key: cord-0311474-t4s25n1g
authors: Kaaijk, P.; Olivo Pimentel, V.; Emmelot, M. E.; Poelen, M.; Cevirgel, A.; Schepp, R. M.; den Hartog, G.; Reukers, D. F. M.; Beckers, L.; van Beek, J.; van Els, C. A. C. M.; Meijer, A.; Rots, N. Y.; de Wit, J.
title: Children and adults with mild COVID-19 symptoms develop memory T cell immunity to SARS-CoV-2
date: 2021-09-15
journal: nan
DOI: 10.1101/2021.09.10.21263333
sha: 4c59911f92f43198703add173d2ee7a15c25a37a
doc_id: 311474
cord_uid: t4s25n1g

Background: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to considerable morbidity/mortality worldwide, but most infections, especially among children, have a mild course. However, it remains largely unknown whether infected children develop cellular immune memory. Methods: To determine whether a memory T cell response is being developed as an indicator for long-term immune protection, we performed a longitudinal assessment of the SARS-CoV-2-specific T cell response by IFN-{gamma} ELISPOT and activation marker expression analyses of peripheral blood samples from children and adults with mild-to-moderate COVID-19. Results: Upon stimulation of PBMCs with heat-inactivated SARS-CoV-2 or overlapping peptides of spike (S-SARS-CoV-2) and nucleocapsid proteins, we found S-SARS-CoV-2-specific IFN-{gamma} T cell responses in most infected children (83%) and all adults (100%) that were absent in unexposed controls. Frequencies of SARS-CoV-2-specific T cells were higher in infected adults, especially in those with moderate symptoms, compared to infected children. The S-SARS-CoV-2 IFN-{gamma} T cell response correlated with S1-SARS-CoV-2-specific serum IgM, IgG, and IgA antibody concentrations. Predominantly, effector memory CD4+ T cells of a Th1 phenotype were activated upon exposure to SARS-CoV-2 antigens, which persisted for 4-8 weeks after symptom onset. We detected very low frequencies of SARS-CoV-2-reactive CD8+ T cells in these individuals. Conclusions: Our data indicate that an antigen-specific memory CD4+ T cell response is induced in children and adults with mild SARS-CoV-2 infection. T cell immunity induced after mild COVID-19 could contribute to protection against re-infection.

Tremendous research efforts have advanced our understanding of immunity to SARS-CoV-2. Most data on the 30 immune response to SARS-CoV-2 was obtained from severe COVID-19 cases [1] [2] [3] [4] . However, the vast majority 31 of infected individuals experience mild symptoms that do not require hospitalization [5] [6] [7] [8] . The question 32 remains whether individuals, including children, with an asymptomatic or mild SARS-CoV-2 infection, develop 33 immune memory which may protect against subsequent SARS-CoV-2 infections. Persons with mild or 34 asymptomatic infections often develop an antibody response, although not all cases do [8] . It has been shown 35 that SARS-CoV-2-induced antibody levels are waning over time [6, [9] [10] [11] . On the other hand, T cell immunity is 36 predicted to persist longer; after SARS-CoV infection in 2003, it was shown that T cell responses can persist for 37 up to 17 years [12] . Some studies investigated the T cell immunity induced after SARS-CoV-2 infection in mild 38 symptomatic adult cases [6, 8, [13] [14] [15] [16] , showing weaker T cell responses in mild than in moderate or severe 39 COVID-19 cases. CD4 + T cell responses against SARS-CoV-2 were more prominent than the CD8 + T cell response 40 in adults with mild-to-moderate infection [8, 15, 16] , while qualitatively impaired CD4 + T cell responses have 41 been reported for critically ill patients [15] . 42

Nevertheless, it remains unclear whether SARS-CoV-2 infection in children, usually showing a mild course, 43 induces substantial T cell immunity. Only a few reports describe the immune responses in children with mild 44 disease or asymptomatic infection, although in these studies T cells specifically reactive to SARS-CoV-2 were 45 not investigated [17] [18] [19] . Recently, a study was published investigating SARS-CoV-2 specific T cell responses in 46 children [20] . Induction of a sustainable T cell response is needed to provide immune memory for long-term 47 protection against re-infections by facilitating an efficient and quick response upon re-exposure. Therefore, 48 knowledge on the induction of memory T cell immunity after a mild course of SARS-CoV-2 infection in children 49 and adults is useful for the consideration of the community mitigation measures needed to protect against 50 COVID-19 and limit the spread of the virus. 51

