Chikungunya virus-specific pre-existing T cell responses in naïve donors and B cell epitopes in seropositive individuals and experimentally infected mice

INTRODUCTION. Chikungunya virus (CHIKV) poses a major public health challenge in endemic regions due to the lack of specific preventive measures and effective antiviral therapies. Understanding and identifying viral epitopes through rigorous research directly supports the development of more effective next-generation vaccines. AIM. This study aimed to investigate the presence of pre-existing CHIKV-specific T cell memory in unexposed individuals and identified the B cell epitopes targeted in both seropositive donors and CHIKV-infected murine models. MATERIALS AND METHODS. Peripheral blood from 34 healthy volunteers and sera from four CHIKV-seropositive individuals, as well as from BALB/c mice immunized with a non-lethal CHIKV strain, were analyzed to assess Tand B-cell responses. Five synthetic peptides (21–29 aa) derived from nsP2, nsP3, nsP4, and E2 proteins were selected using the immunogenicity prediction algorithm from the Immune Epitope Database and synthesized to encompass predicted epitopes with flanking residues. Peripheral blood mononuclear cells from unexposed donors were stimulated with peptides (1.2 µg/mL), and CD4+/CD8+ T cell proliferation was evaluated by carboxyfluorescein succinimidyl ester (CFSE)-based flow cytometry. Human and mouse sera were tested for peptide-specific IgG by enzyme-linked immunosorbent assay (ELISA). Epitope localization was visualized on 3D protein models using UCSF ChimeraX software and web server I-TASSER. RESULTS. T cell proliferation assays with CFSE labeling revealed preexisting CD4+, but not CD8+, T cell responses to peptides from nonstructural (nsP2–4) and structural (E2) CHIKV proteins in seronegative donors ( P <0.05, P <0.01). Linear B cell epitopes within nsP3, nsP4, and E2 were identified by ELISA in sera from CHIKV-seropositive donors and CHIKV-infected mice. These epitopes were subsequently mapped onto 3D models of CHIKV proteins (E2, amino acids 3140–3161; nsP3, amino acids 1801–1823; nsP4, amino acids 2207–2256). CONCLUSIONS. Our findings indicate the presence of preexisting CD4+ T cell responses in antigen-naïve individuals and underscore the importance of experimentally validating in silico –predicted epitopes for advancing serological diagnostics and vaccine development.