1 virions was investigated to secret cytokines in vitro. Our data indicated that the recombinant Nef (∼30 kDa), Hsp27 (∼27 kDa), GFP (∼27 kDa), and Hsp27-Nef (∼53 kDa) proteins were greatly generated in E. coli. Moreover, the modified DCs with the recombinant proteins were prepared in large scale. The results of mice immunization showed the highest levels of antibodies, cytokines, and Granzyme B in heterologous DC prime/protein boost regimen using Hsp27-Nef antigen (DCHsp27-Nef prime/ protein Hsp27-Nef boost regimen). The levels of IFN-γ and IL-10 cytokines in splenocytes isolated from mice immunized with DCHsp27-Nef prime/ protein Hsp27-Nef boost regimen were higher than those in other regimens after exposure to SCR virions. These findings demonstrated the importance of Hsp27 as an adjuvant and heterologous DC prime/ protein boost regimen in improvement of immune responses. Indeed, DC Hsp27-Nef prime/ protein Hsp27-Nef boost regimen can be utilized as a promising candidate for HIV-1 vaccine development.

A comparative study on the activity and antigenicity of truncated and full-length forms of streptokinase.

Application of streptokinase (SK) as a common and cost-effective thrombolytic drug is limited by its antigenicity and undesired hemorrhagic effects. Prior structural/functional and epitope-mapping studies on SK suggested that removal of 59 N-terminal residues led to its fibrin dependency and identified SK antigenic regions, respectively. Following in silico analyses two truncated SK proteins were designed and compared for their fibrin specificity and antigenicity with the full-length SK. Computer-based modeling was used to predict the effect of vector (pET41a)-born protein tags on the conformation of SK fragments. SK60-386, SK143-386 and full-length SK (1-414) were separately cloned, expressed in BL21 E. coli cells and confirmed by Western-blotting. Functional activity of the purified proteins was evaluated with chromogenic and clot lysis assays and their antigenicity was tested by ELISA assay using rabbit anti-streptokinase antibody. As expected, chromogenic bioassay showed a major activity decline for SK60-386 and SK143-386 (83 and 91 percent, respectively), compared to SK1-414. However, in clot lysis assay, which is a fibrin-dependent pharmacopoeia-approved test, SK60-386 and SK143-386 were respectively 35 and 31 percent more active though lysed the clots slower than full-length SK. Antigenic analysis also indicated significant decrease in ELISA signals obtained for truncated proteins compared to SK1-414 (45 and 28 percent less reactivity for SK143-386 and SK60-386, respectively, p < 0.0001). The results of this study for the first time pointed to SK143-386 and SK60-386, as improved SK derivatives with increased fibrin-selectivity and decreased antigenicity as well as acceptable bioactivity profiles in a pharmacopoeia-based analysis, which deserve more detailed pharmacological studies.

Naturally occurring NS5A and NS5B resistant associated substitutions in HCV and HCV/HIV patients in iranian population.

BACKGROUND: The introduction of direct acting antivirals (DAAs) for hepatitis C virus (HCV) treatment promises shorter treatment duration, higher cure rates and fewer side effects. Naturally, occurring Resistance Associated Substitutions (RASs) are major challenge to the success of the HCV antiviral therapy. AIM: To determine the naturally occurring NS5A and NS5B RASs in Iranian HCV and HCV/human immunodeficiency virus (HIV) patients. METHODS: A total of 209 DAA-naïve chronic HCV patients including 104 HCV mono-infected and 105 HCV/HIV co-infected cases were enrolled. Amplification and Sanger population sequencing of NS5A and NS5B regions of HCV genome were carried out. The amino acid sequence diversity of the NS5A and NS5B regions were analyzed using geno2pheno HCV. RESULTS: NS5A RASs were detected in 25.5% of HCV and 16.9% of HCV/HIV subjects. In HCV cases, clinically relevant RASs were L28M followed by M28Vand Q30H and Y93H/N. In HCV/HIV subjects, clinically relevant RASs were Y93H/N followed by L28M and P58T and M28V/T and Q30R. NS5B RASs were observed in 11.8% of HCV and 5.9% of HCV/HIV subjects. Clinically relevant substitutions were included V321A/I, C316Y, S282R and L159F. The major S282T mutation was not observed. CONCLUSION: The emergence of RASs is a growing issue in the setting of current treatment with DAAs. Although currently, screening of RASs is recommended before specific DAA regimens, it should be consider in patients with therapeutic failure and in the cases of retreatment.

