Open Access

Simultaneous assessment of cytotoxic T lymphocyte responses against multiple viral infections by combined usage of optimal epitope matrices, anti- CD3 mAb T-cell expansion and "RecycleSpot"

  • Florian K Bihl1,
  • Elisabetta Loggi3,
  • John V ChisholmIII1,
  • Hannah S Hewitt1,
  • Leah M Henry1,
  • Caitlyn Linde1,
  • Todd J Suscovich1,
  • Johnson T Wong2,
  • Nicole Frahm1,
  • Pietro Andreone3 and
  • Christian Brander1Email author
Journal of Translational Medicine20053:20

DOI: 10.1186/1479-5876-3-20

Received: 09 March 2005

Accepted: 11 May 2005

Published: 11 May 2005

Abstract

The assessment of cellular anti-viral immunity is often hampered by the limited availability of adequate samples, especially when attempting simultaneous, high-resolution determination of T cell responses against multiple viral infections. Thus, the development of assay systems, which optimize cell usage, while still allowing for the detailed determination of breadth and magnitude of virus-specific cytotoxic T lymphocyte (CTL) responses, is urgently needed. This study provides an up-to-date listing of currently known, well-defined viral CTL epitopes for HIV, EBV, CMV, HCV and HBV and describes an approach that overcomes some of the above limitations through the use of peptide matrices of optimally defined viral CTL epitopes in combination with anti-CD3 in vitro T cell expansion and re-use of cells from negative ELISpot wells. The data show that, when compared to direct ex vivo cell preparations, antigen-unspecific in vitro T cell expansion maintains the breadth of detectable T cell responses and demonstrates that harvesting cells from negative ELISpot wells for re-use in subsequent ELISpot assays (RecycleSpot), further maximized the use of available cells. Furthermore when combining T cell expansion and RecycleSpot with the use of rationally designed peptide matrices, antiviral immunity against more than 400 different CTL epitopes from five different viruses can be reproducibly assessed from samples of less than 10 milliliters of blood without compromising information on the breadth and magnitude of these responses. Together, these data support an approach that facilitates the assessment of cellular immunity against multiple viral co-infections in settings where sample availability is severely limited.

Keywords

Cytotoxic T Cells HIV EBV CMV HCV HBV CTL epitope peptide cell expansion anti-CD3 ELISpot peptide matrix

Introduction

Cell-mediated immunity is considered critical for the prevention and control of many viral infections [16]. The approaches developed to detect these responses in vitro have evolved over the years and have provided quantitative and qualitative information on virus-specific T cells for a number of viral infections. These assays include, besides others, lymphoproliferative assays using 3H-thymidine incorporation or CFSE staining, limiting dilution precursor-frequency assays for the enumeration of CTL precursor frequencies, intracellular cytokine staining (ICS) and enzyme-linked immunospot (ELISpot) assays [710]. Although these assays differ in their minimal cell requirements, the detailed, simultaneous analysis of anti-viral immunity against multiple viral infections is often limited by cell availability, regardless of the assay employed.

The ELISpot assay has become widely used for rapidly assessing cellular immune responses to extensive numbers of antigens while using relatively few cells. A number of studies have also employed peptide matrix approaches, where every antigenic peptide is tested in two peptide pools, so that responses to reactive pools sharing a specific peptide can help to identify the targeted peptide [9, 11]. This has reduced the required cell numbers significantly, so that for instance HIV-specific responses can generally be comprehensively assessed using less than 15 × l06 cells [9]. However, despite such advances, the simultaneous enumeration of virus-specific immunity to multiple viral infections still exceeds the required sample size that can routinely be obtained. Sample size may not be of great concern when assessing CTL mediated immune responses against single, small genome viruses such as HIV and HCV, which can be tested in a comprehensive manner using overlapping peptide sets spanning the entire expressed viral genome [9, 12]. Nevertheless, such comprehensive approaches are not feasible for larger viruses, such as DNA-based herpesviruses like EBV, CMV and KSHV [4, 13]. Instead, immune analyses need either to be restricted to a selected number of specific viral proteins, or to the use of previously defined, optimal CTL epitopes. Responses against such optimally defined epitopes can account for a significant part of the total virus-specific immune responses, especially when they represent immunodominant epitopes covering the most immunogenic proteins of specific viral genomes. For well-studied viruses such as HIV, HCV, EBV and CMV, large sets of such optimally defined CTL epitopes, restricted by common HLA alleles, have been described in the past [1417], and provide a valuable alternative to measure pathogen-specific CTL responses without the need to synthesize comprehensive peptide sets spanning the entire viral genomes.

The present study describes an algorithm by which matrices of optimally defined CTL epitopes derived from five different human viral infections are used in the same ELISpot assay. As not all wells of the ELISpot plate contain antigens to which the tested PBMCs will respond, there are consistently some wells with cells that have not been stimulated during this first assay. Theoretically, these cells could be recovered from the ELISpot plate before developing it and re-used in subsequent analyses. Indeed, others have suggested the use of "recycled" cells for DNA isolation[18], however, to our knowledge, no data exist on re-using these cells in functional assays. Since the peptide matrix approach is ideally followed by the subsequent confirmation of single targeted peptides present in two corresponding peptide pools, recycled cells from unstimulated ELISpot wells could be used for these assays. Although this second step could be achieved using in vitro expanded cells, for instance by anti-CD3 monoclonal antibody (mAb) stimulation, expanded cells may lose some of the responses compared directly to cells tested ex vivo [19, 20]. In addition, the absolute and relative magnitude of responses may be distorted during cell expansion and assays can only be run after prolonged in vitro culture[21, 22] Therefore, as long as functionality of recycled cells in secondary assays can be ensured, they may provide a simple way to complete initial ELISpot screenings, yielding reliable information on the magnitude of specific CTL responses. The feasibility of this approach was tested and it was shown that combined use of optimal epitope matrices, in vitro T cell expansion and RecycleSpot can provide relevant immune data on multiple viral infections even when cell availability is severely limited.

Materials and methods

Isolation of fresh PMBCs from whole blood

Whole blood was collected using Citrate Vacutainer tubes (BD, Franklin Lakes, NJ) and peripheral blood mononuclear cells (PBMC) were isolated by Histopaque (Histopaque® 1077, Sigma, St. Louis, MO) density centrifugation as described [9]. Fresh PBMC were either used directly after isolation, after in vitro expansion or after freezing and thawing with and without subsequent in vitro expansion. For in vitro use, cells were re-suspended in R10 medium (RPMI 1640 containg 10% heat inactivated FCS (both Sigma), 2 mM L-glutamine, 50 U/ml penicillin, 50 μg/ml streptomucin and 10 mM HEPES (all Mediatech, Hemdon, VA)) at a concentration of 1 × 106 cells/ml. Cells were thawed using R10 medium containing 50 U/ml DNAse (Deoxyribnuclease I, RNase-free, Sigma), washed twice in the same medium, re-suspended in R10 and incubated at 37°C with 5% CO2 for 3–4 hours before they were counted and re-suspended in R10 at 1 × 106 cells/ml. The thawed cells were then either used directly in ELISpot assays or expanded.

