Introduction
Schistosomiasis, a debilitating parasitic disease affecting millions globally, necessitates effective diagnostic tools for sustained control and eventual eradication. While significant progress has been made in schistosomiasis control through mass drug administration of praziquantel (PZQ), reinfection and the potential for drug resistance remain critical challenges. Furthermore, as disease prevalence decreases in endemic regions, highly sensitive and specific diagnostic tests become paramount for accurate case detection and monitoring of control program efficacy. Current diagnostic methods, such as the Kato-Katz stool smear, often lack the sensitivity required to detect low-intensity infections, particularly crucial in elimination phases. Antibody-based and antigen-based diagnostics offer promising alternatives, yet affordability, ease of use, and point-of-care applicability are essential for widespread implementation in resource-limited settings. This study addresses the urgent need for improved diagnostics by exploring the generation of novel antibody fragments derived from the natural water buffalo host of Schistosoma japonicum, for development into a cost-effective point-of-care diagnostic tool.
Methodology: Harnessing Buffalo Immunity to Generate scFv Antibodies
This research leverages the natural immune response of water buffalo (Bubalus bubalis) to Schistosoma japonicum infection to generate a novel source of diagnostic reagents. Water buffalo are significant reservoirs for S. japonicum, making their immune response particularly relevant to parasite antigens. The study employed phage display technology to create single-chain antibody Fv domain (scFv) libraries from the portal lymph nodes of experimentally infected buffalo. Lymph nodes draining the liver, a primary site of schistosome infection, were harvested 11-12 days post-infection, a time point known to exhibit a robust immune response to developing adult worms.
RNA extracted from these lymph nodes was used to amplify the variable heavy (VH) and variable light (VL) chain genes of buffalo antibodies. These genes were then assembled into scFv fragments and cloned into a phage display vector. This vector displays the scFv antibody fragments on the surface of bacteriophages, creating a library of diverse antibody specificities. This library was then subjected to a process called “panning,” where phages displaying scFvs that bind to S. japonicum antigens are selectively enriched.
Selection and Enrichment of Schistosome-Specific scFv-Phages
The scFv-phage library was panned against two target antigens: whole, formaldehyde-fixed adult Schistosoma japonicum worms and excretory-secretory (ES) products released by adult worms. Panning against adult worms aimed to select antibodies binding to surface antigens, while panning against ES products targeted secreted antigens potentially circulating in the host’s bloodstream. Multiple rounds of panning were performed to enrich for phages displaying scFvs with high affinity for the target antigens. The enrichment process was monitored by comparing phage output titers after each round, demonstrating significant enrichment for both adult worm and ES product-specific scFvs.
Figure 1: Fluorescent Microscopy of scFv-Phage Binding to Adult Schistosomes. CFSE-labeled scFv-phages (Bp-pre, Bp-R3-A, Bp-R3-ES) show enhanced binding to adult Schistosoma japonicum compared to control phage (M13K07), indicating successful selection of parasite-specific antibody fragments.
Results: scFv-Phages Exhibit Specific Binding to Adult Worms and ES Products
The selected scFv-phage libraries (Bp-R3-A and Bp-R3-ES) demonstrated enhanced binding to adult schistosomes and ES products, respectively. Fluorescence microscopy using carboxyfluorescein succinimidyl ester (CFSE) labeled phages visually confirmed increased binding of Bp-R3-A and Bp-R3-ES phages to adult worms compared to the unselected library (Bp-pre) and control phage. Confocal microscopy further revealed that scFv-phage binding was primarily localized to the surface of the adult parasites.
Enzyme-linked immunosorbent assays (ELISAs) quantitatively confirmed the specificity of the enriched libraries. The Bp-R3-ES library exhibited significantly higher binding to ES products compared to other phage libraries, while the Bp-R3-A library, selected against adult worms, also showed increased binding to ES antigens, suggesting shared or cross-reactive epitopes between adult worm surfaces and ES products.
Figure 2: ELISA Quantification of scFv-Phage Binding to Schistosome Extracts. Bp-R3-AES phage library demonstrates significant binding to soluble whole adult preparations (SWAP) of Schistosoma japonicum, indicating recognition of adult worm antigens.
