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The beginning of 2020 has seen the emergence of COVID-19 outbreak caused by a novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). There is an imminent need to better understand this new virus and to develop ways to control its spread. In this study, we sought to gain insights for vaccine design against SARS-CoV-2 by considering the high genetic similarity between SARS-CoV-2 and SARS-CoV, which caused the outbreak in 2003, and leveraging existing immunological studies of SARS-CoV. By screening the experimentally-determined SARS-CoV-derived B cell and T cell epitopes in the immunogenic structural proteins of SARS-CoV, we identified a set of B cell and T cell epitopes derived from the spike (S) and nucleocapsid (N) proteins that map identically to SARS-CoV-2 proteins. As no mutation has been observed in these identified epitopes among the 120 available SARS-CoV-2 sequences (as of 21 February 2020), immune targeting of these epitopes may potentially offer protection against this novel virus. For the T cell epitopes, we performed a population coverage analysis of the associated MHC alleles and proposed a set of epitopes that is estimated to provide broad coverage globally, as well as in China. Our findings provide a screened set of epitopes that can help guide experimental efforts towards the development of vaccines against SARS-CoV-2.

Immunotoxins are cytolytic fusion proteins developed for cancer therapy, composed of an antibody fragment that binds to a cancer cell and a protein toxin fragment that kills the cell. exotoxin A (PE) is a potent toxin that is used for the killing moiety in many immunotoxins. Moxetumomab Pasudotox (Lumoxiti) contains an anti-CD22 Fv and a 38 kDa portion of PE. Lumoxiti was discovered in the Laboratory of Molecular Biology at the U.S. National Cancer Institute and co-developed with Medimmune/AstraZeneca to treat hairy cell leukemia. In 2018 Lumoxiti was approved by the US Food and Drug Administration for the treatment of drug-resistant Hairy Cell Leukemia. Due to the bacterial origin of the killing moiety, immunotoxins containing PE are highly immunogenic in patients with normal immune systems, but less immunogenic in patients with hematologic malignancies, whose immune systems are often compromised. LMB-100 is a de-immunized variant of the toxin with a humanized antibody that targets mesothelin and a PE toxin that was rationally designed for diminished reactivity with antibodies and B cell receptors. It is now being evaluated in clinical trials for the treatment of mesothelioma and pancreatic cancer and is showing somewhat diminished immunogenicity compared to its un modified parental counterpart. Here we review the immunogenicity of the original and de-immunized PE immunotoxins in mice and patients, the development of anti-drug antibodies (ADAs), their impact on drug availability and their effect on clinical efficacy. Efforts to mitigate the immunogenicity of immunotoxins and its impact on immunogenicity will be described including rational design to identify, remove, or suppress B cell or T cell epitopes, and combination of immunotoxins with immune modulating drugs.

Antibody maturation in the lymphoid follicle produces antibodies with improved binding affinity. This process requires iterative rounds of mutation and B cell expansion, supported by T cells that recognize epitopes presented on the B cell's MHC-II. In this comprehensive antibody repertoire analysis, we find that established regulatory T cell epitopes (Tregitopes) decline in maturing antibody sequences as somatic hypermutation (SHM) increases, but potential T effector epitopes do not decline. A previous analysis of B cell receptor (BCR)-derived HLA-DR epitopes present in memory antibody repertoires from seven healthy human donors revealed a decrease in donor-specific epitope content with SHM. Moreover, T cell epitope depletion was associated with class-switching and long-term secretion of antibody into serum. Significant depletion of high-affinity germline-encoded epitopes in high SHM sequences was also observed, but the predicted phenotype of T cells responding to the BCR-derived epitopes (regulatory vs. effector) was not previously evaluated. In this follow-on study, we screened a different set of four donor repertoires to investigate the dynamics of donor-specific HLA-DR T cell epitopes and three subsets of T cell epitope content: previously validated T cell epitopes recognized by thymus-derived Tregs (Tregitopes), potentially tolerated T cell epitopes, and potential effector T cell epitopes. Our results show that Tregitope content reduction is correlated with SHM, suggesting that Tregitopes are removed during maturation. Moreover, T cell epitopes that are likely to be tolerated or tolerogenic were also removed with SHM progression. In contrast, potential T effector epitope content increased with SHM. Tregitope depletion occurred in multiple V-gene pair combinations and was the most frequent T cell epitope change. Furthermore, Tregitope content in IgA and IgG sequences was lower and had greater negative correlation with SHM than IgM, indicating that Tregitope removal is likely associated with class-switching. Tregitope depletion was also associated with maturation to plasmablasts. In vitro, representative Tregitopes inhibited CD4+ T cell proliferation. Mutations introduced by SHM altered Tregitope HLA-DR binding affinities. The correlation of Tregitope depletion with increasing SHM implies that the activity of thymus-derived Treg cells in immune responses to antibodies is diminished with SHM, maturation, and isotype switching, supporting the generation of anti-idiotype responses.