For in vitro expansion, 1 to 5 × 106 PBMC were added to 25 ml culture flasks in 10 ml R10 supplemented with 1 μl of the anti-CD3 specific monoclonal antibody (mAb) 12F6 [23]. Cells were fed twice a week using R10 supplemented with 50 U/ml of recombinant Interleukin 2 (IL-2) for 2 weeks. Before use in ELISpot assays, cells were washed twice in R10 medium and incubated overnight at 37°C with 5% CO2 in the absence of IL-2. This overnight starving step was necessary to eliminate background in the subseqnet ELISpot assay, which was, in our hands, not an issue, regardless of how long the in vitro culture had been maintained.

Design of Optimal Peptide Matrix

A total of 416 optimal epitopes from five different viruses were assembled in 98 different peptide pools and used in 5 peptide matrices each containing peptides from a single virus. The number of pools and total number of peptides contained in each virus-specific peptide matrix are summarized in Table 1. Each peptide was present at a final concentration of 200 μg/ml in the peptide pools. Detailed lists of all optimal epitopes included in this study, along with their sequence and HLA restriction, are given in Tables 2 through 6.
Table 1

Virus specific peptide matrix design using previously defined HLA class I restricted CTL epitopes

Virus

Optimal epitopes

No. of peptide pools

Max. no. of peptides per pools

HIV

173

29

14

EBV

91

23

12

CMV

38

13

7

HCV

77

19

11

HBV

37

14

8

Table 2

Optimal HIV-derived HLA class I restricted CTL epitopes

Protein

HLA Restriction

Sequence

Position

gp120

A02

RGPGRAFVTI

311–320

gp120

A03

TVYYGVPVWK

37–46

gp120

A11l

SVITQACPK

199–207

gp120

A24

LFCASDAKAY

53–62

gp120

A29

SFEPIPIHY

209–217

gp120

A30

HIGPGRAFY

310–318

gp120

A32

RIKQIINMW

419–427

gp120

B07

RPNNNTRKSI

303–312

gp120

B08

RVKEKYQHL

2–10

gp120

B1516/Cw04

SFNCGGEFF

379–387

gp120

B3801

MHEDIISLW

104–112

gp120

B35

VPVWKEATTTL

42–52

gp120

B35

DPNPQEVVL

77–85

gp120

B44

AENLWVTVY

30–38

gp120

B51

LPCRIKQII

416–424

gp120

B55

VPVWKEATTT

42–51

gp120

A33

VFAVLSIVNR

187–196

gp120

A33

EVAQRAYR

320–327

gp41

A01

RRGWEVLKY

787–795

gp41

A02

SLLNATDIAV

818–827

gp41

A0205

RIRQGLERA

335–343

gp41

A03/A30

RLRDLLLIVTR

775–785

gp41

A23/A24

RYLKDQQLL

591–598

gp41

A30

IVNRNRQGY

704–712

gp41

A30

KYCWNLLQY

794–802

gp41

A6802

IVTRIVELL

782–790

gp41

B07

IPRRIRQGL

843–851

gp41

B08

YLKDQQLL

591–598

gp41

B08

RQGLERALL

848–856

gp41

B14

ERYLKDQQL

589–597

gp41

B2705

GRRGWEALKY

791–799

gp41

B35

TAVPWNASW

611–619

gp41

B4001

QELKNSAVSL

810–819

gp41

Cw3/Cw15

RAIEAQQHL

46–54

p17

A02

SLYNTVATL

77–85

p17

A03

KIRLRPGGK

18–26

p17

A03

RLRPGGKKK

20–28

p17

A03

RLRPGGKKKY

20–29

p17

A11

TLYCVHQRI

84–92

p17

A24

KYKLKHIVW

28–36

p17

A30

RSLYNTVATLY

74–86

p17

B08

GGKKKYKL

24–31

p17

B08

ELRSLYNTV

74–82

p17

B2705

IRLRPGGKK

19–27

p17

B35

WASRELERF

36–44

p17

B35

NSSKVSQNY

124–132

p17

B4001

IEIKDTKEAL

92–101

p17

B4002

GELDRWEKI

11–19

p24

A0207

YVDRFYKTL

164–172

p24

A11

ACQGVGGPGHK

349–359

p24

A24/B44

RDYVDRFFKTL

296–306

p24

A25

QAISPRTLNAW

145–155

p24

B07

SPRTLNAWV

148–156

p24

B07/B42/B81/Cw8

TPQDLNTML

48–56

p24

B07

GPGHKARVL

223–231

p24

B07

HPVHAGPIA

84–92

p24

B08

EIYKRWII

260–267

p24

B08

DCKTILKAL

329–337

p24

B14

DRFYKTLRA

298–306

p24

B1501

GLNKIVRMY

267–277

p24

B18

FRDYVDRFYK

293–302

p24

B2703

RRWIQLGLQK

260–269

p24

B2705

KRWIILGLNK

265–274

p24

B35

NPVPVGNIY

245–253

p24

B35

PPIPVGDIY

254–262

p24

B39

GHQAAMQML

193–201

p24

B4001

SEGATPQDL

176–184

p24

B4002

KETINEEAA

70–78

p24

B4002

AEWDRVHPV

78–86

p24

B44

AEQASQDVKNW

174–184

p24

B44

EEKAFSPEV

28–36

p24

B52

RMYSPTSI

143–150

p24

B53

TPYDINQML

48–56

p24

B53/B57

QASQEVKNW

176–184

p24

B57

ISPRTLNAW

15–23

p24

B57

KAFSPEVIPMF

30–40

p24

B57

TSTLQEQIGW