Protein Microarray Screening Identifies Target Antigens
To identify specific schistosome antigens recognized by the selected scFv-phages, a protein microarray containing 232 S. japonicum proteins was screened. Both Bp-R3-A and Bp-R3-ES libraries recognized ten antigens, with overlapping and distinct antigen recognition profiles. Notably, the Bp-R3-ES library exhibited stronger binding to all identified antigens, further supporting the hypothesis that these antigens are likely secreted or excreted by the parasite and accessible to the host immune system. Among the recognized antigens were known schistosome proteins like tropomyosin and Sjp40, as well as several hypothetical proteins, providing potential targets for diagnostic development.
Table 1: Schistosome Protein Microarray Antigen Recognition by Bp-R3 scFv-Phages. The table highlights antigens recognized by the scFv-phage libraries, indicating potential diagnostic targets. (Note: As a text-based response, the actual table is not rendered here, but would be present in the markdown output).
Soluble scFv Clones Recognize Adult Worm Antigens in Mouse Sera
To further refine the selection and identify scFvs suitable for diagnostic applications in serum, the Bp-R3-AES library (a pool of Bp-R3-A and Bp-R3-ES) was panned against sera from S. japonicum-infected mice. This additional panning step aimed to select for scFvs that bind antigens present in the serum of infected hosts, while removing scFvs binding to non-specific serum components. From this serum-panned library (Bp-R3-PIMS), five soluble scFv clones (Bp-scFv-1 to Bp-scFv-5) were generated as alkaline phosphatase (AP) fusion proteins for ease of detection and characterization.
ELISA analysis of these soluble scFv clones demonstrated that all five clones exhibited significantly higher binding to soluble whole adult preparations (SWAP) compared to an irrelevant control protein (ovalbumin). Importantly, two clones (Bp-scFv-1 and Bp-scFv-2) were able to selectively recognize SWAP antigens even when spiked into naïve mouse sera, indicating their potential to detect parasite antigens in a complex biological matrix like blood.
Figure 3: ELISA Binding of Soluble scFv Clones to SWAP Antigens. Bp-scFv-1 to Bp-scFv-5 show significant binding to Schistosoma japonicum SWAP, with Bp-scFv-1 and Bp-scFv-2 demonstrating preferential recognition even in the presence of mouse sera.
Discussion: Towards a Point-of-Care Diagnostic Tool
The generation of these novel buffalo-derived scFv antibody fragments represents a significant step towards developing an improved point-of-care diagnostic test for Schistosoma japonicum infection. The scFvs exhibit several key advantages for diagnostic applications:
- Specificity: Selected scFvs demonstrate specific binding to adult schistosome antigens and ES products, minimizing cross-reactivity with host proteins.
- Sensitivity: Enrichment through phage display and serum panning suggests the ability to detect relevant parasite antigens, even those circulating in low concentrations in serum.
- Point-of-Care Potential: scFv fragments are small, stable, and can be produced cost-effectively in bacteria, making them suitable for incorporation into rapid, point-of-care diagnostic assays.
- Buffalo-Derived Advantage: Utilizing the natural host immune response may lead to the identification of antigens particularly relevant for diagnosis in both human and animal reservoirs of S. japonicum.
The study’s findings highlight the potential of phage display technology and the unique immune repertoire of water buffalo for generating diagnostic reagents against schistosomiasis. The identified soluble scFv clones, particularly Bp-scFv-1 and Bp-scFv-2, warrant further development and validation. Future research will focus on:
- Optimization and Purification: Improving the production and purification of the lead scFv clones for robust diagnostic assay development.
- Assay Development: Incorporating the scFvs into a rapid diagnostic format, such as a lateral flow assay, suitable for point-of-care use.
- Clinical Validation: Evaluating the diagnostic performance of scFv-based assays in detecting S. japonicum infection in both human and water buffalo populations in endemic settings.
Conclusion
This research successfully generated and characterized novel scFv antibody fragments from water buffalo with specific binding to Schistosoma japonicum adult worm antigens and excretory-secretory products. These antibody fragments hold significant promise for the development of a cost-effective and sensitive point-of-care diagnostic tool for schistosomiasis japonica, contributing to improved disease surveillance and control efforts in endemic regions. The buffalo-derived scFvs offer a unique avenue for advancing schistosomiasis diagnostics and addressing the critical need for accurate and accessible tools in the fight against this neglected tropical disease.
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