Immunodominant T-cell Epitopes Research Articles

Rational immunosilencing of a promiscuous T-cell epitope in the capsid of an adeno-associated virus.

Owing to the immunogenicity of adeno-associated viruses (AAVs), gene therapies using AAVs face considerable obstacles. Here, by leveraging ex vivo T-cell assays, the prediction of epitope binding to major histocompatibility complex class-II alleles, sequence-conservation analysis in AAV phylogeny and site-directed mutagenesis, we show that the replacement of amino acid residues in a promiscuous and most immunodominant T-cell epitope in the AAV9 capsid with AAV5 sequences abrogates the immune responses of peripheral blood mononuclear cells to the chimaeric vector while preserving its functions, potency, cellular specificity, transduction efficacy and biodistribution. This rational approach to the immunosilencing of capsid epitopes promiscuously binding to T cells may be applied to other AAV vectors and epitope regions.

A T cell-targeted multi-antigen vaccine generates robust cellular and humoral immunity against SARS-CoV-2 infection

SARS-CoV-2, the etiological agent behind the coronavirus disease 2019 (COVID-19) pandemic, has continued to mutate and create new variants with increased resistance against the WHO-approved spike-based vaccines. With a significant portion of the worldwide population still unvaccinated and with waning immunity against newly emerging variants, there is a pressing need to develop novel vaccines that provide broader and longer-lasting protection. To generate broader protective immunity against COVID-19, we developed our second-generation vaccinia virus-based COVID-19 vaccine, TOH-VAC-2, encoded with modified versions of the spike (S)and nucleocapsid (N)proteins as well as a unique poly-epitope antigen that contains immunodominant Tcell epitopes from seven different SARS-CoV-2 proteins. We show that the poly-epitope antigen restimulates Tcells from the PBMCs of individuals formerly infected with SARS-CoV-2. In mice, TOH-VAC-2 vaccination produces high titers of S- and N-specific antibodies and generates robust Tcell immunity against S, N, and poly-epitope antigens. The immunity generated from TOH-VAC-2 is also capable of protecting mice from heterologous challenge with recombinant VSV viruses that express the same SARS-CoV-2 antigens. Altogether, these findings demonstrate the effectiveness of our versatile vaccine platform as an alternative or complementary approach to current vaccines.

Fast Preparation of Peptide Tools to address new emerging Viruses: Case example SARS-CoV-2

Abstract The emergence of new virus species or variants poses a major challenge for healthcare systems. Countermeasures, including vaccine development, need to be developed without delay to ensure success. Vaccine and antibody development depend on highly efficient tools for antigen discovery, epitope mapping, diagnostics and (clinical) immune monitoring. Here we present a workflow for peptide tool development that can be rapidly adapted to emerging viruses. It combines bioinformatics algorithms and an ultra-high throughput peptide synthesis method alongside high-content assay formats. Data from different SARS-CoV-2 studies are presented addressing elucidation of both cellular and humoral immune responses. Robust spike-specific cellular immune responses were measured using SARS-CoV-2 Spike PepMix ™peptide pools during infection and/or vaccine response monitoring [ 1]. Using epitope mapping (matrix) pools an immunodominant T-cell epitope was identified that may contribute to the optimization of existing vaccines [ 2]. In addition, the immune response of Covid-19 convalescents against all major SARS-CoV-2 proteins was analyzed [ 2]. Humoral immune response analysis by high-density peptide microarrays identified immunodominant B cell epitopes as starting points for the development of alternative vaccines and diagnostic tests [ 3]. In conclusion, we show that readily available and tailored peptide formats, as exemplified for SARS-CoV-2, are important tools that contribute to the fast development and optimization of vaccines and diagnostic tests for emerging viruses of concern.

Hemophilia A subjects with an intron-22 gene inversion mutation show CD4+ T-effector responses to multiple epitopes in FVIII.

