B-Cell Epitope Prediction for Antipeptide Paratopes with the HAPTIC2/HEPTAD User Toolkit (HUT).

B-cell epitope prediction is key to developing peptide-based vaccines and immunodiagnostics along with antibodies for prophylactic, therapeutic and/or diagnostic use. This entails estimating paratope binding affinity for variable-length peptidic sequences subject to constraints on both paratope accessibility and antigen conformational flexibility, as described herein for the HAPTIC2/HEPTAD User Toolkit (HUT). HUT comprises the Heuristic Affinity Prediction Tool for Immune Complexes 2 (HAPTIC2), the HAPTIC2-like Epitope Prediction Tool for Antigen with Disulfide (HEPTAD) and the HAPTIC2/HEPTAD Input Preprocessor (HIP). HIP enables tagging of residues (e.g., in hydrophobic blobs, ordered regions and glycosylation motifs) for exclusion from downstream analyses by HAPTIC2 and HEPTAD. HAPTIC2 estimates paratope binding affinity for disulfide-free disordered peptidic antigens (by analogy between flexible-ligand docking and protein folding), from terms attributed to compaction (in view of sequence length, charge and temperature-dependent polyproline-II helical propensity), collapse (disfavored by residue bulkiness) and contact (with glycine and proline regarded as polar residues that hydrogen bond with paratopes). HEPTAD analyzes antigen sequences that each contain two cysteine residues for which the impact of disulfide pairing is estimated as a correction to the free-energy penalty of compaction. All of HUT is freely accessible online ( https://freeshell.de/~badong/hut.htm ).

Identification and Validation of Peptides Specifying SARS-CoV-2 B-Cell Epitopes Eliciting Neutralizing Antibodies.

Vaccination is an effective means of inducing immune protection to prevent transmissible diseases. During the Covid-19 pandemic, immunizations using traditional and novel vaccine platforms such as the inactivated SARSCo-V-2 vaccine, adenoviral-vectored, and nucleic acid-based mRNA vaccines have been relatively successful in controlling the rates of infection and hospitalizations. Nevertheless, the danger posed by the emergence of SARS-CoV-2 variants would set the stage for the design of next generation vaccines. To overcome the lack of efficacy of current vaccines against emerging SARS-CoV-2 variants, new vaccines must be able to overcome the reduced effectiveness of the current vaccines. Since the current Covid-19 vaccines are dependent on the whole S-protein of Wuhan strain as the antigen, mutations have rendered the current Covid-19 vaccines less effective against variants of concern (VoCs). Instead of using the whole S-protein, peptide-based epitopes could be predicted using immunoinformatic approaches, simulation of the 3D structures, overlapping peptides covering the whole length of the S-protein or peptide arrays based on synthetic peptide combinatorial libraries comprising peptides recognizable by monoclonal antibodies. B-cell epitopes were predicted, and immunogenicity of peptides was validated in mice by immunizing mice with peptides conjugated to keyhole limpet hemocyanin (KLH) mixed with Montanide 51 as an adjuvant. The immunogenicity of epitopes that could elicit peptide specific IgGs was determined by peptide-based ELISA. Neutralizing activities were determined by cPass and pseudovirus-based neutralization assays.

Characterization of Peptide Antibodies by Epitope Mapping Using Resin-Bound and Soluble Peptides.

Characterization of peptide antibodies through identification of their target epitopes is of utmost importance, as information about epitopes provide important knowledge, among others, for discovery and development of new therapeutics, vaccines, and diagnostics.This chapter describes a strategy for mapping of continuous peptide antibody epitopes using resin-bound and soluble peptides. The approach combines three different types of peptide sets for full characterization of peptide antibodies; (i) overlapping peptides, used to locate antigenic regions; (ii) truncated peptides, used to identify the minimal peptide length required for antibody binding; and (iii) substituted peptides, used to identify the key residues important for antibody binding and to determine the specific contribution of key residues. For initial screening, resin-bound peptides are used for epitope estimation, while soluble peptides subsequently are used for final epitope characterization and identification of critical hot spot residues. The combination of resin-bound peptides and soluble peptides for epitope mapping provides a time-saving and straightforward approach for characterization of antibodies recognizing continuous epitopes, which applies to peptide antibodies and occasionally antibodies directed to larger proteins as well.

