The rationale for the project rests on well-recognized shortcomings of current vaccination strategies against lumpy skin disease. Live-attenuated vaccines, while capable of inducing robust immunity, carry lingering safety concerns, particularly in settings where incomplete viral attenuation or reversion could pose risks to herds. Inactivated vaccines, by contrast, tend to deliver incomplete protection and often require repeated dosing. Perhaps most importantly for disease-control programs, neither approach is readily compatible with DIVA-based surveillance, the differentiation of infected from vaccinated animals, which is a cornerstone of modern eradication campaigns. A subunit vaccine containing only carefully selected viral epitopes would, in principle, allow serological assays to distinguish vaccinated animals from naturally infected ones, restoring a critical tool for trade and surveillance authorities.

To build such a candidate, the team turned to an integrated immunoinformatics workflow, a pipeline that mines viral protein sequences for short peptide segments, or epitopes, that the immune system can recognize. The researchers focused on the envelope protein P32 and the surface protein A27L of lumpy skin disease virus, two antigens exposed on the viral surface and therefore attractive targets for neutralizing antibodies and cellular immunity. Using prediction algorithms, they identified candidate cytotoxic T-lymphocyte epitopes, helper T-lymphocyte epitopes and B-cell epitopes from these proteins, and then subjected every candidate to a demanding series of filters. Only epitopes that scored well for antigenicity, showed no allergenic or toxic signatures, did not resemble host proteins in ways that could trigger autoimmunity, and were conserved across viral sequences survived the screening process.

The surviving epitopes were then assembled into a single chimeric construct, stitched together with optimized peptide linkers and augmented with an RS09 adjuvant, a Toll-like receptor agonist designed to sharpen the immune response. The final architecture incorporated two cytotoxic T-lymphocyte epitopes, two helper T-lymphocyte epitopes and two B-cell epitopes. Quantitative assessment of the resulting protein suggested a favorable biochemical profile: an antigenicity score of 0.90, indicating a strong likelihood of provoking an immune response; an instability index of 24.32, well below the threshold that would suggest chemical instability; and a solubility score of 0.87, hinting that the protein could be produced in soluble form during recombinant expression. No allergenic or toxic motifs were detected in the assembled sequence.

Because a vaccine’s amino acid sequence alone says little about its three-dimensional shape, the team next modeled and validated the construct’s structure. Structural quality checks returned an ERRAT score of 99 percent, a measure of overall non-bonded interaction quality in the model, and more than 91 percent of residues fell within favored regions of the Ramachandran plot, the standard gauge of stereochemical plausibility for protein backbone conformations. These metrics indicate a high-quality structural model, providing a credible basis for the downstream interaction studies that followed.

A central question for any adjuvanted subunit vaccine is whether the immune system will actually take notice. The RS09 adjuvant works by engaging Toll-like receptors, and the researchers used molecular docking to test how strongly the vaccine construct binds to bovine Toll-like receptor 9, the relevant innate immune sensor in cattle. The interaction proved to be the strongest among the receptors examined, with a binding free energy of minus 16.2 kilocalories per mole and a predicted dissociation constant of 3.5 times ten to the power of minus twelve, an extraordinarily tight affinity. To confirm that this docking pose was not a fleeting computational artifact, the team ran molecular dynamics simulations lasting 100 nanoseconds, which showed the complex remaining stable throughout, suggesting the interaction could plausibly persist long enough in a real cellular environment to trigger innate immune signaling.

Immune simulation extended the picture to the adaptive arm of immunity. The computational models predicted strong humoral and cellular responses following immunization, characterized by elevated levels of immunoglobulin M, immunoglobulin G1 and immunoglobulin G2, alongside the cytokines interferon-gamma and interleukin-2. That cytokine signature is consistent with a Th1-biased immune profile, the type of response generally associated with robust antiviral defense and effective cytotoxic T-lymphocyte activity. For a virus like lumpy skin disease, where both neutralizing antibodies and cellular killing of infected cells matter, a vaccine that can plausibly elicit both arms of the adaptive response is an encouraging design outcome, even if the predictions remain computational rather than experimental at this stage.

What distinguishes this study from many purely in silico vaccine papers is the step toward experimental feasibility. The researchers codon-optimized the vaccine sequence for bacterial expression and performed in silico cloning to confirm the construct could be accommodated in an expression vector. They then produced the recombinant protein in the laboratory and verified its expression by SDS-PAGE and Western blot analysis, both of which revealed a distinct band at approximately 14.3 kilodaltons, matching the predicted molecular weight of the designed construct. That single band provides tangible, if preliminary, support that the computational design can be translated into a real protein, a hurdle where many multi-epitope candidates have faltered.

