key: cord-0993133-c2rsoqzz authors: Jin, Xiaoxiao; Ding, Yan; Sun, Shihui; Wang, Xinyi; Zhou, Zining; Liu, Xiaotao; Li, Miaomiao; Chen, Xian; Shen, Anran; Wu, Yandan; Liu, Bicheng; Zhang, Jianqiong; Li, Jian; Yang, Yi; Qiu, Haibo; Shen, Chuanlai; He, Yuxian; Zhao, Guangyu title: Screening of HLA-A restricted T cell epitopes of SARS-CoV-2 and induction of CD8+ T cell responses in HLA-A transgenic mice date: 2021-05-24 journal: bioRxiv DOI: 10.1101/2021.04.01.438020 sha: 629b8e432f5c37f7539d1f550ffae33afd9bdcf8 doc_id: 993133 cord_uid: c2rsoqzz While SARS-CoV-2-specific T cells have been characterized to play essential roles in host immune protection in COVID-19 patients, few researches focus on the functional validation of T cell epitopes and development of vaccines inducing specific T cell responses. In this study, 120 CD8+ T cell epitopes from E, M, N, S and RdRp proteins were validated. Among them, 110 epitopes have not been reported previously; 110, 15, 6, 14 and 12 epitopes were highly homologous with SARS-CoV, OC43, NL63, HKU1, and 229E, respectively; 4 epitopes from S protein displayed one amino acid distinct from the current variants of SARS-CoV-2. Thirty-one epitopes restricted by HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C), R848 or polylactic-co-glycolic acid nanoparticles, which elicited robust specific CD8+ T cell responses in wild-type and HLA-A2/DR1 transgenic mice. Seven of the 31 epitopes were found to be cross-presented by HLA-A2 and H-2K/Db molecules. Unlike previous researches, this study established a modified cell co-culture system of DC-peptide-PBL using healthy donor’s PBMCs to validate the CD8+ T cell epitope on-silicon predicted; provided a library of CD8+ T cell epitopes restricted by a series of high-frequency HLA-A allotypes which covering broad Asian populations; identified the HLA-A cross-restrictions of these CD8+ T cell epitopes using competitive binding experiments with HMy2.CIR cell lines expressing indicated HLA-A molecules; and initially confirmed the in vivo feasibility of 9 or 10-mer peptide cocktail vaccines of SARS-CoV2. These data will facilitate the development of vaccines inducing antiviral CD8+ T cell responses. The highly contagious COVID-19 has spread worldwide at an unprecedentedly quick speed 53 since its first identification at December 2019, leading to an ongoing global pandemic 1 . As 22 54 of April, 2021, there have been more than 143 million confirmed cases and over 3.0 million 55 deaths. Although relentless efforts have been paid in effective vaccine race, there are still 56 many risks for a long-term immune protection 2 , since host immunity to Severe Acute 57 Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has not been fully understood. Currently, 58 most vaccines focus on the induction of neutralizing antibodies against spike (S) protein of 59 SARS-CoV-2 3, 4 , which can block the virus from entering and infecting human cells, helping 60 the immune system to clear the virus and to prevent future infections 5 . However, researchers 61 have found that circulating antibodies to SARS-Cov-2 declined rapidly and persisted only 62 around seven months. And, certain patients who are asymptomatic or mildly symptomatic do 63 not have detectable neutralizing antibodies 6 . In addition, growing incidence of COVID-19 64 re-infections emerges since the first reported re-infection in August, 2020. This suggests that than 100% compared with the negative control or proliferation percentage of CD8 + T cells in 140 CD3 + /CD8 + T cell population increased by more than 20% compared with the negative 141 control, the candidate epitope peptide in the co-culture well was identified as positive peptide 142 with immunogenicity. 143 To evaluate whether this procedure is sensitive for identification of peptide 144 immunogenicity, several reference peptides were tested using this DC-peptide-PBL co-culture After the PBMC samples from 156 healthy donors were tested, a total of 120 candidate 156 epitope peptides of SARS-CoV-2 have been defined as antigenic T cell epitopes by using the 157 DC-peptide-PBL procedure, which indicating that they can elicit naive peptide-specific CD8 + 158 T cells to activate and produce IFN-γ or proliferate after 14 days co-stimulation. Some Table 1 . Of them, the number of epitopes derived from E (75aa), M 162 (222aa), N (419aa), S (1273aa) and RdRp (932aa) proteins is 18, 27, 12, 36, and 27, 163 respectively, with a relatively bias distribution. The density of CD8 + T cell epitopes per 10 aa 164 is 2.40, 1.216, 0.286, 0.283, and 0.290, respectively. Their sequence homology between 165 SARS-CoV-2 and other HCoVs were aligned and exhibited in Table S2 . Of the 120 validated 166 SARS-CoV-2 CD8 + T cell epitopes, 110, 15, 6, 14 and 12 epitopes were highly homologous 167 (0-2 amino acids deviation) with SARS-CoV, OC43, NL63, HKU1,and 229E, respectively. 