Peptide Antibodies: Current Status.

Peptide antibodies have become one of the most important classes of reagents in molecular biology and clinical diagnostics. For this reason, methods for their production and characterization continue to be developed, including basic peptide synthesis protocols, peptide-conjugate production and characterization, conformationally restricted peptides, immunization procedures, etc. Detailed mapping of peptide antibody epitopes has yielded important information on antibody-antigen interaction in general and specifically in relation to antibody cross-reactivity and theories of molecular mimicry. This information is essential for detailed understanding of paratope-epitope dynamics, design of antibodies for research, design of peptide-based vaccines, development of therapeutic peptide antibodies, and de novo design of antibodies with predetermined specificity.

Immunoinformatics approach for design novel multiepitope prophylactic and therapeutic vaccine based on capsid proteins L1 and L2 and oncoproteins E6 and E7 of human papillomavirus 16 and human papillomavirus 18 against cervical cancer.

Objectives: This study aimed to identify the optimal protein construction for designing a multi-epitope vaccine with both prophylactic and therapeutic effects against cervical cancer, utilizing an immunoinformatics approach. The construction process involved using capsid epitopes L1 and L2, as well as oncoproteins E5, E6, and E7 from human papillomavirus (HPV) types 16 and 18. Methods: An experimental in silico analysis with an immunoinformatics approach was used to develop 2 multi-epitope vaccine constructs (A and B). Further analysis was then conducted to compare the constructs and select the one with the highest potential against cervical cancer. Results: This study produced 2 antigenic, non-allergenic, and nontoxic multi-epitope vaccine constructs (A and B), which exhibited the ideal physicochemical properties for a vaccine. Further analysis revealed that construct B effectively induced both cellular and humoral immune responses. Conclusion: The multi-epitope vaccine construct B for HPV 16 and 18, designed for both prophylactic and therapeutic purposes, met the development criteria for a cervical cancer vaccine. However, these findings need to be validated through in vitro and in vivo experiments.

Experimental Data on PIRCHE and T-Cell Reactivity: HLA-DPB1-Derived Peptides Identified by PIRCHE-I Show Binding to HLA-A*02:01 in vitro and T-Cell Activation in vivo.

Introduction: Human leukocyte antigen (HLA)-DPB1 mismatches during hematopoietic stem cell transplantation (HSCT) with an unrelated donor result in an increased risk for the development of graft-versus-host disease (GvHD). The number of CD8+ T-cell epitopes available for indirect allorecognition as predicted by the PIRCHE algorithm has been shown to be associated with GvHD development. As a proof of principle, PIRCHE-I predictions for HLA-DPB1 mismatches were validated in vitro and in vivo. Methods: PIRCHE-I analysis was performed to identify HLA-DPB1-derived peptides that could theoretically bind to HLA-A*02:01. PIRCHE-I predictions for HLA-DPB1 mismatches were validated in vitro by investigating binding affinities of HLA-DPB1-derived peptides to the HLA-A*02:01 in a competition-based binding assay. To investigate the capacity of HLA-DPB1-derived peptides to elicit a T-cell response in vivo, mice were immunized with these peptides. T-cell alloreactivity was subsequently evaluated using an interferon-gamma ELISpot assay. Results: The PIRCHE-I algorithm identified five HLA-DPB1-derived peptides (RMCRHNYEL, YIYNREEFV, YIYNREELV, YIYNREEYA, and YIYNRQEYA) to be presented by HLA-A*02:01. Binding of these peptides to HLA-A*02:01 was confirmed in a competition-based peptide binding assay, all showing an IC50 value of 21 µm or lower. The peptides elicited an interferon-gamma response in vivo. Conclusion: Our results indicate that the PIRCHE-I algorithm can identify potential immunogenic HLA-DPB1-derived peptides present in recipients of an HLA-DPB1-mismatched donor. These combined in vitro and in vivo observations strengthen the validity of the PIRCHE-I algorithm to identify HLA-DPB1 mismatch-related GvHD development upon HSCT.

Utilizing the Banana S-Adenosyl-L-Homocysteine Hydrolase Allergen to Identify Cross-Reactive IgE in Ryegrass-, Latex-, and Kiwifruit-Allergic Individuals.

