key: cord-0979331-w2pml51s
authors: da Silva Antunes, Ricardo; Pallikkuth, Suresh; Williams, Erin; Esther, Dawen Yu; Mateus, Jose; Quiambao, Lorenzo; Wang, Eric; Rawlings, Stephen A; Stadlbauer, Daniel; Jiang, Kaijun; Amanat, Fatima; Arnold, David; Andrews, David; Fuego, Irma; Dan, Jennifer M; Grifoni, Alba; Weiskopf, Daniela; Krammer, Florian; Crotty, Shane; Hoffer, Michael E; Pahwa, Savita G; Sette, Alessandro
title: Differential T cell reactivity to endemic coronaviruses and SARS-CoV-2 in community and health care workers
date: 2021-04-02
journal: J Infect Dis
DOI: 10.1093/infdis/jiab176
sha: bb3cc16b42ed147d6fa0f2fb278900f1a41fbd96
doc_id: 979331
cord_uid: w2pml51s

Herein we measured CD4 (+) T cell responses against common cold corona (CCC) viruses and SARS-CoV-2 in high-risk health care workers (HCW) and community controls. We observed higher levels of CCC reactive T cells in SARS-CoV-2 seronegative HCW compared to community donors, consistent with potential higher occupational exposure of HCW to CCC. We further show that SARS-CoV-2 T cell reactivity of seronegative HCW was higher than community controls and correlation between CCC and SARS-CoV-2 responses is consistent with cross-reactivity and not associated with recent in vivo activation. Surprisingly, CCC T cell reactivity was decreased in SARS-CoV-2 infected HCW, suggesting that exposure to SARS-CoV-2 might interfere with CCC responses, either directly or indirectly. This result was unexpected, but consistently detected in independent cohorts derived from Miami and San Diego.

Healthcare workers (HCW) that provide frontline care during the global pandemic of Coronavirus Disease are at increased risk of infection due to frequent close and prolonged exposure to patients with SARS-CoV-2 [1] . SARS-CoV-2 infection rates among HCW are still largely undetermined and highly variable depending on the geographical and temporal distribution among other factors [2] [3] [4] [5] but higher prevalence has been documented during periods of upsurge [6, 7] . Still, only a minority have developed mild to severe disease manifestations and the majority have remained seronegative for SARS-CoV-2 antibodies despite having close contact with SARS-CoV-2 infected patients [2-4, 8, 9] .

Robust T cell immunity has been consistently reported in multiple studies in asymptomatic, acute, and convalescent COVID-19 individuals [8, [10] [11] [12] [13] . Furthermore, we and others have previously reported significant pre-existing immune memory responses to SARS-CoV-2 sequences in unexposed subjects [10, [12] [13] [14] [15] . Here, we aimed to characterize preexisting SARS-CoV-2 T cell responses in this HCW cohort.

Due to close contact with patients, HCW are particularly prone to exposure to respiratory pathogens such as human coronaviruses (HCoVs) and particularly to endemic "common cold" corona virus (CCC) [16] [17] [18] (https://www.cdc.gov/niosh/topics/healthcare/infectious.html). Human CCC are seasonal endemic circulating viruses that cause only mild upper and lower respiratory infections. They are globally distributed with higher incidences in winter months. Little is known about their pattern of infection, transmission rates, or duration of immunity [19] [20] [21] , however detailed analysis of CCC reactivity from healthy donors and COVID-19 patients have been reported in recent studies [22] [23] [24] . As expected, on the basis of their common phylogeny, CCC share varying degrees of sequence homology with SARS-CoV-2 and we and others have shown that cross-reactive CD4 + T cell memory responses against SARS-CoV-2 can be detected in unexposed donors [14, 22, [24] [25] [26] , although pre-existing reactivity cannot solely be explained by prior exposure to CCC [27] .

A c c e p t e d M a n u s c r i p t 5 However, it is still unclear how pre-existing immunity impacts disease severity or clinical outcome after SARS-CoV-2 exposure [28, 29] and if this could translate into a protective effect. While some studies suggest this could be the case [23, [30] [31] [32] , and exposure to CCC concomitantly results in a faster response of pre-existing memory cells to control SARS-CoV-2 infection, it cannot be excluded that CCC cross-reactivity could contribute to drive COVID-19 immunopathogenesis [33] . Thus, it is important to study differences in CCC reactivity and pre-existing immunity in different cohorts, particularly HCW.

