Design of inhibitor peptide sequences based on the interfacial knowledge of the protein G-IgG crystallographic complex and their binding studies with IgG.

Protein-protein interactions (PPI) have emerged as valuable targets in medicinal chemistry due to their key roles in important biological processes. The modulation of PPI by small peptides offers an excellent opportunity to develop drugs against human diseases. Here, we exploited the knowledge of the binding interface of the IgG-protein G complex (PDB:1FCC) for designing peptides that can inhibit these complexes. Herein, we have designed several closely related peptides, and the comparison of results from experiments and computational studies indicated that all the peptides bind close to the expected binding site on IgG and the complexes are stable. A minimal sequence consisting of 11 amino acids (P5) with binding constants in the range of 100 nM was identified. We propose that the main affinity differences across the series of peptides arose from the presence of polar amino acid residues. Further, the molecular dynamic studies helped to understand the dynamic properties of complexes in terms of flexibility of residues and structural stability at the interface. The ability of P5 to compete with the protein G in recognizing IgG can help in the detection and purification of antibodies. Further, it can serve as a versatile tool for a better understanding of protein-protein interactions.

Multi-pathogen based chimeric vaccine to fight against COVID-19 and concomitant coinfections.

BACKGROUND: COVID-19 has proved to be a fatal disease of the year 2020, due to which thousands of people globally have lost their lives, and still, the infection cases are at a high rate. Experimental studies suggested that SARS-CoV-2 interacts with various microorganisms, and this coinfection is accountable for the augmentation of infection severity. METHODS AND RESULTS: In this study, we have designed a multi-pathogen vaccine by involving the immunogenic proteins from S. pneumonia, H. influenza, and M. tuberculosis, as they are dominantly associated with SARS-CoV-2. A total of 8 antigenic protein sequences were selected to predict B-cell, HTL, and CTL epitopes restricted to the most prevalent HLA alleles. The selected epitopes were antigenic, non-allergenic, and non-toxic and were linked with adjuvant and linkers to make the vaccine protein more immunogenic, stable, and flexible. The tertiary structure, Ramachandran plot, and discontinuous B-cell epitopes were predicted. Docking and MD simulation study has shown efficient binding of the chimeric vaccine with the TLR4 receptor. CONCLUSION: The in silico immune simulation analysis has shown a high level of cytokines and IgG after a three-dose injection. Hence, this strategy could be a better way to decrease the disease's severity and could be used as a weapon to prevent this pandemic.

Cloning, expression and in vitro validation of chimeric multi epitope vaccine candidate against visceral leishmaniasis infection.

Visceral Leishmaniasis or Kala-Azar is one of the most severe and deadly neglected tropical disease caused by the Leishmania parasite. A few number of vaccines are going through different phases in clinical trial but failing of these vaccines in successive phase trial or less efficacy, urge to develop highly immunogenic and cost-effective treatment to get rid of deadly VL. This study focuses on the development of more potent vaccine candidate against VL. The recombinant vaccine candidate LeiSp was expressed in Pichia pastoris, followed by purification and characterization. The purified protein was also tested for any post-translation modification, which favors being a potent immunogenic candidate. Further, the expression modulation of different pro-inflammatory and anti-inflammatory cytokines was evaluated in THP1 cell lines. A significant upregulation in the expression of pro-inflammatory cytokines while no significant changes were observed in the expression of anti-inflammatory cytokines. The impact of recombinant vaccine protein candidates in infected conditions were determined. Here, upon treatment with chimeric vaccine protein candidate, we observed a considerable recovery in the expression level of pro-inflammatory cytokines, which were downregulated upon infection alone. In addition to this, we found a significant decrease in the expression of anti-inflammatory cytokines, which were upregulated during infection alone. We further validated our findings in infected hPBMCs and observed similar expression modulation of pro-inflammatory and anti-inflammatory cytokines with and without treatment. Thus, the present study indicates that the chimeric LeiSp protein which was designed using bioinformatics approaches shows a potential inductive efficacy for pro-inflammatory cytokines in Leishmania-infected cells.

Rational designing of peptide-ligand conjugates-based immunotherapy for the treatment of complicated malaria.