108–118

p24

B57

KAFSPEVI

30–37

p24

B58

TSTLQEQIGW

108–117

p24

B58

TSTVEEQIQW

108–117

p24

Cw0I

VIPMFSAL

36–43

p24

A25

ETINEEAAEW

71–80

p24

A26

EVIPMFSAL

35–43

p15

A02

FLGKIWPSYK

1–10

p15

B14

CRAPRKKGC

42–50

p15

B4001

KELYPLTSL

33–41

p15

B4002

TERQANFL

64–71

Protease

A6802/A74

ITLWQRPLV

3–11

Protease

A6802

DTVLEEMNL

30–38

Integrase

A30

KIQNFRVYY

219–227

Integrase

A03/A11

AVFIHNFKRK

179–188

Integrase

B1503

RKAKIIRDY

263–271

Integrase

B42

VPRRKAKII

260–268

Integrase

B57

KTAVQMAVF

173–181

RT

A26

ETKLGKAGY

604–612

RT

A02

ALVEICTEM

33–41

RT

A02

VIYQYMDDL

179–187

RT

A02

ILKEPVHGV

309–317

RT

A03

ALVEICTEMEK

33–43

RT

A03

GIPHPAGLK

93–101

RT

A03/A1 1

AIFQSSMTK

158–166

RT

A03

QIYPGIKVR

269–277

RT

A03

KLVDFRELNK

73–82

RT

A03

RMRGAHTNDVK

356–366

RT

A11

IYQEPFKNLK

341–350

RT

A11

QIIEQLIKK

80–88

RT

B51

TAFTIPSI

128–135

RT

B57

IVLPEKDSW

244–252

RT

B58

IAMESIVIW

375–383

RT

B81

LFLDGIDKA

715–723

RT

B1503

VTDSQYALGI

651–660

RT

A30

KQNPDIVIY

173–181

RT

A30

KLNWASQIY

263–271

RT

A30

RMRGAHTNDV

356–365

RT

A32

PIQKETWETW

392–401

RT

B08

GPKVKQWPL

18–26

RT

B1501

LVGKLNWASQIY

260–271

RT

B1501

IKLEPVHGVY

309–318

RT

B35

TVLDVGDAY

107–115

RT

B35

VPLDEDFRKY

118–127

RT

B35

NPDIVIYQY

175–183

RT

B35

HPDIVIYQY

175–183

RT

B4001

IEELRQHLL

202–210

RT

B42

YPGIKVRQL

271–279

RT

B51

EKEGKISKI

42–50

Vpr

A02

AIIRILQQL

59–67

Vpr

B07/B81

FPRIWLHGL

34–42

Vpr

B51

EAVRHFPRI

29–37

Vpr

B57

AVRHFPRIW

30–38

Tat

A6801

ITKGLGISYGR

39–49

Tat

B1503

FQTKGLGISY

38–47

Tat

B53

EPVDPRLEPW

2–11

Tat

Cw12

CCFHCQVC

30–37

Vif

A03

RIRTWKSLVK

17–26

Vif

A03

HMYISKKAK

28–36

Vif

A03

KTKPPLPSVKK

158–168

Vif

B07

HPRVSSEVHI

48–57

Vif

B18

LADQLIHLHY

102–111

Vif

B57

ISKKAKGWF

31–39

Nef

A02

PLTFGWCYKL

136–145

Nef

A02

VLEWRFDSRL

180–189

Nef

A03/A11

QVPLRPMTYK

73–82

Nef

A03/A11

AVDLSHFLK

84–92

Nef

A11

PLRPMTYK

75–82

Nef

A24

RYPLTFGW

134–141

Nef

A33

TRYPLTFGW

133–141

Nef

B07

FPVTPQVPLR

68–77

Nef

B07

FPVTPQVPL

68–76

Nef

B07

TPQVPLRPM

71–79

Nef

B07

RPMTYKAAL

77–85

Nef

B07

TPGPGVRYPL

128–137

Nef

B07

RQDILDLWIY

106–115

Nef

B08

WPTVRERM

13–20

Nef

B08

FLKEKGGL

90–97

Nef

B1501

TQGYFPDWQNY

117–127

Nef

B1501

RMRRAEPAA

19–27

Nef

B1503

WRFDSRLAF

183–191

Nef

B18/B53

YPLTFGWCY

135–143

Nef

B2705

RRQDILDLWI

105–114

Nef

B35

VPLRPMTY

74–81

Nef

A01/A29/837/857

YFPDWQNYT

120–128

Nef

B40

KEKGGLEGL

92–100

Nef

B42

TPGPGVRYPL

128–137

Nef

B53

YPLTFGWCF

135–143

Nef

B57

HTQGYFPDWQ

116–125

Nef

B57

HTQGYFPDW

116–124

Nef

Cw07

RRQDILDLWIY

105–115

Nef

Cw7

KRQEILDLWVY

105–115

Nef

Cw8

AAVDLSHFL

83–91

Rev

A03

ERILSTYLGR

57–66

Rev

B57/B58

KAVRLIKFLY

14–23

Rev

Cw05

SAEPVPLQL

67–75

Vpu

A33

EYRKILRQR

29–37

All epitopes were referred from the Los Alamos HIV Immunology Database 2004 [24].

Table 6

Optimal HBV-derived HLA class I restricted CTL epitopes

Protein

HLA Restriction

Sequence

Position

Reference

Core

A2

FLPSDFFPSV

18–27

[84]

Core

A2

CLTFGRETV

107–115

[85]

Core

A2

VLEYLVSFGV

115–124

[85]

Core

A2/A24

EYLVSFGVW

117–125

[86, 87]

Core

A2

ILSTLPETTV

139–148

[86]

Core

A33/A68

STLPETTVVRR

141–151

[88]

Core

A2

AILSKTGDPV

152–161

[89]

Env

A2

LLDPRVRGL

131–139

[85]

Env

A2

VLQAGFFLL

177–185

[90]

Env

A2

FLLTRILTI

183–191

[91]

Env

A2

SLNFLGGTTV

201–210

[92]

Env

A2

FLGGTPVCL

204–212

[89]

Env

A2

LLLCLIFLL

250–258

[86]

Env

A2

LLCLIFLLV

251–259

[92]

Env

A2

LLDYQGMLPV

260–269

[92]

Env

A2

LVLLDYQGML

269–278

[85]

Env

A2

VLLDYQGML

270–278

[85]

Env

A2

LLDYQGMLPV

271–280

[85]

Env

A2

WLSLLVPFV

335–343

[92]

Env

A2

LLVPFVQWFV

338–347

[92]

Env

A2

GLSPTVWLSV

348–357

[92]

Env

A2

SIVSPFIPLL

370–379

[89]

Env

A2

LLPIFFCLWV

378–387

[92]

