Design and expression of polytopic construct of cathepsin-L1, SAP-2 and FhTP16.5 proteins of Fasciola hepatica.

The enzyme-linked immunosorbent assay (ELISA) technique can play an important role in the early detection of fascioliasis. However, they have some diagnostic limitations, including cross-reaction with other helminths. It seems that the combination of recombinant parasite proteins as antigen can reduce these problems. Hence, the present study was aimed to design and confirm the antigenic recombinant multi-epitope (rMEP) construct of three protein epitopes (linear and conformational B-cell epitopes) of the parasite using immunoinformatic tools. For this purpose, the tertiary structures of Fasciola hepatica cathepsin-L1, saposin-like protein 2 and 16.5-kDa tegument-associated protein were predicted using the I-TASSER server. Validation of the modelled structures was performed by Ramachandran plots. The antigenic epitopes of the proteins were achieved by analysing the features of the IEDB server. The synthesized gene was cloned into the pET-22b (+) expression vector and transformed into the Escherichia coli BL21. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to verify and analyse the expression of the rMEP protein. Western blotting was utilized to confirm rMEP protein immunogenicity in two forms, one using an anti-His tag antibody and the other with human pooled sera samples (fascioliasis, non-fascioliasis and negative control sera). Our results demonstrated that the rMEP designed for the three proteins of F. hepatica was highly antigenic, and immune-detection techniques confirmed the antigen specificity. In conclusion, the presented antigenic multi-epitope may be very helpful to develop serodiagnostic kits such as indirect ELISA to evaluate the proper diagnosis of fascioliasis in humans and ruminants.

Novel Applications of Immuno-bioinformatics in Vaccine and Bio-product Developments at Research Institutes.

There are many challenges in the field of public health sciences. Rational decisions are required in order to treat different diseases, gain knowledge and wealth regarding research, and produce biological or synthetic products. Various advances in the basic laboratory science, computer science, and the engineering of biological production processes can help solve the occurring problems. Bioinformatics is defined as a field of science combined of biology, mathematics, physics, chemistry, and computer sciences. Recently, bioinformatics has been extensively used in the designing of the epitope, vaccines, antibodies, adjuvants, diagnostic kits, and therapeutic purposes (e.g., proteins, peptides, or small molecules). Moreover, bioinformatics includes chemoinformatics that has been employed to produce various biological or chemical products to target and combat pathogens. Bioinformatics is involved in other areas of data analysis and prediction, such as structural biology, system biology, phylogeny, population genetics, and next-generation data sequencing. To the best of our knowledge, no published study coherently described the benefits of bioinformatics fields applied for medication development or diagnostic aims in bio-productive and pharmaceutical/vaccine companies. Therefore, in the current review, we attempted to present the available bioinformatics resources, practical experiences, and other findings in the mentioned field along with providing a harmonized and applied model(s). The key points presented in the current review may help to elevate production and reduce the costs for the development of novel vaccines, medicines, and antibodies. In addition, these methods can facilitate the identification of organisms and may guarantee the quality of biological products.

Phylogenetic Analysis of bor Gene in an Escherichia coli Strain 1378 (O78:K80) Isolated from an Avian Colibacillosis Case in Tehran, Iran.

