In silico designing of novel epitope-based peptide vaccines against HIV-1.

The HIV-1 virus has been regarded as a catastrophe for human well-being. The global incidence of HIV-1-infected individuals is increasing. Hence, development of effective immunostimulatory molecules has recently attracted an increasing attention in the field of vaccine design against HIV-1 infection. In this study, we explored the impacts of CD40L and IFN-γ as immunostimulatory adjuvants for our candidate HIV-1 Nef vaccine in human and mouse using immunoinformatics analyses. Overall, 18 IFN-γ-based vaccine constructs (9 constructs in human and 9 constructs in mouse), and 18 CD40L-based vaccine constructs (9 constructs in human and 9 constructs in mouse) were designed. To find immunogenic epitopes, important characteristics of each component (e.g., MHC-I and MHC-II binding, and peptide-MHC-I/MHC-II molecular docking) were determined. Then, the selected epitopes were applied to create multiepitope constructs. Finally, the physicochemical properties, linear and discontinuous B cell epitopes, and molecular interaction between the 3D structure of each construct and CD40, IFN-γ receptor or toll-like receptors (TLRs) were predicted. Our data showed that the full-length CD40L and IFN-γ linked to the N-terminal region of Nef were capable of inducing more effective immune response than multiepitope vaccine constructs. Moreover, molecular docking of the non-allergenic full-length- and epitope-based CD40L and IFN-γ constructs to their cognate receptors, CD40 and IFN-γ receptors, and TLRs 4 and 5 in mouse were more potent than in human. Generally, these findings suggest that the full forms of these adjuvants could be more efficient for improvement of HIV-1 Nef vaccine candidate compared to the designed multiepitope-based constructs.

Immunoinformatics Approach to Design a Chimeric CD70-Peptide Vaccine against Renal Cell Carcinoma.

Renal cell carcinoma (RCC) accounts for the majority of cancer-related deaths worldwide. Overexpression of CD70 has been linked to advanced stages of RCC. Therefore, this study aims to develop a multiepitope vaccine targeting the overexpressed CD70 using immunoinformatics techniques. In this investigation, in silico multiepitope vaccines were constructed by linking specific CD70 protein epitopes for helper T lymphocytes and CD8+ T lymphocytes. To enhance immunogenicity, sequences of cell-penetrating peptide (CPP), penetratin (pAntp), along with the entire sequence of tumor necrosis factor-α (TNF-α), were attached to the N-terminal and C-terminal of the CD70 epitopes. Computational assessments were performed on these chimeric vaccines for antigenicity, allergenicity, peptide toxicity, population coverage, and physicochemical properties. Furthermore, refined 3D constructs were subjected to a range of analyses, encompassing structural B-cell epitope prediction and molecular docking. The chosen vaccine construct underwent diverse assessments such as molecular dynamics simulation, immune response simulation, and in silico cloning. All vaccines comprised antigenic, nontoxic, and nonallergenic epitopes, ensuring extensive global population coverage. The vaccine constructs demonstrated favorable physicochemical characteristics. The binding affinity of chimeric vaccines to the TNF receptor remained relatively stable, influenced by the alignment of vaccine components. Molecular docking and dynamics analyses predicted stable interactions between CD70-CPP-TNF and the TNF receptor, indicating potential efficacy. In silico codon optimization and cloning of the vaccine nucleic acid sequence were accomplished using the pET28a plasmid. Furthermore, this vaccine displayed the capacity to modulate humoral and cellular immune responses. Overall, the results suggest therapeutic potential for the chimeric CD70-CPP-TNF vaccine against RCC. However, validation through in vitro and in vivo experiments is necessary. This trial is registered with NCT04696731 and NCT04046445.

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.

Immunoinformatics studies of heat shock proteins 27 and 70: Development of potent therapeutic vaccine constructs against human papillomavirus-related cancers.

Heat shock proteins (HSPs) improve cross-presentation of linked tumor antigens, thus they can be exploited in therapeutic vaccine design. Herein, in silico analyses of different vaccine constructs were performed based on human papillomavirus (HPV)-16 E7 protein linked to Homo sapiens/Mus musculus Hsp27 or Hsp70 in multiepitope and whole sequence forms. Then, computational comparison between different orientations of Hsp/E7 was carried out in both forms. Finally, molecular docking was performed between the designed constructs and signaling (TLRs) or endocytic (CD14, LOX-1 and SREC-1) receptors. Our data represented the high-ranked T-cell epitopes and the potential B-cell epitopes of Homo sapiens/Mus musculus Hsp27 and Hsp70. Moreover, molecular docking showed that whole sequence of Hsp27 had better interaction with all receptors than whole sequence of Hsp70 suggesting likely stronger stimulation of innate and adaptive immunity. All designed Homo sapiens/Mus musculus Hsp27/E7 constructs had better docking scores with the endocytic receptors especially SREC-1 than all designed Homo sapiens/Mus musculus Hsp70/E7 constructs in both orientations. Generally, the multiepitope-/whole sequence-based Homo sapiens/Mus musculus Hsp27-E7 fusion constructs showed more conservancy and immunogenicity than other designed constructs. These fusion constructs were non-allergenic, non-toxic and stable suggesting them as promising vaccine candidates against HPV-related cancers.

Marham-Mafasel decrease joint inflammation and IL-1ß gene expression in rheumatoid arthritis animal model.