Env

A2

ILSPFFFLPLL

382–390

[85]

x-Protein

A2

VLCLRPVGA

15–23

[93]

x-Protein

A2

TLPSPSSSA

36–44

[93]

x-Protein

A2

HLSLRGLFV

52–60

[93]

x-Protein

A2

VLHKRTLGL

92–100

[93]

x-Protein

A2

AMSTTDLEA

102–110

[93]

x-Protein

A2

CLFKDWEEL

115–123

[93]

Pol

A24

LYSSTVPVF

62–70

[90]

Pol

A2

GLSRYVARL

455–463

[90]

Pol

A2

YMDDVVLGA

551–559

[91]

Pol

A2

FLLSLGIHL

575–583

[90]

Pol

A24

KYTSFPWLL

756–764

[87]

Pol

A2

ILRGTSFVYV

773–782

[91]

Pol

A2

SLYADSPSV

816–824

[91]

ELISpot assay

96-well polyvinylidene plates (Millipore, Bedford, MA), pre-coated overnight with 2 μg/ml of anti-interferon gamma (IFN-γ) mAb 1-D1K (Mabtech, Stockholm, Sweden), were washed six times with sterile phosphate buffered saline (DPBS, no Ca & Mg, Mediatech) containing 1% fetal calf serum (FCS) before use. After washing, 30 μl of R10 were added to each well to avoid drying of the membrane, and 100,000 to 200,00 cells per well were added in 100 μl R10. 100,00 cells/well were used to detect responses to HIV, CMV and EBV, whereas responses to HCV and HBV were tested using 200,000 cells/well. Each peptide was added at a final concentration of 14 μg/ml (both single peptides as well as pools). As a negative control, cells were incubated in medium alone, and PHA was added at a concentration of 1.8 μg/ml to serve as a positive control. Plates were incubated for 16 h at 37°C with 5% CO2 before being developed. After washing six times with PBS, 100 μl of biotinylated anti-IFN-γ mAB 7-B6-1 (0.5 μg/ml, Mabtech) were added and plates were incubated for 1 hour at room temperature (RT). The plates were washed again and incubated with a 1:2000 dilution of streptavidin-coupled alkaline phosphatase (Streptavidin-ALP-PQ Mabtech) for 1 hour at RT in the dark. After washing the plates again, IFN-γ production was detected as dark spots after a short incubation of 10–20 minutes with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (BioRad, Hercules, CA). The color reaction was stopped by washing plates with tap water and the plates were air-dried before counting using a AID ELISPOT Reader Unit (Autoimmun Diagnostika GmbH, Strassberg, Germany). Results were expressed as spot forming cells (SFC) per million input cells. Thresholds for positive responses were determined as either 5 spots (50 SFC/106 input cells) or as the mean plus 3 standard deviations of negative control wells, whichever was higher.

RecycleSpot

After overnight incubation in a primary ELISpot assay, cells from all wells of the ELISpot plate were transferred to a 96-well round-bottom plate and incubated at 37°C with 5% CO2 while developing the ELISpot assay. Cells from wells without any spots (including negative control wells) were then pooled, counted and used for secondary ELISpot assays. In control experiments, cells corresponding to wells with positive responses were also pooled, washed extensively (>5 times) and re-used in subsequent, secondary ELISpot assays as well. Cells from positive control wells (PHA stimulated) were not used for subsequent assays.

Results

Design of the optimal epitope matrix for five viral infections

To simultaneously test CTL responses against five different viruses with a limited number of PBMCs, a peptide matrix approach was used that included all previously published, well-defined CTL epitopes in HIV, HCV, HBV, EBV and CMV. The total number of described CTL epitopes for these viruses varied from 37 described optimal epitopes in HBV to more than 170 optimal epitopes in HIV. A list of all the optimal epitopes included in the present study is given in Tables 2 through 6, totaling 416 well-defined, HLA class I-restricted CTL epitopes. The included HIV epitopes were derived from the annually updated list of HIV CTL epitopes at the Los Alamos National Laboratory HIV immunology database[24]. For all the other pathogens, the epitopes listed were those for which, to the best of our knowledge, at least one publication existed showing CTL activity against this epitope in at least one infected individual. While the optimal epitopes in HIV, HCV and HBV cover large parts of their respective viral genomes, the epitopes defined in EBV and CMV represent only a portion of the proteins expressed by these viruses. Given the approximately 100 open reading frames in these large-genome viruses, complete representation of all viral proteins can hardly be achieved and most studies on these pathogens have thus focused on a relatively small number of viral proteins, especially concentrating on those containing serological determinants and those characterized by specific viral gene expression patterns. Thus, described EBV and CMV encoded CTL epitopes are derived from eleven and four different viral proteins respectively, whereas the known HIV, HCV and HBV epitopes cover all the viral proteins in these small-genome pathogens.

As the number of described optimal CTL epitopes varies between pathogens, separate peptide matrices were designed for each virus (Table 1). Importantly, the first set of pools ("protein pools") was designed so that the pools contained all the epitopes derived from the same viral protein, whereas the second half of matrix peptide pools contained the epitopes in a non-protein specific composition ("random pools"). This matrix design allowed assessment of the virus specific immune response at different levels of resolution including i) a "total virus" specific response by adding up all the protein pool specific or random peptide pool specific responses, ii) a "protein" specific responses by focusing on single pools containing all the epitopes of a given protein; and iii) upon single peptide confirmation, on a single epitope level, by comparing responses in pools containing the same epitope. Together, the epitope matrix design facilitated the assessment of T cell responses to more than 400 CTL epitopes from five different viruses simultaneously, using less than 10 × 106 PBMCs while still allowing determination of breadth and magnitude of virus-, protein-, and epitope-specific responses for each virus separately.

Moreover, since each epitope is tested twice in different pools, it should reflect the same magnitude of response in each pool, thus the matrix approach provides its own internal control. Additionally, "protein pools" and "random pools" should theoretically yield the same total magnitude of responses since they, as a whole, contain the same set of peptides. To test this, and rule out the possibility that peptide compositions in the different pools interfered with the detection of specific responses, the magnitude of all "protein pool" and "random pool" specific responses were compared in 19 subjects infected with EBV (n = 19) and co-infected with CMV (n = 14), HIV (12), and HCV (9). These analyses showed a statistically highly significant correlation between total magnitudes of responses detected by either set of peptide pools, indicating that the peptide mixtures in the pools sharing a specific response did not significantly impact the detection of the targeted epitope (Figure 1). Of note, for all four viruses analyzed, the "random pools" detected a slightly higher, statistically not significant total virus-specific response than the "protein pools". This is likely due to the presence of highly reactive epitopes which, when tested in the same peptide pool, can exceed the upper detection limit of the ELISpot assay and may thus underestimate the total virus-specific magnitude of responses. This may be more likely for epitopes in "peptide pools" than "random pools" if some proteins elicit generally stronger immune responses than others. A protein pool accumulating strongly reactive epitopes would result in fewer spots than the total of the respective "random pools" containing these epitopes equally distributed and fully quantitative.
Figure 1

Comparable magnitude of responses detected by "protein" and "random" peptide pools: The magnitude of CTL responses was determined by adding magnitudes for all "protein" or "random" pools for each virus. Responses on the Y-axes represent the total of all virus specific "random pools", the X-axes indicate total responses detected using the "protein pools". Data from 12 HIV-, 19 EBV-, 14 CMV-, 9 HCV-infected individuals were tested against either set of peptide pools for A) HIV, B) HCV, C) EB V, and D) CMV and compared using the non-parametric Wilcoxon matched pairs test.

