Engineered ClearColi™-derived outer membrane vesicles as functional carriers for development of HIV-1 therapeutic vaccine candidate.

Bacteria-derived outer membrane vesicles (OMVs) can be engineered to incorporate foreign antigens. This study explored the potential of ClearColi™-derived OMVs as a natural adjuvant and a carrier (recombinant OMVs or rOMVs) for development of an innovative therapeutic vaccine candidate harboring HIV-1 Nef and Nef-Tat antigens. Herein, the rOMVs containing CytolysinA (ClyA)-Nef and ClyA-Nef-Tat fusion proteins were isolated from ClearColi™ strain. The presence of Nef and Nef-Tat proteins on their surface (rOMVNef and rOMVNef-Tat) was confirmed by western blotting after proteinase K treatment. Immune responses induced by Nef and Nef-Tat proteins emulsified with Montanide® ISA720 or mixed with OMVs, and also rOMVNef and rOMVNef-Tat were investigated in BALB/c mice. Additionally, the potency of splenocytes exposed to single-cycle replicable (SCR) HIV-1 virions was assessed for the secretion of cytokines in vitro. Our findings showed that the rOMVs as an antigen carrier (rOMVNef and rOMVNef-Tat) induced higher levels of IgG2a, IFN-γ and granzyme B compared to OMVs as an adjuvant (Nef + OMV and Nef-Tat + OMV), and also Montanide® ISA720 (Nef + Montanide and Nef-Tat + Montanide). Moreover, IFN-γ level in splenocytes isolated from mice immunized with rOMVNef-Tat was higher than other regimens after exposure to SCR virions. Generally, ClearColi™-derived rOMVs can serve as potent carriers for developing effective vaccines against HIV-1 infection.

LL-37 antimicrobial peptide and heterologous prime-boost vaccination regimen significantly induce HIV-1 Nef-Vpr antigen- and virion-specific immune responses in mice.

OBJECTIVES: HIV infection still remains a leading cause of morbidity and mortality worldwide. The inability of highly-active antiretroviral therapy in HIV-1 eradication led to development of therapeutic vaccines. Exploiting effective immunogenic constructs and potent delivery systems are important to generate effective therapeutic vaccines, and overcome their poor membrane permeability. Among HIV-1 proteins, the Nef and Vpr proteins can be considered as antigen candidates in vaccine design. METHODS: In this study, the immunogenicity of Nef-Vpr antigen candidate in different regimens along with antimicrobial peptide LL-37 (as a DNA carrier) and Montanide 720 (as an adjuvant) was studied in mice. Moreover, the secretion of cytokines was assessed in virion-exposed mice lymphocytes in vitro. RESULTS: Our data indicated that groups immunized with the homologous protein + Montanide regimen (group 1), and also the heterologous DNA + LL-37 prime/protein + Montanide boost regimen (group 2) could significantly generate strong immune responses as compared to groups immunized with the DNA constructs (groups 3 & 4). Moreover, immunization of mice with the homologous DNA + LL-37 regimen in low dose of DNA (5 µg) could induce higher immune responses than the homologous naked DNA regimen in high dose of DNA (50 µg) indicating the role of LL-37 as a cell penetrating peptide. Additionally, the heterologous DNA + LL-37 prime/protein + Montanide boost regimen (group 2) induced significantly IFN-gamma secretion from virion-exposed lymphocytes in vitro. CONCLUSION: Generally, the use of LL-37 for DNA delivery, Montanide 720 as an adjuvant, and heterologous DNA prime/protein boost strategy could significantly increase IgG2a, IFN-gamma, and Granzyme B, and maintain cytokine secretion after exposure to virions. Indeed, the heterologous DNA + LL-37 prime/protein + Montanide boost regimen can be considered as a potent strategy for development of therapeutic HIV vaccines.

Impact of Blood Transfusion on the Prevalence of HHpgV-1, HPgV-1, and B19V Among Iranian HCV-infected Patients With Hemophilia.

