Monitoring the immune response to SARS-CoV-2: The importance of T cells

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    T cells are essential to combat infection with any viral pathogen. Because T-cell responses have traditionally been more challenging to study than antibodies, they have been somewhat neglected compared with their humoral counterparts. The emphasis on antibody response in infection is highlighted by the COVID-19 pandemic; media coverage is dominated by reports on antibody testing and titers to determine prior exposure and potential immunity to SARS-CoV-2.

    Recent advances have allowed T-cell monitoring technology to catch up with antibody tests, and several lines of evidence indicate that T-cell responses may provide a better measure of the immune response to SARS-CoV-2. Technologies that profile the entire T-cell repertoire can identify and study discrete antigen-specific T-cell clones in individuals and across populations, allowing a greater understanding of the role of T cells in COVID-19. These technologies could ultimately provide a way to assess and monitor the efficacy of vaccines and treatments and enable the development of more accurate SARS-CoV-2 diagnostics.

    This article discusses the importance of monitoring T-cell responses in COVID-19 and highlights how immune repertoire profiling can provide critical insights into the role of T cells in SARS-CoV-2 infection and immunity. 

    Why measuring T cells is vital in monitoring COVID-19 

    Methods to test for SARS-CoV-2 infection can detect either the virus itself or an immune response to the virus. The current gold standard and primary method used for assessing active SARS-CoV-2 infection is real-time polymerase chain reaction (RT-PCR), as shown in Figure 1. RT-PCR specifically detects viral RNA, which can be present before the onset of symptoms and in asymptomatic individuals. While RT-PCR can diagnose active infection, it does not offer information about immune response, prior infection, or ongoing immunity. Monitoring the T-cell response gives that necessary insight into the immune system, which is vital to understanding pandemic dynamics and the efficacy of potential vaccines and treatments. 

    Detection of Viral RNA by RT-PCR

    Figure 1. Detection of viral RNA by RT-PCR. A. RNA is extracted from a patient sample. B. Single-stranded RNA is reverse transcribed to double-stranded DNA. C. Probes that are complementary to regions in the viral genome containing a fluorescent dye are added. During PCR amplification, DNA polymerase releases this dye. D. Fluorescence is monitored as a function of PCR cycle. If fluorescence increases above a certain threshold, the sample is considered to have viral RNA.

    The problem with antibodies for monitoring SARS-CoV-2

    Antibody tests measure the levels of SARS-CoV-2-specific antibodies in the blood, meaning they can provide some information on the immune response to SARS-CoV-2. Antibody-based tests detect immune responses to specific parts of the virus, which vary depending on the test used. These tests may detect the spike (S), nucleocapsid (N), or a combination of multiple viral antigens. Because antibodies remain in the bloodstream after the virus is cleared, antibody levels, or titers, can be used to determine if a person was previously infected or exposed to SARS-CoV-2, even if they didn’t develop symptoms. 

    However, recent data have shown that seroconversion (the point at which antibodies become detectable in the blood) and antibody titers can be variable and inconsistent across COVID-19 patients. In a study of 285 patients with COVID-19, the median time from symptom onset to seroconversion was 13 days; however, this ranged from 4 to 22 days (Figure 2). Antibody titers can vary more than 20-fold across patients, with higher titers often correlating with disease severity. (1,2)

    Image showing data on how IgG/IgM seroconversion is highly variable in response to SARS-CoV-2

    Figure 2. The IgG/IgM seroconversion is highly variable in response to SARS-CoV-2. Adapted from Long Q-X, et al. (4)

    Additional studies have shown that some patients with COVID-19 never seroconvert. A longitudinal analysis of 177 individuals who tested positive for SARS-CoV-2 by RT-PCR showed that up to 8.5% never developed antibodies, even weeks after infection. (3) Finally, the persistence of antibody titers is short-lived. Approximately 40% of asymptomatic patients and 12.9% of symptomatic patients lost seropositivity within 8 weeks. (4) 

    Due to the observed variable nature of the antibody response in COVID-19, antibody titers may not be the best factor for determining if an individual has been exposed to SARS-CoV-2 or whether they have developed immunity or not.

    Measuring T cells: A better way to assess the immune response?

    The more robust way to measure the immune response to SARS-CoV-2 is to monitor virus-specific T cells. T-cell responses appear earlier than antibodies and can be detected approximately one week after the onset of COVID-19 symptoms. (5) T-cell responses are also more uniform and consistent than antibody levels across COVID-19 patients. Multiple reports have shown that COVID-19 patients who never seroconvert all have measurable T-cell responses. In one study, 100% of SARS-CoV-2-positive patients had a CD4+ T-cell response, and 70% had a CD8+ response. (6) These responses were seen across disease severity levels, including in asymptomatic patients. Strikingly, T-cell responses were still robust and measurable up to 6 months after SARS-CoV-2 infection or exposure, whereas antibody titers may never develop or can fade after just 8 weeks. (7,8) Finally, T cells respond to more than twice the number of viral antigens as antibodies. While antibody responses are generally limited to a few viral structural proteins, T cells develop responses across the viral genome (Figure 3). This makes T cells a more specific, robust, and comprehensive measure of the immune response. (6)

    Image showing the different viral targets of T cells in COVID 19

    Figure 3. T-cell responses target viral targets across the viral genome whereas antibody responses are typically limited to a few viral structural proteins. Approximately 75% of antibody targets are also recognized by T cells. Based on data from Grifoni et al. and adapted from Poland et al. (6,9)

    Together, these studies show that the T-cell response in SARS-CoV-2-infected individuals appears sooner, is less variable, lasts longer, and is more comprehensive than antibodies, making it a more robust method for monitoring infection and the immune response (Figure 4). For more on the role of T cells in COVID-19, view this webinar.

