Expression and Characterization of Two DNA Constructs Derived from HIV-1-vif in Escherichia coli and Mammalian Cells.

BACKGROUND: Acquired immunodeficiency syndrome (HIV/AIDS) is still a major global concern and no effective therapeutic vaccine has been produced to prevent the problem. Among HIV-1 proteins, vif as a basic cytoplasmic protein of HIV-1 is involved in late stages of viral generation and plays important role in HIV-1 virion replication. It also increases the stability of virion cores, which probably inhibits early degradation of viral entry. Therefore, it seems rational to apply this protein as a vaccine based on its impact on HIV-1 life cycle. This study aimed at cloning, expression and production of vif protein as an HIV-1 vaccine candidate. METHODS: In this study, vif sequence was amplified from pLN4-3 plasmid including HIV-1 vif gene and then cloned in pET23a to generate the recombinant plasmids of pET23a/vif with hexahistidine tags. BL21 competent cells were transformed to obtain the protein of interest. Ni-NTA column was used to purify the protein of interest and western blotting confirmed vif protein using anti-His tag antibody. In order to express the gene of interest in eukaryotic cells, vif was sub-cloned into pEGFP plasmids and HEK 293-T cells were transfected. Flow cytometry was then applied to evaluate GFP expression. RESULTS: vif protein was expressed in BL21(DE3) strain and identified as a23 kDa band in SDS-PAGE and confirmed by anti-His antibody in western blotting. The purified protein concentration was 173.3 µg/ml using Bradford assay. HEK-293T cells were successfully transfected by recombinant pEGFP plasmids and flow cytometry confirmed the cell transfection. CONCLUSION: vif protein can be expressed in mammalian cells and may be a proper protein subunit vaccine candidate against HIV-1.

Evaluation of transduced dendritic cells expressing HIV-1 p24-Nef antigens in HIV-specific cytotoxic T cells induction as a therapeutic candidate vaccine.

Various approaches have been investigated to prevent or eliminate HIV-1 since 1981. However, the virus has been affecting human population worldwide with no effective vaccine yet. The conserved regions among the viral genes are suitable targets in mutable viruses to induce the immune responses via an effective delivery platform. In this study, we aimed at evaluation of p24 and nef in two forms of full and truncated genes as two fusion antigenic forms according to our previous bioinformatics analysis. The designed antigens were then transferred through ex vivo generated dendritic cells and also proteins in BALB/c to assess and compare immunogenicity. p24 and Nef amino acid sequences were aligned, then, the most conserved regions were selected and two fusion forms as the truncated (p24:80-231aa-Nef:120-150aa) and the full from (p24-Nef) were cloned and expressed in prokaryotic and eukaryotic systems. Lentiviral vectors were applied to generate recombinant virions harboring the genes of interest to transduce generated murine dendritic cells. BALB/c mice received the recombinant DCs or recombinant proteins according to the defined schedule. IgG development was assessed to determine humoral immune activity and cellular immune responses were evaluated by IL-5 and IFN-y induction. Granzyme B secretion was also investigated to determine CTL activity in different immunized groups. The data showed high induction of cellular immune responses in dendritic cell immunization specifically in immunized mice with the truncated form of the p24 and Nef by high secretion of IFN-y and strong CTL activity. Moreover, protein/ DC prime-boost formulation led to stronger Th1 pathway and strong CTL activation in comparison with other formulations. The generated recombinant dendritic cells expressing p24-Nef induced humoral and cellular immunity in a Th1 pathway specifically with the in silico predicted truncated antigen which could be of high value as a dendritic cell therapeutic vaccine candidate against HIV-1.

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.

Small Heat Shock Proteins B1 and B6: Which One is the Most Effective Adjuvant in Therapeutic HPV Vaccine?

Therapeutic human papillomaviruse (HPV) vaccines have the potential to inhibit the tumor growth by targeting HPV E6 and E7 oncoproteins. Among different vaccine strategies, DNA and protein-based approaches are the most effective candidates for stimulation of the immune responses against HPV infections. Our study was designed to assess the efficacy of small heat shock proteins B1 (Hsp27) and B6 (Hsp20) as an adjuvant accompanied by HPV16 E7 and hPP10-E7 antigens in tumor mouse model. A major key for successful DNA and protein transfer into cells is the development of delivery systems with high efficiency and low cytotoxicity. Herein, we used hPP10 and MPG cell penetrating peptides (CPPs) for protein and DNA delivery in vivo, respectively. Our data indicated that the combination of Hsp27 with the recombinant hPP10-E7 protein in homologous protein/protein (hPP10-E7 + Hsp27) and heterologous DNA/protein (pcDNA-E7 + MPG/ hPP10-E7 + Hsp27) significantly enhanced the E7-specific T cell responses. Indeed, these regimens induced high levels of IgG2a, IFN-γ and IL-2 directed toward Th1 responses and also Granzyme B secretion as compared to other immunization strategies, and also displayed complete protection more than 60 days after treatment. These data suggest that the use of Hsp27 as an adjuvant and MPG and hPP10 as a gene and protein carrier would represent promising applications for improvement of HPV therapeutic vaccines. © 2018 IUBMB Life, 70(10):1002-1011, 2018.

