key: cord-0944115-syj2edz0 authors: Qiu, Congling; Xiao, Chanchan; Wang, Zhigang; Chen, Xiongfei; Gao, Lijuan; Deng, Jieping; Su, Jun; Su, Huanxing; Fang, Evandro Fei; Zhang, Zhang-Jin; Zhu, Guodong; Zhang, Jikai; Xie, Caojun; Yuan, Jun; Luo, Oscar Junhong; Wang, Pengcheng; Chen, Guobing title: CD8+ T cell epitope variations suggest a potential antigen presentation deficiency for spike protein of SARS-CoV-2 date: 2021-01-24 journal: bioRxiv DOI: 10.1101/2021.01.22.427863 sha: 0a66dbebc2ddd84cb811d037ea4f79e396933ff9 doc_id: 944115 cord_uid: syj2edz0 COVID-19 is caused by a newly identified coronavirus, SARS-CoV-2, and has become a pandemic around the world. The illustration of the immune responses against SARS-CoV-2 is urgently needed for understanding the pathogenesis of the disease and its vaccine development. CD8+ T cells are critical for virus clearance and induce long lasting protection in the host. Here we identified specific HLA-A2 restricted T cell epitopes in the spike protein of SARS-CoV-2. Seven epitope peptides (n-Sp1, 2, 6, 7, 11, 13, 14) were confirmed to bind with HLA-A2 and potentially be presented by antigen presenting cells to induce host immune responses. Tetramers containing these peptides could interact with specific CD8+ T cells from convalescent COVID-19 patients, and one dominant epitope (n-Sp1) was defined. In addition, these epitopes could activate and generate epitope-specific T cells in vitro, and those activated T cells showed cytotoxicity to target cells. Meanwhile, all these epitopes exhibited high frequency of variations. Among them, n-Sp1 epitope variation 5L>F significantly decreased the proportion of specific T cell activation; n-Sp1 epitope 8L>V variant showed significantly reduced binding to HLA-A2 and decreased the proportion of n-Sp1-specific CD8+ T cell, which potentially contributes to the immune escape of SAR-CoV-2. COVID-19 is an emerging pandemic sweeping the world, which is caused by SARS-CoV-2 infection (1) . With no highly effective clinical treatment available for COVID-19 so far, the clearance of SARS-CoV-2 was considered relying on host immune system, especially adaptive immunity (2) . It has been demonstrated that both T and B cell clones are highly expanded in the recovery phase of COVID-19 patients (3) . Besides the discovery of neutralizing antibodies (4), proliferation of CD8+ T cells has been observed in the lungs of patients with mild COVID-19 (5) . Although CD8+ T cell counts were dramatically reduced in severe COVID-19 cases, the revealed antigen specific T cell responses indicated an effective role of CD8+ T cells (6) (7) (8) . T cell epitopes are the basis for the initiation of T cell mediated immune responses. Thus, the identification of epitopes specific to SARS-CoV-2 and illustration of the corresponding T cell responses are of great relevance to the understanding of COVID-19 pathogenesis and vaccine development. Computational analysis has been carried out to predict potential T cell epitopes of SARS-CoV-2 (9) , and T cell responses specific to SARS-CoV-2 have also been tested in convalescent COVID-19 patients by using predicted peptide 'megapools' (8) . Recently, multiple specific CD8+ T cell epitopes have been identified throughout SARS-CoV-2 ORFs including spike protein (10) . These epitopes were widely shared among SARS-CoV-2 isolates and located in regions of the virus that are not subject to mutational variation. Therefore, the above epitope identification is beneficial to the development of With the continuous epidemics worldwide, variations in virus strains have been found in many countries. It's reported recently that CD8+ T cell epitope variants in SARS-CoV-2 spike protein led to persistently variable SARS-CoV-2 infection with different susceptibility and severity (11) . In view of the above phenomenon, we analyzed the epitope variations of the spike protein among SARS-CoV-2 strains from different geographic regions and evaluated the epitope characteristics of these variations. To narrow down the potential candidates of SARS-CoV-2 specific antigen epitopes, we first predicted potential HLA-A2 restricted