Detection of prior SARS-CoV-2 infection by T-cell receptor sequencing

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019, ultimately causing a rapidly spreading wave of infections that affected the world. Spanning a wide spectrum of disease severity, from asymptomatic to a fatal illness, the virus has eluded research into the pathophysiology of coronavirus disease 2019 (COVID-19).

The large-scale rollout of COVID-19 vaccines was intended to protect the population from the most severe symptoms of COVID-19 and related death. However, the need to distinguish vaccine-induced immunity from that elicited by natural infection and by both prior infection and vaccination has led researchers to investigate new methods to diagnose prior COVID-19.

A new JCI Insight study describes a novel technique based on T-cell receptor sequencing.

Study: T-cell Receptor Sequencing Identifies Prior SARS-Cov-2 Infection And Correlates with Neutralizing Antibodies And Disease Severity. Image Credit: Panggabean / Shutterstock.com

Introduction

SARS-CoV-2 initiates both antibody-mediated and T-cell immune responses; however, the relative contribution of each immune response varies with the variant responsible for the infection. Serology is a widely used method to detect prior infection at the population level; however, it is not clear whether seropositivity correlates with durable protective immunity.

Neutralizing antibody assays are better associated with protective immunity but are difficult to perform, require handling of the dangerous live virus, and wane over time in many cases. This has led researchers to instead rely on tests that utilize T-cell receptor (TCR) repertoires to examine the type and extent of immunity elicited by prior infection.

While the vast majority of TCRs are unique to the individual, less than 1% are common to many individuals, especially those who have shared human leukocyte antigens (HLA) alleles. These public TCRs could be due to common antigens.

Using thousands of public TCRs from individuals previously infected with cytomegalovirus, Lyme disease, and SARS-CoV-2, the researchers in this study previously demonstrated that specific classification criteria were present to identify the condition of interest.

Earlier, these scientists found that the TCR classifier for SARS-CoV-2 infection could characterize the T-cell response with respect to magnitude and direction of change. To this end, symptomatic disease was associated with a higher rise in the T-cell response as compared to asymptomatic infection, and the rise was correlated with severity of disease.

In the present study, the researchers compared TCR testing to other techniques using samples collected for up to six months after symptom onset. Based on their earlier demonstration that SARS-CoV-2 immunoglobulin G (IgG) antibodies correlated well with neutralizing antibody titers, the researchers further sought to examine the relationship between cellular and humoral immune responses, as well as between cellular immune responses and clinical disease.

Both the clonal breadth, which represents the number of unique TCRs associated with SARS-CoV-2 among the total repertoire, as well as clonal depth, which is the degree to which these TCRs expanded following infection, were examined. TCRs to the corresponding viral antigens and HLA restrictions were also tracked and correlated with disease severity.

Finally, the researchers compared the detection results of prior infection using TCRs to serology results among both hospitalized and outpatients for up to six months from the first symptoms.

Study findings

The study included about 300 individuals. Among these samples, the clonal breadth and depth were positively correlated with the neutralizing antibody titers.

Additionally, different TCRs could be correlated to the spike, nucleocapsid (NC), and other viral proteins. HLA class II TCRs were related to the spike and NC proteins in both breadth and depth, both of which are related to CD4+ helper T-cells.

Conversely, there were no correlations between class I-associated CD8+ cytotoxic T-cells. This indicates that the CD4+ helper T-cells correlate with the neutralizing antibody response rather than the CD8+ T-cells.

CD8+ T-cells were also activated and showed a more than 10-fold rise in the clonal breadth of virus-specific TCRs as compared to other mononuclear cells. This suggests that the identified TCRs, which responded to the virus when presented by antigen-presenting cells, were functional. The increased clonal depth and breadth of these SARS-CoV-2-specific TCRs also correlated with severe COVID-19.

Virus-specific TCR testing was found to be more sensitive for prior SARS-CoV-2 infection than serologic testing, especially in mild to moderate outpatients and at three or more months from the onset of symptoms. In hospitalized patients, both testing methods showed equivalent detection rates of 93%.

The sensitivity was about 90% for all cases but slightly higher for more severe cases. Even five or more months from symptom onset, more than 95% of samples showed positive results on the assay.

As compared to serologic testing, the TCR assay was more sensitive, with the greatest difference being at five or more months from illness onset. Serologic testing appeared to decrease in sensitivity faster than TCR-based testing, more so in milder cases. This agrees with the known loss of antibodies, or seroreversion, that has been described in some earlier studies.

In fact, some convalescent patients with undetectable antibodies and neutralizing antibodies, most of whom were non-hospitalized, were positive for virus-specific TCRs. This corroborates earlier findings that some patients, especially with mild disease, produce a T-cell response despite the lack of a detectable antibody response.

Implications

The current study shows the utility of TCR sequencing for the diagnosis of prior SARS-CoV-2 infection. The findings also show that durable and strong T cell responses occur and are correlated with the severity of illness. Moreover, these responses are found even in individuals with mild illness who failed to seroconvert or seroreverted.

The class II-associated TCRs on CD4+ T-cells responding to the spike and NC protein antigens are likely responsible for the correlation between the TCR response and neutralizing antibody titers. These cells facilitate the development of a functional antibody response and can predict the development of humoral and CD8+ cytotoxic T-cell responses to two doses of a COVID-19 vaccine.

The researchers also developed a TCR classifier that could differentiate between T-cell responses produced by a vaccine vs natural infection.

We expect that most T-cell responses will not be substantially affected by viral strain variations as the majority of antigens do not overlap with sites of variation.”

This is important for future vaccination strategies and for exploring the immunogenicity of vaccines.

The increased T-cell responses in more severe disease may also contribute to the heightened protective immunity against emerging variants of SARS-CoV-2 in those who have recovered from severe as compared to mild COVID-19. The mechanisms underlying this protection may include a higher viral load, a longer period when the virus is present in the body, or a more intense immune response in severe illness.

The increased sensitivity of the TCR assay for the diagnosis of prior SARS-CoV-2 for six months from symptom onset compared to serologic assays is also shown by these results. The investigators have already validated a similar assay, named T-Detect, for this purpose, that claims 100% specificity and high sensitivity, without cross-reactivity, and produces results better than equivalent commercial serologic tests.

The better performance of the test in outpatients is heartening, as it provides an opportunity to identify past infections in real-world settings. Moreover, this test can help broaden the current understanding of the host immune response to COVID-19. Finally, the superior ease and safety of the test commend it to broader use, despite its higher cost and sophistication.