In Silico Design of a New Multi-Epitope Peptide-Based Vaccine Candidate Against Q Fever

Molecular Biology - Novel types of the vaccines with high immunogenicity and low risks, including epitope-based vaccines, are sought. Among zoonotic disease, Q fever caused by Coxiella burnetii is...     

Designing a Fusion Protein Vaccine Against HCV: An In Silico Approach

Hepatitis C virus (HCV) infection is a major global issue that leads to serious liver disease such as chronic liver inflammation and hepatocellular carcinoma. At present, no approved vaccine is available for control or treatment of HCV infection. Therefore, the development of an efficient vaccine against HCV is an urgent need. Today, designing an effective vaccine against hepatitis C is one of the outmost propriety for researchers. Fusion protein vaccines containing the immunogen proteins and adjuvant molecules are able to stimulate both humoral and cellular responses that are crucial for eradicating HCV infection. Herein, in silico design of fusion forms of vaccine candidates against HCV, including flagellin (fliC) from Pseudomonas aeruginosa and NS5B antigen (NT300) from HCV was performed. First, two forms of fusion protein (NT300-fliC and fliC-NT300) were designed and analyzed using different bioinformatics tools. For this aim, the Iterative threading assembly refinement (I-TASSER) server was used for modeling the fusion forms of protein; namely, NT300-fliC and fliC-NT300, then the high-rank 3D model of fusion protein was selected, subsequently various physico-chemical, and structural parameters were examined bioinformatically. After the selection of the best construct (fliC-NT300), the interaction of flagellin part of vaccine with toll-like receptor 5 (TLR5) was evaluated via docking studies. Our results represented that based on data obtained from various servers, and the docking analyses of two constructs, fliC-NT300 fusion form showed better results than NT300-fliC. For this reason, the fliC-NT300 form was selected for further evaluations. In sum, structural and immunological computational studies showed that the fliC-NT300 can be introduced as a prophylactic or therapeutic candidate vaccine against the HCV, after the efficacy of that was confirmed via in vitro and in vivo assays.

     

In Silico Analysis of Acinetobacter baumannii Phospholipase D as a Subunit Vaccine Candidate

The rate of human health care-associated infections caused by Acinetobacter baumannii has increased significantly in recent years for its remarkable resistance to desiccation and most antibiotics. Phospholipases, capable of destroying a phospholipid substrate, are heterologous group of enzymes which are believed to be the bacterial virulence determinants. There is a need for in silico studies to identify potential vaccine candidates. A. baumannii phospholipase D (PLD) role has been proved in increasing organism’s resistance to human serum, destruction of host epithelial cell and pathogenesis in murine model. In this in silico study high potentials of A. baumannii PLD in elicitation of humoral and cellular immunities were elucidated. Thermal stability, long half-life, non-similarity to human and gut flora proteome and non-allergenicity were in a list of A. baumannii PLD positive properties. Potential epitopic sequences were also identified that could be used as peptide vaccines against A. baumannii and various other human bacterial pathogens.     

In Silico Design of Epitope-Based Allergy Vaccine Against Bellatella germanica Cockroach Allergens

International Journal of Peptide Research and Therapeutics - Asthma is a chronic inflammatory airway disorder that is a subclass of IgE mediated hypersensitivity. Cockroach allergy is one of the...     

In Silico Analyses of Staphylococcal Enterotoxin B as a DNA Vaccine for Cancer Therapy

Immunotherapy has been suggested as a compelling alternative approach for conventional breast cancer treatment methods. Despite the paramount rolesof T cells in this approach, insufficient numbers of them in the combat against progressive tumor growth still remain to be dealt with. Super antigens are a class of antigens, capable of eliciting T cell proliferation response against desired antigens. Staphylococcal enterotoxin B (SEB) is categorized as a super antigen, its anti-tumor properties has been previously reported. However, to the best our knowledge, SEB has not been ever administered as a DNA construct. In the present study, we exploited bioinformatics tools to assess the immunoreactivity of a SEB-coding DNA construct that serves as a DNA vaccine for breast cancer therapy. Potential B and T (MHC class I and II binders) cell epitopes of the hypothetically expressed protein, along with its sub cellular localization were predicted. Moreover, probable glycosylation and phosphorylation sites within the protein sequence were determined. The gene sequence was optimized according to murine model codon bias and its mRNA stability was analyzed. Employing an integrative in silico approach, we revealed that apparently the construct could be efficiently expressed in mouse model. Moreover, the hypothetically expressed protein could act as an amenable adjuvant in cancer immunotherapy.     