Comparison of six cell penetrating peptides with different properties for in vitro and in vivo delivery of HPV16 E7 antigen in therapeutic vaccines.

The high risk human papillomavirus (HPV) types 16 and 18 are globally linked to >50% and 20% of all cervical cancers, respectively. The HPV E7 oncoprotein was determined as a therapeutic vaccine target due to its constitutive expression by HPV-infected cells. The findings demonstrated the efficiency of therapeutic HPV DNA- and protein-based vaccines in preclinical and clinical trials. However, there are limitations for penetration of DNA and protein constructs into the cells without a suitable delivery system. Recently, several cell penetrating peptides (CPPs) have been suggested for delivery of nucleic acids and proteins into cells through covalent or non-covalent fashion. In this study, we determined highly efficient CPPs for the controlled delivery of HPV16 E7 antigen, in vitro and in vivo. Our data indicated the effective delivery of E7 protein by Pep-1, Cady-2, P28 and hPP10, and E7 DNA by MPG and +36 GFP CPPs in HEK-293T cell line at certain ratios. Moreover, immunization with the heterologous MPG + E7 DNA prime/P28 + rE7 protein boost elicited a higher Th1 cellular immune response with a predominant IFN-γ profile and strong Granzyme B secretion than those induced by other groups in a murine tumor model. Indeed, the groups vaccinated with rE7+ P28/rE7+ P28, MPG+ E7 DNA/P28+ rE7, and E7 DNA+ MPG/E7 DNA+ MPG nanovaccines displayed complete protection and remained tumor-free >60 days after treatment. These data suggest P28 and MPG as promising protein and gene delivery systems for development of HPV therapeutic vaccines.

Delivery of molecular cargoes in normal and cancer cell lines using non-viral delivery systems.

OBJECTIVE: In this study, transfection efficiency of human papillomavirus (HPV) E7 DNA and protein constructs into HEK-293T normal cell line, and A549 and TC-1 tumor cell lines was evaluated by four delivery systems including supercharge GFP, hPP10 cell penetrating peptide, TurboFect and Lipofectamine using fluorescence microscopy and flow cytometry. RESULTS: The results indicated that Lipofectamine 2000 and TurboFect produced more effective transfection for GFP and E7-GFP DNA constructs in HEK-293T cells compared to in A549 and TC-1 cells (p < 0.05). In contrast, the supercharge GFP was efficient for E7 DNA and E7 protein delivery in both normal cell (~ 83.94 and ~ 77.01% for HEK-293T), and cancer cells (~ 71.69 and ~ 67.19% for TC-1, and ~ 73.86 and ~ 67.49% for A549), respectively. Indeed, in these cell lines, transfection efficiency by +36 GFP reached ~ 60-80%. Moreover, the hPP10 produced the best transfection result for E7-GFP protein in HEK-293T cells (~ 63.66%) compared to TurboFect (~ 32.95%); however, the efficiency level of hPP10 was only ~ 17.51 and ~ 16.36% in TC-1 and A549 cells. CONCLUSIONS: Our data suggested that the supercharge GFP is the most suitable transfection vehicle for DNA and protein delivery into TC-1 and A549 tumor cell lines compared to other carriers.

Generation of the Fluorescent HPV16 E7 Protein for Detection of Delivery In vitro.

BACKGROUND: Immunotherapies targeting the human papillomavirus (HPV) oncogenic proteins, E6 and E7, are effective to treat HPV-associated cervical malignancies. OBJECTIVE: The main objective of this study was to generate the fluorescent HPV16 E7 protein for detection of delivery in vitro. METHODS: Two types of the fusion E7-GFP proteins (i.e., with or without linker) were expressed in different E. coli strains. Then, the efficiency of GFP and E7-GFP transfection was compared with FITC-antibody protein control using TurboFect reagent in the HEK-293T cell line. RESULTS: Our data indicated that both E7-GFP fusion proteins were efficiently produced in M15 E. coli strain, but not in BL21 or Rosetta strains. The E7-GFP fusion showed a clear band of ~ 50 kDa in SDS-PAGE. Moreover, the E7-GFP protein maintained the fluorescent properties only when there was a distance between E7 and GFP genes, suggesting a promising potential to use GFP fusion protein in generating soluble form of protein. This fluorescent property was stable and could be detected in vitro. Moreover, the HEK-293T cells transfected by GFP/TurboFect and E7- GFP/TurboFect complexes demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the GFP fluorescence was stable even at 24 h post-transfection. CONCLUSION: Briefly, the E7-GFP fusion protein with linker can be useful for the development of protein vaccines against HPV16 infections and detection in vivo.

