Search Results (243)

This study focused on utilizing the double-mutant heat-labile toxin (R192G/L211A) (dmLT) as a backbone protein, into which neutralizing epitopes of ETEC (FaeG, FedF, FanC, FasA, and Fim41a) were embedded. A combination of computational modeling and immunogenicity analysis was conducted to evaluate the dmLT_{(R192G/L211A)} multiepitope fusion antigen (MEFA). Both the computational modeling and experimental results confirmed that all relevant epitopes were clearly exposed on the surface of the MEFA. Subcutaneous immunizations of rabbits with the MEFA protein yielded the development of IgG antibodies that targeted all five fimbriae. Furthermore, these antibodies demonstrated significant inhibition of adhesion for K88+, K99+, 987P+, F18+, and F41+ ETEC strains to porcine small intestinal epithelial cell line IPEC-J2 cells. These results indicated that the dmLT toxoid-based MEFA protein effectively elicits high-titer, functional antibodies capable of neutralizing the attachment of multiple prevalent ETEC fimbrial types, highlighting its potential as a broad-spectrum vaccine candidate. Consequently, it shows promising potential as a broad and effective vaccine against ETEC. Full article

Background/Objectives: Streptococcus suis (SS), an important zoonotic pathogen, has caused significant economic losses to the global pig industry. Existing commercial vaccines for SS mainly provide effective protection against a single serotype. Due to the existence of many serotypes and their robust immune evasion capabilities, the development of multi-component subunit vaccines or multi-epitope vaccines that provide effective cross-protection against different strains of SS is a key focus of current research. Methods: We applied two-dimensional electrophoresis (2-DE) and immunoblotting to screen for candidate immunogens among the immunogenic cell wall proteins of SS. BALB/c mice were immunized intradermally with a multi-component, multi-epitope vaccine. The vaccine’s safety and immunogenicity were assessed via clinical monitoring, antibody titer detection, cytokine assays, and survival curve analyses. Results: In this study, eight immunogenic cell wall proteins (GH25, Pk, PdhA, Ldh, ExoA, Pgk, MalX, and Dnak) were successfully identified using MALDI-TOF-MS, all of which could induce high IgG antibody titers. Based on the conservation and immunoprotection demonstrated by these eight protective antigenic proteins, PdhA, Ldh, and MalX were screened to construct a multi-component subunit vaccine as a candidate vaccine for providing cross-protection against SS isolates of multiple serotypes. Challenge studies showed that mice immunized with the multi-component subunit vaccine (PdhA, Ldh, and MalX) were protected against challenges with the SS2 virulent strain ZY05719 (62.5% protection) and the SSChz virulent strain CZ130302 (75% protection). Subsequently, we utilized immunoinformatics techniques to design a novel multi-epitope vaccine (MVPLM) derived from the immunogenic proteins PdhA, Ldh, and MalX. However, challenge tests revealed that the MVPLM offered limited protection against SS. Conclusions: These data demonstrate that a multi-component subunit vaccine composed of PdhA, Ldh, and MalX proteins shows promise as a candidate universal vaccine against multiple SS serotypes. Full article