In Silico Analysis of Synaptonemal Complex Protein 1 (SYCP1) and Acrosin Binding Protein (ACRBP) Antigens to Design Novel Multiepitope Peptide Cancer Vaccine Against Breast Cancer

Multiepitope cancer vaccines are manifesting as the future of breast cancer immunotherapy. In the present study, high immunogenic fragments of synaptonemal complex protein 1 (SYCP1) and acrosin binding protein (ACRBP) antigens were selected according to MHCs binding affinity and CD8⁺ cytotoxic T lymphocytes’ (CTL) epitopes by various immunoinformatic servers. (RCQHKIAEMVALMEKHKHQYDKI) residue from p702–724 SYCP1 and the (YIQYPNYCSFKSQQCL) residue from p481–496 ACRBP were selected as the immunodominant fragments. Then, the selected fragments were fused together with a furin-sensitive linker to form the final peptide vaccine construct. The cleavage sites, TAP transport efficiency, CTL epitopes, post-translational modifications, and B cells epitopes were predicted for the final construct. The final construct contained appropriate CTLs epitopes and also, several linear and conformational B cell epitopes. Also, it exhibited 90.37% HLA population coverage in the world. Therefore, these preliminary results require validation by in vitro and in vivo experiments.

     

In Silico Design of Multimeric HN-F Antigen as a Highly Immunogenic Peptide Vaccine Against Newcastle Disease Virus

Application of molecular biology techniques to the production of new vaccines against different strains of the Newcastle disease virus (NDV) has been the subject of recent research reports. Development of improved techniques for genome sequencing has led to the recognition of protective mechanisms and the identification of possible candidate antigens. Such procedures could generate meaningful results regarding the virulence determinants of NDV. This study proposed an in silico approach by assembling potential and conserved epitopic regions of hemagglutinin–neuraminidase (HN) and fusion (F) glycoproteins of NDV to induce multiepitopic responses against the virus. Epitope predictions showed that the hypothetical synthetic construct could induce immature B and T cell epitopes that expect a high immune response because of their location in four distinct parts of the construct, namely the head, stalk and the heptad repeated regions known as the HRA and HRB domains. Most regions of the chimeric construct were found to have high antigenic propensity and surface accessibility, which further confirmed the strategy for selection of precise continuous and discontinuous epitopes of HN and F antigens. Thermodynamic folding of mRNA structures revealed correct folding of the RNA construct, indicating good stability of the mRNA to increase the efficiency of translation in the target host. The three-dimensional structure of the native HN-F chimeric protein was successfully generated and validated as a proper model which may define reliability, structural quality and conformation.     

Designing of a Novel Fusion Protein Vaccine Candidate Against Human Visceral Leishmaniasis (VL) Using Immunoinformatics and Structural Approaches

Leishmaniasis is caused by an obligate intracellular protozoan parasite. The clinical forms of leishmaniasis differ from cutaneous leishmaniasis, mucocutaneous leishmaniasis and visceral leishmaniasis (VL) which depend on the parasite species and the host’s immune responses. There are significant challenges to the available anti-leishmanial drug therapy, particularly in severe forms of disease, and the rise of drug resistance has made it more difficult. Currently, no licensed vaccines have been introduced to the market for the control and elimination of VL. A potential target for use in candidate vaccines against leishmaniasis has been shown to be leishmania Kinetoplastid membrane protein-11 (KMP-11) antigen. In this study, we chose KMP-11 antigen as target antigen in our vaccine construct. In addition, B-type flagellin (fliC) was used as an adjuvant for enhancing vaccine immunogenicity. The GSGSGSGSGSG linker was applied to link the KMP-11 antigen and fliC (KMP-11-fliC) to construct our fusion protein. Bioinformatics approaches such as; 3D homology modeling, CTL, B-cell, MHC class I and II epitopes prediction, allergenicity, antigenicity evaluations, molecular docking, fast simulations of flexibility of docked complex and in silico cloning were employed to analysis and evaluation of various properties of the designed fusion construct. Computational results showed that our engineered structure has the potential for proper stimulation of cellular and humoral immune responses against VL. Consequently, it could be proposed as a candidate vaccine against VL according to these data and after verifying the efficacy of the candidate vaccine through in vivo and in vitro immunological tests.

