Paloma Napoleão-Pêgo

Angiostrongyliasis, the primary cause of eosinophilic meningitis, represents an emerging disease caused by Angiostrongylus cantonensis larvae, inadvertently transmitted to humans. The diagnosis of human angiostrongyliasis relies on epidemiological features, clinical symptoms, medical history, and laboratory findings, notably hyper eosinophilia in blood and cerebrospinal fluid. Consequently, accurate diagnosis is challenging and prone to confusion with other parasitic diseases. The quest for an early, rapid, and specific diagnostic test for angiostrongyliasis persists, driven by the imperative for enhanced test specificity. This study focused on mapping IgM epitopes on galectin-1 (Gal-1) and galectin-2 (Gal-2) proteins derived from A. cantonensis. The specificity of the epitopes was assessed using database homology analysis. After selecting specific epitopes, researchers chemically synthesized 12 individual multi-antigen peptides (MAPs4) and one chimeric polypeptide that is 65 amino acids long. The effectiveness of these synthesized peptides was subsequently evaluated using enzyme-linked immunoassay (ELISA). A total of twelve unique IgM epitopes were discovered; five were linked to Gal-1, while seven were linked to Gal-2. An ELISA-peptide method confirmed the twelve epitopes, and then the chimeric polypeptide was employed as an antigen to coat ELISA plates. This setup was evaluated with patients’ sera to diagnose strongyloidiasis in vitro. This study provides a comprehensive representation of the IgM epitopes of Gal-1 and Gal-2 from A. cantonensis. ELISA data utilizing the chimeric polypeptide demonstrate that the selected sequences hold promise for the development of a specific immunological assay tailored for the acute diagnosis of angiostrongyliasis infections.

Brucellosis is a globally prevalent zoonotic disease affecting both humans and animals. Its nonspecific clinical manifestations often complicate diagnosis, underscoring the need for reliable laboratory confirmation. Traditional serological assays, though widely used, suffer from limitations such as inconsistent sensitivity and false-positive results. To address these challenges, this study mapped IgM and IgG epitopes of the Brucella Omp-2a protein using sera from infected patients. Epitope identification was performed through SPOT synthesis on cellulose membranes, followed by assessment of potential cross-reactivity using peptide database analysis and ELISA validation. Three major IgM and seven IgG linear B-cell epitopes were identified, six of which demonstrated strong reactivity in peptide-ELISA. Importantly, no significant cross-reactivity with proteins from other human pathogens was detected. Two chimeric multi-epitope peptides, composed of 50 and 60 amino acids and integrating Brucella-specific IgM and IgG epitopes, exhibited excellent diagnostic performance in ELISA, achieving near 100% sensitivity and specificity. These findings support the potential of synthetic peptides as reliable and cost-effective alternatives to native antigens in serological assays. Further validation in larger, geographically diverse cohorts will be essential to confirm their diagnostic robustness and facilitate their integration into routine brucellosis diagnostics.

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.

Angiostrongyliasis, the primary cause of eosinophilic meningitis, represents an emerging disease caused by Angiostrongylus cantonensis larvae, inadvertently transmitted to humans. The diagnosis of human angiostrongyliasis relies on epidemiological features, clinical symptoms, medical history, and laboratory findings, notably hyper eosinophilia in blood and cerebrospinal fluid. Consequently, accurate diagnosis is challenging and prone to confusion with other parasitic diseases. The quest for an early, rapid, and specific diagnostic test for angiostrongyliasis persists, driven by the imperative for enhanced test specificity. Material and Methods: This study focused on the mapping of IgM epitopes within galectin-1 (Gal-1) and galectin-2 (Gal-2) proteins derived from A. cantonensis. The specificity of the epitopes was assessed using database homology analysis. After selecting specific epitopes, researchers chemically synthesized 12 individual MAPs4 peptides and one chimeric polypeptide that is 58 amino acids long. The effectiveness of these synthesized peptides was subsequently evaluated using ELISA. Results: A total of twelve unique IgM epitopes were discovered; four were linked to Gal-1, while eight linked to Gal-2. An ELISA-peptide method confirmed the twelve epitopes, and then the chimeric polypeptide was employed as an antigen to coat ELISA plates. This setup was evaluated with patients' sera to diagnose strongyloidiasis in vitro. Conclusions: This study provides a comprehensive representation of the IgM epitopes of Gal-1 and Gal-2 from A. cantonensis. ELISA data utilizing chimeric the polypeptide demonstrate that the selected sequences hold promise for the development of a specific immunological assay tailored for the acute diagnosis of angiostrongyliasis infections.