Supercharged green fluorescent protein delivers HPV16E7 DNA and protein into mammalian cells in vitro and in vivo.

Macromolecules including DNA and proteins serve as important human therapeutics but are limited by their general inability to cross cell membranes. Supercharged proteins have been known as potent tools for delivery of macromolecules into mammalian cells. Thus, the use of these delivery systems is important to reduce the human papillomavirus (HPV)-associated malignancies through improvement of vaccine modalities. In this study, we used a supercharged green fluorescent protein (+36 GFP) for delivery of the full-length HPV16 E7 DNA and protein into mammalian cells and evaluated immune responses, and protective/therapeutic effects of different formulations in C57BL/6 tumor mice model. Our results showed that the complexes of E7 DNA/+36 GFP and also E7 protein/+36 GFP form stable nanoparticles through non-covalent binding with an average size of ∼ 200-300 nm. The efficient delivery of E7 DNA or protein by +36 GFP was detected in HEK-293T cell line for 4 h and 24 h post-transfection. Mice immunization with E7 protein/+36 GFP nanoparticles elicited a higher Th1 cellular immune response with the predominant IgG2a and IFN-γ levels than those induced by E7 protein, E7 DNA, E7 DNA/+36 GFP and control groups (p < .05). Moreover, the E7 DNA/+36 GFP and E7 protein/+36 GFP nanoparticles similarly protected mice against TC-1 tumor challenge (∼67%) as compared to E7 DNA and E7 protein (∼33%), respectively. These data suggest that +36 GFP may provide a promising platform to improve protein and DNA delivery in vitro and in vivo.

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.

Comparison of HCV Core and CoreE1E2 Virus-Like Particles Generated by Stably Transfected Leishmania tarentolae for the Stimulation of Th1 Immune Responses in Mice.

BACKGROUND: Virus-like particles (VLPs) could be improved into successful immunogens as well as a potent delivery vehicle, but however, the current expression systems for VLPs production have some limitations. METHOD: Recently, we developed a novel strategy to produce two HCV VLPs containing core or coreE1E2 proteins using stably transfected Leishmania tarentolae promastigotes. Then, BALB/c mice were injected by both viral like particles in different immunization strategies such as homologous DNA-, homologous VLP-, and heterologous DNA/ VLP-based immunizations. RESULTS: TEM microscopy indicated HCV core and HCV coreE1E2 VLP assembly with average size of 30-40 and 40-60 nm after purification, respectively. Our results showed that homologous immunizations with both HCV core or coreE1E2 VLPs significantly induced anti-core or anti- coreE1E2 antibody responses, respectively as well as secretion of IFN-γ cytokine as compared to other strategies. Moreover, DNA-prime/VLP-boost regimens significantly elicited higher levels of IFN-γ and antibody responses in comparison with homologous DNA/DNA regimens. The groups immunized with homologous or heterologous coreE1E2 VLPs showed markedly higher immune responses as compared to groups immunized with core VLP regimens against coreE1E2 protein. CONCLUSION: The crude HCV VLPs generated by Leishmania expression system could elicit a Th1- type response as a promising vaccine candidate against HCV infections.

Small heat shock protein 27: An effective adjuvant for enhancement of HIV-1 Nef antigen-specific immunity.