Food allergies mediated by specific IgE (sIgE) have a significant socioeconomic impact on society. Evaluating the IgE cross-reactivity between allergens from different allergen sources can enable the better management of these potentially life-threatening adverse reactions to food proteins and enhance food safety. A novel banana fruit allergen, S-adenosyl-L-homocysteine hydrolase (SAHH), has been recently identified and its recombinant homolog was heterologously overproduced in E. coli. In this study, we performed a search in the NCBI (National Center for Biotechnology Information) for SAHH homologs in ryegrass, latex, and kiwifruit, all of which are commonly associated with pollen-latex-fruit syndrome. In addition, Western immunoblot analysis was utilized to identify the cross-reactive IgE to banana SAHH in the sera of patients with a latex allergy, kiwifruit allergy, and ryegrass allergy. ClustalOmega analysis showed more than 92% amino acid sequence identity among the banana SAHH homologs in ryegrass, latex, and kiwifruit. In addition to five B-cell epitopes, in silico analysis predicted eleven T-cell epitopes in banana SAHH, seventeen in kiwifruit SAHH, twelve in ryegrass SAHH, and eight in latex SAHH, which were related to the seven-allele HLA reference set (HLA-DRB1*03:01, HLA-DRB1*07:01, HLA-DRB1*15:01, HLA-DRB3*01:01, HLA-DRB3*02:02, HLA-DRB4*01:01, HLA-DRB5*01:01). Four T-cell epitopes were identical in banana and kiwifruit SAHH (positions 328, 278, 142, 341), as well as banana and ryegrass SAHH (positions 278, 142, 96, and 341). All four SAHHs shared two T-cell epitopes (positions 278 and 341). In line with the high amino acid sequence identity and B-cell epitope homology among the analyzed proteins, the cross-reactive IgE to banana SAHH was detected in three of three latex-allergic patients, five of six ryegrass-allergic patients, and two of three kiwifruit-allergic patients. Although banana SAHH has only been studied in a small group of allergic individuals, it is a novel cross-reactive food allergen that should be considered when testing for pollen-latex-fruit syndrome.

Gene and protein sequence features augment HLA class I ligand predictions.

The sensitivity of malignant tissues to T cell-based immunotherapies depends on the presence of targetable human leukocyte antigen (HLA) class I ligands. Peptide-intrinsic factors, such as HLA class I affinity and proteasomal processing, have been established as determinants of HLA ligand presentation. However, the role of gene and protein sequence features as determinants of epitope presentation has not been systematically evaluated. We perform HLA ligandome mass spectrometry to evaluate the contribution of 7,135 gene and protein sequence features to HLA sampling. This analysis reveals that a number of predicted modifiers of mRNA and protein abundance and turnover, including predicted mRNA methylation and protein ubiquitination sites, inform on the presence of HLA ligands. Importantly, integration of such "hard-coded" sequence features into a machine learning approach augments HLA ligand predictions to a comparable degree as experimental measures of gene expression. Our study highlights the value of gene and protein features for HLA ligand predictions.

Exosomes multiplex profiling, a promising strategy for early diagnosis of laryngeal cancer.

BACKGROUND: Exosomes are nanosized vesicles released from all cells into surrounding biofluids, including cancer cells, and represent a very promising direction in terms of minimally invasive approaches to early disease detection. They carry tumor-specific biological contents such as DNA, RNA, proteins, lipids, and sugars, as well as surface molecules that are able to pinpoint the cellular source. By the above criteria, exosomes may be stratified according to the presence of tissue and disease-specific signatures and, due to their stability in such biofluids as plasma and serum, they represent an indispensable source of vital clinical insights from liquid biopsies, even at the earliest stages of cancer. Therefore, our work aimed to isolate and characterize LCa patients' derived exosomes from serum by Flow Cytometry in order to define a specific epitope signature exploitable for early diagnosis. METHODS: Circulating exosomes were collected from serum collected from 30 LCa patients and 20 healthy volunteers by the use of antibody affinity method exploiting CD63 specific surface marker. Membrane epitopes were then characterized by Flow cytometry multiplex analysis and compared between LCa Patients and Healthy donors. Clinical data were also matched to obtain statistical correlation. RESULTS: A distinct overexpression of CD1c, CD2, CD3, CD4, CD11c, CD14, CD20, CD44, CD56, CD105, CD146, and CD209 was identified in LCa patients compared to healthy controls, correlating positively with tumor presence. Conversely, CD24, CD31, and CD40, though not overexpressed in tumor samples, showed a significant correlation with nodal involvement in LCa patients (p < 0.01). CONCLUSION: This approach could allow us to set up a cost-effective and less invasive liquid biopsy protocol from a simple blood collection in order to early diagnose LCa and improve patients' outcomes and quality of life.

