In silico design of a multi-epitope Chimera from Aedes aegypti salivary proteins OBP 22 and OBP 10: A promising candidate vaccine.

Context: Plasmodium knowlesi is an emerging zoonotic malaria species in Southeast Asia, challenging elimination efforts. Aims: To design a safe and immunogenic multi-epitope vaccine (MEV) targeting the merozoite surface protein-1 (MSP-1) of P. knowlesi using a comprehensive immunoinformatics approach. Methods: Conserved regions of the P. knowlesi MSP-1 sequence were retrieved and analyzed to predict helper T lymphocyte (HTL), cytotoxic T lymphocyte (CTL), and B cell epitopes. Epitopes were selected based on predicted immunogenicity, solubility, and safety. Suitable linkers were used to fuse selected epitopes, adjuvants, and sequences into a multi-epitope vaccine (MEV) construct. Physicochemical properties, immunogenicity, allergenicity, and antigenicity of the MEV were assessed in silico. Molecular docking and molecular dynamics (MD) simulation were conducted to evaluate MEV binding affinity and stability with toll-like receptor 4 (TLR4). The immunogenic potential was further evaluated using the C-ImmSim server to simulate host immune responses. Results: The 219-amino acid MEV construct was predicted to be stable, soluble, non-toxic, and non-allergenic, with 98.89% global population coverage. Molecular docking and MD simulations confirmed a stable interaction with TLR4. Immune simulation revealed efficient antigen clearance, significant production of IgM and IgG1/IgG2 antibodies, expansion of plasma and memory B cells, and activation of CD4⁺ and CD8⁺ T cells. A Th1-skewed cytokine response was observed, marked by elevated IFN-γ, IL-2, and IL-12 levels. Conclusions: The in silico-designed MEV demonstrated favorable immunological, structural, and population coverage profiles, supporting its potential as a promising vaccine candidate against P. knowlesi malaria. Further experimental validation is needed.

Brucellosis, caused by the intracellular pathogen Brucella, remains a significant health challenge, alongside substantial economic impacts on livestock industries. Despite antibiotic treatments, the absence of licensed human vaccines necessitates innovative preventive strategies. In this study, we employed reverse vaccinology to design a novel multi-epitope vaccine (MEV) targeting Brucella melitensis. Three immunogenic proteins—outer membrane protein OMP31, LPS assembly protein LptE, and the type IV secretion system protein VirB2—were selected as vaccine candidates. Comprehensive bioinformatics analysis identified six cytotoxic T lymphocyte (CTL) epitopes, nine helper T lymphocyte (HTL) epitopes, seven linear B-cell epitopes, and five conformational B-cell epitopes. The incorporation of molecular adjuvants (cholera toxin B subunit and PADRE) served to further enhance the immunogenicity of the vaccine. Given that Brucella is an intracellular parasite, TAT cell-penetrating peptides were added to further enhance the intracellular delivery of MEV. The constructed MEV has been shown to have excellent antigenicity (VaxiJen score >0.8), stability (instability index <40), solubility (Protein-Sol score: 0.87) and hydrophilicity (GRAVY index: −0.319), and is non-allergenic. Structural optimization, including disulfide bond engineering (11 pairs of residues), improved molecular stability, with molecular docking and dynamics simulations confirming robust interactions with immune cell receptors (docking score: −311.85). Using SnapGene 7.1.2, we performed in silico cloning simulation of the codon-optimized multi-epitope vaccine (MEV) sequence into the pMV261 shuttle vector, generating a recombinant BCG (rBCG) construct. Immunoinformatics simulations (C-ImmSim) demonstrated potent immune activation, with significant increases in cytotoxic T cells (1050 cells/mm³), memory helper T cells (1150 cells/mm³), and IFN-γ production (2 × 10^6 ng/ml), alongside sustained IgG/IgM titers over 350 days(1 × 10^5 cells/mm3) . Furthermore, the recombinant BCG multi-epitope Brucella vaccine, developed through bioinformatics approaches, demonstrates promising characteristics and immunogenicity. Nevertheless, its immunological efficacy requires to further experimental validation.

