As SARS-CoV-2 variants emerged, SPR provided critical real-time data on how antibody cross-reactivity with native and variant spike proteins changes — with direct implications for vaccine strategy.
Introduction
The rise of SARS-CoV-2 variants of concern (VOCs) adds more challenges to the public health system. A thorough understanding of how protective antibodies are produced from the native strain against VOCs would be beneficial in outlining public health guidelines and vaccination programs.
SARS-CoV-2 invades cells in the human respiratory tract by membrane fusion. The receptor-binding domain (RBD) from a subunit of the spike proteins present on the virus binds to the angiotensin-converting enzyme 2 (ACE-2) receptor found on cell membranes in the respiratory tract. Neutralizing antibodies (from natural infection or vaccine) prevent the viruses from binding to the ACE-2 receptors.
A research team led by Joelle Pelletier and Jean-François Masson from Université de Montréal investigated the cross-reactivity of antibodies from COVID-19 positive individuals (non-hospitalized) against the native and beta variant (B.1.351) SARS-CoV-2 spike proteins. ELISAs and cell-based neutralization assays are typically used to study the humoral response against SARS-CoV-2. However, these assays are lengthy and expensive. SPR is becoming an essential technique in this field because it can complement biochemical data from ELISA and serve as a surrogate for cell-based neutralization assays.
Experimental Details
1. Detection of Anti-Spike Antibodies by SPR
The SPR experiment procedures used to determine the level of IgG antibody binding to the native or beta strain spike protein involved using a quad inlet P4SPR™. The final detection step involved adding anti-human IgG as a secondary antibody in a sandwich assay.
2. Dissociation Constants of Antibody-Spike Protein Interactions by SPR
The KDs were determined by constructing a calibration curve using a dual inlet P4SPR. The spike protein from the native or beta variant was immobilized on the SPR chips. Then, decreasing dilutions of the sera containing the antibodies were injected into the P4SPR. The signals at equilibrium were fit into a 1:1 Langmuir model to obtain the KD value for each interaction.
3. SPR Used as a Pseudo-Neutralization Assay
SPR was used as a virus-free, in vitro method to replace cell-based neutralization assay. Spike proteins from the native or beta variant were immobilized on a gold sensor chip, followed by injection of COVID-19 positive or negative sera. The presence of antibodies in COVID-19 positive sera bound to the immobilized spike proteins, inhibiting the ACE-2 receptors from binding.
Major Discoveries
According to both ELISA and SPR results, the antibodies from COVID-19 positive individuals bound to the spike proteins of both native and beta (B.1.351) strains. However, the signals were lower for the beta spike protein. The antibodies' affinity towards the spike proteins was highest for both native and beta strains shortly after infection and decreased afterwards. Nevertheless, sera of individuals who were never exposed to the beta strain contained anti-spike antibodies that cross-reacted with the beta strain spike protein up to 16 weeks post-diagnosis.
Key Advantages of the P4SPR Highlighted from this Study
The dilution factor required for SPR assays was at 1:5 compared to 1:50 for an antigen-down colorimetric ELISA. Other SPR experiments in the study did not even require any dilutions. Furthermore, for SPR assays, both high and low concentrations or high and low-affinity interactions can be observed, versus only high affinity and high concentration of antibodies for ELISA.
SPR can be used to perform a pseudo-neutralization study, negating the need to use live viruses in a cell-based neutralization assay in a BSL3 lab. It is less expensive, takes less time, and the procedure is certainly not as complicated.
Conclusions
This study, assisted by Affinité's P4SPR™ system, revealed important information about the cross-reactivity of anti-spike antibodies from naturally infected individuals. The antibodies were able to bind to the spike proteins of the beta variant, albeit at a lower level than the native spike proteins. They were also able to inhibit the interactions between ACE-2 and the spike protein. The P4SPR was able to quantify the inhibition level of the spike protein-ACE-2 receptor interaction by anti-spike antibodies without performing a complex cell-based neutralization assay in a BSL-3 lab.