Epitope Screening Research Articles

Designing of a Multi-epitope Vaccine against the Structural Proteins of Marburg Virus Exploiting the Immunoinformatics Approach.

Marburg virus disease (MVD) caused by the Marburg virus (MARV) generally appears with flu-like symptoms and leads to severe hemorrhagic fever. It spreads via direct contact with infected individuals or animals. Despite being considered to be less threatening in terms of appearances and the number of infected patients, the high fatality rate of this pathogenic virus is a major concern. Until now, no vaccine has been developed to combat this deadly virus. Therefore, vaccination for this virus is necessary to reduce its mortality. Our current investigation focuses on the design and formulation of a multi-epitope vaccine based on the structural proteins of MARV employing immunoinformatics approaches. The screening of potential T-cell and B-cell epitopes from the seven structural proteins of MARV was carried out through specific selection parameters. Afterward, we compiled the shortlisted epitopes by attaching them to an appropriate adjuvant and linkers. Population coverage analysis, conservancy analysis, and MHC cluster analysis of the shortlisted epitopes were satisfactory. Importantly, physicochemical characteristics, human homology assessment, and structure validation of the vaccine construct delineated convenient outcomes. We implemented disulfide bond engineering to stabilize the tertiary or quaternary interactions. Furthermore, stability and physical movements of the vaccine protein were explored using normal-mode analysis. The immune simulation study of the vaccine complexes also exhibited significant results. Additionally, the protein–protein docking and molecular dynamics simulation of the final construct exhibited a higher affinity toward toll-like receptor-4 (TLR4). From simulation trajectories, multiple descriptors, namely, root mean square deviations (rmsd), radius of gyration (Rg), root mean square fluctuations (RMSF), solvent-accessible surface area (SASA), and hydrogen bonds, have been taken into account to demonstrate the inflexible and rigid nature of receptor molecules and the constructed vaccine. Inclusively, our findings suggested the vaccine constructs’ ability to regulate promising immune responses against MARV pathogenesis.

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

Klebsiella oxytoca is a gram-negative bacterium. It is opportunistic in nature and causes hospital acquired infections. Subtractive proteomics and reverse vaccinology approaches were employed to screen out the best proteins for vaccine designing. Whole proteome of K. oxytoca strain ATCC 8724, consisting of 5483 proteins, was used for designing the vaccine. Total 1670 cytotoxic T lymphocyte (CTL) epitope were predicted through NetCTL while 1270 helper T lymphocyte (HTL) epitopes were predicted through IEDB server. The epitopes were screened for non-toxicity, allergenicity, antigenicity and water solubility. After epitope screening 300 CTL and 250 HTL epitopes were submitted to IFN-γ epitope server to predict their Interferon-γ induction response. The selected IFN-γ positive epitopes were tested for their binding affinity with MHCI-DRB1 by MHCPred. The 15 CTL and 13 HTL epitopes were joined by linkers AAY and GPGPG respectively in vaccine construct. Chain C of Pam3CSK4 (PDB ID; 2Z7X) was linked to the vaccine construct as an adjuvant. A 450aa long vaccine construct was submitted to I-TASSER server for 3D structure prediction. Thirteen Linear B cells were predicted by ABCPred server and 10 sets of discontinues epitopes for 3D vaccine structure were predicted by DiscoTope server. The modeled 3D vaccine construct was docked with human Toll-like receptor 2 (PDB ID: 6NIG) by PatchDock. The docked complexes were refined by FireDock. The selected docked complex showed five hydrogen bonds and one salt bridge. The vaccine sequence was reverse transcribed to get nucleotide sequence for In silico cloning. The reverse transcribed sequence strand was cloned in pET28a(+) expression vector. A clone containing 6586 bp was constructed including the 450 bp of query gene sequence.