Background/Objectives: Influenza A viruses—including highly pathogenic H5N1—remain a global threat due to rapid evolution, zoonoses, and pandemic potential. Strain-specific vaccines targeting variable antigens often yield limited, short-lived immunity. The HA receptor-binding domain (RBD), a functionally constrained and immunologically relevant region, is a promising target for broad and subtype-focused vaccines. We aimed to design multiepitope constructs targeting conserved HA-RBD and adjacent domains to elicit robust, durable, cross-protective responses. Methods: Extensive sequence analyses (>20,000 H5N1 and >190,000 influenza A sequences) were used to derive consensus sequences. Three HA-based candidates were developed: (i) EpitoCore-HA-VX, a multi-epitope construct containing CTL, HTL, and B-cell epitopes from the H5N1 HA-RBD; (ii) StructiRBD-HA-VX, incorporating a conformationally preserved RBD segment; and (iii) FusiCon-HA-VX, targeting the conserved HA fusion peptide shared across subtypes. Two external HA comparators—a 400-aa HA fragment and the literature-reported HA-13–263-Fd-His—were analyzed under the same pipeline. The workflow predicted epitopes; evaluated antigenicity, allergenicity, toxicity, conservation, and HLA coverage; generated AlphaFold models; performed TLR2/TLR4 docking with pyDockWEB; and carried out interface analysis with PDBsum; and C-ImmSim simulations. Results: Models suggested stable, energetically favorable TLR2/TLR4 interfaces supported by substantial binding surfaces and complementary electrostatic/desolvation profiles. Distinct docking patterns indicated receptor-binding flexibility. Immune simulations predicted strong humoral responses with modeled memory formation and, for the H5N1-focused designs, cytotoxic T-cell activity. All candidates and comparators were predicted to be antigenic, non-allergenic, and non-toxic, with combined HLA coverage approaching global breadth. Conclusions: This study compares three design strategies within a harmonized framework—epitope collation, structure-preserved RBD, and fusion-peptide targeting—while benchmarking against two HA comparators. EpitoCore-HA-VX and StructiRBD-HA-VX showed promise against diverse H5N1 isolates, whereas FusiCon-HA-VX supported cross-subtype coverage. As these findings are model-based, they should be interpreted qualitatively; nonetheless, the integrated, structure-guided approach provides an adaptable path for advancing targeted H5N1 and broader influenza A vaccine concepts. Full article

Background/Objectives: Fully personalized peptide vaccines targeting tumor-specific mutations are a promising treatment option for patients in an adjuvant but also advanced/metastatic disease situation in addition to non-personalized standard therapies. Here, we report a patient’s case with advanced metastatic colorectal cancer (mCRC) who was treated with a neoantigen-derived multi-peptide vaccine in addition to standard of care. Methods: Tumor-specific mutations were identified by whole exome and transcriptome sequencing. An individualized peptide vaccine was designed using an in-house developed epitope prediction and vaccine design platform. In this case, the vaccine consisted of 20 peptides targeting 18 distinct mutations. The vaccine was administered according to a prime-boost scheme for a total of 12 vaccinations. Vaccine immunogenicity was determined by stimulation of patient T cells with vaccinated peptides and subsequent intracellular cytokine staining (ICS). Tumor-infiltrating lymphocytes (TIL) were analyzed by ICS and T cell receptor beta chain (TCRβ) sequencing. Results: The patient survived for 41 months since initial diagnosis despite continuous disease progression under all therapeutic interventions. The vaccination induced multiple neoantigen-specific T cell responses in the patient without notable side effects. Two liver metastases were resected five months after the start of vaccination, and TIL were extracted and cultured. Analysis of TIL cultures revealed tumor infiltration by vaccine-induced neoantigen-specific T cells in only one of the metastases. TCRβ sequencing of neoantigen-specific T cells and tumor tissues supported this finding. Vaccine-targeted variants were reduced or absent in the metastasis with vaccine-specific T cell infiltration. Conclusions: This case demonstrates immunogenicity of a neoantigen-derived peptide vaccine and highlights tumor-infiltrating capabilities and potential cytotoxicity of vaccine-induced T cells in mCRC. Full article

