CTL酶联免疫荧光斑点分析仪应用新发表关于“COVID”研究的文献

Fueled additionally by evidence that T cell-mediated immunity is required for immune protection

against SARS-CoV-2 5-7, attention has turned to T cell immunodiagnostics trying to establish whether the

detection of T cell memory may be a more sensitive and reliable indicator of SARS-CoV-2 exposure than

antibodies 8-11. In most studies published so far SARS-CoV-2-specific T cell memory cells were detected in

the majority of infected individuals, but such were also found in 20-81% of control subjects who clearly

could not have been infected by the SARS-CoV-2 virus 9, 12-19. If generalizable, such results would imply

that T cell assays are unsuited to reliably identify who has, or has not, been infected by SARS-CoV-2

providing false positive results in up to 81% of the individuals tested. It should be noted right away,

however, that the notion of cross-reactive SARS-CoV-2 antigen recognition by T cells being common in

unexposed subjects might be related to the T cell assay and the test conditions used as it was not

observed by others 14, 20, 21. Progress with settling the issue of T cell cross-reactivity in SARS-CoV-2

antigen recognition, and identifying suitable test systems, will decide whether T cell diagnostics can

reliably detect specific immune memory to SARS-CoV-2 infection/exposure, and possibly identify the

immune protected status of those subjects.

Next to possible cross-reactivity, T cell immune diagnostics of SARS-CoV-2 infection faces the challenge

of having to reliably detect antigen-specific T cells in blood that occur in very low frequency. The

numbers of SARS-CoV-2-specific T cells in blood post-infection is about one tenth of the numbers of T

cells specific for viruses that induce strong T cell responses, such as influenza, Epstein Barr- (EBV) or

human cytomegalovirus (HCMV) 11, 22, and reliably detecting even the latter is at the border of current

technology. Further complicating matters, the frequencies of SARS-CoV-2-specific T cells are even lower

in subjects who underwent a mild or asymptomatic SARS-CoV-2 infection compared to those who

developed more severe COVID-19 12, 21, 23, 24. Owing to these low T cell frequencies, and the antigen

induced signal being small in magnitude, any contribution of cross-reactive T cell stimulation will

interfere with the reliable detection of genuine SARS-CoV-2-specific T cells. Setting up clear cut-off

criteria for identifying antigen-specific T cell memory is therefore paramount.

Because T cell assays rely upon detecting SARS-CoV-2 antigen-specific T cells in blood via memory T cell

re-activation ex vivo, the choice and formulation of the SARS-CoV-2 antigen itself used for the T cell

recall will critically define the assay result. As the epitope utilization in the T cell response to SARS-CoV-

2 is not known yet, by necessity, the aforementioned T cell diagnostic efforts tailored towards this virus

have relied either on pools of hundreds of peptides that cover the entire proteome of the virus, or on

pools of a multitude of predicted epitopes (mega peptide pools). Traditional T cell immune monitoring

efforts, however, have called for the utilization of select, highly purified individual peptides whose

specificity has been carefully established. Presently it is unproven whether pools of hundreds of

unpurified peptides are even suited for reliable T cell diagnostics, and whether false positive or false

negative results obtained using them are inherent to the recall antigen formulation. The chance for T

cell cross-reactivity can be expected to increase with every peptide added into a pool, multiplying the

chance for false positive results. Conversely, irrelevant peptides (those not recognized by T cells) also

present in the pool can be expected to compete with the actually recognized T cell epitopes for binding

to HLA-molecules, possibly causing false negative results 25. To our knowledge, it has not yet been

systematically addressed whether and how chance cross-reactivity or peptide competition affects T cell

immune monitoring results when mega peptide pools are used for testing. Instead of relying on third

party mega peptide pools as the proper negative control to establish the background noise of the T cell

assay, in all SARS-CoV-2 studies published so far, the mega peptide pool-induced T cell activation has

been compared to PBMC cultured in medium alone, in the absence of any exogenously added peptide. bioRxiv preprint doi: https://doi.org/10.1101/2020.11.29.402677; this version posted November 29, 2020. The copyright holder for this preprint

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

available under aCC-BY-NC-ND 4.0 International license.

In this report we introduce suitable negative control mega peptide pools, and using