An integrated database of experimentally validated major histocompatibility complex epitopes for antigen-specific cancer therapy.

Cancer immunotherapy represents a paradigm shift in oncology, offering a superior anti-tumor efficacy and the potential for durable remission. The success of personalized vaccines and cell therapies hinges on the identification of immunogenic epitopes capable of eliciting an effective immune response. Current limitations in the availability of immunogenic epitopes restrict the broader application of such therapies. A critical criterion for serving as potential cancer antigens is their ability to stably bind to the major histocompatibility complex (MHC) for presentation on the surface of tumor cells. To address this, we have developed a comprehensive database of MHC epitopes, experimentally validated for their MHC binding and cell surface presentation. Our database catalogs 451 065 MHC peptide epitopes, each with experimental evidence for MHC binding, along with detailed information on human leukocyte antigen allele specificity, source peptides, and references to original studies. We also provide the grand average of hydropathy scores and predicted immunogenicity for the epitopes. The database (MHCepitopes) has been made available on the web and can be accessed at https://github.com/jcm1201/MHCepitopes.git. By consolidating empirical data from various sources coupled with calculated immunogenicity and hydropathy values, our database offers a robust resource for selecting actionable tumor antigens and advancing the design of antigen-specific cancer immunotherapies. It streamlines the process of identifying promising immunotherapeutic targets, potentially expediting the development of effective antigen-based cancer immunotherapies.

Elevated serum levels of soluble B-cell maturation antigen as a prognostic biomarker for multiple myeloma.

Serum B-cell maturation antigen (sBCMA) levels can serve as a sensitive biomarker in multiple myeloma (MM). In the research setting, sBCMA levels can be accurately detected by enzyme-linked immunosorbent assay (ELISA), but the approach has not been approved for clinical use. Here, we used a novel chemiluminescence method to assess sBCMA levels in 759 serum samples from 17 healthy donors and 443 patients with plasma cell (PC) diseases including AL amyloidosis, POEMS syndrome and MM. Serum BCMA levels were elevated 16.1-fold in patients with newly diagnosed MM compared to healthy donors and rare PC diseases patients. Specifically, the sBCMA levels in patients with progressive disease were 64.6-fold higher than those who showed partial response or above to treatment. The sBCMA level also correlated negatively with the response depth of MM patients. In newly diagnosed and relapsed MM patients, survival was significantly longer among those subjects whose sBCMA levels are below the median levels compared with those above the median value. We optimized the accuracy of the survival prediction further by integrating sBCMA level into the Second Revised International Staging System (R2-ISS). Our findings provide evidence that the novel chemiluminescence method is sensitive and practical for measuring sBCMA levels in clinical samples and confirm that sBCMA might serve as an independent prognostic biomarker for MM.

Multi-epitope protein production and its application in the diagnosis of opisthorchiasis.