AIMS: Malaria deaths occur primarily due to complicated malaria associated with the sequestration of Plasmodium falciparum-infected erythrocyte (PfIE) in the capillary microvasculature. This study aims to design peptide ligand conjugates (PLCs) for treating complicated malaria using various in silico techniques. The PLC includes a natural ligand for the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1): expressed explicitly on the surface of PfIE, and a highly immunogenic peptide derived from the commonly used peptide vaccines in malaria-endemic countries. The ligand is predicted to prevent the sequestration of PfIE, and the peptide is predicted to eliminate PfIE from circulation by the pre-existing vaccine-induced immunity. MAIN METHODS: The epitope identification from the vaccines and the analysis of physicochemical properties, antigenicity, allergenicity, and toxicity were performed using SVMTriP, ProtParam, VaxiJen, AllerTop, and ToxinPred servers, respectively. The high throughput virtual screening (HTVS) and drug-like properties analysis of natural compound ligands were carried out by Schrodinger-2021 software. The molecular dynamics simulations were performed through the WebGro server. KEY FINDINGS: HTVS revealed three bioactive natural ligands for PfEMP1 from (NPASS) database. Three super immunogenic peptides were identified from malaria-endemic countries' commonly used peptide vaccines. Finally, Nine PLCs were designed with different combinations of peptides and ligands with the suitable non-cleavable triazole linker. SIGNIFICANCE: Antimalarials have been losing efficacy in a time when malaria deaths in 2020 significantly increased than in 2019. In this scenario, further research on the designed PLCs may offer some innovative immune therapeutics for complicated malaria with minimum possibilities of drug resistance.

Febrifugine dihydrochloride as a new oral chemotherapeutic agent against visceral leishmaniasis infection.

Visceral leishmaniasis (VL) is the deadliest form of leishmaniasis without a safer treatment option. This study implies drug repurposing to find a novel antileishmanial compound, namely febrifugine dihydrochloride (FFG) targeting Leishmania antioxidant system. Starting with virtual screening revealed the high binding affinity and lead likeness of FFG against the trypanothione reductase (TR) enzyme of Leishmania donovani, followed by experimental validation. The promastigotes inhibition assay gave the IC50 concentration of FFG and Miltefosine (positive control) as 7.16 ± 1.39 nM and 11.41 ± 0.29 µM, respectively. Their CC50 was found as 451 ± 12.73 nM and 135.9 ± 5.94 µM, respectively. FFG has been shown to increase the reactive oxygen species (ROS), leading to apoptosis-like cell death among L. donovani promastigotes. Spleen touch biopsy resulted in 62% and 55% decreased parasite load with FFG and miltefosine treatment, respectively. Cytokine profiling has shown an increased proinflammatory cytokine response post-FFG treatment. Moreover, FFG is safe on the liver toxicity parameter in mice post-treatment.

Designing of a bispecific antibody against SARS-CoV-2 spike glycoprotein targeting human entry receptors DPP4 and ACE2.

COVID-19 originated in Wuhan city, China, in 2019 erupted a global pandemic that had put down nearly 3 million lives and hampered the socio-economic conditions of all nations. Despite the available treatments, this disease is not being controlled totally and spreading swiftly. The deadly virus commences infection by hACE2 receptor and its co-receptors (DPP4) engagement with the viral spike protein in the lung alveolar epithelial cells, indicating a primary therapeutic target. The current research attempts to design an in-silico Bispecific antibody (BsAb) against viral spike glycoprotein and DPP4 receptors. Regdanvimab and Begelomab were identified to block the D614G mutated spike glycoprotein of SARS-CoV-2 and host DPP4 receptor, respectively. The designed BsAb was modified by using KIH (Knobs into Holes) and CrossMAb techniques to prevent heavy chain and light chain mispairings. Following the modifications, the site-specific molecular docking studies were performed, revealing a relatively higher binding affinity of BsAb with spike glycoprotein and DPP4 co-receptor than control BsAb. Also, for blocking the primary entry receptor, hACE2, an anti-viral peptide was linked to the Fc region of BsAb that blocks the hACE2 receptor by linker cleavage inside the infected host. Thus, the designed BsAb and anti-viral peptide therapy could be a promising triumvirate way to obstruct the viral entry by blocking the receptor engagement.