For the Miami cohorts, peripheral venous blood was collected in EDTA vacutainer tubes and PBMC were isolated by density gradient isolation using Ficoll-Paque (Lymphoprep, Nycomed Pharma, Oslo, Norway) as previously described [34] and stored in liquid nitrogen until use. Serum was collected and stored at -80°C. For the San Diego cohorts, whole blood was collected in heparin coated blood bags (healthy unexposed donors) or in ACD tubes (COVID-19 donors) and PBMCs isolated as above. All samples were obtained after written informed consent from the participants in an anonymous fashion and with protocols approved by the respective institutional review boards (IRB).

The CCC (OC43 spike, 229E spike, NL63 spike or HKU1 spike) ELISAs were performed as previously described [35] and the endpoint titers determined. The SARS-CoV-2 ELISAs for all cohorts with the exception of SIP were performed as previously described in detail [35] following a two-step ELISA protocol and results interpreted in accordance with the manufacturer's cutoff calculations. Limits of detection were set at 1:80 and 1:50 for CCC and SARS-CoV-2 ELISA's respectively. All data below was plotted as 1:25. For the SIP cohort, a A c c e p t e d M a n u s c r i p t 6 N-antigen ELISA assay for IgG and IgM that was purely qualitative was performed. All donors had undetectable levels of antibodies.

To investigate CCC CD4 + T cell responses, we performed prediction of peptides for HLA class II spanning the entire sequence of the 4 CCC strains utilizing the Immune Epitope

Database and Analysis Resource (IEDB) [36] . After selection of promiscuous binders, epitopes composed of 15-mer were generated and further divided into 2 different peptide pools (MP) to encompass epitopes sharing 60% or less homology with SARS-CoV-2 sequences or more than 67% homology (Supplementary Table 1 ). Responses were measured against the two different MPs separately, and summed together for graphic display. The CMV MP is a pool of previously reported Class I and Class II epitopes [37] . To study T cell responses against SARS-CoV-2, we used the entire SARS-CoV-2 genome (GenBank: MN908947) and we generated MPs of 15-mer peptides overlapping by 10 spanning the entire protein sequence (6-253 peptides per pool) or alternatively a MP for the remainder genome consistent of dominant HLA Class II predicted CD4 + T cell epitopes as previously described [36, 38] . Supplementary Table 1 lists the number of peptides pooled for each of the viral proteins. Alternatively, HLA Class I predicted CD8 + T cell epitopes prediction was performed as previously reported, using NetMHC pan EL 4.0 algorithm [39] (Supplementary Table 1 ). All peptides were synthesized as crude material (A&A, San Diego, CA).

Cryopreserved cells were thawed, washed and stimulated for flow cytometry determinations using activation induced cell marker (AIM) assays as previously described [34, 40] . Antibodies used in the AIM assay as well as the gating strategy used to define AIM reactive cells and memory sub-populations is listed in Supplementary 

Data and statistical analyses were done in FlowJo 10 and GraphPad Prism 8.4, unless otherwise stated. Non-parametric Mann-Whitney or Kruskal-Wallis test were applied for unpaired two-group or three-group comparisons, respectively. Correlation analysis were performed using non-parametric Spearman test. Details pertaining to significance are also noted in the respective legends and p<0.05 defined as statistical significant. Additional data analysis details are described in the respective figure legends.

Five different cohorts of subjects were enrolled in the study ( In parallel, seropositivity for the spike proteins of the four endemic common cold coronaviruses (CCC; 229E, NL63, HKU1 and OC43), was also determined in the three donor cohorts from Miami ( Figure 1B) . All donors had detectable titers and variable reactivity for each of the CCC strains and consistent with the majority of the general population having detectable responses for the CCC viruses [19, 20] . In conclusion, these data define the serological status of the donor cohorts for which the T cell reactivity was investigated.

