Immunostimulatory effects of Hsp70 fragments and Hsp27 in design of novel HIV-1 vaccine formulations.

BACKGROUND: Heat shock proteins (HSPs) as an adjuvant induce antigen-specific immunity through facilitating antigen presentation and stimulating T cells. In this study, the immunostimulatory properties of two major fragments of Hsp70 (N-Hsp70(aa 1-387) with ATPase property and C-Hsp70 (aa 508-641) with peptide-binding capacity) and the full length of Hsp27 as vaccine adjuvants were evaluated to boost HIV-1 Nef antigen-specific immunity in both in vitro and in vivo experiments. METHODS: At first, the nanoparticles harbouring DNA fusion constructs (i.e. N-Hsp70-Nef, C-Hsp70-Nef and Hsp27-Nef) complexed with HIV Rev (34-50) cell-penetrating peptide were generated to deliver DNA into the cells. Then, the recombinant Nef, Hsp27-Nef, N-Hsp70-Nef and C-Hsp70-Nef proteins were generated in E.coli expression system. Next, the immunostimulatory properties of these fusion constructs were evaluated in both in vitro and in vivo studies. Finally, the secretion of main cytokines from single-cycle replicable (SCR) HIV-1 virion-exposed splenocytes was investigated. RESULTS: Our data showed that the stable and non-toxic DNA/Rev nanoparticles could successfully deliver the genes of interest into the cells. Moreover, higher secretion of antibodies and cytokines was detected in mice receiving the Hsp-Nef constructs than in mice receiving Nef antigen. The C-Hsp70 was also superior for inducing Nef-specific Th1 and CTL immunity compared with N-Hsp70 and Hsp27. The T-cell activity was maintained in the SCR-exposed splenocytes, especially the splenocytes of mice receiving the C-Hsp70-Nef regimen. CONCLUSION: Altogether, these findings demonstrate the significance of Hsps as enhancers of antigen-specific immunity. Notably, the C-Hsp70 region showed better adjuvant properties for inducing cellular immunity in the improvement of HIV-1 therapeutic vaccines.

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.

HPV co-infections with other pathogens in cancer development: A comprehensive review.

High-risk human papillomaviruses (HR-HPVs) cause various malignancies in the anogenital and oropharyngeal regions. About 70% of cervical and oropharyngeal cancers are caused by HPV types 16 and 18. Notably, some viruses including herpes simplex virus, Epstein-Barr virus, and human immunodeficiency virus along with various bacteria often interact with HPV, potentially impacting its replication, persistence, and cancer progression. Thus, HPV infection can be significantly influenced by co-infecting agents that influence infection dynamics and disease progression. Bacterial co-infections (e.g., Chlamydia trachomatis) along with bacterial vaginosis-related species also interact with HPV in genital tract leading to viral persistence and disease outcomes. Co-infections involving HPV and diverse infectious agents have significant implications for disease transmission and clinical progression. This review explores multiple facets of HPV infection encompassing the co-infection dynamics with other pathogens, interaction with the human microbiome, and its role in disease development.

In Silico and in Vivo Analysis of HIV-1 Rev Regulatory Protein for Evaluation of a Multiepitope-based Vaccine Candidate.

In silico-designed multiepitope conserved regions of human immunodeficiency virus 1 (HIV-1) proteins would be a beneficial strategy for antigen design which induces effective anti-HIV-1 T-cell responses. The conserved multiple HLA-DR-binding epitopes of Rev protein were identified using IEDB MHC-I prediction tools and SYFPEITHI webserver to screen potential T-cell epitopes. We analyzed toxicity, allergenicity, immunogenicity, hemolytic activity, cross-reactivity, cell-penetrating peptide (CPP) potency, and molecular docking of the candidate epitopes using several immune-informatics tools. Afterward, we designed a novel multiepitope construct based on non-toxic and non-allergenic Rev, Nef, Gp160 and P24-derived cytotoxic T cell (CTL) and T-helper cell (HTL) epitopes. Next, the designed construct (Nef-Rev-Gp160-P24) was subjected to three B-cell epitope prediction webservers, ProtParam and Protein-Sol to obtain the physicochemical features. Then, the recombinant multiepitope DNA and polypeptide constructs were complexed with different CPPs for nanoparticle formation and pass them via the cell membranes. Finally, the immunogenicity of multiepitope constructs in a variety of modalities was evaluated in mice. The results demonstrated that groups immunized with heterologous DNA+ MPG or HR9 CPP prime/rNef-Rev-Gp160-P24 polypeptide + LDP-NLS CPP boost regimens could significantly produce higher levels of IFN-γ and Granzyme B, and lower amounts of IL-10 than other groups. Moreover, higher levels of IgG2a and IgG2b were observed in all heterologous prime-boost regimens than homologous DNA or polypeptide regimens. Altogether, the present findings indicated that the Nef-Rev-Gp160-P24 polypeptide meets the criteria to be potentially useful as a multiepitope-based vaccine candidate against HIV-1 infection.

