The vast diversity of the virosphere underscores the need for rapid, adaptable vaccine development infrastructures. Arthropod-borne zoonotic alphaviruses, in particular, continue to pose substantial threats to human and animal health. We present a fast, multitarget vaccine design pipeline integrating machine learning–based epitope prediction, protein modeling, and docking to prioritize viral peptides by immunogenicity, allele coverage, solubility, and stability. T cell epitopes were validated using peptide microarrays and molecular dynamics simulations, confirming receptor binding accuracy. Flow cytometry of murine and human peripheral blood mononuclear cells demonstrated robust T cell activation and cytokine secretion (IFN-γ, TNF-α, or IL-2), dependent on species and HLA allele. Final candidates were selected by composite immunogenicity scores. While this study primarily validates the T cell–specific arm of our predictive pipeline, complementary B cell epitope analyses are ongoing. Our findings support the development of broadly protective pan-alphaviral vaccines and the establishment of efficient, tunable processes for global vaccine development.

Decoding the Glycan Shield: Immune Recognition and Response to the HIV-1 Envelope Trimer.

Adeno-associated virus (AAV) vectors are widely used in gene therapy, but their immunogenicity remains a significant challenge, limiting long-term efficacy and the feasibility of repeated administration. In this study, we combine computational prediction with experimental validation to engineer AAV9 capsids with reduced immunogenicity. To facilitate this, we developed the Epitope Modification and MHC Prediction (EMMP) pipeline, which systematically automates the evaluation of amino acid substitutions for their predicted effects on major histocompatibility complex (MHC) presentability. Using this pipeline, we modify a CD4⁺ T-cell epitope in the AAV9 capsid that is identified and characterized as a proof-of-concept. Two mutant variants, R312H and R312Q, are selected and evaluated for transduction efficiency in vitro and immune response modulation in vivo. Notably, R312Q shows a significant reduction in T-cell activation and anti-AAV9 antibody production, albeit with a slight reduction in transduction at low multiplicities of infection (MOI). These results demonstrate a rational approach for optimizing AAV vector design, with potential applications for improving the safety and efficacy of gene therapy.

Mycoplasma pneumoniae (M. pneumoniae) is the crucial factor of global acquired respiratory infections. Currently, there are no specific disease modification treatments or vaccines available, and the vaccine development for this pathogen lags behind due to the complexity and variability of its antigens. A novel vaccine with broad-spectrum characteristics is essential to provide comprehensive protection against continuously evolving wild-type strains. Here, a broad-spectrum muti-epitope vaccine against M. pneumoniae had been designed through immunoinformatics methods. To ensure its broad-spectrum, we generated consistent sequences from all the antigen proteins of different strains, and then identified potential T cell epitopes. The multi-epitope vaccine (MEV) of M. pneumoniae incorporated 16 CTLs and 7 HTLs from the HMW1–3 and p1 adhesin proteins, which comprised 458 amino acids with adjuvant. The vaccine evaluation showed that the MEV had ideal physicochemical properties, high antigenicity, high immunogenicity, and was non-toxic. Furthermore, there was a strong and stable binding interaction between this vaccine and the toll-like receptors, which could be supported by the normal mode analysis. Finally, codon optimization resulted in the optimal GC content and higher CAI value. The vaccine candidate is expected to induce strong cellular immune responses and may provide protective immunity against the pathogen. We provided a novel in silico vaccine design strategy for vaccine design, which could provide a technical framework for the development of vaccines against other pathogens.

Immunodominance is a poor predictor of vaccine-induced T follicular helper cell quality.

Immunopathogenic role of the HLA-B∗51:01-specific immunopeptidome in Behçet's disease.

Design a multi-epitope vaccine targeting Chlamydia psittaci plasmid proteins through reverse vaccinology.

DNA-based cancer vaccines represent a safe and promising immunotherapeutic strategy, but their clinical efficacy is often limited by weak immunogenicity, primarily due to inefficient antigen cross-presentation. To overcome this challenge, the MHC class I trafficking domain (MITD) can be fused to tumor antigens to enhance their intracellular routing in dendritic cells (DCs), thereby promoting the efficiency of cross-presentation. In addition, incorporation of CD4+ T cell epitopes, such as PADRE or P2P16, can robustly activate CD4+ T cells, further amplifying antitumor immunity. Thus, combining MITD with CD4+ epitopes is expected to synergistically improve DNA vaccine potency. Mesothelin (MSLN), a tumor-associated antigen highly expressed in pancreatic cancer, was selected as the target in this study. We designed MSLN-targeted DNA vaccines incorporating MITD together with either PADRE or P2P16. In a Panc02 murine model, the MITD-PADRE construct, a novel design, elicited stronger immune responses and more effective antitumor activity compared to other formulations. To further counteract immunosuppression, we combined the vaccine with gemcitabine, which enhanced therapeutic efficacy. Together, these findings demonstrate that integrating PADRE with MITD in MSLN-targeted DNA vaccines offers a promising combinatorial strategy for advancing pancreatic cancer immunotherapy.

