key: cord-0796695-05djnz4p authors: Bert, Nina Le; Tan, Anthony T; Kunasegaran, Kamini; Tham, Christine Y L; Hafezi, Morteza; Chia, Adeline; Chng, Melissa; Lin, Meiyin; Tan, Nicole; Linster, Martin; Chia, Wan Ni; Chen, Mark I-Cheng; Wang, Lin-Fa; Ooi, Eng Eong; Kalimuddin, Shirin; Tambyah, Paul Anantharajal; Low, Jenny Guek-Hong; Tan, Yee-Joo; Bertoletti, Antonio title: Different pattern of pre-existing SARS-COV-2 specific T cell immunity in SARS-recovered and uninfected individuals date: 2020-05-27 journal: bioRxiv DOI: 10.1101/2020.05.26.115832 sha: bc7e3204ae6ad430f7ff60fa0da85bbf57b24763 doc_id: 796695 cord_uid: 05djnz4p Memory T cells induced by previous infections can influence the course of new viral infections. Little is known about the pattern of SARS-CoV-2 specific pre-existing memory T cells in human. Here, we first studied T cell responses to structural (nucleocapsid protein, NP) and non-structural (NSP-7 and NSP13 of ORF1) regions of SARS-CoV-2 in convalescent from COVID-19 (n=24). In all of them we demonstrated the presence of CD4 and CD8 T cells recognizing multiple regions of the NP protein. We then show that SARS-recovered patients (n=23), 17 years after the 2003 outbreak, still possess long-lasting memory T cells reactive to SARS-NP, which displayed robust cross-reactivity to SARS-CoV-2 NP. Surprisingly, we observed a differential pattern of SARS-CoV-2 specific T cell immunodominance in individuals with no history of SARS, COVID-19 or contact with SARS/COVID-19 patients (n=18). Half of them (9/18) possess T cells targeting the ORF-1 coded proteins NSP7 and 13, which were rarely detected in COVID-19- and SARS-recovered patients. Epitope characterization of NSP7-specific T cells showed recognition of protein fragments with low homology to “common cold” human coronaviruses but conserved among animal betacoranaviruses. Thus, infection with betacoronaviruses induces strong and long-lasting T cell immunity to the structural protein NP. Understanding how pre-existing ORF-1-specific T cells present in the general population impact susceptibility and pathogenesis of SARS-CoV-2 infection is of paramount importance for the management of the current COVID-19 pandemic. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the cause of the coronavirus disease 2019 (COVID-19) 1 . This disease has spread pandemically placing lives and economies of the world under severe stress. SARS-CoV-2 infection is characterized by a broad spectrum of clinical syndromes, ranging from mild influenza-like symptoms to severe pneumonia and acute respiratory distress syndrome 2 . It is common to observe in human the ability of a single virus to cause different pathological manifestations. This is often due to multiple contributory factors including the quantity of viral inoculum, the genetic background of patients and the presence of concomitant pathological conditions. Moreover, an established adaptive immunity towards closely related or completely different viruses can increase protection 3 or enhance disease severity 4 . SARS-CoV-2 belongs to Coronaviridae, a family of large RNA viruses infecting many animal species. Six other coronaviruses are known to infect human. Four of them are endemically transmitted 5 and cause common cold (OC43, HKU1, 229E and NL63), while SARS-CoV (defined from now as SARS-CoV-1) and MERS-CoV have caused limited epidemics of severe pneumonia 6 . All of them trigger antibody and T cell responses in infected patients: however, antibody levels appear to wane relatively quicker than T cells. In SARS recovered patients, SARS-CoV-specific antibodies dropped below detection limit within 2 to 3 years 7 , while SARS-CoV-specific memory T cells can be detected even at 11 years after infection 8 . Since the sequences of selected structural and nonstructural proteins are highly conserved among different coronaviruses (i.e. NSP7 and NSP13 are 100% and 99% identical, respectively, between SARS-CoV-2, SARS-CoV-1 and the bat-SL-CoVZXC21 9 ), we studied whether crossreactive SARS-CoV-2-specific T cells are present in individuals who resolved from SARS-CoV-1 or SARS-CoV-2 infection. We also studied these T cells in individuals with no history of SARS or COVID-19 and who were also not in contact with SARS-CoV-2 infected cases. Collectively these individuals are hereon referred to as SARS-CoV-1/2 unexposed. SARS-CoV-2-specific T cells have just started to be characterized in COVID-19 patients 10, 11 and their potential protective role has been inferred from studies in SARS 12 and MERS 13 patients. To study SARS-CoV-2 specific T cells associated with viral clearance, we collected peripheral blood of 24 individuals who recovered from mild to severe COVID-19 (demographic, clinical and virological information are summarized in Extended Data Table 1 ) and studied the T cell response against selected structural (nucleocapsid protein-NP) and non-structural proteins (NSP7 and NSP13 of ORF1) of the large SARS-CoV-2 proteome ( Figure 1A) . We selected nucleocapsid protein as it is one of the more abundant structural proteins produced and has large homology between different betacoranaviruses (Extended Data Fig. 1) 14 . NSP7 and NSP13 were selected for their complete homology between SARS-CoV-1, SARS-CoV-2 and other animal coronaviruses belonging to the betacoranavirus genus (Extended Data Fig. 2 ) 9 , and because they are representative of the ORF1a/b polyprotein encoding the replicase-transcriptase complex 15 . This polyprotein is the first to be translated upon coronavirus infection. We synthesized 216 15-mer peptides overlapping by 10 amino acids (aa) covering the whole length of NSP7 (83aa), NSP13 (601aa) and NP (422aa) that were organized in 5 pools of approximately 40 peptides each (NP-1, NP-2, NSP13-1, NSP13-2, NSP13-3) and in a single pool of 15 peptides spanning NSP7 ( Figure 1B) . The unbiased method with overlapping peptides was utilized instead of peptide selection by bioinformatic approaches, since the performance of such algorithms in ethnically-diverse Asians is often suboptimal 16 . Peripheral blood mononuclear cells (PBMC) of 24 recovered COVID-19 patients were stimulated for 18h with the different peptide pools and virusspecific T cell responses were analyzed by IFN-γ ELISpot assay. In all tested individuals (24/24) we detected IFN-γ spots following stimulation with the pools of synthetic peptides covering NP (Figure 1C/D) . In nearly all individuals NPspecific responses could be identified for multiple regions of the protein: 23/24 for region 1-205aa (NP-1) and 24/24 for 206-422aa (NP-2). In sharp contrast, responses to NSP7 and NSP13 peptide pools were detected at low levels only in 3 out of 24 COVID-19 convalescents tested. Direct ex vivo intracellular cytokine staining (ICS) was performed to confirm and define the NP-specific IFN-γ ELISpot response. Due to the low frequency, NP-specific T cells were more difficult to visualize by ICS than by ELISpot, but a clear population of CD4 and/or CD8 T cells producing IFN-γ and/or TNF-α were detectable in 7 out of 9 tested subjects ( Figure 1E) . To confirm and further delineate the multispecificity of the NP-specific T cell response detected ex vivo in COVID-19 recovered patients, we defined in nine individuals, the distinctive sections of NP targeted by T cells. We organized the 82 overlapping peptides covering the entire NP into small peptide pools (7-8 peptides) that were used to stimulate PBMC either directly ex vivo or after an in vitro expansion protocol previously used in HBV 17 or SARS recovered subjects 18 . A schematic representation of the peptide pools is shown in Figure 2A . We found that 8 out of 9 COVID-19 recovered patients possess T cells that recognize multiple regions of NP of SARS-CoV-2 (Figure 2A) . Importantly, we then defined single peptides that were able to activate T cells in 7 patients. Utilizing a peptide matrix strategy 18 , we first deconvolute individual peptides responsible for the detected T cell response by IFN-γ ELISpot. Subsequently, we confirmed the identified single peptide by testing, with ICS, its ability to activate CD4 or CD8 T cells ( Figure 2B ). Figure 2B summarizes the different T cell epitopes defined by both ELISpot and ICS, in 7 COVID-19 recovered individuals. Remarkably, we observed that COVID-19 convalescents developed T cells specific to regions that were also targeted by T cells of SARS recovered subjects. For example, the NP region 101-120 which is a described CD4 T cell epitope in SARS-CoV- To explore this possibility, we tested NP and NSP7/13-specific T cell responses in 18 SARS-CoV-1/2 unexposed donors. The blood samples were collected either before July 2019 or were serologically negative for both SARS-CoV-2 neutralizing antibodies and SARS-CoV-2 NP antibodies 19 . Different coronaviruses known to cause common cold in humans like OC43, HKU1, NL63 and 229E present different degrees of amino acid homology with SARS-CoV-2 (Extended Data Fig. 1, 2) and recent data demonstrated the presence of SARS-CoV-2 cross-reactive CD4 T cells (mainly specific for Spike) in SARS-CoV-2 unexposed donors 11 . Remarkably, we detected NP-specific T cells in some of our SARS-CoV-1/2 unexposed individuals. The pattern of T cell reactivity, however, was different compared to COVID-19 and SARS recovered. T cells from SARS-CoV-1/2 unexposed were directed against a single peptide 7 pool: i.e. none of the 18 donors responded to the NP-2 peptide pool ( Figure 4A ). Moreover, a different pattern was observed for NSP7-and NSP13-specific T cells. These cells were detected in only 3 out of 24 COVID-19 and in 2 out of 23 SARS recovered tested, but were present in 9 out of 18 unexposed donors ( Figure 4A/B) . The cumulative proportion of all studied subjects responding to NP and ORF-1-coded NSP7 and 13 proteins is shown in Figure 4B . These These latter two T cell specificities were particularly intriguing since the homology between the two protein regions of SARS-CoV-1/2 and other "common cold" coronaviruses (OC43, HKU1 NL63 and 229E) was minimal ( Figure 4D) , especially for the CD8 peptide epitope. This may suggest that perhaps not only human "common cold" coronaviruses, but other presently unknown coronaviruses, possibly of animal origin, can induce cross-reactive SARS-CoV-2 memory T cells in the general population. It was remarkable to find that NSP7/13-specific T cells were detected in 9 out of 18 (50%) SARS-CoV-1/2 unexposed donors, despite the fact that our analysis was performed with peptides that cover only 10% (684aa) of the ORF-1 proteome (7096aa). Notably, T cells specific for ORF-1-coded proteins were rarely detected in our SARS and COVID-19 convalescents. This is consistent with the findings of Grifoni et al 11 : using selected peptides, they detected ORF-1 specific T preferentially in some SARS-CoV-2 unexposed donors while T cells of COVID-19 recovered donors preferentially recognized structural proteins. The cause of this observed different pattern of immunodominance is presently unknown. We might speculate that a robust T cell response against structural proteins is induced by a productive infection (occurring in COVID-19 and SARS recovered patients). Individuals exposed to but not infected with possible unknown coronaviruses might just prime ORF-1-specific T cells. 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