PANDAA for universal, pan-lineage molecular detection of filoviruses to enable rapid epidemic response.

Ebolavirus [EBOV] and Marburgvirus [MARV] are filoviruses that cause severe hemorrhagic fevers in humans and primates and are listed among the urgently concerning pathogens prioritized in the WHO R&D Blueprint. Genomic sequences of the EBOV and MARV genera differ by >55%, and within each genera the species sequence diversity

threshold is >23% thus detection by gold standard qPCR-based methods has been encumbered by significant

genetic variability between filoviruses. Clinical diagnosis of EBOV and MARV is difficult as the early stages (i.e., pre- hemorrhagic) present with non-specific symptoms associated with a range of febrile illnesses that are displayed by other infectious agents endemic to affected areas e.g., malaria, yellow fever, dengue fever, Crimean-Congo

hemorrhagic (CCHFV) or Lassa fevers. In order to isolate infected individuals, effective control during an EBOV or MARV outbreak can only be achieved by implementing rapid and accurate diagnostics with consistently reliable performance. Aldatu has pioneered the use of PANDAA technology, which enables probe-based qPCR for target

detection in highly variable genomic regions by simultaneously adapting and amplifying diverse templates. PANDAA uniquely mitigates the presence of genetic polymorphisms to allow otherwise divergent templates to be detected by fluorescent probes. As such, PANDAA-enabled qPCR is an ideal solution for universal detection of pathogens with

significant strain, lineage, and/or sub-type sequence diversity. Aldatu is uniquely positioned to deliver a rapid pan- filovirus qPCR-based assay that is rapid, sensitive molecular diagnostic for the detection and differentiation of EBOV and MARV with superior performance compared to existing diagnostics and won’t be affected by genetic changes

in new viral variants. PANDAA has been successfully applied to subtype-independent detection of more than fifteen drug resistance mutation (DRM) targets in HIV. Recently, we developed the first pan-lineage assay for Lassa fever virus (LASV), another WHO priority pathogen with high outbreak potential. We propose to leverage the unique

capabilities of PANDAA to develop a rapid, sensitive molecular diagnostic assay for filovirus detection, and the first with pan-species coverage of EBOV and MARV, through the following specific aims: (1) development of a pan- filovirus PANDAA assay, leveraging proven techniques and proprietary PANDAA reagent design; (2) analytical

validation including confirmation of species inclusivity and high specificity; (3) multiplexing of the PANDAA-Filovirus assay with existing assays for LASV and CCHFV to produce a viral hemorrhagic fever (VHF) panel, including the thermostabilization of the both PANDAA-Filovirus and PANDAA-VHF assays, to meet the requirements of diagnostics

targeted to LMICs; (4) GMP manufacturing of the thermostabilized PANDAA-Filovirus and PANDAA-VHF assays and (5) assay validation using samples representing a broad variety of circulating species and geographies, with multi- site evaluations of the test kits at reference labs at CDC- and WHO-affiliated partner institutions. As the first pan-

filovirus assay, we will provide a rapid, standardized testing option for all regions that can be deployed using pre- existing qPCR equipment in central labs to radically improve the diagnostic workflow and epidemic preparedness.