Manuscript No. HEP-11-1036
Supporting information to the manuscript of Suneetha et al.:
HEV-specific T cell responses are associated with control
of HEV infection
Pothakamuri Venkata Suneetha1, Sven Pischke1, Verena Schlaphoff1, Jan Grabowski1,
Paraskevi Fytili1, Anna Gronert Alvarez1, Birgit Bremer1, Antoaneta Markova1, Jerzy
Jaroszewicz1,2, Christoph Bara3, Michael Peter Manns1, Markus Cornberg1, Heiner
Wedemeyer1*
1
Department of Gastroenterology Hepatology and Endocrinology, Hannover Medical School,
Hannover, Germany; 2Department of Infectious Diseases and Hepatology, Medical University in
Bialystok, Bialystok, Poland; 3Division of Cardiac, Thoracic Transplantation and Vascular
Surgery, Hannover Medical School, Hannover, Germany.
* Address for correspondence:
Prof. Dr.med. Heiner Wedemeyer
Department of Gastroenterology Hepatology and Endocrinology
Hannover Medical School, Hannover, Germany
Phone: +49-511-532-6814
Fax: +49-511-532-8662
Email: Wedemeyer.Heiner@mh-hannover.de
Supporting methods:
Isolation of Peripheral blood mononuclear cells (PBMCs)
PBMCs were isolated from whole blood samples using standard Ficoll-Hypaque density
gradient centrifugation (Biocoll. BIOCHROM AG, Berlin, Germany) and cryo-preserved in liquid
nitrogen as described earlier (1, 2).
Antibodies
The proliferation of CD4+ and CD8+ T lymphocytes was determined by flow cytometric CFSE
assay using PE-labelled anti-CD4 and PE-Cy7 labelled anti-CD8 antibodies (BD Biosciences,
CA, USA). For determining intracellular cytokine levels anti-IFN-γ -FITC, anti-MIP-1β-PE and
anti-TNF-APC antibodies were used. Surface antibody staining was performed by using antiCD8-APC-eFluor® 780 antibody (eBioscience, CA, USA). Levels of PD-1and CTLA-4 were
measured by using anti-PD1 (BioLegend Inc., San Diego, CA, USA) and anti-CTLA-4 antibodies
(BD Biosciences, CA, USA) antibodies respectively.
Hepatitis E virus peptide pools
HEV-specific overlapping peptides (15mers, overlapping adjacent peptides by 5aa) spanning
ORF2 and ORF3 of the genome corresponding to the amino acid sequences of the Yam67
strain of genotype 1a (Gene Bank accession number: AF459438) were synthesized from
PEPscreen custom peptide library (Proimmune, Oxford, UK). Peptides were dissolved in DMSO
as a stock solution with a concentration of 60 mg/ml. Details of the peptide pools, including
sequences, number of peptides per pool, and composition of each pool are given in Supporting
table 1. A total of 78 overlapping peptides were divided into 6 pools and cells were stimulated
using a final concentration of 5μg/ml with final DMSO concentration of 0.2% per pool. The ORF2
and ORF3 regions of HEV genome are at least 90% conserved among genotype 1 vs genotype
3 strains. All HEV overlapping peptides were synthesised based on standard single resin
synthesis. Peptides purity was tested by MALDI-TOF mass spectrometry and the average purity
of most of the peptides is >90%.
In-vitro lymphocyte proliferation assay
In-vitro proliferation assay was performed by CFSE method as described elsewhere (1, 2). After
staining, 0.3x106 cells were stimulated with HEV overlapping peptide pools at a final
concentration of 5μg/ml in 96 well round bottom plates for 7 days at 37ºC and 5% CO2. On day
4, IL-2 was added (5U/ml). PHA (6μg/ml) and medium with 0.2% of DMSO served as positive
and negative control respectively. On day 7, CFSE labeled cells were stained with CD4+ and
CD8+ antibodies before acquiring the samples by flow-cytometry (FACSCanto II, Becton
Dickinson). Data was analyzed by FlowJo Software (TreeStar, SanCarlos, USA).
T cell proliferation was also measured in all patients with chronic hepatitis E overtime in the
presence or absence of viremia. Stimulation index (SI) was calculated by dividing proliferated
cells in the presence of peptide pools by proliferated cells in the absence of peptide pools. A SI
value more than 2 was considered as positive signal.
Cytometric Bead Array (CBA) and Intracellular cytokine Staining (ICS)
Both the assays were performed in different study groups as described earlier (1). Levels of IL10, IFN-
-17 were quantified in cell culture supernatants and intracellular IFN-γ, TNF
and MIP-1β cytokine levels were measured by FACS.
References
1. Suneetha PV, Schlaphoff V, Wang C, Stegmann KA, Fytili P, Sarin SK, Manns MP, et al.
Effect of peptide pools on effector functions of antigen-specific CD8+ T cells J Immunol
Methods 2009; 342(1-2): 33-48.
