Performance of Keyhole Limpet Hemocyanin (KLH) as an Antigen Carrier for Protein Antigens Depends on KLH Property and Conjugation Route

Abstract KLH naturally exists either as high molecular weight (HMW) cylinders composed of 20 copies of subunits, or as separate subunits (Subunit). The molecular weight of KLH Subunit is about 400kDa. Owing mainly to its large size, and the abundant and complex carbohydrate structures on its peptide chains, KLH has been widely used for decades as an immune stimulant, antigen carrier, and vaccine component. But, how to characterize the properties of KLH as an antigen carrier through functional assays remains to be determined. At Stellar, we conjugated KLH of different formats and sources with ovalbumin (OVA) and fluorescence tag-conjugated OVA through different conjugation routes. Dendritic cells (DCs) derived from mouse bone marrow mononuclear cells and mouse DC line JAWSII were pulsed with these conjugates. By utilizing DC uptake assays and measuring the fluorescence intensity of DC lysate supernatant, the amount of KLH- OVA-fluorescence conjugates uptaken by DCs was quantitatively determined. With flow cytometry, both the DC maturation markers, and the amount of ovalbumin epitopes that are presented by MHC II molecules of DCs, were measured. To further compare the property of KLH-protein conjugates as potential vaccines, we vaccinated mice with OVA. OVA-specific memory T cells extracted from the vaccinated mice were stimulated with DCs that had been pulsed with KLH-OVA conjugates. T cell proliferation and cytokine release from these stimulated T cells were quantitatively measured and compared. To summarize, with the functional assays we designed, we found that the performance of KLH as a carrier for protein antigens depends on KLH property and conjugation route.