A novel and efficient approach to high-throughput production of HLA-E/peptide monomer for T-cell epitope screening

Over the past two decades, there has been a great interest in the study of HLA-E-restricted αβ T cells during bacterial and viral infections, including recently SARS-CoV-2 infection. Phenotyping of these specific HLA-E-restricted T cells requires new tools such as tetramers for rapid cell staining or sorting, as well as for the identification of new peptides capable to bind to the HLA-E pocket. To this aim, we have developed an optimal photosensitive peptide to generate stable HLA-E/pUV complexes allowing high-throughput production of new HLA-E/peptide complexes by peptide exchange. We characterized the UV exchange by ELISA and improved the peptide exchange readout using size exclusion chromatography. This novel approach for complex quantification is indeed very important to perform tetramerization of MHC/peptide complexes with the high quality required for detection of specific T cells. Our approach allows the rapid screening of peptides capable of binding to the non-classical human HLA-E allele, paving the way for the development of new therapeutic approaches based on the detection of HLA-E-restricted T cells.

An immunoinformatics study: designing multivalent T-cell epitope vaccine against canine circovirus

BackgroundCanine circovirus is a deadly pathogen of dogs and causes vasculitis and hemorrhagic enteritis. It causes lethal gastroenteritis in pigs, fox, and dogs. Canine circovirus genome contains two main (and opposite) transcription units which encode two open reading frames (ORFs), a replicase-associated protein (Rep) and the capsid (Cap) protein. The replicase protein and capsid protein consist of 303 amino acids and 270 amino acids respectively. Several immuno-informatics methods such as epitope screening, molecular docking, and molecular-dynamics simulations were used to craft peptide-based vaccine construct against canine circovirus. ResultsThe vaccine construct was designed by joining the selected epitopes with adjuvants by suitable linker. The cloning and expression of the vaccine construct was also performed using in silico methods. Screening of epitopes was conducted by NetMHC server that uses ANN (Artificial neural networking) algorithm. These methods are fast and cost-effective for screening epitopes that can interact with dog leukocyte antigens (DLA) and initiate an immune response. Overall, 5 epitopes, YQHLPPFRF, YIRAKWINW, ALYRRLTLI, HLQGFVNLK, and GTMNFVARR, were selected and used to design a vaccine construct. The molecular docking and molecular dynamics simulation studies show that these epitopes can bind with DLA molecules with stability. The codon adaptation and in silico cloning studies show that the vaccine can be expressed by Escherichia coli K12 strain. ConclusionThe results suggest that the vaccine construct can be useful in preventing the dogs from canine circovirus infections. However, the results need further validation by performing other in vitro and in vivo experiments.

Epitope screening of the major capsid protein within grouper iridovirus of Taiwan and the immunoprotective effect with SWCNTs as the vaccine carrier

Iridovirus can cause a mass of death in grouper, leading to huge economic loss in recent years. At present, practical vaccine is still the best way to control the outbreak of this virus. Many researches had indicated that the major capsid protein (MCP) of grouper iridovirus of Taiwan (TGIV) is an effective antigen to induce a specific immune response in grouper. However, these traditional vaccines that based on large proteins or whole organisms are faced with challenges because of the unnecessary antigenic load. Thus, in this study, we screened the dominant linear epitope within the MCP of TGIV and then, a new peptide vaccine (P2) was developed via prokaryotic expression system. Furthermore, SWCNTs was used as a vaccine carrier to enhance the immunoprotective effect. To evaluate the immunoprotective effect of this vaccine, a total of 245 fish were vaccinated with P2 (5, 10, 20 mg L−1) and SWCNTs-P2 (5, 10, 20 mg L−1) via immersion before being challenged with live TGIV at 28 days post immunization (d.p.i.). Results showed that the serum antibody titer, enzymatic activity, expression level of some immune-related genes (CC chemokine, IgM and TNF-α) and survival rate were significantly increased (SWCNTs-P2, 20 mg L−1, 100%) compared to the control group (0%). These results indicated that this peptide vaccine could effectively induce specific immune response in vaccinated groupers. Functionalized SWCNTs could serve as a carrier of the peptide vaccine to enhance the immunoprotective effect via immersion. To sum up, epitope screening might be a potential way to develop an effective vaccine nowadays, and SWCNTs might provide a practical method that can be used in large-scale vaccination, especially for juvenile fish, to fight against diseases in aquaculture industry.