CD8+ T cell epitopes of SARS-CoV-2, SARS-CoV, MERS-CoV and Coronavirus OC43, respectively. The sequences of all the potential peptides were compared with Clustal Omega and 15 candidate peptides specific to SARS-CoV-2 were selected (Table 1 and Table S1 ). To validate these predicted epitopes, we first checked whether they could be presented by HLA-A2 on the antigen presenting cells (APC). T2A2 is an APC with TAP deficiency, whose peptide-MHC complex would be more stabilized if the epitopes bind suitably ( Figure S1A ). Comparing to the well-known CD8+ T cell epitope GIL (GILGFVFTL) from influenza A virus (IAV), most of the predicted SARS-CoV-2 epitopes showed reasonable HLA-A2 binding ( Figure 1A&B ). We further checked the direct binding of these epitopes to recombinant HLA-A2 molecules. In the UV exchange peptide-MHC assay, peptides n-Sp1, n-Sp2, n-Sp9 and n-Sp10 exhibited strong binding to HLA-A2, similar to IAV epitope GIL. However, n-Sp5 and s-Sp12 could not bind to HLA-A2, while the remaining peptides showed moderate binding capability ( Figure 1C ). We then prepared pMHC tetramers with all the peptides and tested whether they could recognize antigen specific CD8+ T cells from convalescent COVID-19 patients. Overall, 31 patients were enrolled and tested (Table 2 ). Compared to healthy donors, the antigen specific CD8+ T cells were detected in convalescent patients with the tetramers of n-Sp1, 2, 6, 7, 11, 13 and 14, but not others. Among them, n-Sp1 and n-Sp7 based pMHC tetramers demonstrated the highest proportion of antigen specific CD8+ T cells ( Figure 1D&E ). To further analyze whether the epitope-bound T2A2 cells could activate T cells, we tested the expression level of T cell activation marker CD69 and the proportion of peptide-specific CD8+ T cells after stimulation with peptide-bound T2A2 cells bearing peptides n-Sp1, 2, 6, 7, 11, 13 and 14. The results demonstrated that n-Sp1, 2 and 7 had the capability to activate CD8+ T cell. In particular, n-Sp1 induced the strongest response ( Figure 2) . Firstly, the expression of CD69 increased significantly under the stimulation of the peptide-bound T2A2 cells (Figure 2A Altogether, we therefore considered n-Sp1 to be one of the dominate CD8+ T cell epitopes specific to SARS-CoV-2. Phylogenetic analyses had indicated diverse variants of SARS-CoV-2 in global circulation (12) , and therefore we examined the extent to which the above epitopes had been evolving in the 105,029 publicly available full-length SARS-CoV-2 sequences as of Sep 26 th 2020. We observed high frequency of sequence variation within these peptides, with n-Sp1, 2, 6, 7, 11, 13 and 14 bearing 20, 9, 13, 10, 12, 10 and 9 types of variations, respectively ( Figure 3A ). Among all the variations discovered, 614D>G, 613Q>H in n-Sp6 and 5L>F in n-Sp1 were the top three most frequently observed ( Figure 3A ). To examine how these variations might affect epitope properties, we tested the binding capability of mutated epitope peptides to HLA-A2 ( Figure 3B -E). For n-Sp1, the mutant FVFFVLVPLV (5L>F, n-Sp1-m1) showed no change in T2A2 stabilization, but mild decrease in pMHC binding capability. The other mutant Page 9 / 25 FVFLVLVPLV (8L>V, n-Sp1-m2), however, demonstrated a significant decrease in pMHC binding capability. The dual mutant of 5L>F and 8L>V (n-Sp1-m3) further confirmed the importance of L8 for the epitope properties of n-Sp1. Meanwhile, the mutants of n-Sp2 showed various levels of decreased pMHC binding capability, especially FQFCNYPFL (138D>Y, n-Sp2-m2). In addition, the n-Sp6 mutant resulted in decreased pMHC binding capability, however, it showed similar T2A2 stabilization ability to the wild type peptide ( Figure 3C -E). By using tetramers containing the mutated epitopes, no CD8+ T cells specific to n-Sp1 mutation were detected in convalescent COVID-19 patients from Guangzhou, China ( Figure 3F -G). However, n-Sp1 mutants was able to increase the expression of T cell activation marker CD69 ( Figure 4A -B) and generate CD8+ T cells specific to the mutants ( Figure 4C -D), even though the proportion of CD8+ T cells specific to mutation was less