Vaccines are effective and much-needed tools against bacterial infection, which mitigate multidrug resistance; however, selection of bacterial antigens that elicit protection and contribute to effective vaccines remains challenging. Here we use immunopeptidomics to identify CD4+ T cell vaccine targets in methicillin-resistant Staphylococcus aureus, a clinically significant, antibiotic-resistant bacterium that is susceptible to T cell-mediated control. We identified a highly conserved, immunodominant CD4+ T cell epitope in S. aureus that is derived from the core DNA-binding protein Hu (Hup). This epitope was shared across a range of clinically relevant streptococcal and staphylococcal species, and cross-species-reactive Hup-specific CD4+ T cells were found in both mice and humans. Immunization of mice with the Hup epitope resulted in the development of broadly protective CD4+ T cell immunity capable of limiting disease severity following infection with S. aureus and Streptococcus pneumoniae. These findings suggest that vaccines incorporating antigens derived from core genes conserved across species might confer broad-spectrum protection against multiple clinically relevant, antibiotic-resistant streptococcal and staphylococcal strains.

Serologic diagnosis of canine brucellosis caused by Brucella canis remains quite challenging since the currently available methods have considerable limitations, although the relevance and awareness about this zoonotic pathogen is increasing over the past few years. Therefore, the development of novel strategies is highly desirable. In this study, in silico analyses resulted in prediction of B. canis specific B cell epitopes, which were validated by synthesizing the corresponding peptides on a membrane followed by immunobloting with sera from dogs naturally infected with B. canis or uninfected controls as well as by excluding epitopes that cross reacted with sera from cattle and sheep infected with B. abortus and B. ovis, respectively. This approach resulted in the identification of 26 epitopes that reacted exclusively with sera from dogs infected with B. canis, from which the 15 with strongest signal in the immunoblot were selected and synthesized in soluble form for further analyses. The best 10 synthetic peptides (considering the noise to signal ratio) were used in combination as antigens for development of a B. canis specific indirect enzyme-linked immunosorbent assay (iELISA) protocol, which yielded improved specificity to differentiate from other common canine pathogens (Leishmania sp. and Babesia sp.) and an improved performance (100% specificity and 80% sensitivity) when compared to crude bacterial protein extracts used as antigen for iELISA. These selected epitopes were also incorporated in a multi-B. canis epitope protein that was expressed in E. coli and employed as antigen for detection of anti-B. canis IgG and IgM from naturally infected dogs, resulting in analytical performance similar to the iELISA protocol in which synthetic peptides were used as antigens (100% specificity and 75% sensitivity). The results clearly indicate that combination of detection of IgM and IgG may result in higher sensitivity. Therefore, this study provides novel tools for improving the accuracy of serologic diagnosis of canine brucellosis caused by B. canis.

Chikungunya virus (CHIKV) infection is a re-emerging arboviral disease in tropical and subtropical regions. In addition to acute febrile syndrome, CHIKV infection may lead to chronic articular manifestations that significantly affect a long-term quality of life. This study aimed to design a universal vaccine candidate covering all circulating genotypes of CHIKV based on conserved multiepitope platform. We employed a large scale phylogenetic and immunoinformatic approach to identify conserved regions of the open reading frames (ORF2) region encoding viral structural proteins. This study ultimately identified 10high-quality epitopes: 6 MHC-I, 1 MHC-II, and 3 B cell epitopes. The selected epitopes span multiple viral domains, including C, E1, E2, and E3, with high immunogenicity (VaxiJen ≥ 66%), non-toxic, and non-allergenic properties. These selected epitopes were utilized to design multiepitope vaccine constructs (MEV-CHIKV) linked with various linkers in combination with adjuvants (human β-defensin 3) to enhance the immune responses. Structural validation analysis showed high quality and stability of the vaccine construct. Based on molecular docking analysis, the designed vaccine has high binding affinities with the active site of TLR3. In silico immune simulation showed induction of robust adaptive immune responses, characterized by the activation and expansion of B and T cell populations. Codon optimization and rare codon analysis revealed a potentially high expression in bacterial system. Thus, the vaccine cadidate is anticipated to effectively and simultaneously induce robust cellular and humoral immune responses. In addition, it should retain its high protection upon emergence of novel mutations within the CHIKV genome. Since our study is merely in silico-based analysis, further in vitro and in vivo experimental validation to demonstrate the immunogenic properties of the vaccine candidate are still needed.

