The SARS-CoV-2 pandemic poses a serious threat to the world population with dramatic socioeconomic consequences. Immunity after SARS-CoV-2 infection is crucial for individual long-term protection upon virus re-exposure, but even more important to reduce transmission rates and ultimately achieve herd immunity. Moreover, elucidation of the immunological mechanisms underlying the potential development of protective long-term immunity in the course of COVID 19 will guide the design of effective SARS-CoV-2 vaccines and treatment.
Long-term immunity is generally mediated by the adaptive immune system. Memory B and T cells persist after infection and enable more rapid and effective responses upon re-challenge with the same pathogen (1). However, the persistence of cellular and humoral immunological memory differs between pathogens, and experience with the other two zoonotic coronaviruses – SARS CoV-1 and MERS-CoV – revealed early loss of humoral immunity (2, 3). So far, data on long-term immunity to SARS-CoV-2 are lacking. Available reports, up to three months after COVID-19 are partially conflicting, but overall point towards a decrease and even loss of SARS-CoV-2-specific antibody responses (4-8) and thus raise concerns regarding long-term humoral immunity. In SARS-CoV-1, T cell immunity was identified as important determinant for recovery and long-term protection (9-12), with long-lasting memory T cell responses detected in convalescents even 17 years after infection (13). Likewise, T cell immunity also appears to play a key role in COVID-19, with several studies reporting on T cell responses in acute infection up to three months after convalescence. This comprises evidence for potential preexisting immunity mediated by cross-reactive T cells to human common cold coronaviruses (6, 13-20). We and others recently characterized the T cell epitopes mediating these specific and cross-reactive SARS-CoV 2 T cell responses in convalescents as well as unexposed individuals and provided evidence that the development of immunity requires recognition of multiple epitopes (13, 15, 16, 20-22). In the light of the available data on SARS-CoV-2 immune responses, persistence of SARS-CoV-2 T cell immunity may be crucial for long-term protection after COVID-19, with respective consequences for vaccine development. Here we conducted the first longitudinal analysis comparing T cell and antibody responses in SARS-CoV-2 convalescents up to six months post infection. We report on the differential kinetics of cellular and humoral immunity after COVID-19 and delineate dominant T cell epitopes for long-term immunity.
Longitudinal follow-up of SARS-CoV-2 convalescents
Clinical and immunological analysis of convalescents after mild or moderate SARS-CoV-2-infection (SARS donors, n = 51, Tables S1 and S2) was conducted 35 - 56 days (median 40 days, time point 1, T1) (21) and 141 - 183 days (median 159 days, time point 2, T2) after positive SARS-CoV-2 PCR (Fig. 1A). Persisting or newly arisen post-infectious symptoms were reported by 27% of SARS donors at T2, with fatigue (64% of symptomatic donors) as well as anosmia and ageusia (64% of symptomatic donors) being most common (Fig. 1B, Table S1). Kinetics of SARS-CoV-2-directed T cell immunity was determined longitudinally with regard to both, (i) intensity (SARS group A, n = 29) and (ii) diversity (percentage of detected T cell epitopes per donor; SARS group B, n = 23) of CD4+ and CD8+ T cell responses (Fig. 1C). To standardize determination of changes in SARS-CoV-2 T cell response intensity over time, we employed broadly applicable human leukocyte antigens (HLA) class I and HLA-DR SARS-CoV-2 epitope compositions (EC) comprising dominant (recognized by ≥ 50% of SARS donors) and subdominant (recognized by < 50% of SARS donors) specific T cell epitopes recognized exclusively in COVID-19 convalescents or cross-reactive T cell epitopes recognized by both, convalescents and individuals never exposed to SARS-CoV-2 (Table S3) (21). The number of convalescents with detectable SARS-CoV-2 T cell responses was found to increase over time. In detail, the percentage of donors with detectable T cell responses to SARS-CoV-2-specific EC (HLA class I: 45% T1 vs. 69% T2; HLA-DR: 90% T1 vs. 100% T2; Fig. 1D) as well as to cross-reactive EC (HLA class I: 31% T1 vs. 38% T2; HLA-DR: 93% T1 vs. 100% T2; Fig. 1E) increased from 93% (T1) to 100% (T2) as assessed by ex vivo IFN-g ELISPOT assays (Fig. 1, D and E).