Almost half of severe hemophilia A (HA) is caused by an intron 22 inversion mutation (Int22Inv), which disrupts the 26-exon F8 gene. Inverted F8 mRNA exons 1-22 are transcribed, while F8B mRNA, containing F8 exons 23-26, is transcribed from a promoter within intron 22. Neither FVIII activity nor FVIII antigen (cross-reacting material, CRM) are detectable in plasma of patients with an intron-22 inversion. To test the hypothesis that (putative) intracellular synthesis of FVIII proteins encoded by inverted F8 and F8B mRNAs confers T-cell tolerance to almost the entire FVIII sequence, and to evaluate the immunogenicity of the region encoded by the F8 exon 22-23 junction sequence. Peripheral blood mononuclear cells (PBMCs) from 30 severe or moderate HA subjects (17 with an Int22Inv mutation) were tested by ELISPOT assays to detect cytokine secretion in response to FVIII proteins and peptides and to map immunodominant T-cell epitopes. Potential immunogenicity of FVIII sequences encoded by the F8 exon 22-23 junction region was also tested using peptide-MHCII binding assays. Eight of the Int22Inv subjects showed robust cytokine secretion from PBMCs stimulated with FVIII proteins and/or peptides, consistent with earlier publications from the Conti-Fine group. Peptide ELISPOT assays identified immunogenic regions of FVIII. Specificity for sequences encoded within F8 mRNA exons 1-22 and F8B mRNA was confirmed by staining Int22Inv CD4+ T cells with peptide-loaded HLA-Class II tetramers. FVIII peptides spanning the F8 exon 22-23 junction (encoding M2124-V2125) showed limited binding to MHCII proteins and low immunogenicity, with cytokine secretion from only one Int22Inv subject. PBMCs from multiple subjects with an Int22Inv mutation, with and without a current FVIII inhibitor, responded to FVIII epitopes. Furthermore, the FVIII region encoded by the exon 22-23 junction sequence was not remarkably immunoreactive and is therefore unlikely to contain an immunodominant, promiscuous CD4+ T-cell epitope. Our results indicate that putative intracellular expression of partial FVIII proteins does not confer T-cell tolerance to FVIII regions encoded by inverted F8 mRNA or F8B mRNA.

Prediction of Putative Epitope Peptides against BaeR Associated with TCS Adaptation in Acinetobacter baumannii Using an In Silico Approach

Background and Objectives: The BaeR protein is involved in the adaptation system of A. baumannii and is associated with virulence factors responsible for systemic infections in hospitalized patients. This study was conducted to characterize putative epitope peptides for the design of vaccines against BaeR protein, using an immune-informatic approach. Materials and Methods: FASTA sequences of BaeR from five different strains of A. baumannii were retrieved from the UNIPROT database and evaluated for their antigenicity, allergenicity and vaccine properties using BepiPred, Vaxijen, AlgPred, AntigenPro and SolPro. Their physio-chemical properties were assessed using the Expasy Protparam server. Immuno-dominant B-cell and T-cell epitope peptides were predicted using the IEDB database and MHC cluster server with a final assessment of their interactions with TLR-2. Results: A final selection of two peptide sequences (36aa and 22aa) was made from the 38 antigenic peptides. E1 was considered a soluble, non-allergenic antigen, and possessed negative GRAVY values, substantiating the hydrophilic nature of the proteins. Further analysis on the T-cell epitopes, class I immunogenicity and HLA allele frequencies yielded T-cell immuno-dominant peptides. The protein-peptide interactions of the TLR-2 receptor showed good similarity scores in terms of the high number of hydrogen bonds compared to other protein-peptide interactions. Conclusions: The two epitopes predicted from BaeR in the present investigation are promising vaccine candidates for targeting the TCS of A. baumannii in systemic and nosocomial infections. This study also demonstrates an alternative strategy to tackling and mitigating MDR strains of A. baumannii and provides a useful reference for the design and construction of novel vaccine candidates against this bacteria.

Immunoinformatics design of multivalent epitope vaccine against monkeypox virus and its variants using membrane-bound, enveloped, and extracellular proteins as targets.

