High titre neutralizing antibodies in response to SARS–CoV–2 infection require RBD–specific CD4 T cells that include proliferative memory cells

Abstract

BackgroundLong-term immunity to SARS-CoV-2 infection, including neutralizing antibodies and T cell-mediated immunity, is required in a very large majority of the population in order to reduce ongoing disease burden.MethodsWe have investigated the association between memory CD4 and CD8 T cells and levels of neutralizing antibodies in convalescent COVID-19 subjects.FindingsHigher titres of convalescent neutralizing antibodies were associated with significantly higher levels of RBD-specific CD4 T cells, including specific memory cells that proliferated vigorously in vitro. Conversely, up to half of convalescent individuals had low neutralizing antibody titres together with a lack of receptor binding domain (RBD)-specific memory CD4 T cells. These low antibody subjects had other, non-RBD, spike-specific CD4 T cells, but with more of an inhibitory Foxp3+ and CTLA-4+ cell phenotype, in contrast to the effector T-bet+, cytotoxic granzymes+ and perforin+ cells seen in RBD-specific memory CD4 T cells from high antibody subjects. Single cell transcriptomics of antigen-specific CD4+ T cells from high antibody subjects similarly revealed heterogenous RBD-specific CD4+ T cells that comprised central memory, transitional memory and Tregs, as well as cytotoxic clusters containing diverse TCR repertoires, in individuals with high antibody levels. However, vaccination of low antibody convalescent individuals led to a slight but significant improvement in RBD-specific memory CD4 T cells and increased neutralizing antibody titres.InterpretationOur results suggest that targeting CD4 T cell epitopes proximal to and within the RBD-region should be prioritized in booster vaccines.

T‐cell activation–induced marker assays in health and disease

“…Unlike CD40L, which is biased toward expression on T-helper type 1 (Th1) cells, OX40 is expressed equally on all Th cells, including Tregs, potentially reducing bias when analyzing antigen-specific T cells. 16,23,24 It is important to note that OX40 is highly expressed on murine FOXP3 + Tregs,…”

“…101 A study of long-term immunity to SARS-CoV-2 found that increased levels of S receptor-binding domain-specific CD25 + OX40 + CD4 + T cells associated with higher neutralizing antibody titers, with single-cell transcriptomics revealing a heterogenous population. 24 More recently, AIM assays have been used to determine vaccine-induced protection against omicron and other emerging SARS-CoV-2 variants. The CD25/ OX40 assay indicated that individuals receiving ChAdOx-1S, Ad26.COV2.S, mRNA-1273 or BNT162b2 vaccines had no significant differences in the level of antigenspecific T cells when stimulated with ancestral spike protein as compared with delta, beta and omicron variant spike proteins, while antibody levels against omicron were significantly decreased.…”

“…OX40 signaling plays a vital role in T‐cell expansion and survival, as well as T‐helper cell differentiation. Unlike CD40L, which is biased toward expression on T‐helper type 1 (Th1) cells, OX40 is expressed equally on all Th cells, including Tregs, potentially reducing bias when analyzing antigen‐specific T cells 16,23,24 . It is important to note that OX40 is highly expressed on murine FOXP3 + Tregs, making it unsuitable for analysis of peripheral murine CD4 + T cells 25 …”

“…However, CD39 alone also identifies a second population that is FOXP3 neg and produces high levels of IFN‐γ and IL‐17A 61 . CD39 + Treg identification may prove to be a useful alternative to intracellularly staining for FOXP3, especially if CD25 is included in the assay 24 . In addition, it is possible to identify circulating Tfh (cTfh) based on CXCR5 and PD‐1 expression 29 .…”

“…AIM assays using CD40L have identified that, following COVID‐19 vaccination, Th1 and Tfh cells are expanded, with the latter correlating with protective humoral immunity 101 . A study of long‐term immunity to SARS‐CoV‐2 found that increased levels of S receptor–binding domain–specific CD25 + OX40 + CD4 + T cells associated with higher neutralizing antibody titers, with single‐cell transcriptomics revealing a heterogenous population 24 …”