Abstract LB027: Tracking and decoding the antigen specificity of peripherally derived T cells that infiltrate into solid tumors in patients treated with an autologous T cell therapy, PRIME IL-15 (RPTR-147)

Abstract PRIME IL-15 (RPTR-147) is a novel non-genetically modified, autologous, multi-clonal T cell product loaded with an IL-15Fc nanogel. The product is derived from peripheral T cells that are primed and expanded against five tumor associated antigens (TAAs) known to be overexpressed in multiple different tumor types: PRAME, NY-ESO, SSX-2, WT-1 and Survivin. 17 patients with advanced or metastatic solid-tumor cancers were dosed in a Phase I dose escalation study. Where samples were available, we characterized the reactivity and specificity of the cellular product, and tracked its persistence and localization post administration in patients. We report cellular composition of the product, along with phenotype and TAA-specificity of the T cells. Antigen specificity of patient-derived product samples was determined by Repertoire Immune Medicines' proprietary DNA-barcoded pMHC tetramer platform (CIPHERTM), as well as by functional immunological methods including IFNg ELISpot and flow cytometry assessment of activation induced markers (AIM). The CIPHER platform was used to identify TAA-specific CD8 T cell clonotypes, allowing decoding of T cell receptor (TCR) sequences and their cognate epitope/MHC. Distinct peptides binding from all five TAAs were observed across patients, and were presented across multiple HLA alleles. The phenotype of antigen-specific T cells identified via CIPHER was also assessed via single cell gene expression. Consistent with our previous studies on healthy donors, reactivity via IFNg ELISpot and AIM was also observed to all targeted TAAs. TCR CDR3 sequences were used as molecular signatures of antigen-specific cells to track the product post administration. Bulk sequencing of TCRVβ was performed on tumors pre- and post-treatment. Several product-derived, TAA-specific CD8+ T cell clonotypes were observed in the post-, but not pre-treatment biopsies. We identified additional CD4+ and CD8+ clones that were enriched in the post-treatment biopsies, and some of these clonotypes were also enriched in the product. To de-orphan these tumor infiltrating lymphocytes (TIL) of interest, we cloned selected TIL TCRs to assess their reactivity using Repertoire Immune Medicines' proprietary MCR epitope screening platform. In summary, characterization of antigen-specific T cells in the PRIME IL-15 product was performed by a variety of functional and molecular immune-based methods. Antigen specific T cell reactivities and clonotypes were identified in the peripheral repertoire. We confirmed that T cells were successfully expanded against TAAs and that these T cells do infiltrate the tumors as evidenced by bulk TCR sequencing of post-treatment tumor biopsies. Citation Format: John David Pajerowski, William Gordon, Phil Bardwell, Christine McInnis, Ji Young Hwang, Tabasum Huseni, Parul Agnihotri, Josh Francis, Christina Tarr, Florian Renoux, Sebastian Heer, Nastassja Cerreghetti-Terraneo, Marisa Loi, Erika Hamilton, Sarah Nikiforow, George Blumenschein, Mihaela Christea, Keren Osman, Anthony Shields, Harriet Kluger, Franz-Josef Obermair, Daniel Pregibon, Jan Kisielow, Anthony Coyle, Mojca Skoberne. Tracking and decoding the antigen specificity of peripherally derived T cells that infiltrate into solid tumors in patients treated with an autologous T cell therapy, PRIME IL-15 (RPTR-147) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB027.

PEG-modified subunit vaccine encoding dominant epitope to enhance immune response against spring viraemia of carp virus.

Spring viraemia of carp (SVC) caused by spring viraemia of carp virus (SVCV) can infect almost all fish of cyprinids, which bring huge economic losses to aquaculture. Glycoprotein (G), as the most important antigenic determinant protein of SVCV, is widely considered as an effective method against SVCV. In our previous study, we found that G3 (131 aa) is the potential dominant antigen epitope that induces strong immune responses similar to G protein (510 aa). Here, in order to further improve the immune effect, we reported a subunit vaccine (PEG-G3) constructed by PEG-modified dominant epitope protein (G3). The results of serum antibody production, enzyme activities and immune-related genes expression showed that PEG-G3 induces significantly stronger immune protective responses against SVCV than G3. PEG modification significantly increased the serum antibody level of the vaccine, which increased significantly after immunization and reached the peak at 21day post-vaccination. T-AOC and AKP activities in the lowest concentration group (5μg) of PEG-G3 were significantly higher than those in the highest concentration group (20μg) of G3. In PEG-G3 group, the expression of almost all genes increased at least 4 times compared with the control group. After 14-day challenge, the RPS (relative percentage survival) of the highest concentration of PEG-G3 group was 53.6%, while that of G3 group is 38.9%. Therefore, this work shows that PEG modification and dominant epitope screening may be effective methods to improve the immune protective effect of vaccines and to resist the infection of aquatic animal viral diseases.

