Rational Design and In Silico Evaluation of a Multiepitope Vaccine Targeting the uPAR for Cancer Immunotherapy

Chlamydia trachomatis is an obligate intracellular Gram-negative pathogen that causes sexually transmitted infections (STIs) and trachoma. Current interventions are limited due to the widespread nature of asymptomatic infections, and the absence of a licensed vaccine exacerbates the challenge. In this study, we predicted outer membrane β-barrel (OMBB) proteins and designed a multi-epitope vaccine (MEV) construct using identified proteins. We employed a consensus-based computational framework on the C. trachomatis D/UW-3/CX proteome and identified 17 OMBB proteins, including well-known Pmp family members and MOMP. Eight OMBB proteins were computationally characterized, showing significant structural homology with known outer membrane proteins from other bacteria. Sequence-based annotation tools were used to determine their putative functions. B-cell and T-cell epitopes were predicted from the selected proteins. The MEV construct was designed using four cytotoxic T-lymphocyte (CTL) epitopes and 29 helper T-lymphocyte (HTL) epitopes from six OMBB proteins, which were conserved across 106 C. trachomatis serovars. To enhance its immunogenicity, the vaccine was supplemented with the Cholera toxin B subunit and PADRE sequence at the N-terminus. The MEV construct, of length 780 amino acids, was predicted to be antigenic, non-allergenic, non-toxic, and soluble. Secondary structure analysis revealed 95% random coils. A three-dimensional structural model of the MEV was generated and subsequently validated. Molecular docking between MEV and toll-like receptor 4 (TLR4) revealed strong and stable binding interactions. The MEV-TLR4 complex was found to be structurally compact and stable using molecular dynamics simulation. Immune simulation of the MEV construct elicited a strong immune response. This study highlights OMBB proteins as promising immunogenic targets and presents a computationally designed MEV candidate for C. trachomatis infection.

The inability of existing vaccines to cope with the mutation rate has highlighted the need for effective preventative strategies for COVID-19. Through the secretion of immunoglobulin A, mucosal delivery of vaccines can effectively stimulate mucosal immunity for better protection against SARS-CoV-2 infection. In this study, various immunoinformatic tools were used to design a multi-epitope oral vaccine against SARS-CoV-2 based on its receptor-binding domain (RBD) and heptad repeat (HR) domains. T and B lymphocyte epitopes were initially predicted from the RBD and HR domains of SARS-CoV-2, and potential antigenic, immunogenic, non-allergenic, and non-toxic epitopes were identified. Epitopes that are highly conserved and have no significant similarity to human proteome were selected. The epitopes were joined with appropriate linkers, and an adjuvant was added to enhance the vaccine efficacy. The vaccine 3D structure constructs were docked with toll-like receptor 4 (TLR-4) and TLR1-TLR2, and the binding affinity was calculated. The designed multi-epitope vaccine construct (MEVC) consisted of 33 antigenic T and B lymphocyte epitopes. The results of molecular dockings and free binding energies confirmed that the MEVC effectively binds to TLR molecules, and the complexes were stable. The results suggested that the designed MEVC is a potentially safe and effective oral vaccine against SARS-CoV-2. This in silico study presents a novel approach for creating an oral multi-epitope vaccine against the rapidly evolving SARS-CoV-2 variants. These findings offer valuable insights for developing an effective strategy to combat COVID-19. Further preclinical and clinical studies are required to confirm the efficacy of the MEVC vaccine.