Which one of the thermal approaches (heating DNA or cells) enhances the gene expression in mammalian cells?

OBJECTIVES: Heat treatment as a physical method could increase the cellular uptake of nucleic acids. In this study, the effects of heat shock were evaluated to enhance the transfection efficiency of three plasmid DNAs into HeLa and TC-1 cancerous, and HEK-293 T and Vero non-cancerous cell lines using lipofectamine 2000 reagent. METHODS: Two methods of cell- and DNA-based heat treatment were used. Heating DNA solution was performed at 94 °C for 5, 10 and 15 min, and also 72 °C for 30, 60 and 120 min, individually. Moreover, heating the cells was done by incubation at 42 °C for 2 h in different times such as before, during and after DNA transfection. RESULTS: Our data showed that the conformation of plasmid DNAs was changed at different temperatures with increasing time. The heat-treated plasmid DNAs (94 °C for 10 min or 72 °C for 30 min) indicated higher transfection efficiency than untreated plasmid DNAs (p < 0.05). Furthermore, heat treatment of cells before and during the transfection was higher than untreated cells (p < 0.01). Our results demonstrated that DNA transfection efficiency in cancerous cells was less than non-cancerous cells (p < 0.01). CONCLUSION: Generally, these findings showed that transfection mediated by thermal stimulation could enhance gene transfection in mammalian cell lines.

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.

Investigation of Immunostimulatory Effects of IFN-γ Cytokine and CD40 Ligand Costimulatory Molecule for Development of HIV-1 Therapeutic Vaccine Candidate.

The most crucial disadvantage of DNA-based vaccines is their low immunogenicity; therefore, finding an effectual adjuvant is essential for their development. Herein, immunostimulatory effects of IFNγ cytokine and a CD40 ligand (CD40L) costimulatory molecule are evaluated as combined with an antigen, and also linked to an antigen in mice. For this purpose, after preparation of the HIV-1 Nef, IFNγ, and CD40L DNA constructs, and also their recombinant protein in an Escherichia coli expression system, nine groups of female BALB/c mice are immunized with different regimens of DNA constructs. About 3 weeks and also 3 months after the last injection, humoral and cellular immune responses are assessed in mice sera and splenocytes. Additionally, mice splenocytes are exposed to single-cycle replicable (SCR) HIV-1 virions for evaluating their potency in the secretion of cytokines in vitro. The data indicate that the linkage of IFNγ and CD40L to Nef antigen can significantly induce the Th-1 pathway and activate cytotoxic T lymphocytes compared to other regimens. Moreover, groups receiving the IFNγ-Nef and CD40L-Nef fusion DNA constructs show higher secretion of IFNγ and TNF-α from virion-infected lymphocytes than other groups. Therefore, the IFNγ-Nef and CD40L-Nef fusion DNA constructs are suggested to be a potential option for development of an efficient HIV-1 vaccine.

Engineered dendritic cells-derived exosomes harboring HIV-1 Nefmut-Tat fusion protein and heat shock protein 70: A promising HIV-1 safe vaccine candidate.

