key: cord-327862-zcg3baym
authors: Luo, Yiqi Ruben; Chakraborty, Indrani; Yun, Cassandra; Wu, Alan H B; Lynch, Kara L
title: Kinetics of SARS-CoV-2 Antibody Avidity Maturation and Association with Disease Severity
date: 2020-09-14
journal: Clin Infect Dis
DOI: 10.1093/cid/ciaa1389
sha: 
doc_id: 327862
cord_uid: zcg3baym

The kinetics of IgG avidity maturation during SARS-CoV-2 infection was studied. The IgG avidity assay used a novel label-free immunoassay technology. It was found that there was a strong correlation between IgG avidity and days since symptom onset, and peak readings were significantly higher in severe than mild disease cases.

A c c e p t e d M a n u s c r i p t 3 

In the race to identify correlates of COVID-19 immunity, the focus has been on characterizing the production of SARS-CoV-2 antibodies. Most immunocompetent individuals with symptomatic infections develop detectable SARS-CoV-2 antibodies within 2 weeks of symptom onset. 1 The SARS-CoV-2 IgG antibody response is more robust in severe cases of COVID-19 at all time-points after seroconversion. 1 Preliminary findings suggest that the magnitude of the antibody response correlates with virus neutralizing power, but some hypothesize that the antibodies could promote disease progression. 2 Influential factors in the distinction between protective antibodies and those that promote pathology include antibody isotype, concentration, specificity, post-translational modifications, and avidity for the virus.

Most SARS-CoV-2 antibody studies to date have focused on the quantity of specific isotypes rather than the quality of the antibodies. Antibody avidity, or functional affinity, is a measure of the maturation of the humoral immune response after both infection and vaccination and typically increases over time. For some viruses, the measurement of antibody avidity is used to differentiate acute infection from past exposure or vaccination (e.g. toxoplasmosis, SARS-CoV infection) and to detect re-infection in the presence of prolonged virus-shedding. 3, 4 Here we report the development of a method to characterize SARS-CoV-2 IgG avidity maturation in COVID-19 patients from initial diagnosis through convalescence.

The IgG avidity assay was established on a novel label-free immunoassay platform Gator Analyzer (Gator Bio, Palo Alto, CA) to measure SARS-CoV-2 IgG avidity to the virus spike protein receptor-binding domain (RBD). The platform utilizes thin-film interferometry and can measure the entire time course of formation of an immune complex on a sensing probe without attaching a reporter. 5 The sensing probe in use was pre-coated with recombinant RBD (His-A c c e p t e d M a n u s c r i p t 4 tagged, Sino Biological, Wayne, PA). Serum samples were diluted 10-fold in running buffer (PBS with 0.02% Tween 20, 0.2% BSA, and 0.05% NaN 3 ) for analysis. The steps of the IgG avidity assay included 1) dipping the sensing probe in running buffer for a baseline measurement, 2) formation of RBD-IgG immune complex on the sensing probe, 3) dissociation of loosely bound IgG using either running buffer or 3 M urea in running buffer, and 4) formation of RBD-IgG-Anti-IgG immune complex using 10 μg/ml anti-IgG in running buffer (goat antihuman IgG antibody, Jackson Immunoresearch, West Grove, PA). The sensing probe was washed in running buffer for 1 min between the steps, and regenerated in glycine pH 2.0. The signal increase in the final step, which is proportional to the quantity of RBD-IgG-Anti-IgG immune complex on the sensing probe, was measured. As other isotypes of antibodies might bind to RBD in the second step, the measurement of the RBD-IgG-Anti-IgG immune complex enhanced the assay specificity. The IgG avidity index was calculated as the ratio of the readout with the dissociation agent (urea) to the reference (running buffer), presented as a percentage.

The performance of the IgG avidity assay was verified for precision and specificity. Precision was evaluated using a spiked serum sample prepared by adding a standard anti-RBD IgG (Absolute Antibody, Oxford, UK) in a negative serum sample. The spiked serum sample was measured simultaneously by a row of 7 sensing probes, and the assay was repeated 4 times with regeneration of the sensing probes. The CV among the sensing probes was 4.5%, and the CV among regeneration cycles was 10.4%. The spiked serum sample was also measured in 4 consecutive days and the CV was 13.7%. Specificity was evaluated using a set of 28 serum samples from individuals tested RT-PCR negative for SARS-CoV-2 and positive for other respiratory viruses (4 coronavirus HKV1, 1 coronavirus 229E, 2 coronavirus OC43, 11 human rhinovirus/enterovirus, 4 human metapneumovirus, 3 respiratory syncytial virus, 2 parainfluenza A c c e p t e d M a n u s c r i p t 5 type 1 virus, 1 adenovirus). No formation of RBD-IgG-Anti-IgG immune complex was observed for these samples, providing negative results in the IgG avidity assay.

The study was approved by the Institutional Review Board of the University of California. All testing was performed on remnant serum samples collected for routine clinical testing and stored at -20°C prior to testing. Individual and serial serum samples (n = 168) from patients with PCR-confirmed COVID-19 (n = 90, 51% male, median age 49, 24% admitted to the ICU) were analyzed. Based on least squares linear regression analysis, there was a strong correlation between IgG avidity and days since symptom onset (P < 0.0001) ( Figure 1A ). SARS-CoV-2 IgG avidity did not correlate with IgG concentration, as measured previously using a quantitative immunoassay (1) . The kinetics of IgG avidity maturation in 13 patients for whom serial samples (≥ 3) were available is shown in Figure 1B . In all patients, IgG avidity increased over time. Peak measurements, which included the last measurement per 7-day interval per patient, were used to compare IgG avidity by disease severity. Peak readings were significantly higher for specimens from ICU than non-ICU patients for the first month after symptom onset (1-4 weeks) and thereafter, using the t-statistic (P < 0.0001) ( Figure 1C ). 

AW reports scientific advisory board fees from ET Healthcare, outside the submitted work. No conflict for any other authors.

A c c e p t e d M a n u s c r i p t 7

Magnitude and kinetics of anti-SARS-CoV-2 antibody responses and their relationship to disease severity

Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study

Clinical utility of avidity assays

Use of antibody avidity assays for diagnosis of severe acute respiratory syndrome coronavirus infection

Development of label-free immunoassays as novel solutions for the measurement of monoclonal antibody drugs and antidrug antibodies

Antibody Avidity Maturation during Severe Acute Respiratory Syndrome-Associated Coronavirus Infection