The authors are careful to frame the work as a rationally designed and experimentally supported candidate rather than a finished vaccine. No animal immunization or challenge data are presented, and the immune simulations, however detailed, remain predictions that must be tested in cattle or suitable animal models. Nonetheless, the study establishes what the authors describe as a promising platform for safer, targeted and potentially DIVA-compatible next-generation capripoxvirus vaccines. Because the construct contains only defined epitopes from P32 and A27L, companion diagnostic assays could in principle be developed to distinguish vaccinated animals from infected ones, addressing one of the most persistent obstacles to controlling this economically damaging disease.

The broader significance of the work lies in its demonstration of a fully integrated pipeline, from epitope prediction and filtering through structural modeling, receptor docking, dynamics simulation, immune simulation and finally recombinant expression. As transboundary livestock diseases continue to threaten food security and international trade, such computational-first approaches offer a way to move from viral genome to testable vaccine candidate in a fraction of the time required by traditional empirical methods. The research was supported by funding from the National Natural Science Foundation of China, the National Key R&D Program of China and the Shenzhen Medical Research Funds, among other sources, and the article is available open access under a Creative Commons license.

Subject of Research: Immunoinformatics-guided design of a multi-epitope subunit vaccine candidate against lumpy skin disease virus

Article Title: Integrated immunoinformatics-guided development and validation of an immunodominant multi-epitope vaccine candidate against lumpy skin disease virus

Article References: Farrukh, M., Munawar, A., Hussain, N., Pan, F., & Riaz, F. (2026). Integrated immunoinformatics-guided development and validation of an immunodominant multi-epitope vaccine candidate against lumpy skin disease virus. Virology Journal. https://doi.org/10.1186/s12985-026-03326-8

Image Credits: AI Generated

DOI: 10.1186/s12985-026-03326-8

Keywords: lumpy skin disease virus, immunoinformatics, multi-epitope vaccine, Capripoxvirus, P32 protein, A27L protein, RS09 adjuvant, TLR9, molecular dynamics, Th1 immune response, DIVA-compatible vaccines, livestock vaccinology

In a study published in International Microbiology, Rasha Assad Assiri of Princess Nourah bint Abdulrahman University and colleagues describe a computational systems immunology framework that integrates subtractive proteomics, reverse vaccinology and immunoinformatics to design a multi-epitope vaccine, or MEV, against K. michiganensis strain ATCC 8724. The approach begins with the bacterium’s complete proteome of 5,483 proteins and systematically filters it down to a handful of promising vaccine targets. Essentiality screening with the Geptop 2.0 server identified 398 proteins the bacterium cannot live without, and comparison against the human proteome using BLASTP, with sequences showing at least 30 percent identity excluded, left 199 non-homologous essential candidates. Subcellular localization prediction, antigenicity scoring with VaxiJen, allergenicity screening with AllerTOP and transmembrane-helix analysis with TMHMM narrowed the field further, ultimately yielding four envelope-associated proteins: the outer-membrane beta-barrel proteins BamA and LptD, the periplasmic lipopolysaccharide transport component LptA, and the outer-membrane lipoprotein LolB.

The choice of targets is biologically deliberate. LptA shuttles lipopolysaccharide, the inflammatory outer-membrane molecule of Gram-negative bacteria, across the periplasm, while LptD delivers LPS to the cell surface. BamA anchors the beta-barrel assembly machinery that builds outer-membrane proteins, and LolB sorts lipoproteins into the outer membrane. All four are essential for bacterial survival, predicted to be antigenic and non-allergenic, and lack similarity to human proteins, reducing the risk of autoimmune cross-reaction. Homologs of these proteins have been reported as conserved and immunogenic targets in several other Gram-negative pathogens, lending external support to their selection. By focusing on envelope-associated machinery rather than variable surface structures, the designers aimed for a vaccine less vulnerable to the antigenic drift that undermines single-antigen subunit vaccines.

From these four proteins, the team predicted cytotoxic T-lymphocyte, helper T-lymphocyte and linear B-cell epitopes using the Immune Epitope Database analysis tools. Eight CTL epitopes survived filtering for non-toxicity, antigenicity, MHC-I binding within the top two percent of predicted binders and non-allergenicity, although six of them carried negative immunogenicity scores on the IEDB scale and were retained only because they met the other predefined criteria. Four HTL epitopes were selected, three of which were predicted to induce interferon-gamma, a cytokine central to cellular immunity against intracellular pathogens; the fourth, ARFNIDSTQVSLTPD, was kept despite its non-inducer prediction because it satisfied all other binding and safety thresholds. Three linear B-cell epitopes, predicted with an artificial-neural-network-based server, completed the immunological repertoire. Population coverage analysis across 78 global population groups estimated that the combined MHC class I and II epitope set would cover 73.80 percent of the world’s population, peaking at 83.22 percent in South Africa and 82.90 percent in England, but dipping to 67.86 percent in South Asia, a regional variation the authors suggest could inform future vaccination prioritization.