Harnessing bioinformatics for the development of a promising multi-epitope vaccine against tuberculosis: The ZL9810L vaccine

Brucellosis is a common zoonosis, which is caused by Brucella infection, and Brucella often infects livestock, leading to abortion and infertility. At present, human brucellosis remains one of the major public health problems in China. According to previous research, most areas in northwest China, including Xinjiang, Tibet, and other regions, are severely affected by Brucella. Although there are vaccines against animal Brucellosis, the effect is often poor. In addition, there is no corresponding vaccine for human Brucellosis infection. Therefore, a new strategy for early prevention and treatment of Brucella is needed. A multi-epitope vaccine should be developed. In this study, we identified the antigenic epitopes of the Brucella type IV secretion system VirB8 and Virb10 using an immunoinformatics approach, and screened out 2 cytotoxic T lymphocyte (CTL) epitopes, 9 helper T lymphocyte (HTL) epitopes, 6 linear B cell epitopes, and 6 conformational B cell epitopes. These advantageous epitopes are spliced together through different linkers to construct a multi-epitope vaccine. The silico tests showed that the multi-epitope vaccine was non-allergenic and had a strong interaction with TLR4 molecular docking. In immune simulation results, the vaccine construct may be useful in helping brucellosis patients to initiate cellular and humoral immunity. Overall, our findings indicated that the multi-epitope vaccine construct has a high-quality structure and suitable characteristics, which may provide a theoretical basis for the development of a Brucella vaccine.

Fatty acids can activate proinflammatory pathways leading to the development of insulin resistance, but the mechanism is undiscovered. Toll like receptor 2 (TLR2) recognizes lipids, activates proinflammatory pathways, and is genetically associated with inflammatory diseases. This study aimed to examine the role of TLR2 in palmitate-induced insulin resistance in C2C12 myotubes. Treatment with palmitate rapidly induced the association of myeloid differentiation factor 88 (MyD88) with the TLR2 receptor, activated the stress-linked kinases p38, JNK, and protein kinase C, induced degradation of IkappaBalpha, and increased NF-kappaB DNA binding. The activation of these pathways by palmitate was sensitive and temporally regulated and occurred within the upper physiologic range of saturated fatty acid concentrations in vivo, suggesting a receptor-mediated event and not simple lipotoxicity. When compared with an equimolar concentration of palmitate, fibroblast-stimulating lipopeptide-1, a known TLR2 ligand, was a slightly more potent activator of signal transduction and interleukin (IL)-6 production. Palmitate inhibited insulin signal transduction in C2C12 cells beginning 1-2 h after exposure and reached a maximum at 12-16 h. An antagonist TLR2 antibody, mAb 2.5, led to a 50-60% decrease in palmitate-induced IL-6 production and partially restored insulin signal transduction, whereas an isotype-matched control antibody had no effect. RNA interference-mediated inhibition of TLR2 and MyD88 expression in C2C12 muscle cells resulted in a near complete inhibition of palmitate-induced insulin resistance and IL-6 production. This study provides strong evidence that TLR2 mediates the initial events of fatty acid-induced insulin resistance in muscle.

BackgroundMiddle East respiratory syndrome (MERS) is caused by MERS coronavirus (MERS-CoV). Thus far, MERS outbreaks have been reported from Saudi Arabia (2013 and 2014) and South Korea (2015). No specific vaccine has yet been reported against MERS.PurposeTo address the urgent need for an MERS vaccine, in the present study, we have designed two multi-epitope vaccines (MEVs) against MERS utilizing several in silico methods and tools.MethodsThe design of both the multi-epitope vaccines (MEVs) are composed of cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) epitopes, screened form thirteen different proteins of MERS-CoV. Both the MEVs also carry potential B-cell linear epitope regions, B-cell discontinuous epitopes as well as interferon-γ-inducing epitopes. Human β-defensin-2 and β-defensin-3 were used as adjuvants to enhance the immune response of MEVs. To design the MEVs, short peptide molecular linkers were utilized to link screened most potential CTL epitopes, HTL epitopes and the adjuvants. Tertiary models for both the MEVs were generated, refined, and further studied for their molecular interaction with toll-like receptor 3. The cDNAs of both MEVs were generated and analyzed in silico for their expression in a mammalian host cell line (human).ResultsScreened CTL and HTL epitopes were found to have high propensity for stable molecular interaction with HLA alleles molecules. CTL epitopes were also found to have favorable molecular interaction within the cavity of transporter associated with antigen processing. The selected CTL and HTL epitopes jointly cover upto 94.0% of worldwide human population. Both the CTL and HTL MEVs molecular models have shown to have stable binding and complex formation propensity with toll-like receptor 3. The cDNA analysis of both the MEVs have shown high expression tendency in mammalian host cell line (human).ConclusionAfter multistage in silico analysis, both the MEVs are predicted to elicit humoral as well as cell mediated immune response. Epitopes of the designed MEVs are predicted to cover large human population worldwide. Hence both the designed MEVs could be tried in vivo as potential vaccine candidates against MERS.