OBJECTIVE: Blood-derived products from patient with hemophilia treated by factor VIII concentrates are potential sources of transfusion-transmitted infections, including human immunodeficiency virus, hepatitis, human pegivirus-1 (HPgV-1), B19 virus, and also human hepegivirus-1 (HHpgV-1). In the current study, we investigated the impact of blood transfusion on the prevalence of HHpgV-1, HPgV-1, and B19 virus in plasma of Iranian patient with hemophilia after direct-acting antiviral treatment of hepatitis C virus (HCV) infections for the first time. MATERIALS AND METHODS: A total of 170 patients with hemophilia who received direct-acting antivirals were enrolled in this study. Among them, 92 patients had a history of blood transfusion. The presence of HHpgV-1, HPgV-1, and B19 virus was detected by nested polymerase chain reaction analysis using the conserved primers. The plasmids harboring 5'-UTR and NS3 were used as positive controls for HPgV-1 and HHpgV-1, respectively. RESULTS: Our data identified 3 individuals with HHpgV-1 viremia (1.76%), 11 individuals with HPgV-1 viremia (6.47%), and 33 individuals with B19 viremia (19.4%). All patients were negative for hepatitis B virus, human immunodeficiency virus, and HCV infections. These findings indicated lower transmissibility or higher rates of virus clearance for HHpgV-1, HPgV-1, and B19 virus as compared with other bloodborne human flaviviruses such as HCV. However, the prevalence of B19 virus was significantly higher than the other 2 viruses. CONCLUSION: In general, these findings showed that the history of blood transfusion could increase the risk of viral transmission of bloodborne viruses among patient with hemophilia.

Development of HPV16,18,31,45 E5 and E7 peptides-based vaccines predicted by immunoinformatics tools.

OBJECTIVES: Viral oncoproteins are ideal targets in therapeutic vaccines for functional inhibition of human papillomaviruses (HPVs). Herein, we designed the peptide constructs derived from E5 and E7 oncoproteins of high-risk HPV types 16, 18, 31 and 45 using the bioinformatics tools and investigated their potency in mice. RESULTS: The framework of the combined in silico/in vivo analysis included (1) to determine physicochemical properties of the designed constructs, (2) to identify potential IFN-γ-inducing epitopes, (3) to assess allergenicity, (4) to recognize linear and discontinuous B cell epitopes using modeling and validation of 3D structure of the designed constructs, and (5) to evaluate immune responses and tumor growth in vivo. Our in silico data determined high potency of the HPV16,18,31,45 E5 and HPV16,18,31,45 E7 peptides for trigger B- and T-cell responses, and IFN-γ secretion. In vivo study indicated that the mixture of E5 and E7 immunodominant peptides from four types of high-risk HPV could induce Th1 immune response, and protect completely mice against TC-1 tumor cells. CONCLUSION: Generally, the combined in silico/in vivo approaches showed the ability of the designed E5 and E7 peptide constructs from four major high-risk HPV types for development of therapeutic vaccines.

B1 protein: a novel cell penetrating protein for in vitro and in vivo delivery of HIV-1 multi-epitope DNA constructs.

OBJECTIVES: Enhancement of the potential ability of biomacromolecules to cross cell membranes is a critical step for development of effective therapeutic vaccine especially DNA vaccine against human immunodeficiency virus-1 (HIV-1) infection. The supercharged proteins were known as powerful weapons for delivery of different types of cargoes such as DNA and protein. Hence, we applied B1 protein with + 43 net charges obtained from a single frameshift in the gene encoding enhanced green fluorescent protein (eGFP) for delivery of two multi-epitope DNA constructs (nef-vpu-gp160-p24 and nef-vif-gp160-p24) in vitro and in vivo for the first time. For this purpose, B1 protein was generated in bacterial expression system under native conditions, and used to interact with both DNA constructs. RESULTS: Our data indicated that B1 protein (~ 27 kDa) was able to form a stable nanoparticle (~ 80-110 nm) with both DNA constructs at nitrogen: phosphate (N: P) ratio of 1:1. Moreover, the transfection efficiency of B1 protein for DNA delivery into HEK-293T cell line indicated that the cellular uptake of nef-vif-gp160-p24 DNA/ B1 and nef-vpu-gp160-p24 DNA/ B1 nanoparticles was about 32-35% with lower intensity as compared to TurboFect commercial reagent. On the other hand, immunization of BALB/c mice with different modalities demonstrated that B1 protein could enhance the levels of antibody, IFN-gamma and Granzyme B eliciting potent and strong Th1-directed cellular immunity. CONCLUSION: Generally, our findings showed the potency of B1 protein as a promising gene delivery system to improve an effective therapeutic vaccine against HIV-1 infection.