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. Full article

Background: Bacterial Chondronecrosis with Osteomyelitis (BCO) is a significant issue affecting the welfare and economy of the broiler industry, causing substantial revenue losses annually. This disease is frequently associated with Staphylococcus spp. and Enterococcus spp. infections and necrosis of leg and vertebral bones. The typical annual lameness incidence of approximately 3–5% may increase to 30% during outbreaks. Neither the etiology or pathogenesis of the disease has been comprehended, nor have effective preventative measures been identified. Electron beam (eBeam) technology is renowned for producing efficient whole-cell vaccines by preventing bacterial multiplication through irreversible DNA shredding while preserving the integrity of membrane proteins (immunogenic epitopes). This study aims to reduce BCO-induced lameness in broiler chickens via in ovo immunization using eBeam-inactivated multi-strain Staphylococcus. Methods: A total 1080 birds were assigned to four vaccination groups: eBeam-inactivated, formalin-inactivated, combination of eBeam- and formalin-inactivated, and sham (vehicle). The birds were directly exposed to aerosolized, natural BCO challenge until 56 days of age. Results: Birds vaccinated with the eBeam-inactivated Staphylococcus vaccine showed a significant reduction (>50%) in daily cumulative lameness compared to other groups and a decrease in Staphylococcus colonization was observed in the leg joints of treated birds. Conclusions: the eBeam-inactivated Staphylococcus vaccine successfully prevented BCO lameness in broiler chickens. Full article

A novel skin test for an in vivo assessment of SARS-CoV-2-specific T-cell immunity was developed using CoronaDermPS, a multiepitope recombinant polypeptide encompassing MHC II–binding CD4+ T-cell epitopes of the SARS-CoV-2 structural proteins (S, E, M) and full length nucleocapsid (N). In silico epitope prediction and modeling guided antigen design, which was expressed in Escherichia coli, was purified (>95% purity) and formulated for intradermal administration. Preclinical evaluation in guinea pigs, mice, and rhesus macaques demonstrated a robust delayed type hypersensitivity (DTH) response at optimal doses (10–75 µg), with no acute or chronic toxicity, mutagenicity, or adverse effects on reproductive organs. An integrated clinical analysis included 374 volunteers stratified by vaccination status (EpiVacCorona, Gam-COVID-Vac, CoviVac) prior to COVID-19 infection (Wuhan/Alpha, Delta, Omicron variants), and SARS-CoV-2–naïve controls. Safety assessments across phase I–II trials recorded 477 adverse events, of which >88% were mild and self-limiting; no severe or anaphylactic reactions occurred. DTH responses were measured at 24 h, 72 h, and 144 h post-injection by papule and hyperemia measurements. Overall, 282/374 participants (75.4%) exhibited a positive skin test. Receiver operating characteristic analysis yielded an overall AUC of 0.825 (95% CI: 0.726–0.924), sensitivity 79.5% (95% CI: 75.1–83.3%), and specificity 85.5% (95% CI: 81.8–88.7%), with comparable diagnostic accuracy across vaccine, and variant subgroups (AUC range 0.782–0.870). CoronaDerm-PS–based skin testing offers a simple, reproducible, and low-cost method for qualitative evaluation of T-cell–mediated immunity to SARS-CoV-2, independent of specialized laboratory equipment (Eurasian Patent No. 047119). Its high safety profile and consistent performance across diverse cohorts support its utility for mass screening and monitoring of cellular immune protection following infection or vaccination. Full article