Cells from negative ELISpot wells can be used in secondary ELISpot assays (RecycleSpot)

In order to maximize cell use in samples with limited cell availability, we investigated whether cells from initial ELISpot matrix screens could be re-used in subsequent functional assays. Specifically, cells from wells that did not respond to peptides added in the first assay as well as the cells in the negative control wells may be used for secondary ELISpot assays. To assess the feasibility of this strategy, all wells from the initial ELISpot plate were transferred to a 96-well plate and incubated at 37°C with 5% CO2 while the ELISpot plate was developed. Cells from negative ELISpot wells were then used to confirm the identity of the epitope(s) targeted in the matrix peptide pools. In separate experiments, cells from initially positive wells were also tested in subsequent assays to determine if continuing IFN-γ production in these cells would prevent them from being used in further ELISpot assays. The analyses also compared ELISpot results in plates that were either undisturbed, or from which cells were transferred for later use.

Representative RecyleSpot assays using PBMC and recovered cells from initial ELISpot assays from three individuals are shown in Figure 2. In all cases, negative wells from initial peptide matrix ELISpot assays were re-used to reconfirm the identity of the presumed, single targeted epitope shared by the two pools. Further, initially positive pools were re-tested to assess whether recycled cells responded with a different magnitude compared to the initial assay. The data show that sufficient cells were recovered from initial assays to perform reconfirmations of single targeted epitopes in the RecycleSpot, and that background activity and magnitude of responses were not significantly different between the first and the subsequent assays. RecycleSpot assays that used initially positive wells, or mixtures of initially positive and negative wells, showed high background in the secondary assay, indicating ongoing IFN-γ production and thus precluding these cells from use in the RecycleSpot (data not shown). No effects on the quality and the number of spots between the manipulated and non-manipulated wells were observed, indicating that harvesting cells from the ELISpot plate did not negatively interfere with the quality of the assay, at least when cells are removed by careful pipetting using a 12-channel pipetor Furthermore, RecycleSpot assays were performed using both fresh and frozen/thawed cells and showed that HIV-and EBV-specific responses were maintained in recycled cells in both cases (data not shown). Together, the data indicate that RecycleSpot can provide sufficient numbers of cells from initial assays and that these cells maintain functional capacity for use in subsequent assays, without raising background activity. Also, the data show that re-using the cells form negative wells after an overnight incubation did not reduce the magnitude of responses to a statistically significant level.
Figure 2

RecycleSpot using recycled cells for the de-convolution of positive peptide pools: Wells of primary ELISpot and secondary RecycleSpot are shown. Line A shows the data from the initial ELISpot assay, including two positive wells indicating cellular response to EBV peptide pools, three negative and one positive control wells. Line B shows the same outline as in A, this time with recycled cells in a secondary ELISpot analysis and, one separate well, using the predicted targeted epitopes from the matrix analysis. The numbers indicate the spot forming cells per million PBMC.

In vitro expanded T cells mount responses detected in fresh ex vivo PBMC samples

Even though rational optimal epitope matrix design and RecycleSpots may help in reducing the required cell numbers for in vitro analyses, cell availability may still be limiting in settings where only very small biological samples can be obtained. In such instances, investigators have resorted to the use of in vitro expanded cells [19, 20, 25]. However, despite its potential usefulness in situations of small sample size (e.g. tissue biopsies or small volume peripheral blood samples), relatively little is known on how in vitro expansion impacts magnitude and breadth of detectable responses [20, 25]. Furthermore, CTL responses to pathogens like HIV, for which a defect in their proliferative capacity has been shown, may be severely distorted by in vitro expansion, even when stimulated unspecifically [7]. To address this issue and to investigate whether stimulation of PBMC with an anti-CD3 mAb (12F6) expands CTL of different specificity equally well, we tested cells either directly or after expansion against peptide sets of described HIV- and EBV-specific epitopes restricted by the individual's HLA alleles.

These analyses included twelve subjects, of which seven were tested for responses to HIV and EBV epitopes, while the remaining five were tested for EBV-specific responses only (Figure 3).
Figure 3

In vitro expansion of thawed cells increases the magnitude and breadth of HIV and EBV specific responses: Thawed PBMC from 12 individuals were tested against HIV and EBV peptide pools (n = 7 subjects) or against EBV peptide pools only (n = 5). Cells were used either directly after thawing or after thawing and a subsequent two-week in vitro expansion using the anti-CD3 mAb 12F6. A) The breadth of the detected responses (number of peptide pools reacting) and B) the total magnitude (sum of all positive peptide pools) is compared between the two cell preparation using the non-parametric Wilcoxon matched pairs test. C) PBMC from 5 EBV infected individuals were used either directly after isolation of after a two-week in vitro expansion or as frozen/thawed cells with and without in vitro expansion, and compared for the breadth (number of pools recognized) and D) total magnitude of the EBV specific responses.

In a first analysis, frozen PBMC were either tested directly or after a 2-week stimulation using 12F6 and the number of targeted HIV or EBV epitopes were compared, resulting in 19 data points (seven individuals tested for HIV and EBV responses and five subjects tested for EBV-specific responses). Flow cytometry in nine individuals showed preferential expansion of CD8 T cells, as CD4 expressing T cells ranged between 0.5% and 14% only, independent of HIV infection and starting CD4 T cell counts (data not shown). The Elispot results revealed no difference in the breadth of responses (number of targeted epitopes) between the directly tested and the expanded cells, as a median of 6.4 and 6.9 positive responses were detected for HIV and EBV, respectively (Figure 3A). The recognition of HIV- and EBV-derived epitopes was equally frequent by the two different cell preparations (data not shown). When the magnitude of responses was compared between directly used and expanded cells, expanded cells responded with a slightly higher magnitude than unexpanded cells. This trend was more prominent when HIV and EBV responses were analyzed separately. The HIV responses in directly tested cells showed a median of 185 SFC/106 PBMC, as compared to 285 SFC/106 PBMC in expanded cells (p = 0.0005); whereas the median EBV-specific responses had a magnitude of 170 SFC/106 in unexpanded PBMC compared to 190 SFC/106PBMC in expanded cells (p > NS).