Inflammatory and immune response genes: A genetic analysis of inhibitor development in Iranian hemophilia A patients.

A major problem of hemophilia A (HA) treatment is the development of factor VIII (FVIII) inhibitor, which usually occurs shortly after initiating replacement therapy. Several studies showed the correlation between inhibitor development and polymorphisms in inflammatory and immune response genes of HA patients; however, literature data are not available to prove this association in Iranian population. The aim of this study was to investigate a possible association between FVIII inhibitor formation and the polymorphisms of 16 inflammatory and immune response genes in Iranian severe HA patients (FVIII activity < 1%). This case-control study was performed on 55 patients with severe HA inhibitors and 45 samples without inhibitors from Iranian Comprehensive Hemophilia Care center. After extraction of whole genomic DNA from blood samples and design of primers for 16 genes, the genotyping was performed by Tetra primer ARMS PCR, and the validation of single nucleotide polymorphisms was determined by DNA sequencing. The data indicated that there was a significant association between inhibitor development, and F13A1 (TT), DOCK2 (CC& CT), and MAPK9 (TT) genotypes. Moreover, a considerably increased inhibitor risk carrying T, C, and T allele for F13A1, DOCK2, and MAPK9 genes was observed in patients with inhibitors, respectively. In contrast, there was no statistically significant difference between the genotypic and allelic frequencies for other genes in patients with inhibitors compared to patients without inhibitors. These results demonstrate that only polymorphisms in F13A1, DOCK2, and MAPK9 genes are associated with the risk of developing FVIII inhibitors in Iranian HA patients.

Protein vaccination with HPV16 E7/Pep-1 nanoparticles elicits a protective T-helper cell-mediated immune response.

Two human papillomavirus (HPV) viral oncoproteins, E6 and E7 represent ideal targets for development of a therapeutic HPV vaccine. It is important to reduce the rate of HPV-associated malignancies through improvement of vaccine modalities. In this study, we used a short amphipathic peptide carrier, Pep-1, for delivery of the full-length HPV16 E7 protein into mammalian cells and evaluated immune responses and protective effects of different formulations in C57BL/6 tumor mice model. Our results showed that the complexes of E7/Pep-1 protein form stable nanoparticles through noncovalent binding with an average size of 120 to 250 nm. The efficient delivery of E7 protein by Pep-1 at molar ratio of 1:20 was detected in HEK-293T cell line for 1 h and 3 h post-transfection. Immunization with E7/Pep-1 nanoparticles at a ratio of 1:20 induced a higher Th1 cellular immune response with the predominant IgG2a and IFN-γ levels than those induced by E7 protein in a murine tumor model. These data suggest that Pep-1 peptide would indicate promising applications for improvement of HPV therapeutic vaccines. © 2016 IUBMB Life, 68(6):459-467, 2016.

VLP production in Leishmania tarentolae: A novel expression system for purification and assembly of HPV16 L1.

