TZ1391: a computationally designed circular mRNA multi-epitope vaccine candidate against Mycobacterium tuberculosis via TLR3 immunomodulation.

One of the deadliest malignant cancer in women globally is cervical cancer. Specifically, cervical cancer is the second most common type of cancer in Indonesia. The main infectious agent of cervical cancer is the human papilloma virus (HPV). Although licensed prophylactic vaccines are available, cervical cancer cases are on the rise. Therapy using multiepitope-based vaccines is a very promising therapy for cervical cancer. This study aimed to develop a multiepitope vaccine based on the E1 and E2 proteins of HPV 16, 18, 45, and 52 using in silico. In this study, we develop a novel multiepitope vaccine candidate using an immunoinformatic approach. We predicted the epitopes of the cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) and evaluated their immunogenic properties. Population coverage analysis of qualified epitopes was conducted to determine the successful use of the vaccine worldwide. The epitopes were constructed into a multiepitope vaccine by using AAY linkers between the CTL epitopes and GPGPG linkers between the HTL epitopes. The tertiary structure of the multiepitope vaccine was modeled with AlphaFold and was evaluated by Prosa-web. The results of vaccine construction were analyzed for B-cell epitope prediction, molecular docking with Toll like receptor-4 (TLR4), and molecular dynamics simulation. The results of epitope prediction obtained 4 CTL epitopes and 7 HTL epitopes that are eligible for construction of multiepitope vaccines. Prediction of the physicochemical properties of multiepitope vaccines obtained good results for recombinant protein production. The interaction showed that the interaction of the multiepitope vaccine-TLR4 complex is stable based on the binding free energy value -106.5 kcal/mol. The results of the immune response simulation show that multiepitope vaccine candidates could activate the adaptive and humoral immune systems and generate long-term B-cell memory. According to these results, the development of a multiepitope vaccine with a reverse vaccinology approach is a breakthrough to develop potential cervical cancer therapeutic vaccines.

Computational construction of a glycoprotein multi-epitope subunit vaccine candidate for old and new South-African SARS-CoV-2 virus strains

An in-silico design of a multi-epitope vaccine candidate against human metapneumovirus (HMPV) through prediction of B- and T-cell epitopes and molecular dynamics simlation.

Endometrial cancer (EC) is one of the most common cancers of the female reproductive system. Multi-epitope vaccine may be a promising and effective strategy against EC. In this study, we designed a novel multi-epitope vaccine based on the antigenic proteins PRAME and TMPRSS4 using immunoinformatics and bioinformatics approaches. After a rigorous selection process, 14 cytotoxic T lymphocyte (CTL) epitopes, 6 helper T lymphocyte (HTL) epitopes, and 8 B cell epitopes (BCEs) were finally selected for vaccine construction. To enhance the immunogenicity of the vaccine candidate, the pan HLA DR-binding epitope was included in the vaccine design as an adjuvant. The final vaccine construct had 455 amino acids and a molecular weight of 49.8 kDa, and was predicted to cover 95.03% of the total world population. Docking analysis showed that there were 10 hydrogen bonds and 19 hydrogen bonds in the vaccine-HLA-A*02:01 and vaccine-HLA-DRB1*01:01 complexes, respectively, indicating that the vaccine has a good affinity to MHC molecules. This was further supported by molecular dynamics (MD) simulation. Immune simulation showed that the designed vaccine was able to induce higher levels of immune cell activity, with the secretion of numerous cytokines. The codon adaptation index (CAI) value and GC content of the optimised codon sequences of the vaccine were 0.986 and 54.43%, respectively, indicating that the vaccine has the potential to be highly expressed. The in silico analysis suggested that the designed vaccine may provide a novel therapeutic option for the individualised treatment of EC patients in the future. Communicated by Ramaswamy H. Sarma