BACKGROUND: Opisthorchiasis and cholangiocarcinoma (CCA) continue to be public health concerns in many Southeast Asian countries. Although the prevalence of opisthorchiasis is declining, reported cases tend to have a light-intensity infection. Therefore, early detection by using sensitive methods is necessary. Several sensitive methods have been developed to detect opisthorchiasis. The immunological detection of antigenic proteins has been proposed as a sensitive method for examining opisthorchiasis. METHODS: The Opisthorchis viverrini antigenic proteins, including cathepsin B (OvCB), asparaginyl endopeptidase (OvAEP), and cathepsin F (OvCF), were used to construct multi-antigenic proteins. The protein sequences of OvCB, OvAEP, and OvCF, with a high probability of B cell epitopes, were selected using BepiPred 1.0 and the IEDB Analysis Resource. These protein fragments were combined to form OvCB_OvAEP_OvCF recombinant DNA, which was then used to produce a recombinant protein in Escherichia coli strain BL21(DE3). The potency of the recombinant protein as a diagnostic target for opisthorchiasis was assessed using immunoblotting and compared with that of the gold standard method, the modified formalin-ether concentration technique. RESULTS: The recombinant OvCB_OvAEP_OvCF protein showed strong reactivity with total immunoglobulin G (IgG) antibodies against light-intensity O. viverrini infections in the endemic areas. Consequently, a high sensitivity (100%) for diagnosing opisthorchiasis was reported. However, cross-reactivity with sera from other helminth and protozoan infections (including taeniasis, strongyloidiasis, giardiasis, E. coli infection, enterobiasis, and mixed infection of Echinostome spp. and Taenia spp.) and no reactivity with sera from patients with non-parasitic infections led to a reduced specificity of 78.4%. In addition, the false negative rate (FNR), false positive rate (FPR), positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy were 0%, 21.6%, 81.4%, 100%, and 88.9%, respectively. CONCLUSIONS: The high sensitivity of the recombinant OvCB_OvAEP_OvCF protein in detecting opisthorchiasis demonstrates its potential as an opisthorchiasis screening target. Nonetheless, research on reducing cross-reactivity should be undertaken by detecting other antibodies in other sample types, such as saliva, urine, and feces.

Identification of HLA-A*11:01 and A*02:01-Restricted EBV Peptides Using HLA Peptidomics.

Epstein-Barr Virus (EBV) is closely linked to nasopharyngeal carcinoma (NPC), notably prevalent in southern China. Although type II latency of EBV plays a crucial role in the development of NPC, some lytic genes and intermittent reactivation are also critical for viral propagation and tumor progression. Since T cell-mediated immunity is effective in targeted killing of EBV-positive cells, it is important to identify EBV-derived peptides presented by highly prevalent human leukocyte antigen class I (HLA-I) molecules throughout the EBV life cycle. Here, we constructed an EBV-positive NPC cell model to evaluate the presentation of EBV lytic phase peptides on streptavidin-tagged specific HLA-I molecules. Utilizing a mass spectrometry (LC-MS/MS)-based immunopeptidomic approach, we characterized eleven novel EBV peptides as well as two previously identified peptides. Furthermore, we determined these peptides were immunogenic and could stimulate PBMCs from EBV VCA/NA-IgA positive donors in an NPC endemic southern Chinese population. Overall, this work demonstrates that highly prevalent HLA-I-specific EBV peptides can be captured and functionally presented to elicit immune responses in an in vitro model, which provides insight into the epitopes presented during EBV lytic cycle and reactivation. It expands the range of viral targets for potential NPC early diagnosis and treatment.

Regional Variation of the CD4 and CD8 T Cell Epitopes Conserved in Circulating Dengue Viruses and Shared with Potential Vaccine Candidates.

As dengue expands globally and many vaccines are under trials, there is a growing recognition of the need for assessing T cell immunity in addition to assessing the functions of neutralizing antibodies during these endeavors. While several dengue-specific experimentally validated T cell epitopes are known, less is understood about which of these epitopes are conserved among circulating dengue viruses and also shared by potential vaccine candidates. As India emerges as the epicenter of the dengue disease burden and vaccine trials commence in this region, we have here aligned known dengue specific T cell epitopes, reported from other parts of the world with published polyprotein sequences of 107 dengue virus isolates available from India. Of the 1305 CD4 and 584 CD8 epitopes, we found that 24% and 41%, respectively, were conserved universally, whereas 27% and 13% were absent in any viral isolates. With these data, we catalogued epitopes conserved in circulating dengue viruses from India and matched them with each of the six vaccine candidates under consideration (TV003, TDEN, DPIV, CYD-TDV, DENVax and TVDV). Similar analyses with viruses from Thailand, Brazil and Mexico revealed regional overlaps and variations in these patterns. Thus, our study provides detailed and nuanced insights into regional variation that should be considered for itemization of T cell responses during dengue natural infection and vaccine design, testing and evaluation.

Repeated human leukocyte antigens eplets, importance of typing the partner.

