key: cord-0796800-1isw68a6
authors: Murugesan, Kanagavel; Jagannathan, Prasanna; Altamirano, Jonathan; Maldonado, Yvonne A; Bonilla, Hector F; Jacobson, Karen B; Parsonnet, Julie; Andrews, Jason R; Shi, Run-Zhang; Boyd, Scott; Pinsky, Benjamin A; Singh, Upinder; Banaei, Niaz
title: Long Term Accuracy of SARS-CoV-2 Interferon-γ Release Assay and its Application in Household Investigation
date: 2022-01-25
journal: Clin Infect Dis
DOI: 10.1093/cid/ciac045
sha: 2c4fb61accd6811aa8aaf97dd6b5b308bdfc4f10
doc_id: 796800
cord_uid: 1isw68a6

BACKGROUND: An immunodiagnostic assay that sensitively detects a cell-mediated immune response to SARS-CoV-2 is needed for epidemiological investigation and for clinical assessment of T cell-mediated immune response to vaccines, particularly in the context of emerging variants that might escape antibody responses. METHODS: The performance of a whole blood interferon-gamma (IFN-γ) release assay (IGRA) for the detection of SARS-CoV-2 antigen-specific T cells was evaluated in COVID-19 convalescents tested serially up to 10 months post-infection and in healthy blood donors. SARS-CoV-2 IGRA was applied in contacts of households with index cases. Freshly collected blood in the lithium heparin tube was left unstimulated, stimulated with a SARS-CoV-2 peptide pool, and stimulated with mitogen. RESULTS: The overall sensitivity and specificity of IGRA were 84.5% (153/181; 95% confidence interval [CI] 79.0-89.0) and 86.6% (123/142; 95% CI;80.0-91.2), respectively. The sensitivity declined from 100% (16/16; 95% CI 80.6-100) at 0.5-month post-infection to 79.5% (31/39; 95% CI 64.4-89.2) at 10 months post-infection (P<0.01). The IFN-γ response remained relatively robust at 10 months post-infection (3.8 vs. 1.3 IU/mL, respectively). In 14 households, IGRA showed a positivity rate of 100% (12/12) and 65.2% (15/23), and IgG of 50.0% (6/12) and 43.5% (10/23) in index cases and contacts, respectively, exhibiting a difference of +50% (95% CI +25.4-+74.6) and +21.7% (95% CI, +9.23-+42.3), respectively. Either IGRA or IgG was positive in 100% (12/12) of index cases and 73.9% (17/23) of contacts. CONCLUSIONS: The SARS-CoV-2 IGRA is a useful clinical diagnostic tool for assessing cell-mediated immune response to SARS-CoV-2.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has demanded the development of sensitive laboratory diagnostics to detect active and remote infections to control the pandemic [1] . In the arena of immunodiagnostics, serologic assays that probe antibody responses to SARS-CoV-2 are far more available than assays that measure T cell responses [1] . However, serological tests for anti-SARS-CoV-2 antibodies may not accurately predict the magnitude, functionality, and durability of T cell-mediated immune response to SARS-CoV-2 [2, 3] , particularly in immunocompromised patients with impaired B cell function [3] [4] [5] [6] . Recent studies have shown that T cell responses are more sensitive markers of past SARS-CoV-2 infection compared with antibody responses and postulated to represent a correlate of protective immunity [5, [7] [8] [9] [10] [11] . Furthermore, T cell responses are more robust than antibody responses in convalescents with mild or asymptomatic COVID-19 infection [10] [11] [12] [13] . Thus, clinical T cell assays for SARS-CoV-2 are needed to evaluate individuals for the cell-mediated immune response against SARS-CoV-2 as evidence for past infection and immune response to vaccination [14] .

Interferon gamma release assay (IGRA) is an in vitro blood diagnostic used clinically to measure IFN-γ released by antigen-specific T cells after stimulation with pathogen-specific peptides. IGRA is best known for its role in diagnosing latent Mycobacterium tuberculosis infection [15, 16] in which either purified or whole blood mononuclear cells are stimulated overnight followed by IFN-γ enzyme-linked immunosorbent spot or ELISA, respectively, to measure IFN-γ response from sensitized T cells [9, 17] . Recently, we and another group reported on the accuracy of a laboratory-developed whole blood IGRA for the detection of the SARS-CoV-2 specific T cell responses in convalescents weeks after infection [18, 19] .

Since then, our assay has been further optimized with commercial peptide pools and M a n u s c r i p t 6 simplified with in-tube stimulation, and offered clinically in a CLIA laboratory at Stanford Health Care. In this study, we report the longitudinal accuracy of SARS-CoV-2 IGRA up to 10 months after infection and evaluated its utility in household contacts exposed to an index case. We show that IGRA response in convalescents is durable and it identifies more infected household contacts compared with antibody testing.

