Removing negative impacts from inevitable non-reproducible and non-specific antibody-probe interactions in viral serology (preprint)

Serological assays are indispensable tools in public health. Presently deployed serological assays, however, largely overlook research progress made in the last two decades that jeopardize the conceptual foundation of these assays, i.e., antibody (Ab) specificity. Challenges to traditional understanding of Ab specificity include Ab poly-specificity, and most recently non-reproducible Ab-probe interactions (NRIs). Here, using SARS-CoV-2 and 4 common livestock viruses as a test bed, we developed a new serological platform that integrates recent understanding about Ab specificity. We first demonstrate that the response rate (RR) from a large sized serum pool (~100) is not affected by NRIs or by non-specific Ab-probe interactions (NSIs), so RR can be incorporated into the diagnostic probe selection process. We subsequently used multiple probes (configured as a “protein peptide hybrid microarray”, PPHM) to generate a digital microarray index (DMI), and finally demonstrate that DMI-based analysis yields an extremely robust probabilistic trend that enables accurate diagnosis of viral infection that overcomes multiple negative impacts exerted by NSI/NRI. Thus, our study with SARS-CoV-2 confirms that the PPHM-RR-DMI platform enables very rapid development of serological assays that outperform traditional assays (for both sensitivity and specificity) and supports that the platform is extendable to other viruses.

Previously unrecognized non-reproducible antibody-antigen interactions and their implications for diagnosis of viral infections including COVID-19 (preprint)

Antibody-antigen (Ab-Ag) interactions are canonically described by a model which exclusively accommodates non-interaction (0) or reproducible-interaction (RI) states, yet this model is inadequate to explain often-encountered non-reproducible signals. Here, by monitoring diverse experimental systems and confirmed COVID-19 clinical sera using a peptide microarray, we observed that non-specific interactions (NSI) comprise a substantial proportion of non-reproducible antibody-based results. This enabled our discovery and capacity to reliably identify non-reproducible Ab-Ag interactions (NRI), as well as our development of a powerful explanatory model ("0-RI-NRI-Hook four-state model") that is [mAb]-dependent, regardless of specificity, which ultimately shows that both NSI and NRI are not predictable yet certain-to-happen. In experiments using seven FDA-approved mAb drugs, we demonstrated the use of NSI counts in predicting epitope type. Beyond challenging the centrality of Ab-Ag interaction specificity data in serology and immunology, our discoveries also facilitated the rapid development of a serological test with uniquely informative COVID-19 diagnosis performance.

A diagnostic decision-making protocol combines a new-generation of serological assay and PCR to fully resolve ambiguity in COVID-19 diagnosis (preprint)

The capacity to accurately diagnosis COVID-19 is essential for effective public health measures to manage the ongoing global pandemic, yet no presently available diagnostic technologies or clinical protocols can achieve full positive predictive value (PPV) and negative predictive value (NPV) performance. Two factors prevent accurate diagnosis: the failure of sampling methods (e.g., 40% false negatives from PCR testing of nasopharyngeal swabs) and sampling-time-dependent failures reflecting individual humoral responses of patients (e.g., serological testing outside of the sero-positive stage). Here, we report development of a diagnostic protocol that achieves full PPV and NPV based on a cohort of 500 confirmed COVID-19 cases, and present several discoveries about the sero-conversion dynamics throughout the disease course of COVID-19. The fundamental enabling technology for our study and diagnostic protocol-termed SANE, for Symptom (dpo)-Antibody-Nucleic acid-Epidemiological history-is our development of a peptide-protein hybrid microarray (PPHM) for COVID-19. The peptides comprising PPHMCOVID-19 were selected based on clinical sample data, and give our technology the unique capacity to monitor a patient's humoral response throughout the disease course. Among other assay-development related and clinically relevant findings, our use of PPHMCOVID-19 revealed that 5% of COVID-19 patients are from an "early sero-reversion" subpopulation, thus explaining many of the mis-diagnoses we found in our comparative testing using PCR, CLIA, and PPHMCOVID-19. Accordingly, the full SANE protocol incorporates orthogonal technologies to account for these patient variations, and successfully overcomes both the sampling method and sampling time limitations that have previously prevented doctors from achieving unambiguous, accurate diagnosis of COVID-19

AT-527 is a potent in vitro replication inhibitor of SARS-CoV-2, the virus responsible for the COVID-19 pandemic (preprint)

AT-527, an orally administered double prodrug of a guanosine nucleotide analog, has been shown previously to be highly efficacious and well tolerated in HCV-infected subjects. Herein we report the potent in vitro activity of AT-511, the free base form of AT-527, against several coronaviruses, including SARS-CoV-2, the causative agent of COVID-19. In normal human airway epithelial (HAE) cell preparations, the average concentration of AT-511 required to inhibit replication of SARS-CoV-2 by 90% (EC90) was 0.5 {micro}M, very similar to the EC90 for AT-511 against HCoV-229E, HCoV-OC43 and SARS-CoV in Huh-7 cells. No cytotoxicity was observed for AT-511 in any of the antiviral assays up to the highest concentration tested (100 {micro}M). Surprisingly, AT-511 was 30-fold less active against MERS-CoV. This differential activity may provide a clue to the apparent unique mechanism of action of the guanosine triphosphate analog formed from AT-527.

Effectiveness of Convalescent Plasma for Treatment of COVID-19 Patients (preprint)

Background and objective: The outbreak of COVID-19 has become a global health concern. In this study, we evaluate the effectiveness and safety of convalescent plasma therapy in patients with severe and critically ill COVID-19. Methods: Sixteen COVID-19 patients received transfusion of anti-COVID-19 antibody-positive convalescent plasma. The main outcome was time for viral nucleic acid amplification (NAA) test turning negative. Clinical laboratory parameters were measured at the baseline (d0) before plasma transfusion, and day 1 (d1), day 3 (d3) after transfusion as well. Results: Among the 16 patients, 10 of them had a consistently positive result of viral NAA test before convalescent plasma transfusion. Eight patients (8/10) became negative from day 2 to day 8 after transfusion. Severe patients showed a shorter time for NAA test turning negative after transfusion (mean rank 2.17 vs 5.90, P = 0.036). Two critically ill patients transfused plasma with lower antibody level remained a positive result of NAA test. CRP level demonstrated a decline 1 day after convalescent plasma treatment, compared with the baseline (P = 0.017). No adverse events were observed during convalescent plasma transfusion. Conclusions: Viral NAA test of most patients with COVID-19 who received convalescent plasma transfusion turned negative on the 2nd to 8th days after transfusion, and the negative time of severe patients was shorter than that of critically ill patients.