than that to wild type in the same host ( Figure 4D ). More importantly, although n-Sp1-m1 could activate T cells, the wild type n-Sp1 tetramer could not recognize antigen specific CD8+ T cells induced by n-Sp1 mutants ( Figure 4E -F). Overall, these results indicated that these emerged variations might have caused a deficiency in the antigen presentation of the dominant epitopes, which was required to rebuild a new CD8+ T cell immune response in COVID-19 patients. induce T cell responses mainly to spike protein (13) . Our study identified HLA-A2 restricted CD8+ T cell epitopes on SARS-CoV-2 spike protein. We first predicted 15 potential HLA-A2 restricted CD8+ T cell epitopes. Among them, n-Sp11, n-Sp13 and n-Sp15 have also been reported as SARS-CoV-specific epitopes and have been shown to stimulate specific CTL responses (14, 15) . In the study of Ferretti et al., memory CD8+ T cells from convalescent COVID-19 patients could be activated by 29 epitope peptides, among which only 3 peptides were in spike protein, including n-Sp3 (10). In our study, n-Sp3 also showed a certain HLA-A2 binding capability, but it was much lower than that of the dominate epitope n-Sp1. Although their ability to activate memory CD8+ T cells from convalescent COVID-19 patients were not tested, our results did show that mixed epitope-loaded antigen presentation cells could activate T cells from healthy donors. Notably, the proportion of n-Sp1-specific T cells produced by mixed epitope activation was even higher than that of the positive control (Influenza A M1 peptide GILGFVFTL). Combining the results of HLA-A2 binding capability, the proportion of antigen-specific CD8+ T cells in convalescent COVID-19 patients and the ability to activate T cell, we therefore proposed n-Sp1 as the dominate CD8+ T cell epitope specific to SARS-CoV-2. An artificial antigen presenting cell (aAPC) system was used in this study, providing a convenience protocol to validate T cell epitopes with no need of a high Page 11 / 25 biosafety level laboratory. T2A2 cell line was selected for our study with following reasons. Firstly, its intrinsic deficiency in endogenous antigen presentation made it more reliable to evaluate the presentation of exogenous epitope by HLA-A2, without the interference of endogenous epitopes. Secondly, it demonstrated an excellent capability to stimulate CD8+ T cells, with the high proportion of antigen specific CD8+ T cells generated upon the primary immune challenge. Thirdly, it could also be a target cell for cytotoxic assay after labelled with given epitope. Altogether, it is an ideal model to identify T cell epitopes for novel antigen protein, especially for the low biosafety level laboratories. By using this cell model, we demonstrated that the novel epitopes from spike protein of SARS-CoV-2 had the capability to initiate CD8+ T cell response, even in the unexposed donors. These epitopes might be candidates for vaccine development in future. Since the virus variants have been emerging geographically around the world ( Figure S2 and Table S2 ), especially the ongoing mutation of these epitopes, it is critical to explore how the variations could affect the transmissibility and pathogenicity of the virus. Among all the variations reported so far, 614D>G and 613Q>H in n-Sp6, and 5L>F in n-Sp1 were the three most frequently observed ( Figure 3A ). However, n-Sp6 showed a low proportion of antigen-specific T cells in convalescent COVID-19 patients and a lack of T cell activation ability (Figure 3 , Figure S3 ), so it's not considered as the dominate CD8+ T cell epitope. Although 614D>G replacement has been reported as the dominant pandemic form in an epidemiological analysis (16) , the G614 peptide mediated T cell activation was not significantly altered compared to D614 peptide. The possible explanation for this phenomenon is that G614 has a stronger binding ability with Angiotensin-converting Enzyme 2 (ACE2) (17) but it's not involved in the induction of T cell immune responses. Our result was also in accordance with the report that although G614 variant showed higher viral loads in patients, it didn't