Mariana Silva Freitas

Lyme borreliosis (LB) is a tick-borne disease caused by a diverse and expanding group of spirochetes characterized by complex biology and advanced immune evasion mechanisms. It presents a wide range of clinical symptoms affecting multiple organ systems and can lead to persistent complications. The pathogenesis of LB remains incompletely understood, and diagnosis typically relies on serologic assays to detect antibodies against LB. However, the standard two-tiered testing (STTT) algorithm is limited by cross-reactivity, low sensitivity, and delayed results, hindering timely and accurate diagnosis. Although molecular tests are considered the gold standard, their reliance on centralized laboratories can delay critical treatment decisions. This underscores the urgent need for rapid, reliable diagnostic tools, particularly for use at the point of hospital admission. Point-of-care serological and direct antigen testing can provide actionable information, supporting decentralized healthcare systems in diagnosing complex diseases, such as LB. In this study, we developed two fluorescent polarization immunoassays (FPIAs) using IgM and IgG LB-specific synthetic epitopes/peptides to evaluate their diagnostic potential. These FPIAs showed high sensitivity and specificity in detecting IgM or IgG anti-LB antibodies in patient sera within minutes. The fluorescently labeled synthetic peptides produced significant polarization differences between infected and healthy samples, allowing clear discrimination. The FPIA-LB functions as a one-step assay, eliminating the need for secondary antibodies or complex protocols. This work highlights the FPIA technique as a robust, rapid, and efficient tool for LB diagnosis, offering a promising advance in improving early detection and patient outcomes, which could save lives and reduce long-term health complications.

Background: Pertussis toxin (Ptx) is a major virulence factor and protective antigen of Bordetella pertussis. Understanding its antigenic landscape is essential for improving vaccine design. This study aimed to compare the linear epitope profiles of Ptx recognized by antibodies from vaccinated children and mice, identifying conserved and species-specific immune targets across subunits S1–S5. Methods: Two libraries of overlapping 14-mer peptides spanning the full-length Ptx sequence were synthesized. Sera from children and mice immunized with the whole-cell pertussis vaccine were analyzed to map antibody-binding regions. Comparative and structural analyses were performed to evaluate epitope distribution and recognition patterns. Results: Murine sera recognized 12 major epitopes, whereas children’s sera identified 24. Eleven epitopes were shared between species, mainly in subunits S1 (Ep3–5, 7, 9, 10), S3 (Ep20, 21, 25, 26), and S5 (Ep32), although minor positional shifts were observed. Eight epitopes were unique to children’s sera, located in S1 (Ep1, 6, 8), S3 (Ep22–24), and S4 (Ep27, 29–30). In the S2 subunit, four distinct epitopes were identified for each species, while only one mouse-specific epitope was detected in S4 (Ep28). Structural analysis revealed non-uniform antibody recognition, with dominant targeting of S3 and conserved antigenic hotspots, as well as selective recognition of the catalytic S1 subunit. Fourteen novel epitopes were identified. Conclusions: These findings highlight both shared and species-specific Ptx epitopes, revealing differences between murine and human immune responses. The identified conserved regions and novel epitopes provide a basis for improved pertussis vaccine design.

Pertussis toxin (Ptx) is a key virulence factor and protective antigen of Bordetella pertussis. Understanding its antigenic landscape is critical for improving vaccine formulations. This study compared the antigenic mapping of continuous epitopes recognized by children and murine antibodies against Ptx subunits S1–S5. Two libraries of nine overlapping 14-mer peptides, each encompassing the full-length Ptx sequence, were synthesized to identify linear antibody-binding regions. Antibodies from children and mice immunized with whole-cell pertussis vaccine recognized several common, though not identical, epitopes. Some regions were species-specific, while shared epitopes exhibited minor positional shifts, indicating subtle differences in recognition. Eighteen major epitopes were recognized by vaccinated mice and twenty-six by the serum of vaccinated children. Eleven present shared sequences [S1 (Ep3-5,7,9,10); S3 (20,21,25,26); S5 (Ep 30)], and eight were recognized uniquely by the serum of children’s [S1 (Ep1,6,8); S3 (Ep22-Ep24) and S4 (Ep27,29-30)]. Four unique and distinct epitopes were identified in the S2 subunit for each species [mice Ep11,14,16,17 and children Ep12,13,15,18, and only one in the S4 subunit [mice Ep 28]. Structural analysis revealed non-uniform antibody recognition of pertussis toxin, with dominant targeting of the S3 subunit and conserved epitope hotspots, alongside a distinct subset of antibodies that engage the catalytic S1 subunit, providing structural evidence for multiple neutralization mechanisms. These findings provide a mechanistic basis for differences in toxin neutralization and highlight limitations in extrapolating murine epitope data to human immunity. The conserved antigenicity of the subdomains provides valuable insights into Ptx immunogenicity and represents promising candidates for inclusion in next-generation synthetic pertussis vaccine design.