Detection of neutralizing antibodies against multiple SARS-CoV-2 strains in dried blood spots using cell-free PCR

Abstract

An easily implementable serological assay to accurately detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies is urgently needed to better track herd immunity, vaccine efficacy and vaccination rates. Herein, we report the Split-Oligonucleotide Neighboring Inhibition Assay (SONIA) which uses real-time qPCR to measure the ability of neutralizing antibodies to block binding between DNA-barcoded viral spike protein subunit 1 and the human angiotensin-converting enzyme 2 receptor protein. The SONIA neutralizing antibody assay using finger-prick dried blood spots displays 91-97% sensitivity and 100% specificity in comparison to the live-virus neutralization assays using matched serum specimens for multiple SARS-CoV-2 variants-of-concern. The multiplex version of this neutralizing antibody assay, using easily collectable finger-prick dried blood spots, can be a valuable tool to help reveal the impact of age, pre-existing health conditions, waning immunity, different vaccination schemes and the emergence of new variants-of-concern.

Fig. 1. Principle of SONIA neutralization PCR test.

a Viral entry of SARS-CoV-2 is mediated by the binding of the spike protein to the human receptor angiotensin-converting enzyme 2 (ACE2). Disruption of this interaction forms the basis of neutralization by antibodies (NAb). b SONIA Neutralization PCR test reconstructs this interaction using a combination of S1 subunits of the spike protein- and ACE2-DNA conjugates. In the absence of NAb, S1 and ACE2 engage with strong affinity, thereby positioning the two DNA barcodes in proximity for subsequent ligation and PCR-amplification. On the other hand, binding of NAb blocks S1 subunit from binding ACE2, leaving the two DNA barcodes separated. Since each barcode has only one PCR primer binding site, they cannot be separately amplified. Therefore, the quantities of NAb are correlated with the decrease of PCR amplicon formation.

Fig. 2. Selection of antigens for the SONIA neutralization PCR test.

Both S1 subunit and receptor binding domain (RBD) of spike protein have the ability to engage with the ACE2. To evaluate suitability for the neutralization PCR assay, we tested convalescent COVID-19 patient sera (N = 2, blue circle) and healthy donor sera (N = 4, red circle) collected prior to the outbreak. The COVID-19 sera were confirmed to harbor NAb by a pseudo-virus neutralization assay. The y-axis is the SONIA neutralization PCR test signal Δ$\overline{Ct}$ calculated by subtracting the Ct value of the sample from that of a buffer only blank control.

Fig. 3. NAb reactivity in convalescent COVID-19 and control sera with non-COVID-19 infection by the SONIA neutralization PCR test.

Convalescent COVID-19 sera (N = 144) were from patients 14 days after symptom onset (blue circle), and control sera (N = 43) from patients with HCV, flavivirus infection and Lyme disease (pink square). The y-axis is the SONIA neutralization PCR test signal Δ$\overline{Ct}$ calculated by subtracting the Ct value of the sample from that of a buffer only blank control.

Fig. 4. Correlation between the SONIA neutralization test and VNT titers.

The SONIA neutralization PCR test signals were averaged for samples with the same PRNT titers and plotted on the y-axis. Considering the PCR signals were logarithmic in nature, the VNT titers were log-transformed and plotted on the x-axis. The VNT was performed by the Mayo Clinic. The y-axis is the SONIA neutralization PCR test signal Δ$\overline{Ct}$ calculated by subtracting the Ct value of the sample from that of a buffer only blank control.

Fig. 5. Evaluation of kinetics of NAb development by the SONIA neutralization PCR test.

Sera from COVID-19 patients after different days post symptom onset were tested by the neutralization PCR assay. All serum samples (N = 28) were from unique individuals. The y-axis is the SONIA neutralization PCR test signal Δ$\overline{Ct}$ calculated by subtracting the Ct value of the sample from that of a buffer only blank control. The boxplots show medians (middle line) and 75th and 25th quartiles (upper and lower boxes), while the whiskers show the minimum and maximum of the data.

Fig. 6. Analysis of neutralizing antibodies in at-home collected dried blood spot specimens.

Dried blood spot specimens from convalescent COVID-19 patients (n = 27, green circle), vaccinated (n = 9, purple diamond) and controls (n = 20, pink square) were analyzed by the SONIA neutralization PCR test for NAb. The y-axis is the SONIA neutralization PCR test signal Δ$\overline{Ct}$ calculated by subtracting the Ct value of the sample from that of a buffer only blank control. The same samples were also analyzed for total spike (S1) antibodies with a previously reported method and plotted in the x-axis. The x-axis is the ADAP assay signals ΔCt calculated by subtracting the buffer only blank control Ct from the Ct value of the sample.

Fig. 7. Multiplex SONIA neutralization test for analysis of NAb against variant of concern (VOC).

a Comparison of multiplex SONIA neutralization test for wild type against live wild type virus neutralizing assay (COVID-19 samples in purple diamond and controls in red square). b Comparison of multiplex SONIA neutralization test for delta against live delta virus neutralizing assay (COVID-19 samples in purple diamond and controls in red square). The vertical and horizontal dot lines are the cutoff of positivity for the live virus assay and the multiplex SONIA neutralization assay. The y-axis is the SONIA neutralization PCR test signal Δ$\overline{Ct}$ calculated by subtracting the Ct value of the sample from that of a buffer only blank control.