Novel vaccine modalities have been designed to improve the efficiency of vaccines against HIV infections. In this way, the HIV-1 Nef protein has been known as an attractive antigenic candidate in therapeutic vaccine development. Moreover, the endogenous adjuvants such as heat shock proteins (HSPs) and high mobility group box 1 protein (HMGB1) have been suggested effectively to induce antigen-specific humoral and cellular immune responses. In this study, different Nef DNA and protein constructs were produced in eukaryotic and prokaryotic expression systems, and their immunostimulatory properties were evaluated using small heat shock protein 27 (Hsp27) and the HMGB1-derived peptide (Hp91) in a mouse model. Generally, our results indicated that the Hsp27-Nef fusion DNA or protein could significantly elicit higher humoral and cellular immune responses than Nef DNA or protein, respectively. Analysis of the immune responses demonstrated that the Hsp27-Nef fusion protein, and also the mixture of Nef and Hp91 significantly enhanced the Nef-specific T cell responses. Indeed, these regimens induced high levels of IgG2a and IFN-γ directed toward Th1 responses and also Granzyme B secretion as compared to other immunization strategies. The immunostimulatory properties of Freund's adjuvant were significantly less than Hsp27 and Hp91 peptide in various immunization strategies. These findings showed that the use of Hsp27 and Hp91 in protein strategy could improve HIV-1 Nef-specific B- and T-cell immune responses, and also represent a promising HIV-1 vaccine candidate in future.

Conjugated anionic PEG-citrate G2 dendrimer with multi-epitopic HIV-1 vaccine candidate enhance the cellular immune responses in mice.

Multi-epitope vaccines might cause immunity against multiple antigenic targets. Four immunodominant epitopes of HIV-1 genome were used to construct a polytope vaccine, formulated by dendrimer. Two regimens of polytopes mixture with dendrimer were utilized to immunize BALB/c mice. Adjuvants were also used to boost immune responses. The conjugated polytope could arouse significant cellular immune responses (P < 0.05) and Th1 response showed higher intensity compared to Th2 (P < 0.05). Our study depicted that conjugated dendrimer with multi-epitopic rHIVtop4 would efficiently induce cell-mediated immune responses and might be considered as promising delivery system for vaccines formulation.

The structural HCV genes delivered by MPG cell penetrating peptide are directed to enhance immune responses in mice model.

One of the significant problems in vaccination projects is the lack of an effective vaccine against hepatitis C virus (HCV). The goal of the current study is to evaluate and compare two DNA constructs encoding HCV core and coreE1E2 genes alone or complexed with MPG peptide as a delivery system for stimulation of antibody responses and IFN-γ secretion in Balb/c mice model. Indeed, MPG cell penetrating peptide was used to improve DNA immunization in mice. Our results demonstrated that MPG forms stable non-covalent nanoparticles with pcDNA-core and pcDNA-coreE1E2 at an N/P ratio of 10:1. The in vitro transfection efficiency of core or coreE1E2 DNA using MPG and TurboFect delivery systems was confirmed by western blot analysis. The results indicated the expression of the full-length core (∼21 kDa), and coreE1E2 (∼83 kDa) proteins using an anti-His monoclonal antibody. In addition, the expression of HCV core and coreE1E2 proteins was performed in bacteria and the purified recombinant proteins were injected to mice with Montanide 720 adjuvant. Our data showed that the immunized mice with HCV core and coreE1E2 proteins generated the mixture of sera IgG1 and IgG2a isotypes considerably higher than other groups. Furthermore, DNA constructs encoding core and coreE1E2 complexed with MPG could significantly induce IFN-γ secretion in lower concentrations than the naked core and coreE1E2 DNAs. Taken together, the DNA formulations as well as protein regimens used in this study triggered high-level IFN-γ production in mice, an important feature for the development of Th1 immune responses.

Venom Components of Iranian Scorpion Hemiscorpius lepturus Inhibit the Growth and Replication of Human Immunodeficiency Virus 1 (HIV-1).

