Epitope profiling of cow's milk allergen-specific antibodies with determining IgE content in epitopes-ALL, a 14-epitopes mixture.

Allergen-specific antibodies (Abs), IgE, and IgG4 increase during the early phase of oral immunotherapy (OIT) of allergen food in patients; subsequently, IgE levels decrease and specific IgG4 levels increase after successful OIT treatment. The detailed profile of these Abs during OIT remains largely unclear. We developed a diagnostic tool to assess the OIT efficacy and extent of responsiveness based on a profiling method by identifying epitopes recognized by the Ab classes of IgE or IgG4. A peptide microarray followed by microplate analysis using synthetic peptides was used to identify 14 epitopes widely recognized by IgE and/or IgG4 in the serum samples of patients with OIT among the amino acid sequences of five major cow's milk allergens. The set of defined 14 epitopes clarified different epitope profiles of allergen-specific IgE and IgG4 in each patient's serum samples. Moreover, the total signal of Abs recognizing all 14 epitopes was equal to the sum of all individual epitope-specific Abs. It was further observed that the quantitative value of IgE concentrations of 14 epitopes-ALL correlated with the ImmunoCAP IgE value. These findings strongly imply that the quantity of IgE and IgG4 recognizing epitopes-ALL may easily be used to measure allergy severity. To investigate this potential, we developed an immunochromatographic method that can detect IgE and IgG4 levels in patient samples. This study clearly demonstrated the usefulness of the defined 14 epitopes and their mixture, "epitopes-ALL," and that the simple and reliable methods of immunochromatography and microplate analyses demonstrating the epitope profile of allergen-specific Abs are applicable for diagnostic use at multiple disease stages and the OIT-treatment course in patients with cow's milk allergy.

Screening of Diabetes-Associated Autoantigens and Serum Antibody Profiles Using a Phage Display System.

Aims/Introduction: Phage display method is a crucial tool to find novel clinically valuable diabetes-associated autoantigens and identify known autoantigen epitopes that are associated with diabetes and could provide scientific support and guidance for the artificial construction and synthesis of Type I diabetes mellitus (T1DM) novel biomarkers. Materials and Methods: The phage display system was used for the "biopanning" of T1DM serum. Following the sequencing of the phage DNAs, the homologous sequences of the above fusion heptapeptide were further investigated by BLAST to track the origin of the polypeptide sequences. The antibody spectrum revealed new T1DM-associated epitopes and antibodies. Results: A total of 1200 phage DNA were sequenced and 9 conserved polypeptide sequences were collected. It was confirmed that the zinc transporter and islet amyloid protease were among them. The conserved polypeptide sequence 8 and another three distinctive polypeptide sequences derived from Proteus were discovered. Furthermore, we expressed recombinant proteins with homologous polypeptide sequences for the human islet amyloid polypeptide (IAPP) and polypeptide precursor human zinc transporter 8 (ZNT8). Through clinical sample detection for the serum from T1DM (n = 100) and T2DM (n = 200) patients, results demonstrate the importance and relevance of these polypeptides in the recognition and classification of various forms of diabetes. Conclusion: Human pancreatic and concurrent bacterial-derived protein antigens and their epitopes were identified in this research by the phage display system, which is crucial for distinguishing different types of diabetes.

Epitope prime editing shields hematopoietic cells from CD123 immunotherapy for acute myeloid leukemia.

Acute myeloid leukemia (AML) is a malignant cancer characterized by abnormal differentiation of hematopoietic stem and progenitor cells (HSPCs). While chimeric antigen receptor T (CAR-T) cell immunotherapies target AML cells, they often induce severe on-target/off-tumor toxicity by attacking normal cells expressing the same antigen. Here, we used base editors (BEs) and a prime editor (PE) to modify the epitope of CD123 on HSPCs, protecting healthy cells from CAR-T-induced cytotoxicity while maintaining their normal function. Although BE effectively edits epitopes, complex bystander products are a concern. To enhance precision, we optimized prime editing, increasing the editing efficiency from 5.9% to 78.9% in HSPCs. Epitope-modified cells were resistant to CAR-T lysis while retaining normal differentiation and function. Furthermore, BE- or PE-edited HSPCs infused into humanized mice endowed myeloid lineages with selective resistance to CAR-T immunotherapy, demonstrating a proof-of-concept strategy for treating relapsed AML.