INTRODUCTION: Antibody-mediated rejection (AMR) is the most common cause of immune-mediated allograft failure after kidney transplant and impacts allograft survival. Previous sensitization is a major risk factor for development of donor specific antibodies (DSA). AMR can have a wide range of clinical features such as impaired kidney function, proteinuria/hypertension or can be subclinical. HLA molecules have specific regions of antigens binding antibodies called epitopes and eplets are considered essential components responsible for immune recognition. We present a patient with subclinical AMR 1 week post transplantation. CASE REPORT: A 48-year-old, caucasian woman with end-stage kidney disease (ESKD) secondary to autosomal dominant polycystic kidney disease (ADPKD) on peritoneal dialysis was registered in deceased donor waitlist. She was a hypersensitized patient from 3 prior pregnancies with a calculated panel reactive antibody of 93,48%. She was transplanted through kidney paired exchange donation with no evidence of DSA pre transplantation. Surgery and post-op were unremarkable with excellent and immediate graft function. Per protocol DSA levels on the 5th day was DR1 of 3300 MFI, with an increase in MFI by day 13 with 7820 MFI and a new B41 1979MFI. Allograft kidney biopsy findings were diagnostic of AMR and she was treated with immunoglobulin and plasmapheresis. As early onset AMR post transplantation was observed an anamnestic response was hypothesized from a previous exposure to allo-HLA. We decided to type her husband, her son's father, which was presented with DSA. Mismatch eplet analysis revealed a shared 41 T and 67LQ eplets between the donor and husband, responsible for the reactivity and new HLA class I B41 and HLA class II DR1 DSA, respectively. DISCUSSION: Shared eplets between the patient husband and donor was responsible for the alloimmune response and early development of DSAs. This case highlights the importance of early monitoring DSA levels in highly sensitized patients after transplant in order to promptly address and lower inflammatory damage. Mismatch eplet analysis can provide a thorough and precise evaluation of immune compatibility providing a useful technique to immune risk stratification, donor selection and post-transplant immunosuppressive therapy and monitoring.

A novel chimeric vaccine containing multiple epitopes for simulating robust immune activation against Klebsiella pneumoniae.

BACKGROUND: Due to antibiotic resistance, the Klebsiella genus is linked to morbidity and death, necessitating the development of a universally protective vaccine against Klebsiella pathogens. METHODS: Core sequence analysis prioritized non-redundant host molecules and expected lipid bilayer peptides from fully sequenced Klebsiella genomes. These proteins were refined to identify epitopes, examining their immunogenicity, toxicity, solubility, and interaction with MHC alleles. Epitopes were linked to CPG ODN C274 via EAAAK, HEYGAEALERAG, and GGGS linkers to enhance immunological responses. The vaccine's tertiary structure was modelled and docked with MHC-I and MHC-II. RESULTS: Fifty-five proteins were recognized in the Vaxign collection as having remarkable features. Twenty-three proteins with potential pathogenicity were then identified. Eight options for vaccines emerged after the immunogenicity of proteins was examined. The best antigens were three proteins: MrkD, Iron-regulated lipid membrane polypeptides, and RmpA. These compounds were selected for their sensitivity. The structural protein sequences of K. pneumoniae were utilized to identify seven CTL epitopes, seven HTL epitopes, and seven LBL epitopes, respectively. The produced immunization displayed a stable contact with the receptors, based on molecular dynamic simulations lasting 250 nanoseconds. Intermolecular binding free energies also indicated the dominance of the van der Waals and electrostatic energies. CONCLUSION: In summary, the results of this study might help scientists develop a novel vaccine to prevent K. pneumoniae infections.

Designing a smallpox B-cell and T-cell multi-epitope subunit vaccine using a comprehensive immunoinformatics approach.

Smallpox is a highly contagious human disease caused by the variola virus. Although the disease was eliminated in 1979 due to its highly contagious nature and historical pathogenicity, with a mortality rate of up to 30%, this virus is an important candidate for biological weapons. Currently, vaccines are the critical measures to prevent this virus infection and spread. In this study, we designed a peptide vaccine using immunoinformatics tools, which have the potential to activate human immunity against variola virus infection efficiently. The design of peptides derives from vaccine-candidate proteins showing protective potential in vaccinia WR strains. Potential non-toxic and nonallergenic T-cell and B-cell binding and cytokine-inducing epitopes were then screened through a priority prediction using special linkers to connect B-cell epitopes and T-cell epitopes, and an appropriate adjuvant was added to the vaccine construction to enhance the immunogenicity of the peptide vaccine. The 3D structure display, docking, and free energy calculation analysis indicate that the binding affinity between the vaccine peptide and Toll-like receptor 3 is high, and the vaccine receptor complex is highly stable. Notably, the vaccine we designed is obtained from the protective protein of the vaccinia and combined with preventive measures to avoid side effects. This vaccine is highly likely to produce an effective and safe immune response against the variola virus infection in the body. IMPORTANCE: In this work, we designed a vaccine with a cluster of multiple T-cell/B-cell epitopes, which should be effective in inducing systematic immune responses against variola virus infection. Besides, this work also provides a reference in vaccine design for preventing monkeypox virus infection, which is currently prevalent.