The emergence or reemergence of monkeypox (Mpox) and Ebola virus (EBOV) agents causing zoonotic diseases remains a huge threat to human health. Our study aimed at designing a multi-epitope vaccine (MEV) candidate to target both the Mpox and EBOV agents using immunoinformatics tools. Viral protein sequences were retrieved, and potential nonallergenic, nontoxic, and antigenic epitopes were obtained. Next, cytotoxic and helper T-cell (CTL and HTL, respectively) and B-cell (BCL) epitopes were predicted, and those potential epitopes were fused utilizing proper linkers. The in silico cloning and expression processes were implemented using Escherichia coli K12. The immune responses were prognosticated using the C-ImmSim server. The MEV construct (29.53kDa) included four BCL, two CTL, and four HTL epitopes and adjuvant. The MEV traits were pertinent in terms of antigenicity, non-allergenicity, nontoxicity, physicochemical characters, and stability. The MEV candidate was also highly expressed in E. coli K12. The strong affinity of MEV-TLR3 was confirmed using molecular docking and molecular dynamics simulation analyses. Immune simulation analyses unraveled durable activation and responses of cellular and humoral arms alongside innate immune responses. The designed MEV candidate demonstrated appropriate traits and was promising in the prediction of immune responses against both Mpox and EBOV agents. Further experimental assessments of the MEV are required to verify its efficacy.

An unusual pneumonia infection, named COVID-19, was reported on December 2019 in China. It was reported to be caused by a novel coronavirus which has infected approximately 220 million people worldwide with a death toll of 4.5 million as of September 2021. This study is focused on finding potential vaccine candidates and designing an in-silico subunit multi-epitope vaccine candidates using a unique computational pipeline, integrating reverse vaccinology, molecular docking and simulation methods. A protein named spike protein of SARS-CoV-2 with the GenBank ID QHD43416.1 was shortlisted as a potential vaccine candidate and was examined for presence of B-cell and T-cell epitopes. We also investigated antigenicity and interaction with distinct polymorphic alleles of the epitopes. High ranking epitopes such as DLCFTNVY (B cell epitope), KIADYNKL (MHC Class-I) and VKNKCVNFN (MHC class-II) were shortlisted for subsequent analysis. Digestion analysis verified the safety and stability of the shortlisted peptides. Docking study reported a strong binding of proposed peptides with HLA-A*02 and HLA-B7 alleles. We used standard methods to construct vaccine model and this construct was evaluated further for its antigenicity, physicochemical properties, 2D and 3D structure prediction and validation. Further, molecular docking followed by molecular dynamics simulation was performed to evaluate the binding affinity and stability of TLR-4 and vaccine complex. Finally, the vaccine construct was reverse transcribed and adapted for E. coli strain K 12 prior to the insertion within the pET-28-a (+) vector for determining translational and microbial expression followed by conservancy analysis. Also, six multi-epitope subunit vaccines were constructed using different strategies containing immunogenic epitopes, appropriate adjuvants and linker sequences. We propose that our vaccine constructs can be used for downstream investigations using in-vitro and in-vivo studies to design effective and safe vaccine against different strains of COVID-19.

Dental caries, a persistent oral health challenge primarily linked to Streptococcus mutans, extends its implications beyond dental decay, affecting over 4 billion individuals globally. Despite its historical association with childhood, dental caries often persists into adulthood with prevalence rates ranging from 60 to 90% in children and 26 to 85% in adults. Currently, there is a dearth of multiepitope vaccines (MEVs) specifically designed to combat S. mutans. To address this gap, we employed an immunoinformatics approach for MEV design, identifying five promising vaccine candidates (PBP2X, PBP2b, MurG, ATP-F, and AGPAT) based on antigenicity and conservation using several tools including CELLO v.2.5, Vaxign, v2.0, ANTIGENpro, and AllerTop v2.0 tools. Subsequent identification of linear B-cell and T-cell epitopes by SVMTrip and NetCTL/NetMHC II tools, respectively, guided the construction of a MEV comprising 10 Cytotoxic T Lymphocyte (CTL) epitopes, 5 Helper T Lymphocyte (HTL) epitopes, and 5 linear B-cell epitopes, interconnected by suitable linkers. The resultant MEV demonstrated high antigenicity, solubility, and structural stability. In silico immune simulations showcased the MEV's potential to elicit robust humoral and cell-mediated immune responses. Molecular docking studies revealed strong interactions between the MEV construct and Toll-Like Receptors (TLRs) and Major Histocompatibility Complex (MHC) molecules. Remarkably, the MEV-TLR-4 complexes exhibited a low energy score, high binding affinity, and a low dissociation constant. The Molecular Dynamic (MD) simulation analysis suggested that MEV-TLR-4 complexes had the highest stability and minimal conformational changes indicating equilibrium within 40 nanosecond time frames. Comprehensive computational analyses strongly support the potential of the proposed MEV to combat dental caries and associated infections. The study's computational assays yielded promising results, but further validation through in vitro and in vivo experiments is needed to assess its efficacy and safety.