Mining the Mycobacterium tuberculosis proteome for identification of potential T-cell epitope based vaccine candidates

Identification of protective antigens for designing a high-efficacy tuberculosis vaccine is the need of the hour. Till date only 7% of the Mycobacterium tuberculosis proteome has been explored for discovering antigens capable of activating T-cell responses. Therefore, it becomes crucial to screen the remaining Mycobacterium tuberculosis proteome for more immunodominant T-cell epitopes. An extensive knowledge of the epitopes recognized by our immune system can aid this process of finding potential T cell antigens for development of a better TB vaccine. In the present in-silico study, 237 proteins belonging to the ‘virulence, detoxification, and adaptation’ category of Mycobacterium tuberculosis proteome were targeted for T-cell epitope screening. 50825 MHC Class I and 49357 MHC Class II epitopes were generated using NetMHC3.4 and IEDB servers respectively and tested for their antigenicity and cytokine stimulation. The highest antigenic epitopes were analyzed for their world population coverage and epitope conservancy. Molecular docking and molecular dynamics simulation studies were performed to corroborate the binding affinities and structural stability of the peptide-MHC complexes. We predicted a total of 3 MHC Class I (ILLKMCWPA, FAVGMNVYV, and SLAGNSAKV) and 7 MHC Class II (DLTIGFFLHIPFPPV, RPDLTIGFFLHIPFP, LTIGFFLHIPFPPVE, VLVFALVVALVYLQF, LVFALVVALVYLQFR, PNLVAARFIQLTPVY, and LVLVFALVVALVYLQ) epitopes that can be promising vaccine candidates. These predicted epitopes belong to 6 distinct proteins: Rv0169 (mce1a), Rv3490 (ostA), Rv3496 (mce4D), Rv1085c, Rv0563 (HtpX), Rv3497c (mce4C). All these proteins are expressed at different stages in the life cycle of Mycobacterium tuberculosis and thus, the predicted epitopes could be employed as candidates for designing a multistage-multiepitopic vaccine.

T cell epitope screening of Epstein-Barr virus fusion protein gB.

Glycoprotein B (gB) is an essential fusion protein for the Epstein-Barr virus (EBV) infection of both B cells and epithelial cells and is thus a promising target antigen for a prophylactic vaccine to prevent or reduce EBV-associated disease. T cell responses play key roles in the control of persistent EBV infection and in the efficacy of a vaccine. However, to date, T cell responses to gB have been characterized for only a limited number of human leukocyte antigen (HLA) alleles. Here, we screened gB T cell epitopes in 23 healthy EBV carriers and ten patients with nasopharyngeal cancer (NPC) using a peptide library spanning the entire gB sequence. We identified twelve novel epitopes in the context of seven new HLA restrictions that are common in Asian populations. Two epitopes, gB214-223 and gB840-849, restricted by HLA-B*58:01 and B*38:02, respectively, elicited specific CD8+ T cell responses to inhibit EBV-driven B cell transformation. Interestingly, gB-specific CD8+ T cells were more frequent in healthy viral carriers with EBV reactivation than in those without EBV reactivation, indicating that EBV reactivation in vivo stimulates both humoral (VCA-gp125-IgA) and cellular responses to gB. We further found that most gB epitopes are conserved among different EBV strains. Our study broadens the diversity and HLA restrictions of gB epitopes and suggests that gB is a common target of T cell responses in healthy viral carriers with EBV reactivation. In particular, the precisely mapped and conserved gB epitopes provide valuable information for prophylactic vaccine development.ImportanceT cells are crucial for the control of persistent EBV infection and the development of EBV-associated diseases. The EBV gB protein is essential for virus entry into B cells and epithelial cells and is thus a target antigen for vaccine development. Understanding T cell responses to gB is important for subunit vaccine design. Herein, we comprehensively characterized T cell responses to full-length gB. Our results expand the available gB epitopes and HLA restrictions, particularly those common in Asian populations. Furthermore, we showed that gB-specific CD8+ T cells inhibit B cell transformation ex vivo and that gB-specific CD8+ T cell responses in vivo may be associated with intermittent EBV reactivation in asymptomatic viral carriers. These gB epitopes are highly conserved among geographically separated EBV strains. Precisely mapped and conserved T cell epitopes may contribute to immune monitoring and to the development of a gB subunit vaccine.

