We report here on antigens from the SARS-CoV-2 virus spike protein, that when presented by Class I MHC, can lead to cytotoxic CD8⁺ T cell anti-viral responses in COVID-19 patients. We present a method in which the SARS-CoV-2 spike protein is converted into a library of peptide antigen-Major Histocompatibility Complexes (pMHCs) as single chain trimers that contain the peptide antigen, the MHC HLA allele, and the β-2 microglobulin sub-unit. That library is used to detect the evolution of virus-specific T cell populations from two COVID-19 patients, at two time points over the course of infection. Both patients exhibit similar virus-specific T cell populations, but very different time-trajectories of those populations. These results can be used to track those virus-specific T cell populations over the course of an infection, thus providing deep insight into the variations in immune system trajectories observed in different COVID-19 patients

Algren, Heather A.

Berrington, William R.

Chaffee, Mary E.

Choi, Jongchan

Chour, William

Delucia, Diane C.

Duvvuri, Venkata R.

Edmark, Rick A.

Goldman, Jason D.

Greenberg, Philip D.

Heath, James R.

Jones, Lesley C.

Lee, John K.

Murray, Kim M.

Ng, Alphonsus H. C.

O'Mahoney, D. Shane

Peng, Songming

Schmitt, Thomas M.

Wallick, Julie

Xie, Jingyi

Xu, Alexander M.

Yuan, Dan

English

Caltech Authors - Main

Shared Antigen-specific CD8+ T cell Responses Against the SARS-COV-2 Spike Protein in HLA 
A*02:01 COVID-19 Patients 
Supplemental Materials 
Table S1.  Top HLA A*02.01 9-mer to 11-mer epitopes as predicted by NetMHC4.0 algorithm. 
Table S2. Selected bacterial and viral A*02:01 peptides used for SCT expression. 
Table S3. Metadata on participants InCov002 and InCov005. 
Table S4. HLA haplotypes of COVID-19 participants (InCov002 and InCov005) and healthy donor 
sample. 
Fig. S1. SCT library expression of reported A*02:01 bacterial and viral peptides. 
Fig. S2. SCT design and optimization. 
Fig. S3. Expression of HLA-A*02:01 SCTs containing SARS-CoV-2 spike protein-derived epitopes. 
Fig. S4. Supplemental clinical data for participant InCov002. 
Fig. S5. Supplemental clinical data for participant InCov005. 
Fig. S6. Immunogenicity of spike protein peptides compared to predicted binding affinity. 
   
 
Table S1. Top HLA-A*02:01 9-mer to 11-mer epitopes as predicted by NetMHC4.0 algorithm. 
The peptides selected for expression as SCTs are listed in this table, indexed according to 
binding affinity. ‘Yield’ column denotes the relative expression of each SCT based on ratio of 
protein gel band intensity to positive control band (see Fig. S3). Relative yield is also shaded in 
grayscale in the column to the right. 
 