Moreover, to determine whether freshly isolated cells could also be expanded without drastic changes in their response patterns, PBMC from five EBV-infected subjects were tested directly after isolation, or after freezing, and with or without in vitro expansion. In agreement with the data from frozen samples, no significant difference in the number of pools targeted or the median magnitude of these responses was observed (Figure 3 C and 3 D). Despite concordance among the response patterns between the different cell preparations that was as low as 80%, the overall breadth and magnitude of these responses did not change. In addition, when comparing the magnitudes of the responses between each other, the relative magnitude of the responses was maintained between the four different cell preparations (data not shown). Combined, the data demonstrate that anti-CD3 expanded cells maintain their specificity and relative magnitudes when compared to unexpanded cells (both when used fresh or after thawing) indicating that in vitro expansion could be employed when the breadth, but not the absolute magnitude of responses, is being assessed. This was the case for the assessment of HIV- as well as the EBV-specific responses, suggesting that cells specific for HIV do not significantly differ from EBV specific cells in their ability to undergo in vitro expansion using a non-antigenic stimulus.

Discussion

Cell availability can severely hamper in vitro analyses of antigen specific immune responses, hence approaches which optimize cell use are urgently needed. This is especially true for assays requiring extensive sets of antigens to be tested while only a limited number of cells can be obtained. However, logistic considerations may prevent repetitive sample collection for larger trials, and re-use of fresh or frozen samples could provide more effective ways to perform necessary analyses. The present study introduces a novel approach by which some of the sample limitations can be overcome, and may prove helpful in routine laboratory tests that currently do not make optimal use of available cells. This may not only facilitate currently performed assays, but may open possibilities to expand analyses to simultaneous assessment of even larger sets of antigens and additional functional aspects.

In the present study, we have designed and tested an approach that allows the assessment of the CTL mediated immunity against five different viral infections, including HIV, HCV, HBV, EBV and CMV. We provide an up-to-date listing of currently determined viral epitopes for which the minimal length and HLA restriction have been established. In the case of the small genome viruses HIV and HCV, these optimal epitopes represent a large portion of the respective immune targets [26]. Although they do not include all responses detected in OLP screenings, our comparative analyses of HIV-specific responses from 100 individuals detected by either overlapping peptide (OLP) sets or optimal epitopes show that on average 68% of the observed OLP responses are covered by previously established HIV optimal epitopes (data not shown).

The present data also show that PBMC recycled from negative wells from an ELISpot assay can be re-used for subsequent functional assays. Depending on the analyses performed in the subsequent assay, such as reconfirmation of single epitope responses predicted in the initial matrix analyses, relatively small numbers of cells maybe required. Thus, although individuals with broad responses in the initial ELISpot assay will not yield many negative wells from which to recycle cells, the wells with non-targeted peptide in addition to the negative control wells often provide sufficient quantities of recycled cells to complete the matrix based analyses. Since the responses in the RecycleSpot are not significantly diminished as compared to the initial assay (Figure 2), the magnitude of responses in the subsequent assay can still provide adequate data at the single epitope level.

In vitro expanded cells have been used in a number of studies where cell availability has been the limiting factor[21, 22]. However, no study has directly compared for instance biopsy and PBMC-derived responses in a systematic manner and on a single epitope level, and it is unclear whether the in vitro expansion provides identical data. In the present report, we have compared the response patterns to EBV- and HIV-derived antigens in directly ex vivo and in vitro expanded PBMC preparations. No significant differences were observed, although some responses are lost or gained upon expansion. As no difference in the concordance between EBV- and HIV-specific responses was observed, the data indicate that responses to both viruses are equally well expandable in vitro using an antigen-unspecific stimulus, despite the ongoing viral replication in most HIV infected subjects tested here.

Thus, optimal epitope matrices, RecycleSpot and in vitro expansion of cells can be combined to achieve maximal information on an extensive set of antigens, even if sample availability is limited. As a practical approach, expanded cells from frozen PBMC aliquots can be used initially to screen a large number of antigens to determine the approximate breadth of responses within the set of antigens used. Subsequent studies using unexpanded cells and antigen matrices in conjunction with RecycleSpot would then allow determination of the true breadth and, more importantly, the true magnitude of these responses while requiring minimal cell numbers. Furthermore, cells can be successfully recovered from the RecycleSpot once more to be used for genetic analyses such as HLA typing. This combined approach should facilitate future work in settings in which cell availability is of constant concern.
Table 3

Optimal EBV-derived HLA class I restricted CTL epitopes

Protein

HLA Restriction

Sequence

Position

Reference

BMLF1

A1

LVSDYCNVLNKEFT

25–39

[27]

BMLF1

A2

GLCTLVAML

280–288

[28]

BMLF1

B18

DEVEFLGHY

397–405

[28]

BMLF1

n.d.*

KDTWLDARM

265–273

[29]

BMLF1

A24

DYNFVKQLF

320–328

[30]

BHRF

A2

LLWAARPRL

204–212

[31]

BZLF1

B7

LPCVLWPVL

44–52

[13]

BZLF1

B8

RAKFQLL

190–197

[32]

BZLF1

Cw6

RKCCRAKFKQLLQH

186–201

[1]

BMRF1

Cw6

YRSGIIAW

268–276

[16]

BMRF1

Cw3

FRNLAYGRTCVLGK

86–100

[16]

BRLF1

A2

YVLDHLIVV

109–117

[33]

BRLF1

A2

RALIKTLPRASYSSH

225–239

[27]

BRLF1

A3

RVRAYTYSK

148–156

[1]

BRLF1

A11

ATIGTAMYK

134–142

[16]

BRLF1

A24

DYCNVLNKEF

28–37

[27]

BRLF1

A24

TYPVLEEMF

198–206

[30]

BRLF1

B61

QKEEAAICGQMDLS

529–543

[1]

BRLF1

Cw4

ERPIFPHPSKPTFLP

393–407

[1]

gp110

A2

ILIYNGWYA

106–114

[1]

gp110

B35

VPGSETMCY

544–552

[1]

gp110

B35

APGWLIWTY

190–198

[1]

gp85

A2

TLFIGSHVV

420–428

[1]

gp85

A2

LMPIIPLINV

542–550

[1]

gp85

A2

SLVIVTTFV

225–233

[1]

gp350

A2

VLQWASLAV

863–871

[1]

gp350

A2

VLTLLLLLV

871–879

[34]

gp350

A2

LIPETVPYI

152–160

[34]

gp350

A2

QLTPHTKAV

67–75

[34]

