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Journal of Translational Medicine logoLink to Journal of Translational Medicine
. 2005 May 11;3:20. doi: 10.1186/1479-5876-3-20

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 Bihl 1, Elisabetta Loggi 3, John V Chisholm III 1, Hannah S Hewitt 1, Leah M Henry 1, Caitlyn Linde 1, Todd J Suscovich 1, Johnson T Wong 2, Nicole Frahm 1, Pietro Andreone 3, Christian Brander 1,
PMCID: PMC1164435  PMID: 15888204

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 [1-6]. 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 [7-10]. 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 [14-17], 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.

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.

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.

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]

Acknowledgments

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.

Contributor Information

Florian K Bihl, Email: fbihl@partners.org.

Elisabetta Loggi, Email: epalab@med.unibo.it.

John V Chisholm, III, Email: jchisholmiii@partners.org.

Hannah S Hewitt, Email: hshewitt@partners.org.

Leah M Henry, Email: lmhenry@partners.org.

Caitlyn Linde, Email: clinde1@partners.org.

Todd J Suscovich, Email: tsuscovich@partners.org.

Johnson T Wong, Email: jwong1@partners.org.

Nicole Frahm, Email: nfrahm@partners.org.

Pietro Andreone, Email: andreone@med.unibo.it.

Christian Brander, Email: brander@helix.mgh.harvard.edu.

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