ID peptide domain span length predicted nM affinity yield ID ID peptide domain span length predicted nM affinity yield ID
1 YLQPRTFLL NTD 269-277 9 5.4 0.33 1 49 VLYENQKLI HR1 915-923 9 194.4 0.16 49
2 FQFCNDPFL NTD 133-141 9 9.2 1.19 2 50 KLIANQFNSA HR1 921-930 10 195.6 1.09 50
3 FIAGLIAIV TM 1220-1228 9 10.3 0.09 3 51 VTWFHAIHV NTD 62-70 9 202 0.17 51
4 FVSNGTHWFV CD 1095-1104 10 13.1 0.53 4 52 DLLFNKVTLA FP 820-829 10 202 0.08 52
5 SIIAYTMSL S2 691-699 9 13.5 0.88 5 53 QSIIAYTMSL S2 690-699 10 207.4 0.00 53
6 KLNDLCFTNV RBD 386-395 10 15.3 0.21 6 54 SRLDKVEAEV HR1/CH 982-991 10 221.2 0.08 54
7 RLQSLQTYV CH 1000-1008 9 16.7 0.18 7 55 QYIKWPWYIWL S2/TM 1208-1218 11 223.4 0.00 55
8 YHLMSFPQSA S2 1047-1056 10 17.9 0.08 8 56 TLLALHRSYL NTD 240-249 10 227.8 0.33 56
9 YIKWPWYIWL S2/TM 1209-1218 10 18.8 0.00 9 57 MFVFLVLLPLV S 1-11 11 229 0.00 57
10 YIWLGFIAGL TM 1215-1224 10 20.5 0.09 10 58 GLIAIVMVT TM 1223-1231 9 242 0.06 58
11 FELLHAPATV RBD/S1 515-524 10 21 0.16 11 59 YTNSFTRGV NTD 28-36 9 245.1 0.18 59
12 FTISVTTEI S2 718-726 9 25.4 1.74 12 60 ITSGWTFGA S2 882-890 9 265.1 0.11 60
13 LLFNKVTLA FP 821-829 9 25.4 1.74 13 61 KNLNESLIDL HR2 1191-1200 10 268.8 0.07 61
14 HLMSFPQSA S2 1048-1056 9 26.4 1.24 14 62 GVVFLHVTYV S2 1059-1068 10 272.4 0.12 62
15 FVFLVLLPLV S 2-11 10 32.6 0.00 15 63 LLIVNNATNV NTD 117-126 10 272.8 0.16 63
16 VLNDILSRL HR1 976-984 9 33.6 0.11 16 64 SLSSTASAL HR1 937-945 9 273.3 0.22 64
17 FIEDLLFNKV FP 817-826 10 36 0.11 17 65 KIYSKHTPI NTD 202-210 9 288.5 0.36 65
18 GYLQPRTFLL NTD 268-277 10 36.1 0.13 18 66 FVSGNCDVV CD 1121-1129 9 290.4 0.12 66
19 KIADYNYKL RBD 417-425 9 36.1 0.50 19 67 FQFCNDPFLG NTD 133-142 10 296.2 1.17 67
20 VVFLHVTYV S2 1060-1068 9 36.6 0.08 20  68 KLNDLCFTNVY RBD 386-396 11 302.6 0.16 68
21 RLDKVEAEV HR1/CH 983-991 9 39 0.23 21 69 VLSFELLHA RBD 512-520 9 302.6 0.11 69
22 FVFLVLLPL S 2-10 9 41.7 0.00 22 70 AIPTNFTISV S2 713-722 10 324.5 0.11 70
23 GFIAGLIAIV TM 1219-1228 10 56.9 0.15 23 71 VLLPLVSSQCV S/NTD 6-16 11 325.3 0.49 71
24 YQDVNCTEV S1 612-620 9 57.8 0.83 24 72 LLALHRSYL NTD 241-249 9 329.1 0.23 72
25 FLHVTYVPA S2 1062-1070 9 65.2 0.73 25 73 IITTDNTFV CD 1114-1122 9 338.8 0.10 73
26 MIAQYTSAL S2 869-877 9 69 0.92 26 74 IMLCCMTSC TM/CT 1232-1240 9 349 0.13 74
27 KLPDDFTGCV RBD 424-433 10 77.1 0.26 27 75 VFLHVTYVPA S2 1061-1070 10 377.3 0.00 75
28 GLIAIVMVTI TM 1223-1232 10 80.8 0.00 28 76 ELLHAPATV RBD/S1 516-524 9 380.7 0.99 76
29 KQIYKTPPI S2 786-794 9 85.6 0.61 29 77 VLHSTQDLFL NTD 47-56 10 382.2 0.00 77
30 EFQFCNDPFL NTD 132-141 10 87.8 0.00 30 78 VLYNSASFST RBD 367-376 10 388.9 0.29 78
31 TKLNDLCFTNV RBD 385-395 11 91.2 0.00 31 79 YRVVVLSFEL RBD 508-517 10 391.5 0.00 79
32 SVTTEILPV S2 721-729 9 113.1 0.90 32 80 SFIEDLLFNKV FP 816-826 11 394.6 0.05 80
33 YLQPRTFLLK NTD 269-278 10 118.8 0.54 33 81 FLPFFSNVT NTD 55-63 9 398.9 0.15 81
34 MQMAYRFNGI S2/HR1 900-909 10 120.9 0.39 34 82 SVLNDILSRL HR1 975-984 10 399.3 0.22 82
35 FQFCNDPFLGV NTD 133-143 11 126.8 1.09 35 83 ALEPLVDLPI NTD 222-231 10 405.6 0.07 83
36 GRLQSLQTYV CH 999-1008 10 128.4 0.13 36 84 ALQIPFAMQM S2 893-902 10 415.7 1.76 84
37 GLTVLPPLL S2 857-865 9 130.4 0.13 37 85 RVVVLSFEL RBD 509-517 9 419.8 0.12 85
38 FIAGLIAIVMV TM 1220-1230 11 136.3 0.09 38 86 FTISVTTEIL S2 718-727 10 419.8 1.66 86
39 VFVSNGTHWFV CD 1094-1104 11 137.7 0.00 39 87 FELLHAPATVC RBD/S1 515-525 11 435.6 0.09 87
40 WYIWLGFIAGL TM 1214-1224 11 140.1 0.00 40 88 YIWLGFIAGLI TM 1215-1225 11 436.3 0.00 88
41 SFELLHAPATV RBD/S1 514-524 11 140.8 0.10 41 89 KTSVDCTMYI S2 733-742 10 438.1 0.00 89
42 KQLSSNFGA HR1 964-972 9 141.2 0.22 42 90 GIYQTSNFRV S1 311-320 10 449 0.09 90
43 FIAGLIAIVM TM 1220-1229 10 143 0.10 43 91 MIAQYTSALL S2 869-878 10 453 1.10 91
44 TLDSKTQSL NTD 109-117 9 153.6 0.12 44 92 LGFIAGLIAIV TM 1218-1228 11 457.3 0.08 92
45 MFVFLVLLPL S 1-10 10 157.8 0.00 45 93 VGYLQPRTFLL NTD 267-277 11 461.4 0.06 93
46 RLITGRLQSL CH 995-1004 10 167 1.06 46 94 WTAGAAAYYV NTD 258-267 10 463.3 0.08 94
47 GYHLMSFPQSA S2 1046-1056 11 172.6 0.09 47 95 SWMESEFRV NTD 151-159 9 475.3 0.09 95
48 NLNESLIDL HR2 1192-1200 9 177.3 0.24 48 96 NFTISVTTEI S2 717-726 10 501.6 0.13 96
 