Background: Whooping cough (pertussis), caused by Bordetella pertussis, remains a major public health concern worldwide despite high vaccination coverage. Resurgent outbreaks underscore the need for continued epidemiological and immunological monitoring to evaluate population immunity. To assess the humoral immune protection in children aged 1–14 years vaccinated with DTP/Hib/HB between January and December 2022 in Duque de Caxias, Rio de Janeiro, Brazil. Methods: A total of 220 serum samples were analyzed using commercial ELISA kits to detect circulating IgG antibodies against pertussis toxin (PTx) and B. pertussis antigens. Antibody levels were compared across age groups using the Kruskal–Wallis test followed by Dunn’s multiple comparisons. Results: Anti-PTx antibody levels were low across all age groups, with only 2.17% of children showing seropositive levels (>40 IU/mL). Broader reactivity to B. pertussis antigens (PTx + FHA) was detected in 36.7% of samples, but antibody titers declined significantly with increasing age (p < 0.05). These findings indicate waning vaccine-induced immunity and potential susceptibility to reinfection. Conclusions: The study reveals low levels of circulating IgG antibodies against pertussis among vaccinated children, emphasizing the need to reassess the current immunization schedule. Introduction of adolescent booster doses and expanded access to acellular pertussis vaccines are recommended to enhance long-term protection.

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.

Brucellosis is one of the most serious zoonotic diseases worldwide, affecting both human and animal health. In humans, the disease often presents with diverse and nonspecific symptoms, making laboratory confirmation essential for accurate diagnosis and effective treatment. However, traditional diagnostic methods, including serological tests, suffer from limitations such as low sensitivity and high false-positive rates, underscoring the need for improved diagnostic strategies. This underscores the need for improved assays that enable rapid and reliable laboratory detection. In this study, we performed comprehensive IgM and IgG epitope mapping of the Omp-2a protein using sera from Brucella-infected patients. We identified epitopes, created chimeric peptides, and tested their diagnostic value with ELISA. Materials and Methods: The IgM and IgG epitopes of the Omp-2a protein were identified through SPOT synthesis on cellulose membranes, utilizing sera obtained from seropositive individuals. Potential cross-reactive epitopes were screened using peptide database searches and ELISA. Long, chimeric, multi-epitope peptides were synthesized and tested using ELISA on sera from 40 patients to evaluate their diagnostic performance. Results: Three major IgM and seven IgG linear B-cell epitopes were identified. None of the selected epitopes showed cross-reactivity with proteins from other significant human pathogenic organisms, as determined by analysis of peptide databases. Six peptide epitopes were confirmed using peptide-ELISA. Two chimeric peptides (50 and 60 amino acids) containing Brucella-specific IgM and IgG epitopes demonstrated high diagnostic performance, with a sensitivity of 99.96% and a specificity of 100%. Conclusion: Our study identified and validated IgM and IgG epitopes of the transmembrane Omp-2a protein from Brucella abortus. The use of these specific epitopes as biomarkers presents a promising strategy for developing accurate serological assays, potentially enhancing Brucellosis diagnosis and facilitating better monitoring of infection-induced immunity.