A minigene DNA vaccine encoding peptide epitopes derived from Galectin-1 has protective antitumoral effects in a model of neuroblastoma.

We recently identified Galectin-1 (Gal-1), a β-galactoside-binding lectin, as a novel immune regulator in neuroblastoma (NB). Here, we characterized the tolerogenic function of Gal-1 within the CD8+ T cell compartment and further evaluated its relevance as an antigen for effective DNA vaccination against NB in a mouse model. NB cells with Gal-1 knockdown (NXS-2L) exhibited significantly reduced tumor growth compared to NXS-2 NB cells. Administration of anti-CD8 antibodies prevented this antitumor effect, with primary tumor growth comparable to that from Gal-1 (G1)-sufficient NB cells. Peptide epitope screening with online databases and in silico docking experiments predicted the sequences "FDQADLTI" (#1), "GDFKIKCV" (#2), and "AHGDANTI" (#3) to have superior H2-KK binding affinities and "KFPNRLNM" (#4), "DGDFKIKCV" (#5), and "LGKDSNNL" (#6) to have superior H2-DD binding affinities. Minigenes encoding G1-KK (#1-#2-#3), G1-DD (#4-#5-#6) and the triplet with the highest affinity, G1-H (#1-#2-#4), were generated and cloned into a ubiquitin-containing plasmid (pU). Mice receiving pU-G1-KK or pU-G-1H presented a reduction in the s.c. tumor volume and weight of up to 80% compared to control mice; this reduction was associated with increased cytotoxicity of isolated splenocytes from vaccinated animals. Vaccination with pUG1-DD showed a lower capability to suppress primary tumor progression. In conclusion, Gal-1 expression by NB negatively regulates CD8+ T cells. Vaccination with DNA plasmids encoding Gal-1 epitopes overcomes immune escape, enhances CD8+ T cell-dependent immunity and displays effective antitumor activity against NB.

The Crystal Structure of the MHC Class I (MHC-I) Molecule in the Green Anole Lizard Demonstrates the Unique MHC-I System in Reptiles.

The reptile MHC class I (MCH-I) and MHC class II proteins are the key molecules in the immune system; however, their structure has not been investigated. The crystal structure of green anole lizard peptide-MHC-I-β2m (pMHC-I or pAnca-UA*0101) was determined in the current study. Subsequently, the features of pAnca-UA*0101 were analyzed and compared with the characteristics of pMHC-I of four classes of vertebrates. The amino acid sequence identities between Anca-UA*0101 and MHC-I from other species are <50%; however, the differences between the species were reflected in the topological structure. Significant characteristics of pAnca-UA*0101 include a specific flip of ∼88° and an upward shift adjacent to the C terminus of the α1- and α2-helical regions, respectively. Additionally, the lizard MHC-I molecule has an insertion of 2 aa (VE) at positions 55 and 56. The pushing force from 55-56VE triggers the flip of the α1 helix. Mutagenesis experiments confirmed that the 55-56VE insertion in the α1 helix enhances the stability of pAnca-UA*0101. The peptide presentation profile and motif of pAnca-UA*0101 were confirmed. Based on these results, the proteins of three reptile lizard viruses were used for the screening and confirmation of the candidate epitopes. These data enhance our understanding of the systematic differences between five classes of vertebrates at the gene and protein levels, the formation of the pMHC-I complex, and the evolution of the MHC-I system.