    Comparison of the antibody-based detection, T-cell-based detection and PCR/Viral Antigen based detection of SARS-CoV-2

    Figure 4. Comparison of PCR/Antigen, antibody-based, and T-cell-based detection of SARS-CoV-2. Based on data from Gallais et al., Peng et al., Snyder et al., Subbarao et al., Channappanavar et al., and Zuo et al.  (8, 10–14)

    Methods for monitoring the immune response in COVID-19  

    Antibody-based tests

    There are several methods to measure antibody titers. While we discussed above some limitations in using antibody titers in monitoring immune responses in COVID-19 patients, these tests are further limited in that they often measure antibodies only against particular known antigens, not the virus as a whole. (15)

    ELISpot

    A common way to measure T-cell response is enzyme-linked immunosorbent spot (ELISpot). ELISpot has historically been used in vaccine research to measure the immune response by detecting specific proteins secreted by immune cells (Figure 5). ELISpot suffers from low sensitivity and, like antibody-based testing, is often limited to specific known antigens. This may underreport immune responses as research has shown that ~50% of CD4+ T-cell responses were against antigens other than the commonly studied spike protein and may be missed by ELISpot assays. (6) Another distinct disadvantage of ELISpot is that it relies on functional T-cell responses and therefore requires viable T cells, limiting standardization and throughput. Therefore, the reliability of the results is highly dependent on proper sample handling, transport, and storage.

    Overview of ELISpot

    Figure 5. Overview of how the ELISpot method works. ELISpot monitors T-cell activation by detecting excreted analytes (e.g. cytokines). A. Analyte-specific antibodies are coated onto a plate. B. Live T cells, antigen-presenting cells and antigen (e.g. SARS-CoV-2 spike protein/peptide) are added and incubated to allow T-cell activation and analyte secretion to occur. C. Cells and stimuli are removed, and a secondary biotinylated antibody is added. D. ‘Spots’ are visualized via fluorescence by binding of avidin- or streptavidin-conjugated enzyme to the secondary antibody. 

    Immune repertoire sequencing

    A more comprehensive way to monitor T-cell response is via T-cell repertoire sequencing. This method can assess the entire T-cell repertoire, allowing evaluation of high-level repertoire metrics and individual clone-level data in single samples or longitudinal series. The immunoSEQ® Assay is an immune repertoire sequencing technology that uses multiplex PCR to accurately, quantitatively, and reproducibly measure the T-cell immune response (Figure 6). 

    The CDR3 region of the T-cell receptor.

    Figure 6. The immunoSEQ Assay uses multiplex PCR and Illumina sequencing to sequence the CDR3 region of the T-cell receptor. 

    The immunoSEQ Assay is highly sensitive, with a resolution down to 1 x 10-6 cells. It amplifies genomic DNA from a range of samples, including peripheral blood mononuclear cells (PBMCs), whole blood, or fresh or formalin-fixed paraffin-embedded (FFPE) tissue. The ability to assess the entire T-cell repertoire, not just T cells that respond to known targets, is especially crucial given that T-cell receptors that are stimulated by viral components other than the spike glycoprotein have been identified. These notably include the matrix and nucleocapsid proteins along with other open reading frames (ORFs). (6) Since the T-cell response to SARS-CoV-2 lasts for months—in contrast to antibodies—T-cell profiling can map, track, and monitor the T-cell responses to assorted SARS-CoV-2 variants over time. (16) The immunoSEQ Assay combined with this groundbreaking insight into T-cell specificity in the context of COVID-19 is available to researchers as immunoSEQ® T-MAP™ COVID.

    Table 1: Methods to detect immune responses to SARS-CoV-2

    Antibody-based testELISpotimmunoSEQ T-MAP COVID
    What it detectsAntibodies against SARS-CoV-2Proteins secreted by immune cellsT-cell response
    Sample typeBloodFresh cellsFlexible sample input, including FFPE
    Number of detectable viral epitope targets1–2 specific targets (IgG/IgM)2–3100s of viral targets recognized by complete T-cell repertoire
    Quantitative assessment?NNY
    Longitudinal assessment?NNY
    Other considerations
    • Cannot capture the comprehensive immune response
    • Cross-reactivity with other targets
    • Antibody response may wane over time
    • Sample processing and storage can be challenging
    • May not be scalable for large-scale studies
    • May not be accurately reproducible
    • Can map, track, and assess T-cell responses to SARS-CoV-2 to specific antigens
    • Provides a comprehensive view of the T-cell immune response
    • Reproducible and scalable

    FFPE, formalin-fixed paraffin-embedded; IgG, Immunoglobulin G; IgM, Immunoglobulin M; N, No; Y, Yes.