Developing a multiepitope vaccine for the prevention of SARS-CoV-2 and monkeypox virus co-infection: A reverse vaccinology analysis.

BackgroundThe novel coronavirus disease (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to the ongoing 2019-2020 pandemic. SARS-CoV-2 is a positive-sense single-stranded RNA coronavirus. Effective countermeasures against SARS-CoV-2 infection require the design and development of specific and effective vaccine candidates.ObjectiveTo address the urgent need for a SARS-CoV-2 vaccine, in the present study, we designed and validated one cytotoxic T lymphocyte (CTL) and one helper T lymphocyte (HTL) multi-epitope vaccine (MEV) against SARS-CoV-2 using various in silico methods.MethodsBoth designed MEVs are composed of CTL and HTL epitopes screened from 11 Open Reading Frame (ORF), structural and nonstructural proteins of the SARS-CoV-2 proteome. Both MEVs also carry potential B-cell linear and discontinuous epitopes as well as interferon gamma–inducing epitopes. To enhance the immune response of our vaccine design, truncated (residues 10-153) Onchocerca volvulus activation-associated secreted protein-1 was used as an adjuvant at the N termini of both MEVs. The tertiary models for both the designed MEVs were generated, refined, and further analyzed for stable molecular interaction with toll-like receptor 3. Codon-biased complementary DNA (cDNA) was generated for both MEVs and analyzed in silico for high level expression in a mammalian (human) host cell line.ResultsIn the present study, we screened and shortlisted 38 CTL, 33 HTL, and 12 B cell epitopes from the 11 ORF protein sequences of the SARS-CoV-2 proteome. Moreover, the molecular interactions of the screened epitopes with their respective human leukocyte antigen allele binders and the transporter associated with antigen processing (TAP) complex were positively validated. The shortlisted screened epitopes were utilized to design two novel MEVs against SARS-CoV-2. Further molecular models of both MEVs were prepared, and their stable molecular interactions with toll-like receptor 3 were positively validated. The codon-optimized cDNAs of both MEVs were also positively analyzed for high levels of overexpression in a human cell line.ConclusionsThe present study is highly significant in terms of the molecular design of prospective CTL and HTL vaccines against SARS-CoV-2 infection with potential to elicit cellular and humoral immune responses. The epitopes of the designed MEVs are predicted to cover the large human population worldwide (96.10%). Hence, both designed MEVs could be tried in vivo as potential vaccine candidates against SARS-CoV-2.