EBNA1

A2

FMVFLQTHI

562–570

[13]

EBNA1

B7

RPQKRPSCI

72–80

[35]

EBNA1

B7

IPQCRLTPL

528–536

[35]

EBNA1

B53

HPVGEADYF

407–415

[35]

EBNA2

A2/B51

DTPLIPLTIF

42–50

[36]

EBNA3A

A2

SVRDRLARL

596–604

[37]

EBNA3A

A3

RLRAEAQVK

603–611

[38]

EBNA3A

A24

RYSIFFDY

246–253

[37]

EBNA3A

A29

VFSDGRVAC

491–499

[16]

EBNA3A

A30

AYSSWMYSY

176–184

[1]

EBNA3A

B7

RPPIFIRRL

379–387

[39]

EBNA3A

B7

VPAPAGPIV

502–510

[16]

EBNA3A

B8

QAKWRLQTL

158–166

[37]

EBNA3A

B8

FLRGRAYGL

325–333

[40]

EBNA3A

B35

YPLHEQYGM

458–466

[37]

EBNA3A

B46

VQPPQLTLQV

617–625

[41]

EBNA3A

B62

LEKARGSTY

406–414

[16]

EBNA3A

n.d.*

HLAAQGMAY

318–326

[16]

EBNA3B

A1l

NPTQAPVIQLHAVY

101–115

[40]

EBNA3B

A1l

AVFDRKSDAK

399–408

[16]

EBNA3B

A1l

LPGPQVTAVLLHEES

481–495

[40]

EBNA3B

A1l

DEPASTEPVHDQLL

551–563

[40]

EBNA3B

Al1

IVTDFSVIK

416–424

[40]

EBNA3B

A24

TYSAGIVQI

217–225

[16]

EBNA3B

A27

RRARSLSAERY

243–253

[42]

EBNA3B

B35

AVLLHEESM

488–496

[1]

EBNA3B

B44

VEITPYKPTW

657–666

[16]

EBNA3B

B58

VSFIEFVGW

279–287

[43]

EBNA3B

B62

GQGGSPTAM

831–839

[16]

EBNA3C

B7

QPRAPIRPI

881–889

[39]

EBNA3C

B27

RRIYDLIEL

258–266

[44]

EBNA3C

B27

HRCQAIRK

149–157

[16]

EBNA3C

B27

FRKAQIQGL

343–351

[16]

EBNA3C

B27

RKIYDLIEL

258–266

[45]

EBNA3C

B27

RRIFDLIEL

258–266

[45]

EBNA3C

B27

LRGKWQRRYR

249–258

[44]

EBNA3C

B37

LDFVRFMGV

285–293

[46]

EBNA3C

B39

HHIWQNLL

271–278

[16]

EBNA3C

B44

KEHVIQNAF

335–343

[47]

EBNA3C

B44

EENLLDFVRF

281–290

[40]

EBNA3C

B44

EGGVGWRHW

163–171

[48]

EBNA3C

B62

QNGALAINTF

213–222

[49]

EBNALP

A2

SLREWLLRI

284–292

[43]

LMP1

A2

YLQQNWWTL

159–167

[50]

LMP1

A2

YLLEMLWRL

125–133

[50]

LMP1

A2

LLVDLLWLL

167–175

[50]

LMP1

A2

TLLVDLLWL

166–174

[50]

LMP1

A2

LLLIALWNL

92–100

[50]

LMP1

B51

DPHGPVQLSYYD

393–404

[51]

LMP2

A2

FLYALALLL

356–364

[52]

LMP2

A2

LLWTLWLL

329–337

[53]

LMP2

A2

CLGGLLTMV

426–434

[54]

LMP2

A2

LTAGFLIFL

453–461

[53]

LMP2

A11

SSCSSCPLSKI

340–350

[53]

LMP2

A23

PYLFWLAAI

131–139

[55]

LMP2

A2

LLSAWILTA

447–455

[43]

LMP2

A24

TYGPVFMCL

419–427

[53]

LMP2

A24

IYVLVMLVL

222–230

[30]

LMP2

A25

VMNSNTLLSAW

442–451

[16]

LMP2

A27

RRRWRRLTV

236–244

[44]

LMP2

B40

IEDPPFNSL

200–208

[53]

LMP2

B63

WTLWLLI

331–338

[1]

*not determined

Table 4

Optimal CMV-derived HLA class I restricted CTL epitopes

Protein

HLA Restriction

Sequence

Position

Reference

pp65

B35

IPSINVHHY

123–131

[56]

pp65

B35

DDVWTSGSDSDEELV

397–411

[57]

pp65

B35

VFPTKDVAL

187–195

[17]

pp65

B38

PTFTSQYRIQGKL

367–379

[17]

pp65

B7

TPRVTGGGAM

417–426

[57]

pp65

B7

RPHERNGFTVL

265–275

[17]

pp65

A1

YSEHPTFTSQY

363–373

[17]

pp65

A1101

SVLGPISGHVLK

13–24

[17]

pp65

A2402

FTSQYRIQGKL

369–379

[17]

pp65

A68

FVFPTKDVALP

186–196

[17]

pp65

A2

NLVPMVATV

495–503

[57]

pp65

A2

VLGPISGHV

14–22

[58]

pp65

A2

MLNIPSINV

120–128

[58]

pp65

B44

EFFWDANDIY

512–521

[57]

pp65

A2402

VYALPLKML

113–121

[59]

pp65

A2402/Cw0401

QYDPVAALF

341–349

[60, 61]

pp65

B5201

QMWQARLTV

155–163

[62]

pp65

A0207

RIFAELEGV

522–530

[61]

pp65

A1101

ATVQGQNLK

501–509

[61]

pp65

B1501

KMQVIGDQY

215–223

[61]

pp65

B4001

CEDVPSGKL

232–240

[61]

pp65

B40

HERNGFTVL

267–275

[61]

pp65

B4006

AELEGVWQPA

525–534

[61]

pp65

B4403

SEHPTFTSQY

364–373

[61]

pp65

B5101

DALPGPCI

545–552

[61]

pp65

Cw0102

RCPEMISVL

7–15

[61]

pp65

Cw0801

VVCAHELVC

198–206

[61]

pp65

Cw1202

VAFTSHEHF

294–302

[61]

pp65

A33

SVNVHNPTGR

91–100

[63]

pp150

A0301

TTVYPPSSTAK

945–955

[17]

pp150

A68

QTVTSTPVQGR

792–802

[17]