Background: Helcococcus kunzii, a facultative anaerobe and Gram-positive coccus, has been documented as a cunning pathogen, mainly in immunocompromised individuals, as evidenced by recent clinical and microbiological reports. It has been associated with a variety of polymicrobial infections, comprising diabetic foot ulcers, prosthetic joint infections, osteomyelitis, endocarditis, and bloodstream infections. Despite its emerging clinical relevance, no licensed vaccine or targeted immunotherapy currently exists for H. kunzii, and its rising resistance to conventional antibiotics presents a growing public health concern. Objectives: In this study, we employed an integrated subtractive proteomics and immunoinformatics pipeline to design a multi-epitope subunit vaccine (MEV) candidate against H. kunzii. Initially, pan-proteome analysis identified non-redundant, essential, non-homologous, and virulent proteins suitable for therapeutic targeting. Methods/Results: From these, two highly conserved and surface-accessible proteins, cell division protein FtsZ and peptidoglycan glycosyltransferase FtsW, were selected as promising vaccine targets. Comprehensive epitope prediction identified nine cytotoxic T-lymphocyte (CTL), five helper T-lymphocyte (HTL), and two linear B-cell (LBL) epitopes, which were rationally assembled into a 397-amino-acid-long chimeric construct. The construct was designed using appropriate linkers and adjuvanted with the cholera toxin B (CTB) subunit (NCBI accession: AND74811.1) to enhance immunogenicity. Molecular docking and dynamics simulations revealed persistent and high-affinity ties amongst the MEV and essential immune receptors, indicating a durable ability to elicit an immune reaction. In silico immune dynamic simulations predicted vigorous B- and T-cell-mediated immune responses. Codon optimization and computer-aided cloning into the E. coli K12 host employing the pET-28a(+) vector suggested high translational efficiency and suitability for bacterial expression. Conclusions: Overall, this computationally designed MEV demonstrates favorable immunological and physicochemical properties, and presents a durable candidate for subsequent in vitro and in vivo validation against H. kunzii-associated infections. Full article

The presently used vaccines do not offer solid immunity/protection against the currently circulating strains of the H5N1 viruses. We aim to design a pan-H5N1 vaccine that protects birds against the presently circulating clade 2.3.4.4b in chickens. We used AI tools, including epitope mapping, molecular docking, and immune simulation, to design a multiepitope DNA vaccine including the top-ranked B and T cell epitopes within four major proteins (HA, NA, NP, and M2) of H5N1 clade 2.3.4.4b. We selected the top-ranked 12 epitopes and linked them together using linkers. The designed vaccine is linked to IL-18 as an adjuvant. The molecular docking results showed a high binding affinity of those predicted epitopes from the MHC I and MHC II classes of molecules with chicken alleles. The immune simulation results showed that the designed vaccine has the potential to stimulate the host immune response, including antibody and cell-mediated immunity in chickens and other birds. We believe this vaccine is going to be a universal vaccine that offers good protection against HPAI-H5N1 clade 2.3.4.4b. We are reporting the successful molecular cloning of a recombinant multiepitope-based vaccine spanning some key epitopes within some key proteins of the currently circulating H5N1 clade 2.3.4.4b. These designed vaccines could be a great positive impact on the protection of birds and various species of animals, as well as humans, against the HP-H5N1 influenza virus. Further studies are required to validate this vaccine candidate in chickens. Full article

Background: While most Escherichia coli strains are harmless members of the gastrointestinal microbiota, certain pathogenic variants can cause severe intestinal and extraintestinal diseases. A notable outbreak of E. coli O104:H4, involving both enteroaggregative (EAEC) and enterohemorrhagic (EHEC) strains, occurred in Europe, resulting in symptoms ranging from bloody diarrhea to life-threatening colitis and hemolytic uremic syndrome (HUS). Since treatment options remain limited and have changed little over the past 40 years, there is an urgent need for an effective vaccine. Such a vaccine would offer major public health and economic benefits by preventing severe infections and reducing outbreak-related costs. A multiepitope vaccine approach, enabled by advances in immunoinformatics, offers a promising strategy for targeting HUS-causing E. coli (O104:H4 and O157:H7 serotypes) with minimal disruption to normal microbiota. This study aimed to design an immunogenic multiepitope vaccine (MEV) construct using bioinformatics and immunoinformatic tools. Methods and Results: Comparative proteomic analysis identified 672 proteins unique to E. coli O104:H4, excluding proteins shared with the nonpathogenic E. coli K-12-MG1655 strain and those shorter than 100 amino acids. Subcellular localization (P-SORTb) identified 17 extracellular or outer membrane proteins. Four proteins were selected as vaccine candidates based on transmembrane domains (TMHMM), antigenicity (VaxiJen), and conservation among EHEC strains. Epitope prediction revealed ten B-cell, four cytotoxic T-cell, and three helper T-cell epitopes. Four MEVs with different adjuvants were designed and assessed for solubility, stability, and antigenicity. Structural refinement (GALAXY) and docking studies confirmed strong interaction with Toll-Like Receptor 4 (TLR4). In silico immune simulations (C-ImmSim) indicated robust humoral and cellular immune responses. In Conclusions, the proposed MEV construct demonstrated promising immunogenicity and warrants further validation in experimental models. Full article