The current monkeypox (MPX) outbreak, caused by the monkeypox virus (MPXV), has turned into a global concern, with over 59,000 infection cases and 23 deaths worldwide. Herein, we aimed to exploit robust immunoinformatics approach, targeting membrane-bound, enveloped, and extracellular proteins of MPXV to formulate a chimeric antigen. Such a strategy could similarly be applied for identifying immunodominant epitopes and designing multi-epitope vaccine ensembles in other pathogens responsible for chronic pathologies that are difficult to intervene against. A reverse vaccinology pipeline was used to select 11 potential vaccine candidates, which were screened and mapped to predict immunodominant B-cell and T-cell epitopes. The finalized epitopes were merged with the aid of suitable linkers, an adjuvant (Resuscitation-promoting factor), a PADRE sequence (13 aa), and an HIV TAT sequence (11 aa) to formulate a multivalent epitope vaccine. Bioinformatics tools were employed to carry out codon adaptation and computational cloning. The tertiary structure of the chimeric vaccine construct was modeled via I-TASSER, and its interaction with Toll-like receptor 4 (TLR4) was evaluated using molecular docking and molecular dynamics simulation. C-ImmSim server was implemented to examine the immune response against the designed multi-epitope antigen. The designed chimeric vaccine construct included 21 immunodominant epitopes (six B-cell, eight cytotoxic T lymphocyte, and seven helper T-lymphocyte) and is predicted non-allergen, antigenic, soluble, with suitable physicochemical features, that can promote cross-protection among the MPXV strains. The selected epitopes indicated a wide global population coverage (93.62%). Most finalized epitopes have 70%-100% sequence similarity with the experimentally validated immune epitopes of the vaccinia virus, which can be helpful in the speedy progression of vaccine design. Lastly, molecular docking and molecular dynamics simulation computed stable and energetically favourable interaction between the putative antigen and TLR4. Our results show that the multi-epitope vaccine might elicit cellular and humoral immune responses and could be a potential vaccine candidate against the MPXV infection. Further experimental testing of the proposed vaccine is warranted to validate its safety and efficacy profile.

Employing an immunoinformatics approach revealed potent multi-epitope based subunit vaccine for lymphocytic choriomeningitis virus

BackgroundLymphocytic choriomeningitis virus (LCMV) infects many individuals worldwide and causes severe infection in the immunosuppressant recipient, spontaneous abortion, and congenital disabilities in infants. ObjectivesThere is no specific vaccine or therapeutics available to protect against LCMV infection; thus, there is a need to design a potential vaccine to combat the virus by developing immunity in the population. Herein, we attempted to design a potent multi-epitope vaccine for LCMV using immunoinformatics methods. MethodsThe whole proteome of the virus was screened and mapped to extract immunodominant B-cell and T-cell epitopes which were fused with appropriate linkers (EAAAK, GGGS, AAY, GPGPG, and AAY), PADRE sequence (13aa) and an adjuvant (50 S ribosomal protein L7/L12) to formulate a multi-epitope vaccine ensemble. Codon adaptation and in silico cloning of the constructed vaccine were carried out using bioinformatics tools. The secondary and tertiary structure of the vaccine construct was predicted and refined. The physicochemical profile of the designed vaccine was analyzed, and the multi-epitope vaccine's potential to bind Toll-like receptors (TLR2 and TLR4) was evaluated through molecular docking and molecular dynamics simulations. Computational immune simulation of the designed vaccine antigen was performed using the C-ImmSim server. ResultsThe designed multi-epitope-based vaccine (613 aa) comprised 26 immunodominant (six B-cell, nine cytotoxic T lymphocytes, and 11 helper T lymphocytes) epitopes and is predicted antigenic, non-toxic, non-allergen, soluble, and topographically accessible with a suitable physicochemical profile. The designed vaccine is expected to cover a broad worldwide population (96.35 %) and stimulate a robust adaptive immune response against the virus upon administration. In silico cloning of the constructed vaccine in PET28a (+) vector ensured its optimal expression in the Escherichia coli system. Molecular docking, molecular dynamics simulation, and binding free energy estimation collectively support the stability and energetically favourable interaction of the modeled vaccine–TLR2/4 complexes. ConclusionThe designed multi-epitope vaccine in the present study could serve as a potential vaccine candidate to protect against LMCV infection; however, the experimental validation and safety testing of the vaccine is warranted to validate the study’s outcomes.

SARS-CoV-2 epitopes inform future vaccination strategies.

