Passive Immunization with Anti-Ov-TSP-2-LEL IgM mAbs Induced Partial Protection Against O. the control IgM) blocked the uptake of fluke EVs by human bile duct epithelial cells in vitro. This is the first report of passive immunization against human liver fluke contamination, and the findings portend the feasibility of antibody-directed therapies for liver fluke contamination, bolstering the selection of TSPs as components of a subunit vaccine for opisthorchiasis and fluke infections generally. Keywords:passive immunization, monoclonal antibody,Opisthorchis viverrini, tetraspanin == 1. Introduction == The human liver fluke,Opisthorchis viverrini, resides in the bile ducts within the biliary tree, where it establishes chronic contamination that VGX-1027 drives long-term inflammation, hepatobiliary damage, and bile duct obstruction, leading to cholangiocarcinoma (CCA) [1]. Humans become infected by ingesting the infective stage metacercariae from natural or undercooked cyprinoid fish dishes [2,3]. We recently showed that subunit vaccines targeting tetraspanins (TSPs) on the surface of fluke-secreted extracellular vesicles (EVs) confer protection in a hamster challenge model ofO. viverriniinfection, and that the antibodies against the TSPs present around the EV surface block the uptake of fluke EVs by the host bile duct epithelial cells [4,5,6]. TSPs are a family of transmembrane proteins which interact with other transmembrane proteins to form a web that stabilizes membranes [7]. Structurally, the tetraspanin protein includes two extracellular loops, a short extracellular loop (SEL) and a large extracellular loop (LEL), both of which decorate the extracellular surface of the cell membrane. TSPs are abundant on the surface of the tegument of the fluke, and EVs, derived from the tegumental surface, VGX-1027 are actively internalized by adjacent cholangiocytes, the epithelial cells lining the bile duct [8,9]. RecombinantOv-TSP-2 shows promise as a vaccine candidate in that it confers protection to vaccinated hamsters against challenge contamination withO. viverrinimetacercariae when administered in an adjuvanted form VGX-1027 via the parenteral route [4,6]. In addition, oral administration to hamsters of spores of recombinantBacillus subtilis, expressingOv-TSP-2-LEL on the surface of the bacilli, induced serum and bile IgG and IgA responses, and anti-Ov-TSP-2 IgG blocked the uptake ofO. viverriniEVs (Ov-EVs) in a human cholangiocyte cell line. Moreover, vaccination provided up to 56% reductions in both worm and egg burdens after challenge contamination FGF22 [10]. To understand the role of antibodies againstOv-TSP-2 in conferring protection againstO. viverriniinfection, we raised two monoclonal antibodies (mAbs) to recombinantOv-TSP-2-LEL, both of which significantly blocked the uptake ofOv-EVs by human cholangiocytes in vitro, and one of which, after passive transfer to hamsters, conferred highly significant protection against challenge contamination withO. viverrinicompared to a control mAb. == 2. Materials and Methods == == 2.1. Ethics Statement == Vertebrate animal protocols were approved by the Animal Ethics Committee of Khon Kaen University (approval number ACUC-KKU-121/62) according to the Ethics of Animal Experimentation of the National Research Council of Thailand. Monoclonal antibody production was approved by the James Cook University Animal Care and Use Committee (A2629). == 2.2. Preparation of Recombinant Ov-TSP-2-LEL Antigen == The recombinant protein corresponding to the large extracellular loop (LEL) of theOv-TSP-2 (GenBank accessionJQ678707.1) was produced as a fusion protein with thioredoxin (TRX) using the plasmid pET32a+ (Novagen, Madison, WI, USA), which was expressed byEscherichia colistrain BL21DE3. The recombinant fusion protein was purified with a Ni2+affinity column as previously described [8]. The purified protein was dialyzed into PBS before the treatment of mice. == 2.3. Mouse Immunization == Five-week aged male BALB/c mice were immunized a total of 5 occasions with 50 L of recombinantOv-TSP-2-LEL at a concentration of 1 1 mg/mL in an equal volume of Freunds complete (first immunization) or incomplete (second, third, and fourth immunizations) adjuvants administered subcutaneously at two-week intervals. The fifth (final) immunization was undertaken with 50 g of recombinantOv-TSP-2-LEL without adjuvant. Serum samples were collected 2 days prior to the third, fourth, and fifth immunizations for the analysis of antibody titers. Mice were housed in the specified pathogen-free animal facility at James Cook University, Cairns, Queensland, Australia. == 2.4. Hybridoma Generation == Hybridomas were generated.