Project Summary Liver fluke infection with Opisthorchis viverrini remains problematic in East Asia and is endemic in Thailand and Laos, where ~10 million people are infected. The public health implications of this situation are substantial since there is no stronger link between a human malignancy and a eukaryotic pathogen than that between cholangiocarcinoma (CCA, bile duct cancer) and infection with O. viverrini. Northeast Thailand reports the highest incidence of CCA worldwide, with the 2014 CCA age standardized incidence rate (ASR) of 85 per 100,000, which equates to 26,000 CCA-related deaths annually. The contrast with countries without liver flukes is stark given that incidence of CCA is less than 3 per 100,000 elsewhere (USA, 1.67 ASR, 2014). To survive in the biliary tract, the liver fluke actively releases excretory/secretory (ES) proteins and extracellular vesicles (EVs) that facilitate fluke feeding and manipulation of the host immune response, and ultimately modify cellular homeostasis that contributes to malignant transformation. Our hypothesis is that targeting proteins that are essential for several discrete facets of parasitism and/or are known carcinogens via a multi-valent vaccine will deliver a novel anti-infection/ anti-cancer therapy. We will address this hypothesis through the following aims: Aim 1. Explore the importance of key fluke ES proteins in host-parasite communication and leverage the findings to prioritize their selection as vaccine antigens. Aim 2. Determine whether mRNA and/or protein subunit vaccines induce antibodies that reduce both fluke burdens and CCA incidence in the hamster infection-cancer model. This proposal targets liver fluke ES proteins which drive the phenotypic hallmarks of cancer in the biliary epithelium: 1) the granulin-like growth factor, Ov-GRN-1, known to induce rampant cholangiocyte proliferation; 2) the tetraspanin, Ov-TSP-2, a key element of EVs and host-cell communication; 3) the Ov-catF cysteine protease, a key component of the protein digestion cascade; and 4) the Ov-M60mucinase which degrades host defensive mucus in the bile ducts. The conceptual innovation we utilize is a rodent model of human carcinogenesis where liver fluke infection is a known risk factor. Technical innovations include gene knockout in the liver fluke, targeting key pathogenic and nutritional processes, and combining the findings to develop vaccination against fluke infection and the infection-induced cancer. These studies will determine whether fluke proteins that communicate at the host-parasite interface represent the Achilles’ heel of this carcinogenic parasite. Targeting fluke-host communication in combination with nutrient acquisition pathways may ultimately combat liver fluke-induced bile duct cancer in the form of an anti-fluke, and indeed anti-cancer vaccine, a public health development with the potential to benefit millions of (often impoverished) residents of endemic regions in East Asia.