Chemical system biology approach to identify multi-targeting FDA inhibitors for treating COVID-19 and associated health complications.

In view of many European countries and the USA leading to the second wave of COVID-19 pandemic, winter season, the evolution of new mutations in the spike protein, and no registered drugs and vaccines for COVID-19 treatment, the discovery of effective and novel therapeutic agents is urgently required. The degrees and frequencies of COVID-19 clinical complications are related to uncontrolled immune responses, secondary bacterial infections, diabetes, cardiovascular disease, hypertension, and chronic pulmonary diseases. It is essential to recognize that the drug repurposing strategy so far remains the only means to manage the disease burden of COVID-19. Despite some success of using single-target drugs in treating the disease, it is beyond suspicion that the virus will acquire drug resistance by acquiring mutations in the drug target. The possible synergistic inhibition of drug efficacy due to drug-drug interaction cannot be avoided while treating COVID-19 and allied clinical complications. Hence, to avoid the unintended development drug resistance and loss of efficacy due to drug-drug interaction, multi-target drugs can be promising tools for the most challenging disease. In the present work, we have carried out molecular docking studies of compounds from the FDA approved drug library, and the FDA approved and passed phase -1 drug libraries with ten therapeutic targets of COVID-19. Results showed that known drugs, including nine anti-inflammatory compounds, four antibiotics, six antidiabetic compounds, and one cardioprotective compound, could effectively inhibit multiple therapeutic targets of COVID-19. Further in-vitro, in vivo, and clinical studies will guide these drugs' proper allocation to treat COVID-19.Communicated by Ramaswamy H. Sarma.

Pharmacophore based virtual screening, molecular docking, molecular dynamics and MM-GBSA approach for identification of prospective SARS-CoV-2 inhibitor from natural product databases.

COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily appeared in Wuhan, China, in December 2019. At present, no proper therapy and vaccinations are available for the disease, and it is increasing day by day with a high mortality rate. Pharmacophore based virtual screening of the selected natural product databases followed by Glide molecular docking and dynamics studies against SARS-CoV-2 main protease was investigated to identify potential ligands that may act as inhibitors. The molecules SN00293542 and SN00382835 revealed the highest docking score of -14.57 and -12.42 kcal/mol, respectively, when compared with the co-crystal ligands of PDB-6Y2F (O6K) and 6W63 (X77) of the SARS-CoV-2 Mpro. To further validate the interactions of top scored molecules SN00293542 and SN00382835, molecular dynamics study of 100 ns was carried out. This indicated that the protein-ligand complex was stable throughout the simulation period, and minimal backbone fluctuations have ensued in the system. Post-MM-GBSA analysis of molecular dynamics data showed free binding energy-71.7004 +/- 7.98, -56.81+/- 7.54 kcal/mol, respectively. The computational study identified several ligands that may act as potential inhibitors of SARS-CoV-2 Mpro. The top-ranked molecules SN00293542, and SN00382835 occupied the active site of the target, the main protease like that of the co-crystal ligand. These molecules may emerge as a promising ligands against SARS-CoV-2 and thus needs further detailed investigations. Communicated by Ramaswamy H. Sarma.

Cognizance of posttranslational modifications in vaccines: A way to enhanced immunogenicity.

