Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals AUTHORS Alba Grifoni1, Daniela Weiskopf1, Sydney I. Ramirez1,2, Jose Mateus1, Jennifer M. Dan1,2, Carolyn Rydyznski Moderbacher1, Stephen A. Rawlings2, Aaron Sutherland1, Lakshmanane Premkumar3, Ramesh S. Jadi3, Daniel Marrama1, Aravinda M. de Silva3, April Frazier1, Aaron Carlin2, Jason A. Greenbaum1, Bjoern Peters1,2, Florian Krammer4, Davey M. Smith2, Shane Crotty1,2*, Alessandro Sette1,2* AFFILIATIONS 1 Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology (LJI), La Jolla, CA 92037, USA 2 Department of Medicine, Division of Infectious Diseases and Global Public Health, University of California, San Diego (UCSD), La Jolla, CA 92037, USA 3 Department of Microbiology and Immunology, University of North Carolina School of Medicine, USA. 4 Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York * indicates equal contributions Correspondence: [email protected] (A.S.) and [email protected] (S.C.). Lead Contact: [email protected] (A.S.) SUMMARY Understanding adaptive immunity to SARS-CoV-2 is important for vaccine development, interpreting coronavirus disease 2019 (COVID-19) pathogenesis, and calibration of pandemic control measures. Using HLA class I and II predicted peptide ‘megapools’, circulating SARS-CoV-2−specific CD8+ and CD4+ T cells were identified in ~70% and 100% of COVID-19 convalescent patients, respectively. CD4+T cell responses to spike, the main target of most vaccine efforts, were robust and correlated with the magnitude of the anti-SARS-CoV-2 IgG and IgA titers. The M, spike and N proteins each accounted for 11-27% of the total CD4+ response, with additional responses commonly targeting nsp3, nsp4, ORF3a and ORF8, among others. For CD8+ T cells, spike and M were recognized, with at least eight SARS-CoV-2 ORFs targeted. Importantly, we detected SARS-CoV-2−reactive CD4+ T cells in ~40-60% of unexposed individuals, suggesting cross-reactive T cell recognition between circulating ‘common cold’ coronaviruses and SARS-CoV-2. INTRODUCTION COVID-19 is a world-wide emergency. The first cases occurred in December 2019 and now more than 240,000 deaths and 3,000,000 cases of SARS-CoV-2 infection have been reported globally as of May 1st, (Dong et al., 2020; Wu and McGoogan, 2020). Vaccines against SARS-CoV-2 are just beginning development (Amanat and Krammer, 2020; Thanh Le et al., 2020). An understanding of human T cell responses to SARS-CoV2 is lacking, due to the rapid emergence of the pandemic. There is an urgent need for foundational information about T cell responses to this virus. The first steps for such an understanding are the ability to quantify the virus-specific CD4+ and CD8+ T cells. Such knowledge is of immediate relevance, as it will provide insights into immunity and pathogenesis of SARS-CoV-2 infection, and the same knowledge will assist vaccine design and evaluation of candidate vaccines. Estimations of immunity are also central to epidemiological model calibration of future social distancing pandemic control measures (Kissler et al., 2020). Such projections are dramatically different depending on whether SARS-CoV-2 infection creates substantial immunity, and whether any crossreactive immunity exists between SARS-CoV-2 and circulating seasonal ‘common cold’ human coronaviruses. Definition and assessment of human antigen-specific SARS-CoV2 T cell responses are best made with direct ex vivo T cell assays using broad-based epitope pools 2 and assays capable of detecting T cells of any cytokine polarization. Herein, we have completed such an assessment with blood samples from COVID-19 patients. There is also great uncertainty about whether adaptive immune responses to SARS-CoV-2 are protective or pathogenic, or whether both scenarios can occur depending on timing, composition, or magnitude of the adaptive immune response. Hypotheses range the full gamut (Peeples, 2020), based on available clinical data from severe acute respiratory disease syndrome (SARS) or MERS (Alshukairi et al., 2018; Wong et al., 2004; Zhao et al., 2017) or animal model data with SARS in mice (Zhao et al., 2016; Zhao et al., 2010; Zhao et al., 2009), SARS in NHPs (Liu et al., 2019; Takano et al., 2008) or FIPV