To test the various Miami cohorts for CD4 + T cell reactivity, we performed Activation Induced Marker (AIM) assays [34, 40] , previously utilized to characterize viral responses including SARS-CoV-2 CD4 + T cell responses [12, 13, 15] , using sets of predicted dominant Class II-restricted T cell peptides, for each of the four CCCs (Supplementary Table 1 ). This epitope prediction strategy was previously applied in multiple studies [34, 36, 40] and was envisioned to capture the top 50% of the predicted response.

The CD4 + T cell reactivity to the 229E, NL63, HKU1 and OC43 viruses was higher in the NHCW cohort, as compared to the SIP cohort (Figures 2a-b show absolute magnitude A c c e p t e d M a n u s c r i p t 9 and stimulation index (SI) plots). This difference was most pronounced for NL63 and least pronounced for HKU1 (p values ranged from 0.03 to 0.0005 by the Kruskal-Wallis test).

By contrast, NHCW CD4 + T cell reactivity was significantly higher compared to PHCW against 229E, NL63 and OC43 (p values ranging from 0.004 to 0.002). For HKU1

there was a trend toward higher responses (p=0.12). No difference was noted with a control MP composed of epitopes derived from the unrelated ubiquitous cytomegalovirus (CMV)

pathogen [37] . Representative flow cytometry plots with CCC-specific and CMV CD4 + T cell responses are shown in Figure 2c . The SARS-CoV-2 infected donors analyzed were associated with either mild or asymptomatic disease ( Table 1) . We have analyzed responses to CCC in the cohort of PHCW segregating asymptomatic individuals (n=7) vs.

individuals with mild disease (n=19) and no differences were observed (data not shown).

To validate these results further, we assessed CCC responses in two additional cohorts recruited in the San Diego region, selected on the basis of being asymptomatic and seronegative (NSD) or symptomatic and seropositive (COVID-19SD) for SARS-CoV-2

infection. (Table 1) . Both cohorts were recruited between March and July of 2020, similar to the Miami cohort.

The 229E, NL63, HKU1 and OC43 epitope pools displayed higher CD4 + T cell reactivity in the unexposed donors, as compared to the COVID-19 diagnosed donors (Figures 3a-b) . No differences between groups were observed in the responses against the CMV control MP. These results indicate that healthy unexposed donors demonstrate higher CD4 + T cell reactivity against CCC than COVID-19 donors.

A c c e p t e d M a n u s c r i p t 10

Next, we tested the various cohorts from the Miami area for SARS-CoV-2 CD4 + T cell reactivity, using the AIM assay as before and previously described MPs, one encompassing overlapping peptides spanning the entire sequence of the SARS-CoV-2 spike protein (S), and one encompassing predicted CD4 + T cell epitopes from the remainder of the genome (CD4R) [12, 36] (Supplementary Table 1 CD4 + T cell responses from PHCW cohort were highest, in accordance with their recent exposure to SARS-CoV-2, followed by responses measured in the NHCW and then the SIP cohort. More specifically, the total CD4 + T cell reactivity of the PHCW cohort to the SARS-CoV-2 pools was significantly higher than both NHCW (p =0.03 and p =0.003 by the Kruskal-Wallis test for absolute and SI readouts, respectively) and SIP (p =0.002 and p <0.0001 by the Kruskal-Wallis test for absolute and SI readouts, respectively). Of further interest, the total CD4 + T cell reactivity of NHCW was also higher than that observed in the SIP cohort (p=0.04 for both absolute and SI readouts). No difference was noted in the case of the CMV MP. As shown in Supplementary Figure 3 , the differences noted above, are further confirmed by assessing the total reactivity obtained by summing the responses to the various individual SARS-CoV-2 antigen pools as previously reported [12] . Analysis of the expression of the CCR7 and CD45RA memory markers confirmed that the CD4 + T cell reactivity in all three cohorts was mediated by memory T cell subsets (Supplementary

A c c e p t e d M a n u s c r i p t 11

We also analyzed the AIM + CD4 + T cells for expression of the HLA-DR/CD38 markers, which have been found increased in donors from mild to acute SARS-CoV-2 infection, and therefore to be associated with recent in vivo activation [11, 41] . The data shown in Figure 5 , demonstrates that the CD4 + T cell reactivity to SARS-CoV-2 peptides is 

Finally, we measured CD8 + T cell reactivity to SARS-CoV-2 epitopes (Supplementary Table 1 ) in the various cohorts as previously described [12, 13] , utilizing a pool of overlapping peptides spanning the S antigen, and two MPs containing SARS-CoV-2 predicted HLA binders for the 12 most common HLA A and B alleles (CD8A and CD8B MPs) Table 1 ). Figure 6 shows CD8 + T cell responses plotted as background subtracted data, or plotted as stimulation index, against the S pool, the two different CD8A

A c c e p t e d M a n u s c r i p t 12 and CD8B epitope summed together, and the control CMV pool. A representative flow cytometry AIM + gating is shown in Supplementary Figure 6 .