Vaccination is a significant advancement or preventative strategy for controlling the spread of various severe infectious and noninfectious diseases. The purpose of vaccination is to stimulate or activate the immune system by injecting antigens, i.e., either whole microorganisms or using the pathogen's antigenic part or macromolecules. Over time, researchers have made tremendous efforts to reduce vaccine side effects or failure by developing different strategies combining with immunoinformatic and molecular biology. These newly designed vaccines are composed of single or several antigenic molecules derived from a pathogenic organism. Although, whole-cell vaccines are still in use against various diseases but due to their ineffectiveness, other vaccines like DNA-based, RNA-based, and protein-based vaccines, with the addition of immunostimulatory agents, are in the limelight. Despite this, many researchers escape the most common fundamental phenomenon of protein posttranslational modifications during the development of vaccines, which regulates protein functional behavior, evokes immunogenicity and stability, etc. The negligence about post translational modification (PTM) during vaccine development may affect the vaccine's efficacy and immune responses. Therefore, it becomes imperative to consider these modifications of macromolecules before finalizing the antigenic vaccine construct. Here, we have discussed different types of posttranslational/transcriptional modifications that are usually considered during vaccine construct designing: Glycosylation, Acetylation, Sulfation, Methylation, Amidation, SUMOylation, Ubiquitylation, Lipidation, Formylation, and Phosphorylation. Based on the available research information, we firmly believe that considering these modifications will generate a potential and highly immunogenic antigenic molecule against communicable and noncommunicable diseases compared to the unmodified macromolecules.

Immunoglobulin interface redesigning to enhance lebrikizumab mediated immunomodulation of IL-13 hyper-response.

The overexpression of interleukin-13 (IL-13) leads to autoimmune and inflammatory diseases. These adverse responses can be neutralized by using lebrikizumab as a therapeutic monoclonal antibody (mAb). Herein, we have attempted to modulate the lebrikizumab mAb to enhance its binding affinity towards IL-13. The interface residues of the lebrikizumab-IL-13 complex were determined by the PyMOL and verified by the artificial neural network-based B-cell epitope prediction server (ABCpred server) and the Paratome web server. The Cologne University Protein Stability Analysis Tool (CUPSAT) web server based mutational approach was used to identify the stable and favorable interface mutations in the lebrikizumab. Only 40 mutations were selected to generate a single mutant library, and their binding affinity for IL-13 was analyzed by using the Z-Dock server. Based on high Z-score, mutants having a better affinity with IL-13 were selected to create a multi-mutant library. The multi-mutant library was again subjected to the Z-Dock server, and their binding affinity was determined. The highest-scoring ten mAb mutants were validated by using PatchDock and ClusPro servers. The best two potential mAb mutants were identified and subjected to molecular dynamics (MD) simulations to ensure its structural stability at the microscopic level. The changes in the different bonds as the effect of mutation were assessed by LigPlot + v2.1. The AllerTOP and ToxinPred web servers were used to analyze the non-allergic and nontoxic nature of the selected mutants. Therefore, these redesigned mAb could be used for potential treatment against IL-13 associated diseased conditions.Communicated by Ramaswamy H. Sarma.

High throughput and comprehensive approach to develop multiepitope vaccine against minacious COVID-19.

The ongoing enigmatic COVID-19 outbreak, first reported from Wuhan, China, on last day of the year 2019, which has spread to 213 countries, territories/areas till 28th April 2020, threatens hundreds of thousands human souls. This devastating viral infection has stimulated the urgent development of viable vaccine against COVID-19 across the research institutes around the globe. The World Health Organization (WHO) has also confirmed that the recent pandemic is causing Public Health Emergency of International apprehension. Moreover, the earlier two pathogenic SARS-CoV and MERS-CoV and many others yet to be identified pose a universal menace. Here, in this piece of work, we have utilized an in silico structural biology and advanced immunoinformatic strategies to devise a multi-epitope subunit vaccine against ongoing COVID-19 infection. The engineered vaccine sequence is adjuvanted with ß-3 defensin and comprised of B-cell epitopes, HTL epitopes and CTL epitopes. This is very likely that the vaccine will be able to elicit the strong immune response. Further, specific binding of the engineered vaccine and immune cell receptor TLR3 was estimated by molecular interaction studies. Strong interaction in the binding groove as well as good docking scores affirmed the stringency of engineered vaccine. The interaction is stable with minimal deviation in root-mean square deviation and root-mean-square fluctuation was confirmed by the molecular dynamics simulation experiment. The immune-simulation by C-ImmSim server, which mimics the natural immune environment, yielded more potent immune response data of B-cells, Th cells, Tc cells and IgG for vaccine. The encouraging data obtained from the various in-silico works indicated this vaccine as an effective therapeutic against COVID-19.

High throughput virtual screening reveals SARS-CoV-2 multi-target binding natural compounds to lead instant therapy for COVID-19 treatment.

