SARS-CoV-2 Neutralization Assays Used in Clinical Trials: A Narrative Review

Abstract

Since the emergence of COVID-19, extensive research efforts have been undertaken to accelerate the development of multiple types of vaccines to combat the pandemic. These include inactivated, recombinant subunit, viral vector, and nucleic acid vaccines. In the development of these diverse vaccines, appropriate methods to assess vaccine immunogenicity are essential in both preclinical and clinical studies. Among the biomarkers used in vaccine evaluation, the neutralizing antibody level serves as a pivotal indicator for assessing vaccine efficacy. Neutralizing antibody detection methods can mainly be classified into three types: the conventional virus neutralization test, pseudovirus neutralization test, and surrogate virus neutralization test. Importantly, standardization of these assays is critical for their application to yield results that are comparable across different laboratories. The development and use of international or regional standards would facilitate assay standardization and facilitate comparisons of the immune responses induced by different vaccines. In this comprehensive review, we discuss the principles, advantages, limitations, and application of different SARS-CoV-2 neutralization assays in vaccine clinical trials. This will provide guidance for the development and evaluation of COVID-19 vaccines.

Keywords: SARS-CoV-2; clinical trials; correlation of protection; neutralizing antibodies; standards.

Figure 1

Mechanism of neutralizing antibody detection using live viruses. In the absence of a neutralizing antibody, the SARS-CoV-2 virus binds ACE2, followed by membrane fusion and cell entry; the release of its genetic material leads to virus propagation. The presence of neutralizing antibody blocks ACE2 interaction with the virus. (Figure created with BioRender.com accessed on 27 April 2024).

Figure 2

Schematic diagram of neutralizing antibody detection using pseudoviruses. (a). The 293T cells are transfected with a plasmid encoding the lentiviral backbone and a plasmid expressing envelope protein. The transfected cells produce recombined pseudoviruses. (b). The 293T cells are first transfected with an envelope protein expression plasmid; 24 h post-transfection, the cells are infected with VSV*∆G encoding firefly luciferase. The transfected cells produce recombined pseudoviruses. (c). The 293T cells are co-transfected with an envelope protein-encoding plasmid, an MLV packaging transfer vector encoding a luciferase reporter. The transfected cells produce pseudo-typed MLV particles, like the HIV system. SARS-CoV-2 pseudoviruses are capable of binding ACE2 and entering the infected cells; non-replicative pseudovirus binds ACE2 and is internalized. Neutralizing antibody blocks ACE2 interaction with the pseudovirus. (Figure created with BioRender.com accessed on 27 April 2024).

Figure 3

Principle of neutralizing antibody detection using competitive ELISA. The HRP coupling fragment of RBD or S1 binds to samples containing neutralizing antibodies. This mixture is added to an ELISA well plate that has been coated with ACE2 receptors. If the antibodies in the sample have neutralizing antibody activity, the binding between HRP-RBD/S1 and ACE2 will be broken, with cleaning to remove HRP-RBD/S1. The signal generated using a light absorption microplate reader will be lower in the presence of neutralizing antibodies. (Figure created with BioRender.com accessed on 27 March 2024).