In the present study, we examined the frequency and the phenotypic/functional characteristics of SARS-CoV-2-52 reactive T cells in infected children and adults with mild to moderate symptoms. In addition, T cell responses 53 correlated with SARS-CoV-2-specific serum IgM, IgG, and IgA antibody concentrations. 54 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted September 15, 2021. ; https://doi.org/10.1101/2021.09.10.21263333 doi: medRxiv preprint 4 was subtracted from total spot numbers induced by antigen-specific stimulation; more than 5 spots, after 81 background subtraction, was considered positive. Supernatants and cells from ELISPOT plates were harvested 82 for cytokine release assay and analysis of activation marker expression by T cells, respectively ( is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint days after T1 (referred to as 'T2') and only for adults also at 4-6 weeks after T1 (referred to as 'T3'). No 106 significant differences in major immune cell types were found over time after infection neither in children nor 107 adults, i.e. frequencies of total T cells, B cells, monocytes, or NK cells were comparable between infected 108 groups and healthy age-matched unexposed groups (Supplementary Figure 1) . 109 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 15, 2021. ; https://doi.org/10.1101/2021.09.10.21263333 doi: medRxiv preprint 6 adults with moderate symptoms. Nevertheless, the IFN-ɣ response of mild symptomatic adults also tended to 125 be slightly higher than infected children ( Figure 1E -F). The pre-existing T cell response against S-HCoV-OC43 of 126 infected children was very low, although S-HCoV-OC43-specific T cell frequency was slightly higher at T2 127 compared to the age-matched unexposed control group (9.0 vs 1.0 SFU/2.10 5 PBMCs; P=0.037) ( Figure 1D , left 128 panel). In infected adults, no difference in numbers of S-HCoV-OC43-specific T cells was found between SARS-129

CoV-2-infected and unexposed adults ( Figure 1D , right panel). In both SARS-CoV-2-infected children and 130 adults, the frequency of IFN-ɣ + T cells was 4 to 60-fold lower after stimulation with S-HCoV-OC43 ( Figure 1D is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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Serum antibody concentrations against the SARS-CoV-2 Spike S1 protein (S1-SARS-CoV-2) above the previously 171 established cutoff level [10] at any of the two sampling time points were found in 83.3% (IgM), 79.2% (IgG), 172 and 75.0% (IgA) of the infected children. From the four children without detectable S-SARS-CoV-2-specific T 173 cell responses, two did have S1-SARS-CoV-2-specific IgM, IgG, and IgA antibodies; the other two did not. In 174 adults, 100%, 96.3%, and 88.9% were seropositive for respectively, IgM, IgG, and IgA antibodies to S1-SARS-175

CoV-2 at any of the sampling time points. Interestingly, in children good correlations were observed between 176 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted September 15, 2021. ; https://doi.org/10.1101/2021.09.10.21263333 doi: medRxiv preprint 8 S-SARS-CoV-2 IFN-ɣ + T cell frequency and S1-SARS-CoV-2-specific serum IgM, IgG and IgA concentrations, 177 though this was only observed at T1 (for IgM, Rs=0.73 (P<0.0001); IgG, rs=0.74 (P<0.0001); IgA, rs=0.66 178 (P=0.0005)) ( Figure 5A ). In adults, frequency of S-SARS-CoV-2-specific T cells was also correlated with S1-SARS-179

CoV-2-specific serum IgM concentrations at T2 and T3 (respectively, rs=0.42 (P=0.03) and rs=0.49 (P=0.009)), 180 with S1-SARS-CoV-2-specific serum IgG concentrations at T2 and T3 (for both time points, rs=0.47 (P=0.01)), 181 and with S1-SARS-CoV-2-specific serum IgA concentrations at T3 (rs=0.44 (P=0.02)) ( Figure 5B ). 182

. CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted September 15, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint [20], we found that the SARS-CoV-2 activated T cells mainly belonged to the CD4 + effector memory subset 211 (TEM: CD45RO + /CCR7 -). Data on the SARS-CoV-2-specific IFN-ɣ + T cell response and CD4 + T cell activation 212 correlated, suggesting that IFN-ɣ was produced by antigen-specific CD4 + T cells. Apart from IFN-ɣ, SARS-CoV-2-213 specific T cells produced IL-2, suggesting a Th1 phenotype of the CD4 + TEM. 214