Viral like particles (VLPs) have been used as immunogen for improvement of preventive vaccines against several viral infections in preclinical and clinical trials. These constructs can stimulate both cellular and humoral immunity. Two prophylactic HPV L1 VLP vaccines known as Gardasil and Cervarix were commercialized worldwide. However, there are main problems for expression and purification of VLPs in eukaryotic expression systems such as baculovirus and yeast leading to high cost of these vaccines. A novel Leishmania protozoan system has been applied to produce different recombinant proteins due to unique properties including generation of similar proteins with mammalian, easy handling, and large-scale culture. In the current study, we developed a novel strategy to produce HPV L1 VLP using stably transfected Leishmania cells. The positive transfectants were analyzed by SDS-PAGE and Western blot analysis. The assembly of purified L1 protein was detected by TEM microscopy. Finally, C57BL/6 mice were immunized by crude VLPs and antibody responses were assessed. The results of electronic microscopy revealed average 55-60 nm for L1 VLP. Furthermore, high IgG1 and IgG2a antibody responses were generated by L1 VLPs in mice similar to L1 VLPs produced in baculovirus-infected insect cells. Regarding the results, the amount of recombinant protein generated by Leishmania was 2-3mg/500 ml media, suggesting further optimization of this system for using in large animals and human.

Chemo-immunotherapy using saffron and its ingredients followed by E7-NT (gp96) DNA vaccine generates different anti-tumor effects against tumors expressing the E7 protein of human papillomavirus.

Saffron and its components have been suggested as promising candidates for cancer prevention. Carotenoids and monoterpene aldehydes are two potent ingredients of saffron. The goal of the current study was to investigate the anti-tumor effect of chemo-immunotherapy using saffron and its ingredients followed by E7-NT (gp96) DNA vaccine against tumors expressing the E7 protein of human papillomavirus. The in vitro cytotoxic and apoptotic effects of aqueous saffron extract and its components were evaluated in malignant TC-1 and non-malignant COS-7 cell lines. Then, multimodality treatments using E7-NT (gp96) DNA vaccine combined with saffron extract and its ingredients as well as single-modality treatments were tested for their efficacy in inhibiting large and bulky tumor growth. Saffron and its components exerted a considerable anti-tumor effect through prevention of cell growth and stimulation of programmed cell death. Furthermore, 100 % of mice treated with crocin were tumor-free, in contrast to DNA vaccine alone (~66.7 %) and DNA + crocin (~33.3 %) indicating the high potency of crocin as a chemotherapeutic agent. Interestingly, the multimodality treatment using DNA vaccine along with picrocrocin augmented the anti-tumor effects of picrocrocin. Thus, the combination of DNA vaccine with saffron extract and crocin at certain concentrations did not potentiate protective and therapeutic effects compared to mono-therapies for the control of TC-1 tumors.

Comparison of Adjuvant Effects of Montanide ISA-720 and Heat Shock Protein 27 in Increasing Immunostimulatory Properties of HIV-1 Nef-Vif Fusion Protein Construct.

INTRODUCTION: Effective T-cell-mediated immunity has emerged as an essential component of human immunodeficiency virus-1 (HIV-1) vaccination. Thus, inducing an immune response against HIV proteins such as Nef and Vif, two major accessory proteins with critical roles in HIV pathogenesis and immune evasion, may lead to an effective approach. AIM: Our goal is to evaluate and compare Montanide ISA-720 and heat shock protein 27 in increasing immunostimulatory properties of HIV-1 Nef-Vif fusion protein as a vaccine candidate. METHODS: In this study, the nef-vif fusion gene with and without the heat shock protein 27 (hsp27) gene was cloned in the prokaryotic pET24a (+) vector. Then, the recombinant Nef-Vif and Hsp27-Nef- Vif proteins were generated in the E. coli system. Finally, their immunostimulatory properties were evaluated in mice. Indeed, the potency of Hsp27 as an endogenous natural adjuvant was investigated to enhance HIV-1 Nef-Vif antigen-specific immunity compared to Montanide ISA-720 as a commercial adjuvant in protein-based immunization strategy. RESULTS: Our results approved the role of Hsp27 as an effective adjuvant in the stimulation of B- and T-cell immunity. The linkage of Hsp27 to antigen could elicit higher levels of IgG1, IgG2a, IFN-γ, IL- 5 and Granzyme B than antigen mixed with Montanide ISA-720. Moreover, the ratios of IFN-γ/IL-5 and IgG2a/IgG1 were significantly increased in groups receiving Nef-Vif protein + Montanide ISA- 720 and Hsp27-Nef-Vif protein indicating the direction of the immune response pathway toward strong Th1 response. These ratios were higher in the group receiving Hsp27-Nef-Vif protein than in the group receiving Nef-Vif protein + Montanide ISA-720. CONCLUSION: Our findings suggest that Hsp27 can be used as an effective adjuvant to enhance antigenspecific immune responses in HIV-1 infectious models for therapeutic vaccine development.

