Enterovirus 71 (EV71) is the major etiological agent contributing to the development of hand-foot-mouth disease (HFMD). There are not any global available vaccines or antibody drugs against EV71 released yet. In this study, we perform the virus immunization in a cost-effective and convenient approach by preparing virus particles from size exclusion and immunization of chicken. Polyclonal yolk-immunoglobulin (IgY) was simply purified from egg yolk and monoclonal single-chain variable fragments (scFv) were selected via phage display technology with two scFv libraries containing 6.0 × 10⁶ and 1.3 × 10⁷ transformants. Specific clones were enriched after 5 rounds of bio-panning and four identical genes were classified after the sequence analysis. Moreover, the higher mutation rates were revealed in the CDR regions, especially in the CDR3. IgY showed specific binding activities to both EV71-infected and Coxsackievirus 16-infected cell lysates and high infectivity inhibitory activity of EV71. However, while IgY detected a 37 kDa protein, the selected scFv seemingly detected higher size proteins which could be cell protein instead of EV71 proteins. Despite the highly effective chicken antibody generation, the purity of virus particles prepared by size exclusion is the limitation of this study, and further characterization should be carried out rigorously.

Background: Keratoconus is a progressive corneal ectasia characterised by extracellular matrix (ECM) loss and an emerging inflammatory component, for which no disease-modifying molecular therapy exists. Exosomes derived from limbal and mesenchymal stem cells are an attractive cell-free therapeutic modality, but the cargo that should be delivered is undefined, and no curated limbal stem-cell (LSC) exosome cargo dataset currently exists. Methods: We reanalysed a public keratoconus corneal RNA-sequencing dataset (GEO: GSE77938; discovery and replication cohorts) with DESeq2, defined a replicated differentially expressed gene (DEG) set, and performed Gene Ontology, KEGG and Reactome enrichment. A high-confidence protein-protein interaction (PPI) network (STRING) identified hub genes. We integrated keratoconus disease-gene evidence (Open Targets Platform) and documented extracellular-vesicle cargo (ExoCarta, Vesiclepedia) and computed a transparent Cargo Prioritization Score (CPS) to nominate candidate LSC-exosome therapeutic cargo. Results: 1677 DEGs were detected in discovery (152 up, 1525 down) and 1380 were replicated. Enrichment was dominated by extracellular matrix organization; adaptive immune response; mononuclear cell differentiation. Network analysis nominated ECM and immune hub genes. The CPS prioritised COL1A1, FN1, COL4A1, COL3A1, COL5A1, MMP1 as leading restoration-cargo candidates, all documented as EV cargo and present in the mesenchymal stem-cell EV reference proteome. Conclusions: This fully reproducible, real-data framework provides a ranked, evidence-traceable shortlist of candidate LSC-exosome cargo for keratoconus and an explicit account of current data gaps to guide experimental validation.

Background: Keratoconus is a progressive corneal ectasia characterised by extracellular matrix (ECM) loss and an emerging inflammatory component, for which no disease-modifying molecular therapy exists. Exosomes derived from limbal and mesenchymal stem cells are an attractive cell-free therapeutic modality, but the cargo that should be delivered is undefined, and no curated limbal stem-cell (LSC) exosome cargo dataset currently exists. Methods: We reanalysed a public keratoconus corneal RNA-sequencing dataset (GEO: GSE77938; discovery and replication cohorts) with DESeq2, defined a replicated differentially expressed gene (DEG) set, and performed Gene Ontology, KEGG and Reactome enrichment. A high-confidence protein–protein interaction (PPI) network (STRING) identified hub genes. We integrated keratoconus disease-gene evidence (Open Targets Platform) and documented extracellular-vesicle cargo (ExoCarta, Vesiclepedia) and computed a transparent Cargo Prioritization Score (CPS) to nominate candidate LSC-exosome therapeutic cargo. Results: A total of 1677 DEGs were detected in discovery (152 up, 1525 down) and 1380 were replicated. Enrichment was dominated by extracellular matrix organisation; adaptive immune response; and mononuclear cell differentiation. Network analysis nominated ECM and immune hub genes. The CPS prioritised COL1A1, FN1, COL4A1, COL3A1, COL5A1, MMP1 as leading restoration-cargo candidates, all documented as EV cargo and present in the mesenchymal stem-cell EV reference proteome. Conclusions: This fully reproducible, real-data framework provides a ranked, evidence-traceable shortlist of candidate LSC-exosome cargo for keratoconus and an explicit account of current data gaps to guide experimental validation.