(H-299) Ultra-sensitive point-of-care immunoassay for secreted glycoprotein detects Ebola infection earlier than PCR in multiple non-human primate models of the disease.

Introduction::
Ebola virus (EBOV) outbreaks are a cause for grave concern due to disease severity and their increased frequency. Recent development of vaccines and therapies have improved disease outcome, but widespread diagnostics capabilities to detect those infected in early stages of the disease where treatment is most successful hasn’t significantly improved over the years. To address the need for a rapid, low-cost, and sensitive point-of-care test to detect EBOV infected patients, we developed the EBOV D4 assay, which targets the viral secreted glycoprotein (sGP), a novel diagnostic biomarker detectable in circulation prior to viremia and onset of symptoms. The EBOV D4 assay employs high affinity antibodies that target non-overlapping sites on sGP. All Ab reagents are dispensed onto a “non-fouling”—protein and cell-resistant—polymer brush on glass that eliminates virtually all non-specific adsorption, and thereby provides an extremely low —femtomolar— limit-of-detection (LoD). Therefore, the EBOV D4 assay is over 1000-fold more sensitive than FDA approved rapid detection tests and in two non-human primate models of the disease –intramuscular and conjunctival challenges– was more sensitive than PCR.




Materials and Methods::
In this study we developed a new point-of-care test to diagnose EBOV by detecting sGP in circulation. To this end, we first generated sGP targeting high-affinity monoclonal antibodies from phage immune libraries. Next, a panel of antibody candidates were characterized with SPR and screened with the D4 assay to identify the best pairs compatible with the assay platform. Next, we optimized the assay fabrication parameters and evaluated its performance with analyte spiked fetal bovine serum, pooled human serum, pooled human blood and rhesus monkey serum. The analytical performance of the test was evaluated with a high sensitivity tabletop and a handheld, in-house developed fluorescence scanner. Finally, we employed samples from a total of 16 non-human primates from two cohorts challenged with Makona EBOV through intramuscular (IM) and conjunctival (CNJ) routes that were collected to cover a 6 (IM) and 10 (CNJ) days period.




Results, Conclusions, and Discussions::
The generation of EBOV D4 assay first required the development of high-affinity antibodies that target non-overlapping regions of sGP. Antibody phage display yielded several candidates to be further explored which were characterized by surface plasmon resonance. All antibodies had binding affinity (K_D) in the low nM range for EBOV sGP, except A1F3-1, which presented K_D~ 200pM. Next, we employed the D4 to identify the best capture and detection antibody pairs that target sGP. Interestingly, A1F3-1, which presented the best KD, was identified as the best capture antibody and when paired with C2bA5-2, had a limit-of-detection as low as 0.05 ng/mL of sGP in multiple sample matrixes. When testing non-human primates with the EBOV D4 assay we were able to detect EBOV infection at least one day before PCR in both IM and CNJ NHP models. Interesting, IM models present a faster progression with high sGP levels as soon as 3 days after challenge. CNJ models had high sGP levels only after the 5th day of infection and better simulate the natural progression of the disease. These results indicate that circulating sGP can reliably predict the progression of the disease and that immunoassays targeting sGP can outperform RNA-targeting PCR.




Acknowledgements (Optional): :

References (Optional): : Cassio M. Fontes, et al., Ultrasensitive point-of-care immunoassay for secreted glycoprotein detects Ebola infection earlier than PCR.Sci. Transl. Med.13,eabd9696(2021).DOI:10.1126/scitranslmed.abd9696


Cross RW, Prasad AN, Woolsey CB, Agans KN, Borisevich V, Dobias NS, Comer JE, Deer DJ, Geisbert JB, Rasmussen AL, Lipkin WI, Fenton KA, Geisbert TW. Natural history of nonhuman primates after conjunctival exposure to Ebola virus. Sci Rep. 2023 Mar 13;13(1):4175. doi: 10.1038/s41598-023-31027-7. PMID: 36914721; PMCID: PMC10011569.