Computational study designs multi-epitope vaccine candidate against Lassa virus

Researchers have used a purely computational, reverse vaccinology workflow to design and evaluate a multi-epitope subunit vaccine candidate against Lassa virus, the arenavirus responsible for recurrent outbreaks of Lassa fever in West Africa. The study, published open access in Discover Viruses, reports no laboratory or clinical testing; every finding is a prediction generated by bioinformatics servers, molecular docking, molecular dynamics simulation and immune modelling. The authors, Akeem Babatunde Sikiru, Ibrahim Shuaibu Harande and Nurulfiza Bint Mat Isa, describe the construct as a computationally validated candidate that now requires experimental validation before any claims about real-world performance can be made.

The rationale for the work rests on the epidemiology of Lassa fever. The disease is endemic in Nigeria, Sierra Leone, Liberia, Guinea and Benin, with epidemiological estimates suggesting between 100,000 and 300,000 infections and approximately 5,000 deaths each year, figures the authors note are widely considered underestimates because of limited surveillance in rural, resource-constrained settings. Around 80 percent of infections are estimated to be asymptomatic or mild, but the remaining cases can progress to severe multisystem disease. The case-fatality rate among hospitalised patients is frequently cited at around 15 percent and can exceed that during major outbreaks. In pregnancy, particularly the third trimester, infection is associated with maternal mortality above 30 percent and foetal or neonatal loss in more than 80 percent of cases.

No licensed vaccine exists against Lassa virus, and the authors argue that traditional development is difficult for a Biosafety Level 4 pathogen, since working with live virus requires expensive high-containment facilities. Candidate vaccines are in development, including rVSVΔG-LASV-GPC, the DNA vaccine INO-4500 and the inactivated rabies virus platform LASSARAB, but the pipeline still lacks a licensed product. The World Health Organization has designated Lassa fever a priority pathogen in its Research and Development Blueprint. The study targeted the viral L protein, the RNA-dependent RNA polymerase encoded by the L segment, selected for its high expression during replication, its role as a source of T-cell epitopes in arenaviruses and its higher conservation across strains compared with surface glycoproteins.

The design workflow began with the complete genomic sequence of the Lassa virus L segment from the Josiah strain, retrieved from GenBank under accession number NC_004297.1. The L protein spans 2,218 amino acids. Linear B-cell epitopes were predicted with BepiPred 2.0, discontinuous epitopes with ElliPro, cytotoxic T-lymphocyte epitopes with NetMHCpan 4.1 against 27 common HLA class I alleles, and helper T-lymphocyte epitopes with NetMHCIIpan 4.0 against 27 HLA class II alleles. Predicted epitopes then passed through sequential filtering for antigenicity with VaxiJen 2.0, immunogenicity with the IEDB Class I predictor, allergenicity with AllerTop 2.0, toxicity with ToxinPred, conservation across 50 LASV strains using Clustal Omega, and human homology screening with BLASTp against the human proteome.

Selected epitopes were assembled into a chimeric construct using defined linkers: an EAAAK rigid linker connecting the N-terminal adjuvant, the 50S ribosomal protein L7/L12 from Mycobacterium tuberculosis, GPGPG flexible linkers between helper T-cell epitopes, AAY linkers between cytotoxic T-cell epitopes and KK linkers between B-cell epitopes. A 6×His tag was added at the C-terminus for purification, and the final sequence was checked for internal stop codons and frameshifts. Physicochemical characterisation with ExPASy ProtParam yielded a molecular weight of 24.38 kilodaltons, a theoretical isoelectric point of 11.82 indicating a strongly basic protein, and a grand average of hydropathicity of −1.70, signifying a highly hydrophilic character that the authors consider favourable for solubility.

The same analyses flagged significant stability concerns. The instability index of 72.57 classifies the protein as unstable, and the low aliphatic index of 18.10 suggests it is likely to be thermolabile, which the authors acknowledge would complicate cold-chain storage in endemic regions. They frame this as an engineering problem addressable through stabilising excipients, lyophilisation or site-directed mutagenesis, rather than a fatal flaw. On safety-related predictions, the construct scored 0.385 for antigenicity with VaxiJen and was predicted to be non-allergenic, an assessment the authors base on its lack of cysteine residues and glycine content of 18.18 percent, below the 20 percent threshold sometimes associated with allergenicity. Solubility on overexpression in Escherichia coli was predicted with the Protein-Sol server.

To probe whether the construct could engage an innate immune receptor, the team docked the vaccine model against the human TLR4/MD2 complex using ClusPro 2.0 after preparing structures in UCSF Chimera. The docking analysis identified hotspot residues on the vaccine, including R15, R10 and R18, contributing electrostatic and hydrogen-bond interactions with TLR4, with the vaccine chain contributing approximately 9.5 kilocalories to electrostatic stability. A 100-nanosecond molecular dynamics simulation in GROMACS with the CHARMM36m force field showed the complex reaching equilibrium after roughly 30 nanoseconds, with backbone RMSD stabilising around 2.56 angstroms, a radius of gyration stabilising at 4.13 nanometres, an average of approximately 150 hydrogen bonds over the final 60 nanoseconds, and a persistent salt bridge between TLR4-Lys42 and Vaccine-Asp67 at a distance below 1.0 nanometre. These are simulated stability metrics, not measurements of a physical molecule.

Immune simulation with the C-IMMSIM server, run for 1,050 steps over a simulated 350 days with a three-dose prime-boost schedule at weeks 0, 4 and 8, predicted a primary IgM response followed by class-switched IgG, activation and clonal expansion of both CD4+ and CD8+ T-cell populations, a Th1-polarised cytokine profile dominated by IFN-γ and IL-2, and a lasting memory T-cell population. Population coverage analysis with the IEDB tool predicted combined coverage of 95.30 percent for the West African population, spanning Benin, Ghana, Guinea, Liberia, Mali, Nigeria and Sierra Leone, compared with 92.10 percent globally. Gene Ontology enrichment analysis with g:Profiler additionally associated the construct with nucleic acid binding and transcription factor activity terms, leading the authors to hypothesise, without experimental support, that the strongly basic construct might possess intrinsic self-adjuvanting properties.

The authors are explicit that the study’s central limitation is its computational nature: in silico predictions, however sophisticated, remain hypotheses requiring rigorous experimental validation. The construct’s predicted instability, its reliance on predicted epitope immunogenicity and the absence of any antibody binding, T-cell activation or protection data mean the candidate sits at the earliest stage of the development pathway. What the study delivers is a rationally designed sequence, a documented filtering pipeline and a set of testable predictions, including the Th1-oriented immune profile and the self-adjuvancy hypothesis, that the authors argue warrant advancement into preclinical evaluation and in vivo testing as part of the broader effort to develop a Lassa fever vaccine.

Subject of Research: In silico design of a multi-epitope subunit vaccine candidate against Lassa virus

Article Title: Design and in silico validation of a novel multi-epitopes subunit vaccine candidate against Lassa virus using reverse vaccinology approach

Article References: Sikiru, A. B., Harande, I. S., & Isa, N. B. M. (2026). Design and in silico validation of a novel multi-epitopes subunit vaccine candidate against Lassa virus using reverse vaccinology approach. Discover Viruses, 3(1), Article 2. https://doi.org/10.1007/s44370-026-00037-0

Image Credits: AI Generated

DOI: 10.1007/s44370-026-00037-0

Keywords: Lassa virus, Lassa fever, reverse vaccinology, multi-epitope vaccine, immunoinformatics, TLR4 docking, molecular dynamics simulation, epitope prediction, West Africa, subunit vaccine, in silico study, vaccine design