In contrast, very low frequencies of activated (CD25 + /CD137 + ) CD8 + T cells were detected after SARS-CoV-2-215 specific stimulation. An explanation for this may be that CD8 + T cells have migrated to the local sites of 216 infection to attack virus-infected cells or might be a result of the antigenic stimulation used in our study. 217 Smaller peptides (9 to 10-mers instead of 15-mers) or live SARS-CoV-2 may be more suitable to measure CD8 + 218 T cell responses. Sekine et al. also observed proportionately larger SARS-CoV-2-specific CD4 + T cell responses 219 than CD8 + T cell responses to different sets of overlapping peptides in the convalescent phase of both mild and 220 severe COVID-19 cases, although in that study also IFN-ɣ + CD8 + T cell responses were detected [8] . 221 Pre-existing cross-reactive T cell immunity generated by common cold human coronaviruses (HCoV) has been 222 suggested to affect clinical outcomes of SARS-CoV-2 infection. T cell lines from unexposed healthy donors 223 specific for S-HCoV-229E and -OC43 were cross-reactive to S-SARS-CoV-2 [29]. Based on these findings, the 224 authors suggested that children may have higher HCoV prevalence due to more frequent social contacts, 225 explaining their lower risk for severe . In the present study, we did, however, not find a 226 significant difference in the low frequencies of S-HCoV-OC43-reactive IFN-ɣ + T cells between unexposed 227 children and unexposed adults. In another study with mild COVID-19 adult patients, also low T cell frequencies 228 recognizing S-HCoV-229E/S-HCoV-OC43 peptide pools were found [15] . It cannot be excluded that pre-existing 229 cross-reactive immunity to other conserved parts of SARS-CoV-2 played a role, or that pre-existing immunity to 230 other HCoV played a role. We, however, found no evidence that pre-existing S-HCoV-OC43-reactive T cells 231 boosted upon SARS-CoV-2 infection could explain the mild course of infection. 232

We found positive correlations between SARS-CoV-2-specific IFN-ɣ + T cell frequency and serum-IgG, -IgM, and -233

IgA antibody concentrations to S1-SARS-CoV-2 in both children and adults. Although, it should be taken into 234 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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

The copyright holder for this this version posted September 15, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 15, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 15, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 15, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 15, 2021. ; https://doi.org/10.1101/2021.09.10.21263333 doi: medRxiv preprint Table 1 . Demographic and clinical characteristics of unexposed participants and the cohort of PCR confirmed SARS-CoV-2 infection 341 A Disease severity was classified as asymptomatic (absence of symptoms), mild (at least one symptom but absence of shortness of breath), moderate (presence of shortness of breath with or without other symptoms, including 342 hospitalized cases). None of the hospitalized patients were admitted to an intensive care unit, thus not considered severe cases. B Three children were remained asymptomatic during the study but tested PCR positive. n, 343 number of subjects in specific group; Spike-S1, SARS-CoV-2 Spike S1 protein; Spike, overlapping peptides of SARS-CoV-2 spike protein; N/A, not applicable 344 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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Longitudinal analyses reveal immunological misfiring in severe COVID-272 19

Deep immune profiling of COVID-19 patients reveals distinct 274 immunotypes with therapeutic implications

Complex Immune Dysregulation in COVID-19

Patients with Severe Respiratory Failure

Comprehensive mapping of immune perturbations 278 associated with severe COVID-19

Characteristics of and Important Lessons From the Coronavirus Disease

COVID-19) Outbreak in China: Summary of a Report of 72314 Cases From the Chinese Center for 281

Disease Control and Prevention

Discordant neutralizing antibody and T cell responses in 283 asymptomatic and mild SARS-CoV-2 infection

Functional SARS-CoV-2-Specific Immune Memory Persists after 285

Dot plots summarizing the percentages of CD25 + /CD137 + activated CD4 + T cells responding to SARS-CoV-2 and is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted September 15, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted September 15, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted September 15, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted September 15, 2021. ; https://doi.org/10.1101/2021.09.10.21263333 doi: medRxiv preprint