Influenza vaccines targeting hemagglutinin require annual updates due to viral variability, limiting protection against pandemic strains. The conserved matrix protein 2 ectodomain (M2e) peptide offers cross‐strain immunity but suffers from low immunogenicity. Here, M2e peptides are conjugated onto nanostructured lipid carriers (NLCs) using bioorthogonal click chemistry, preserving key epitopes and enhancing antigen presentation. Immunization in mice demonstrates that NLC‐M2e induces focused and robust anti‐M2e antibody responses, which are significantly amplified by incorporating a pan‐DR epitope helper T cell epitope, achieving antibody levels compatible with protection. The formulation elicits balanced IgG subclasses and T‐helper 1 cell‐type cellular responses, supporting effective immune mechanisms. These findings suggest that NLC‐based delivery of M2e peptides, especially with helper epitopes, represents a promising universal influenza vaccine strategy warranting further preclinical and clinical investigation.

Previous Staphylococcus aureus vaccines have been unsuccessful, but it seems that including T cell immunity in the vaccine is essential for its success. This study used Metal ABC transporter substrate-binding protein (MABC), Nickel ABC transporter (NABC), and Phosphatidylinositol phosphodiesterase (PIc) B and T cell epitopes as a vaccine in a mouse bacteremia model. Mice immunized with PIc and epitope mixture groups showed robust humoral immunity and high survival rates in the bacteremia model. The highest protection level of mice was shown in the peptide mixture group, which was correlated with robust humoral response, the highest level of interferon-gamma (INF-γ) and the lowest levels of interleukin-2 (IL-2). The peptide mixture group also showed the highest count of CD8 cells. These results demonstrate that including B and T cell epitopes improved both the humoral and cellular immunity and resulted in the best outcome in the S. aureus bacteremia model. This finding has important implications for the development of future S. aureus vaccines.

The recent outbreak of the Monkeypox virus (MPXV) across multiple regions has highlighted the need for effective vaccine. This study employed an in-silico immunoinformatics approach to design a multi-epitope vaccine against monkeypox virus. The IMV heparin binding surface protein (H3L) of MPV was selected as vaccine target. B and T cell epitopes were predicted and evaluated for their toxicity, allergenicity, antigenicity and global population coverage. A multi-epitope construct was assembled using adjuvant and appropriate linkers. The 3D vaccine structure was modelled, enhanced and then checked for its overall structure and physiochemical properties. The in-silico docking vaccine construct and TLR3 receptor was performed to check affinity and stability of vaccine-TLR3 complex. The vaccine's ability to express and activate the immune system was demonstrated by its cloning into the E. coli plasmid pET-28b (+). The selected H3L protein exhibited 92.22% sequence conservation across Monkeypox virus isolates of different continents, supporting its suitability as broad-spectrum vaccine target. The molecular docking and dynamics analysis confirmed both the stability and strong binding affinity of the vaccine construct with the TLR3. Immune stimulation predicted robust humoral and cellular responses, with evidence of immunological memory formation. Overall, we proposed a potential vaccine construct that has the potential to effectively prevents monkeypox. However, in-vivo and in-vitro investigations are needed to validate the biological effectiveness and safety of the suggested vaccine.

BackgroundThe recent outbreak of the Monkeypox virus (MPXV) across multiple regions has highlighted the need for effective vaccine. This study employed an in-silico immunoinformatics approach to design a multi-epitope vaccine against monkeypox virus.MethodsThe IMV heparin binding surface protein (H3L) of MPV was selected as vaccine target. B and T cell epitopes were predicted and evaluated for their toxicity, allergenicity, antigenicity and global population coverage. A multi-epitope construct was assembled using adjuvant and appropriate linkers. The 3D vaccine structure was modelled, enhanced and then checked for its overall structure and physiochemical properties. The in-silico docking vaccine construct and TLR3 receptor was performed to check affinity and stability of vaccine-TLR3 complex. The vaccine’s ability to express and activate the immune system was demonstrated by its cloning into the E. coli plasmid pET-28b (+).ResultsThe selected H3L protein exhibited 92.22% sequence conservation across Monkeypox virus isolates of different continents, supporting its suitability as broad-spectrum vaccine target. The molecular docking and dynamics analysis confirmed both the stability and strong binding affinity of the vaccine construct with the TLR3. Immune stimulation predicted robust humoral and cellular responses, with evidence of immunological memory formation.ConclusionOverall, we proposed a potential vaccine construct that has the potential to effectively prevents monkeypox. However, in-vivo and in-vitro investigations are needed to validate the biological effectiveness and safety of the suggested vaccine.