in cats (Vennema et al., 1990). Protective immunity, immunopathogenesis, and vaccine development for COVID-19 are each briefly discussed below, related to introducing the importance of defining T cell responses to SARS-CoV-2. Based on data from SARS patients in 2003-2004 (caused by SARS-CoV, the most closely related human betacoronavirus to SARS-CoV-2), and based on the fact that most acute viral infections result in development of protective immunity (Sallusto et al., 2010), a likely possibility has been that substantial CD4+ T cell, CD8+ T cell, and neutralizing antibody responses develop to SARS-CoV-2 and all contribute to clearance of the acute infection; and, as a corollary, some of the T and B cells areretained long-term (i.e., multiple years) as immunological memory and protective immunity against SARS-CoV-2 infection (Guo et al., 2020b; Li et al., 2008). However, a contrarian viewpoint is also legitimate. While most acute infections result in the development of protective immunity, available data for human coronaviruses suggest the possibility that substantive adaptive immune responses can fail tooccur (Choe et al., 2017; Okba et al., 2019; Zhao et al., 2017) and robust protective immunity can fail to develop (Callow et al., 1990). A failure to develop protective immunity could occur due to a T cell and/or antibody response of insufficient magnitude or durability, with the neutralizing antibody response being dependent on the CD4+ T cell response (Crotty, 2019; Zhao et al., 2016). Thus, there is urgent need to understand the magnitude and composition of the human CD4+ and CD8+ T cell responses to SARS-CoV-2. If natural infection with SARS-CoV-2 elicits potent CD4+ and CD8+ T cell responses commonly associated with protective antiviral immunity, COVID-19 is a strong candidate for rapid vaccine development. Immunopathogenesis in COVID-19 is a serious concern (Cao, 2020; Peeples, 2020). It is most likely that an early CD4+ and CD8+ T cell response against SARS-CoV-2 is protective, but an early response is difficult to generate because of efficient innate immune evasion mechanisms of SARS-CoV-2 in humans (Blanco-Melo et al., 2020). Immune evasion by SARS-CoV-2 is likely exacerbated by reduced myeloid cell antigen presenting cell (APC) function or availability in the elderly (Zhao et al., 2011). In such cases, it is conceivable that late T cell responses may instead amplify pathogenic inflammatory outcomes in the presence of sustained high viral loads in the lungs, by multiple hypothetical possible mechanisms (Guo et al., 2020a; Li et al., 2008; Liu et al., 2019). Critical (ICU) and fatal COVID-19 (and SARS) outcomes are associated with elevated levels of inflammatory cytokines and chemokines, including IL-6 (Giamarellos-Bourboulis et al., 2020; Wong et al., 2004; Zhou et al., 2020) Vaccine development against acute viral infections classically focuses on vaccine-elicited recapitulation of the type of protective immune response elicited by natural infection. Such foundational knowledge is currently missing for COVID-19, including how the balance and the phenotypes of responding cells vary as a function of disease course and severity. Such knowledge can guide selection of vaccine strategies most likely to elicit protective immunity against SARS-CoV-2. Furthermore, knowledge of the T cell responses to COVID-19 can guide selection of appropriate immunological endpoints for COVID-19 candidate vaccine clinical trials, which are already starting. Limited information is also available about which SARS-CoV-2 proteins are recognized by human T cell immune responses. In some infections, T cell responses are strongly biased towards certain viral proteins, and the targets can vary substantially between CD4+ and CD8+ T cells (Moutaftsi et al., 2010; Tian et al., 2019). Knowledge of SARS-CoV-2 proteins and epitopes recognized by human T cell responses is of immediate relevance, as it will allow for monitoring of COVID-19 immune responses in laboratories worldwide. Epitope knowledge will also assist candidate vaccine design and facilitate evaluation of vaccine candidate immunogenicity. Almost all of the current COVID-19 vaccine candidates are focused on the spike protein.