Antigen presenting cells (APCs)-derived exosomes are nano-vesicles that can induce antigen-specific T cell responses, and possess therapeutic effects in clinical settings. Moreover, dendritic cells (DCs)-based vaccines have been developed to combat human immunodeficiency virus-1 (HIV-1) infection in preclinical and clinical trials. We investigated the immunostimulatory effects (B- and T-cells activities) of DCs- and exosomes-based vaccine constructs harboring HIV-1 Nefmut-Tat fusion protein as an antigen candidate and heat shock protein 70 (Hsp70) as an adjuvant in mice. The modified DCs and engineered exosomes harboring Nefmut-Tat protein or Hsp70 were prepared using lentiviral vectors compared to electroporation, characterized and evaluated by in vitro and in vivo immunological tests. Our data indicated that the engineered exosomes induced high levels of total IgG, IgG2a, IFN-γ, TNF-α and Granzyme B. Moreover, co-injection of exosomes harboring Hsp70 could significantly increase the secretion of antibodies, cytokines and Granzyme B. The highest levels of IFN-γ and TNF-α were observed in exosomes harboring Nefmut-Tat combined with exosomes harboring Hsp70 (Exo-Nefmut-Tat + Exo-Hsp70) regimen after single-cycle replicable (SCR) HIV-1 exposure. Generally, Exo-Nefmut-Tat + Exo-Hsp70 regimen can be considered as a promising safe vaccine candidate due to high T-cells (Th1 and CTL) activity and its maintenance against SCR HIV-1 exposure.

Development of novel HPV therapeutic vaccine constructs based on engineered exosomes and tumor cell lysates.

AIMS: Human papillomavirus (HPV) infections are highly prevalent globally. While preventive HPV vaccines exist, therapeutic vaccines are needed to treat existing HPV lesions and malignancies. This study evaluated the immunostimulatory and anti-tumor effects of three therapeutic vaccine candidates based on the recombinant protein, tumor cell lysate (TCL), and engineered exosome (Exo) harboring the heat shock protein 27 (Hsp27)-E7 fusion construct in mouse model. MAIN METHODS: At first, the recombinant Hsp27-E7 protein was generated in E. coli expression system. Then, tumor cell lysates-based and engineered exosomes-based vaccine constructs harboring green fluorescent protein (GFP) and Hsp27-E7 were produced using lentiviral system. Finally, their immunological and antitumor effects were investigated in both prophylactic and therapeutic experiments. KEY FINDINGS: Our data showed that the recombinant Hsp27-E7 protein, TCL-Hsp27-E7 and Exo-Hsp27-E7 regimens can induce the highest level of IFN-γ, TNF-α and Granzyme B, respectively. The percentage of tumor-free mice was identical for three vaccine strategies (survival rate: 75 %) in both prophylactic and therapeutic experiments. Generally, the TCL-Hsp27-E7, Exo-Hsp27-E7 and recombinant Hsp27-E7 protein regimens induced effective immune responses toward Th1 and CTL activity, and subsequently antitumor effects in mouse model. SIGNIFICANCE: Regarding to higher Granzyme B secretion, lower tumor growth and more safety, the Exo-Hsp27-E7 regimen can be considered as the most promising HPV vaccination strategy.

Stability of Neutralizing Antibody of PastoCoAd Vaccine Candidates against a Variant of Concern of SARS-CoV-2 in Animal Models.

Background: Since the beginning of the SARS-CoV-2 pandemic, there have been mutations caused by new SARS-CoV-2 variants, such as Alpha, Beta, Gamma, Delta, and Omicron, recognized as the variants of concern (VOC) worldwide. These variants can affect vaccine efficacy, disease control, and treatment effectiveness. The present study aimed to evaluate the levels of total and neutralizing antibodies produced by PastoCoAd vaccine candidates against the VOC strains at different time points. Methods: Two vaccine candidates were employed against SARS-CoV-2 using adenoviral vectors: prime only (a mixture of rAd5-S and rAd5 RBD-N) and heterologous prime-boost (rAd5-S/SOBERANA vaccine). The immunogenicity of these vaccine candidates was assessed in mouse, rabbit, and hamster models using ELISA assay and virus neutralization antibody test. Results: The immunogenicity results indicated a significant increase in both total and neutralizing antibodies titers in the groups receiving the vaccine candidates at various time points compared to the control group (p < 0.05). The results also showed that the PastoCoAd vaccine candidates Ad5 S & RBD-N and Ad5 S/SOBERANA could neutralize the VOC strains in the animal models. Conclusion: The ability of vaccine candidate to neutralize the VOC strains in animal models by generating neutralizing antibodies at different time points may be attributed to the use of the platform based on the Adenoviral vector, the N proteins in the Ad5 S & RBD-N vaccine candidate, and the SOBERANA Plus booster in the Ad5 S/SOBERANA vaccine candidate.

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.