Immunological investigation of a multiepitope peptide vaccine candidate based on main proteins of SARS-CoV-2 pathogen.

Multiepitope vaccines could induce multiantigenic immunity against large complex pathogens with different strain variants. Herein, the in silico, in vitro and in vivo studies were used to design and develop a novel candidate antigenic multiepitope vaccine against SARS-CoV-2 pathogen. The designed multiepitope construct targets the spike glycoprotein (S), membrane protein (M), and nucleocapsid phosphoprotein (N) of SARS-CoV-2 (i.e., the S-N-M construct). This construct contains the cytotoxic T lymphocyte (CTL)-, helper T lymphocyte (HTL)-, and linear B lymphocyte (LBL)-inducing epitopes. The multiepitope s-n-m fusion gene was subcloned in prokaryotic (pET24a) and eukaryotic (pcDNA3.1) expression vectors. Its expression was evaluated in mammalian cell line using LL37 cell penetrating peptide. Moreover, the recombinant multiepitope S-N-M peptide was produced in E. coli strain. Finally, mice were immunized using homologous and heterologous regimens for evaluation of immune responses. Our data indicated that the multiepitope S-N-M peptide construct combined with Montanide 720 in homologous regimen significantly stimulated total IgG, IgG2a, IFN-γ, TNF-α, IL-15, IL-21 and IL-6, and Granzyme B secretion as compared to other groups. Moreover, the pcDNA-s-n-m/ LL37 nanoparticles significantly induced higher immune responses than the naked DNA in both homologous and heterologous regimens. In general, our designed multiepitope vaccine construct can be considered as a vaccine candidate in SARS-CoV-2 infection model.

Genotypes and phenotypes characterization of 17 Iranian patients with inherited factor X deficiency: identification of a novel mutation: Leu487Phe.

Factor X deficiency is a rare bleeding disorder that affects almost 1 : 1000 000 people worldwide. It derives from multiple mutational changes in the factor X gene (F10). The main objective of the present study was to determine a consistent correlation between the clinical presentations and causative genotype. The phenotype and genotype of 17 Iranian patients with reduced factor X activity (FX:C) from 14 unrelated families were analyzed to screen factor X gene expression for any possible mutations and function alteration. Analysis of the sequencing results led to the identification of eight different mutations besides a single nucleotide variation. One of the mutations was novel (Leu487Phe) as studied by means of online analysis programs and molecular modeling. Eight patients were homozygote; three were heterozygote, while six out of 17 patients were symptomatic cases without any mutations. The Arg40Thr missense mutation was detected in three patients including two siblings and was associated with severe bleeding symptoms. Also, two patients were identified with Gly262Asp missense mutation which commonly presented with bleeding disorder. Each of the other patients was associated with a unique missense mutation including one novel mutation in which the tentative relation of the mutation to bleeding symptoms is reported. Mutations leading to a FX:C of less than 1% are associated with severe bleeding symptoms confirming the strong correlation between clinical severity and FX:C. The novel Leu487Phe mutation with FX:C of 13% may have possible negative effects on factor X protein function resulting in minor clinical manifestation.

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.

Immunopotentiation by linking Hsp70 T-cell epitopes to Gag-Pol-Env-Nef-Rev multiepitope construct and increased IFN-gamma secretion in infected lymphocytes.