Background: Multi-epitope vaccines have become the preferred strategy for protection against infectious diseases by integrating multiple MHC-restricted T-cell and B-cell epitopes that elicit both humoral and cellular immune responses against pathogens. Computational methods address various aspects independently, yet their orchestration is technically challenging, as most bioinformatics tools are accessible through heterogeneous interfaces and lack interoperability features. The present work proposes a novel framework for rationalized multi-epitope vaccine design that streamlines end-to-end analyses through an integrated web-based environment. Results: VaccineDesigner is a comprehensive web-based framework that streamlines the design of protective epitope-based vaccines by seamlessly integrating computational methods for B-cell, CTL, and HTL epitope prediction. VaccineDesigner incorporates single-epitope prediction and evaluation as well as additional analyses, such as multi-epitope vaccine generation, estimation of population coverage, molecular mimicry, and proteasome cleavage. The functionalities are transparently integrated into a modular architecture, providing a single access point for rationalized, multi-epitope vaccine generation in a time- and cost-effective manner. Conclusions: VaccineDesigner is a web-based tool that identifies and evaluates candidate B-cell, CTL, and HTL epitopes and constructs a library of multi-epitope vaccines that combine strong immunogenic responses, safety, and broad population coverage. The source code is available under the academic license and freely accessible. Full article

Monkeypox virus (MPXV) has caused 148,892 confirmed cases and 341 deaths from 137 countries worldwide, as reported by the World Health Organization (WHO), highlighting the urgent need for effective vaccines to prevent the spread of MPXV. Traditional vaccine development is low-throughput, expensive, time consuming, and susceptible to reversion to virulence. Alternatively, a reverse vaccinology approach offers a rapid, efficient, and safer alternative for MPXV vaccine design. Here, MPXV proteins associated with viral infection were analyzed for immunogenic epitopes to design multi-epitope vaccines based on B-cell, CD4+, and CD8+ epitopes. Epitopes were selected based on allergenicity, antigenicity, and toxicity parameters. The prioritized epitopes were then combined via peptide linkers and N-terminally fused to various protein adjuvants, including PADRE, beta-defensin 3, 50S ribosomal protein L7/12, RS-09, and the cholera toxin B subunit (CTB). All vaccine constructs were computationally validated for physicochemical properties, antigenicity, allergenicity, safety, solubility, and structural stability. The three-dimensional structure of the selected construct was also predicted. Moreover, molecular docking and molecular dynamics (MD) simulations between the vaccine and the TLR-4 immune receptor demonstrated a strong and stable interaction. The vaccine construct was codon-optimized for high expression in the E. coli and was finally cloned in silico into the pET21a (+) vector. Collectively, these results could represent innovative tools for vaccine formulation against MPXV and be transformative for other infectious diseases. Full article