The present-day world is severely suffering from the recently emerged SARS-CoV-2. The lack of prescribed drugs for the deadly virus has stressed the likely need to identify novel inhibitors to alleviate and stop the pandemic. In the present high throughput virtual screening study, we used in silico techniques like receptor-ligand docking, Molecular dynamic (MD), and ADME properties to screen natural compounds. It has been documented that many natural compounds display antiviral activities, including anti-SARS-CoV effect. The present study deals with compounds of Natural Product Activity and Species Source (NPASS) database with known biological activity that probably impedes the activity of six essential enzymes of the virus. Promising drug-like compounds were identified, demonstrating better docking score and binding energy for each druggable targets. After an extensive screening analysis, three novel multi-target natural compounds were predicted to subdue the activity of three/more major drug targets simultaneously. Concerning the utility of natural compounds in the formulation of many therapies, we propose these compounds as excellent lead candidates for the development of therapeutic drugs against SARS-CoV-2.

Vaccinomics strategy to concoct a promising subunit vaccine for visceral leishmaniasis targeting sandfly and leishmania antigens.

Visceral leishmaniasis (VL) has been declared as one of the most severely neglected tropical diseases by the World Health Organization report 2017. Cumulative incidences of treatment failure and drug resistance, demanding a potential treatment and preventive strategy for VL. In this study, we have devised a multi-epitope vaccine by targeting sandfly saliva and parasite-derived membrane and secretory antigens. We have predicted the immunogenic B-cell, HTL, and CTL epitopes from all the selected protein sequences. The epitopes were then linked to the spacer sequences for providing stability and flexibility, and the construct was linked with a synthetic TLR-4 agonist namely RS09 as an adjuvant. The 3D structure of vaccine was modelled, refined and validated by generating a Ramachandran plot. Later, molecular docking was performed between the TLR-4 receptor and vaccine. The obtained docked complex was then checked for their stability by performing MD simulation. The immune dynamics simulation was done to check the probable immune response generated when the host will be exposed to the vaccine candidate. This novel vaccine strategy will provide functional and mechanistic evidence on parasite and vector-derived epitopes that could activate B- and T-cells and potentially elicit a long-lasting memory cell response.

Immunoselective algorithm to devise multi-epitope subunit vaccine fighting against human cytomegalovirus infection.

BACKGROUND: Human cytomegalovirus (HCMV) generally causes asymptomatic infection, but sometimes it may cause severe complications among immunocompromised individuals. It may also promote various malignancies like prostate cancer and breast cancer. However, even after having this severe illness, there is no effective cure yet. This situation urges the need for effective chemotherapeutics or vaccination to tackle this severe complication. METHODS: A combinatorial screening algorithm was applied to design a subunit vaccine consisting of B-cell epitopes, CTL- and HTL epitopes along with a suitable adjuvant (TLR-4 agonist) and linkers. The conservancy of CTL, HTL, and B-cell epitopes was also determined. Further, physicochemical characterization, antigenicity, and allergenicity were determined to check the safety and immunogenic behavior of the designed vaccine candidate. Later on, the 3D structure of the vaccine protein was determined, followed by molecular docking and molecular dynamics simulation with TLR-4 to check their binding free energy and complex stability. RESULT: A subunit vaccine of 964 amino acid residues was developed, having good immunogenicity and non-allergenicity behavior. The designed subunit vaccine has HTL epitopes with their ability to induce the release of IFN-γ cytokine. The sorted HTL and CTL epitopes were found to be conserved among two available strains of HCMV. It has also shown an excellent binding affinity with the TLR-4 receptor along with the formation of the stable complex as determined by a molecular dynamics simulation study. CONCLUSION: The designed subunit vaccine may have the ability to induce an immunogenic response and memory cell formation to protect against the HCMV mediated disease conditions.

Strategic Development of a Next-Generation Multi-Epitope Vaccine To Prevent Nipah Virus Zoonotic Infection.

