Recombinant antigens derived from Rickettsia prowazekii GltA: from in silico design to preliminary serological evaluation

Epidemic typhus, caused by Rickettsia prowazekii , remains a re-emerging public health concern in resource-limited and conflict-affected regions, underscoring the need for improved diagnostic tools. Although the citrate synthase (GltA) gene is widely used as a molecular diagnostic marker, it has not been explored as an immunological marker. We sought to exploit this by designing recombinant antigens that harbor multiple epitopes or immunodominant regions of Rickettsia prowazekii GltA. As proof of concept, we employed an integrated immunoinformatics and molecular engineering strategy to design, produce, and evaluate recombinant GltA-derived immunogens. Two constructs, MA3 and MA4, were designed and computationally characterized for antigenicity, immunogenicity, physicochemical properties, structural features, and heterologous expression potential. MA3 showed the most favourable predicted profile and was successfully expressed and purified in Escherichia coli. Immunization of mice elicited robust MA3-specific antibody responses, experimentally supporting its predicted immunogenicity. Importantly, preliminary evaluation of human sera demonstrated stronger MA3 reactivity with Rickettsia prowazekii -positive samples than with negative controls, indicating its potential as a candidate antigen for serodiagnostic assay development. These findings establish a proof of concept for GltA-derived recombinant antigens as immunodiagnostic targets and identify MA3 as a promising candidate for further development. Larger, clinically characterized serum panels, systematic assessment of cross-reactivity with related Rickettsia species, and rigorous assay optimization and validation are required to determine its diagnostic accuracy and clinical applicability.