All currently approved COVID-19 vaccines utilize the spike protein as their immunogen. SARS-CoV-2 variants of concern (VOCs) contain mutations in the spike protein, enabling them to escape infection- and vaccination-induced immune responses to cause reinfection. New vaccines are hence being researched intensively. Studying SARS-CoV-2 epitopes is essential for vaccine design, as identifying targets of broadly neutralizing antibody responses and immunodominant T-cell epitopes reveal candidates for inclusion in next-generation COVID-19 vaccines. We summarize the major studies which have reported on SARS-CoV-2 antibody and T-cell epitopes thus far. These results suggest that a future of pan-coronavirus vaccines, which not only protect against SARS-CoV-2 but numerous other coronaviruses, may be possible. The T-cell epitopes of SARS-CoV-2 have gotten less attention than neutralizing antibody epitopes but may provide new strategies to control SARS-CoV-2 infection. T-cells target many SARS-CoV-2 antigens other than spike, recognizing numerous epitopes within these antigens, thereby limiting the chance of immune escape by VOCs that mainly possess spike protein mutations. Therefore, augmenting vaccination-induced T-cell responses against SARS-CoV-2 may provide adequate protection despite broad antibody escape by VOCs.

In Silico-Based Vaccine Design Against Hepatopancreatic Microsporidiosis in Shrimp

Enterocytozoon hepatopenaei (EHP) is a spore-forming intracellular parasite that produces an economically devastating disease in farmed shrimp called hepatopancreatic microsporidiosis (HPM), known to slow the growth of hosts in aquaculture. Spore wall proteins are implicated in the recognition of host cells in microsporidia. Many parasites have been demonstrated to detect heparin, a glycosaminoglycan (GAG) molecule present on cell surfaces, as a host. The sequencing and characterization of several EHP strains started to reveal information regarding pathogen biology, pathogenicity, and spore wall proteins must bind to the shrimp cell surface to infect it, and failure to do so results in ineffective infection. Despite these devastating complications, there is still no cure or vaccine for the parasites. In this regard, an immunoinformatics method was used to generate an epitope-based vaccine against this pathogen. The immunodominant T-cell and B-cell epitopes were identified using the spore wall proteins of EHP. The final constructed vaccine sequence was developed to be immunogenic, non-allergenic as well as have better solubility. Molecular dynamics simulation revealed significant binding stability and structural compactness. Finally, using Escherichia coli K12 as a model, codon optimization yielded ideal GC content and a higher CAI value, which was then included in the cloning vector pET2+ (a). Altogether, our outcomes imply that the proposed peptide vaccine may be a good option for HPM prophylaxis. HIGHLIGHTS Hepatopancreatic microsporidiosis (HPM) is known to slow the growth of shrimp There are approved drugs or vaccines to control this disease In-silico approach and immunoinformatics has some advantage previously in fishes The design vaccine candidate would have some advantages to combat the disease for sustainable shrimp production GRAPHICAL ABSTRACT

Molecular Mimicry of the Rheumatoid Arthritis-Related Immunodominant T-Cell Epitope within Type II Collagen (CII260-270) by the Bacterial L-Asparaginase

The etiology of most autoimmune diseases, including rheumatoid arthritis (RA), remains unclear. Both genetic and environmental factors are believed to be involved in pathogenesis. Molecular mimicry is considered one of the mechanisms for the occurrence of autoimmune diseases. The aim of the study was to determine whether the bacterial peptide L-ASNase67-81, which mimics the immunodominant T-cell epitope CII259-273, can induce T-cell reactivity in blood samples from RA patients and healthy subjects through molecular mimicry. Using bioinformatic molecular modeling methods, we first determined whether the L-ASNase67-81 peptide binds to the HLA-DRB1*04:01 molecule and whether the formed MHCII–peptide complex interacts with the corresponding T-cell receptor. To validate the obtained results, leukocytes isolated from early RA patients and healthy individuals were stimulated in vitro with L-ASNase67-81 and CII259-273 peptides as well as with bacterial L-asparaginase or human type II collagen (huCII). The activated T cells (CD4+CD154+) were analyzed by flow cytometry (FACS), and the levels of cytokines produced (IL-2, IL-17A/F, and IFN-γ) were measured by ELISA. Our in silico analyses showed that the bacterial peptide L-ASNase67-81 binds better to HLA-DRB1*04:01 compared to the immunodominant T-cell epitope CII259-273, mimicking its structure and localization in the binding groove of MHCII. Six contact points were involved in the molecular interaction of the peptide with the TCR. FACS data showed that after in vitro stimulation with the L-ASNase67-81 peptide, the percentage of activated T cells (CD154+CD4+) was significantly increased in both cell cultures isolated from ERA patients and those isolated from healthy individuals, as higher values were observed for the ERA group (9.92 ± 0.23 vs. 4.82 ± 0.22). Furthermore, the ELISA assays revealed that after stimulation with L-ASNase67-81, a significant increase in the production of the cytokines IL-2, IL-17A/F, and IFN-γ was detected in the group of ERA patients. Our data showed that the bacterial L-ASNase67-81 peptide can mimic the immunodominant T-cell epitope CII259-273 and activate HLA-DRB1*04:01-restricted T cells as well as induce cytokine production in cells isolated from ERA patients. These results are the first to demonstrate that a specific bacterial antigen could play a role in the pathogenesis of RA, mimicking the immunodominant T-cell epitope from type II collagen.