168 The common epitopes with common-cold HCoVs mainly locate in RdRp protein (39/47). In (Table S2) . 172 Figure S1 showed the phenotypes of mature DC as verified by flow cytometry. (Table 3) . Surprisingly, all epitopes derived from E protein 192 displayed low affinity with corresponding HLA-A allotypes in this HLA-A competitive 193 binding assay (Table 3) , but these data are inconsistent with the results from DC-peptide-PBL 194 costimulation and vaccine immunization in HLA-A2/DR1 transgenic mice. Figure S6 ). According to the fluorescence index (FI), 18 epitopes showed high affinity (FI > 1.0), 7 208 epitopes displayed intermediate affinity (1.0 ≥ FI >0.5), and 6 epitopes exhibited low or no 209 binding (FI ≤ 0.5) with HLA-A0201 molecules (Table S5) . Finally, HLA-A0201 +/+ /DR1 +/+ 210 transgenic and H-2-β2m -/-/I-Aβ -/-C57BL/6 mice were immunized with the three vaccines 211 respectively. After three rounds in vivo stimulation, splenocytes of primed mice were tested 212 for peptide-specific T cell responses by IFN-γ-ELSPOT, IFN-γ-ICS and IFN-γ-ELISA. The 31 positive peptides were grouped into eight pools (Table S3) splenocytes from each mouse in the three vaccine groups was 400-500 times more than that 218 from control group ( Figure 3A ). Interestingly, splenocytes in all vaccine groups showed 219 almost the strongest T cell responses to E protein and weakest T cell responses to N protein 220 relative to other antigens ( Figure 3B ). Figure 4 showed that the frequencies of IFN-γ + in CD3 + CD8 + T cell populations from the three vaccine 230 groups were about 20-30 times higher than that in control mice ( Figure 3C ). Splenocytes in 231 vaccine A group showed the strongest CD8 + T cell responses to RdRP while splenocytes in 232 vaccine C group showed the strongest CD8 + T cell responses to E protein ( Figure 3D ). To investigate whether the HLA-A2 molecules-restricted 9 or 10-mer peptides can also be in the control group ( Figure 6C ). The robust CD8 + T cell responses were mainly against the 262 epitopes from M, N and S protein ( Figure 6D ). Figure 7A presented the flow plots of all mice. To uncover whether the peptide-based vaccine immunizations cause organ toxicity, the heart, 273 liver, lung and kidney from each mouse were checked at day 28 after the mice were 274 inoculated three times with Vaccine A, Vaccine B or Vaccine C. The organs were immersed 275 and stained with Hematoxylin-Eosin. As the scanning copy showed, no visible organ toxicity 276 was found in all organs in each group ( Figure S7 ). and CD8 + T cells in coordination with neutralizing antibodies will generate more robust and 288 durable protective immunity 20 SARS-CoV-2 in a relatively equal distribution 9 , here four structural proteins and the RdRp 318 consisted of nsp7, nsp8 and nsp12 were screened for the identification of CD8 + T cell epitopes to be further elucidated. In order to further confirm the sensitivity and reliability of this co-culture system, some 352 reference CD8 + T cell epitope peptides, which were derived from HCC-associated tumor Of the unexposed cohorts from the United States, Netherlands, Germany, Singapore, 420 and United Kingdom, 20-50% could detect specific memory CD4 + T cells that showed 421 cross-reactivity to SARS-CoV-2 antigens, but the frequency was about 10 times lower (0.1% 422 vs.1%) than that of those infected with SARS-CoV-2, presumably due to the common T cell (20 IU/mL) was added at day 11. At day 14, the corresponding peptide (20μg/mL) was added. At day 17, rhIL-2 was added again (10 IU/ mL). At day 21, cells were harvested and followed 498 by ICS or T cell proliferation assay. (Table S3) . For IFN-γ-ELISPOT assay, the 31 antigenic peptides were grouped 557 into eight pools according to their derived protein and the feature of acid and alkalinity (Table 558 S3). For IFN-γ ICS and ELISA, the 31 antigenic peptides were grouped into five pools 559 according to their derived proteins (Table S3) . groups, vaccines formula and vaccination scheme were described in Table S4 . The sponsors had no role in study design, data collection and analysis, preparation of the 639 manuscript, or decision to submit the article for publication. The authors declare no competing financial interests related to this study. T cells in CD3 + /CD8 + population was analyzed according to the reduction of CFSE-staining brightness. 