Table S2. Selected bacterial and viral A*02:01 peptides used for SCT expression. High affinity 
epitopes as predicted by NetMHC4.0 and experimentally reported from IEDB are 
(www.iedb.org) were selected for initial testing of SCT library expression platform.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ID peptide antigen organism length predicted nM affinity
1 LLFGYPVYV Protein Tax-1 HTLV-1 9 2.72
2 KLVALGINAV Genome polyprotein Hepatitis C virus 10 40.72
3 YVLDHLIVV Replication & transcription activatoEpstein Barr virus 9 4.13
4 FLLSLGIHL Protein P Hepatitis B virus 9 9.41
5 SLFNTVATL Gag polyprotein HIV-1 9 39.13
6 YLLFEVFDV Ad11 Hexon Adenovirus 9 4.00
7 FLDKGTYTL Envelope glycoprotein B Epstein Barr virus 9 3.79
8 YLQQNWWTL Latent membrane protein 1 Epstein Barr virus 9 9.46
9 AIMDKNIIL Non-structural protein 1 Influenza A virus 9 53.78
10 KLIANNTRV Ag85A protein M. tuberculosis 9 25.06
11 NLVPMVATV 65 kDa phosphoprotein Human CMV 9 25.85
12 FMYSDFHFI Polymerase acidic protein Influenza A virus 9 2.66
Patient:     InCoV002    InCoV005  
Time frame:   Admit: Time 1: Time 2  Admit: Time 1:  Time 2:  
Study Blood Draw 
Hospital Day & 
Time: 
 