Colibacillosis is known as a fatal bacterial disease resulting in a high level of commercial loss worldwide. This study amid to elucidate the sequence, genetic characteristics, and phylogeny of the bor gene in Escherichia coli (E. coli) strain c1378 (O78:K80) isolated from avian colibacillosis in Iran and develop a rapid and optimal polymerase chain reaction (PCR) molecular-based technique with specific primers to detect this gene in E. coli. A virulent avian E. coli (i.e., laboratory designation E. coli strain c1378) isolated from a chicken with systemic colibacillosis from a broiler farm in Tehran, Iran, in 2004 was used as a source of the bor gene. After DNA extraction, PCR method was used to amplify the bor gene. A 658 bp fragment of the bor gene was amplified, sequenced, blasted, and phylogenetically studied. The most similar sequences to the bor gene in E. coli strain c1378 were E. coli APEC O78, Enterobacteria phage HK630, and Escherichia coli BW2952, respectively. There was a high similarity between the bor gene in E. coli bacteria with their phage and plasmid. Moreover, a high similarity was observed between the bor and iss genes (approximately 92%) showing that they were homologous genes. In addition, the similarity analysis of different bacterial species, as well as their plasmid and bacteriophage, to the bor gene indicated that the highest similarity to O78:K80 was related to Paracoccidioides brasiliensis, Bacillus thuringiensis CT43 plasmid pBMB0558, and Salmonella enterica subsp. enterica serovar Kentucky strain CVM29188 plasmid, respectively. Altogether, the results of the present study confirmed the presence of the bor gene in the studied isolates and clarified its sequence, phylogenetic relationship, and similarities of E. coli strain c1378 (O78:K80) isolated from avian colibacillosis.

Designing a polytopic complex vaccine candidate against Gallibacterium anatis: an In-silico study.

The haemolytic biovar of Gallibacterium anatis (G. anatis) is responsible for urogenital, gastrointestinal, and respiratory diseases in chickens. There are numerous reports on the resistance of G. anatis to antibiotics and recurrence of the disease, which raise concerns about antimicrobial treatment efficiency. Vaccination has been considered as the most feasible procedure of prevention in high risk farms. Subunit vaccines containing immunogenic components can have practical protective value in preventive measures regarding the infection. The present study aimed to introduce a polytopic vaccine candidate based on epitope detection. All registered sequences of four immunogenic proteins, includig Flfa, GTxA, Gab_1309, and Gab_2348 were retrieved and directed for variational analysis. A vaccine isolate was selected for each protein and tested for B-cell epitope mapping using different tools. Furthermore, consensus selected immunogenic regions with special patterns fused together by flexible linkers were integrated into two constructs and checked for the best status of proteasomal cleavage sites, as well as hydropathy plot. Moreover, back translations, along with codon optimization were performed, and then some tags were added to the constructs. The selected consensus B-cell immunogenic epitopes were for 12656: AA114-181, 7990: AA114-181, Avicor: AA42-77, 134-197, and IPDH: 61-155 for Flfa protein, AA185-235, AA372-457, and AA807-941 for GtxA-N, AA260-305, AA340-400, and AA110-146 for Gab-1309, and AA125-AA175 for Gab-2348. Two suitable patterns of attachment were selected from the different fusion patterns of epitopes in B-cell polytopic vaccinal constructs. Finally, the examination of these constructs showed their effect and efficacy for immune system stimulation. Based on bioinformatics results, these immunogens could be utilized as potential candidates to develop polytopic protective vaccines and design diagnostic kits.

Sequencing and In Silico Multi-aspect Analysis of S1 Glycoprotein in 793/B Serotype of Infectious Bronchitis Virus Isolated From Iran in 2003 and 2011.