Application of reverse vaccinology for designing of an mRNA vaccine against re-emerging marine birnavirus affecting fish species

Marine birnavirus (MABV) has been the most pathogenic virus for marine and shellfish species across the world in terms of production and financial benefits. An outbreak of the disease may lead to entire mortality within a short period under standard culture conditions. The sequencing and characterization of several MABV strains have begun to reveal information regarding pathogen biology and pathogenicity. The major capsid protein VP2 and RNA-dependent RNA polymerase (RdRp), as well as polyprotein of Lates calcarifer birnavirus, was determined from several marine birnavirus (MABV) strains from different host or geographic origins. Despite the devastating complications, there is very limited prevention or control for the virus. In this regard, an immunoinformatics method was used to generate an epitope-based vaccine against this pathogen. The immunodominant T-cell epitopes were identified using the most antigenic and pathogenic proteins of MABV. The final constructed vaccine sequence was developed to be immunogenic, non-allergenic as well as to have better solubility. Molecular dynamics simulation revealed significant binding stability and structural compactness. Finally, using Escherichia coli K12 as a model, codon optimization yielded ideal GC content and a higher CAI value, which was then included in the cloning vector pET2+ (a). Overall, our findings suggest that the suggested peptide vaccine might be a viable approach for MABV prevention.

Specific T cells targeting Staphylococcus aureus fibronectin-binding protein 1 induce a type 2/type 1 inflammatory response in sensitized atopic dermatitis patients.

Atopic dermatitis (AD) is one of the most common inflammatory skin diseases worldwide and Staphylococcusaureus colonization and secondary infections occur in the majority of AD patients. Allergic sensitizations against microbial antigens have been discussed as possible trigger factors of AD. Recently, we reported IgE sensitization against fibronectin-binding protein 1 (FBP1), an essential virulence component in S.aureus, in a subgroup of patients suffering from AD. To expand these findings by investigating delayed-type immune reactions, the objective of this study was to detect and phenotypically characterize FBP1-specific T cells as possible trigger factors in AD. Immunodominant T-cell epitopes were mapped by proliferation testing of patient-derived FBP1-specific T-cell lines after stimulation with single 15mer peptides, which were derived from different functional domains of the FBP1 sequence. Major histocompatibility complex class II tetramers carrying immunodominant epitopes successfully stained T helper cells in 8 out of 8 HLA-matched, IgE-sensitized AD patients. Cytokine profiling of multimer-sorted cells revealed that predominantly the type 2 cytokines IL-13 and IL-4 were secreted by these cells. In contrast, IL-17, the marker cytokine for response to extracellular pathogens, was scarcely detectable. We demonstrate that FBP1 contains immunodominant peptides that induce a specific pro-inflammatory T helper cell response with increased Th2 levels that can drive an allergic inflammation in sensitized AD patients.

FVIII Proteins with a Modified Immunodominant T-Cell Epitope Exhibit Reduced Immunogenicity and Normal Procoagulant Activity