Differential patterns of antibody response against SARS-CoV-2 nucleocapsid epitopes detected in sera from patients in acute phase of COVID-19, convalescents and pre-pandemic individuals.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have already infected more than 0.7 billion people and caused over 7 million deaths worldwide. At the same time, our knowledge about this virus is still incipient. In some cases, there is a pre-pandemic immunity, however its source is unknown. The analysis of patients' humoral responses might shed a light on this puzzle. In this paper, we evaluated the antibody recognition of nucleocapsid protein, one of the structural proteins of SARS-CoV-2. For this purpose, we used pre-pandemic, acute COVID-19 and convalescent patients' sera to identify and map nucleocapsid protein epitopes. We identified a common epitope KKSAAEASKKPRQKRTATKA recognized by sera antibodies from all three groups. Some motifs of this sequence are widespread among various coronaviruses, plant or human proteins indicating that there might be more sources of nucleocapsid-reactive antibodies than previous infection with seasonal coronavirus. The two sequences MSDNGPQNQRNAPRITFGGP and KADETQALPQRQKKQQTVTL were detected as specific for sera from patients in acute phase of infection and convalescents making them suitable for future development of vaccine against SARS-CoV-2. Knowledge of the humoral response to SARS-CoV-2 infection is essential for the design of appropriate diagnostic tools and vaccine antigens.

Prolonged Omicron-specific B cell maturation alleviates immune imprinting induced by SARS-CoV-2 inactivated vaccine.

SARS-CoV-2 ancestral strain-induced immune imprinting poses great challenges to updating vaccines for new variants. Studies showed that repeated Omicron exposures could override immune imprinting induced by inactivated vaccines but not mRNA vaccines, a disparity yet to be understood. Here, we analyzed the immune imprinting alleviation in inactivated vaccine (CoronaVac) cohorts after a long-term period following breakthrough infections (BTI). We observed in CoronaVac-vaccinated individuals who experienced BA.5/BF.7 BTI, the proportion of Omicron-specific memory B cells (MBCs) substantially increased after an extended period post-Omicron BTI, with their antibodies displaying enhanced somatic hypermutation and neutralizing potency. Consequently, the neutralizing antibody epitope distribution encoded by MBCs post-BA.5/BF.7 BTI after prolonged maturation closely mirrors that in BA.5/BF.7-infected unvaccinated individuals. Together, these results indicate the activation and expansion of Omicron-specific naïve B cells generated by first-time Omicron exposure helped to alleviate CoronaVac-induced immune imprinting, and the absence of this process should have caused the persistent immune imprinting seen in mRNA vaccine recipients.

Allergies to Allergens from Cats and Dogs: A Review and Update on Sources, Pathogenesis, and Strategies.

Inhalation allergies caused by cats and dogs can lead to a range of discomforting symptoms, such as rhinitis and asthma, in humans. With the increasing popularity of and care provided to these companion animals, the allergens they produce pose a growing threat to susceptible patients' health. Allergens from cats and dogs have emerged as significant risk factors for triggering asthma and allergic rhinitis worldwide; however, there remains a lack of systematic measures aimed at assisting individuals in recognizing and preventing allergies caused by these animals. This review provides comprehensive insights into the classification of cat and dog allergens, along with their pathogenic mechanisms. This study also discusses implementation strategies for prevention and control measures, including physical methods, gene-editing technology, and immunological approaches, as well as potential strategies for enhancing allergen immunotherapy combined with immunoinformatics. Finally, it presents future prospects for the prevention and treatment of human allergies caused by cats and dogs. This review will improve knowledge regarding allergies to cats and dogs while providing insights into potential targets for the development of next-generation treatments.