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

Background: Klebsiella pneumoniae is one of the bacteria that causes pneumonia infection. Even though the number of pneumonia cases is relatively high and has become a global problem, there is still no vaccine available to prevent this disease. This study was aimed to design a multi-epitope vaccine design through an in silico approach, against K. pneumoniae.Materials and method: Vaccine candidate was constructed based on proteins of K. pneumoniae. These proteins were analyzed to identify the antigens sequence for multi-epitope vaccine design. The constructed vaccine was predicted for allergenicity, toxicity, population coverage, and its physicochemical properties. The vaccine structure was then docked with the toll like receptor 2 (TLR2) molecule to show the interaction. Expression analysis and cloning of the constructed vaccine was carried out in the pET-28a vector using SnapGene.Results: The vaccine was 567 amino acids long, consisting of Cholera Toxin Subunit B as an adjuvant, 6 B-cell epitopes, 11 cytotoxic T-cell epitopes, and 10 helper T-cell epitopes connected with the appropriate linker. Epitopes analysis showed that the vaccine will be a non-toxic, has high antigenicity, but non-allergenic. The vaccine was predicted to be stable, hydrophilic, and had a low risk of triggering autoimmune response. The vaccine molecule was compatible to humans TLR2 molecule. Furthermore, visualization of the candidate vaccine protein on pET-28a showed that the vaccine protein might be expressed correctly.Conclusion: The construction of multi-epitope vaccine has been developed, which might be a good vaccine candidate, containing 6 B-cell epitopes, 11 CTL epitopes, and 10 HTL epitopes. The construct may help scientists to experimentally formulate multi-epitope vaccine against K. pneumoniae in the future.Keywords: in silico, Klebsiella pneumoniae, multi-epitope, vaccine

Nipah virus (NiV) is an emerging zoonotic virus that caused several serious outbreaks in the south asian region with high mortality rates ranging from 40 to 90% since 2001. NiV infection causes lethal encephalitis and respiratory disease with the symptom of endothelial cell-cell fusion. No specific and effective vaccine has yet been reported against NiV. To address the urgent need for a specific and effective vaccine against NiV infection, in the present study, we have designed two Multi-Epitope Vaccines (MEVs) composed of 33 Cytotoxic T lymphocyte (CTL) epitopes and 38 Helper T lymphocyte (HTL) epitopes. Out of those CTL and HTL combined 71 epitopes, 61 novel epitopes targeting nine different NiV proteins were not used before for vaccine design. Codon optimization for the cDNA of both the designed MEVs might ensure high expression potential in the human cell line as stable proteins. Both MEVs carry potential B cell linear epitope overlapping regions, B cell discontinuous epitopes as well as IFN-γ inducing epitopes. Additional criteria such as sequence consensus amongst CTL, HTL and B Cell epitopes was implemented for the design of final constructs constituting MEVs. Hence, the designed MEVs carry the potential to elicit cell-mediated as well as humoral immune response. Selected overlapping CTL and HTL epitopes were validated for their stable molecular interactions with HLA class I and II alleles and in case of CTL epitopes with human Transporter Associated with antigen Processing (TAP) cavity. The structure based epitope cross validation for interaction with TAP cavity was used as another criteria choosing final epitopes for NiV MEVs. Finally, human Beta-defensin 2 and Beta-defensin 3 were used as adjuvants to enhance the immune response of both the MEVs. Molecular dynamics simulation studies of MEVs-TLR3 ectodomain (Human Toll-Like Receptor 3) complex indicated the stable molecular interaction. We conclude that the MEVs designed and in silico validated here could be highly potential vaccine candidates to combat NiV infections, with great effectiveness, high specificity and large human population coverage worldwide.

Chlamydiosis is a widespread ailment affecting humans, livestock, and wildlife, caused by C. abortus, a member of the Chlamydia genus. This disease leads to reproductive disorders in bovines and poses a zoonotic risk, resulting in adverse outcomes such as abortion, stillbirths, weak offspring, endometritis, repeat breeding, and perinatal mortality. However, current chlamydiosis vaccines have limitations in terms of safety, efficacy, and stability, necessitating the development of effective and safe alternatives. In this study, our objective was to design a multi-epitope vaccine (MEV) targeting all strains of C. abortus using bioinformatics and immunoinformatics approaches. We identified highly antigenic and non-allergic proteins (yidC, yajC, secY, CAB503, and CAB746) using VaxiJen and AlgPred tools. Physicochemical analyses and secondary structure predictions confirmed protein stability through ProtParam and SOPMA methods. Furthermore, we employed IEDB-AR, NETMHCpan, and ToxinPred2 tools to predict cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B-cell epitopes, resulting in the identification of conserved epitopes for further analysis. The MEV construct, consisting of 545 amino acids, incorporated the adjuvant Beta defensin-3, along with 9 CTL epitopes and 21 HTL epitopes linked by EAAAK, KK, and AAY linkers. We assessed the safety and immunogenicity of the vaccine through comprehensive evaluations of antigenicity, toxicity, allergenicity, and physicochemical properties. Structural stability and quality were examined using 3D modeling via the ab initio approach with the Robetta platform. Molecular docking analysis explored the compatibility of the MEV with Toll-like receptor 9 (TLR9) using ClusPro, while molecular dynamics simulation with the DESMOND Maestro software predicted the stability and flexibility of the docked complex. Despite promising in silico findings, further wet lab investigations are crucial to validate the safety and efficacy of the MEV. Successful development and validation of this MEV hold significant potential in combatting chlamydiosis in both animal and human populations. Communicated by Ramaswamy H. Sarma

