A systematic evaluation of sample preparation and 2-D gel electrophoresis protocol for mosquito proteomic profiling.

-Mosquito act as the carrier insect to transfer pathogens into hosts for various vector-borne diseases.To identify the pathogenesis causing determinant, comprehensive knowledge of the protein expression in different tissues and physiological conditions is very important. The most widely used technique is 2-D gel electrophoresis to study the protein expression in mosquitoes. 2-D gel electrophoresis is the multistep process to resolve intact protein with similar molecular weight. It is also useful to separate post-translational modified protein, which are not distinguished through shotgun proteomic analysis. Here, we optimized the protocol for 2-D gel electrophoresis that can effectively resolve the protein in mosquitoes and some other insects, to target immunogenic protein to fight against the vector borne disease. The optimized 2-D protocol helps to resolve complex proteomic data which is very difficult to analyze in mosquitoes.The updated protocol improved the protein solubility, resolution and visualization that help in comparative analysis of protein expression.

An analytical review of vector- and pathogen-based transmission-blocking vaccine for malaria control.

Malaria is a vector borne disease, considered to be one of the most serious public health problems. The present review focused on the blocking of parasite development in mosquito vectors; one broad strategy for achieving this is Transmission Blocking Vaccines (TBV). The TBVs usually rely on immunization of vertebrate hosts with molecules derived from the vector or pathogen to reduce pathogen transmission from infected to uninfected hosts. Most of the studies on the TBVs are based on the antibodies targeted against the surface antigens of sexual stages of malaria parasite, but it is meagre to develop mosquito-based vaccine in this regard. Vector-based TBVs include surface proteins that are expressed by the mosquito midgut digestive enzymes which are induced upon blood-feeding, and receptors expressed on the epithelial line of the tissue. Many proteins are reported that can act as candidates for transmission-blocking vaccines. This review aims to summarize the vector midgut-based proteins identified till date, that can block the development and maturity of sexual stages of the parasite within mosquitoes as targets for transmission-blocking vaccine development. The TBVs intervention can block transmission of different malaria parasite species in various species of mosquitoes with future application perspective worldwide.

Identification and Characterization of Aminopeptidase N 1 Gene of the Indian Malaria Vector Anopheles culicifacies (Diptera: Culicidae).

Aminopeptidase N1 (APN) is one of the important enzymes involved in blood digestion and is up-regulated along with several other enzymes in response to bloodmeal ingestion. APN is a zinc metalloprotease that cleaves one amino acid residue at a time from the amino terminus of the protein. The APN1 gene of the Indian malaria vector Anopheles culicifacies Giles was cloned and characterized. The An. culicifacies APN1 (AcAPN1) gene has an Open Reading Frame of 3084 basepairs which encodes a putative protein of 1027 amino acids. The coding region of the gene shares 81% and 78% similarity to the APN1 genes found in An. stephensi (Diptera: Culicidae) and An. gambiae (Diptera: Culicidae), respectively. The organization of the APN1 gene was studied in available mosquito genomes and a three-dimensional structure of AcAPN1 modeled using homology structure modeling. The enzymatic active site was predicted to consist of HEYAH and GAMEN amino acid residues, and a comparison of the protein sequences among different genera revealed the conservation of zinc-binding residues. The expression pattern of AcAPN1 showed that the gene was expressed rapidly in response to the ingestion of the bloodmeal and therefore this gene may be used to exploit its promoter region as an antiparasite candidate molecule.

An Immunoinformatics Study to Predict Epitopes in the Envelope Protein of SARS-CoV-2.

COVID-19 is a new viral emergent disease caused by a novel strain of coronavirus. This virus has caused a huge problem in the world as millions of people are affected by this disease. We aimed at designing a peptide vaccine for COVID-19 particularly for the envelope protein using computational methods to predict epitopes inducing the immune system. The envelope protein sequence of SARS-CoV-2 has been retrieved from the NCBI database. The bioinformatics analysis was carried out by using the Immune Epitope Database (IEDB) to predict B- and T-cell epitopes. The predicted HTL and CTL epitopes were docked with HLA alleles and binding energies were evaluated. The allergenicity of predicted epitopes was analyzed, the conservancy analysis was performed, and the population coverage was determined throughout the world. Some overlapped CTL, HTL, and B-cell epitopes were suggested to become a universal candidate for peptide-based vaccine against COVID-19. This vaccine peptide could simultaneously elicit humoral and cell-mediated immune responses. We hope to confirm our findings by adding complementary steps of both in vitro and in vivo studies to support this new universal predicted candidate.

Designing spike protein (S-Protein) based multi-epitope peptide vaccine against SARS COVID-19 by immunoinformatics.