IE

B7

CRVLCCYVL

309–317

[64]

IE

A2

YILEETSVM

315–323

[65]

IE

B18

ELKRKMIYM

199–207

[65]

IE

B18

CVETMCNEY

279–287

[65]

IE

B18

DEEDAIVAY

379–387

[65]

IE

B18

SDEEEAIVAYTL

378–389

[56]

GB

A2

FIAGNSAYEYV

618–628

[66]

Table 5

Optimal HCV-derived HLA class I restricted CTL epitopes

Protein

HLA Restriction

Sequence

Position

Reference

Core

B60

GQIVGGVYLL

28–37

[67]

Core

A0201

YLLPRRGPRL

35–44

[68]

Core

B7

GPRLGVRAT

41–49

[69]

Core

B44

NEGCGWAGW

88–96

[70]

Core

A0201

DLMGYIPLV

132–140

[71]

Core

A0201

ALAHGVRAL

150–158

[15]

Core

A0201

LLALLSCLTV

178–187

[72]

Core

A11

MSTNPKPQK

1–9

[73]

P7

A29

FYGMWPLLL

790–798

[15]

P7

Cw7

FYGMWPLL

790–797

[15]

E1

A0201

ILHTPGCV

220–227

[74]

E1

B35

NASRCWVAM

234–242

[25]

E1

A0201

QLRRHIDLLV

257–266

[74]

E1

A23

FLVGQLFTF

285–293

[15]

E1

A0201

MMMNWSPTT

322–330

[15]

E1

A0201

SMVGNWAKV

363–371

[74]

E1

B35

CPNSSIVY

207–214

[15]

E2

A0201

SLLAPGAKQNV

401–411

[74]

E2

B53

CRPLTDFDQGW

460–469

[69]

E2

B51

YPPKPCGI

489–496

[73]

E2

B60

GENDTDVFVL

530–539

[75]

E2

B50

CVIGGAGNNT

569–578

[73]

E2

A0201

RLWHYPCTV

614–622

[76]

E2

A11

TINYTIFK

621–628

[69]

E2

B60

LEDRDRSEL

654–662

[75]

E2

A2402

EYVLLLFLL

717–725

[77]

E2

B57

NTRPPLGNWF

541–550

[15]

NS2

A29

MALTLSPY

827–834

[25]

NS2

A25

SPYYKRYISW

832–841

[78]

NS2

A23

YISWCLWWL

838–845

[69]

NS3

A24

AYSQQTRGL

1031–1039

[79]

NS3

A0201

CINGVCWTV

1073–1081

[68]

NS3

A0201

LLCPAGHAV

1169–1177

[68]

NS3

A0201

LLCPSGHAV

1169–1177

[68]

NS3

A11

TLGFGAYMSK

1261–1270

[80]

NS3

A0201

ATLGFGAYM

1260–1268

[81]

NS3

A0201

TLHGPTPLL

1617–1625

[81]

NS3

A0201

TGAPVTYSTY

1287–1296

[79]

NS3

A2402

TYSTYGKFL

1292–1300

[77]

NS3

B35

HPNIEEVAL

1359–1367

[82]

NS3

B8

HSKKKCDEL

1395–1403

[69]

NS3

A0201

KLVALGINAV

1406–1415

[68]

NS3

B8

LIRLKPTL

1611–1618

[75]

NS3

A11

TLTHPVTK

1636–1643

[80]

NS3

A68

HAVGLFRAA

1175–1184

[15]

NS3

A0201

GLLGCIITSL

1038–1047

[81]

NS4

A2402

FWAKHMWNF

1760–1768

[77]

NS4

B35

IPDREVLY

1695–1712

[15]

NS4

A24

VIAPAVQTNW

1745–1754

[15]

NS4

B57

LTTSQTLLF

1801–1809

[15]

NS4B

A25

EVIAPAVQTNW

1744–1754

[78]

NS4B

A25

ETFWAKHMW

1758–1766

[78]

NS4B

A0201

SLMAFTAAV

1789–1797

[68]

NS4B

A0201

LLFNILGGWV

1807–1816

[72]

NS4B

A0201

ILAGYGAGV

1851–1859

[72]

NS4B

B37

SECTTPCSGSW

1966–1976

[78]

NS4B

B38

AARVTAIL

1941–1948

[75]

NS5

A2

VLSDFKTWL

1987–1995

[83]

NS5

B35

EPEPDVAVL

2162–2170

[15]

NS5

B57

LGVPPLRAWR

2912–2921

[15]

NS5A

B60

HEYPVGSQL

2152–2160

[75]

NS5A

B35

PCEPEPDVAVL

2161–2171

[75]

NS5A

B38

NHDSPDAEL

2218–2226

[75]

NS5A

A2

SPDAELIEANL

2221–2231

[75]

NS5A

A25

ELIEANLLW

2225–2233

[78]

NS5A

A0201

ILDSFDPLV

2252–2260

[68]

NS5A

B60

REISVPAEIL

2267–2275

[80]

NS5B

A3

SLTPPHSAK

2510–2518

[80]

NS5B

A3

RVCEKMALY

2588–2596

[69]

NS5B

A2

ALYDWTKL

2594–2602

[78]

NS5B

B57

KSKKTPMGF

2629–2637

[80]

NS5B

A0201

GLQDCTMLV

2727–2735

[72]

NS5B

B38

HDGAGKRVYL

2794–2804

[80]

NS5B

A25

TARHTPVNSW

2819–2828

[78]

NS5B

A2402

RMILMTHFF

2841–2849

[77]

NS5B

A2402

CYSIEPLDL

2870–2878

[77]

NS5B

A31

VGIYLLPNR

3003–3011

[80]

Declarations

Acknowledgements

This work was supported by a grant of the Swiss National Science Foundation to FKB (SNF-PBSKB-102686) and by the Solid Organ Transplantation in HIV: Multi-Side Study (AI052748) funded by the National Institute of Allergy and Infectious Diseases.

Authors’ Affiliations

(1)
Partners AIDS Research Center, Massachusetts General Hospital, Harvard Medical School
(2)
Department of Pathology, Massachusetts General Hospital, Harvard Medical School
(3)
Dipartimento di Cardioangiologia ed Epatologia, Ospedale S. Orsola-Malpighi, Università degli Studi di Bologna

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© Bihl et al; licensee BioMed Central Ltd. 2005

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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