Unlocking Hope: Paving the Way for a Cutting-Edge Multi-Epitope Dengue Virus Vaccine.

Dengue fever is a significant health issue in Pakistan, demanding a vaccine effective against all the viral strains. This study employs reverse vaccinology to develop potential dengue vaccine candidates (DVAX I-III). The study thoroughly examined conserved areas of dengue virus serotypes 1-4's structural and non-structural proteins. Key viral proteins were analyzed to find antigenic peptides, which were incorporated into vaccine candidates and potentiated with adjuvants. Computational methods predicted peptide structures and evaluated their binding to immune receptors TLR 2, TLR 4, HLA *A1101, and DRB*401. A molecular dynamics simulation lasting 100 ns of the DVAX II-TLR4 complex at different time intervals clearly indicated that the ligand is attached to the receptor. Normal mode analysis assessed the stability and flexibility of these interactions. Encouragingly, all three vaccine candidates demonstrated favorable interactions with these immune receptors and the potential to induce a robust immune response. These findings suggest their safety and warrant further in vivo studies to evaluate their efficacy for clinical development.

From clones to immunopeptidomes: New developments in the characterization of permissive HLA-DP mismatches in hematopoietic cell transplantation.

Mismatching at the HLA-DPB1 locus occurs frequently in hematopoietic cell transplantation with unrelated donors. Despite this, HLA-DPB1 allelic mismatches have traditionally not been considered in patient-donor matching. A T-cell epitope (TCE) model for the functional assessment of permissive mismatches at this locus has nevertheless been adopted in clinical practice. While initially based on a hierarchical immunogenicity elucidated from allorecognition by T-cell clones isolated from a patient, newer developments in the understanding of this model's biological basis, including a central role for immunopeptidome divergence between mismatched allotypes, have prompted changes in the assignment of permissiveness, providing the opportunity for a more granular evaluation of graft-versus-host disease and relapse risks according to the nature and directionality of permissive mismatches. How these advances impact the assessment of permissiveness at HLA-DPB1 and potentially the intelligent selection of donors according to the main clinical goal for different patients is the subject of the present review.

An epitope imprinted electrochemical sensor for highly sensitive and selective determination of prostate-specific antigen.

A simple method for highly selective and sensitive prostate-specific antigen (PSA) detection using a molecularly imprinted electrochemical sensor is presented. The sensor was developed through an epitope imprinted strategy combined with electrochemical measurement techniques. An epitope molecularly imprinted polymer (EMIP) film was constructed on a AuNPs-coated gold electrode surface through electropolymerization, utilizing the C-terminus epitope of PSA (KWIKDTIVANP) as the template molecular and o-phenylenediamine as the functional monomer. The characteristics of EMIP film were investigated by using a scanning electron microscope and electrochemical test methods, including electrochemical impedance spectroscopy and cyclic voltammetry. Key parameters such as electropolymerization cycles, elution and rebinding times, and the molar ratio of template molecular to functional monomer were systematically optimized. The sensor demonstrated a detection limit (LOD) of 0.31 fg/mL and exhibited an excellent linear response towards PSA concentration ranging from 1.0 fg/mL to 0.1 µg/mL. Furthermore, the EMIP sensor showed excellent selectivity against other biological macromolecules, such as bovine serum albumin, human serum albumin, alpha-fetoprotein, and carcinoembryonic antigen. With recoveries between 95.89 and 106.04% for PSA detection in human serums the EMIP/AuNPs/AuE electrochemical sensor showed great potential in real sample analysis.

Integrating machine learning to advance epitope mapping.