Background: Chromobacterium violaceum is an emerging multidrug-resistant (MDR) Gram-negative bacterium associated with severe septicemia, abscess formation, and high mortality, particularly in immunocompromised individuals. Increasing antimicrobial resistance and the absence of approved vaccines underscore the urgent need for alternative preventive strategies. Traditional vaccine approaches are often inadequate against genetically diverse MDR pathogens, prompting the use of computational immunology and reverse vaccinology for vaccine design. Objectives: This study aimed to design and characterize a novel multi-epitope subunit vaccine (MEV) candidate against C. violaceum using a comprehensive pangenome-guided subtractive proteomics and immunoinformatics pipeline to identify conserved antigenic targets capable of eliciting strong immune responses. Methods: Comparative genomic analysis across eight C. violaceum strains identified 3144 core genes. Subtractive proteomics filtering yielded two essential, non-homologous, surface-accessible, and antigenic proteins-penicillin-binding protein 1A (Pbp1A) and organic solvent tolerance protein (LptD)-as vaccine targets. Cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes were predicted and integrated into a 272-amino-acid MEV construct adjuvanted with human β-defensin-4A using optimal linkers. The construct was evaluated through structural modeling, molecular docking with TLR4, molecular dynamics simulation, immune simulation, and in silico cloning into the pET-28a(+) vector. Results: The MEV construct exhibited strong antigenicity, non-allergenicity, and non-toxicity, with stable tertiary structure and favorable physicochemical properties. Docking and dynamics simulations demonstrated high binding affinity and stability with TLR4 (ΔG = -16.2 kcal/mol), while immune simulations predicted durable humoral and cellular immune responses with broad population coverage (≈89%). Codon optimization confirmed high expression potential in E. coli K12. Conclusions: The pangenome-guided immunoinformatics approach enabled the identification of conserved antigenic proteins and rational design of a promising multi-epitope vaccine candidate against MDR C. violaceum. The construct exhibits favorable immunogenic and structural features, supporting its potential for experimental validation and future development as a preventive immunotherapeutic against emerging MDR pathogens.

Chlamydia trachomatis (C. trachomatis) is an obligate intracellular bacterium which causes eye and sexually transmitted infections. During pregnancy, the bacterium is associated with preterm complications, low weight of neonates, fetal demise and endometritis leading to infertility. The aim of our study was design of a multi-epitope vaccine (MEV) candidate against C. trachomatis. After protein sequence adoption from the NCBI, potential epitopes toxicity, antigenicity, allergenicity, MHC-I and MHC-II binding, cytotoxic T lymphocytes (CTLs), Helper T lymphocytes (HTLs) and interferon-γ (IFN-γ)- induction were predicted. The adopted epitopes were fused together using appropriate linkers. In the next step, the MEV structural mapping and characterization, three-dimensional (3D) structure homology modeling and refinement were also performed. The MEV candidate interaction with the toll-like receptor 4 (TLR4) was also docked. The immune responses simulation was assessed using the C-IMMSIM server. Molecular dynamic (MD) simulation verified the structural stability of the TLR4-MEV complex. The Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) approach demonstrated the MEV high affinity of binding to the TLR4, MHC-I and MHC-II. The MEV construct was also stable and water soluble and had enough antigenicity and lacked allergenicity with stimulation of T cells and B cells and INF-γ release. The immune simulation confirmed acceptable responses of both the humoral and cellular arms. It is proposed that in vitro and in vivo studies are needed to evaluate the findings of this study. Communicated by Ramaswamy H. Sarma