A conserved motif in the immune-subdominant RAP-1 related antigen of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle.

Introduction: Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis, as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins. Methods and results: Sequence comparisons among the RRA sequences of several B. bovis strains and other Babesia spp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the NT of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinct Babesia species. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence. Discussion and conclusion: The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. The demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of the B. bovis RRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines against B. bovis.

Tumor Antigens beyond the Human Exome.

With the advent of immunotherapeutics, a new era in the combat against cancer has begun. Particularly promising are neo-epitope-targeted therapies as the expression of neo-antigens is tumor-specific. In turn, this allows the selective targeting and killing of cancer cells whilst healthy cells remain largely unaffected. So far, many advances have been made in the development of treatment options which are tailored to the individual neo-epitope repertoire. The next big step is the achievement of efficacious "off-the-shelf" immunotherapies. For this, shared neo-epitopes propose an optimal target. Given the tremendous potential, a thorough understanding of the underlying mechanisms which lead to the formation of neo-antigens is of fundamental importance. Here, we review the various processes which result in the formation of neo-epitopes. Broadly, the origin of neo-epitopes can be categorized into three groups: canonical, noncanonical, and viral neo-epitopes. For the canonical neo-antigens that arise in direct consequence of somatic mutations, we summarize past and recent findings. Beyond that, our main focus is put on the discussion of noncanonical and viral neo-epitopes as we believe that targeting those provides an encouraging perspective to shape the future of cancer immunotherapeutics.

AutoPepVax, a Novel Machine-Learning-Based Program for Vaccine Design: Application to a Pan-Cancer Vaccine Targeting EGFR Missense Mutations.

The current epitope selection methods for peptide vaccines often rely on epitope binding affinity predictions, prompting the need for the development of more sophisticated in silico methods to determine immunologically relevant epitopes. Here, we developed AutoPepVax to expedite and improve the in silico epitope selection for peptide vaccine design. AutoPepVax is a novel program that automatically identifies non-toxic and non-allergenic epitopes capable of inducing tumor-infiltrating lymphocytes by considering various epitope characteristics. AutoPepVax employs random forest classification and linear regression machine-learning-based models, which are trained with datasets derived from tumor samples. AutoPepVax, along with documentation on how to run the program, is freely available on GitHub. We used AutoPepVax to design a pan-cancer peptide vaccine targeting epidermal growth factor receptor (EGFR) missense mutations commonly found in lung adenocarcinoma (LUAD), colorectal adenocarcinoma (CRAD), glioblastoma multiforme (GBM), and head and neck squamous cell carcinoma (HNSCC). These mutations have been previously targeted in clinical trials for EGFR-specific peptide vaccines in GBM and LUAD, and they show promise but lack demonstrated clinical efficacy. Using AutoPepVax, our analysis of 96 EGFR mutations identified 368 potential MHC-I-restricted epitope-HLA pairs from 49,113 candidates and 430 potential MHC-II-restricted pairs from 168,669 candidates. Notably, 19 mutations presented viable epitopes for MHC I and II restrictions. To evaluate the potential impact of a pan-cancer vaccine composed of these epitopes, we used our program, PCOptim, to curate a minimal list of epitopes with optimal population coverage. The world population coverage of our list ranged from 81.8% to 98.5% for MHC Class II and Class I epitopes, respectively. From our list of epitopes, we constructed 3D epitope-MHC models for six MHC-I-restricted and four MHC-II-restricted epitopes, demonstrating their epitope binding potential and interaction with T-cell receptors. AutoPepVax's comprehensive approach to in silico epitope selection addresses vaccine safety, efficacy, and broad applicability. Future studies aim to validate the AutoPepVax-designed vaccines with murine tumor models that harbor the studied mutations.