Performance analyses of highly efficient inverted all-perovskite bilayer solar cell.

Numerical simulation of an all-perovskite bilayer solar cell has been conducted by the SCAPS-1D. The presented structure employs MAPbI3 as a relatively wide bandgap (1.55 eV) top absorber and FA0.5MA0.5Pb0.5Sn0.5I3 as a narrow bandgap (1.25 eV) bottom absorber. The viability of the proposed design is accomplished in two steps. First, to validate this study, two inverted solar cells in standalone conditions are simulated and calibrated to fit previously reported state-of-the-art results. Second, both these devices are appraised for the bilayer configuration to boost their performances. Affecting parameters such as the thickness of perovskite absorbers, the work function of front and rear contacts, and the effect of temperature have been studied because solar cells are temperature-sensitive devices, and also carrier concentration and their mobility get overwhelmingly influenced as temperature increases. It is manifested that using bilayer structures could easily widen the absorption spectrum to the near-infrared region and significantly enhance the performance of the device which is mainly affected by the thickness of the FA0.5MA0.5Pb0.5Sn0.5I3 layer. Also, it has been found that the work function of the front contact has a prominent role with its optimal values being above 5 eV. Finally, the optimized inverted all-perovskite bilayer solar cell delivers a power conversion efficiency of 24.83%, fill factor of 79.4%, open circuit voltage of 0.9 V, and short circuit current density of 34.76 mA/cm2 at 275 K and a thickness of 100 nm and 600 nm for MAPbI3 and FA0.5MA0.5Pb0.5Sn0.5I3, respectively.

Gene delivery in adherent and suspension cells using the combined physical methods.

Physical methods are widely utilized to deliver nucleic acids into cells such as electro-transfection or heat shock. An efficient gene electro-transfection requires the best conditions including voltage, the pulse length or number, buffer, incubation time and DNA form. In this study, the delivery of pEGFP-N1 vector into two adherent cell lines (HEK-293 T and COS-7) with the same origin (epithelial cells), and also mouse bone marrow-derived dendritic cells (DCs) was evaluated using electroporation under different conditions alone and along with heat treatment. Our data showed that the highest green fluorescent protein (GFP) expression in HEK-293 T and COS-7 cells was observed in serum-free RPMI cell culture medium as electroporation buffer, voltage (200 V), the pulse number (2), the pulse length (15 ms), the circular form of DNA, and 48 h after electro-transfection. In addition, the highest GFP expression in DCs was detected in serum-free RPMI, voltage (300 V), the pulse number (1), the pulse length (5 ms), and 48 h after electro-transfection. The use of sucrose as electroporation buffer, the pulse number (2), and the pulse length (25 ms) led to further cytotoxicity and lower transfection in HEK293T and COS-7 cells than other conditions. Moreover, the high voltage (700 V) increased the cell cytotoxicity, and decreased electro-transfection efficiency in DCs. On the other hand, the best conditions of electroporation along with heat treatment could significantly augment the transfection efficiency in all the cells. These data will be useful for gene delivery in other cells with the same properties using physical methods. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00524-4.

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.

In vitro Anti-HIV-1 Activity of the Recombinant HIV-1 TAT Protein Along With Tenofovir Drug.

BACKGROUND: HIV-1 TAT protein is essential for the regulation of viral genome transcription. The first exon of TAT protein has a fundamental role in the stimulation of the extrinsic and intrinsic apoptosis pathways, but its anti-HIV activity is not clear yet. METHODS: In the current study, we firstly cloned the first exon of the TAT coding sequence in the pET-24a expression vector and then protein expression was done in the Rosetta expression host. Next, the expressed TAT protein was purified by Ni-NTA column under native conditions. After that, the protein yield was determined by Bradford kit and NanoDrop spectrophotometry. Finally, the cytotoxicity effect and anti-Scr-HIV-1 activity of the recombinant TAT protein alone and along with Tenofovir drug were assessed by MTT and ELISA, respectively. RESULTS: The recombinant TAT protein was successfully generated in E. coli, as confirmed by 13.5% SDS-PAGE and western blotting. The protein yield was ~150-200 µg/ml. In addition, the recombinant TAT protein at a certain dose with low toxicity could suppress Scr-HIV replication in the infected HeLa cells (~30%) that was comparable with a low toxic dose of Tenofovir drug (~40%). It was interesting that the recombinant TAT protein could enhance anti-HIV potency of Tenofovir drug up to 66%. CONCLUSION: Generally, a combination of TAT protein and Tenofovir drug could significantly inhibit HIV-1 replication. It will be required to determine their mechanism of action in the next studies.