Identifying epitopes, or the segments of a protein that bind to antibodies, is critical for the development of a variety of immunotherapeutics and diagnostics. In vaccine design, the intent is to identify the minimal epitope of an antigen that can elicit an immune response and avoid off-target effects. For prognostics and diagnostics, the epitope-antibody interaction is exploited to measure antigens associated with disease outcomes. Experimental methods such as X-ray crystallography, cryo-electron microscopy, and peptide arrays are used widely to map epitopes but vary in accuracy, throughput, cost, and feasibility. By comparing machine learning epitope mapping tools, we discuss the importance of data selection, feature design, and algorithm choice in determining the specificity and prediction accuracy of an algorithm. This review discusses limitations of current methods and the potential for machine learning to deepen interpretation and increase feasibility of these methods. We also propose how machine learning can be employed to refine epitope prediction to address the apparent promiscuity of polyreactive antibodies and the challenge of defining conformational epitopes. We highlight the impact of machine learning on our current understanding of epitopes and its potential to guide the design of therapeutic interventions with more predictable outcomes.

Using reverse vaccinology techniques combined with B-cell epitope prediction to screen potential antigenic proteins of the bovine pathogen Clostridium perfringens type A.

Clostridium perfringens type A frequently causes necrohaemorrhagic enteritis in cattle, a rapidly progressing disease with a high mortality rate, thus inflicting substantial economic losses in the cattle industry. Effective prevention and control of this disease rely on rapid detection and vaccination strategies, making the screening of antigenic proteins with diagnostic and vaccine potential particularly crucial. In this study, we conducted a pangenomic analysis of 15 bacterial strains, grounded in traditional reverse vaccinology and supplemented with B-cell linear and conformational epitope analysis tools. This approach led to the identification of 2304 core genes and 3606 accessory genes, among which 58 surface-exposed proteins, encoded by core genes, were identified Proteins lacking tertiary structure information were predicted via AlphaFold2, ultimately identifying four target proteins and 14 candidate proteins enriched with linear and conformational epitopes, including virulence proteins such as alpha-toxin, theta-toxin, and alpha-clostripain, and extracellular solute-binding proteins, rhodanese-like proteins, and the accessory gene-encoded lysozyme inhibitor LprI family protein. Our findings demonstrate that the combined use of multiple B-cell epitope analysis tools can help overcome the limitations of any single tool. The proteins selected in this study offer valuable references for rapid diagnostics and the development of genetically engineered vaccines.

Preventative Cancer Vaccine-Elicited Human Anti-MUC1 Antibodies Have Multiple Effector Functions.

BACKGROUND/OBJECTIVES: Mucin-1 (MUC1) is a transmembrane glycoprotein that is overexpressed and hypoglycosylated in premalignant and malignant epithelial cells compared to normal cells, creating a target antigen for humoral and cellular immunity. Healthy individuals with a history of advanced colonic adenomas and at high risk for colon cancer were enrolled in a clinical trial to evaluate the feasibility of using a MUC1 peptide vaccine to prevent colon cancer. Anti-MUC1 antibodies elicited by this vaccine were cloned using peripheral blood B cells and sera collected two weeks after a one-year booster. Twelve of these fully human monoclonal antibodies (mAb) were tested for binding to MUC1+ target cells, and three with the highest binding were further evaluated for various effector functions important for tumor rejection. METHODS: Immune cells were incubated together with target cells expressing variations in the number, distance, and membrane anchoring properties of the MUC1 epitope in the presence of each mAb. RESULTS: All three mAbs mediated antibody-dependent cytokine release (ADCR), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). Two also mediated antibody-dependent trogocytosis/trogoptosis (ADCT). None were capable of complement-dependent cytotoxicity (CDC). CONCLUSIONS: ADCP and ADCT functions were more efficient when antibodies bound epitopes proximal to and anchored to the membrane, providing insight for future therapeutic antibody validation strategies.

HERV-Derived Syncytin-1 and Syncytin-2 as Sources of Linear and Discontinuous Epitopes in Antiphospholipid Syndrome: A Pivotal Computational Study.

