An evaluation of a FluoroSpot assay as a diagnostic tool to determine SARS-CoV-2 specific T cell responses

Abstract

Numerous assays evaluating serological and cellular responses have been developed to characterize immune responses against SARS-CoV-2. Serological assays are both cost- and time-effective compared to cellular assays, but cellular immune responses may provide a diagnostic value to determine previous SARS-CoV-2 infection in seronegative individuals. However, potential cross-reactive T cell responses stemming from prior encounters with human coronaviruses (HCoVs) may affect assay specificity. In this study, we evaluated the specificity and sensitivity of a SARS-CoV-2 IFN-γ Release Assay (IGRA) based on the FluoroSpot method employing commercially available SARS-CoV-2-specific peptide pools, as well as an in-house designed SARS-CoV-2 peptide pool restricted to 5 amino acid stretches or less aligning with endemic HCoVs. Blood samples were obtained from healthcare workers (HCW) 5–6 months post SARS-CoV-2 spike (S) IgG and nucleocapsid (N) IgG dual seroconversion (n = 187) and HCW who had been S IgG and N IgG dual seronegative at repeated occasions, including the current sampling time point (n = 102). In addition, samples were obtained 4 to 5 months post infection from 55 polymerase chain reaction (PCR)-confirmed COVID-19 patients. Assay specificity and sensitivity were calculated with serology as a reference standard for HCW. The in-house generated peptide pool displayed a specificity of 96.1%, while the commercially available peptide pools displayed specificities of 80.4% and 85.3%, respectively. Sensitivity was higher in a cohort of previously hospitalized COVID-19 patients (96.4% and 84.0% for the commercially available peptide pools and 92.7% for the in-house generated peptide pool) compared to the HCW cohort (92.0% and 66.8% for the commercially available peptide pools and 76.0% for the in-house generated peptide pool). Based on these findings, the individual diagnostic value of T cell immune responses against SARS-CoV-2 currently appears to be limited but remain an important research tool ahead.

Duration of SARS-CoV-2 Immune Responses Up to Six Months Following Homologous or Heterologous Primary Immunization with ChAdOx1 nCoV-19 and BNT162b2 mRNA Vaccines

“…T-cell responses were assessed 2 weeks ( n = 190) and 3 months ( n = 337) post second vaccine dose. Whole blood was stimulated with an in-house generated peptide pool containing in total 16 peptides based on the spike, nucleocapsid, membrane and open reading frame 3 and 7 proteins [ 29 , 30 ]. The peptides have an estimated HLA coverage of 97% (class I and II combined) and have shown to provide a specificity of 96.1% when using it as a read-out for specific cellular responses against SARS-CoV-2 [ 29 , 30 ].…”

Long‐term SARS‐CoV‐2‐specific and cross‐reactive cellular immune responses correlate with humoral responses, disease severity, and symptomatology

“…These seronegative individuals were not expected to have a memory response against neither of the two peptide pools, due to their serology status. Using dual S and N directed serology to identify a SARS‐CoV‐2 naïve and exposed group we have previously reported the specificity and sensitivity of each peptide pool and noted a high sensitivity but poor specificity with the commercial TB47 pool 25 . This prompted us to determine the overlap of immunogenic regions between the two pools and endemic HCoV.…”

“…Peptides with sequences overlapping with the proteome of endemic HCoVs included in the two peptide pools have previously been reported in Mangsbo et al, 25 and are also summarized in Table S3 . The TS16 pool contains no more than five amino acid sequences matching endemic HCoVs.…”

“…In addition, studies have mainly focused on IFNγ as the main determinant for an effector response, skewing the measurement towards a specific memory T cell subset. Here, we examined both IL‐2 and IFNγ responses using a highly selective SARS‐CoV‐2‐specific peptide pool (TS16) without any identified overlapping T cell epitopes with endemic HCoV 25 . Interestingly, the cross‐reactive response identified in SARS‐CoV‐2 seronegative HCWs was associated with increased IFNγ secretion, while SARS‐CoV‐2‐specific stimulation resulted in the release of both IL‐2 and IFNγ.…”

“…Here, we examined both IL‐2 and IFNγ responses using a highly selective SARS‐CoV‐2‐specific peptide pool (TS16) without any identified overlapping T cell epitopes with endemic HCoV. 25 Interestingly, the cross‐reactive response identified in SARS‐CoV‐2 seronegative HCWs was associated with increased IFNγ secretion, while SARS‐CoV‐2‐specific stimulation resulted in the release of both IL‐2 and IFNγ. This difference in the cytokine production profile could indicate different subsets of circulating memory T cells.…”