Identification of immunogenic proteins of Ascaris lung stage larvae through immunoproteomics: towards recombinant-based serodiagnostic assays for humans and pigs.

Soil-transmitted helminthiases are recognised by the World Health Organization as one of the 20 neglected tropical diseases, primarily affecting communities with socioeconomic disadvantages in tropical and subtropical regions. Of the four soil-transmitted helminths, Ascaris stands out as the most widespread, affecting more than 700 million people globally. Today, the diagnostic standard for ascariasis is based on microscopic examination of stool, which faces important limitations. Although serological diagnosis is a promising alternative, the current landscape of well-validated commercial serological diagnostics is sobering. An ELISA based on homogenate from Ascaris suum lung stage larvae (AsLungL3-ELISA) showed significant potential to inform human and veterinary prevention and control programs against ascariasis. Therefore, this study aimed to identify the immunogenic proteins in Ascaris lung stage larval homogenate and investigate the antibody response towards recombinantly expressed versions of these proteins. Given the potential of recombinant-based assays for both human and veterinary applications, the study encompasses experiments involving both humans and pigs. First, immuno-affinity purifications were coupled with liquid chromatography-tandem mass spectrometry, resulting in three lists of immunogenic proteins (for children, adults, and pigs). As a proof of concept, four promising immunogenic proteins (polyprotein ABA-1, paramyosin, apolipophorin and an S60 ribosomal protein) were recombinantly produced in Escherichia coli and the antibody response against these recombinants was evaluated using ELISA. While the results for pigs were inconclusive due to non-specific binding of antibodies, the findings for potential human serodiagnostic applications detecting IgG4 appeared promising. For both polyprotein ABA-1 and paramyosin, a notable difference in OD values was observed between children and adults who were AsLungL3-ELISA negative and positive. In conclusion, this study is a steppingstone towards the development of new serodiagnostic assays and demonstrates that recombinant protein production offers an efficient method to produce diagnostic Ascaris antigens without requiring pig studies.