BACKGROUND: To date, no studies have investigated the potential reactivation of human endogenous retroviruses (HERVs) in the pathogenesis of antiphospholipid syndrome (APS). HERV-derived syncytin-1 and syncytin-2 are localized in the plasma membrane of cells and physiologically expressed during pregnancy. The current study aimed to determine whether the epitopes of syncytins can trigger an immune response leading to APS in genetically predisposed individuals. METHODS: The TepiTool, ABCpred, and DiscoTope servers were utilized to predict T-cell and B-cell epitopes by inputting the FASTA sequences and 3D structures of syncytin-1, syncytin-2, and ß2-glycoprotein I (ß2GPI), which served as a reference antigen for APS. T-cell epitopes were selected based on their binding to a panel of human leukocyte antigen (HLA) class II alleles associated with APS according to the literature. Epitope predictions for the different proteins were statistically compared using GraphPad Prism. RESULTS: For syncytin-1, we identified a total of 721 T-cell epitopes, 51 linear B-cell epitopes, and up to 40 conformational epitopes. For syncytin-2, we predicted 705 T-cell epitopes and 28 linear B-cell epitopes, but a lower number of conformational epitopes, which also exhibited lower B-cell receptor (BCR)-binding scores. The predicted T-cell and B-cell conformational epitopes of both syncytin-1 and syncytin-2 demonstrated significantly higher binding affinity to selected HLA alleles and BCR compared with ß2GPI. Furthermore, syncytin-1 exhibited significantly higher immunogenicity than syncytin-2. CONCLUSIONS: Both syncytin-1 and syncytin-2 are computationally endowed with potential epitopes that may activate either T cells or B cells in individuals genetically predisposed to APS. While these findings may illuminate the possible role of HERVs in the development of APS, they warrant validation in further laboratory studies.

Self-Tumor Antigens in Solid Tumors Turned into Vaccines by α-gal Micelle Immunotherapy.

A major reason for the failure of the immune system to detect tumor antigens (TAs) is the insufficient uptake, processing, and presentation of TAs by antigen-presenting cells (APCs). The immunogenicity of TAs in the individual patient can be markedly increased by the in situ targeting of tumor cells for robust uptake by APCs, without the need to identify and characterize the TAs. This is feasible by the intra-tumoral injection of α-gal micelles comprised of glycolipids presenting the carbohydrate-antigen "α-gal epitope" (Galα1-3Galß1-4GlcNAc-R). Humans produce a natural antibody called "anti-Gal" (constituting ~1% of immunoglobulins), which binds to α-gal epitopes. Tumor-injected α-gal micelles spontaneously insert into tumor cell membranes, so that multiple α-gal epitopes are presented on tumor cells. Anti-Gal binding to these epitopes activates the complement system, resulting in the killing of tumor cells, and the recruitment of multiple APCs (dendritic cells and macrophages) into treated tumors by the chemotactic complement cleavage peptides C5a and C3a. In this process of converting the treated tumor into a personalized TA vaccine, the recruited APC phagocytose anti-Gal opsonized tumor cells and cell membranes, process the internalized TAs and transport them to regional lymph-nodes. TA peptides presented on APCs activate TA-specific T cells to proliferate and destroy the metastatic tumor cells presenting the TAs. Studies in anti-Gal-producing mice demonstrated the induction of effective protection against distant metastases of the highly tumorigenic B16 melanoma following injection of natural and synthetic α-gal micelles into primary tumors. This treatment was further found to synergize with checkpoint inhibitor therapy by the anti-PD1 antibody. Phase-1 clinical trials indicated that α-gal micelle immunotherapy is safe and can induce the infiltration of CD4+ and CD8+ T cells into untreated distant metastases. It is suggested that, in addition to converting treated metastases into an autologous TA vaccine, this treatment should be considered as a neoadjuvant therapy, administering α-gal micelles into primary tumors immediately following their detection. Such an immunotherapy will convert tumors into a personalized anti-TA vaccine for the period prior to their resection.

Design of an Epitope-Based Vaccine Against MERS-CoV.