CD8 + T cells are a pivotal part of the immune response to viruses, playing a key role in disease outcome and providing long-lasting immunity to conserved pathogen epitopes. Understanding CD8 + T cell immunity in humans is complex due to CD8 + T cell restriction by highly polymorphic Human Leukocyte Antigen (HLA) proteins, requiring T cell epitopes to be defined for different HLA allotypes across different ethnicities. Here we evaluate strategies that have been developed to facilitate epitope identification and study immunogenic T cell responses. We describe an immunopeptidomics approach to sequence HLA-bound peptides presented on virus-infected cells by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Using antigen presenting cell lines that stably express the HLA alleles characteristic of Indigenous Australians, this approach has been successfully used to comprehensively identify influenza-specific CD8 + T cell epitopes restricted by HLA allotypes predominant in Indigenous Australians, including HLA-A*24:02 and HLA-A*11:01. This is an essential step in ensuring high vaccine coverage and efficacy in Indigenous populations globally, known to be at high risk from influenza disease and other respiratory infections.

•AHSV4 VP1, -2, -4, -7 and NS3 contain regions with CD8+ T cell epitopes.•Overlapping peptides spanning these AHSV4 proteins were synthesized.•Recall immune assays identified T cell and linear B cell epitopes.•Conserved T cell and linear B cell epitopes will be used in NGV. The viral proteins VP1-1, VP2, VP4, VP7 and NS3, of African horse sickness virus serotype 4 (AHSV4), have previously been identified to contain CD8+ T cell epitopes. In this study, overlapping peptides spanning the entire sequences of these AHSV4 proteins were synthesized and used to map epitopes. Peripheral blood mononuclear cells (PBMC) isolated from five horses immunized with an attenuated AHSV4 were stimulated in vitro with the synthesized peptides. Various memory immune assays were used to identify the individual peptides that contain CD8+ T cell epitopes, CD4+ T cell epitopes and linear B cell epitopes. The newly discovered individual peptides of AHSV4 proteins VP1-1, VP4, VP7 and/or NS3 that contain CD8+ T cell, CD4+ T cell or linear B cell epitopes could contribute to the design and development of new generation AHS peptide-based vaccines and therapeutics.

The sodium iodide symporter (NIS) is a key protein in thyroid function responsible for iodine uptake, and it may be involved in the pathogenesis of autoimmune thyroiditis. However, it is also expressed in the salivary glands, the primary target of autoreactive cells in Sjögren’s syndrome (SS). Given the common link between the two diseases, we computationally investigated whether the epitopes of NIS can trigger an immune response leading to SS in Hashimoto’s thyroiditis (HT) patients genetically predisposed to both diseases. The TepiTool 2016, ABCpred 2006, and DiscoTope 2.0 servers were used to predict T-cell and B-cell epitopes by inputting the FASTA sequences and 3D structures of NIS, thyroid peroxidase (TPO) and Ro60 Y RNA-binding protein (Ro60), which served as reference antigens for HT and SS, respectively. T-cell epitopes were selected based on their binding to a panel of human leukocyte antigen (HLA) alleles associated with both SS and HT. We identified a total of 376 linear T-cell epitopes, 64 linear B-cell epitopes and 68 conformational B-cell epitopes of NIS. Compared to TPO, NIS T-cell epitopes showed significantly lower affinity for HLA alleles (p < 0.0001), while no significant difference was found compared to Ro60. While linear B-cell epitopes of NIS, TPO, and Ro60 showed similar binding affinity, conformational epitopes of NIS were predicted to have higher immunogenicity than Ro60 (p = 0.04), while no significant difference was found compared to TPO. These pivotal findings, discovered by the methods of computer modeling, suggest that NIS can potentially activate T cells and B cells in patients with genetic predisposition to SS and HT and need to be confirmed by further laboratory studies.

