Rapid quantification of SARS-CoV-2 Spike-specific T cells by direct peptide stimulation of whole peripheral blood

The immune response that protects an individual from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 (coronavirus disease 2019), involves the activation of neutralizing antibodies, antigen-binding antibodies, and antiviral T cells, among other reactions.

There is evidence that individuals with SARS-CoV-2 infection but not severe clinical COVID-19 symptoms present antibodies and T cells, which are able to help them control the infection.

There is, however, still some uncertainty about the level of antibodies and/or T cells that are necessary for such protection. Is immunity mediated by differences in the antibody levels and the T cell levels among vaccinated subjects?

Correlates of protection

To understand both the correlates of protection induced by vaccinations and the necessary immune responses to manage the COVID-19, researchers from Singapore used an alternative method to closely evaluate the kinetics and magnitude of specific T cell responses to SARS-CoV-2 infection. Their work was posted to the bioRxiv* preprint server while awaiting peer review.

Study: Rapid determination of the wide dynamic range of SARS-CoV-2 Spike T cell responses in whole blood of vaccinated and naturally infected. Image Credit: NIAID

In this study, the researchers tested the sensitivity and performance of a simple, rapid SARS‐CoV‐2 Spike‐specific T cell test based on stimulation of whole blood with peptides covering the SARS‐CoV‐2 Spike protein. The Spike protein is a structural glycoprotein that envelopes the virus, binds to the host receptor, and enables viral entry and fusion.

Spike‐specific T cells

The researchers detected and quantified the Spike‐specific T cells in vaccinated individuals through the simple addition of Spike peptide pools to whole blood. Importantly, the cohorts in the study included 1) BNT162b2 vaccinated, 2) convalescent asymptomatic and 3) symptomatic COVID‐19 patients, and 4) SARS‐CoV‐1 convalescent individuals. The test included cytokine (IFN‐γ, IL‐2) measurement.

“In this work, by sequentially testing vaccinated and SARS‐CoV‐2 convalescent individuals, we show that IL‐2 and IFN‐γ  quantification in whole blood measured Spike‐specific T cell response with an accuracy equivalent to ELISPOT assays performed in freshly purified PBMC.”  

A wide dynamic range of Spike‐specific T cell responses after vaccination was revealed in this study. However, the researchers noted that these results could not be predicted from neutralizing antibody quantities.

“Efforts to define the protective threshold of antibodies through mathematical modeling have shed some light on this issue, but such work on T cell responses have so far been absent.”

The researchers collected whole fresh blood from individuals before and after vaccinations. The blood was either directly stimulated with peptides for a cytokine release assay (CRA) or processed to isolate PBMCs by Ficoll density gradient centrifugation.

The researchers demonstrated that assessing the Spike‐specific T cell response directly from the fresh whole blood yields reliably comparable results to the classical T cell assays.

Further, they demonstrated that the whole blood CRA could be used to rapidly define T cell immunogenic regions of the whole Spike protein.

“Taken together, these data show that direct analysis of cytokines secreted in whole blood pulsed with different peptides constitutes a reliable method to gauge the presence and magnitude of functional T cells specific for epitopes covered by the utilized peptides,” observed the researchers in the study.

The researchers analyzed the Spike‐specific T cell response in a larger cohort of BNT162b2 vaccinated individuals and in individuals who have recovered from SARS‐CoV‐2 and SARS‐CoV‐1 infection using the whole blood CRA with SpG peptide pool as a stimulant. Importantly, using the rapid cytokine assay, they detected the Spike‐specific T cells in ~84% of the SARS‐CoV‐2 individuals one year after the infection and also in 8 out of 12 SARS‐CoV‐1 patients 18 years after infection.

In their paper, the researchers compare the specific T-cell responses to the two viruses using different cohorts and demonstrate the wide dynamic range and heterogeneous nature of Spike‐specific T-cell responses. They also pointed out that quantifying IL‐2 secretion provides better sensitivity over IFN‐γ in detecting individuals with a long‐term Spike‐specific memory T cell response.

“Though speculative at the moment, this lack of difference between IFN‐γ and IL‐2 secretion could reflect a better functionality of Spike‐specific T cell response and hence plausibly contribute to the benign disease trajectory in these individuals.”

According to the researchers, no significant correlations between neutralizing antibodies and Spike-specific T cell responses were found among the analyzed groups.

“The rapidity, simplicity, and accuracy of the cytokine release assay (CRA) in whole blood can allow a routine measurement of SARS‐CoV‐2 T cells in large populations and thus help in understanding the role of antiviral T cells during the current COVID‐19 pandemic.”

Importantly, this study has shown that the tests of cytokine release in the stimulated whole blood do not detect the mere presence of T cells but also their functionality. The measurement of the wide dynamic range of functional Spike‐specific T cells helps evaluate the protective ability of T cells after infection or vaccination.

Because the quantity of Spike‐specific T cells cannot be predicted by simple antibodies measurement, this higher throughput and simple assay represent a feasible approach to implement in routine testing. In addition, this technique will complement existing measurements of antibodies and provide valuable information for formulating vaccine strategies, the researchers say.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Dr. Ramya Dwivedi

Ramya has a Ph.D. in Biotechnology from the National Chemical Laboratories (CSIR-NCL), in Pune. Her work consisted of functionalizing nanoparticles with different molecules of biological interest, studying the reaction system and establishing useful applications.