T cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses

Journal/preprint name: medRxiv 

Paper DOI: https://doi.org/10.1101/2020.09.28.20202929 

Tags:  Immunology/Immunity, Clinical, T cells 

Summary

Ogbe et al developed T cell assays to distinguish between SARS-CoV-2 specific responses and cross-reactive responses. Strong ex vivo ELISPOT and proliferation responses to multiple antigens (including M, NP and ORF3) were found in those who had been infected by SARS-CoV-2. Unexposed subjects had a more restricted proliferative response to spike antigens only, (likely a cross-reactive response to common cold coronaviruses). Seronegative doctors with high occupational exposure and recent COVID-19 compatible illness had broad T cell responses, indicating that a T cell response to SARS-CoV-2 can be detected after infection even in the absence of antibodies. Memory responses to specific non-spike proteins provide a method to distinguish between recently infected individuals and those with pre-existing immunity in exposed populations. 

Research Highlights: 

1. High magnitude and broad T cell responses to SARS-CoV-2 structural proteins are detected in convalescent subjects by ex vivo ELISPOT and T cell proliferation assays 

2. In contrast, the T cell proliferation assay demonstrated memory T cell responses only to spike protein in the T cell repertoires of unexposed, SARS-CoV-2 seronegative individuals (but not M, NP, ORF3, ORF6, ORF7 or ORF8). 

3. In convalescent patients, T cell responses to either M or NP correlate with the global response to spike, structural and accessory proteins indicating that an assay to measure responses to M or NP could reflect the global effector T cell response.  

4. IFNγ responses (measured by ELISPOT) appear to peak a month after symptom onset and then tend to decline 

5. Symptoms, particularly self-reported fever, were associated with stronger memory T cell responses in recovery 

6. A high frequency of proliferating cells and broad targeting of SARS-CoV-2-specific CD4+ and CD8+ T cells were identified in a FACS-based assay of central memory T cell responses. This assay appeared more sensitive than ELISPOT 

7. SARS-CoV-2-specific CD4+ T cells (and to a lesser extent CD8+ T cells) were polyfunctional in recovery. 

8. Using a T cell proliferation assay, T cell responses to SARS-CoV-2 proteins with high breadth and magnitude were detected in convalescent doctors that were seronegative, indicating that a memory T cell response may exist in the absence of a T cell response. The most sensitive assays in this case was M and/ or NP-specific T cell responses in the proliferation assay. 

9. Significantly higher magnitude of CD4+ but not CD8+ T cells proliferating in response to the M, N, ORF3, 6, 7 and 8 was detected in these doctors. 

Impact for COVID-19 research:  

• The study provides an insight to different ex-vivo assays to measure T cell responses and show for the first time an optimized method to distinguish between SARS-CoV-2 specific T cell responses from pre-existing cross-reactive responses to other coronaviruses 

• Suggest central memory T cell responses to M and NP in the proliferation assay as the best marker of SARS-CoV-2 specific response. 

• Lays the groundwork for assessing T cell responses to SARS-CoV-2 vaccines 

Methodologies: 

• Key Techniques:  

1. IFN-γ enzyme-linked immunospot (ELISPOT) assay  

2. Intracellular cytokine stimulation (ICS) assay  

3. Proliferation assay using CellTrace® Violet 

4. Flow cytometry analysis 

5. ELISA 

6. Lactate measurements 

7. SARS-CoV-2 pseudotype micro-neutralisation assay 

• Assays were validated between multiple groups 

• Large cohorts were used 

 Limitations: 

• The correlation data for T cell responses in M or NP protein and other structural and accessory parts included donors with low responses which may limit the conclusion. 

• There are isolated examples of seronegative 2020 controls which appear to have a SARS-CoV-2 specific response