Ethics. This study was conducted under two Institutional Review Board-approved protocols (IRB-55619 and IRB-55479) named "A Phase 2 Randomized, Single-Blind Study of a Single Dose of Peginterferon Lambda-1a Compared with Placebo in Outpatients with Mild COVID-19" and "Natural History of Shedding and Household Transmission of COVID-19:

Constructing Patterns of Viral Spread and Evolution" which collected blood from infected and exposed individuals, respectively. Informed consent was obtained from participants before blood collection.

This was a case-control study to evaluate the longitudinal sensitivity and specificity of SARS-CoV-2 IGRA ( Figure 1 ). Additionally, the utility of SARS-CoV-2 IGRA was assessed in household contacts of COVID-19 index cases. Blood samples from outpatient COVID-19 patients with positive SARS-CoV-2 reverse transcriptase (RT)-PCR enrolled in an IFN-λ vs. placebo therapeutic clinical trial (referred to as cases) and healthy blood donors with no COVID-19 symptoms (referred to as controls) were tested with SARS-CoV-2 IGRA and IgG ELISA. Cases were tested at 0.5, 1, 4, 7 and 10 months post-infection between April 6, 2020 and April 30, 2021. Controls were tested once between May 11, 2020 and November 11, 2021. None of the samples were tested in a previous study [18] . Based on A c c e p t e d M a n u s c r i p t 7 prior studies, IFN-λ has been shown to not negatively impact adaptive response to SARS-CoV-2 [20] . Household contacts of RT-PCR-positive COVID-19 index cases with mild COVID-19 from 14 households were tested with SARS-CoV-2 IGRA and IgG ELISA days after the index case was diagnosed. Members of each household were tested on the same day.

Whole blood was collected in lithium heparin blood collection tube and transported to the Stanford Health Care Clinical Microbiology Laboratory at room temperature for IGRA testing. Blood from healthy blood donors was purchased from Stanford Blood Center.

Antigen and Mitogen. The SARS-CoV2 peptide pools consisting of PepTivator SARS-CoV-2 Prot S, S1, N and M were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Per the manufacturer, S1 pool consists of 15-mer peptides overlapping by 11-residues covering the S1, while the remaining pools contain 15-mer peptides corresponding to immunodominant epitopes. Peptides were developed for stimulation of both CD4 and CD8 T cells. Each 60nmoL vial, which contains 60 peptides, was reconstituted aseptically in 2mL of sterile de-ionized water and all 4 vials were combined into a single mega pool. For in-tube stimulation 80µL of the mega pool was dispensed into a BD vacutainer no additive tube (Beckton Dickenson; city, state). Optimization studies were performed with an individual S1 peptide pool preparation and in combination with S, and S, M, N (Supplementary Figure 1A) .

Phytohemagglutinin PHA-P Mitogen (Sigma) was dissolved in sterile de-ionized water at 1mg/mL. For in-tube stimulation, 50µL was dispensed into a BD Vacutainer No Additive Tube. Prepared tubes and peptide reagents were stored at -80 :C.

A c c e p t e d M a n u s c r i p t 8 Interferon Gamma Release Assay (IGRA). SARS-CoV-2 IGRA was performed as described previously [18] . One mL of freshly collected blood was transferred to a 24 well tissue culture plate or BD vacutainer with no additive tubes at 1mL per well/tube. One well or tube was left unstimulated (nil), one well or tube was stimulated with SARS-CoV-2 antigen mega pool at 7.5 nmol/mL (approximately 1 µg/mL), and one well or tube was stimulated with mitogen at 50 µL/mL. Antigen concentration was based on optimization studies stimulating with 3.75 and 7.5 nmol/mL (Supplementary Figure 1B) . In-tube stimulation was validated using inplate stimulation as the reference method (Supplementary Figure 2 ) and used only for clinical testing after implementation in the clinical laboratory [21] . The blood samples were mixed gently and incubated at 37 :C with a relative humidity of 95% for 20 to 24 h. Plasma A c c e p t e d M a n u s c r i p t 9 Statistical Analysis. The Mann-Whitney U test was used to compare median IFN-γ response between groups. Adjusted P values were determined as per the Bonferroni's multiple comparison test. Fisher's exact test was used to analyze differences between proportions.

Statistical analysis was done with GraphPad Prism 8.0.1 software (San Diego, CA, USA).

To assess the accuracy of SARS-CoV-2 IGRA longitudinally after natural infection, a total of 94 unique adult COVID-19 convalescents were tested at 0.5 (n=16), 1 (n=8), 4 (n=48), 7 Table 1 and Supplementary Table 1A Six contacts (26.1%) were RT-PCR positive after exposure to index case, but four of them were RT-PCR positive on the day of sample collection for this study.

