Immunization of BALB/c mice with BAB1-0278: An initial investigation of a novel potential vaccine for brucellosis based on Lactococcus Lactis vector.

The gram-negative intracellular bacterium Brucella abortus causes bovine brucellosis, a zoonotic disease that costs a lot of money. This work developed a vector vaccine against brucellosis utilizing recombinant L. lactis expressing Brucella outer membrane protein BAB1-0278. Gene sequences were obtained from GenBank. The proteins' immunogenicity was tested with Vaxijen. The target vector was converted into L. lactis after enzymatic digestion and PCR validated the BAB1-0278 gene cloning in the pNZ8148 vector. The target protein was extracted using a Ni-NTA column and confirmed using SDS-PAGE and western blot. After vaccination with the target vaccine, the expression of IgG subclasses was evaluated by the ELISA method. Cytokine production was also measured by the qPCR method in the small intestine and spleen. Lymphocyte proliferation and innate immune response (NLR, CRP, and PLR) were also assessed. Finally, after the challenge test, the spleen tissue was examined by H&E staining. BAB1-0278 was chosen because of its antigenicity score of 0.5614. A 237-bp gene fragment was discovered using enzymatic digestion and PCR. The presence of a 13 kDa protein band was confirmed by SDS-PAGE and western blot. In comparison to the PBS group, mice given the L. lactis-pNZ8148-BAB1-0278-Usp45 vaccine 14 days after priming had substantially greater levels of total IgG, IgG1, and IgG2a (P < 0.001). Also, the production of cytokines (IFN-γ, TNFα, IL-4, and IL-10) indicating cellular immunity increased compared to the control group (P < 0.001). The target group had a lower inflammatory response, morphological impairment, alveolar edema, and lymphocyte infiltration. An efficient probiotic-based oral brucellosis vaccination was created. These studies have proven that the recommended immunization gives the best protection, which supports its promotion.

In-silico design of peptides for inhibition of HLA-A*03-KLIETYFSK complex as a new drug design for treatment of multiples sclerosis disease.

Multiple sclerosis is recognized as a chronic inflammatory disease. Human leukocyte antigen (HLA) plays an important role in initiating adaptive immune responses. HLA class I is present in almost all nucleated cells and presents the cleaved endogenous peptide antigens to cytotoxic T cells. HLA-A*03 is one of the HLA class I alleles, which is reported as substantially related HLA to MS disease. In 2011, the structure of the HLA-A*03 in complex was identified with an immunodominant proteolipid protein (PLP) epitope (KLIETYFSK). This complex has been reported as an important autoantigen-presenting complex in MS pathogenesis. In this study, new peptides were designed to bind to this complex that may prevent specific pathogenic cytotoxic T cell binding to this autoantigen-presenting complex and CNS demyelination. Herein, 14 new helical peptides containing 19 amino acids were designed and their structures were predicted using the PEP-FOLD server. The binding of each designed peptide to the mentioned complex was then performed. A mutation approach was used by the BeAtMuSiC server to improve the binding affinity of the designed peptide. In each position, amino acid substitutions leading to an increase in the binding affinity of the peptide to the mentioned complex were determined. Finally, the resulting complexes were simulated for 40 ns using AMBER18 software. The results revealed that out of 14 designed peptides, "WRYWWKDWAKQFRQFYRWF" peptide exhibited the highest affinity for binding to the mentioned complex. This peptide can be considered as a potential drug to control multiple sclerosis disease in patients carrying the HLA-A*03 allele.

In silico evaluation of the downstream effect of mutated glucagon is consistent with higher blood glucose homeostasis in Galliformes and Strigiformes.

In contrast to mammals, glucagon is reported as a much more potent blood glucose modulator in birds. Interestingly, we have found p.Thr16Ser mutation, a variation in the highly conserved glucagon hormone, in Galliformes as well as Strigiformes. To check the effect of this mutation on the receptor binding of glucagon, we predicted the ancestral glucagon receptor sequence of all available Galliformes and Strigiformes species. Subsequently, we analysed their binding to the mutated and wild type glucagon (ancestral) by molecular dynamics simulation. At first, we made a model of ancestral glucagon receptor and ancestral mutated, and wild type glucagon in the order Galliformes and Strigiformes. Then we performed molecular dynamics for each Galliformes and Strigiformes receptor as well as each glucagon peptide, respectively. The final structures were used for docking simulation of glucagon to their receptors. The results of the docking simulations showed a stronger binding affinity of mutated glucagon to glucagon receptors. Afterward, we obtained blood glucose concentrations of all available Galliformes members, as well as all available members of its only taxonomic neighbour (order Anseriformes) in superorder Galloanserae. Interestingly the p.Thr16Ser mutation could finely cluster these two orders into two groups: higher blood glucose concentration (order Galliformes, 17.64 ± 1.66 mMol/L) and lower blood glucose concentration (order Anseriformes, 11.34 ± 1.11 mMol/L). Strigiformes which carry the mutated glucagon peptide show also high blood glucose concentrations (17.40 ± 1.51 mMol/L). Therefore, the results suggest this mutation, which leads to stronger binding affinity of mutated glucagon to its receptor, may be a driving force for higher blood glucose homeostasis in the related birds.