BACKGROUND: During the recent years, significant progress has been achieved on development of novel anti-viral drugs. Natural products are assumed as the potential sources of novel anti-viral drugs; therefore, there are some previous studies reporting the anti-viral compounds from venomous animals. Based on the significant value for tracing of non-toxic anti-viral agents from natural resources, this study was aimed to investigate the anti-viral activity of some HPLC purified fractions derived from the venom of Iranian scorpion, Hemiscorpius lepturus, against human immunodeficiency virus 1 (HIV-1) and herpes simplex virus 1 (HSV-1). METHODS: H. Lepturus crude venom was subjected to reverse phase HPLC analysis to determine its active components precisely where four dominant fractions obtained at retention time of 156-160 minutes. The phospholipase A2 and hemolytic activities of the purified fractions were first evaluated. Then the anti-viral activity was measured using single cycle HIV (NL4-3) replication and HSV (KOS) plaque reduction assays. RESULTS: The H. lepturus crude venom inhibited HIV replication by 73% at the concentration of 200 µg/ml, while it did not show significant anti-HSV activity. It also inhibited the cell-free viral particles in a virucidal assay, while it showed no toxicity for the target cells in a proliferation assay. The four HPLC fractions purified from H. lepturus inhibited HIV with IC50 of 20 µg/ml. CONCLUSION: H. lepturus venom contains components with considerable anti-HIV activity insofar as it has virucidal activity that offers a novel therapeutic approach against HIV infection. Our results suggest a promising pilot for anti-HIV drug discovery with H. lepturus scorpion venom.

A Live Vector Expressing HPV16 L1 Generates an Adjuvant-Induced Antibody Response In-vivo.

BACKGROUND: The association between human papillomavirus (HPV) infections and cervical cancer has suggested the design of prophylactic and therapeutic vaccines against genital warts. The HPV capsid has made of two L1 and L2 coat proteins that have produced late in viral infections. Regarding to the recent studies, two commercial prophylactic vaccines have based on L1 viral like particles (VLPs) could strongly induce antibody responses, and protect human body from HPV infections. However, the use of these HPV vaccines has hindered due to their high cost and some limitations. Currently, among various vaccination strategies, live vector-based vaccines have attracted a great attention. OBJECTIVES: Herein, a non-pathogenic strain of the protozoan organism known as Leishmania tarentolae has utilized to induce potent humoral immunity in mice model. MATERIALS AND METHODS: At first, cloning of HPV16 L1 gene into Leishmania expression vector has performed and confirmed by PCR and digestion with restriction enzymes. The promastigotes of Leishmania tarentolae (L.tar) have transfected with linearized DNA construct by electroporation. Protein expression has analyzed by SDS-PAGE and western blotting. Then, the immunogenicity of leishmania expressing L1 protein (L.tar-L1) has assessed in mice model. RESULTS: Our data has indicated that subcutaneous immunization of mice with the recombinant L.tar-L1 has led to enhance the levels of IgG1 and lgG2a in comparison with control groups. Furthermore, there was no significant increase in antibody levels between two and three times of immunizations. CONCLUSIONS: The recombinant live vector was able to induce humoral immunity in mice without need of any adjuvant. However, further studies have required to increase its efficiency.

Design and expression of fusion protein consists of HBsAg and Polyepitope of HCV as an HCV potential vaccine.

BACKGROUND: Hepatitis C virus (HCV) infection is a serious public health threat worldwide. Cellular immune responses, especially cytotoxic T-lymphocytes (CTLs), play a critical role in immune response toward the HCV clearance. Since polytope vaccines have the ability to stimulate the cellular immunity, a recombinant fusion protein was developed in this study. MATERIALS AND METHODS: The designed fusion protein is composed of hepatitis B surface antigen (HBsAg), as an immunocarrier, fused to an HCV polytope sequence. The polytope containing five immunogenic epitopes of HCV was designed to induce specific CTL responses. The construct was cloned into the pET-28a, and its expression was investigated in BL21 (DE3), BL21 pLysS, BL21 pLysE, and BL21 AI Escherichia coli strains using 12% gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Finally, the identity of expressed fusion protein was confirmed by Western blotting using anti-His monoclonal antibody and affinity chromatography was applied to purify the expressed protein. RESULTS: The accuracy of the construct was confirmed by restriction map analysis and sequencing. The transformation of the construct into the BL21 (DE3), pLysS, and pLysE E. coli strains did not lead to any expression. The fusion protein was found to be toxic for E. coli DE3. By applying two steps inhibition, the fusion protein was successfully expressed in BL21 (AI) E. coli strain. CONCLUSION: The HBsAg-polytope fusion protein expressed in this study can be further evaluated for its immunogenicity in animal models.