    Profiling the T-cell repertoire in COVID-19 with the immunoSEQ T-MAP COVID

    The power and utility of the immunoSEQ Assay in studying the T-cell response in COVID-19 is highlighted in a recent study by Snyder et al. (12) Here, the immunoSEQ Assay was used to characterize the CD8+ and CD4+ T-cell repertoires of ~1,000 blood samples from COVID-19 patients, collected as part of the ImmuneCODE™ project. To assess the T-cell response dynamics over time, investigators segmented patients based on the number of days since their last PCR-confirmed diagnosis. Consistent with previous studies of T-cell dynamics, the authors showed that most patients experience an expansion of their T-cell repertoire, peaking between days 8 and 28 before contracting slightly. Notably, samples taken from patients up to 43 days after diagnosis still showed higher SARS-CoV-2-specific clones, demonstrating the persistence of the T-cell response.

    Snyder et al. also identified public enhanced T-cell receptor (TCR) sequences shared in most patients with COVID-19, but not in control subjects. These sequences may be a useful biomarker for determining past infection. The authors compared the ability of this T-cell signature with antibody serology testing to identify COVID-19 in patients enrolled in ImmuneRACE (a prospective virtual study that is enrolling patients who were exposed to or actively infected with, or who had recovered from, SARS-CoV-2 across the United States). 

    Among 100 subjects for whom immunoSEQ Assay and serology assays were performed, the T-cell signature had the highest rate of positivity for detecting SARS-CoV-2 infection (Figure 7).

    Positivity rates for previous SARS-CoV-2 infection as assessed by T-cell signature, a multi-antibody serology test, and an IgG antibody test

    Figure 7. Positivity rates for previous SARS-CoV-2 infection as assessed by T-cell signature, a multi-antibody serology test, and an IgG antibody test among 100 COVID-19 patients. Based on data from Snyder et al. (12)

    In addition, of the 23 subjects who enrolled shortly after being exposed to SARS-CoV-2, only one positive infection was determined by serology testing, while two were classified as positive using the T-cell signature. Both subjects identified using the T-cell signature later tested positive by RT-PCR. These results are based on a small number of subjects, but they suggest that T-cell-based approaches may be more consistent with RT-PCR results than antibody serology in determining SARS-CoV-2 infection and may be better able to determine recent infection.

    A second study by Gittelman et al. also demonstrates that T-cell responses can provide a sensitive, reliable, and persistent way to measure past SARS-CoV-2 infection. (17) The authors analyzed T-cell and antibody signatures in over 2,200 individuals in Vo’, Italy. In patients with a confirmed PCR diagnosis, T-cell responses were detected by the immunoSEQ Assay up to 60 days after diagnosis in 97% (68/70) of subjects. In comparison, only 77% (54/70) of patients still had detectable antibodies at this time. T-cell responses also identified 45 additional suspected infections (Figure 8). Individuals who reported symptoms or were exposed to someone with a PCR-confirmed diagnosis were more likely to demonstrate a positive T-cell response. (17)

    A T-cell signature identified with the immunoSEQ Assay was more sensitive and specific than antibody-based tests, highlighting the role of T cells in COVID-19.

    Figure 8. A T-cell signature identified with the immunoSEQ Assay was more sensitive and specific than antibody-based tests. In one study, the T-cell signature correctly identified 97% of COVID-19 patients at 2 months after a confirmed PCR diagnosis and 95% of patients at 5 months or more after diagnosis. In comparison, leading antibody tests correctly identified 77–97% of patients at 2 months and 52–71% of patients at 5 or more months. The specificity of the T-cell signature was >99%. Based on data from Gittelman et al. and Dalai et al. (17, 18) *Adaptive Data on File

    Assessing and monitoring immunity in COVID-19 summarized

    Monitoring T cells in COVID-19 is a robust way to measure and monitor infection response given the early, lasting, and consistent nature of the T-cell response compared with antibody production. While several well-known methods for assessing and measuring T cells exist, the immunoSEQ Assay offers a reproducible, quantitative, and powerful method to evaluate and track the T-cell repertoire in all types of COVID-related research.

    Along with the immunoSEQ Assay, Adaptive Biotechnologies has developed a suite of tools to help researchers work with the data. The immunoSEQ Analyzer allows researchers to analyze, visualize, compare, and share their data. immunoSEQ T-MAP COVID provides not only the SARS-CoV-2-specific T-cell sequences, but also full repertoires of patients exposed to, infected with, or recovered from COVID-19, all of which are incorporated into the Analyzer for easy, seamless analysis. Please reach out to Adaptive Biotechnologies to see if immunoSEQ T-MAP COVID can help you address your research needs. Scientists are available to help with everything from experimental design to data analysis and publication support.

    For Research Use Only. Not for use in diagnostic procedures.

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