N/A HD3 09:30 
HD8 
10:05 
 
N/A Day 2 13:00 
Day 6 
06:20 
Vital Signs & Clinical Status (most recent, range in 24 hour prior): 
Temp (oC):  38.9 37.3 36.8  37.1 36.8 36.6 
HR (/min):  101 59 80  120 116 105 
BP (mmHg):  139/88 96/57 125/74  104/60 126/105 130/79 
RR (/min):  18 20 20  20 26 31 
SpO2 (%):  90 96 91  93 93 91 
O2 delivery device:   NC HFNC VENT  NC NC HFNC 
WHO Ordinal Scale:   4 5 6  4 4 5 
Laboratory Data (closest to time frame): 
WBC (103/mm3):   6.9 6.0 8.9  11.5 8.0 5.0 
ANC (103/mm3):  5.38 4.56 5.34  10.46 6.88 3.8 
ALC (103/mm3):  1.10 1.08 2.40  0.58 0.56 0.60 
Cr (mg/dL):  0.79 0.76 0.73  1.23 0.83 0.86 
ALT (U/L):  16 16 1291  - 39 31 
AST (U/L):  26 27 1194  - 33 39 
PCT (ng/mL):  0.07 0.29 0.07  0.22 0.17 - 
CRP (mg/L):  129 260 10.0  203.1 178 40 
Treatments (number of doses, units per dose, during the interval prior to blood draw time 
frame, and subsequent to previous draw). 
RDV, # x dose:   0 0 1x200mg 3x100mg 
 0 0 1x200mg 3x100mg 
RDV, cumulative:   0 0 500 mg  0 0 500 mg 
HCQ, # x dose:   0 1x400mg 0  0 1x400mg 0 
HCQ, cumulative:   0 400mg  400 mg  0 400 mg 400 mg 
AZM, # x dose:   0 3x500mg 0  0 1x500mg 0 
AZM, cumulative:   0 1500 mg 1500 mg  0 500 mg 500 mg 
Steroid, # x dose:   0 0 0  * 0 0 
Steroid, cumulative:   0 0 0  * * * 
Toci, # x dose:   0 1x8mg/kg 2x8mg/kg  0 0 3x8mg/kg 
Toci, cumulative:   0 8 mg/kg 24 mg/kg  0 0 24 mg/kg 
* Steroid taper finished 8 days prior to admission, see Fig S5.    
Table S3. Metadata on participants InCov002 and InCov005. 
Abbreviations: Temp = temperature, HR = heart rate, BP = blood pressure, RR = respiratory rate, SpO2 = 
peripheral capillary oxygen saturation, O2 = oxygen, NC = nasal cannula, HFNC = high flow nasal cannula, 
VENT = mechanical ventilation, WHO = World Health Organization, WBCs = white blood cells, ANC = 
absolute neutrophil count, ALC = absolute lymphocyte count, ALT = alanine aminotransferase, AST = 
aspartate aminotransferase, PCT = procalcitonin, CRP = C-reactive protein, RDV = Remdesivir, HCQ = 
hydroxychloroquine, AZM = azithromycin, Toci = tocilizumab.   
 
 
 
