Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jul 1:532:130-136.
doi: 10.1016/j.cca.2022.05.025. Epub 2022 Jun 8.

T-cell proliferation assay for the detection of SARS-CoV-2-specific T-cells

Chang Chu  1 , Anne Schönbrunn  2 , Saban Elitok  3 , Florian Kern  4 , Karsten Schnatbaum  5 , Holger Wenschuh  5 , Kristin Klemm  3 , Volker von Baehr  2 , Bernhard K Krämer  6 , Berthold Hocher  7
Affiliations

T-cell proliferation assay for the detection of SARS-CoV-2-specific T-cells

Chang Chu et al. Clin Chim Acta. .

Abstract

Both infection with and vaccination against SARS-CoV-2 trigger a complex B-cell and T-cell response. Methods for the analysis of the B-cell response are now well established. However, reliable methods for measuring the T-cell response are less well established and their usefulness in clinical settings still needs to be proven. Here, we have developed and validated a T-cell proliferation assay based on 3H thymidine incorporation. The assay is using SARS-CoV-2 derived peptide pools that cover the spike (S), the nucleocapsid (N) and the membrane (M) protein for stimulation. We have compared this novel SARS-CoV-2 lymphocyte transformation test (SARS-CoV-2 LTT) to an established ELISA assay detecting Immunoglobulin G (IgG) antibodies to the S1 subunit of the SARS-CoV-2 spike protein. The study was carried out using blood samples from both vaccinated and infected health care workers as well as from a non-infected control group. Our novel SARS-CoV-2 LTT shows excellent discrimination of infected and/or vaccinated individuals versus unexposed controls, with the ROC analysis showing an area under the curve (AUC) of > 0.95. No false positives were recorded as all unexposed controls had a negative LTT result. When using peptide pools not only representing the S protein (found in all currently approved vaccines) but also the N and M proteins (not contained in the vast majority of vaccines), the novel SARS-CoV-2 LTT can also discriminate T-cell responses resulting from vaccination against those induced by infection.

Keywords: Humoral and cellular immune responses; Lymphocyte transformation test; SARS-CoV-2; T-cell response.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
ROC curves showing discrimination between vaccinated and unvaccinated health care workers (A)-(C), or infected and uninfected health-care workers (D)-(F), based on detection of IgG against spike S1 (A, D) or LTT with the C-terminal (B, E) or N-terminal (C, F) spike peptide pools.
Fig. 2
Fig. 2
SARS-CoV-2 antigen-specific T-cell responses in vaccinated or infected volunteers (cohort 3) were examined using the SARS-CoV-2 LTT based on peptide pools covering the S, N, and M proteins. (A) All vaccinated participants (cohort 3b, n = 11) showed positive responses (SI ≥ 2) to the S peptide pools but no responses to the N or M pools (SI < 2). (B) All previously infected participants (cohort 3a, n = 12) showed positive responses to the S and, in addition, to both the N and M pools. (C) Participants without infection and without vaccination (cohort 3c, n = 12) showed no LTT response (SI < 2) to any of the peptide pools used for stimulation.
Fig. 3
Fig. 3
SARS-CoV-2-specific T cell-response is more prominent for CD4 + T cells. After five-days stimulation PBMCs were stained for CD45, CD3, CD4 and CD8. CD45 + CD3 + blasts were calculated for CD4+- and CD8 + proportion. A: PBMCs were stimulated with Pokeweed mitogen. B: PBMCs were stimulated with SARS-CoV-2 specific peptide pools spanning different total virus specific proteins.
See this image and copyright information in PMC

References

    1. Mahajan S., Kode V., Bhojak K., Karunakaran C., Lee K., Manoharan M., Ramesh A., Hv S., Srivastava A., Sathian R., Khan T., Kumar P., Gupta R., Chakraborty P., Chaudhuri A. Immunodominant T-cell epitopes from the SARS-CoV-2 spike antigen reveal robust pre-existing T-cell immunity in unexposed individuals. Sci. Rep. 2021;11(1) doi: 10.1038/s41598-021-92521-4. - DOI - PMC - PubMed
    1. Winkler E.S., Gilchuk P., Yu J., Bailey A.L., Chen R.E., Chong Z., Zost S.J., Jang H., Huang Y., Allen J.D., Case J.B., Sutton R.E., Carnahan R.H., Darling T.L., Boon A.C.M., Mack M., Head R.D., Ross T.M., Crowe J.E., Diamond M.S. Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions for optimal therapeutic protection. Cell. 2021;184(7):1804–1820.e16. - PMC - PubMed
    1. Zuo J., Dowell A.C., Pearce H., Verma K., Long H.M., Begum J., Aiano F., Amin-Chowdhury Z., Hoschler K., Brooks T., Taylor S., Hewson J., Hallis B., Stapley L., Borrow R., Linley E., Ahmad S., Parker B., Horsley A., Amirthalingam G., Brown K., Ramsay M.E., Ladhani S., Moss P. Robust SARS-CoV-2-specific T cell immunity is maintained at 6 months following primary infection. Nat. Immunol. 2021;22(5):620–626. - PMC - PubMed
    1. Cassaniti I., Percivalle E., Bergami F., Piralla A., Comolli G., Bruno R., et al. SARS-CoV-2 specific T-cell immunity in COVID-19 convalescent patients and unexposed controls measured by ex vivo ELISpot assay. Clin. Microbiol. Infect. 2021;27(7):1029–1034. - PMC - PubMed
    1. Jung M.K., Shin E.-C. Phenotypes and Functions of SARS-CoV-2-Reactive T Cells. Mol. Cells. 2021;44(6):401–407. - PMC - PubMed