Intensity of SARS-CoV-2 T cell responses over time
Longitudinal ex vivo IFN-g ELISPOT analysis of T cell responses at T1 and T2 with standardized EC (SARS group A, n = 29, Fig. 2A, Fig. S1) revealed robust intensities of HLA class I SARS-CoV‑2-specific and cross-reactive T cell responses (median calculated spot counts: 20 (T1) vs. 18 (T2) and 74 (T1) vs. 61 (T2), respectively), whereas the intensities of SARS-CoV-2 T cell responses to HLA-DR specific and cross-reactive EC (median calculated spot counts: 29 (T1) vs. 53 (T2) and 35 (T1) vs. 75 (T2), respectively) was significantly increased over time (Fig. 2, B and C). A high inter-individual heterogeneity of longitudinal T cell response intensity was observed (52% and 45% of donors with new or ≥ 2-fold increased T cell responses, 24% and 45% with stable (fold-change 0.6-1.9) T cell responses, as well as 24% and 9% showed ≥ 2-fold decreased or lost T cell responses to HLA class I specific and cross-reactive EC at T2, respectively (Fig. 2D, Fig. S2A)). For HLA-DR, longitudinal increase of T cell response intensity in individual donors was even more pronounced (66% and 55% of donors with new or ≥ 2-fold increased T cell responses, 24% and 31% with stable (fold-change 0.6-1.9) T cell responses, as well as 10% and 14% showed ≥ 2-fold decreased or lost T cell responses to HLA-DR specific and cross-reactive EC at T2, respectively (Fig. 2E, Fig. S2B)). Interestingly, the three SARS donors showing the most pronounced decrease of T cell responses to the HLA-DR specific EC all still suffered from post-infectious symptoms (Fig. 2E). Characterization of long-term SARS-CoV-2-directed T cells at T2 using ex vivo flow cytometry-based assessment of surface markers and intracellular cytokine staining (ICS) revealed that T cell responses to HLA class I cross-reactive EC were predominantly mediated by CD8+ T cells, whereas T cell responses to HLA-DR specific and cross-reactive EC were mainly mediated by CD4+ T cells. The vast majority of T cell responses to HLA class I SARS-CoV-2-specific EC was mediated by both, CD8+ and CD4+ T cells (Fig. 2, F and G, Fig. S3). CD8+ T cells targeting HLA class I specific EC were mainly positive for CD107a, whereas CD4+ and CD8+ T cells targeting HLA-DR SARS-CoV-2-specific as well as HLA-DR and HLA class I cross-reactive EC displayed positivity for several of the markers IL-2, TNF, IFN-g, and CD107a (Fig. 2, H and I, Fig. S3).
Dynamics of SARS-CoV-2 antibodies in relation to T cell responses and post-infectious clinical status
Two independent assays were employed to longitudinally assess SARS-CoV-2 antibody responses in convalescents (n = 51) at T1 and T2 to determine (i) ratios of IgG and IgA antibodies targeting the S1 domain of the spike protein including the immunologically relevant receptor binding domain (RBD, EUROIMMUN; Fig. 3, A and B, Fig. S4, A and B) as well as (ii) anti-nucleocapsid antibody titers (Elecsys® immunoassay including IgG; Fig. 3C, Fig S4C). Both, anti-S1 IgG and IgA response significantly decreased over time (median 3.8 vs. 2.6 and 2.6 vs. 1.6, respectively; Fig. 3, A and B, Fig. S4, A and B), whereas anti-nucleocapsid antibody titers remained stable from T1 to T2 (median 29 vs. 25; Fig. 3C, Fig. S4C). Loss or ≥ 2-fold decrease of anti-S1 IgG and IgA was observed in 31% and 44% of SARS donors, respectively, whereas loss or ≥ 2-fold decrease of anti-nucleocapsid antibody titers was documented in only 13% of convalescents. Among the convalescents still suffering from post-infectious symptoms at T2, 36% (5/14) and 50% (7/14) presented with ≥ 2-fold decrease or loss of anti-S1 IgG and IgA, respectively, whereas none showed ≥ 2-fold decrease of anti-nucleocapsid antibody titers (Fig. 3, D to F). Anti-S1 IgG antibody responses moderately correlated with the intensity of T cell responses to SARS-CoV-2-specific and cross-reactive HLA-DR EC as well as cross-reactive HLA class I EC at T2 (Fig. S5). Longitudinal T cell and antibody responses as well as symptoms during and after COVID-19 varied among the donors (Fig. 3G). Neither the intensity of SARS-CoV-2-specific nor that of cross-reactive T cell responses to HLA class I or HLA-DR EC at T2 correlated with demographics (gender, age, or BMI; Table S4). High anti-nucleocapsid antibody titers at T2 were associated with a higher prevalence of post-infectious symptoms (Fig. 3, H and I). In contrast, neither intensity nor longitudinal kinetics of SARS-CoV-2 T cell responses were associated with post-infectious symptoms (Fig. 3, J and K).
Diversity of SARS-CoV-2 T cell immunity identifies epitopes mediating long-term T cell responses
In various viral diseases including SARS-CoV-2, diversity of T cell responses, i.e. the recognition of multiple T cell epitopes, has been implicated as a prerequisite for effective immunity (21, 23). We longitudinally analyzed the diversity of SARS-CoV-2 T cell responses by single epitope mapping using dominant and subdominant HLA-DR (n = 20) and HLA-A*24 (n = 6) SARS-CoV-2 T cell epitopes (21). To enable detection of low-frequent peptide-specific T cell populations, we used in vitro 12-day pre-stimulation for expansion of peptide-specific T cells. Longitudinal diversity of HLA-DR and HLA-A*24 T cell responses decreased across all donors and T cell epitopes over time (median T cell recognition per donor 59% and 50% at T1, 48% and 17% at T2, respectively; Fig. 4A, Fig. S6A). The decrease in HLA-DR T cell diversity was confirmed in subgroup analyses for specific and cross-reactive (Fig. S7A), dominant and subdominant T cell epitopes (Fig. S7B) as well as for epitopes derived from structural or non-structural (Fig. S7C) and nucleocapsid vs. non-nucleocapsid viral open reading frames (ORF, Fig. S7D). However, donor- and epitope-specific assessment identified a subset of T cell epitopes derived from different ORFs, which sustain a persisting T cell response (10/20 HLA-DR T cell epitopes; 2/6 HLA-A*24 T cell epitopes; Fig. 4, B and C, Fig. S6, B and C, Tables S5 and S6). In particular the eight dominant T cell epitopes (7 HLA-DR T cell epitopes, 1 HLA-A*24 T cell epitope) identified to mediate a persisting T cell response in up to 100% of convalescents appear to be essential for long-term T cell immunity to SARS-CoV-2 and may thus enable the development of effective vaccination approaches.