Human papillomavirus (HPV), which is transmitted through sexual activity, is the primary cause of cervical cancer and the fourth most common type of cancer in women. In this study, an immunoinformatics approach was employed to predict immunodominant epitopes from a diverse array of antigens with the ultimate objective of designing a potent multiepitope vaccine against multiple HPV types. Immunodominant B cell, cytotoxic T cell (CTL), and helper T cell (HTL) epitopes were predicted using bioinformatics tools These epitopes were subsequently analyzed using various immunoinformatics tools, and those that exhibited high antigenicity, immunogenicity, non-allergenicity, non-toxicity, and excellent conservation were selected. The selected epitopes were linked with appropriate linkers and adjuvants to formulate a broad-spectrum multiepitope vaccine candidate against HPV. The stability of the multiepitope vaccine candidate was confirmed through structural analysis, and docking results indicated a high affinity for Toll-like receptors (TLR2 and TLR4). Molecular dynamics simulations demonstrated a persistent interaction of TLR2 and TLR4 with the multiepitope vaccine candidate. In silico immunological simulations showed that three injections of the multiepitope vaccine candidate resulted in high levels of B- and T-cell immune responses. Moreover, the in silico cloning results indicated that the multiepitope vaccine candidate could be expressed in substantial amounts in E. coli. The results of this study imply that designing a broad-spectrum vaccine against various HPV types using computational methods is plausible; however, experimental validation and safety testing to confirm the findings is essential.

In-silico design and evaluation of a novel mRNA vaccine against human bocavirus 1: A neglected viral pathogen.

Immunization With a Bivalent Adenovirus-vectored Tuberculosis Vaccine Provides Markedly Improved Protection Over Its Monovalent Counterpart Against Pulmonary Tuberculosis

CD4 Tcells are rapidly depleted from tuberculosis granulomas following acute SIV co-infection.

Lomentospora prolificans is an emerging opportunistic pathogen that predominantly affects immunocompromised individuals, as well as healthy individuals, often leading to disseminated disease with high mortality rates. Effective treatment is challenging due to its high intrinsic resistance to antifungal agents. To address this, we employed subtractive proteomics and reverse vaccinology approaches to identify potential antigenic proteins for the design of an mRNA-based multi-epitope vaccine (MEV). Our study identified four antigenic proteins as promising vaccine targets. A vaccine construct was developed using a combination of twelve cytotoxic T lymphocyte (CTL), nine helper T lymphocyte (HTL), and five linear B lymphocyte (LBL) epitopes. These epitopes were connected using appropriate linkers (AAY, GPGPG, and KK) and adjuvants to enhance antigenicity and immunogenicity. The vaccine construct was rigorously evaluated for its physicochemical properties, demonstrating high antigenicity, non-toxicity, non-allergenicity, stability, and solubility. Molecular docking studies were conducted to validate the interactions between the vaccine construct and the human toll-like receptor (TLR4). Immune simulation studies further confirmed the vaccine’s potential to elicit a robust immune response. Additionally, molecular dynamics (MD) simulations, principal component analysis (PCA), dynamic cross-correlation matrix (DCCM) analysis, and binding free energy calculations were performed to assess the stability and efficacy of the vaccine-receptor complex. Codon optimization and in-silico cloning were carried out to ensure efficient expression of the vaccine in Escherichia coli strain K12. The findings of this study suggest that the proposed vaccine construct holds significant promise as a novel mRNA-based therapeutic candidate against L. prolificans infections. Further experimental validation is recommended to advance this vaccine toward clinical application.

