Title : Peptide-protein conjugates as vaccine candidates against hookworm infection
Abstract:
Background: Human hookworm infection, primarily caused by the hematophagous nematode Necator americanus, remains a leading neglected tropical disease (NTD) characterized by chronic iron-deficiency anaemia and malnutrition. While current anthelmintic drug treatments provide temporary relief, their long-term efficacy is significantly hindered by high reinfection rates and emerging drug resistance; moreover, no commercial vaccine has been successfully developed to date. Herein, we propose a novel vaccine strategy targeting a highly conserved neutralizing epitope on the surface of the Na APR-1 aspartic protease, an enzyme essential for parasite survival that initiates host hemoglobin degradation within the hookworm gut. This study investigates a vaccine candidate targeting the P3-derived epitope—a functionally vital domain of the Na APR-1 catalytic site—conjugated to the immunogenic carrier protein Keyhole Limpet Hemocyanin (KLH). By formulating this conjugate with adjuvant systems, we aim to elicit potent neutralizing antibodies that inhibit APR-1 enzymatic activity and disrupt the parasite's primary nutrient acquisition pathway.
Methods: Vaccine candidates were prepared by formulation the epitope-KLH conjugate with diverse adjuvating platforms, including the commercial oil-in-water emulsion MF59 and a proprietary polyleucine system, using homogenization to ensure nanostructure integration. Structural integrity was characterized via Dynamic Light Scattering (DLS) to determine the formed nanoparticles size and polydispersity index (PDI). Morphology of the nanoparticle was visualized through Transmission Electron Microscopy (TEM). Then the vaccine candidates were examined toward their ability to induce humoral immune repones following two dose immunization in female BALB/c mice. Systemic humoral immunity was evaluated using indirect Enzyme-linked Immunosorbent Assays.
Results: All produced formulations formed nanoparticles with distinct size and polydispersity as confirmed by DLS. TEM micrographs corroborated these findings, visualizing successful integration of conjugate clusters within the adjuvant matrix while preserving the carrier protein's multimeric structure. In BALB/c mice, the MF59, and Emulsion-CPG-adjuvanted formulation elicited a robust antigen-specific IgG titers, with a significant boost after the secondary dose. These antibodies effectively target Na APR-1, a protease critical for hookworm haemoglobin digestion. In addition, we examined antibody responses to KLH carrier itself.
Conclusion: We have demonstrated that P3-based epitope conjugated with KLH potent carrier, demonstrating enhanced structural integrity and immunogenicity when formulated with advanced adjuvant platforms. This precision engineered system offers a scalable and stable strategy for neutralizing Na APR-1 and disrupting the nutritional pathways of N. americanus.