Factor VIII (FVIII) neutralizing antibodies (inhibitors) are a serious complication in hemophilia A (HA). The peptide FVIII2194-2213 (legacy FVIII numbering, sequence: SYFTNMFATWSPSKARLHLQ) has been shown to contain an immunodominant HLA-DRA*01-DRB1*01:01 (DRB1*01:01)-restricted epitope recognized by CD4+ T-effector cells isolated from HA subjects. The aim of this study was to identify amino acid substitutions to de-immunize this epitope while retaining pro-coagulant function and expression levels comparable to those of wild-type (WT) FVIII proteins. The minimal DRB1*01:01-binding motif was mapped to FVIII2194-2205 by quantitative peptide-MHC Class II (MHCII) binding assays. Further binding assays with sequence-modified FVIII peptides established that the residues important for DRB1*01:01 affinity were F2196, M2199, A2201, and S2204, corresponding to MHCII anchor positions 1, 4, 6 and 9. T-cell proliferation experiments with Ala-substituted FVIII2194-2205 peptides identified F2196A as a substitution that abrogated proliferation and cytokine secretion of five clones specific for FVIII2194-2213. The substitution F2196A was then engineered into a recombinant (r)FVIII-C2 protein. T-cell clones that were strongly stimulated by wild-type rFVIII-C2 did not proliferate when cultured with rFVIII-C2-F2196A, indicating the immunogenic synthetic peptide includes a naturally processed T-cell epitope. The measured and predicted effects of additional amino acid substitutions within this binding motif were further evaluated by peptide-MHCII binding assays, CD4+ T-cell proliferation assays, and MHCII-binding prediction algorithms. Amino acid homologs within this binding motif were evaluated with sequence homologies to FVIII and FV proteins from different species. On the basis of all these results, six rB-domain-deleted (BDD)-FVIII proteins with amino acid substitutions F2196A, F2196L, F2196K, M2199A, M2199W or M2199R, respectively, were designed and produced; all but the F2196A variant had specific activities comparable to that of rWT-BDD-FVIII. The F2196A variant also had the lowest expression level among the rBDD-FVIIII proteins produced. Peptide-MHCII binding assays confirmed that FVIII2194-2205 peptides with substitutions F2196A, F2196K, M2199A and M2199R did not bind to DRB1*01:01 or to an additional 9 HLA-DR proteins tested, with the exception of the M2199A peptide binding to DRB1*09:01. The predicted effects of these FVIII sequence modifications on binding to additional MHCII alleles included in reference sets representing ~99% of the human population HLA diversity were evaluated using ProPred and Immune Epitope Database algorithms. These programs correctly predicted that the substitutions would abrogate binding to DRB1*01:01 and also indicated that affinities for additional MHCII would be reduced by each substitution. The substitutions were predicted to increase peptide-MHCII affinities for a smaller number of MHCII alleles, indicating a possible risk of creating neo-epitopes restricted to these specific HLA alleles. Importantly, proliferation of patient-derived T-cell clones in response to rBDD-FVIII-F2196K and rBDD-FVIII-M2199A proteins was dramatically reduced compared with responses to rWT-BDD-FVIII. We have previously demonstrated that a severe HA inhibitor subject had an oligoclonal T-cell response to only one T-cell epitope within the FVIII A2, C1 and C2 domains and that one epitope was FVIII2194-2213 (Ettinger et al., Blood 2106; 128(16):2043). This observation, together with the results presented here, further suggests that modification of only a small number of immunodominant epitopes may be sufficient to significantly de-immunize FVIII and decrease FVIII inhibitors in HA patients. The present results provide proof-of-principle for the design of less immunogenic FVIII proteins targeted to specific subsets of HA patients. DisclosuresEttinger:Bloodworks NW: Patents & Royalties: Inventor on FVIII patents. James:Bloodworks NW: Patents & Royalties: inventor on FVIII patent(s). Pratt:Bloodworks NW: Patents & Royalties: inventor on FVIII patents.

Immunodominant T-cell epitopes from the SARS-CoV-2 spike antigen reveal robust pre-existing T-cell immunity in unexposed individuals

The COVID-19 pandemic has revealed a range of disease phenotypes in infected patients with asymptomatic, mild, or severe clinical outcomes, but the mechanisms that determine such variable outcomes remain unresolved. In this study, we identified immunodominant CD8 T-cell epitopes in the spike antigen using a novel TCR-binding algorithm. The predicted epitopes induced robust T-cell activation in unexposed donors demonstrating pre-existing CD4 and CD8 T-cell immunity to SARS-CoV-2 antigen. The T-cell reactivity to the predicted epitopes was higher than the Spike-S1 and S2 peptide pools in the unexposed donors. A key finding of our study is that pre-existing T-cell immunity to SARS-CoV-2 is contributed by TCRs that recognize common viral antigens such as Influenza and CMV, even though the viral epitopes lack sequence identity to the SARS-CoV-2 epitopes. This finding is in contrast to multiple published studies in which pre-existing T-cell immunity is suggested to arise from shared epitopes between SARS-CoV-2 and other common cold-causing coronaviruses. However, our findings suggest that SARS-CoV-2 reactive T-cells are likely to be present in many individuals because of prior exposure to flu and CMV viruses.