Preparation of a New Nanobody and Its Usage in Treatment of Spontaneous Hypertension

In order to improve the controllability of spontaneous hypertension, it was necessary to study a new nanobody that can inhibit calcium flow and reduce hypertension by considering the specificity and long half-life of antibody. Firstly, the antibody epitope screening technology was adopted to select a short peptide (CN8) in the E3 region of the first domain of Cav1.2 α1c subunit, the full-length complementary deoxyribonucleic acid (DNA) (cDNA) clone of which was undertaken as a template. The gene fragment in extracellular region of CN8 was amplified by polymerase chain reaction (PCR), and the pET30a-CN8 recombinant plasmid was constructed. Ribose nucleic acids (RNAs) in peripheral lymphocytes of 5 camels were extracted, and the reverse transcribed cDNA was taken as a template to realize the amplification of VHH gene fragment. After the double enzyme digestion, the pMECS vector was connected and TG1 was transformed to form a natural VHH antibody library. The phage display library was screened by CN8 protein to obtain the new nanobody (VHH-CN8). Western blotting (WB) and immunofluorescence (IF) were used to analyze the specific binding of antibody, and the calcium flux test was adopted to test the effect of antibody on calcium channels. A rat model of spontaneous hypertension and a mice model of spontaneous hypertension induced by angiotensin II (Ang II) were constructed to verify the antihypertensive effect of the prepared nanobody materials. In vitro, the VHH-CN8 antibody could realize specific binding to the Cav1.2 channel, inhibit the transfected human embryonic kidney (HEK293) cells, and prevent the increase in intracellular calcium concentration of arterial smooth muscle by adjusting the concentration and potential; in vivo, the VHH-CN8 antibody could reduce the blood pressure level of spontaneously hypertensive rats (SHRs) and mice in Ang II model, which was 196±3/186±4 mmHg (P &lt; 0.05) and 187±5/158±3 mmHg (P &lt; 0.01), respectively. Thus, it proved that the prepared nanobody could reduce the blood pressure level of spontaneous hypertension.

Neoantigen‐based vaccines as a promising strategy in cancer immunotherapeutics

AbstractThe development of cancer vaccines is based on the premise that tumor cells are potentially targetable by host immunity through the effective presentation of tumor‐associated antigens to reactive T‐cell populations. Vaccine efficacy may be limited by the functional properties of effector cells including the lack of high‐affinity T cells to target self‐antigens. In contrast, neoantigens arise from tumor‐specific mutational events that generate epitopes that are potentially seen as foreign by host immunity. As such, neoantigen‐targeted vaccination provides a high level of tumor specificity, promotes greater T‐cell effector function, and minimizes off‐target toxicities. Next‐generation sequencing and high‐throughput computational algorithms have allowed for the identification of neoantigens in solid tumor and hematologic malignancies. Vaccine generation involves the screening of potential epitopes based on HLA restriction and reactivity by the T‐cell repertoire. Early phase studies have demonstrated feasibility of vaccine production and resultant potent immunologic responses. The clinical impact of neoantigen vaccination and its incorporation into combinatorial immunotherapeutic strategies is currently being explored.

A Potent Cancer Vaccine Adjuvant System for Particleization of Short, Synthetic CD8+ T Cell Epitopes.

Short major histocompatibility complex (MHC) class I (MHC-I)-restricted peptides contain the minimal biochemical information to induce antigen (Ag)-specific CD8+ cytotoxic T cell responses but are generally ineffective in doing so. To address this, we developed a cobalt-porphyrin (CoPoP) liposome vaccine adjuvant system that induces rapid particleization of conventional, short synthetic MHC-I epitopes, leading to strong cellular immune responses at nanogram dosing. Along with CoPoP (to induce particle formation of peptides), synthetic monophosphoryl lipid A (PHAD) and QS-21 immunostimulatory molecules were included in the liposome bilayer to generate the "CPQ" adjuvant system. In mice, immunization with a short MHC-I-restricted peptide, derived from glycoprotein 70 (gp70), admixed with CPQ safely generated functional, Ag-specific CD8+ T cells, resulting in the rejection of multiple tumor cell lines, with durable immunity. When cobalt was omitted, the otherwise identical peptide and adjuvant components did not result in peptide binding and were incapable of inducing immune responses, demonstrating the importance of stable particle formation. Immunization with the liposomal vaccine was well-tolerated and could control local and metastatic disease in a therapeutic setting. Mechanistic studies showed that particle-based peptides were better taken up by antigen-presenting cells, where they were putatively released within endosomes and phagosomes for display on MHC-I surfaces. On the basis of the potency of the approach, the platform was demonstrated as a tool for in vivo epitope screening of peptide microlibraries comprising a hundred peptides.

