Evaluation of SARS-CoV-2-Specific T-Cell Activation with a Rapid On-Chip IGRA

Abstract

Interferon-gamma release assays (IGRAs) that measure pathogen-specific T-cell response rates can provide a more reliable estimate of protection than specific antibody levels but have limited potential for widespread use due to their workflow, personnel, and instrumentation demands. The major vaccines for SARS-CoV-2 have demonstrated substantial efficacy against all of its current variants, but approaches are needed to determine how these vaccines will perform against future variants, as they arise, to inform vaccine and public health policies. Here we describe a rapid, sensitive, nanolayer polylysine-integrated microfluidic chip IGRA read by a fluorescent microscope that has a 5 h sample-to-answer time and uses ∼25 μL of a fingerstick whole blood sample. Results from this assay correlated with those of a comparable clinical IGRA when used to evaluate the T-cell response to SARS-CoV-2 peptides in a population of vaccinated and/or infected individuals. Notably, this streamlined and inexpensive assay is suitable for high-throughput analyses in resource-limited settings for other infectious diseases.

Keywords: COVID-19; COVID-19 vaccine; IGRA; T-cell response; rapid test; whole blood assay.

Scheme 1. Microfluidic Chip IGRA Test

Fingertip whole blood can be directly used for SARS-CoV-2 spike protein peptide pool stimulation and positive T-cell detection (left). All PMBC will be captured by the polylysine nanolayer coated on the glass surface, and then, antigen-presenting cells such as dendritic cells will present peptides to CD4 or CD8 T-cells. IFNγ in activated T-cells will be stained with fluorescent antibody and imaged with this microfluidic chip.

Figure 1

Slide-based PBMC activation analyses. (A) Thawed PBMC aliquots stimulated with or without PMA/ionomycin were cultured for 24 h in glass bottom wells coated with IFNγ-specific antibody then incubated with a biotinylated secondary antibody, streptavidin-HRP, and a chromogenic (red) HRP substrate. (B–E) PBMCs (∼2 × 10⁵) were seeded on microplate wells coated with and without polylysine and stained with Hoechst 33342 to quantify the cell density of captured cells, or (D, E) induced with PMA/ionomycin for 4 h, stained with Hoechst 33342 (B, C) and specific antibodies to IFNγ, OX40, and 4-1BB (D, E), after which total cell numbers and activated T-cell percentages were quantified using a fluorescent plate reader. Positive control (PC) wells were not washed to remove nonadherent or weakly adherent cells. One-way two-sided parametric ANOVAs with Tukey’s post-test were performed to analyze differences between the polylysine-coated and uncoated well values and (E) PMA-stimulated and unstimulated well values. White size bars indicate 75 μm. Data indicate mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, no significant difference when analyzed by two-sided Mann–Whitney U-test. The schematics in A, B and D were created with BioRender.com.

Figure 2

T-cell activation with SARS-CoV-2 spike peptide pool. (A, B) Cryopreserved PBMCs from individuals who had received zero (unvaccinated), two, or three SARS-CoV-2 vaccine doses were simulated by incubation with a SARS-CoV-2 peptide pool for 24 h, after which IFNγ+ cells were detected by (A) flow cytometry or (B) ELISpot. (C) Representative images and (D) quantification results from freshly isolated PBMCs from individuals who were unvaccinated and or had received three vaccine doses after 24 h incubation with or without a SARS-CoV-2 peptide pool. (E) Summary of the IFNγ+ cell ratios detected in PBMCs of SAR-CoC-2-vaccine recipients (three doses) following 24 h incubation with peptides from SARS-CoV-2, the M. tuberculosis (Mtb) CFP-10 and ESAT-6 proteins, HIV-1 p24, or Ebola VP40 protein. Data indicate mean ± SD; symbols denote p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), or nonsignificant (ns) differences between indicated groups by (C) one-way parametric ANOVA with Tukey’s post-test or (D, E) Mann–Whitney U-test.

Figure 3

Evaluation of on-chip ELISpot assay results in vaccinated HIV– individuals. (A) On-chip ELISpot, (B) flow cytometry, and (C) ELISpot assay produced after stimulating PBMCs isolated from individuals without a history of HIV infection who had received three vaccine doses with SARS-CoV-2 spike or HIV-1 p24 (nonspecific control) peptides. (D) Correlation of flow cytometry and on-chip ELISpot data. Data indicate mean ± SD; symbols indicate p < 0.05 (*), p < 0.01 (**), or ns (nonsignificant) differenced by two-sided Mann–Whitney U-test.

Figure 4

Evaluation of on-chip ELISpot assay with whole blood samples. (A) Scheme of whole blood T-cell evaluation with an on-chip ELISpot test. (B, C) On-chip ELISpot assays result from fingerstick whole blood samples (B) from one subject, with and without RBC lysis, and (C) from eight HIV negative individuals >6 months after receipt of two or three vaccine doses, without RBC lysis. Data indicate mean ± SD; symbols indicate p < 0.05 (*), p < 0.01 (**), or ns (nonsignificant) differenced by two-sided Mann–Whitney U-test. The schematic in A was created with BioRender.com.