2. Schlaphoff V, Lunemann S, Suneetha PV, Jaroszewicz J, Grabowski J, Dietz J, Helfritz F, et
al. Dual function of the NK cell receptor 2B4 (CD244) in the regulation of HCV-specific CD8+ T
cells. PLoS Pathog 2011; 7(5): e1002045.
Supporting figure 1
Supporting Figure 1: HEV-specific antibody (IgG) titres: HEV-specific IgG antibodies were
relatively higher in immune-suppressed patients who developed chronic HEV infection as
compared to resolved transplant subjects (p=0.014) sero-positive healthy controls (p=0.02).
Supporting figure 2
Gated on CD4+ T cells
PD1
CTLACTLA-4
Gated on CD8+ T cells
PD1
CTLACTLA-4
Supporting figure 2: Ex vivo expression levels of PD-1 and CTLA-4 was studied in patients
with chronic HEV infection by surface or intracellular staining respectively, and the expression
was detectable in all the patients included in the study.
Supporting table 1: Details of amino acid sequences of peptides spanning entire region of
ORF2 (peptides 1-66) and ORF3 (peptides 67-76) regions of the HEV genome.
Pool number
1
Peptide sequence
1. MRPRPILLLFLMFLP
2. LMFLPMLPAPPPGQP
3. PPGQPSGRRRGRRSG
4. GRRSGGSGGGFWGDR
5. FWGDRVDSQPFAPYI
6. FAPYIHPTNPFAPDV
7. FAPDVTAAAGAGPRV
8. AGPRVRQPVRPLGSA
9. PLGSAWRDQAQRPAA
10. QRPAAASRRRPTTAG
11. PTTAGAAPLTAVAPA
12. AVAPAHDTPPVPDVD
13. VPDVDSRGAILRRQY
2
14. LRRQYNLSTSPLTSS
15. PLTSSVATGTNLVLY
16. NLVLYAAHLSPLLPL
17. PLLPLQDGTNTHIMA
18. THIMATEASNYAQYR
19. YAQYRVARATIRYRP
20. IRYRPLVPNAVGGYA
21. VGGYAISISFWPQTT
22. WPQTTPTPTSVDMNS
23. VDMNSVTSTGVRILV
24. VRILVQPGIASELVI
25. SELVIPSERLHYRNQ
26. HYRNQGWRPVETSGV
3
27. ETSGVAEEEATSGLV
28. TSGLVMLCIHGSPVN
29. GSPVNSYTNTPYTGA
30. PYTGALGLLDFALEL
31. FALELEFRNLTPGNT
32. TPGNTNTRVSRYSST
33. RYSSTARHRLRRGAD
34. RRGADGTAELTTTAA
35. TTTAATRFMKDLYFT
36. DLYFTSTNGVGEIGR
37. GEIGRGIALTLFNLA
38. LFNLADTLLGGLPTE
39. GLPTELISSAGGQLF
40. GGQLFYSRPVVSANG
41. VSANGEPTVKLYTSV
Genome region
ORF2
4
42. LYTSVENAQQDKGIA
43. DKGIAIPHDIDLGES
44. DLGESRVVIQDYDNQ
45. DYDNQHEQDRPTPSP
46. PTPSPAPSRPFSVLR
47. FSVLRANDVLWLSLT
48. WLSLTGAEYDQSTYG
49. QSTYGSSTGPVYVSD
50. VYVSDSVTLVNVATG
51. NVATGAQAVARSLDW
52. RSLDWTKVTLDGRPL
5
53. DGRPLSTIQQYSKTF
54. YSKTFFVLPLRGKLS
55. RGKLSFWEAGTTKAG
56. TTKAGYPYNYNTTAS
57. NTTASDQLLVENAAG
58. ENAAGHRVAISTYTT
59. STYTTSLGAGPVSIS
60. PVSISAVAVLAPHSA
61. APHSALALLEDTLDY
62. DTLDYPARAHTFDDF
63. TFDDFCPECRPLGLQ
64. PLGLQGCAFQSTVAE
65. STVAELQRLKMKVGK
6
66. ELQRLKMKVGKTREL
67. MNNMSFAAPMGSRPC
68. GSRPCALGLFCCCSS
69. CCCSSCFCLCCPRHR
70. CPRHRPVSRLAAVVG
71. AAVVGGAAAVPAVVS
72. PAVVSGVTGLILSPS
73. ILSPSPPIFIQPTPS
74. QPTPSPPMSPLRPGL
75. LRPGLDLVFANPSDH
76. NPSDHSAPLGATRPS
77. ATRPSAPPLPHVVDL
78. PLPHVVDLPQLGPRR
ORF3