The current research investigated the development of a multi-epitope mRNA vaccine against the rabies virus on the basis of viral proteomes via the use of bioinformatic tools and reverse vaccinology. The aim of this study was to address the limitations of the currently available rabies vaccine by eliciting strong and long-lasting humoral and cellular immune responses. The cytotoxic T lymphocytes (CTLs), helper T lymphocytes (HTLs), and linear B-cell epitopes (LBLs) were mapped and prioritized from four top-ranking vaccine targets (nucleoprotein, phosphoprotein, matrix, and glycoprotein) that were highly antigenic, nonallergenic, nontoxic, and nonhuman homologs. The selected epitopes exhibited strong binding affinity to high-frequency HLA alleles, as evidenced by highly negative ΔG values and low dissociation constants, predicting efficient T-cell recognition and broad population coverage (96.01% globally). A single mRNA construct encompassing 21 shortlisted epitopes (four CTL, four HTL, and thirteen LBL epitopes) was designed with appropriate linkers and the immunostimulatory 50 S ribosomal protein L7/L12 adjuvant. Physicochemical analysis revealed stable, soluble, and hydrophobic properties, with an overall Ramachandran score of 93.2%, an ERRAT quality factor of 94.724%, and a Z score of -5.39. Additionally, molecular docking and normal mode analysis demonstrated the strong binding affinity of the vaccine construct-TLR-4 complex, with a minimum energy of -1655.0 kcal/mol, which was maintained by 23 hydrogen bonds and 2 salt bridge interactions, indicating significant structural stability and stiffness. The structural integrity and stable interaction of the complex were validated through 200 ns molecular dynamics simulations, as evidenced by stable RMSD and radius of gyration values, minimal fluctuations in RMSF, consistent solvent-accessible surface area (SASA), and well-defined conformational transitions observed in principal component analysis (PCA). In silico immune simulation revealed the capacity of the vaccine to stimulate the release of high levels of immunoglobulin, TH, and TC and the release of cytokines. It also has the ability to produce long-lasting memory cells, induce macrophage activity, and promote natural killer cell and neutrophil production. Moreover, further validation, including codon optimization and mRNA secondary structure prediction, confirmed the stable structure and high level of expression in the host. Overall, this study proposed a promising multi-epitope-based mRNA vaccine as an innovative therapeutic candidate against rabies. However, experimental validations are needed with systemic animal studies.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-16143-w.

An integrated multi-pronged reverse vaccinology and biophysical approaches for identification of potential vaccine candidates against Nipah virus

Bioinformatics analysis, immunogenicity, and therapeutic efficacy evaluation of a novel multi-stage, multi-epitope DNA vaccine for tuberculosis.

Background: Tuberculosis (TB) remains a global health priority, with current interventions like the Bacille Calmette-Guérin (BCG) vaccine lacking efficacy against latent infection and drug-resistant strains. Novel vaccines targeting both latent and active TB are urgently needed. Objective: This study aims to design a multi-epitope vaccine (MEV) and evaluate its immunogenicity, structural stability, and interactions with toll-like receptor 2/4 (TLR-2/4) via computational biology approaches. Methods: We designed MEV using bioinformatics tools, prioritizing immunodominant epitopes from Mycobacterium tuberculosis antigens. Structural stability was optimized through disulfide engineering, and molecular docking/dynamics simulations were used to analyze interactions and conformational dynamics with TLR-2/4. Antigenicity, immunogenicity, population coverage, and immune responses were computationally assessed. Results: The MEV candidate, CP91110P, exhibited 86.18% predicted global human leukocyte antigen (HLA)-I/II coverage, high antigenicity (VaxiJen: 0.8789), and immunogenicity (IEDB: 4.40091), with favorable stability (instability index: 33.48) and solubility (0.485). Tertiary structure analysis indicated that 98.34% residues were located in favored regions. Molecular docking suggested strong TLR-2 (-1535.9 kcal/mol) and TLR-4 (-1672.5 kcal/mol) binding. Molecular dynamics simulations indicated stable TLR-2 interactions (RMSD: 6-8 Å; Rg: 38.50-39.50 Å) and flexible TLR-4 binding (RMSD: 2-6 Å; Rg: 33-36 Å). Principal component analysis, free energy landscapes, and dynamic cross-correlation matrix analyses highlighted TLR-2's structural coherence versus TLR-4's adaptive flexibility. Immune simulations predicted potential robust natural killer cell activation, T helper 1 polarization (interferon-gamma/interleukin-2 dominance), and elevated IgM/IgG levels. Conclusions: CP91110P is predicted to stably bind to TLR-2 and flexibly interact with TLR-4, with prediction of its high antigenicity and broad coverage across immune populations. However, this conclusion requires confirmation through experimental validation. Therefore, it may provide a promising candidate for experimental validation in the development of tuberculosis vaccines.