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.

Prevention of EAE by tolerogenic vaccination with PEGylated antigenic peptides.

Background:Therapeutic treatment options for chronic autoimmune disorders such as multiple sclerosis (MS) rely largely on the use of non-specific immunosuppressive drugs, which are not able to cure the disease. Presently, approaches to induce antigen-specific tolerance as a therapeutic approach; for example, by peptide-based tolerogenic ‘inverse’ vaccines have regained great interest. We have previously shown that coupling of peptides to carriers can enhance their capacity to induce regulatory T cells in vivo.Method:In this present study, we investigated whether the tolerogenic potential of immunodominant myelin T-cell epitopes can be improved by conjugation to the synthetic carrier polyethylene glycol (PEG) in an experimental autoimmune encephalomyelitis (EAE) mouse model for chronic MS (MOG C57BL/6).Results:Preventive administration of the PEGylated antigenic peptide could strongly suppress the development of EAE, accompanied by reduced immune cell infiltration in the central nervous system (CNS). Depletion of regulatory T cells (Tregs) abrogated the protective effect indicating that Tregs play a crucial role in induction of antigen-specific tolerance in EAE. Treatment during the acute phase of disease was safe and did not induce immune activation. However, treatment at the peak of disease did not affect the disease course, suggesting that either induction of Tregs does not occur in the highly inflamed situation, or that the immune system is refractory to regulation in this condition.Conclusion:PEGylation of antigenic peptides is an effective and feasible strategy to improve tolerogenic (Treg-inducing) peptide-based vaccines, but application for immunotherapy of overt disease might require modifications or combination therapies that simultaneously suppress effector mechanisms.

Variations in BK Polyomavirus Immunodominant Large Tumor Antigen-Specific 9mer CD8 T-Cell Epitopes Predict Altered HLA-Presentation and Immune Failure.

Failing BK polyomavirus (BKPyV)-specific immune control is underlying onset and duration of BKPyV-replication and disease. We focused on BKPyV-specific CD8 T-cells as key effectors and characterized immunodominant 9mer epitopes in the viral large tumor-antigen (LTag). We investigated the variation of LTag-epitopes and their predicted effects on HLA-class 1 binding and T-cell activation. Available BKPyV sequences in the NCBI-nucleotide (N = 3263), and the NCBI protein database (N = 4189) were extracted (1368 sequences) and analyzed for non-synonymous aa-exchanges in LTag. Variant 9mer-epitopes were assessed for predicted changes in HLA-A and HLA-B-binding compared to immunodominant 9mer reference. We identified 159 non-synonymous aa-exchanges in immunodominant LTag-9mer T-cell epitopes reflecting different BKPyV-genotypes as well as genotype-independent variants altering HLA-A/HLA-B-binding scores. Decreased binding scores for HLA-A/HLA-B were found in 27/159 (17%). This included the immunodominant LPLMRKAYL affecting HLA-B*07:02-, HLA-B*08:01- and HLA-B*51:01-presentation. In two healthy BKPyV-seropositive HLA-B*07:02 blood donors, variant LSLMRKAYL showed reduced CD8 T-cell responses compared to LPLMRKAYL. Thus, despite LTag being highly conserved, aa-exchanges occur in immunodominant CD8 T-cell epitopes of BKPyV-genotypes as well as of genotypes -independent variants, which may contribute to genotype-dependent and genotype-independent failure of cellular immune control over BKPyV-replication. The data warrant epidemiological and immunological investigations in carefully designed clinical studies.

T-Cell Epitope Immunotherapy in Mouse Models of Food Allergy.