Nipah virus (NiV) is an emerging zoonotic pathogen, reported for the recent severe outbreaks of encephalitis and respiratory illness in humans and animals, respectively. Many antiviral drugs have been discovered to inhibit this pathogen, but none of them were that much efficient. To overcome the complications associated with this severe pathogenic virus, we have designed a multi-epitope subunit vaccine using computational immunology strategies. Identification of structural and nonstructural proteins of Nipah virus assisted in the vaccine designing. The selected proteins are known to be involved in the survival of the virus. The antigenic binders (B-cell, HTL, and CTL) from the selected proteins were prognosticated. These antigenic binders will be able to generate the humoral as well as cell-mediated immunity. All the epitopes were united with the help of suitable linkers and with an adjuvant at the N-terminal of the vaccine, for the enhancement of immunogenicity. The physiological characterization, along with antigenicity and allergenicity of the designed vaccine candidates, was estimated. The 3D structure prediction and its validation were performed. The validated vaccine model was then docked and simulated with the TLR-3 receptor to check the stability of the docked complex. This next-generation approach will provide a new vision for the development of a high immunogenic vaccine against the NiV.

Emerging role of circulating microRNA in the diagnosis of human infectious diseases.

The endogenic microRNAs (miRNA) are evolutionary, conserved, and belong to a group of small noncoding RNAs with a stretch of 19-24 nucleotides. The miRNAs play an indispensable role in gene modulation at the posttranscriptional level, inclusive of stem-cell differentiation, embryogenesis, hematopoiesis, metabolism, immune responses, or infections. The miRNAs secreted from the cells and their presence in the biological fluids signifies the regulatory role of circulating miRNAs in the pathogenesis. The phenomenal expression levels of circulating miRNAs in serum or plasma during infection makes them the potential therapeutic biomarkers for the diagnosis of assorted human infectious diseases. In this article, we have accentuated the methods for the profiling of circulating miRNA as well as the importance of miRNA as biomarkers for the diagnosis of human infectious diseases. To date, numerous biomarkers have been identified for the diagnostic or prognostic purpose; for instance, miR-182, miR-486, and miR15a in sepsis; miR-320 and miR505 in inflammatory bowel disease; miR-155 and miR-1260 in influenza; miR-12, miRVP-3p, and miR-184 in arboviruses; and miR-29b and miR-125 in hepatitis infection. Nevertheless, the noninvasive diagnostic approach, with the aid of biomarkers, currently plays a decisive role in the untimely diagnosis of human infections. So, in the near future, the exploitation of circulating miRNAs as therapeutic biomarkers for the diagnosis of human infections will help us to cure the associated diseases promptly and effectively.

Contriving multiepitope subunit vaccine by exploiting structural and nonstructural viral proteins to prevent Epstein-Barr virus-associated malignancy.

Cancer is one of the common lifestyle diseases and is considered to be the leading cause of death worldwide. Epstein-Barr virus (EBV)-infected individuals remain asymptomatic; but under certain stress conditions, EBV may lead to the development of cancers such as Burkitt's and Hodgkin's lymphoma and nasopharyngeal carcinoma. EBV-associated cancers result in a large number of deaths in Asian and African population, and no effective cure has still been developed. We, therefore, tried to devise a subunit vaccine with the help of immunoinformatic approaches that can be used for the prevention of EBV-associated malignancies. The epitopes were predicted through B-cell, cytotoxic T lymphocytes (CTL), and helper T lymphocytes (HTL) from the different oncogenic proteins of EBV. A vaccine was designed by combining the B-cell and T-cell (HTL and CTL) epitopes through linkers, and for the enhancement of immunogenicity, an adjuvant was added at the N-terminal. Further, homology modeling was performed to generate the 3D structure of the designed vaccine. Moreover, molecular docking was performed between the designed vaccine and immune receptor (TLR-3) to determine the interaction between the final vaccine construct and the immune receptor complex. In addition, molecular dynamics was performed to analyze the stable interactions between the ligand final vaccine model and receptor TLR-3 molecule. Lastly, to check the expression of our vaccine construct, we performed in silico cloning. This study needed experimental validation to ensure its effectiveness and potency to control malignancy.

Exploratory algorithm to devise multi-epitope subunit vaccine by investigating Leishmania donovani membrane proteins.