In Silico Comparison of Separate or Combinatorial Effects of Potential Inhibitors of the SARS-CoV-2 Binding Site of ACE2.

BACKGROUND: The COVID-19 is a pandemic viral infection with a high morbidity rate, leading to many worldwide deaths since the end of 2019. The RBD (Receptor Binding Domain) of SARS-CoV-2 through its spike utilizes several host molecules to enter host cells. One of the most important ones is the angiotensin-converting enzyme 2 (ACE2), an enzyme normally engaged in renin angiotensin pathway and is responsible for hypertension regulation. As different articles have analyzed separate compounds which can bind ACE2 as the potential virus entry blockers, and each one with a different molecular docking algorithm, in this study we compared all candidate compounds individually as well as their combinations using a unique validated software to introduce most promising ones. METHODS: We collected and prepared a list of all available compounds which potentially can inhibit RBD binding site of the ACE2 from different studies and then reanalyzed and compared them using the Patchdock (ver. 1.3) as a suitable molecular docking algorithm for analysis of separate compounds or their combinations. RESULTS: Saikosaponin A (e.g. in Bupleurum chinense), Baicalin (e.g. in several species in the genus Scutellaria), Glycyrrhizin (Glycyrrhiza glabra), MLN-4760 and Umifenovir better occupied ACE2 to inhibit viral RBD binding and are suggested as the top five inhibitors of the SARS-CoV-2 binding site of ACE2. Their combinatory effects were also inspiring concurrent ACE2 blockade. CONCLUSION: The results propose greatest compounds and their combinatory anti-SARS-CoV-2 effects in order to decrease the time and expenses required for further experimental designs.

In silico study of the association of the HLA-A*31:01 allele (human leucocyte antigen allele 31:01) with neuroantigenic epitopes of PLP (proteolipid protein), MBP (myelin basic protein) and MOG proteins (myelin oligodendrocyte glycoprotein) for studying the multiple sclerosis disease pathogenesis.

The main pathologic hallmark of multiple sclerosis is a demyelinating plaque that contains a prominent immunologic response dominated by T cells of the immune system. PLP (proteolipid protein), MPB (myelin basic protein), and Myelin oligodendrocyte glycoprotein (MOG) proteins are important autoantigens for the demyelinating of CNS in multiple sclerosis. There is good evidence indicating that T CD8+ cells and MHC class I molecules play an important role in this disease. The HLA-A*31:01 allele of MHC class I is a member of HLA-A3 superfamily and there is no clear report concerning the relationship of this allele with MS. Feeling this gap, we studied the possible association of the HLA-A*31:01 with MS by prediction of neuroantigenic epitopes of human MBP, PLP, and MOG proteins of myelin sheath using in silico methods. PLP did not show any neuroantigenic epitope, but the two epitopes of MBP and seven epitopes of MOG for HLA-A*31:01 were determined via bioinformatics servers. In silico study of the nine epitope showed that MOG195-204 (LIICYNWLHR) peptide of the membrane-associated/cytoplasmic part of human MOG has suitable binding affinity to the HLA-A*31:01 allele as a potential neuroantigenic epitope. Further investigations of this peptide revealed that the binding of C-terminal residue of this peptide has a more significant effect on binding to this allele than the N-terminal part of the peptide. Altogether, this combination of "LIICYNWLHR/A*31:01 allele "may play an important role in MS pathogenesis and this complex is suggested for further studies such as T cell receptor.Communicated by Ramaswamy H. Sarma.

Design and molecular dynamic simulation of a new double-epitope tolerogenic protein as a potential vaccine for multiple sclerosis disease.

One of the debilitating diseases affecting the central nervous system is multiple sclerosis (MS). As there is no definitive treatment for MS, researchers have mainly consented with optimization of strategies which slows down the progression of the disease such as specific auto-antigens tolerance induction. In this regard, the aim of this study was design of a new double-epitope protective vaccine based on interleukin (IL)-16-neuroantigens fusion proteins. First, we selected highly antigenic epitopes of myelin basic protein (MBP) (aa 84-104) and myelin oligodendrocyte glycoprotein (MOG) (aa 99-107) from available literature and our bioinformatics analysis. The correct cleavage of our constructs and major histocompatibility complex class II binding affinities of cleaved epitopes were checked and evaluated using Pepcleave and IEDB servers, respectively. Then, different combination of MOG and MBP epitopes with or without fusion to C-terminal active part of IL-16 were designed as constructs. Afterward, Modeller and Gromacs softwares used for the investigation of the MBP, and MOG epitopes antigenicity in these constructs. The results of molecular dynamics simulations showed that IL-16 in MOG + linker + MBP + IL-16 construct does not interfere with final epitopes antigenicity of MOG + linker + MBP construct. To sum up, the construct with IL-16 is suggested as a new double-epitope tolerogenic vaccine for prevention and amelioration of MS in human.