Table S4. HLA haplotypes of COVID-19 participants (InCov002 and InCov005) and healthy donor 
sample. 
Sample ID Locus Allele 1 Allele 2
Diploid 
Ambiguities
Allele 1 
Ambiguities
Allele 2 
Ambiguities
InCov002 A A*01:01:01 A*02:01:01
A*02:01:130, 
A*02:01:159, 
A*02:844
B B*15:17:01 B*44:02:01
C C*05:01:01 C*07:01:02
DPA1 DPA1*01:03:01 DPA1*01:03:01
DPB1 DPB1*02:01:02 DPB1*20:01:01
DPB1*352:01:02 
+ DPB1*905:01 DPB1*02:01:19
DQA1 DQA1*01:02:01 DQA1*03:03:01
DQB1 DQB1*03:01:01 DQB1*06:04:01
DRB1 DRB1*04:01:01 DRB1*13:02:01
DRB345 DRB3*03:01:01 DRB4*01:03:01
InCov005 A A*01:01:01 A*02:01:01
A*02:01:130, 
A*02:01:159, 
A*02:844
B B*08:01:01 B*18:01:01
C C*07:01:01 C*07:01:01
DPA1 DPA1*01:03:01 DPA1*01:03:01
DPB1 DPB1*03:01:01 DPB1*04:02:01
DPB1*351:01 + 
DPB1*463:01:01
DQA1 DQA1*05:01:01 DQA1*05:05:01
DQB1 DQB1*02:01:01 DQB1*03:01:01
DRB1 DRB1*03:01:01 DRB1*11:04:01
DRB1*03:01:08 + 
DRB1*11:04:20 DRB1*03:147
DRB345 DRB3*01:01:02 DRB3*02:02:01
healthy donor A A*02:01 A*24:02
B B*39:05 B*51:01
C C*02:02 C*07:02
DPA1 DPA1*01:03 DPA1*01:03
DPB1 DPB1*04:01G DPB1*04:02G
DQA1 n/a n/a
DQB1 DQB1*03:02 DQB1*03:04
DRB1 DRB1*04:03 DRB1*04:07
DRB345 DRB4*01:03 DRB4*01:03
 
 
Fig. S1. SCT library expression of reported A*02:01 bacterial and viral peptides. A. Reduced SDS-
PAGE of SCT library. Transfection was conducted as a biological triplicate. Each unique SCT 
library is numbered according to peptide ID from Table S2. Leftmost lane of each gel is protein 
ladder (kDa masses indicated). -, supernatant from mammalian cells transfected without 
plasmid; +, purified WT1 SCT as mass reference and positive control. B. Quantified SCT 
expression. Average protein band intensity of each SCT library element relative to WT1 control 
band is plotted to demonstrate reproducibility of antigen-dependent SCT expression. Peptide 
IDs correspond to those of Table S2 and to numbered lanes in (A).  
 
 
Fig. S2. SCT design and optimization. A. Axial view of crystal structure of HLA-A*02:01 SCT.41 
Highlighted regions of interest: H74 (blue), Y84 (green), A139 (orange), first three amino acids 
of L1 linker (purple). Peptide is loaded into pocket in N-to-C direction (left-to-right). B. Summary 
of L1 and HLA amino acid modifications for each of the 6 SCT templates tested. C. Flow 
cytometry assay to identify optimized SCT-TCR capture. WT1 (RMFPNAPYL) SCTs constructed 
according to each of the 6 designs in (B) were paired with a MART-1 (ELAGIGILTV) SCT 
(constructed with design 1) to identify their cognate TCR-transduced cells in a 95/5 mixture of 
C4 TCR-transduced primary T cells and MART-1 Jurkat T cells. Number at top left of each plot 
indicates the SCT template used for WT1 SCT in the flow assay. D. Histogram of WT1 SCT 
tetramer-APC signal from flow assay. Percentages indicate the proportion of total cell 
population captured between the dashed lines by each of the six WT1 SCT designs. 
 
 
 
 
 
Fig. S3. Expression of HLA-A*02:01 SCTs containing SARS-CoV-2 spike protein-derived epitopes. 
A. Reduced SDS-PAGE of each transfected library element. Numbers above lanes correspond to 
peptide ID (Table S1). +, purified WT1 SCT as mass reference and positive control. See Table S1 
for quantified relative intensities of spike protein SCTs against WT1 SCT. B. Scatterplot 
comparing relative SCT expression of spike protein SCTs against NetMHC4.0 predicted binding 
affinity for each peptide. C. Boxplot comparing relative SCT expression of spike protein SCTs 
against the length of each peptide. 
 