In the case of the S pool, the CD8 + T cell response to SARS-CoV-2 spike protein was highest in PHCW (and similar between SIP and NHCW). More specifically, the total CD8 + T cell reactivity of the PHCW cohort to the SARS-CoV-2 pools was significantly higher than Figure 7) . Overall these data suggest that the higher reactivity observed in NHCW as compared to SIP is largely confined to CD4 + T cell responses and only marginally seen in the case of CD8 + T cell responses, further suggesting that it is not resulting from infected individuals rapidly becoming seronegative.

Here we present evidence for differential reactivity to endemic CCC and SARS-CoV-2 epitopes. Although previous reports studied responses to CCC or SARS-CoV-2 in either unexposed or COVID-19 survivors [22] [23] [24] , this is the first study, to the best of our knowledge, investigating T cell and antibody responses measured simultaneously for both CCC and SARS-CoV-2 and in particular by presenting evidence for differentially T cell reactivity among high-risk HCW and community workers. In particular, we show that a cohort of HCW with presumed exposure to respiratory viruses is associated with higher levels of Samples from SARS-CoV-2 infected subjects were associated with lower levels of CCC reactivity as compared to non-exposed donors. This result was unexpected, but consistently detected in independent cohorts derived from Miami and San Diego. Several 

other CCCs but not unrelated viruses such as CMV. Impaired responses particularly associated with type I interferon activity in COVID-19 patients were also described in a recent report [42] , suggesting that SARS-CoV-2 might interfere with innate immunity. SARS-CoV-2 infection may also result in expansion of SARS-CoV-2-specific, non-CCC reactive T cells, competing with the pre-existing CCC specificities [22, 43] . Pre-existing CCC reactivity and different pre-exposure history can also influence disease severity and infection [28] .

Indeed, the repertoire of cross-reactive T cells in HCW might have a protective effect against SARS-CoV-2 infection as suggested in other studies [23, 30, 31] . Based on our current understanding of viral dynamics, it appears unlikely that CD4 + T cells might be able to prevent disease, but it is possible that their presence may lead to rapid termination of infection and only transient seropositivity ( [26, 44] and see above). It is also possible that CD8 + T cells might mediate or contribute to rapid termination of infection as described for SARS-CoV [45, 46] and other viral infection diseases [47, 48] .

All recruited SARS-CoV-2 infected donors were associated with mild or asymptomatic disease, and the small sample size of the study does not allow to address whether levels of preexisting cross-reactive CCC T cell responses might influence disease severity [28, 30] .

Also, it would be expected that HCW would wear PPE during the pandemic period, so it seems unlikely that they would be exposed to CCC to a great extent, at least in the workplace. It is therefore possible that, despite our effort to balance HCW and SIP control cohort, other demographic differences could explain the results. Larger sample sizes will be required to analyze this issue in this type of cross-sectional design, but it is likely that a prospective longitudinal design might be necessary to firmly address this point based on 

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We would like to thank Kizzmekia Corbett and Barney Graham at the Vaccine Research Center at NIH for providing plasmids for CCC spike expression. We wish to acknowledge all subjects for their participation and for donating their blood and time for this study. We thank the Clinical Core of the La Jolla Institute for Immunology and UCSD for sample collection and Allan Watson from Biorad for technical support with ZE5 Cell analyzer.

A c c e p t e d M a n u s c r i p t 18 A c c e p t e d M a n u s c r i p t 19 M a n u s c r i p t Mar-Jun 2020Apr-Jul 2020

Ab(-) Ab(+) or PCR(+) Ab(-) Ab(-) Ab(+)