Therapeutic human immunodeficiency virus (HIV) vaccines can boost the anti-HIV host immunity to control viral replication and eliminate viral reservoirs in the absence of anti-retroviral therapy. In this study, two computationally designed multiepitope Gag-Pol-Env-Nef-Rev and Hsp70-Gag-Pol-Env-Nef-Rev constructs harboring immunogenic and highly conserved HIV T cell epitopes were generated in E. coli as polypeptide vaccine candidates. Furthermore, the multiepitope gag-pol-env-nef-rev and hsp70-gag-pol-env-nef-rev DNA vaccine constructs were prepared and complexed with MPG cell-penetrating peptide. The immunogenicity of the multiepitope constructs were evaluated using the homologous and heterologous prime/boost strategies in mice. Moreover, the secretion of IFN-γ was assessed in infected lymphocytes in vitro. Our data showed that the homologous polypeptide regimens could significantly induce a mixture of IgG1 and IgG2a antibody responses, activate T cells to secret IFN-γ, IL-5, IL-10, and generate Granzyme B. Moreover, IFN-γ secretion was significantly enhanced in single-cycle replicable (SCR) HIV-1 virions-infected splenocytes in these groups compared to uninfected splenocytes. The linkage of heat shock protein 70 (Hsp70) epitopes to Gag-Pol-Env-Nef-Rev polypeptide in the homologous regimen increased significantly cytokines and Granzyme B levels, and IFN-γ secretion in virions-infected splenocytes. Briefly, both designed constructs in the homologous regimens can be used as a promising vaccine candidate against HIV infection.

HIV-1 Accessory Proteins: Which one is Potentially Effective in Diagnosis and Vaccine Development?

BACKGROUND: The combination antiretroviral therapy (cART) could increase the number of circulating naive CD4 T lymphocytes, but was not able to eradicate human immunodeficiency virus-1 (HIV-1) infection. OBJECTIVE: Thus, induction of strong immune responses is important for control of HIV-1 infection. Furthermore, a simple and perfect serological method is required to detect virus in untreated-, treated- and drug resistant- HIV-1 infected individuals. METHODS: This study was conducted to assess and compare immunogenic properties of Nef, Vif, Vpr and Vpu accessory proteins as an antigen candidate in mice and their diagnostic importance in human as a biomarker. RESULTS: Our data showed that in mice, all heterologous prime/ boost regimens were more potent than homologous prime/ boost regimens in eliciting Th1 response and Granzyme B secretion as CTL activity. Moreover, the Nef, Vpu and Vif proteins could significantly increase Th1 immune response. In contrast, the Vpr protein could considerably induce Th2 immune response. On the other hand, among four accessory proteins, HIV-1 Vpu could significantly detect treated group from untreated group as a possible biomarker in human. CONCLUSION: Generally, among accessory proteins, Nef, Vpu and Vif antigens were potentially more suitable vaccine antigen candidates than Vpr antigen. Human antibodies against all these proteins were higher in HIV-1 different groups than healthy group. Among them, Vpu was known as a potent antigen in diagnosis of treated from untreated individuals. The potency of accessory proteins as an antigen candidate in an animal model and a human cohort study are underway.

Pharmaceutical Approaches for Treatment of Hepatitis C virus.

Many studies have been performed to develop an antiviral therapy against the hepatitis C virus (HCV) infections. The usual treatment for HCV infection is a combination of PEGylated interferon and ribavirin which offer restricted efficiency and major side effects. Thus, recent development in molecular biology of HCV and its life cycle led to the design of many drugs that target viral proteins and host factors required for viral replication. These drugs were named as direct-acting antivirals (DAAs) that were specifically designed for inhibition of viral life cycle, promising tolerability, short duration of treatment, higher barrier to resistance, and fewer drug interactions. The use of DAAs for the treatment of HCV infection resulted in high virological cure rates in patients. However, the use of combined DAA regimens may present drug interactions especially in patients under treatment for other co-morbidities. On the other hand, drug resistance against virus infection determines the success of long-term therapy. High genetic diversity among HCV virions due to error-prone polymerase activity led to the reduced susceptibility to DAA-therapy. Therefore, preclinical and clinical analysis of HCV resistance to novel drugs is needed. In this review, we describe pharmaceutical approaches for HCV treatment, structural and functional properties of DAAs, the principles of HCV drug-drug interaction, and finally HCV resistance to DAAs.