Visceral leishmaniasis (VL) is a deadly parasitic infection which affects poorest to poor population living in the endemic countries. Increasing resistant to existing drugs, disease burden and a significant number of deaths, necessitates the need for an effective vaccine to prevent the VL infection. This study employed a combinatorial approach to develop a multi-epitope subunit vaccine by exploiting Leishmania donovani membrane proteins. Cytotoxic T- and helper T-lymphocyte binding epitopes along with suitable adjuvant and linkers were joined together in a sequential manner to design the subunit vaccine. The occurrence of B-cell and IFN-γ inducing epitopes approves the ability of subunit vaccine to develop humoral and cell-mediated immune response. Physiochemical parameters of vaccine protein were also assessed followed by homology modeling, model refinement and validation. Moreover, disulfide engineering was performed for the increasing stability of the designed vaccine and molecular dynamics simulation was performed for the comparative stability purposes and to conform the geometric conformations. Further, molecular docking and molecular dynamics simulation study of a mutated and non-mutated subunit vaccine against TLR-4 immune receptor were performed and respective complex stability was determined. In silico cloning ensures the expression of designed vaccine in pET28a(+) expression vector. This study offers a cost-effective and time-saving way to design a novel immunogenic vaccine that could be used to prevent VL infection. Communicated by Ramaswamy H. Sarma.

Combinatorial screening algorithm to engineer multiepitope subunit vaccine targeting human T-lymphotropic virus-1 infection.

Human T-lymphotropic virus (HTLV), the first human retrovirus has been discovered which is known to cause the age-old assassinating disease HTLV-1 associated myelopathy. Cancer caused by this virus is adult T cell leukemia/lymphoma which targets 10-20 million throughout the world. The effect of this virus extends to the fact that it causes chronic disease to the spinal cord resulting in loss of sensation and further causes blood cancer. So, to overcome the complications, we designed a subunit vaccine by the assimilation of B-cell, cytotoxic T-lymphocyte , and helper T-lymphocyte epitopes. The epitopes were joined together along with adjuvant and linkers and a vaccine was fabricated which was further subjected to 3D modeling. The physiochemical properties, allergenicity, and antigenicity were evaluated. Molecular docking and dynamics were performed with the obtained 3D model against toll like receptor (TLR-3) immune receptor. Lastly, in silico cloning was performed to ensure the expression of the designed vaccine in pET28a(+) expression vector. The future prospects of the study entailed the in vitro and in vivo experimental analysis for evaluating the immune response of the designed vaccine construct.

Examination of antigenic proteins of Trypanosoma cruzi to fabricate an epitope-based subunit vaccine by exploiting epitope mapping mechanism.

Chagas disease is a protozoan parasitic disease caused by Trypanosoma cruzi. This injurious disease spread by the circulation of the blood sucking triatomine insects and transmitted to humans. Chagas disease is endemic in Latin America and also known as American trypanosomasis. Currently, 7 million people are infected by T. cruzi infection and about 22,000 death cases were reported per year throughout the Americas. Various immunization approaches against T. cruzi infection have been examined and some of the developed vaccine showed efficacy in animal models but there is no effective and safe vaccines for humans have been developed yet. Since, the drug resistance is increasing day by day because the developed drug (nifurtimox and benznidazole) to control T. cruzi infection, failed to activate a prodrug and still no drug and vaccine have been established. To control the infection of chagas disease, here in this study we use immunoinformatics method to design multi-epitope subunit vaccine against T. cruzi infection. Moreover, on the basis of immunogenicity B and T cell epitopes were evaluated. The allergenicity, antigenicity was predicted to ensure the safety of vaccine constructs whereas, the physiochemical property showing the stable nature of final vaccine model. Further, molecular docking was performed to optimize the interaction between TLR-2 and TLR-4 (receptor) and vaccine model (ligand) complex. Molecular dynamics simulation was performed to evaluate the energy minimization; RMSD and RMSF plot which confirm the stability of TLR-2 and TLR-4 (receptor) present on immune cells and vaccine model (ligand) complex. This study needed the experimental validation for the safety and immunogenic behavior of designed vaccine protein and it may be helpful in future to control T. cruzi infection.