BACKGROUND: Rheumatoid arthritis (RA) is a systemic chronic disease with synovial membrane, tendon and articular tissue inflammation. Current treatments of RA have many side effects and are quite expensive. Today, new treatments procedures and inexpensive herbal drugs are developed. Marham-Mafasel is mainly made out of two traditional herbs (Arnebia euchroma and Martricaria chamomilla). OBJECTIVE: In this study, for the first time, the impact of Marham-Mafasel on joint inflammation, histopathological changes and IL-1ß gene expression was evaluated in RA animal model. METHODS: The RA was induced by a single s.c. injection of 0.1 ml Freund's complete adjuvant into the left hind footpad. In continuous, 15 RA male Wistar rats were used in three groups: I: Control; II: Treatment I (Piroxicam) and III: Treatment II (Marham-Mafasel). The volume of the hind paw was measured every day from 0 to 19 using water changed volume approach. The inflammation in the joint was evaluated using histopathology assay and gene expression of IL-1ß was evaluated with use of Real-Time PCR. RESULTS: Hind paw swelling of Marham-Mafasel at days 10th and 19th was reduced compared with the control group (p < 0.05). There was no statistically difference in histological degrading and changes index in three groups (p ≥ 0.05). Relative expression of IL-1ß in Marham-Mafasel group was significantly decreased compared with other groups. CONCLUSION: The co-administration of M. Chamomile and A. euchroma, called Marham-Mafasel, decreases IL-1ß gene expression that leads to a reduction in inflammation in rheumatoid arthritis (RA) animal model.

Development of high-affinity monoclonal antibody using CD44 overexpressed cells as a candidate for targeted immunotherapy and diagnosis of acute myeloid leukemia.

AIM: CD44s antigens have been suggested as an efficient biomarker for cancer stem cells. Current study aimed to develop a hybridoma that producing a high affinity murine anti-human CD44 monoclonal antibody for early diagnostic laboratory tests of some cancer. MATERIALS AND METHODS: To make hybridoma against CD44, mice were immunized with MDA-MB-468 cells. Resulted hybridomas using three culture media were screened by indirect ELISA, then cloned by limiting dilution, and isotype was determined after obtaining ascitic fluid and antibody purification. RESULTS: We obtained a stable secreting clone, capable of secreting a high-affinity monoclonal antibody against CD44 protein, IgG2a kappa, with the affinity of 5.4 × 10-8 M without cross-reactivity. CONCLUSION: We could establish a hybridoma in a native form. This stable and high-affinity anti-CD44 mAb has a potential for diagnostic procedures and laboratory research. Thus, it could be exploited as a suitable tool for target-specific diagnosis and even treatment in several cancers.

Anti-proliferative activity of Fumaria vaillantii extracts on different cancer cell lines.

Plant-derived natural products are known to have cancer chemo-preventive and chemo-therapeutic properties. Plant extracts or their active constituents are used as folk medicine in traditional therapies by 80% of the world population. The aim of the present study was to determine the anti-proliferative potential of Fumaria vaillantii extracts on three different cancer cell lines including malignant melanoma SKMEL-3, human breast adenocarcinoma MCF-7 and human myelogenous leukemia K562 as well as human gingival fibroblast (HGF) as normal cell line. Anti-proliferative activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flowcytometry and annexin methods. Total phenolics and flavonoids were determined by Folin-Ciocalteu and aluminum chloride methods. Chloroform fraction had the lowest IC50 value at 72 h (0.1 µg/ml) in MCF-7 cells. Flowcytometry and annexin-V analysis indicated that the chloroform fraction induced necrosis in MCF-7 cells. In addition, the colorimetric methods showed that the methanolic fraction possessed the highest amount of total phenolics (33.03 ± 0.75 mg/g of dry powder) and flavonoids (10.5 ± 2.0 mg/g of dry powder). The collective data demonstrated that F. vaillantii chloroform fraction may contain effective compounds with chemo-therapeutic potential act through an apoptotic independent pathway.

Evaluation of CD4+ CD25+ FoxP3+ Regulatory T cells and FoxP3 and CTLA-4 gene Expression in Patients wwith Newly Diagnosed Tuberculosis in Northeast of Iran.

BACKGROUND: Tuberculosis (TB) is the world's second most common infectious disease after Human Immunodeficiency Virus Infection/Acquired Immunodeficiency Syndrome (HIV/AID) and the most frequent cause of mortality especially in developing countries. T regulatory (Treg) cells, which have suppressive activity and express forkhead winged-helix family transcriptional repressor p3 (FoxP3), suppress the immune responses against pathogens such as Mycobacterium tuberculosis. There are controversial results regarding the role of FoxP3 expressing cells in the blood of patients with TB. OBJECTIVES: The aim of this study was to evaluate the frequency CD4+ CD25+ Treg cells, and FoxP3 and Cytotoxic T Lymphocyte Antigen 4 (CTLA-4) gene expressions in peripheral blood of patients with tuberculosis and patients with positive tuberculin skin test before and after Peripheral Blood Mononuclear Cells (PBMCs) activation with Purified Protein Derivative (PPD). PATIENTS AND METHODS: In this cross-sectional study, Peripheral Mononuclear Cells (PBMCs) were isolated from peripheral blood of 29 patients with newly diagnosed pulmonary TB and 19 patients with positive tuberculin skin test. The PBMCs were activated with PPD for 72 hours. Activated cells were harvested, RNA was extracted and cDNA was synthesized. A real-time Taqman method was designed and optimized for evaluation of Foxp3 gene expression and SYBR Green method was used and optimized for evaluation of CTLA-4 gene expression. A flow cytometry analysis was used to evaluate the frequency of CD4+ CD25+ Foxp3+ regulatory T cells in both groups. RESULTS: There was no significant difference in the frequency of CD4+ CD25+ FoxP3+ regulatory T cells between the two groups. Expression of FoxP3 and CTLA-4 in peripheral blood of patients with newly diagnosed TB was significantly lower than the control group after and before activation with PPD. CONCLUSIONS: The expression of FoxP3 and CTLA-4 in PBMCs of patients with newly diagnosed TB was low, which might suggest that Treg cells may be sequestered in the lungs.