Biomimetic nanoplasmonic sensor for rapid evaluation of neutralizing SARS-CoV-2 monoclonal antibodies as antiviral therapy

Abstract

Monoclonal antibody (mAb) therapy is one of the most promising immunotherapies that have shown the potential to prevent or neutralize the effects of COVID-19 in patients at very early stages, with a few formulations recently approved by the European and American medicine agencies. However, a main bottleneck for their general implementation resides in the time-consuming, laborious, and highly-specialized techniques employed for the manufacturing and assessing of these therapies, excessively increasing their prices and delaying their administration to the patients. We propose a biomimetic nanoplasmonic biosensor as a novel analytical technique for the screening and evaluation of COVID-19 mAb therapies in a simpler, faster, and reliable manner. By creating an artificial cell membrane on the plasmonic sensor surface, our label-free sensing approach enables real-time monitoring of virus-cell interactions as well as direct analysis of antibody blocking effects in only 15 min assay time. We have achieved detection limits in the 10² TCID50/mL range for the study of SARS-CoV-2 viruses, which allows to perform neutralization assays by only employing a low-volume sample with common viral loads. We have demonstrated the accuracy of the biosensor for the evaluation of two different neutralizing antibodies targeting both Delta and Omicron variants of SARS-CoV-2, with half maximal inhibitory concentrations (IC₅₀) determined in the ng/mL range. Our user-friendly and reliable technology could be employed in biomedical and pharmaceutical laboratories to accelerate, cheapen, and simplify the development of effective immunotherapies for COVID-19 and other serious infectious diseases or cancer.

Keywords: COVID-19; Immunotherapy; Label-free analysis; Neutralization assay; Supported lipid bilayer; Surface plasmon resonance.

Fig. 1

Schematic illustration of the biomimetic plasmonic biosensor approach for mAb therapy evaluation via neutralization assays. 1) panel shows direct capture and detection of SARS-CoV-2 viruses over an artificial cell membrane displaying ACE-2 receptors created on the plasmonic sensor. 2) panel shows how therapeutic monoclonal antibodies bind to the Spike protein of the SARS-CoV-2 virus, blocking and inhibiting its interaction with the host cell.

Fig. 2

(a) Schematic illustration of the ACE-2 functional supported lipid bilayer formed on SiO₂-coated plasmonic sensor surface; (b) SPR sensorgram showing the formation of a lipid bilayer from the disruption of small unilamellar vesicles (SUV), followed by a NaOH cleaning step; (c) SPR sensorgram showing the ACE-2 receptor immobilization on the COOH-functional SLB.

Fig. 3

Standard calibration curve for a) SARS-CoV-2 viral antigens (Delta and Omicron) obtained with triplicate measurements of different concentrations over a range between 30 ng/mL to 5000 ng/mL for each specific protein variant; and b) UV-inactivated SARS-CoV-2 virus different variants (delta and omicron) obtained with triplicate measurements of different virus titters over a range between 10³ TCID50/mL to 5 × 10⁵ TCID50/mL for each specific virus variant.

Fig. 4

a) binding curves for Delta-S protein with NAb1 and NAb2, respectively; and b) Standard binding curves for Omicron-S protein with NAb1 and NAb2, respectively.

Fig. 5

Competitive immunoassay with SARS-CoV-2 viral antigens and virus variants: a) Schematic illustration of the neutralization assay performed with SARS-CoV-2 viral antigens and neutralizing antibodies (NAb) over an ACE-2 receptor anchored to a SLB-functionalized SPR biosensor; b) Inhibition curves obtained for the Delta-S protein at a fixed concentration (500 ng/mL) incubated with different concentrations of both neutralizing antibodies (NAb1 and NAb2); c) Inhibition curves obtained for the Omicron-S protein at a fixed concentration (500 ng/mL) incubated with different concentrations of both neutralizing antibodies (NAb1 and NAb2). All sensor signals are normalized according to maximum signal (100%) and minimum signal (0%) to facilitate comparison; d) Schematic illustration of the neutralization assay performed with SARS-CoV-2 viruses and neutralizing antibodies (NAb) over an ACE-2 receptor anchored to a SLB-functionalized SPR biosensor; e) Inhibition curves obtained for the Delta SARS-CoV-2 at a fixed concentration (5 × 10⁴ TCID50/mL) incubated with different concentrations of both neutralizing antibodies (NAb1 and NAb2); f) Inhibition curves obtained for the Omicron SARS-CoV-2 viruses at a fixed concentration (5 × 10⁴ TCID50/mL) incubated with different concentrations of both neutralizing antibodies (NAb1 and NAb2). All sensor signals are normalized according to maximum signal (100%) and minimum signal (0%) to facilitate comparison.