Tuesday, May 13, 2025
5/13/2025
Mechanism and Evolution of Filoviral Monoclonal Affinity Reagent Sandwich Assays - DESCRIPTION (provided by applicant): Rapidly detecting viral signatures is important in diagnosing Filovirus disease to hasten quarantine, limit the spread of contagion, and contain an outbreak. Achieving this efficiently and economically in an outbreak setting is not trivial, especially when virus numbers are low early in the course of infection. Using llama single domain antibodies (sdAb) specific for the C-terminus of nucleoprotein (NP), we are able to detect low titers of both Marburg and Ebola viruses though not yet at the levels achievable by qRT-PCR. Each sdAb is able to act as both captor and tracer in an antigen capture assay, relying avidity or Velcro-like effect with polyvalent antigen. Structural analysis of the anti-Marburg sdAb-NP complexes has defined the epitopes and shown that they are conserved among all virus strains documented since the first emergence in 1967. Our broad long term goal is to leverage knowledge of the interaction between sdAb and highly conserved Filoviral NP epitopes, to develop durable immunoassays that not only match nucleic acid based detection sensitivities but will have a high probability of recognizing and categorizing Filoviral strains ye to emerge. We will achieve this with three specific aims: SA1, simplify the assay format as we hypothesize our existing assays can be streamlined without losing sensitivity or specificity from a 4 step to a 3 step capture and novel 1 step solution phase assay. We will benchmark the sensitivity, specificity, reproducibility and durability of our new immunoassays against qRT-PCR; SA2, improve Marburg virus assay sensitivity as we hypothesize our sdAb have un-optimized NP recognition mechanisms that can be fine-tuned to improve limits of detection while retaining Marburg virus strain cross-reactivity. Structure guided design will be used to evolve sdAb paratopes, ensure any altered epitopes are still conserved, and gain of function mutants fed into the streamlined assays and benchmarking of SA1; SA3, modulate Ebola virus assay cross-reactivity as we hypothesize the five Ebola virus NP C-terminal domains have sufficient epitope conservation to enable a pan-reactive sdAb to be engineered yet enough diversity for species specific sdAb to be generated. The structure of a partially cross- reactive sdAb will be solved and the information used to guide the evolution of a broadly cross-reactive sdAb, while subtractive selection of a poly-NP immune library will be used to generate species specific sdAb. Epitopes of the new sdAb will be checked to ensure they are completely conserved among the relevant species and fed into streamlining and benchmarking as in SA1. Pioneering simple, fast and inexpensive sdAb based Filoviral immunoassays that match qRT-PCR sensitivity and specificity, will help safeguard human health by providing a comprehensive diagnostic toolkit for outbreak settings. The innovative approaches and new insights into immunoassay assembly will accelerate diagnostic countermeasures against other infectious disease agents, particularly rapidly evolving emerging negative strand RNA viruses with multiple serotypes.
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