Helper CD4+ T cells play a central role in orchestrating protective immune responses during infection and vaccination. Activation of naïve CD4+ T cells is initiated by the recognition of antigenic peptides presented on MHC class II molecules, leading to their differentiation into distinct effector subsets. In the context of influenza virus infection, the quality of CD4+ T cell reponses is a critical determinant of both cellular and humoral immunity. Given the limited breadth of protection conferred by current influenza vaccines, which primarily induce strain-specific neutralizing antibodies, there is growing interest in harnessing CD4+ T cell responses targeting conserved viral epitopes to achieve broader and more durable protection. Identifying potent T cell epitopes within viral antigens is therefore critical for designing effective vaccines that elicit robust, durable, and cross-protective helper T cell and antibody responses. In this study, using a mouse model of influenza virus infection, we identified CD4+ T cell epitopes within the hemagglutinin (HA) and nucleoprotein (NP) of the H1N1 strain. We generated two NP peptide/MHC class II tetramers, NP264-274/I-Ab and NP418-428/I-Ab, which enabled in vivo tracking and characterization of epitope-specific CD4+ T cells during infection. These epitope-specific T cells exhibited distinct TCR repertoires and differentiation patterns toward either Th1 or T follicular helper (Tfh) lineages, suggesting that epitope specificity shapes the quality of the T cell response. Our findings provide new insights into the epitope-dependent functional diversity of helper T cell responses and offer valuable tools for the rational design of broadly protective influenza vaccines.

Immunization with radiation-attenuated sporozoites (RAS) drives effective sterilizing immunity against liver-stage Plasmodium infection. However, protection is compromised in individuals living in malaria endemic regions and the mechanisms of vaccine failure are unclear. Here we show that previous blood-stage exposure in a mouse model of Plasmodium yoelii infection compromises Plasmodium berghei RAS-induced essential CD8+ T cell responses and subsequent protection. The persisting malarial pigment haemozoin mediates impaired CD8+ T cell responses owing to impaired antigen uptake by dendritic cells, leading to reduced T cell activation. We designed a lipid nanoparticle-encapsulated mRNA vaccine that encodes a string of Plasmodium CD8+ T cell epitopes, which overcomes the defective T cell response and restores protection in Plasmodium-exposed mice. A combined RAS-plus-mRNA vaccine regimen enhances liver-resident memory T cells and protection in murine malaria-experienced hosts. The identification of haemozoin as a potential obstacle to vaccine efficacy in malaria endemic areas can inform the design of more effective malaria vaccines.

Novel methods for the discovery of disease-associated T cell epitopes in autoimmunity.

Autoreactive T cells identified in patients with anti-Jo1+ antisynthetase syndrome recognise a new epitope on histidyl t-RNA synthetase.

Enteroendocrine cells (EECs) are dispersed along the intestinal mucosa and transduce luminal stimuli into hormonal signals. EECs exhibit neuron-like features and express both α-synuclein and tau, two proteins pathologically and genetically linked to Parkinson disease (PD). These observations support the hypothesis that EECs may be involved in disease development in the "body-first" PD subtype. Cellular models represent invaluable tools for studying the role of α-synuclein and tau in PD pathogenesis. However, the sensitivity and specificity of commercial antibodies for detecting α-synuclein and tau in EEC cell lines remain unclear. We tested by immunoblot a panel of commercial total-α-synuclein and total-tau antibodies on protein lysates from three EEC cell lines: GLUTag, NCI-H716, and STC-1. Pharmacological and biochemical manipulations were applied to assess the specificity of antibodies against phosphorylated α-synuclein and tau. Five antibodies detected total α-synuclein in NCI-H716 lysates, whereas the antibodies D1M9X and Tau12 detected total tau in GLUTag and NCI-H716 lysates, respectively. α-synuclein and tau protein levels were comparable between naïve and differentiated NCI-H716 cells. Four phospho-specific antibodies revealed phospho-α-synuclein S129 in NCI-H716. Of the nine phospho-tau antibodies tested, six recognized tau phosphorylated at specific epitopes (T181, S199, T231, S356, S396, and S404) in GLUTag cells. Our results indicate that NCI-H716 cells represent an ideal EEC model for studying α-synuclein expression and phosphorylation, whereas GLUTag cells are preferable for investigating tau protein biology. This work provides a comprehensive antibody toolbox to dissect the physiological and pathological role of α-synuclein and tau in EECs.