     

In Silico Prediction of Epitopes in Virulence Proteins of Mycobacterium ulcerans for Vaccine Designing

Background Mycobacterium ulcerans is the fundamental agent of the third most common Mycobacterial disease known as Buruli Ulcer (BU). It is an infection of the skin and soft tissue affecting the human population worldwide. Presently, the vaccine is not available against BU.ObjectiveThis study aimed to investigate the vaccine potential of virulence proteins of M. ulcerans computationally.MethodsChromosome encoded virulence proteins of Mycobacterium ulcerans strain Agy99 were selected, which were available at the VFDB database. These proteins were analyzed for their subcellular localization, antigenicity, and human non-homology analysis. Ten virulence factors were finally chosen and analyzed for further study. Three-dimensional structures for selected proteins were predicted using Phyre2. B cell and T cell epitope analysis was done using methods available at Immune Epitope Database and Analysis Resource. Antigenicity, allergenicity, and toxicity analysis were also done to predict epitopes. Molecular docking analysis was done for T cell epitopes, those showing overlap with B cell epitopes.ResultsSelected virulence proteins were predicted with B cell and T cell epitopes. Some of the selected proteins were found to be already reported as antigenic in other mycobacteria. Some of the predicted epitopes also had similarities with experimentally identified epitopes of M. ulcerans and M. tuberculosis which further supported our predictions.ConclusionIn-silico approach used for the vaccine candidate identification predicted some virulence proteins that could be proved important in future vaccination strategies against this chronic disease. Predicted epitopes require further experimental validation for their potential use as peptide vaccines.     

One Step Membrane Incorporation of Viral Antigens as a Vaccine Candidate Against HIV

Liposomes can been used as potential immunoadjuvants, because they have the ability to elicit both a cellular mediated immune response and a humoral immune response. Studies have shown liposomes to be effective immunopotentiators in hepatitis A and influenza vaccines. For all these purposes, liposomes can be prepared by different methods. After disperging suitable membrane lipids in an aqueous phase and spontaneous formation of multilamellar large vesicles (MLV), mechanical procedures such as ultrasonication, homogenization by a French press or by other high pressure devices and, or extrusion through polycarbonate membranes with defined pore sizes lead to a reduction in size and number of lamellae of the vesicles. A second group of preparation procedures uses suitable detergents, e.g., bile salts or alkylglycosides. A third group of procedures starts with dissolving the lipids in an organic solvent and mixing it with an aqueous phase. The concentration of the organic solvent is then reduced by suitable procedures.

Here we present a new technique for the preparation of liposomes with associated membrane proteins, where lipid vesicles are formed immediately after injection into a micellar protein solution. The model membrane protein used for these studies is a truncated recombinant gp41 produced in E. coli. This viral membrane antigen is a possible candidate protein for the establishment of HIV-vaccines.

The data presented here, show an efficient and reproducible one step membrane protein encapsulation procedure into liposomes in a closed and sterile containment. We examined encapsulation efficiency, membrane protein conformation and immunogenicity of this possible liposomal vaccine candidate, which can be produced in GMP-compliant quality with the described technique.     

Subtractive Proteomics and Immuno-informatics Approaches for Multi-peptide Vaccine Prediction Against Klebsiella oxytoca and Validation Through In Silico Expression

International Journal of Peptide Research and Therapeutics - Klebsiella oxytoca is a gram-negative bacterium. It is opportunistic in nature and causes hospital acquired infections....     

In Silico Identification of Epitope-Based Peptide Vaccine for Nipah Virus

International Journal of Peptide Research and Therapeutics - Nipah virus was first appeared in Malaysian 1998, which further found be outburst in neighbor countries i.e. Bangladesh, Singapore, and...     

Immunoinformatics Driven Prediction of Multiepitopic Vaccine Against Klebsiella pneumoniae and Mycobacterium tuberculosis Coinfection and Its Validation via In Silico Expression

International Journal of Peptide Research and Therapeutics - Klebsiella pneumoniae and Mycobacterium tuberculosis coinfection is one of the most lethal combinations that has been becoming frequent...     

