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.

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.

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.

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.

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.

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.

Combination of cell penetrating peptides and heterologous DNA prime/protein boost strategy enhances immune responses against HIV-1 Nef antigen in BALB/c mouse model.

To develop a strong HIV specific T-cell response, the HIV-1 Tat and Nef regulatory proteins have been known as attractive antigenic candidates in vaccine design. A peptide transduction domain of Tat (48-60 aa) could act to deliver other therapeutic molecules into different cells. In this line, several cell-penetrating peptides (CPPs) have been designed to transfer DNA, siRNA, polypeptides and proteins into cells through non-covalent approach such as CADY and PEP families. Some studies showed that the endogenous adjuvants including heat shock protein Gp96 could stimulate antigen-specific T cell immune responses. In this study, different Nef DNA and protein constructs were generated, and their abilities were evaluated to induce T cell immune responses and humoral immunity in mouse model. A novel prime-boost immunization approach was also used in which the priming injections consisted of Nef/Tat (PTD)-Nef DNA plus Gp96 DNA followed by Nef/Tat (PTD)-Nef protein formulated in Freund's adjuvant or the Pep-1 and Cady-2 CPPs. Generally, our results indicated that Tat (PTD)-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 Tat (PTD)-Nef+Gp96 DNA prime/Tat (PTD)-Nef protein+Cady-2 boost regimen significantly enhanced the Nef/Tat (PTD)-Nef-specific T cell responses. This modality induced high levels of IgG2a and IFN-γ directed toward Th1 responses, and also Cytotoxic T Lymphocytes (CTLs) activity as compared to other immunization strategies. The immunostimulatory properties of Pep-1 and Freund's adjuvant were almost similar in different immunization strategies. These findings showed that the use of Tat (PTD)-Nef antigen in prime-boost strategy along with Gp96 adjuvant and Cady-2 CPP may have practical implications for developing HIV-1 vaccine in large animal model.

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.

Hp91 immunoadjuvant: An HMGB1-derived peptide for development of therapeutic HPV vaccines.

BACKGROUND: High percentage of human cervical malignancy is related to human papillomavirus (HPV) infections. Thus, it is important to find novel non-invasive treatment strategies among various therapeutic HPV vaccines. In current study, we investigated the protective and therapeutic effects of DNA- and protein-based vaccines using HPV16 E7 as a model antigen in tumor mice model. In this line, the full length of high-mobility group box 1 (HMGB1) protein as well as an HMGB1-derived short peptide (Hp91) was used as an adjuvant for stimulating adaptive immunity and developing the potency of these vaccines. METHODS: DNA vaccination of HPV16 E7 with HMGB1 was performed as the complexed and conjugated forms. The immunostimulatory properties of Hp91 peptide along with Hp121 control peptide were compared to Montanide 720 in protein vaccination. RESULTS: Our data showed that co-immunization of HPV16 E7 protein with Hp91 peptide or Hp91+Hp121 peptides significantly increased the secretion of IFN-γ, IgG2a antibody response, and protected 100% of mice against a TC-1 tumor challenge. Furthermore, the linkage of HMGB1 with E7 antigen led to enhance the immunogenicity of DNA vaccine especially in combination with Hp91 and Hp121 peptides. CONCLUSIONS: These findings suggest that Hp91 peptide, and the full length of HMGB1 gene could be an efficient adjuvant for improvement of therapeutic HPV protein- and DNA-based vaccines, respectively.

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.

Whole recombinant Pichia pastoris expressing HPV16 L1 antigen is superior in inducing protection against tumor growth as compared to killed transgenic Leishmania.

The development of an efficient vaccine against high-risk HPV types can reduce the incidence rates of cervical cancer by generating anti-tumor protective responses. Traditionally, the majority of prophylactic viral vaccines are composed of live, attenuated or inactivated viruses. Among them, the design of an effective and low-cost vaccine is critical. Inactivated vaccines especially heat-killed yeast cells have emerged as a promising approach for generating antigen-specific immunotherapy. Recent studies have indicated that yeast cell wall components possess adjuvant activities. Moreover, a non-pathogenic protozoan, Leishmania tarentolae (L.tar) has attracted a great attention as a live candidate vaccine. In current study, immunological and protective efficacy of whole recombinant killed Pichia pastoris and Leishmania tarentolae expressing HPV16 L1 capsid protein was evaluated in tumor mice model. We found that Pichia-L1, L.tar-L1 and Gardasil groups increase the IgG2a/IgG1 ratio, indicating a relative preference for the induction of Th1 immune responses. Furthermore, subcutaneous injection of killed Pichia-L1 generated the significant L1-specific IFN-γ immune response as well as the best protective effects in vaccinated mice as compared to killed L.tar-L1, killed Pichia pastoris, killed L.tar and PBS groups. Indeed, whole recombinant Leishmania tarentolae could not protect mice against C3 tumor mice model. These data suggest that Pichia-L1 may be a candidate for the control of HPV infections.