The rapid outbreak of the new coronavirus SARS-COV-2 has created a major public health challenge. Immunoinformatics tools had a clear effect in tracking the genetic sequence of the virus and monitoring mutations and design vaccines that are effective enough to produce antibodies. In our study, we resorted to the emerging discipline of immunoinformatics in order to design a multi-epitope mRNA vaccine against the spike glycoprotein of SARS-CoV-2. We screened the B cell and T cell epitopes of the Spike glycoprotein. we used ABC pred server to predict B cell epitope in the spike glycoprotein sequence and we used NetMHC-4.1 server to predict the T-cell epitope. Then we selected the B cell and T cell epitopes that fulfilled the antigenicity, non-toxicity, non-allergenicity, induction of both IL4 and IFN gamma. Finally, we designed multi-epitope mRNA Vaccine construct by linking 6 B lymphocytes epitopes (BL) with 6 cytotoxic T lymphocytes epitopes (CTL) together with helper T lymphocyte (HTL) epitope up-streamed by 5’ cap and down-streamed by poly A tail. The vaccine was found to be antigenic, non-toxic, non-allergenic, capable of generating a robust immune response. Based on these parameters, this design can be considered a promising choice for a vaccine against SARS-CoV-2.

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

BackgroundHuman metapneumovirus (hMPV) is a significant etiological agent of acute respiratory infections in children and immunocompromised individuals. Despite its growing clinical impact, no approved vaccines or targeted antiviral therapies are currently available.MethodsAn immunoinformatic approach was employed to design a chimeric multi-epitope vaccine candidate against hMPV. Conserved and virulence-associated proteins were analyzed to predict highly antigenic B cell, cytotoxic T lymphocyte (CTL), and helper T lymphocyte (HTL) epitopes. The selected epitopes were screened for antigenicity, non-toxicity, non-allergenicity, and lack of homology to human proteins. The final construct included six B cell epitopes, six CTL epitopes, and two HTL epitopes, linked with appropriate adjuvants and Toll-like receptor (TLR) agonists. Structural modeling, molecular docking, and molecular dynamics simulations were conducted to evaluate the stability and receptor binding. Immunogenicity and expression potential were assessed through in silico immune simulation and codon optimization for expression in Escherichia coli.ResultsAll selected epitopes showed high antigenicity with no allergenic or toxicity. Structural validation indicated a stable vaccine construct with favorable physicochemical properties. Molecular docking analysis predicted a high binding affinity between the vaccine construct and TLR2/TLR4 receptors. Molecular dynamics (MD) simulations suggested that the docked complexes maintained stable interactions under simulated physiological conditions. In silico immune simulations predicted strong B- and T-cell responses following three doses. Codon adaptation analysis supported the high-level expression in E. coli.ConclusionThe proposed multi-epitope vaccine demonstrates strong potential against hMPV, as supported by comprehensive computational analyses. Further experimental studies are required to validate its efficacy and safety.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12879-025-11339-x.