 
 
 
 
A. Chest Radiograph.  B. Cytokine Profile:  
Name: Result 
(pg/mL): 
Ref 
Range: 
IL-2 <5 ≤5 
IL-2R 836 ≤1033 
IL-12 <5 ≤6 
INF-γ <5 ≤5 
IL-4 <5 ≤5 
IL-5 <5 ≤5 
IL-10 <5 ≤18 
IL-13 <5 ≤5 
IL-17 <5 ≤5 
IL-1β <5 ≤5 
IL-6 7  ≤5 
IL-8 <5 ≤5 
Cytokine profile (ARUP), on 
hospital day #2 at 14:00, 
prior to tocilizumab showing 
mildly elevated IL-6 levels.   
C. Time course with relevant clinical biomarkers 
 
Fig. S4. Supplemental clinical data for participant InCov002. A. Chest radiograph at arrival in the 
emergency room showing patchy bilaterally pulmonary infiltrates. B. Cytokine profile prior to 
tocilizumab. C. Clinical time course with relevant clinical biomarkers. T1 and T2 refer to the two 
time points of study blood collection. Abbreviations: RDV = remdesivir, Toci = tocilizumab, HCQ 
= hydroxychloroquine, AZM = azithromycin.  
 
 
A. CT angiography on arrival to the Emergency Room:   
 
B. Cytokine Profile:  
Name: Result (pg/mL): 
Ref 
Range: 
IL-2 <5 ≤12 
IL-2R 203 ≤1033 
IL-12 <5 ≤6 
INF-γ <5 ≤5 
IL-4 <5 ≤5 
IL-5 <5 ≤5 
IL-10 <5 ≤18 
IL-13 <5 ≤5 
IL-17 <5 ≤13 
IL-1β <5 ≤36 
IL-6 74  ≤5 
IL-8 <5 ≤5 
Cytokine profile (ARUP), on 
hospital day #7 at 15:32 after 
tocilizumab, showing 
persistent elevation in IL-6 
levels.   
C. Pulmonary Function Testing: Baseline %Pred Post-BD %Pred 
FVC (L) 3.25 77% 3.42 81% 
FEV1 (L) 1.98 63% 2.05 65% 
FEV1/FVC 61 81% 60 79% 
FEF 25-75% (L/sec) 0.88 38% 1.27 55% 
TLC (Pleth) (L) 7.24 100%   
DLCOunc (mL/min/mmHg) 21.4 82%   
D. Partial Medication List: albuterol 90 mcg/puff 2 puffs inhaled q 4-6 hours prn; albuterol 1.25 
mg/3 mL nebulizer inhaled q 6 hours prn; azithromycin 250 mg PO 3 times weekly; fluticasone-
umeclidinium-vilanterol 100-62.5-25 mcg/puff, 1 puff inhaled daily; predniSONE 40 mg daily X 3 
days, 30 mg daily X 3 days, 20 mg daily X 3 days, 10 mg daily X 3 days starting 3/4/20; losartan 
50 mg PO daily.  
Fig. S5. Supplemental clinical data for participant InCov005. A. Cut from computed tomography 
with angiography at arrival in the emergency room showing patchy geographic well-
circumscribed regions of ground glass opacities predominantly throughout the periphery 
bilaterally with superimposed moderate to severe emphysematous changes. B. Cytokine 
profile. C. Pulmonary function testing 24 days prior to arrival at the emergency room with 
spirometry, lung volumes and diffusion capacity. D. Relevant home medications prior to 
hospitalization. 
 
Fig. S6. Immunogenicity of spike protein peptides compared to predicted binding affinity. 
Scatterplot of counts per 104 CD8+ T cells as quantified by NP-NACS for each participant sample 
against each peptide’s NetMHC4.0 predicted binding affinity to HLA-A*02:01. 
 


Shared Antigen-specific CD8⁺ T cell Responses Against the SARS-COV-2 Spike Protein in HLA A*02:01 COVID-19 Participants

https://authors.library.caltech.edu/106749/3/2020.05.04.20085779-1.pdf

Shared Antigen-specific CD8⁺ T cell Responses Against the SARS-COV-2 Spike Protein in HLA A*02:01 COVID-19 Participants

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