In silico design and in vitro expression of novel multiepitope DNA constructs based on HIV-1 proteins and Hsp70 T-cell epitopes

Objectives

Epitope-driven vaccines carrying highly conserved and immunodominant epitopes have emerged as promising approaches to overcome human immunodeficiency virus-1 (HIV-1) infection.

Methods

Two multiepitope DNA constructs encoding T cell epitopes from HIV-1 Gag, Pol, Env, Nef and Rev proteins alone and/or linked to the immunogenic epitopes derived from heat shock protein 70 (Hsp70) as an immunostimulatory agent were designed. In silico analyses were applied including MHC-I and MHC-II binding, MHC-I immunogenicity and antigen processing, population coverage, conservancy, allergenicity, toxicity and hemotoxicity. The peptide-MHC-I/MHC-II molecular docking and cytokine production analyses were carried out for predicted epitopes. The selected highly immunogenic T-cell epitopes were then used to design two multiepitope fusion constructs. Next, prediction of the physicochemical and structural properties, B cell epitopes, and constructs-toll-like receptors (TLRs) molecular docking were performed for each construct. Finally, the eukaryotic expression plasmids harboring totally 12 cytotoxic T Lymphocyte (CTL) and 10 helper T lymphocytes (HTL) epitopes from HIV-1 proteins (i.e., pEGFP-N1-gag-pol-env-nef-rev), and linked to 2 CTL and 2 HTL epitopes from Hsp70 (i.e., pEGFP-N1-hsp70-gag-pol-env-nef-rev) were generated and transfected into HEK-293 T cells for evaluating the percentage of multiepitope peptides expression using flow cytometry and western blotting.

Results

The designed DNA constructs could be successfully expressed in mammalian cells. The expression rates of Gag-Pol-Env-Nef-Rev-GFP and Hsp70-Gag-Pol-Env-Nef-Rev-GFP were about 56–60% as the bands of?~?63 and?~?72 kDa confirmed in western blotting, respectively.

Conclusion

The combined in silico/in vitro methods indicated two multiepitope constructs can be produced and used as probable effective immunogens for HIV-1 vaccine development.

     

Exposure to Leishmania major modulates the proportion of CD4+ T cells without affecting cellular immune responses

The immune responses of individuals exposed to Leishmania major were evaluated and compared with those of non-exposed volunteers. Forty-one patients with active lesion(s), 43 healed individuals, 15 vaccinees 1 month or 1 year post vaccination, and 15 non-exposed volunteers were studied. Leishmanin skin test (LST) response, proliferative response of lymphocyte (PRL) to L. major antigen, IFN-gamma and IL-4 production, and percentage of L. major-specific CD4+, CD8+ and CD16+/CD56+ cells in peripheral blood mononuclear cells were assessed. Data showed positive LST (>5 mm) in 92% of patients, 98% of healed, and 80% or 43% of vaccinees 1 month and 1 year post vaccination, respectively. Positive PRL (SI>2.5) was displayed in 90%, 84%, 46% and 7% of patients, healed, vaccinated (post 1 year) and non-exposed donors, respectively. The mean +/-S.E. of IFN-gamma was 924 +/- 149, 1,278 +/- 185, 470 +/- 282 or 258 +/- 82 pg/ml in patients, healed cases and vaccinees after 1 month or 1 year, respectively. Positive IFN-gamma responders (>300 pg/ml) were shown in 72% of patients, 81% of healed cases, 31% or 39% of vaccinees and 0% of non-exposed donors. A reduced percentage of CD4+ T-cells and an increased percentage of NK cells were found in exposed individuals compared to non-exposed donors. The data indicated that exposure to L. major modulates the proportion of CD4+ T cells and increases NK cells percentage. However, the cellular immune responses including induction of LST, and IFN-gamma production are increased in exposed individuals.     

Recombinant Interleukin-1 Promotes Leishmaniasis in Susceptible Mice

Interleukin-1 exacerbates leishmanial lesions in Leishmania major-infected BALB/c mice. Indomethacin can modulate the effect of IL-1, so at least part of the IL-1 effect on disease progression is due to the induction of prostaglandin synthesis.     

Lack of association of Toll-like receptor 2 Arg753Gln with cutaneous leishmaniasis

The aim of the present study was to investigate the frequency of Arg753Gln and Arg677Trp polymorphisms of TLR2 in patients with cutaneous leishmaniasis (CL) compared to healthy controls. The polymorphisms were genotyped by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system assay (ARMS-PCR). The results showed that the frequency of Arg753Gln genotype was 14.3% and 10.1% in CL patients and normal controls, respectively. No one in either group was homozygous for the mutation. There was no significant difference in the genotype frequency. In contrast to the results for Arg753Gln polymorphism, we did not detect any case with Arg677Trp polymorphism in either control or patient group. In conclusion the TLR2 mutations are found equally in CL patients and healthy subjects.     

Recombinant Expression of a Plant-Derived Dimeric Antifungal Peptide (DiSkh-AMP1) Joined by a Flexible Linker in Escherichia coli and Evaluation of Its Biological Activity In Vitro

International Journal of Peptide Research and Therapeutics - DiSkh-AMP1, a novel dimeric antifungal peptide contained 65 amino acid residues was recombinant produced by a flexible linker to improve...     

Toll-like receptor 4 polymorphisms predispose to cutaneous leishmaniasis

The clinical spectrum of cutaneous leishmaniasis (CL) is extremely variable. Studies in experimental leishmaniasis have revealed a role for TLR4 in control of infection. In the present study the associations between TLR4 mutations (Asp299Gly and Thr399Ile) with outcome of CL have been investigated. Genotyping for Asp299Gly and Thr399Ile was performed in patients with chronic (N?=?22) and acute (N?=?61) CL, asymptomatic (N?=?45) and healthy leishmanin skin test negative individuals (N?=?75). The results showed the frequency of the Asp299Gly genotype was increased in patients with chronic disease (OR 25.3, 95% CI 5.2-115.6, P?相似文献   

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The destruction of cancer cells with chemotherapeutic agents is normally achieved through apoptosis. We previously introduced two synthetic halogenated flavanone derivatives, 3^′,7-dichloroflavanone (3′-7 DCF) and 3^′,6-dichloroflavanone (3′-6 DCF), as potential apoptosis-inducing agents. In the current study, we investigated the ability of these compounds in triggering intrinsic or/and extrinsic pathway of apoptosis in breast and prostate cancer cells. Also, the synergistic effect of 3′-7 DCF with TLR3 (Toll-like receptor 3) agonist in apoptosis induction was evaluated on PC3 and LNCaP human prostate cancer cells. The involved pathway of apoptosis in the treated cells was delineated by caspase-3 activity assay, PARP-1 (poly(ADP-ribose)polymerase-1) cleavage, and procaspase-9 cleavage as markers of the intrinsic pathway and procaspase-8 cleavage as the marker of the extrinsic pathway. With the exception of the normal cells, treatment of all cell lines with both 3′-7 DCF and 3′-6 DCF triggered the cleavage of procaspase-8 and procaspase-9. These results indicate that the intrinsic and the extrinsic pathways of apoptosis are the mechanisms of the toxicity of flavanones in these cancer cell lines. However, the cytoxicity of the compound 3′-7 DCF was not synergistic with TLR3 agonist. Interestingly, the activation of caspases-9 preceeded that of caspase-8 suggesting that the intrinsic pathway is the primary reason for apoptosis induction by the flavanones.