Secreted Glycoprotein Targeting Antibodies and Ultra-Potent Synthetic Nanobodies Yield PCR like Sensitivity for Point-of-Care Assays to Detect Ebolavirus Infection

Introduction: Ebolaviruses are highly infectious and acutely deadly with mortality rates that can surpass 90%. Outbreaks of the disease have increased in size and frequency due to climate change and encroaching on wildlife habitats. While significant developments have been made in vaccines and therapies, lack of a low-cost, sensitive, and specific point-of-care test to diagnose those in the early stages of the infection has plagued outbreak responses and is key to enable containment, contact tracing and efficacious clinical management. To address the need for such a test we developed a set highly sensitive and specific monoclonal antibodies and rationally designed nanobodies that bind non-overlapping epitopes of ebolavirus secreted glycoprotein (sGP), a novel diagnostic target that is present in circulation prior to onset of symptoms and active viremia. Using the antibodies we engineered a novel protein microarray assay, the Ebolavirus D4 assay, that was able to detect the presence of sGP up to 2 days before PCR in two distinct primate models of the infection. Finally, we deployed a combination of all 3 reagents (2 antibodies and 1 nanobody) to engineer a multicolor lateral flow assay that has analytical sensitivity comparable to that of the D4 assay and can perform differential diagnosis (Zaire vs Sudan ebolavirus) and will be now evaluated with samples from ebola infected non-human primates.

Materials and

Methods: In this study, we first developed a new point-of-care test, the D4 assay, a single-step protein microarray to detect and quantify ebolavirus sGP in biological samples. To this end, we first isolated several antibodies that bind to non-overlapping sites of sGP through mouse immunizations and phage display technology. These new binders were then characterized through SPR and integrated into the D4 assay to deliver a final assay with pg/mL sensitivity. The ebolavirus D4 assay was then analytically validated with analyte spiked pooled human serum, human whole blood, and monkey serum. Next, we used this assay to test samples from 16 non-human primates from two cohorts challenged with Makona ebolavirus through intramuscular (10 monkeys) and conjunctival (6 monkeys) over a respectively 6- and 10-day period. Using these antibodies we also developed a Lateral-flow assay that presented a 10 ng/mL limit-of-detection that was able to detect sGP in most PCR positive samples. Next, we rationally engineered synthetic nanobodies isolated through yeast display and affinity matured them with autonomous hypermutation in yeast to target a specific site of Zaire sGP that was complementary to the previously identified antibodies. Deploying all 3 reagents (2 antibodies and 1 nanobody) we engineered a novel Multicolor Lateral Flow Assay that can perform differential diagnosis (Zaire and Sudan) with sub ng/mL sensitivity. This new assay is currently undergoing analytical validation and will be soon tested with samples from ebolavirus challenged non-human primates.

Results, Conclusions, and Discussions: IgGs and integrated into a final D4 assay device. This test was analytically tested with pooled human serum, human whole blood and monkey serum and displayed a limit-of-detection of 0.05 ng/mL. This assay was then used to quantify circulating sGP in non-human primates challenged with Makona ebolavirus through intramuscular and conjunctival routes. sGP was detected prior to viremia and onset of symptoms and rapidly rose to ng/mL within 2-3 days post exposure. This indicated this biomarker could be used to detect infection prior to PCR with the D4 assay and potentially with a lateral flow assay. To build a lateral flow assay for sGP we next isolated several nanobodies with high nM affinities but bound to a site complementary to the previously isolated antibodies. To generate a reagent compatible with in vitro diagnostic applications, we performed affinity maturation on our most promising nanobody which improved its KD by ~10-fold from ~200nM to ~20nM. This nanobodies was fused to a mouse Fc, which enabled simultaneous engagement of 2Nbs per sGP (a homodimer), resulting in a sub pM effective binding affinity. Finally, all 3 reagents were integrated into a new lateral flow assay that employs gold nanoparticles and gold nanoshells to achieve Zaire and Sudan ebolavirus differential diagnosis and sub ng/mL analytical sensitivity in analyte spiked human and monkey samples. Samples from non-human primate models of ebolavirus infection will be tested in the coming months to determine the true performance of this new assay, which we believe can meaningfully impact how this disease is diagnosed and outbreaks are curbed.

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