The outbreak of COVID-19 was originated from China, responsible for Several Acute Respiratory Syndrome (SARS). Scientists are forced to develop vaccine and effective drugs to control COVID-19 infection. To develop effective vaccine for SARS - COVID 19, immunoinformatics and computational approaches could helps to design successful vaccine against this biggest danger for humanity. Here we used various in - silico approaches to designed vaccine against COVID-19. To develop vaccine, we target S- protein, expressed on the virus surface plays important role in COVID-19 infection. We identified 12 B-cell, 9 T-helper and 20 Cytotoxic T-cell epitope based on criteria of selection. The predicted epitopes were link simultaneously with GPGPG & AAY linkers. The ß-defensin was used as adjuvant, linked with selected epitope by using EAAAK linker. For vaccine construct justification we analysed its immunogenicity, allergenicity and physiochemical properties. Our study revealed that vaccine was non toxic, immunogenic and antigenic in nature and covers 98.6% of world population, important for vaccine effectively. In- silico cloning was used to analyse its expression in vector. Molecular docking was performed to study the interaction of construct with TLR (TLR3, TLR4, and TLR9) molecules. The immune simulation was conducted and conformed that our vaccine constructs can induces both acquired and humoral immunity effectively against COVID-19 at very low concentration, but along with bioinformatics study we need to conduct experiment in laboratory to validate its safety and effectiveness.

3CL hydrolase-based multiepitope peptide vaccine against SARS-CoV-2 using immunoinformatics.

The present study provides the first multiepitope vaccine construct using the 3CL hydrolase protein of SARS-CoV-2. The coronavirus 3CL hydrolase (Mpro) enzyme is essential for proteolytic maturation of the virus. This study was based on immunoinformatics and structural vaccinology strategies. The design of the multiepitope vaccine was built using helper T-cell and cytotoxic T-cell epitopes from the 3CL hydrolase protein along with an adjuvant to enhance immune response; these are joined to each other by short peptide linkers. The vaccine also carries potential B-cell linear epitope regions, B-cell discontinuous epitopes, and interferon-γ-inducing epitopes. Epitopes of the constructed multiepitope vaccine were found to be antigenic, nonallergic, nontoxic, and covering large human populations worldwide. The vaccine construct was modeled, validated, and refined by different programs to achieve a high-quality three-dimensional structure. The resulting high-quality model was applied for conformational B-cell epitope selection and docking analyses with toll-like receptor-3 for understanding the capability of the vaccine to elicit an immune response. In silico cloning and codon adaptation were also performed with the pET-19b plasmid vector. The designed multiepitope peptide vaccine may prompt the development of a vaccine to control SARS-CoV-2 infection.

A comprehensive analysis of amino-peptidase N1 protein (APN) from Anopheles culicifacies for epitope design using Immuno-informatics models.

Analysis of the Amino-peptidase N (APN) protein from Anopheles culicifacies as a vector based Transmission Blocking Vaccines (TBV) target has been considered for malaria vaccine development. Short peptides as potential epitopes for B cells and cytotoxic T cells and/or helper T cells were identified using prediction models provided by NetCTL and IEDB servers. Antigenicity determination, allergenicity, immunogenicity, epitope conservancy analysis, atomic interaction with HLA allele specific structure models and population coverage were investigated in this study. The analysis of the target protein helped to identify conserved regions as potential epitopes of APN in various Anopheles species. The T cell epitopes like peptides were further analyzed by using molecular docking to check interactions against the allele specific HLA models. Thus, we report the predicted B cell (VDERYRL) and T cell (RRYLATTQF for HLA class I and LKATFTVSI for HLA class II) epitopes like peptides from APN protein of Anopheles culicifacies (Diptera: Culicidae) for further consideration as vaccine candidates subsequent to in vitro and in vivo analysis.

Clinco-epidemiologic study of cutaneous leishmaniasis in Bikaner, Rajasthan, India.

Cutaneous leishmaniasis is endemic to the Thar Desert of Rajasthan, Bikaner, India. The present study describes clinico-epidemiologcial data of all cases of cutaneous leishmaniasis CL in this region during 2001-2011. A total of 1,379 patients with 2,730 lesions were reported during the study period. Ages of patients ranged from 3 months to 86 years, and there was a predominance of infections in males. Most patients were from urban areas and lower middle socioeconomic groups. Lesions were dry, ulcerated nodules or plaques of different sizes commonly over face and upper limb. Skin smears were positive for parasites in 958 (69.5%) patients, and the remaining 45.8% (193 of 421) patients were positive by skin biopsy. Histopathologic analysis of the skin showed mixed granulomas consisting of macrophages, lymphocytes, epitheloid, and plasma cells. Species identification was conducted for 45 randomly selected patients by polymerase chain reaction, the infective species was Leishmania tropica. Most patients were treated with intra-lesional injections of sodium stibogluconate.