Efficacy of tenofovir disoproxil fumarate therapy in nucleoside-analogue naive Iranian patients treated for chronic hepatitis B.

BACKGROUND: Tenofovir disoproxil fumarate (TDF) is a new effective treatment option for patients with chronic hepatitis B (CHB). OBJECTIVES: To evaluate TDF efficacy in nucleos(t)ide analogues (NAs)-naive Iranian patients with CHB. PATIENTS AND METHODS: The NA-naive patients received TDF for at least six months. The primary endpoint was the proportion of patients achieving a complete virological response (CVR) during the treatment. Multivariate Cox regression analysis determined predictive factors independently associated with the time to CVR. The secondary endpoints were biochemical and serological responses, frequency of virological breakthrough, genotypic resistance development, safety and tolerability. RESULTS: In all, 93 patients (64.5% hepatitis B e antigen [HBeAg]-negative) were eligible. Of these, 70 patients completed 24 months of treatment. The cumulative CVR rates in HBeAg-negative and HBeAg-positive patients were 87% versus 53% at 24 months, respectively. The multivariate Cox regression model showed only HBeAg positivity at baseline and a high baseline HBV DNA level were independent factors predicting a CVR. No patient achieved hepatitis B surface antigen (HBsAg) and HBeAg loss or seroconversion and no virologic breakthrough occurred. A new amino acid substitution (rtD263E) was observed to develop in 60% of patients with viremia. CONCLUSIONS: The cumulative CVR rates showed that patients with HBeAg-negative have better virologic respond than those with HBeAg-positive during the same period. The rtD263E mutation might be associated with partial resistance to TDF.

Evaluation of cellular responses for a chimeric HBsAg-HCV core DNA vaccine in BALB/c mice.

BACKGROUND: Fusion of Hepatitis B virus surface antigen (HBsAg) to a DNA construct might be considered as a strategy to enhance cellular and cytotoxic T-lymphocytes (CTL) responses of a Hepatitis C Virus core protein (HCVcp)-based DNA vaccine comparable to that of adjuvanted protein (subunit) immunization. MATERIALS AND METHODS: pCHCORE vector harboring coding sequence of HBsAg and HCVcp (amino acids 2-120) in tandem within the pCDNA3.1 backbone was constructed. The corresponding recombinant HCVcp was also expressed and purified in Escherichia coli. Mice were immunized either by adjuvanted HCVcp (pluronic acid + protein) or by pCHCORE vector primed/protein boosted immunization regimen. The cellular immune responses (proliferation, In vivo CTL assay and IFN-γ/IL-4 ELISpot) against a strong and dominant H2-d restricted, CD8(+)-epitopic peptide (C39) (core 39-48; RRGPRLGVRA) of HCVcp were compared in immunized animals. RESULT: Proper expression of the fused protein by pCHCORE in transiently transfected HEK 293T cells and in the expected size (around 50 kDa) was confirmed by western blotting. The immunization results indicated that the pCHCORE shifted the immune responses pathway to Th1 by enhancing the IFN-γ cytokine level much higher than protein immunization while the proliferative and CTL responses were comparable (or slightly in favor of DNA immunization). CONCLUSION: Fusion of HBsAg to HCVcp in the context of a DNA vaccine modality could augment Th1-oriented cellular and CTL responses toward a protective epitope, comparable to that of HCVcp (subunit HCV vaccine) immunization.