Background and Objectives: Middle East Respiratory Syndrome (MERS) is a viral respiratory illness caused by a coronavirus called Middle East respiratory syndrome. In the current study, immunoinformatics studies were applied to design an epitope-based vaccine construct against Middle East Respiratory Syndrome. Materials and Methods: In this study, epitopes base vaccine construct was designed against MERS using immunoinformatics approach. Results: In this approach, the targeted proteins were screened, and probable antigenic, non-allergenic, and good water-soluble epitopes were selected for vaccine construction. In vaccine construction, the selected epitopes were joined by GPGPG linkers, and a linear multi-epitope vaccine was constructed. The vaccine construct underwent a physiochemical property analysis. The 3D structure of the vaccine construct was predicted and subjected to refinement. After the refinement, the 3D model was subjected to a molecular docking analysis, TLRs (TLR-3 and TLR-9) were selected as receptors for vaccine construct, and the molecular docking analysis study determined that the vaccine construct has binding ability with the targeted receptor. Conclusions: The docking analysis also unveils that the vaccine construct can properly activate immune system against the target virus however experimental validation is needed to confirm the in silico findings further.

Designing a multi-epitope influenza vaccine: an immunoinformatics approach.

Influenza continues to be one of the top public health problems since it creates annual epidemics and can start a worldwide pandemic. The virus's rapid evolution allows the virus to evade the host defense, and then seasonal vaccines need to be reformulated nearly annually. However, it takes almost half a year for the influenza vaccine to become accessible. This delay is especially concerning in the event of a pandemic breakout. By producing the vaccine through reverse vaccinology and phage display vaccines, this time can be reduced. In this study, epitopes of B lymphocytes, cytotoxic T lymphocytes, and helper T lymphocytes of HA, NA, NP, and M2 proteins from two strains of Influenza A were anticipated. We found two proper epitopes (ASFIYNGRL and LHLILWITDRLFFKC) in Influenza virus proteins for CTL and HTL cells, respectively. Optimal epitopes and linkers in silico were cloned into the N-terminal end of M13 protein III (pIII) to create a multi-epitope-pIII construct, i.e., phage display vaccine. Also, prediction of tertiary structure, molecular docking, molecular dynamics simulation, and immune simulation were performed and showed that the designed multi-epitope vaccine can bind to the receptors and stimulate the immune system response.

Epitope Spreading in Immune-Mediated Glomerulonephritis: The Expanding Target.

Epitope spreading is a critical mechanism driving the progression of autoimmune glomerulonephritis. This phenomenon, where immune responses broaden from a single epitope to encompass additional targets, contributes to the complexity and severity of diseases such as membranous nephropathy (MN), lupus nephritis (LN), and ANCA-associated vasculitis (AAV). In MN, intramolecular spreading within the phospholipase A2 receptor correlates with a worse prognosis, while LN exemplifies both intra- and intermolecular spreading, exacerbating renal involvement. Similarly, ANCA reactivity in AAV highlights the destructive potential of epitope diversification. Understanding these immunological cascades reveals therapeutic opportunities-targeting early epitope spreading could curb disease progression. Despite promising insights, the clinical utility of epitope spreading as a prognostic tool remains debated. This review provides a complete overview of the current evidence, exploring the dual-edged nature of epitope spreading, the intricate immune mechanisms behind it, and its therapeutic implications. By elucidating these dynamics, we aim to pave the way for more precise, targeted interventions in autoimmune glomerular diseases.

Design of the conserved epitope peptide of SARS-CoV-2 spike protein as the broad-spectrum COVID-19 vaccine.