Food allergy has been rising in prevalence over the last two decades, affecting more than 10% of the world population. Current management of IgE-mediated food allergy relies on avoidance and rescue medications; research into treatments that are safer and providing guaranteed and durable curative effects is, therefore, essential. T-cell epitope-based immunotherapy holds the potential for modulating food allergic responses without IgE cross-linking. In this chapter, we describe the methods in evaluating the therapeutic capacities of immunodominant T-cell epitopes in animal models of food allergy. Moreover, we explain in detail the methods to measure the allergen-specific antibody levels, prepare single-cell suspension from spleen, and prepare small intestine for immunohistochemical analysis of eosinophils and Foxp3+ cells.

Clostridium bolteae is elevated in neuromyelitis optica spectrum disorder in India and shares sequence similarity with AQP4.

ObjectiveTo understand the role of gut microbiome in influencing the pathogenesis of neuromyelitis optica spectrum disorders (NMOSDs) among patients of south Indian origin.MethodsIn this case-control study, stool and blood samples were collected from 39 patients with NMOSD, including 17 with aquaporin 4 IgG antibodies (AQP4+) and 36 matched controls. 16S ribosomal RNA (rRNA) sequencing was used to investigate the gut microbiome. Peripheral CD4+ T cells were sorted in 12 healthy controls, and in 12 patients with AQP4+ NMOSD, RNA was extracted and immune gene expression was analyzed using the NanoString nCounter human immunology kit code set.ResultsMicrobiota community structure (beta diversity) differed between patients with AQP4+ NMOSD and healthy controls (p < 0.001, pairwise PERMANOVA test). Linear discriminatory analysis effect size identified several members of the microbiota that were altered in patients with NMOSD, including an increase in Clostridium bolteae (effect size 4.23, p 0.00007). C bolteae was significantly more prevalent (p = 0.02) among patients with AQP4-IgG+ NMOSD (n = 8/17 subjects) compared with seronegative patients (n = 3/22) and was absent among healthy stool samples. C bolteae has a highly conserved glycerol uptake facilitator and related aquaporin protein (p59-71) that shares sequence homology with AQP4 peptide (p92-104), positioned within an immunodominant (AQP4 specific) T-cell epitope (p91-110). Presence of C bolteae correlated with expression of inflammatory genes associated with both innate and adaptive immunities and particularly involved in plasma cell differentiation, B cell chemotaxis, and Th17 activation.ConclusionOur study described elevated levels of C bolteae associated with AQP4+ NMOSD among Indian patients. It is possible that this organism may be causally related to the immunopathogenesis of this disease in susceptible individuals.

Delineating the Immuno-Dominant Antigenic Vaccine Peptides Against gacS-Sensor Kinase in Acinetobacter baumannii: An in silico Investigational Approach

ObjectivesTo predict the novel vaccine peptide candidates against gacS protein involved with the citrate utilization in the two-component system of A. baumannii-associated virulence as an alternative strategy to combat the multi-drug resistant strains using an immuno-informatic approach.MethodsThe study is designed as an observational in silico study design with the application of BepiPred, AlgPred, VaxiJen, AntigenPro, SolPro, Expasy ProtParam server, IEDB database, and MHC cluster analytical tools and servers to predict the immuno-dominant B-cell and T-cell epitopes from gacS FASTA sequences retrieved from UNIPROT database. Further peptide interactions with TLR-4 was assessed based on the number of hydrogen bonds.ResultsNine peptides (20aa) with the highest score of 1 were selected from the 137 epitopes, and five were predicted as antigenic epitopes (E1–E5). E3 was selected as the potent antigen (score: 0.939537) and E1 as the best vaccine candidate (score: 0.9803) under AntigenPro and Vaxijen server, respectively. SolPro predicted all epitopes as soluble peptides. ProtParam predictions showed E3 and E5 as stable proteins with a shelf life of 3.5 and 1.9 h and possessed negative GRAVY values. PsortB server predicted a final localization score of 7.88 for the gacS protein sequence as a cytoplasmic membrane protein. IEDB conservancy analysis showed 100% conserved sequences within the gacS sequence, and class I conservancy yielded positive values for all epitopes. Cluster analysis showed strong interactions, and the protein-peptide interactions with TLR-2 finally detected E5 as the best interacting peptide (H bonds = 14) followed by E3 (H bonds = 12).ConclusionThe study suggests five antigenic peptides as promiscuous vaccine candidates to target the gacS of A. baumannii using immuno-informatic approach toward the peptide synthesis and in vitro analysis. However, the study recommends further experimental validation for immunological response and memory through in vivo studies.