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.

     

Culture Independent Diversity of Bacterial Communities Indigenous to Lower Altitude at Laohugou Glacial Environment

Abstract

High-throughput sequencing approach of the 16S rRNA gene was employed to evaluate the bacterial diversity inhabit in melted water, snow, soil, and rocks samples at the lower altitudes of the Laohugou glacial environment. Bioinformatics tools were used to process millions of Illumina reads for alpha and beta diversities of bacterial communities. The diversity indices such as Chao, Shannon, and Simpson were different in the collected samples and solid samples (soil and rocks) showed higher taxon richness and evenness. Taxonomic diversity was unexpectedly higher and the major portion of sequences was assigned to Proteobacteria, Actinobacteria, and Acidobacteria. Higher variation in community structure was reported at the class level and Alphaproteobacteria was dominant. The solid niches were occupied by a higher number of phyla compared with liquid. The physicochemical variables acted as spatial gradients and associated with the bacterial structural communities of the glacial ecosystem. Findings showed that both Proteobacteria and Actinobacteria in solid samples influenced the bacterial community structure in downstream liquid samples. Interestingly, the metagenomic biomarkers were higher in liquid samples. This study provides precious datasets to understand the bacterial community in a better way under the influence of spatial, physical and environmental factors.     

Caligus tunisiensis n. sp. (Copepoda: Caligidae) parasitic on the painted comber Serranus scriba (L.) (Perciformes: Serranidae) from the Mediterranean Sea,off the Tunisian coast

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First DNA Barcoding-based Inventory of Suez Gulf Fishes in Egypt and its Implication for Species Diversity

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Focus: Zoonotic Disease: Molecular Detection of Zoonotic Pathogens in the Blood and Tissues of Camels (Camelus dromedarius) in Central Desert of Iran

Dromedary camels (Camelus dromedarius) play a major economic role in many countries in Africa and Asia. Although they are resistant to harsh environmental conditions, they are susceptible to a wide range of zoonotic agents. This study aimed to provide an overview on the prevalence of selected zoonotic pathogens in blood and tissues of camels in central Iran. Blood, liver, portal lymph node, and brain were collected from 100 apparently healthy camels at a slaughterhouse in Qom city to assess the presence of DNA of Brucella spp., Trypanosoma spp., Coxiella burnetii, and Bartonella spp. PCR products were sequenced bidirectionally and phylogenetic analyses were performed. Eleven percent of camels tested positive for Brucella abortus (3%) and Trypanosoma evansi (8%). Coxiella burnetii and Bartonella spp. DNA was not detected. Our data demonstrate that camels from Iran contribute to the epidemiology of some zoonotic pathogens. Performing proper control strategies, such as vaccination of camels and humans in contact with them, test-and-slaughter policy, and education of the general population is necessary for minimizing the risk of zoonotic infection.     

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MicroRNA-424-5p enhances chemosensitivity of breast cancer cells to Taxol and regulates cell cycle,apoptosis, and proliferation

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Identification of copy number variants in children and adolescents with autism spectrum disorder: a study from Turkey

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Cold atmospheric plasma induced genotoxicity and cytotoxicity in esophageal cancer cells

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