Whole proteome screening and identification of potential epitopes of SARS-CoV-2 for vaccine design-an immunoinformatic, molecular docking and molecular dynamics simulation accelerated robust strategy

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the most cryptic pandemic outbreak of the 21st century, has gripped more than 1.8 million people to death and infected almost eighty six million. As it is a new variant of SARS, there is no approved drug or vaccine available against this virus. This study aims to predict some promising cytotoxic T lymphocyte epitopes in the SARS-CoV-2 proteome utilizing immunoinformatic approaches. Firstly, we identified 21 epitopes from 7 different proteins of SARS-CoV-2 inducing immune response and checked for allergenicity and conservancy. Based on these factors, we selected the top three epitopes, namely KAYNVTQAF, ATSRTLSYY, and LTALRLCAY showing functional interactions with the maximum number of MHC alleles and no allergenicity. Secondly, the 3D model of selected epitopes and HLA-A*29:02 were built and Molecular Docking simulation was performed. Most interestingly, the best two epitopes predicted by docking are part of two different structural proteins of SARS-CoV-2, namely Membrane Glycoprotein (ATSRTLSYY) and Nucleocapsid Phosphoprotein (KAYNVTQAF), which are generally target of choice for vaccine designing. Upon Molecular Docking, interactions between selected epitopes and HLA-A*29:02 were further validated by 50 ns Molecular Dynamics (MD) simulation. Analysis of RMSD, Rg, SASA, number of hydrogen bonds, RMSF, MM-PBSA, PCA, and DCCM from MD suggested that ATSRTLSYY is the most stable and promising epitope than KAYNVTQAF epitope. Moreover, we also identified B-cell epitopes for each of the antigenic proteins of SARS CoV-2. Findings of our work will be a good resource for wet lab experiments and will lessen the timeline for vaccine construction. Communicated by Ramaswamy H. Sarma

Comprehensive epitope mapping using polyclonally expanded human CD8 T cells and a two-step ELISpot assay for testing large peptide libraries

The genetic diversity of circulating HIV-1 strains poses a major barrier to the design, development and evaluation of HIV-1 vaccines. The assessment of both vaccine- and natural infection-elicited T cell responses is commonly done with multivalent peptides that are designed to maximally capture the diversity of potential T cell epitopes (PTEs) observed in natural circulating sequences. However, depending on the sequence diversity of viral subtypes and number of the HIV immunogens under investigation, PTE estimates, including HLA-guided computational methods, can easily generate enormous peptide libraries. Evaluation of T cell epitope specificity using such extensive peptide libraries is usually limited by sample availability, even for high-throughput and robust epitope mapping techniques like ELISpot assays. Here we describe a novel, two-step protocol for in-vitro polyclonal expansion of CD8 T cells from a single vial of frozen PBMC, which facilitated the screening 441 HIV-1 Gag peptides for immune responses among 32 HIV-1 positive subjects and 40 HIV-1 negative subjects for peptide qualification. Using a pooled-peptide mapping strategy, epitopes were mapped in two sequential ELISpot assays; the first ELISpot screened 33 large peptide pools using CD8 T cells expanded for 7 days, while the second step tested pool-matrix peptides to identify individual peptides using CD8 T cells expanded for 10 days. This comprehensive epitope screening established the breadth and magnitude of HIV-1 Gag-specific CD8 T cells and further revealed the extent of immune responses to variable/polymorphic epitopes.

Bioinformatics features and immunogenic epitopes of Echinococcus granulosus Myophilin as a promising target for vaccination against cystic echinococcosis.