Assembling fifteen epitopes into a single molecule requires architectural finesse. Isolated epitopes are weak immunogens, so the researchers grafted the 130-amino-acid 50S ribosomal protein L7/L12 from Mycobacterium tuberculosis, a known immunostimulatory adjuvant, onto the N-terminus of the construct via a rigid, alpha-helical EAAAK linker that spatially separates the adjuvant from the epitope payload. Within the epitope region, AAY linkers separate CTL epitopes to support proteolytic processing, flexible GPGPG linkers rich in glycine and proline space out the HTL epitopes, and short lysine-rich KK linkers keep the B-cell epitopes accessible. The result is a 386-amino-acid chimeric protein with a predicted molecular weight of 40.53 kilodaltons, a theoretical pI of 8.97, an instability index of 28.71 and a GRAVY value of minus 0.334 indicating overall hydrophilicity. VaxiJen assigned the full construct an antigenicity score of 0.9564, comfortably above the 0.5 threshold, and the in silico safety screens classified it as non-toxic and non-allergenic.

Structural credibility was assessed with AlphaFold2 through the ColabFold implementation, followed by refinement in GalaxyRefine. The best model placed 95.6 percent of residues in favored regions of the Ramachandran plot, with 3.8 percent in allowed regions and only 0.3 percent in disallowed regions. ProSA-web returned a Z-score of minus 4.62, consistent with experimentally determined protein structures, and ERRAT reported a quality factor of 99.219. Secondary-structure prediction estimated 34.46 percent alpha helix, 47.93 percent random coil and 17.62 percent beta strand. Notably, the linker regions showed lower pLDDT confidence scores than the structured domains, a caveat the authors carried forward when interpreting downstream docking and dynamics, since flexible segments are inherently harder to model.

To probe whether the vaccine could engage innate immunity, the team docked the construct against chain B of the TLR1-TLR2 heterodimer, a pattern-recognition receptor complex, using ClusPro 2.0. The model drawn from the largest cluster, containing 72 members, was selected for analysis. PDBsum identified 24 hydrogen bonds at the interface, and the PRODIGY server estimated a binding free energy of minus 14.8 kilocalories per mole with a dissociation constant of 1.5 times ten to the minus eleventh molar, values consistent with a favorable predicted interaction. Normal mode analysis with iMODS described a comparatively rigid core with mobile peripheral regions, and a 100-nanosecond molecular dynamics simulation in GROMACS with the CHARMM36m force field showed the complex settling after roughly the first 10 nanoseconds, with the radius of gyration contracting from about 4.2 to 3.7-3.8 nanometers and the solvent-accessible surface area declining from about 580 to 500 square nanometers before stabilizing. The authors are careful to note that these metrics describe structural compatibility under simulated conditions and do not demonstrate receptor activation or downstream immune signaling.

Immune simulation with the C-ImmSim agent-based platform, run with HLA-DRB101:01, HLA-B07:02 and HLA-A*01:01, predicted a coordinated response to a single simulated vaccine dose: rapid antigen clearance accompanied by rising IgM, IgG1 and IgG2 levels, early increases in IL-2 and interferon-gamma, expansion of active and duplicating CD8-positive and CD4-positive T-cell populations, and the persistence of memory-associated helper T cells. The modeled Th1 response predominated over Th2, Th17 and regulatory populations. Codon optimization with JCat produced a Codon Adaptation Index of 0.94 and a GC content of 50 percent, both favorable for expression in Escherichia coli, and in silico cloning into the pET-30a(+) vector between NcoI and XhoI sites yielded a recombinant plasmid of 4,483 base pairs, supporting the theoretical feasibility of recombinant production.