CD4 T cells have a major role in regulating immune responses. They are activated by recognition of peptides mostly generated from exogenous antigens through the major histocompatibility complex (MHC) class II pathway. Identification of epitopes is important and computational prediction of epitopes is used widely to save time and resources. Although there are algorithms to predict binding affinity of peptides to MHC II molecules, no accurate methods exist to predict which ligands are generated as a result of natural antigen processing. We utilized a dataset of around 14,000 naturally processed ligands identified by mass spectrometry of peptides eluted from MHC class II expressing cells to investigate the existence of sequence signatures potentially related to the cleavage mechanisms that liberate the presented peptides from their source antigens. This analysis revealed preferred amino acids surrounding both N- and C-terminuses of ligands, indicating sequence-specific cleavage preferences. We used these cleavage motifs to develop a method for predicting naturally processed MHC II ligands, and validated that it had predictive power to identify ligands from independent studies. We further confirmed that prediction of ligands based on cleavage motifs could be combined with predictions of MHC binding, and that the combined prediction had superior performance. However, when attempting to predict CD4 T cell epitopes, either alone or in combination with MHC binding predictions, predictions based on the cleavage motifs did not show predictive power. Given that peptides identified as epitopes based on CD4 T cell reactivity typically do not have well-defined termini, it is possible that motifs are present but outside of the mapped epitope. Our attempts to take that into account computationally did not show any sign of an increased presence of cleavage motifs around well-characterized CD4 T cell epitopes. While it is possible that our attempts to translate the cleavage motifs in MHC II ligand elution data into T cell epitope predictions were suboptimal, other possible explanations are that the cleavage signal is too diluted to be detected, or that elution data are enriched for ligands generated through an antigen processing and presentation pathway that is less frequently utilized for T cell epitopes.

Reactivation of Human Cytomegalovirus (HCMV) and Human Adenovirus (HAdV) in immunocompromised patients following stem cell transplantation (SCT) or solid organ transplantation (SOT) is associated with high morbidity and mortality. The adoptive transfer of virus-specific CD8 + and CD4 + T cells has been shown to re-establish the antiviral T-cell response and improve clinical outcome. The viral load in immunocompromised patients can efficiently be reduced solely by the infusion of virus-specific CD4 + T cells. The identification of CD4 + T-cell epitopes has mainly focused on a limited number of viral proteins that were characterized as immunodominant. Here, we used in silico prediction to determine promiscuous CD4 + T-cell epitopes from the entire proteomes of HCMV and HAdV. Immunogenicity testing with enzyme-linked immuno spot (ELISpot) assays and intracellular cytokine staining (ICS) revealed numerous novel CD4 + T-cell epitopes derived from a broad spectrum of viral antigens. We identified 17 novel HCMV-derived and seven novel HAdV-derived CD4 + T-cell epitopes that were recognized by > 50% of the assessed peripheral blood mononuclear cell (PBMC) samples. The newly identified epitopes were pooled with previously published, retested epitopes to stimulate virus-specific memory T cells in PBMCs from numerous randomly selected blood donors. Our peptide pools induced strong IFNγ secretion in 46 out of 48 (HCMV) and 31 out of 31 (HAdV) PBMC cultures. In conclusion, we applied an efficient method to screen large viral proteomes for promiscuous CD4 + T-cell epitopes to improve the detection and isolation of virus-specific T cells in a clinical setting.