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.

Evaluation of HIV-1 Regulatory and Structural Proteins as Antigen Candidate in Mice and Humans.

BACKGROUND: The diagnosis of HIV infection is important among different groups. Moreover, combination antiretroviral therapy is used to treat HIV-1, but it cannot eradicate the infection. Thus, the development of therapeutic vaccines, along with antiretroviral therapy, is recommended. This study evaluates the values of four HIV proteins as antigen candidates in therapeutic vaccine design as well as a possible diagnostic marker for HIV infection in humans. METHODS: In this study, the HIV-1 Tat and Rev regulatory proteins and structural Gp120 and p24 proteins were generated in E. coli expression system. Their immunogenicity was evaluated in BALB/ c mice using homologous and heterologous prime/boost strategies. Moreover, the detection of anti- HIV IgG antibodies against these recombinant proteins was assessed in untreated (Naïve/ HIV-infected), treated, and drug-resistant patients compared to the healthy (control) group as a possible diagnostic marker for HIV infection. RESULTS: In humans, our results showed that among HIV-1 proteins, anti-Gp120 antibody was not detected in treated individuals compared to the healthy (control) group. The levels of anti-Gp120 antibody were significantly different between the treated group and Naïve as well as drug-resistant subjects. Moreover, the level of anti-p24 antibody was significantly lower in the treated group than the Naive group. In mice, the results of immunization indicated that the Rev antigen could significantly induce IgG2a, IgG2b, and IFN-γ secretion aimed at Th1 response as well as Granzyme B generation as CTL activity in comparison with other antigens. Furthermore, the heterologous DNA prime/ protein boost regimen was more potent than the homologous regimen for stimulation of cellular immunity. CONCLUSION: Briefly, the levels of both anti-Gp120 and anti-p24 antibodies can be considered for the diagnosis of the HIV-infected individuals in different groups compared to the healthy group. Moreover, among four recombinant proteins, Rev elicited Th1 cellular immunity and CTL activity in mice as an antigen candidate in therapeutic vaccine development.

Small Interfering RNAs and their Delivery Systems: A Novel Powerful Tool for the Potential Treatment of HIV Infections.

Small interfering RNAs (siRNAs) have rapidly developed into biomedical research as a novel tool for the potential treatment of various human diseases. They are based on altered gene expression. In spite of the availability of highly active antiretroviral therapy (HAART), there is a specific interest in developing siRNAs as a therapeutic agent for human immunodeficiency virus (HIV) due to several problems including toxicity and drug resistance along with long term treatment. The successful use of siRNAs for therapeutic goals needs safe and effective delivery to specific cells and tissues. Indeed, the efficiency of gene silencing depends on the potency of the carrier used for siRNA delivery. The combination of siRNA and nano-carriers is a potent method to prevent the limitations of siRNA formulation. Three steps were involved in non-viral siRNA carriers such as the complex formation of siRNA with a cationic carrier, conjugation of siRNA with small molecules, and encapsulation of siRNA within nanoparticles. In this mini-review, the designed siRNAs and their carriers are described against HIV-1 infections both in vitro and in vivo.

Production and characterization of freeze-dried banana slices pretreated with ascorbic acid and quince seed mucilage: Physical and functional properties.

The objective of this investigation was to illustrate the effects of quince seed mucilage (QSM) and ascorbic acid pretreatments to prevent the quality of freeze-dried banana slices. The studied parameters were moisture content, antioxidant activity, total phenol, color properties, structural properties, and sensory evaluation. Both treatments were effective in protecting total phenolic content and antioxidant activity in dried banana slices (P Ë‚ .05). The control slices showed greater increase in browning index (BI) and greater decrease in lightness (L*) than pretreated dried samples. Ascorbic acid and QSM treatments can be effective in the control of the enzymatic browning along with maintaining the quality properties of banana chips. Therefore, using of immersion pretreatment with 0.25% QSM and 0.05% ascorbic acid is recommended to prevent enzymatic browning as well as maintain the quality of banana chips before the drying process.