In Silico Analysis of L1/L2 Sequences of Human Papillomaviruses: Implication for Universal Vaccine Design.

The aim of this study was to design a multiepitope universal vaccine for major human papillomavirus (HPV) structural proteins, L1/L2, by bioinformatics models. For this purpose, we predicted the most probable immunogenic epitopes of L1 and L2 from common high-risk HPV 16, 18, 31, and 45 beside high prevalent type 6 and 11 based on BCPREDS defaulted model, while solvent accessibility of structure was extrapolated. The three-dimensional molecular model of L1 protein was constructed by Swiss Model server, whereas sequence alignment provided model for prediction of L2 protein epitopes. After that, N-glycosylation sites were excluded from estimated epitope regions. Then, by other bioinformatics analyses, 20 epitopes were selected and fused in tandem repeats, reverse translated, and codon optimized to relevant sequence. The final protein parameters such as antigenicity were analyzed by protean program. Evaluation of new recombinant protein sequence indicated a molecular weight of 41.8 kDa with 400 amino acids beside positive charge. The computed isoelectric point (pI) value indicated the acidic nature of final product. The aliphatic index showed low thermal stability of this construct and the Grand Average Hydropathicity value was negative (-0.494). Analyzed plot showed that major parts of new protein construct had hydrophilic property, thus harboring antigenic potency. After all, sequence of final construct reverse translated to DNA and this codon-optimized sequence showed Codon Adaptation Index (CAI) of >0.8 for expression in Escherichia coli. Finally, this sequence ligated into pET28a bacterial expression vector. The new recombinant proteins harboring 20 B cell epitope seem to be suitable antigens based on computational methods as a universal vaccine candidate for HPVs.

A comparative approach to strategies for cloning, expression, and purification of Mycobacterium tuberculosis mycolyl transferase 85B and evaluation of immune responses in BALB/c mice.

Protein antigens have drawn a lot of attention from investigators working on tuberculosis vaccines. These proteins can be used to improve the immunogenicity of the new generation BCG vaccines or even replace them completely. Recombinant technology is used to insure the production of pure mycobacterial antigens in high quantities. Mycolyl transferase 85B (Ag85B) is a potent, mycobacterial antigen that significantly stimulates immune responses. Since Ag85B is an apolar protein, production of the water-soluble antigen is of interest. In this work, we report a systematic optimization strategy concerning cloning systems and purification methods, aiming at increasing the yield of recombinant Ag85B. Our optimized method resulted in a yield of 8 mg of recombinant Ag85B from 1 liter of induced culture (400 µg/ml) by using pET32a(+), Escherichia coli Rosseta-gami™(DE3) pLysS and a Ni-NTA agarose-based procedure and on-column re-solubilization. The purified recombinant Ag85B showed strong immunostimulating properties by inducing high levels of TNF-α, IFN-γ, IL-12, and IgG2a in immunized mice, therefore it can effectively be applied in TB vaccine researches.

Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins.

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj)]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.

Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj) ]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.

Invasive aspergillosis promotes tumor growth and severity in a tumor-bearing mouse model.

Invasive aspergillosis increases in chronic immunosuppressive diseases such as cancer. There is little information about the mechanisms by which Aspergillus infection affects the immune regulation and microenvironment of cancer cells. Hence, this study was aimed at investigating the effect of invasive aspergillosis on immunosurveillance, metastasis, and prognosis of cancer in tumor-bearing mice. After implantation of mouse mammary tumor in BALB/c mice, they were infected with Aspergillus conidia intravenously. For comparison, groups of mice were experimentally infected with Aspergillus conidia or implanted with tumor cells separately. Seven days after Aspergillus infection, the serum levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) were measured by ELISA, and subsequently regulatory T lymphocytes were analyzed by flow cytometry. The survival of animals and mean tumor size were then determined. Our results indicated that tumor sizes in mice increased significantly after infection with Aspergillus conidia. Moreover, invasive aspergillosis enhanced the population of regulatory lymphocytes and level of TIMP-1. This study supports the idea that massive Aspergillus infection could stimulate tumor growth and increases the possibility of a bad prognosis. As a result, treatment of Aspergillus infection could be considered an important issue for efficient cancer therapy.

Leukemia markers expression of peripheral blood vs bone marrow blasts using flow cytometry.

BACKGROUND: Flow cytometric techniques are widely used in clinical hematology. Characterization of leukemias by immunotyping is particularly helpful when the morphology is difficult to interpret. The major advantage of using immune markers by flow cytometry is the identification of particular leukemia subtype, not recognized by morphologic criteria, which may have prognostic significance. Current literature suggests when peripheral blood (PB) is consisted of 30% blasts or higher diagnosis of acute leukemia is most likely. However, bone marrow aspiration may also be performed as a confirmatory diagnosis. Immunotyping of PB and BM in leukemias not only determine the decision making for a specific therapeutic regimen, but also is a practical prognostic indicator. MATERIAL/METHODS: We evaluated 18 patients with acute myeloid Leukemia (AML) and 13 patients with acute lymphoid leukemia (ALL). In all cases, the amount of blasts in PB was 30% or higher. Two ml PB and BM samples from each patient was collected. Following the preparation process, expression of markers was detected by using flow cytometry. The panel of monoclonal antibodies used in this study were consisted of CD3, CD7, CD5 (for T lymphocytes lineage); CD19, CD22, CD20, CD10 (for B lymphocytes lineage); CD13, CD14, CD33 (for myeloid subsets); and TDT, HLA-DR, CD45 (non lineage restricted). Expression levels of PB and BM markers were compared by using statistical analysis. RESULTS: The results showed apparent discrepancies for some markers in ALL group. However, in AML patients most of the selected markers have shown considerable correlation between PB and BM samples. Only four markers (CD13, CD14, CD45, and HLA-DR) showed positive correlation. In contrast, most markers (CD3, CD5, CD13, CD14, CD19, CD45, HLA-DR, and TdT) showed strong correlation between PB and BM samples in AML group. CONCLUSIONS: The findings of this study suggests that targeted gating strategy for blast population as well as selection of a suitable panel of monoclonal antibodies may be essential for diagnosis of leukemia resulting in similar immunotyping pattern in PB and BM. Although our results are preliminary, this can minimize the necessity of BM aspiration for leukemia patients.