Graphical Abstract Highlight Research The study aims to develop a multi-epitope vaccine (MEV) against A. hydrophila by targeting the aerolysin toxin, a key virulence factor responsible for infections in fish and humans. Computational methods identified and optimized B-cell and T-cell epitopes, focusing on their ability to trigger immune responses without causing toxicity or allergenicity. In silico simulations demonstrated that the MEV has a strong binding affinity to immune receptors like TLR-4, MHC-I, and MHC-II, indicating its potential to induce robust cellular and humoral immunity. Structural analysis of the MEV showed a stable 3D conformation, with most residues in favorable regions, ensuring stability during immune activation. The MEV could enhance disease control in aquaculture and reduce human infection risks, offering a promising solution to address antibiotic resistance and the absence of effective vaccines. Abstract Aeromonas hydrophila, gram-negative, is a major pathogen responsible for various diseases in mammals, reptiles, amphibia, and vertebrates, including fish and humans. Targeting the specific toxin aerolysin in A. hydrophila is crucial to address antibiotic resistance and the lack of adequate and protective vaccines against this intracellular pathogen. This study aimed to identify a multi-epitope vaccination (MEV) candidate targeting A. hydrophila aerolysin toxin to combat the disease effectively. Standard biochemical characterization methods and sequencing of the 16S rRNA, rpoB, and aerA genes identified the isolate AHSA1 as A. hydrophila. Subsequently, we identified B and T cell epitopes on the aerolysin protein and separately predicted MHC-I and MHC-II epitopes. The epitopes are then evaluated for toxicity, antigenicity, allergenicity, and solubility. The vaccine design integrated multi-epitope-based constructs, utilizing specialized linkers (GPGPG) and EAAAK linkers to connect epitope peptides with adjuvants in the cholera toxin B component, thereby enhancing immunogenicity. Ramachandran plots showed that 85.25% of the residues were located in the most favorable regions, which was followed by the generously allowed zone (1.30%), the additional allowed regions (10.80%), and the forbidden regions (2.65%), thus confirming the feasibility of the modeled vaccine design. Based on docking simulations, MEV had the highest binding and interaction energies with TLR-4, TLR-9, MHC-I, and MHC-II (-1081.4, -723.2, 866.2, -9043.3 kcal/mol). Based on computational modelling, we expect the Aerolysin MEV candidate design to activate diverse immune mechanisms, stimulate robust responses against A. hydrophila, and maintain safety. The significant solubility, absence of toxicity or allergic response, and minimal side effects in animal testing all contribute to the potential clinical utility of this vaccine candidate.

Background: The Zika virus is an infectious virus that belongs to the Flaviviridae family. It is transmitted to humans through mosquito vectors and poses a serious threat to human health. Despite available resources, there is currently no effective and secure vaccine against the Zika virus to prevent such infections Objective: Observation of symptoms including fever, rash, joint pain, and conjunctivitis (red eyes) along with the possibility of severe complications during pregnancy, including birth defects such as microcephaly, is noted in infected individuals. So, it is the need of the hour to develop a potential vaccine that can immunize the human beings against this virus and protect them. For the purpose of vaccine development against the Zika virus, bioinformatics and immunoinformatics approaches are utilized. Materials and methods: Different epitopes from RdRp Protein of Zika virus were predicted including B-Cell and T- Cell epitopes. The selected epitopes were analyzed further in order to check their antigenicity, immunogenicity, solubility and toxicity in correspondence to their ability to initiate immune response in the individuals. The potential epitopes having the ability to trigger a good immune response and having non- toxic nature were utilized for vaccine development. By combining the different epitopes, a potential multi-epitope vaccine construct was developed. Combining of epitopes was done using EAAAK, AAY and GPGPG linkers. A 50S ribosomal protein was added at N terminal to improve the immunogenicity of the vaccine and 6-His tag protein also added at C terminal. Results: The constructed vaccine was found to have good population coverage all across the world. In molecular docking, strong interactions were found between the constructed vaccine and Toll-like receptor 3 (-346.16), suggesting their potential relevance in the immune response to the vaccine and dynamical stabilities were inspected through molecular dynamic (MD) simulation methods. The developed vaccine construct also exhibited good results of immune response in immune simulation and revealed that the constructed vaccine activates B and T lymphocytes which induce high levels of antibodies and cytokines to combat Zika infection. The constructed vaccine is an effective biomarker with non-toxicity, non-allergic properties, good immunogenicity, and antigenicity. However, experimental assays are required to verify the results of the present study. Conclusion: These findings suggest that design vaccine candidate effectively trigger immune response with no side effects against Zika virus. Keywords:- Zika virus vaccine, Epitope prediction, immunoinformatics, Multiepitope vaccine, Molecular docking.