892 IFN-γ: After DC-peptide-PBLs co-cultures, the frequency of IFN-γ + /CD8 + T cells in CD3 + /CD8 + 893 population was analyzed by flow cytometry. Enhance%: The increased percentage when the frequency of 894 IFN-γ + /CD8 + T cells or proliferation percentage of CD8 + T cells in the DC-peptide-PBL co-culture wells 895 was compared with that in the DC-PBL co-culture well without peptide. 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The Characteristics of Peripheral Lymphocyte Subset Alteration in COVID-19 COVID-19 pneumonia: CD8(+) T and NK cells are decreased in number 687 but compensatory increased in cytotoxic potential Systems-Level Immunomonitoring from Acute to Recovery Phase of 689 32 / 58 Severe COVID-19 Deep immune profiling of COVID-19 patients reveals distinct 691 immunotypes with therapeutic implications Imbalance of Regulatory and Cytotoxic SARS-CoV-2-Reactive 693 CD4(+) T Cells in COVID-19 Single-cell landscape of immunological responses in patients with 695 COVID-19 An Effective COVID-19 Vaccine Needs to Engage T Cells Mapping and role of T cell response in SARS-CoV-2-infected mice Computational perspectives revealed prospective vaccine candidates from 701 five structural proteins of novel SARS corona virus 2019 (SARS-CoV-2) Designing a next generation multi-epitope based peptide vaccine candidate 704 against SARS-CoV-2 using computational approaches Designing spike protein (S-Protein) based multi-epitope peptide vaccine 706 against SARS COVID-19 by immunoinformatics Immunoinformatic design of a COVID-19 subunit vaccine using entire 708 structural immunogenic epitopes of SARS-CoV-2 Immuno-informatics approach for multi-epitope vaccine designing 710 against SARS-CoV-2 Immune and bioinformatics identification of T cell and B cell 713 epitopes in the protein structure of SARS-CoV-2: A systematic review COVID-19 coronavirus vaccine T cell epitope prediction analysis based on 716 distributions of HLA class I loci (HLA-A, -B, -C) across global populations Unbiased Screens Show CD8+ T Cells of COVID-19 Patients 719 Recognize Shared Epitopes in SARS-CoV-2 that Largely Reside outside the Spike Protein Selective and cross-reactive SARS-CoV-2 T cell epitopes in unexposed 722 humans Identification and validation of 174 COVID-19 vaccine candidate 724 epitopes reveals low performance of common epitope prediction tools SARS-CoV-2-specific CD8+ T cell responses in convalescent COVID-19 727 individuals Antigen-Specific Adaptive Immunity to SARS-CoV-2 in Acute 729 COVID-19 and Associations with Age and Disease Severity Lack of peripheral memory B cell responses in recovered patients with 732 severe acute respiratory syndrome: a six-year follow-up study Virus-specific memory CD8 T cells provide substantial 735 protection from lethal severe acute respiratory syndrome coronavirus infection Neoepitopes-based vaccines: challenges and perspectives Personalized vaccines for cancer immunotherapy An immunogenic personal neoantigen vaccine for patients with melanoma. 742 Personalized RNA mutanome vaccines mobilize poly-specific therapeutic 744 immunity against cancer Rapid tumor regression in an Asian lung cancer patient following personalized 746 neo-epitope peptide vaccination Identification of an HLA-A*0201-restricted CD8+ T-cell epitope SSp-1 of 748 SARS-CoV spike protein Characterization of immunologic properties of a second HLA-A2 CML patients and HLA-A2 transgenic mice Identification of HLA-A*02:01-restricted candidate epitopes derived 753 from the non-structural polyprotein 1a of SARS-CoV-2 that may be natural targets of 754 CD8(+) T cell recognition in vivo D56 A2402 A2402 D62 A2402 A2402 D64 A2402 A2402 D65 A3001 A3001 D71 A3001 A3001 D72 A3001,A1102,A0201, A0207 A1101 A3001 A0207>A0201 D76 A3303 A3303 D77 A3303, A1102 A3303 A1101 A1102 D78 A3303, A0203, A0206 A0206 >A0203 > A3303 Figure 3: 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 Figure 4: 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 Figure 5: 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 Figure 7: 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264