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.

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.

Antiviral Effects of Saffron and its Major Ingredients.

BACKGROUND: The lack of an effective vaccine against viral infections, toxicity of the synthetic anti-viral drugs and the generation of resistant viral strains led to discover novel inhibitors. Recently, saffron and its compounds were used to treat different pathological conditions. METHOD: In this study, we tested the anti-HSV-1 and anti-HIV-1 activities of Iranian saffron extract and its major ingredients including crocin and picrocrocin as well as cytotoxicity in vitro. The data showed that the aqueous saffron extract was not active against HIV-1 and HSV-1 virions at certain doses (i.e., a mild activity), but crocin and picrocrocin indicated significant anti-HSV-1 and also anti-HIV-1 activities. Crocin inhibited the HSV replication at before and after entry of virions into Vero cells. Indeed, crocin carotenoid suppressed HSV penetration in the target cells as well as disturbed virus replication after entry into the cells. Picrocrocin was also effective for inhibiting virus entry and also its replication. RESULTS: This monoterpen aldehyde showed higher anti-HSV effects after virus penetrating in the cells. Generally, these sugar-containing compounds extracted from saffron showed to be effective antiherpetic drug candidates. CONCLUSION: The recent study is the first report suggesting antiviral activities for saffron extract and its major ingredients. Crocin and picrocrocin could be a promising anti-HSV and anti-HIV agent for herbal therapy against viral 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.

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.

Carotenoids: biochemistry, pharmacology and treatment.

Carotenoids and retinoids have several similar biological activities such as antioxidant properties, the inhibition of malignant tumour growth and the induction of apoptosis. Supplementation with carotenoids can affect cell growth and modulate gene expression and immune responses. Epidemiological studies have shown a correlation between a high carotenoid intake in the diet with a reduced risk of breast, cervical, ovarian, colorectal cancers, and cardiovascular and eye diseases. Cancer chemoprevention by dietary carotenoids involves several mechanisms, including effects on gap junctional intercellular communication, growth factor signalling, cell cycle progression, differentiation-related proteins, retinoid-like receptors, antioxidant response element, nuclear receptors, AP-1 transcriptional complex, the Wnt/ß-catenin pathway and inflammatory cytokines. Moreover, carotenoids can stimulate the proliferation of B- and T-lymphocytes, the activity of macrophages and cytotoxic T-cells, effector T-cell function and the production of cytokines. Recently, the beneficial effects of carotenoid-rich vegetables and fruits in health and in decreasing the risk of certain diseases has been attributed to the major carotenoids, ß-carotene, lycopene, lutein, zeaxanthin, crocin (/crocetin) and curcumin, due to their antioxidant effects. It is thought that carotenoids act in a time- and dose-dependent manner. In this review, we briefly describe the biological and immunological activities of the main carotenoids used for the treatment of various diseases and their possible mechanisms of action. LINKED ARTICLES: This article is part of a themed section on Principles of Pharmacological Research of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.11/issuetoc.

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.

Prime-boost vaccine strategy against viral infections: Mechanisms and benefits.

The essential goal of vaccination is to generate potent and long-term protection against diseases. Among different vaccine modalities, prime-boost vaccine strategies could enhance cellular and also humoral immunity in several animal models. These strategies have been applied for the development of vaccines against important infectious diseases such as HIV, SIV, HCV, HSV, and HBV indicating promising results even in clinical trials. Several factors including selection of antigen, type of vector, delivery route, dose, adjuvant, boosting regimen, the order of vector injection, and the intervals between different vaccinations influence the outcome of prime-boost immunization approaches. The reported data suggest that the prime-boost strategy as a combination of vaccines (i.e., heterologous prime-boost) may be better than a single vaccine for protection against infectious diseases. Indeed, in many cases, heterologous prime-boost can be more immunogenic than homologous prime-boost strategy. This review discusses the recent advances in prime-boost immunization strategies as well as their benefits and mechanisms of action.