Fowl adenovirus (FAdV) is a significant pathogen in poultry, causing various diseases such as hepatitis-hydropericardium, inclusion body hepatitis, and gizzard erosion. Different serotypes of FAdV are associated with specific conditions, highlighting the need for targeted prevention strategies. Given the rising prevalence of FAdV-related diseases globally, effective vaccination and biosecurity measures are crucial. In this study, we explore the potential of structural proteins to design a multi-epitope vaccine targeting FAdV. We employed an in silico approach to design the multi-epitope vaccine. Essential viral structural proteins, including hexon, penton, and fiber protein, were selected as vaccine targets. T-cell and B-cell epitopes binding to MHC-I and MHC-II molecules were predicted using computational methods. Molecular docking studies were conducted to validate the interaction of the multi-epitope vaccine candidate with chicken Toll-like receptors 2 and 5. Our in silico methodology successfully identified potential T-cell and B-cell epitopes within the selected viral structural proteins. Molecular docking studies revealed strong interactions between the multi-epitope vaccine candidate and chicken Toll-like receptors 2 and 5, indicating the structural integrity and immunogenic potential of the designed vaccine. The designed multi-epitope vaccine presents a promising approach for combating FAdV infections in chickens. By targeting essential viral structural proteins, the vaccine is expected to induce a robust immunological response. The in silico methodology utilized in this study provides a rapid and cost-effective means of vaccine design, offering insights into potential vaccine candidates before experimental validation. Future studies should focus on in vitro and in vivo evaluations to further assess the efficacy and safety of the proposed vaccine.

Nocardia genus, a complex group of species classified to be aerobic actinomycete, can lead to severe concurrent infection as well as disseminated infection, typically in immunocompromised patients. With the expansion of the susceptible population, the incidence of Nocardia has been gradually growing, accompanied by increased resistance of the pathogen to existing therapeutics. However, there is no effective vaccine against this pathogen yet. In this study, a multi-epitope vaccine was designed against the Nocardia infection using reverse vaccinology combined with immunoinformatics approaches. First, the proteomes of 6 Nocardia subspecies Nocardia subspecies (Nocardia farcinica, Nocardia cyriacigeorgica, Nocardia abscessus, Nocardia otitidiscaviarum, Nocardia brasiliensis and Nocardia nova) were download NCBI (National Center for Biotechnology Information) database on May 1st, 2022 for the target proteins selection. The essential, virulent-associated or resistant-associated, surface-exposed, antigenic, non-toxic, and non-homologous with the human proteome proteins were selected for epitope identification. The shortlisted T-cell and B-cell epitopes were fused with appropriate adjuvants and linkers to construct vaccines. The physicochemical properties of the designed vaccine were predicted using multiple online servers. The Molecular docking and molecular dynamics (MD) simulation were performed to understand the binding pattern and binding stability between the vaccine candidate and Toll-like receptors (TLRs). The immunogenicity of the designed vaccines was evaluated via immune simulation. 3 proteins that are essential, virulent-associated or resistant-associated, surface-exposed, antigenic, non-toxic, and non-homologous with the human proteome were selected from 218 complete proteome sequences of the 6 Nocardia subspecies epitope identification. After screening, only 4 cytotoxic T lymphocyte (CTL) epitopes, 6 helper T lymphocyte (HTL) epitopes, and 8 B cell epitopes that were antigenic, non-allergenic, and non-toxic were included in the final vaccine construct. The results of molecular docking and MD simulation showed that the vaccine candidate has a strong affinity for TLR2 and TLR4 of the host and the vaccine-TLR complexes were dynamically stable in the natural environment. The results of the immune simulation indicated that the designed vaccine had the potential to induce strong protective immune responses in the host. The codon optimization and cloned analysis showed that the vaccine was available for mass production. The designed vaccine has the potential to stimulate long-lasting immunity in the host, but further studies are required to validate its safety and efficacy.

Next-generation multi-epitope subunit vaccine design: A computational approach utilizing two stable proteins to combat Human Metapneumovirus (HMPV).