Fasciola gigantica (F. gigantica) is a vital parasite that causes fasciolosis. Liver fluke infections affect livestock animals, and the Fasciola species (Fasciola spp.) vaccine has been tested for many types of these diseases. Currently, computer-based vaccine design represents an attractive alternative for constructing vaccines. Thus, this study aimed to design the epitopes of linear B-cells (BCL) and helper T lymphocytes (HTL) using an immunoinformatic approach and to investigate in silico and the mice’s immune response. A non-conserved host region, overlapping F. gigantica cathepsin B proteins (FgCatB), and the highest conserved residue percentages were the criteria used to construct epitopes. The GPGPG linker was used to link epitopes in the multi-epitope Fasciola gigantica cathepsin B (MeFgCatB) peptide. The MeFgCatB peptide has high antigenicity, non-allergenicity, non-toxicity, good solubility, and a high-quality structure. The molecular docking between the MeFgCatB peptide and Toll-like receptor 2 (TLR-2) was evaluated. The IgM, IgG1, and IgG2 levels were elevated in silico. In mice, the MeFgCatB peptide was synthesized and administered as an injection. The MeFgCatB-specific IgG1 and IgG2a levels were elevated after week 2, showing a predominance of IgG1. The rFgCatB1, rFgCatB2, and rFgCatB3 were detected using the MeFgCatB peptide-immunized sera. The MeFgCatB peptide-immunized sera were detected at approximately 28–34 kDa in the whole body. In addition, the MeFgCatB immunized sera can positively signal at the caecal epithelium in the NEJ, 4WKJ, and adult stages. In summary, the MeFgCatB peptide is able to induce mixed Th1/Th2 immune responses with Th2 dominating and to detect the native protein of F. gigantica. The MeFgCatB peptide should help against F. gigantica in future experiments. Full article

Recent outbreaks caused by hMPXV, especially hMPXV lineages/sub-lineages, represent public health threats necessitating stringent prophylactic measures to ameliorate their colossal impact. The current study annotated the EEV form of hMPXV’s target proteins to formulate a reverse vaccinology-dependent hMPXV multiepitope vaccine. Epitope determination, followed by vaccine formulation, was undertaken. The promising formulation was validated for its potential to trigger immune responses immunoinformatically. The MPXV-1-Beta formulation was characterised as a promising candidate based on antigenicity score, physicochemical properties, solubility score, ProSA Z-score, and Ramachandran plot. Docking, normal mode analysis, and molecular dynamic simulation of MPXV-1-Beta with TLRs and MHCs authenticated rigid docking and its efficacy in enhancing immune receptor activation under physiological conditions. MPXV-1-Beta was discerned to trigger a sustained immune response (IR) with a broader average population coverage of 97.526, SD = 12.44. The proposed MPXV-1-Beta candidate showed significant potential. The findings of this study provide a preliminary framework for developing an efficacious hMPXV vaccine; however, extensive in vitro, in vivo, and clinical evaluations are required to substantiate the computational insights. Full article

Background/Objectives: Leishmaniasis is a prevailing infectious disease transmitted via infected phlebotomine sandflies. The lack of an efficient vaccine with respect to immunogenic antigens and adjuvanted delivery systems impedes its control. Following the induction of immune responses in mice vaccinated with multi-epitope Leishmania peptides (LeishPts) encapsulated in doubly adjuvanted self-nanoemulsifying drug delivery systems (ST-SNEDDSs), this study aims to assess ST-SNEDDS-based nanoemulsions as vehicles for the delivery of antigenic proteins. Methods: Model antigens (e.g., BSA-FITC, OVA) were encapsulated in ST-SNEDDS after being complexed with the cationic phospholipid dimyristoyl phosphatidylglycerol (DMPG) via hydrophobic ion pairing. The nanoemulsions were characterized with respect to droplet diameter, zeta potential, stability, protein loading, protein release from the nanodroplets in different release media and cell uptake. Results: Both model antigens exhibited high encapsulation efficiency (>95%) and their release from the nanodroplets was shown to be strongly affected by the type of release medium (e.g., PBS, FBS 10% v/v) and the ratio of its volume to that of the oily phase, in agreement with predictions of protein release. Protein-loaded nanoemulsion droplets labeled with Cy-5 were found to be efficiently taken up by macrophages (J774A.1) in vitro. However, no colocalization of the labeled nanodroplets and BSA-FITC could be observed. Conclusions: It was revealed that in contrast with LeishPts, whole protein molecules may not be appropriate antigenic cargo for ST-SNEDDS formulations due to the rapid protein release from the nanodroplets in release media simulating in vitro culture and in vivo conditions such as FBS 10% v/v. Full article