Background Salmonella enterica serovar Typhimurium ( S. Typhimurium) is a leading cause of salmonellosis, gastroenteritis, sepsis, and reactive arthritis. Transmission primarily occurs through contaminated water, eggs, meat, and dairy products. The disease disproportionately affects developing nations, where young children, the elderly, and immunocompromised individuals face high risks of severe morbidity and mortality. Its ability to evade host immune defenses and acquire multidrug resistance (MDR) exacerbates global public health challenges. Currently, no licensed human vaccine is available, underscoring the urgent need for targeted vaccine development. Methods This study utilized a reverse vaccinology approach and in silico strategies to identify highly immunogenic membrane proteins as potential vaccine candidates. The complete proteome of S. Typhimurium was screened for membrane-associated candidates using the SOSUI server. Antigenicity was evaluated using VaxiJen v2.0 (threshold ≥ 0.9), and allergenicity was assessed using AllerTOP v1.1. To ensure vaccine safety, homologous proteins were excluded based on PSI-BLAST analysis against the human proteome, and toxicity was predicted using ToxinPred. The immunogenic potential was further evaluated through C-ImmSim immune simulation software. B-cell and T-cell epitopes were predicted using ABCpred and the Immune Epitope Database (IEDB). Physicochemical characteristics were analyzed with ProtParam and TMHMM 2.0. Finally, BLASTp analysis was used to confirm the conservation of the selected proteins across MDR clinical isolates. Results Nine membrane proteins were prioritized based on strong antigenicity, non-allergenicity, non-toxicity, favorable epitope profiles, and physicochemical stability. All proteins were highly conserved in MDR isolates, supporting their utility for broad-spectrum vaccine development. Conclusion These targets show promising potential for developing a broadly protective multi-epitope vaccine against S. Typhimurium. However, in vitro and in vivo experimental validation is essential to confirm their immunogenicity and protective efficacy.

Dengue virus (DENV) remains a major global health challenge, with its incidence increasing sharply over the past two decades. More than 390 million infections occur annually, affecting nearly half of the world’s population. Despite progress in vaccine research, the coexistence of four antigenically distinct dengue virus serotypes (DENV1-4) complicates vaccine and diagnostic development, emphasizing the urgent need for improved preventive and diagnostic strategies. Immunoinformatics and computational vaccinology are powerful tools to elucidate host–pathogen interactions and accelerate vaccine and diagnostic discovery. As vaccination remains the most effective strategy to reduce the dengue burden and accurate diagnostics are essential for surveillance, this study identified conserved and serotype-specific dengue epitopes to support vaccine and diagnostic development. We conducted a comprehensive in silico analysis of the DENV envelope (E) protein across all four serotypes to identify conserved and serotype-specific T-cell and B-cell epitopes. Molecular docking was performed to evaluate HLA-binding affinity between predicted epitopes and their corresponding alleles, and intrinsically disordered regions of the dengue envelope protein were analyzed to identify flexible segments potentially involved in immune recognition. Finally, normal mode analysis was carried out to assess the structural flexibility and stability of the resulting epitope–HLA complexes. Our analysis revealed highly conserved CD8+ (CD4 +) epitopes, such as E 57 , E 135 , E 175 , E 313 and E 417 (E 133 , E 134 , and E 234 ) that remain antigenic despite sequence variation, suggesting their potential use in a universal dengue vaccine. We also identified distinct serotype-specific epitopes, which could serve as molecular signatures for precise immunodiagnostic assays (DENV-2: E 236 , E 267 , and E 424 ; DENV-3: E 204 , E 229 , E 274 , E 350 , and E 410 ; and DENV-4: E 402 , E 403 , E 65 , and E 446 ). Population coverage analysis predicted a global reach of 66.87%, with high representation in East Asia, Europe, and the Americas. Molecular docking and normal mode analyses confirmed stable peptide–HLA interactions with favorable binding energies, particularly for LTDYGALTL–HLA-A 01:01 and DTAWDFGSI–HLA-A 26:01 complexes. Collectively, this integrative in silico framework identifies epitope candidates that can inform next-generation multivalent dengue vaccines and enhance serotype-specific diagnostic platforms.