Development of multiepitope therapeutic vaccines against the most prevalent high-risk human papillomaviruses.

Aim: Our goal was the development of DNA- or peptide-based multiepitope vaccines targeting HPV E7, E6 and E5 oncoproteins in tumor mouse model. Materials & methods: After designing the multiepitope E7, E6 and E5 constructs from four types of high risk HPVs (16, 18, 31 & 45) using bioinformatics tools, mice vaccination was performed by different homologous and heterologous modalities in a prophylactic setting. Then, anti-tumor effects of the best prophylactic strategies were studied in a therapeutic setting. Results: In both prophylactic and therapeutic experiments, groups receiving homologous E7+E6+E5 polypeptide, and heterologous E7+E6+E5 DNA prime/polypeptide boost were successful in complete rejection of tumors. Conclusion: The designed multiepitope constructs can be considered as promising candidates to develop effective therapeutic HPV vaccines.

Heat shock proteins in infection.

Heat shock proteins (HSPs) are constitutively expressed under physiological conditions in most organisms but their expression can significantly enhance in response to four types of stimuli including physical (e.g., radiation or heat shock), chemical and microbial (e.g., pathogenic bacteria, viruses, parasites and fungi) stimuli, and also dietary. These proteins were identified for their role in protecting cells from high temperature and other forms of stress. HSPs control physiological activities or virulence through interaction with various regulators of cellular signaling pathways. Several roles were determined for HSPs in the immune system including intracellular roles (e.g., antigen presentation and expression of innate receptors) as well as extracellular roles (e.g., tumor immunosurveillance and autoimmunity). It was observed that exogenously administered HSPs induced various immune responses in immunotherapy of cancer, infectious diseases, and autoimmunity. Moreover, virus interaction with HSPs as molecular chaperones showed important roles in regulating viral infections including cell entry and nuclear import, viral replication and gene expression, folding/assembly of viral protein, apoptosis regulation, and host immunity. Viruses could regulate host HSPs at different levels such as transcription, translation, post-translational modification and cellular localization. In this review, we attempt to overview the roles of HSPs in a variety of infectious diseases.

Modified DCs and MSCs with HPV E7 antigen and small Hsps: Which one is the most potent strategy for eradication of tumors?

Immunotherapy with DCs as antigen-presenting vehicles have already improved patients' outcome against a variety of tumors. Moreover, MSCs were recently used to develop anti-cancer therapeutic or anti-microbial prophylactic vaccines. The current study evaluated immune responses and anti-tumor effects generated by DCs and MSCs derived from mouse bone marrow which were modified with small heat shock proteins 27 and 20 (sHsp27 and sHsp20) and also E7 oncoprotein in tumor mouse model. Two vaccination strategies were utilized including homologous DC or MSC prime/ DC or MSC boost, and heterologous MSC or DC prime/ protein boost vaccinations. Our data revealed that DCs pulsed with E7+Hsp27 and/or E7+Hsp20 in homologous and heterologous prime/ boost vaccinations could stimulate high levels of IgG2a, IgG2b, IFN-γ and IL-10 directed toward Th1 responses. Moreover, these regimens induced an increased level of Granzyme B, and displayed complete protection more than 60 days after treatment. On the other hand, MSCs transfected with E7+Hsp27 DNA in homologous and heterologous prime/ boost vaccinations could significantly enhance the E7-specific T-cell responses and suppress tumor growth in mice. However, MSCs transfected with E7+Hsp20 DNA did not induce a complete protection against TC-1 tumor compared to DCs pulsed with E7+Hsp20 protein complexes. These results indicated that DC- and MSC-based vaccinations with specific modalities will be a useful approach for immunotherapy and protection against HPV-associated cancers.

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.