increase disease severity (18) . Although the n-Sp1 5L>F mutant could induce T cell activation, its specific T cells were undetectable in the convalescent COVID-19 patients. The possible reason was that the patients tested here were infected with wild type virus strain during the early stage of the epidemic, and the rebuild of a new CD8 + T cell immune response to the 5L>F mutant was needed. In addition, n-Sp1 8L>V mutation not only significantly reduced its binding to HLA-A2, but also showed a decreased proportion of n-Sp1-specific CD8+ T cell, which might contribute to the immune escape of SAR-CoV-2. Taken together, our data indicated that the variation of a dominant epitope might cause the deficiency in antigen presentation, which subsequently required the rebuild of a new CD8+ T cell immune response in COVID-19 patients. The Institutional Review Board of the Affiliated Huaqiao Hospital of Jinan University approved this study. Unexposed donors were healthy individuals enrolled in Guangzhou Blood Center and confirmed with a negative report for SARS-CoV-2 RNA RT-PCR assay. These donors had no known history of any significant systemic diseases, including, but not limited to, hepatitis B or C, HIV, diabetes, kidney or liver diseases, malignant tumors, or autoimmune diseases. Convalescent donors included subjects who were hospitalized for COVID-19 or confirmed SARS-CoV-2 infection with RNA RT-PCR assay ( Table 2 ). All subjects provided informed consent at the time of enrollment that their samples could be used for this study. Complete blood samples were collected in acid citrate dextrose tubes and stored at room temperature prior to PBMC isolation and plasma collection. Graphic Prism 7 software was used for statistical analysis. One-way ANOVA was performed for group analysis. P values less than 0.05 were considered to be statistically significant. Ctrl: tetramer with UV-sensitive peptide. Mitomycin pretreated T2A2 cells were loaded with n-Sp2, 6, 7, 11, 13, 14, n-Sp1 and n-Sp1 mutant peptides, respectively. Next, n-Sp1 or n-Sp1 mutant peptide loaded T2A2 cells were mixed with other peptide loaded T2A2 cells and co-cultured with CD8+ T cells from health donors at 1:1 ratio. Activation, cytotoxicity and generation of n-Sp1 and its mutant specific CD8+ T cells were evaluated. A Novel Coronavirus from Patients with Pneumonia in China Adaptive immune responses to SARS-CoV-2 infection in severe versus mild individuals Immune cell profiling of COVID-19 patients in the recovery stage by single-cell sequencing Human neutralizing antibodies elicited by SARS-CoV-2 infection Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19 SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls Phenotype and kinetics of SARS-CoV-2-specific T cells in COVID-19 patients with acute respiratory distress syndrome Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals Bioinformatic prediction of potential T cell epitopes for SARS-Cov-2 Unbiased Screens Show CD8(+) T Cells of COVID-19 Patients Recognize Shared Epitopes in SARS-CoV-2 that Largely Reside outside the Spike Protein CD8 T cell epitope generation toward the continually mutating SARS-CoV-2 spike protein in genetically diverse human population: Implications for disease control and prevention Phylogenetic network analysis of SARS-CoV-2 genomes Immunogenicity of a DNA vaccine candidate for COVID-19 Perception of native and non-native affricate-fricative contrasts: cross-language tests on adults and infants Screening and identification of severe acute respiratory syndrome-associated coronavirus-specific CTL epitopes Tracking COVID-19 Virus D614G variant confers enhanced replication and transmissibility. bioRxiv : the preprint server for biology Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity Allele frequency net 2015 update: new features for HLA epitopes, KIR and disease and HLA adverse drug reaction associations HLA common and well-documented alleles in China. HLA H.S., E.F.F. and Z.Z. assisted with data analysis. C.Q., P.W. and G.C. wrote the manuscript. The epitopes and tetramers from this study are the subject of a patent application.