In COVID-19 convalescents tested with SARS-CoV-2 IGRA, the overall sensitivity over 10 

In household members tested one time after recruitment, 100% (12/12) Figure 3D ).

In the household cohort, concordance between IGRA and IgG results was 50.0% (6/12) and 60.9% (14/23) 

Accurate immunodiagnostics are needed to assess T cell-mediated immune response to SARS-CoV-2 after natural infection and after vaccination to inform providers on possible protective immunity to SARS-CoV-2 [2] and to accurately understand transmission dynamics of emerging variants [9] . Furthermore, T cell responses may be important for understanding immunity to SARS-CoV-2 infection, particularly in the context of emerging variants that might escape antibody responses [22, 23] . Using a whole blood SARS-CoV-2 IGRA, we show that T cell-mediated immune response to SARS-CoV-2 was sustained longitudinally with 85% and 80% sensitivity at 4-and 10-month, respectively, post-infection in convalescents who had mild or asymptomatic COVID-19. Our findings are consistent with a prior study showing a robust memory T-cell response months after SARS-CoV-2 infection in individuals with mild or asymptomatic infection [10, 11, 13, 24, 25] , and similar to prior studies we observed a mild decline in the cellular and humoral immune response in convalescents [6, 26, 27] .

Consistent with prior studies we also found a higher proportion of household contacts with positive SARS-CoV-2 IGRA compared with IgG [11, 12] . These findings indicate that the SARS-CoV-2 IGRA used in this study is more sensitive than IgG testing for detection of asymptomatic or mild infection amongst close contacts, at least in the early period, and thus may be an important tool for epidemiological studies aimed at acutely understanding the transmission dynamics of emerging SARS-CoV-2 variant such as the delta variant [28] .

SARS-CoV-2 IGRA may also serve an important role in the clinical assessment of protective Tcell-mediated immune response to the SARS-CoV-2 vaccines [9, 29, 30] . Humoral immune response has been shown to decline after SARS-CoV-2 vaccination [26, 27, 31] . Furthermore, it was recently shown that the antibody response rate to the SARS-CoV-2 vaccine in A c c e p t e d M a n u s c r i p t 14 transplant patients was low after the second dose (40%) and third dose (68%) [32] .

Preliminary analysis of SARS-CoV-2 vaccine response in immunocompromised patients at our health system using the in-tube version of our whole blood SARS-CoV-2 IGRA, which allows immediate stimulation and provides higher sensitivity [18] , has revealed a significantly higher IGRA positivity rate compared with IgG positivity rate [21] . These findings support the role of SARS-CoV-2 IGRA in vaccine response assessment, particularly in elderly and immunocompromised patients given that immunosenescence and immunosuppression, respectively, may dampen adaptive immune responses and leave the host vulnerable to subsequent infection [4, 33, 34] . Thus, IGRA and IgG serology may serve a role in informing providers on the status of cell-mediated and humoral response to SARS-CoV-2 vaccine and the need for revaccination.

Although the findings are promising, this study has several limitations. First, the whole blood SARS-CoV-2 IGRA did not distinguish between CD4 and CD8 T cell responses. Prior studies in convalescents have shown that IFN-γ response in IGRA is predominantly CD4 T cell-derived [18, 35] . However, a more complex assay design that allows measurement of CD4 and CD8 T cells response is possible if clinically indicated. Second, given that healthy blood donors were recruited during the pandemic, we would not know whether the 16% positivity rate with SARS-CoV-2 IGRA in this group was due to true-positive results due to past SARS-CoV-2 infection, false-positive results, or cross-reactivity due to past infection with seasonal CoV [12, 36, 37] . Third, the IgG response in this study was not further assessed for its ability to neutralize the virus. Such characterization was not relevant in the context of investigating IGRA accuracy. Forth, SARS-CoV-2 IGRA was not applied to the investigation of vaccine response in this study. Studies are underway to measure the vaccine response with IGRA in A c c e p t e d M a n u s c r i p t 15 immunocompromised patients. Lastly, the household study had a small sample size and lacked multiple time points to more accurately assess the performance of IGRA vs IgG, and we did not perform risk assessment in household contacts to correlate IGRA positivity with exposure risk.

In summary, the whole blood SARS-CoV-2 IGRA was shown to maintain sensitivity in convalescents up to 10 months post-infection and was shown to have a higher positivity rate than IgG in household contacts of COVID-19 cases. SARS-CoV-2 IGRA, including commercial options [38] , is a simple and robust clinical immunodiagnostic test that can be applied to accurately understand the transmission of emerging variants and to assess vaccine response in vulnerable populations. A c c e p t e d M a n u s c r i p t 28 Figure 3 

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