Avian mycoplasma is a bacterial disease causing chronic respiratory disease (CRD) in poultry industries with high economic losses. The eradication of this disease still remains as a challenge. A multi-epitope prophylactic vaccine aiming the antigenic proteins of Mycoplasma gallisepticum can be a capable candidate to eradicate this infection. The present study is focused to design a multi-epitope vaccine candidate consisting of cytotoxic T-cell (CTL), helper T-cell (HTL), and B-cell epitopes of antigenic proteins, using immunoinformatics strategies. The multi-epitopic vaccine was designed, and its tertiary model was predcited, which was further refined and validated by computational tools. After initial validation, molecular docking was performed between multi-epitope vaccine construct and chicken TLR-2 and 5 receptors, which predicted effective binding. The in silico results specify the structural stability, precise specificity, and immunogenic response of the designed multi-epitope vaccine, and it could be an appropriate vaccine candidate for the M. gallisepticum infection.

Computational approach for identifying immunogenic epitopes and optimizing peptide vaccine through in-silico cloning against Mycoplasma genitalium

Clear cell sarcoma (CCS) is a rare and aggressive soft tissue malignancy characterized by a high metastatic propensity and recurrence rate, with conventional non-specific treatment modalities often yielding limited efficacy and substantial adverse effects. This study aimed to design novel vaccine candidates: a multi-epitope vaccine (MEV) and an mRNA-based vaccine, both targeting the EWSR1-ATF1 and EWSR1-CREB1 fusion proteins, along with the PRAME antigen, to stimulate robust tumor-specific immune responses. Advanced immunoinformatics approaches were employed to identify highly antigenic B-cell and T-cell epitopes while minimizing potential allergenicity and toxicity, ensuring optimal safety and efficacy. The MEV construct was engineered with GM-CSF as an adjuvant to enhance dendritic cell activation and antigen presentation, with EAAK and AAY linkers incorporated to improve structural integrity and epitope processing. For the mRNA vaccine, the MEV was codon-optimized and incorporated into a stable mRNA construct with a 5' cap, Kozak sequence, and poly(A) tail to enhance the translation efficiency and prolong antigen expression. Structural and molecular dynamics simulations confirmed strong and stable interactions of both vaccine constructs with Toll-Like Receptor-3 (TLR-3), supporting their potential for effective immune activation. Furthermore, population coverage analysis demonstrated a global reach of 99.48%, ensuring broad immunogenicity across diverse genetic backgrounds. In silico immune response simulations predicted a sustained immune activation lasting over 417days, characterized by robust cytokine secretion, strong memory cell formation, and high antibody titers following a three-dose regimen. These findings suggest that both the multi-epitope and mRNA-based vaccine candidates hold substantial promise as novel and precise immunotherapeutic interventions for CCS, potentially overcoming the limitations of existing treatment approaches and significantly enhancing long-term patient prognosis.

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. 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. 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.

Ebola virus is the primary causative agent of viral hemorrhagic fever that is an epidemic disease and responsible for the massive premature deaths in humans. Despite knowing the molecular mechanism of its pathogenesis, to date, no commercial or FDA approved multiepitope vaccine is available against Ebola infection. The current study focuses on designing a multi-epitope subunit vaccine for Ebola using a novel immunoinformatic approach. The best predicted antigenic epitopes of Cytotoxic-T cell (CTL), Helper-T cells (HTL), and B-cell epitopes (BCL) joined by various linkers were selected for the multi-epitope vaccine designing. For the enhanced immune response, two adjuvants were also added to the construct. Further analysis showed the vaccine to be immunogenic and non-allergenic, forming a stable and energetically favorable structure. The stability of the unbound vaccine construct and vaccine/TLR4 was elucidated via atomistic molecular dynamics simulations. The binding free energy analysis (ΔG Bind = −194.2 ± 0.5 kcal/mol) via the molecular mechanics Poisson-Boltzmann docking scheme revealed a strong association and thus can initiate the maximal immune response. Next, for the optimal expression of the vaccine construct, its gene construct was cloned in the pET28a + vector system. In summary, the Ebola viral proteome was screened to identify the most potential HTLs, CTLs, and BCL epitopes. Along with various linkers and adjuvants, a multi-epitope vaccine is constructed that showed a high binding affinity with the immune receptor, TLR4. Thus, the current study provides a highly immunogenic multi-epitope subunit vaccine construct that may induce humoral and cellular immune responses against the Ebola infection. Communicated by Ramaswamy H. Sarma