Computational comparison of two new fusion proteins for multiple sclerosis.

Multiple sclerosis (MS), as one of the human autoimmune diseases, demyelinates the neurons of the central nervous system (CNS). Activation of the T cells which target the CNS antigens is the first autoimmune event in MS. Myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP) are two proteins of the myelin sheath and have been shown to be among the high antigens contributing to the pathogenesis of MS. Production of the drugs with high specificity for the immune system diseases is a concern for various researchers. Therefore, tolerogenic vaccines are considered as a new strategy for the treatment of MS by presenting specific antigens. This study aimed to design and compare two fusion proteins by a combination of two neuroantigens linked to interleukin-16 (IL-16) (MOG-Linker-MBP-IL16 and MBP-Linker-MOG-IL16) as vaccines for MS. In this study, at first two models MOG (aa 11-30) linked to MBP (aa 13-32) was made by Modeler 9.10 and simulated for 20 ns via Gromacs 5.1.1 package. Then simulated antigen domains connected to the N-terminal domain of IL-16 and obtained structures simulated for 50 ns. The results revealed that both constructs had stable structures and the linker could keep two antigenic fragments separate enough, preventing undesired interactions. While MOG-Linker-MBP-IL16 showed better solubility, more accessible surface areas, more flexibility of its IL-16 domain, and better functionality of its IL-16 domain as well as more specific cleavage of its related epitopes after endocytosis lead to a better presentation of its antigenic property.

Y-chromosomal Status of Six Indo-European-speaking Arab Subpopulations in Chaharmahal and Bakhtiari Province, Iran.

BACKGROUND: We analyzed the Y-chromosome haplogroups of six documented Arab subpopulations that accommodated separately in different counties of Chaharmahal and Bakhtiari Province but nowadays speak Indo-European language (Luri and Farsi). METHODS: This was an outcome study conducted in 2015 to test whether there was any genetic relatedness among some Indo-European-speaking Arab subpopulation accommodated in a geographically similar region, Chaharmahal and Bakhtiari Province, Iran. Seven main Y-chromosome bi-allelic markers were genotyped in six documented Arab subpopulations. Therefore, after DNA extraction from blood samples, PCR reaction carried out by designed primers for J1-M267, J2-M172, and J-M304, I-M170, IJ-M429, F-M89 and K-M9 markers. Then PCR products after quality control on agarose gel were sequenced. RESULTS: Most subjects (83.3%) belonged to F-M89 haplogroup. These subjects belonged to K-M9 (40%), J2-M172 (40%) and I-M170 (20%). Generally, there were at least three genetically distinct ancestors with a divergence date of about 22200 yr for I, 429000 for J and 47400 before present for K haplogroup and may show separate historical migrations of studied populations. As the most recent common ancestor (MRCA) of most of these populations, haplogroup F, lived about 40000-50000 yr ago, the data do not support a nearly close genetic relationship among all of these populations. However, there were populations with same haplogroups J2 (n=2), K (n=2), or with a closer MRCA, IJ haplogroups, among I and J2 haplogroups. Finding haplogroup I, a specific European haplogroup, among Arab populations was not expected. CONCLUSION: Identification of various haplogroups in Arab subpopulations despite its small area and geographically conserved region of this part of Iranian plateau was unexpected.

The c.305del3 in IL-2 gene in Homonoidea theoretically affects IL-2/IL-2Rα interaction as well as lymphocyte homeostasis.

Interleukin-2 (IL-2) is a well-known monomeric T-cell growth factor that is produced primarily by activated CD4+ T cells following exposure to antigen. IL-2 structural analysis among primates showed a few polymorphisms as well as a 3-nucleotide deletion (c.305del3) in Hominoidea superfamily including Homo sapiens. On the other hand, the interaction of IL-2 with its alpha subunit of the receptor (IL-2Rα) is the first step for assembly of the whole IL-2R and considered as a species-specific phase. Four models of human IL-2, IL-2Rα, and their ancestral forms were made and were used for molecular dynamics (MD) simulation. Subsequently, the final structures were docked to each other and finally, the complexes were used for MD simulation. Our results showed that the above mentioned deletion led to weaker interaction of human IL-2 to its receptor relative toancestral IL-2. Association study of lymphocyte counts, as an indicator of IL-2 function, in 78 primate species with or without this deletion showed significant association of this deletion with their overall lymphocyte counts (P < .01). Therefore, it can be suggested that p.81delThr in IL-2 in Hominides superfamily interfered with interaction of IL-2 and IL-2Rα and led to overall decrease in lymphocyte counts in this superfamily of primates in comparison with other primates.