Genetic Variation In Vitro and In Vivo of an Attenuated Lassa Vaccine Candidate

The attenuated Lassa vaccine candidate ML29 is a laboratory-produced reassortant between Lassa and Mopeia viruses, two Old World arenaviruses that differ by 40% in nucleic acid sequence. In our previous studies, ML29 elicited sterilizing immunity against Lassa virus challenge in guinea pigs and marmosets and virus-specific cell-mediated immunity in both simian immunodeficiency virus (SIV)-infected and uninfected rhesus macaques. Here, we show that ML29 is stable after 12 passages in vitro without losing its plaque morphology or its attenuated phenotype in suckling mice. Additionally, we used deep sequencing to characterize the viral population comprising the original stock of ML29, the stock of ML29 after 12 passages in Vero cells, and the ML29 isolates obtained from vaccinated animals. Twenty-seven isolates bore approximately 77 mutations that exceeded 20% of the single-nucleotide polymorphism (SNP) changes at any single locus. Of these 77 mutations, 5 appeared to be host specific, for example, appearing in mice but not in primates. None of these mutations were reversions of ML29 to the sequences of the parental Lassa and Mopeia viruses. The host-specific mutations indicate viral adaptations to virus-host interactions, and such interactions make reasonable targets for antiviral approaches. Variants capable of chronic infection did not emerge from any of the primate infections, even in immune-deficient animals, indicating that the ML29 reassortant is reasonably stable in vivo. In conclusion, the preclinical studies of ML29 as a Lassa virus vaccine candidate have been advanced, showing high levels of protection in nonhuman primates and acceptable stability both in vitro and in vivo.     

Designing a Novel Multi-epitope Peptide Vaccine Against Pathogenic Shigella spp. Based Immunoinformatics Approaches

International Journal of Peptide Research and Therapeutics - Shigella spp. causes severe diarrhea and dysenteric disease, which known as shigellosis. Until now, no licensed vaccine is available for...     

稳定、无抗药的痢疾福氏2a和宋内双价菌苗候选株的构建

通过体内外基因重组,将大肠杆菌粘附因子cs3基因定位整合到痢疾杆菌福氏2a疫苗株T32菌染色体的asd基因内,使asd基因灭活;将来内O抗原基因克隆至无抗药性表达载体pXL378,获得重组质粒pXL390,将其转化asd-的T32受体菌,构建成福氏2a和宋内双价苗苗株FS01。实验表明:重组质粒pXL390在不带任何抗菌素基因的情况下,在asd-的T32受体菌内是稳定的。FS01株遗传稳定,能表达两种痢疾菌的PLS-O抗原,无明显毒性作用。动物试验表明,以FS01株皮下免疫的小鼠对福氏2a和宋内有毒株的腹腔攻击有100％的保护。     

In Silico Analysis of Bacteriocins from Lactic Acid Bacteria Against SARS-CoV-2

Probiotics and Antimicrobial Proteins - The COVID-19 pandemic caused by a novel coronavirus (SARS-CoV-2) is a serious health concern in the twenty-first century for scientists, health workers, and...     

Preclinical Testing of a Vaccine Candidate against Tularemia

Tularemia is caused by a gram-negative, intracellular bacterial pathogen, Francisella tularensis (Ft). The history weaponization of Ft in the past has elevated concerns that it could be used as a bioweapon or an agent of bioterrorism. Since the discovery of Ft, three broad approaches adopted for tularemia vaccine development have included inactivated, live attenuated, or subunit vaccines. Shortcomings in each of these approaches have hampered the development of a suitable vaccine for prevention of tularemia. Recently, we reported an oxidant sensitive mutant of Ft LVS in putative EmrA1 (FTL_0687) secretion protein. The emrA1 mutant is highly sensitive to oxidants, attenuated for intramacrophage growth and virulence in mice. We reported that EmrA1 contributes to oxidant resistance by affecting the secretion of antioxidant enzymes SodB and KatG. This study investigated the vaccine potential of the emrA1 mutant in prevention of respiratory tularemia caused by Ft LVS and the virulent SchuS4 strain in C57BL/6 mice. We report that emrA1 mutant is safe and can be used at an intranasal (i. n.) immunization dose as high as 1x10⁶ CFU without causing any adverse effects in immunized mice. The emrA1 mutant is cleared by vaccinated mice by day 14–21 post-immunization, induces minimal histopathological lesions in lungs, liver and spleen and a strong humoral immune response. The emrA1 mutant vaccinated mice are protected against 1000–10,000LD₁₀₀ doses of i.n. Ft LVS challenge. Such a high degree of protection has not been reported earlier against respiratory challenge with Ft LVS using a single immunization dose with an attenuated mutant generated on Ft LVS background. The emrA1 mutant also provides partial protection against i.n. challenge with virulent Ft SchuS4 strain in vaccinated C57BL/6 mice. Collectively, our results further support the notion that antioxidants of Ft may serve as potential targets for development of effective vaccines for prevention of tularemia.