Lyme borreliosis (LB) is a tick-borne infection of global relevance that remains underrecognized, hindering effective surveillance and diagnosis. This lack of awareness and the limited specificity and low antibody titters of current serological assays underscore the need for improved diagnostic tools. Here, we investigated the molecular fine specificity of IgM antibody responses to five proteins of Borrelia burgdorferi. Materials and Methods: We employed peptide arrays on cellulose support (SPOT synthesis) to screen IgM epitopes and assess cross-reactivity through databank searches and Enzyme-Linked Immunosorbent Assay (ELISA). Validation was performed using ELISA and Receiver Operating Characteristic (ROC) curve analysis. Results: We identified ten IgM epitopes, of which four were classified as specific. The ELISA peptide assay demonstrated a sensitivity of ≥87.3%, specificity of ≥56.2%, and accuracy of ≥66.6%. A bi-specific peptide was subsequently synthesized and evaluated by ELISA using a panel of patient sera representing different pathologies. This result showed a sensitivity of 85.0% and a specificity of 100.0%, with significant differences in cross-reactivity between the leptospirosis and syphilis groups. Conclusions: These findings indicate that the identified peptide combinations could facilitate the development of new, highly specific serodiagnostic assays, thereby enhancing public health initiatives and epidemiological studies.

The first immune response controls many bacterial and viral inflammatory diseases. Oral immunization with cholera toxin (CT) elicits antibodies and can prevent cholerae in endemic environments. While the IgG immune response to the toxin is well-documented, the IgA and IgM epitopes responsible for the initial immune reaction to the toxin remained uncharted. In this study, our objective was to identify and characterize immunologically and structurally these IgA and IgM epitopes. We conducted SPOT synthesis to create two libraries, each containing one hundred twenty-two 15-mer peptides, encompassing the entire sequence of the three chains of the CT protein. We could map continuous IgA and IgM epitopes by testing these membrane-bound peptides with sera from mice immunized with an oral vaccine (Schankol™). Our approach involved topological studies, peptide synthesis, and the development of an ELISA. We successfully identified seven IgA epitopes, two in CTA, two in CTB, and three in protein P. Additionally, we discovered eleven IgM epitopes, all situated within CTA. Three IgA-specific and three IgM-specific epitopes were synthesized as MAP4 and validated using ELISA. We then used two chimeric 45-mer peptides, which included these six epitopes, to coat ELISA plates and screened them with sera from immunized mice. This yielded sensitivities and specificities of 100%. Our findings have unveiled a significant collection of IgA and IgM-specific peptide epitopes from cholera toxins A, B, and P. These epitopes, along with those IgG previously identified by our group, reflect the immunoreactivity associated with the dynamic of the immunoglobulins switching associated with the cholera toxin vaccination.

Dengue is a neglected disease mainly affecting tropical and subtropical countries. The diagnosis of dengue fever is still a problem since most of it is made from whole or recombinant DENV proteins, which present cross-reactions with other members of the Flavivirus family. Therefore, there is still a huge demand for new diagnostic methods that provide rapid, low-cost, easy-to-use confirmation. Thus, in this study, we developed an affordable electrochemical biosensor for rapidly detecting immunoglobulin G (IgG) serological antibodies in the sera of DENV-infected patients. An identified linear B-cell epitope (DENV/18) specific for DENV 1–4 serotypes recognized by IgG in patient sera was selected as a target molecule after a microarray of peptides using the SPOT-synthesis methodology. After chemical synthesis, the DENV/18-peptide was immobilized on the surface of the working electrode of a commercially available screen-printed gold electrode (SPGE). The capture of DENV-specific IgG allowed for the formation of an immunocomplex that was measured by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) using a potassium ferrocyanide/ferricyanide ([Fe(CN)₆]^3−/4−) electrochemical probe. An evaluation of the biosensor’s performance showed a detection limit of 100 µg mL⁻¹ for the synthetic peptides (DENV/18) and 1.21 ng mL⁻¹ in CV and 0.43 ng mL⁻¹ in DPV for human serum, with a sensitivity of 7.21 µA in CV and 8.79 µA in DPV. The differentiation of infected and uninfected individuals was possible even at a high dilution factor that reduced the required sample volumes to a few microliters. The final device proved suitable for diagnosing DENV by analyzing real serum samples, and the results showed good agreement with molecular biology diagnostics. The flexibility to conjugate other antigenic peptides to SPEs suggests that this technology could be rapidly adapted to diagnose other pathogens.