Cystic echinococcosis (CE) is a neglected zoonosis especially in underdeveloped countries around the world. Hence, immunization strategies are beneficial to avert the infection. The present investigation was aimed to predict the primary biochemical characteristics of the EgMyophilin and its potential B-cell and human leukocyte antigen (HLA)-binding epitopes as a promising vaccine candidate. Different web servers were used to predict physico-chemical, antigenic and allergenic profiles, transmembrane domain, subcellular localization, post-translational modification (PTM) sites, secondary and 3D structure, tertiary model refinement and validations. B-cell and HLA-binding epitopes were predicted and screened in terms antigenicity, allergenicity, solubility (B-cell) or hydrophobicity (T-cell). The 89.82 KDa protein was non-allergenic, hydrophilic, stable, with improved thermotolerance and 94 post-translational modification sites. The secondary structure included 42.94% alpha helix, 42.82% random coil and 41.23% extended strand. Based on Ramachandran plot output for refined model, 96.2%, 99.5%, and 0.45% of amino acid residues were incorporated in the favored, allowed, and outlier regions of the refined model, respectively. After epitope screening, four B-cell and five HLA-binding epitopes possessed the highest antigenic index in the protein sequence. This paper is a premise for further researches, and provides insights for the development of a suitable vaccine against CE. More empirical studies are required using the EgMyophilin alone or in combination with other antigens/epitopes in the future.

Leishmanolysin gp63: Bioinformatics evidences of immunogenic epitopes in Leishmania major for enhanced vaccine design against zoonotic cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) is a prevalent disease in the Old World territories especially in the underdeveloped countries of the Middle East. The present study was aimed at prediction of primary biochemical features of the Leishmania major gp63 as a vaccine candidate and its potent linear B-cell epitopes as well as those epitopes with highest binding capacity to mouse and human MHC molecules. A wide range of servers were employed for prediction of physico-chemical, antigenic and allergenic traits, post-translational modifications (PTM), transmembrane domain and subcellular localization, 2D and 3D structures, tertiary model rehashing and validations. The 63.95 kDa protein was antigenic, non-allergenic, hydrophilic and stable, with improved thermotolerance and 43 PTM sites. The secondary structure encompassed 48.67% random coil, 31.4% alpha helix and 19.93% extended strand. Ramachandran analysis of the refined model showed 98.5% and 100% of residues in favored and allowed regions, respectively. After epitope screening, two linear B-cell epitopes and a number of mouse and human MHC-specific epitopes were predicted, which possessed highest antigenic index. This paper provides insights into the bioinformatics characteristics of the L. major gp63 protein as a promising vaccine candidate.

Preliminary screening of dominant epitopes of truncated p72 protein of African swine fever virus

African swine fever (ASF) causes huge economic losses in the absence of vaccines with protective efficacy. Diagnostic tools are critical for the early detection, prevention, and control of African swine fever virus (ASFV). The protein p72 is a good serological target for conducting ASF detection and surveillance. This study aimed to adopt a multiparameter analytic method to predict the dominant epitopes for p72 protein of ASFV based on a comprehensive analysis of the secondary structure, hydrophilicity, surface accessibility, and effects of the index of proteins using the software. The results showed that the area of the dominant epitope of protein p72 contained the N-terminal residues aa137–286. This study laid the foundation for developing vaccines and immunodiagnostic techniques.

Immunoinformatics aided design of peptide-based vaccines against ebolaviruses.

Ebolaviruses are at the forefront of emerging viruses and present a very perceptible threat to global peace and harmony. In the last decade, Ebola virus disease has claimed more than 90% of total lives since its inception in 1976. Owing to multiple host immune evasion methods employed by the virus and the limitations of traditional vaccine development approaches, finding a globally effective and reliable counter measure against Ebola virus remains a challenge. Highly conserved peptide fragments belonging to critical viral proteins and containing multiple epitopes which have the capacity to interact with a wide array of HLA molecules present a viable solution. Immunoinformatics or computational immunology enables rapid screening and shortlisting of plausible epitopes with a high immunogenic potential, thus, supporting expeditious elucidation of efficacious vaccine candidates. In light of above facts, we describe a computational methodology in this chapter for identification of potent peptide vaccine candidates against human infecting viruses. By applying this stringent methodology, we were able to identify multiple, immunogenic ebolavirus peptide fragments which, after verification in animal models, might be considered as part of future synthetic Ebola vaccine.