LAMPoles for Dengue Diagnosis

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 DESCRIPTION (provided by applicant): Dengue is a major public health threat throughout the tropics and subtropics will considerable morbidity and mortality It is the most widespread vector-borne disease after malaria and cannot be prevented by vaccination now Dengue can progress rapidly from benign febrile illness to dengue hemorrhage fever (DHF) and/or dengue shock syndrome (DSS) and death; supportive care is important to limit morbidity and mortality from complicated dengue. Hence, the ability to diagnose dengue early and accurately is important for clinical management of the disease. This proposal is innovative in that links 3 powerful technologies - LAMP, `tadpoles' and Nanobodies (Nbs) - to create a dynamic protein-DNA chimera-based diagnostic for DF/DHF with high specificity and sensitivity compared with existing technologies. We successfully created protein-DNA chimeras, termed `LAMPoles', by fusing DNA oligonucleotide tails based on a sequence of a plant enzyme gene with a previously described protein L/G-DNA chimera whose protein moiety binds most classes of mammalian antibodies to create a protein L/G LAMPole for ultrasensitive detection of essentially any mammalian IgG. For proof-of-concept we show that protein LG-LAMPoles enhanced the sensitivity of ELISA to detect host anti-African trypanosome antibodies in blood several hundred-fold leading to our hypothesis that improved detection of acute dengue is also possible. The discovery of single chain camelid antibody fragments called Nanobodies (Nb) is a major breakthrough in antibody engineering that will enable measurement of DV Ags even in the presence of host Abs. By creating LAMPoles from Nbs targeting both human IgM and DV antigens, we will create an ultrasensitive and specific assay for the diagnosis of acute dengue. Our team has archived serum and blood from patients in Malaysia, Sri Lanka, and Nicaragua with confirmed and excluded dengue. We will generate LAMPoles from camelid nanobodies (Nbs) specific for DV antigens (Ags) and evaluate the sensitivity and specificity of LAMPoles versus state-of-the-art diagnosis of acute dengue (vs. past and no dengue) using archived clinical samples.