Our previous study has found that monoclonal antibodies targeting a conserved epitope peptide spanning from residues 1144 to 1156 of SARS-CoV-2 spike (S) protein, namely S(1144-1156), can broadly neutralize all of the prevalent SARS-CoV-2 strains, including the wild type, Alpha, Epsilon, Delta, and Gamma variants. In the study, S(1144-1156) was conjugated with bovine serum albumin (BSA) and formulated with Montanide ISA 51 adjuvant for inoculation in BALB/c mice to study its potential as a vaccine candidate. Results showed that the titers of S protein-specific IgGs and the neutralizing antibodies in mouse sera against various SARS-CoV-2 variants, including the Omicron sublineages, were largely induced along with three doses of immunization. The significant release of IFN-γ and IL-2 was also observed by ELISpot assays through stimulating vaccinated mouse splenocytes with the S(1144-1156) peptide. Furthermore, the vaccination of the S(1143-1157)- and S(1142-1158)-EGFP fusion proteins can elicit more SARS-CoV-2 neutralizing antibodies in mouse sera than the S(1144-1156)-EGFP fusion protein. Interestingly, the antisera collected from mice inoculated with the S(1144-1156) peptide vaccine exhibited better efficacy for neutralizing Omicron BA.2.86 and JN.1 subvariants than Omicron BA.1, BA.2, and XBB subvariants. Since the amino acid sequences of the S(1144-1156) are highly conserved among various SARS-CoV-2 variants, the immunogen containing the S(1144-1156) core epitope can be designed as a broadly effective COVID-19 vaccine. KEY POINTS: • Inoculation of mice with the S(1144-1156) peptide vaccine can induce bnAbs against various SARS-CoV-2 variants. • The S(1144-1156) peptide stimulated significant release of IFN-γ and IL-2 in vaccinated mouse splenocytes. • The S(1143-1157) and S(1142-1158) peptide vaccines can elicit more SARS-CoV-2 nAbs in mice.

Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

The interaction of sclerostin (Scl) with the low-density lipoprotein receptor-related protein 4 (LRP4) leads to a marked reduction in bone formation by inhibiting the Wnt/ß-catenin pathway. To characterize the Scl-LRP4 binding interface, we sorted a combinatorial library of Scl variants and isolated variants with reduced affinity to LRP4. We identified Scl single-mutation variants enriched during the sorting process and verified their reduction in affinity toward LRP4-a reduction that was not a result of changes in the variants' secondary structure or stability. We found that Scl positions K75 (loop 1) and V136 (loop 3) are critical hotspots for binding to LRP4. Our findings establish the foundation for targeting these hotspots for developing novel therapeutic strategies to promote bone formation.

Design of a novel EmTSP-3 and EmTIP based multi-epitope vaccine against Echinococcus multilocularis infection.

Background: Current treatments and prevention strategies for echinococcosis are inadequate. Recent advancements in molecular vaccine development show promise against Echinococcus granulosus; however, Echinococcus multilocularis remains a challenge. A Multi-epitope Vaccine could potentially induce specific B and T lymphocyte responses, thereby offering protection against Echinococcus multilocularis infection. Methods: This study aimed to develop a MEV against alveolar echinococcosis. Key epitopes from the Echinococcus multilocularis proteins EmTSP3 and EmTIP were identified using immunoinformatics analyses. These analyses were conducted to assess the MEV feasibility, structural characteristics, molecular docking, molecular dynamics simulations, and immune simulations. The immunogenicity and antigenicity of the vaccine were evaluated through in vitro and in vivo experiments, employing ELISA, Western blotting, FCM, challenge infection experiments, and ELISPOT. Results: The effective antigenicity and immunogenicity of MEV were demonstrated through immunoinformatics, as well as in vitro and in vivo experiments. In vitro experiments revealed that MEV increased the secretion of IFN-γ and IL-4 in PBMC and successfully bound to specific antibodies in patient serum. Furthermore, mice immunized with MEV developed a robust immune response, characterized by elevated levels of CD4+ and CD8+ T-cells, increased secretion of IFN-γ and IL-4 by specific Th1 and Th2 cells, and heightened serum antibody levels. Importantly, MEV reduced the weight of cysts by conferring resistance against echinococcosis. These findings suggest that MEV is a promising candidate for the prevention of Echinococcus multilocularis infection. Conclusion: A total of 7 CTL, 7 HTL, 5 linear B-cell, and 2 conformational B-cell epitopes were identified. The vaccine has demonstrated effective antigenicity and immunogenicity against AE through molecular docking, immune simulation, molecular dynamics studies, and both in vitro and in vivo experiments. It provides effective protection against Echinococcus multilocularis infection, thereby laying a foundation for further development.