The authors are explicit about the limits of the work. Multi-strain epitope conservation, MM-PBSA binding-energy decomposition, epitope-level cross-reactivity screening against human and microbiome proteins, replicate immune simulations and multi-dose modeling were not performed, and no recombinant expression, in vitro assay or animal study was carried out. Whether responses to the mycobacterial adjuvant might compete with pathogen-directed immunity, a phenomenon known as adjuvant immunodominance, remains an open experimental question. Still, the study represents an early application of an integrated subtractive-proteomics and immunoinformatics pipeline specifically aimed at K. michiganensis, and it arrives amid growing evidence that reverse-vaccinology-designed MEVs can be validated successfully in vitro and in vivo against related pathogens. As carbapenem resistance spreads through hospital and environmental reservoirs alike, computational designs like this one may increasingly serve as the blueprint from which the next generation of antibacterial vaccines is built, provided the laboratory work now required can keep pace with the algorithms.

Subject of Research: Computational immunoinformatics design of a multi-epitope vaccine against multidrug-resistant Klebsiella michiganensis

Article Title: Computational systems immunology and multi-scale modeling for the design of a Multi-Epitope Vaccine (MEV) against emerging multidrug-resistant Klebsiella michiganensis

Article References: Assiri, R. A., Saleh, F. M., Muhammad, S., Fenibo, E. O., & Matambo, T. (2026). Computational systems immunology and multi-scale modeling for the design of a Multi-Epitope Vaccine (MEV) against emerging multidrug-resistant Klebsiella michiganensis. International Microbiology. https://doi.org/10.1007/s10123-026-00903-3

Image Credits: AI Generated

DOI: 10.1007/s10123-026-00903-3

Keywords: Klebsiella michiganensis, multi-epitope vaccine, reverse vaccinology, immunoinformatics, subtractive proteomics, antimicrobial resistance, TLR1 docking, molecular dynamics simulation, epitope prediction, BamA, LptD, codon optimization

The rationale for targeting dense granule antigens, or GRAs, rests on their central role in the parasite’s survival strategy. Once T. gondii invades a host cell, it seals itself inside a parasitophorous vacuole and secretes more than 70 GRA proteins that decorate the vacuole membrane, the intravacuolar network and even the host cell nucleus. These effectors allow the parasite to manipulate host cell function and evade destruction. The research team focused on six GRAs selected for their recent discovery and documented roles in virulence or immunogenicity: GRA15, a well-characterised immunogen that activates the NF-κB pathway; GRA60, a virulence factor acting alongside ROP18; GRA76, identified in 2024 as a growth factor; GRA83, which provokes innate immune responses; and two novel effectors, GRA-α and GRA-β, shown to suppress parasite replication and cyst formation.

No approved vaccine exists for human toxoplasmosis. The only licensed product, the live-attenuated Toxovax used in sheep, cannot be given to people because of its residual pathogenic potential. Existing drug therapies for the acute tachyzoite stage often carry side effects and do not eliminate the dormant tissue cysts that cause lifelong infection. This therapeutic gap has pushed researchers toward reverse vaccinology, the computational discipline that screens pathogen genomes for the most promising antigenic fragments before any experiment begins. By filtering candidates in silico first, laboratories can save years of work, reduce costs and avoid animal testing of constructs destined to fail.

The epitope-mapping stage was deliberately stringent. The team retrieved the amino acid sequences of the six GRAs from the ToxoDB database and predicted linear B-cell epitopes using three independent web servers, retaining only fragments identified by at least two tools. Each surviving epitope was then scored for antigenicity with VaxiJen, screened for allergenicity with AllergenFP and checked for water solubility. In parallel, the researchers predicted cytotoxic T-lymphocyte epitopes of nine or ten residues bound by MHC class I molecules, and helper T-lymphocyte epitopes of fifteen residues bound by MHC class II, selecting the strongest allele-epitope combinations by percentile rank. CTL candidates underwent additional testing for immunogenicity and interferon-gamma induction potential, while HTL candidates were screened for toxicity as well.

From this filtering emerged eighteen final epitopes: six B-cell, six CTL and six HTL segments drawn from the six GRAs. The team then assembled four vaccine constructs, each combining the same epitope payload with a different immunostimulatory adjuvant. RS09-Toxo carried the synthetic TLR-4 agonist peptide RS-09; RSFN-Toxo was double-adjuvanted with RS-09 and human interferon-gamma; 50S-Toxo used a Mycobacterium tuberculosis ribosomal protein; and Rpf-Toxo employed the M. tuberculosis resuscitation-promoting factor E, RpfE. Flexible glycine-serine linkers, proteasomal cleavage sites and the rigid EAAAK spacer held the domains apart, and a C-terminal histidine tag enabled purification. The adjuvant choices were not arbitrary: all favour the Th1-type immunity that is critical for clearing intracellular pathogens such as T. gondii.