Infectious bronchitis (IB) is an acute, highly contagious, and economically important viral disease of chickens. The S1 subunit from Spike (S) protein plays the major role in protective immunity and is involved in the host-virus interactions, as well as infectious bronchitis virus (IBV) serotyping. Aim of the present study was multi-aspect analysis of the molecular and immunological features of 5' part belonging to the S1 glycoprotein sequence of Iranian 793/B IBV strain isolates. This might ideally help in characterization, prevention, and vaccine development. The tissue samples were prepared, followed by virus isolation, reverse transcription polymerase chain reaction and restriction fragment length polymorphism analysis. In addition, sequencing and registration of the sequences in the National Center for Biotechnology Information were performed. Moreover, 12 sequences were retrieved from Fars province, Iran. The next steps included evaluation of conservation/variability along the sequences, phylogenetic analysis, estimation of the average evolutionary divergence over all the sequence pairs, predicting the phosphorylation/N-glycosylation/palmitoylation sites, and the final analysis of antigenicity. The findings of alignment, entropy plot, and pairwise similarity analysis revealed 17 hypervariable regions. The isolates belonging to Tehran were clustered in phylogenetic tree, and the most similar isolates to them were ADW11182 and ADW11183. Location of some of the N-glycosylation/phosphorylation/palmitoylation points indicated that these sites were conserved among the isolates. Furthermore, the frequency of epitopes and their scores reflect the high immunogenicity of S1 protein in 793/B serotype. Analysis of the primary and secondary structures demonstrated that their parameters had variable values and were different regarding the number and location of α-helix, β-strand, and coils. According to our findings, the Iranian isolates of 793/B serotype change their molecular characteristics during time and in different geographical regions. These alterations might account for failure in prevention programs and differences in virulence and pathogenicity.

Production of a Human Recombinant Polyclonal Fab Antivenom against Iranian Viper Echis carinatus.

Venomous snakebite is a life-threatening injury in many tropical and subtropical areas including Iran. The gold standard treatment option for human envenomation is the use of antivenoms. Despite the unique effects of horse-derived antivenoms on the treatment of snakebite, they are not fully perfect and need improvements. In this study, human recombinant Fab fragment antivenom was produced in Rosetta-g bacterium using a gene library constructed in the previous study. The prepared Fab was purified in several steps, desalted, and lipopolysaccharide-depleted using ammonium sulfate solution and dialysis against phosphate buffer and Triton X-114 solution, respectively. Subsequently, the product was initially confirmed by the sodium dodecyl sulfate polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. Finally, the neutralization potency of the product was investigated in laboratory Syrian Mice. The obtained results showed corresponding reduced bands to Fab fragment with the molecular weight of about 28 kDa at a concentration of 3.1 mg/ml. There was a significant difference between the groups in terms of ELISA test (P<0.05). The neutralization potency of the product against the venom of Echis carinatus (E. carinatus) was about 7 LD50/ml (54.6 µg/ml) when tested on mice. Based on the results, the Fab fragment antivenom had the ability to neutralize the in vivo biological activity of the venom of Iranian E. carinatus. However, further studies are recommended to reach a suitable concentration of antivenom fragment.

Analysis of variations, structures, and phylogenic characteristics of bovine leukocyte antigen DRB3 exon2.

Bovine leukocyte antigen (BoLA) DRB3 is a highly polymorphic gene in major histocompatibility complex (MHC) class II that plays a central role in immune responses and production factors. As of yet, molecular and evolutionary characteristics of BoLA-DRB3.2* have not been as fully understood as human and mouse. Therefore, we attempted to analyze variability and phylogeny of BoLA-DRB3.2* and illustrate some novel practical evidence on interspecies diversity, the resistance /susceptibility points in cattle breeding, and vaccine design. Initially, BoLA-DRB3.2* alleles and orthologous exons in the selected livestock were retrieved and checked. In the next step, the secondary/tertiary structure of BoLA-DRB3.2*24 gene product was modeled and validated. Then, hypervariable regions (HVRs) of alleles were identified by hybrid approaches. In the last step, interspecies relationship, allele’s phylogeny/grouping, and estimate of average evolutionary divergence were explored. Shannon entropy variation analysis showed eight HVRs and three semi-variable regions in BoLADRB3.2* alleles. These HVRs were present in all the three sub-structures and dominantly existed in alpha helix. In addition, strong relationships and little diversity were noted in phylogenetic trees of cattle, buffaloes, sheep, and goats. Furthermore, there was some evidence on divergence of DRB3 before speciation among the mentioned species and possibility of cross prediction resistance/susceptibility alleles. Finally, DRB3 alleles were grouped into seven clusters, and older and newer alleles were identified. The results show that similar studies should be done in other animals to better understand the nature of the DRB3 attributes.