Biochemical profiling separated the field quickly. All four constructs scored highly for antigenicity, with values ranging from 0.9148 for Rpf-Toxo to 2.1188 for RS09-Toxo, and none was predicted to be allergenic. However, the ProtParam analysis revealed a critical weakness in the two most antigenic designs: RS09-Toxo and RSFN-Toxo carried instability indices of 60.23 and 50.51, well above the threshold of 40 that marks a protein as unstable in laboratory conditions. Only 50S-Toxo, with an instability index of 24.83, and Rpf-Toxo, at 35.41, passed the stability test, and both showed respectable aliphatic indices suggesting moderate thermal tolerance. All four constructs were hydrophilic, as indicated by negative GRAVY scores, and all exceeded the solubility threshold of the Protein-Sol predictor.

With Rpf-Toxo selected as the lead candidate, the team turned to structural biology. Homology modelling with I-TASSER produced a three-dimensional model that was then refined with the GalaxyRefine server, yielding improved geometry: 86.1 percent of residues fell into favoured Ramachandran regions, the MolProbity score dropped to 2.145 and the clash score fell to 9.6. The researchers candidly note that this figure remains below the 90 percent expected of high-resolution crystal structures, a limitation they attribute to the flexible spacers inherent in multi-epitope constructs. Molecular docking against the human TLR-4 receptor, the innate immune sensor central to Th1-polarised responses against the parasite, produced a stable predicted complex with a binding energy of −20.6 kcal/mol, although the authors caution that the corresponding dissociation constant of 7.3 × 10⁻¹⁶ M likely reflects overestimation by the PRODIGY algorithm rather than a physically realistic affinity.

Immune simulation with the C-ImmSim server, modelling a three-dose schedule at days 1, 84 and 186, painted an encouraging picture. Combined IgG and IgM titres peaked above 170,000 at day 65, memory B-cells reached 500 cells per cubic millimetre and settled at a stable set-point of around 200, and the helper T-cell population included roughly 1,800 memory cells that persisted through day 350. Interferon-gamma levels climbed to approximately 380,000 ng/mL, a signature consistent with the Th1-skewed response needed to eliminate intracellular tachyzoites, and natural killer cell activity peaked near 380 cells per cubic millimetre. Six conformational B-cell epitopes were also predicted on the folded surface of the construct, with ElliPro scores between 0.515 and 0.859, suggesting multiple antibody-accessible sites.

Safety and manufacturability checks completed the computational dossier. A BLASTp search against the human proteome found no significant homology, reducing the risk of autoimmune cross-reactivity, and the sequence was reverse-translated and codon-optimised for expression in E. coli K12, raising the codon adaptation index toward 1.00 and adjusting GC content to 65.63 percent. Restriction sites for Eco53kI and EcoRV, a Shine-Dalgarno ribosome-binding sequence and a stop codon were added to enable directional cloning into the pET-28a(+) vector for recombinant production.

The authors are emphatic that every result remains theoretical until confirmed experimentally. They list a series of limitations, including algorithmic bias in epitope prediction, the simplified nature of immune simulations, the absence of molecular dynamics analysis of the vaccine-receptor complex, and the lack of conservation analysis across genetically diverse T. gondii strains, which will be essential for assessing broad cross-protection. They also flag the inherent risks of multi-epitope design, such as epitope competition or immune masking, which flexible linkers can mitigate but not guarantee against. The proposed next steps include molecular dynamics simulations of the TLR-4 complex, advanced delivery platforms such as liposomes and nanoparticles, and challenge studies in murine models. If those experiments succeed, Rpf-Toxo could represent a meaningful step toward a long-sought human vaccine against one of medicine’s most persistent parasites.

Subject of Research: Immunoinformatic design of a multi-epitope vaccine candidate against Toxoplasma gondii based on dense granule antigens

Article Title: Rpf‐Toxo: A Preliminary Computationally Designed Dense Granule Antigen‐Based Multi‐Epitope Vaccine Against Toxoplasma gondii

Article References: Safari, M. H., Hosseini, S. A., Ghiabi, S., Ghiabi, S., Siamian, D., Majidiani, H., Irannejad, H., & Asghari, A. (2026). Rpf‐Toxo: A Preliminary Computationally Designed Dense Granule Antigen‐Based Multi‐Epitope Vaccine Against Toxoplasma gondii. Veterinary Medicine and Science, 12(5), Article e71189. https://doi.org/10.1002/vms3.71189

Image Credits: AI Generated

DOI: 10.1002/vms3.71189

Keywords: Toxoplasma gondii, toxoplasmosis, multi-epitope vaccine, dense granule antigens, reverse vaccinology, immunoinformatics, TLR-4, RpfE adjuvant, molecular docking, immune simulation, epitope prediction, vaccine design