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Transplant Infectious Disease . ISSN 1398 -2273 Ex vivo monitoring of human cytomegalovirus- specific CD8 1 T-cell responses usingQuantiFERONs-CMV  S.Walker, C. Fazou,T. Crough, R. Holdsworth, P. Kiely, M.Veale, S. Bell,  S. Walker1, C. Fazou1, T. Crough1,  A. Gailbraith, K. McNeil, S. Jones, R. Khanna. Ex vivo monitoring of  human cytomegalovirus-specific CD8 1 T-cell responses using  Transpl Infect Dis 2007. All rights reserved Tumour Immunology Laboratory and Co-Operative Centre for  Vaccine Technology, Division of Infectious Diseases and  Abstract:We have developed a novel diagnostic technology to monitor Immunology, Queensland Institute of Medical Research,  Herston, Queensland, Australia, 2Victorian Transplantation  the human cytomegalovirus (HCMV)-specific CD8 1 T-cell responses   and Immunogenetics Service, 3Virus Serology, Australian Red that is based on the detection of secreted interferon-gamma (IFN-g) in  Cross Blood Transfusion Service, Southbank, Victoria,  the whole blood (referred to as QuantiFERONs-CMV). Evaluation of  Australia, 4Cellestis Ltd., Carnegie, Victoria, Australia, 5The  QuantiFERONs-CMV in healthy individuals revealed that this  Prince Charles Hospital and Department of Medicine,  technology was at least as sensitive and with some HCMVepitopes  University of Queensland, Brisbane, Queensland, Australia  more sensitive than the ELISPOT for detecting ex vivo IFN-g. Results  from QuantiFERONs-CMVassays showed 97% (36/37 individuals)  Key words: virus; immune monitoring; transplant; T cells agreement with the anti-HCMV serology test in healthy individuals.
 Furthermore, we also show that this technology can be used to assess   HCMV-specificT-cell responses in transplant patients. This study  shows that QuantiFERONs-CMV is a simple, reproducible, and reliable  Rajiv Khanna, Clive Berghofer Cancer Research Centre,  test for the detection of IFN-g in response to HCMV CD8 1 T-cell  Queensland Institute of Medical Research, 300 Herston Road, epitopes, and may be a valuable diagnostic test for the detection of  HCMV infection and a useful clinical tool for monitoring the immune response in immunosuppressed patients during therapy.
 Received 16 May 2006, revised 26 July 2006, accepted for Human cytomegalovirus (HCMV) is a herpes virus that in- phocytes (CTLs) can protect against virus-associated path- fects between 50% and 85% of adults in the population (1).
ogenesis (2, 6, 7 ). Recent studies have shown that the HCMV is a frequently occurring complication of immuno- enumeration of CD8 1 HCMV-specific T cells in immuno- suppression, particularly after transplantation, and can suppressed patients and the production of interferon-gam- signi¢cantly contribute to morbidity and mortality in ma (IFN-g) can be predictive of the risk of developing CMV transplant recipients (2^4). Current immunosuppressive disease (8). IFN-g production can be a functional surrogate therapies used to prevent the rejection of a transplanted or- for the identi¢cation of HCMV-specific CTLs (9).
gan have detrimental effects upon the T lymphocytes and HCMV proteins have different roles in infection and the cell-mediated immune responses, resulting in increased pathogenesis of disease. A number of HCMV protein anti- susceptibility to viral infections posttransplant (5). The im- gens may therefore give rise to protective CTL responses.
portance of T-cell function in suppressing HCMV replica- Most of the previous studies have focused on CTL re- tion is also highlighted by the fact that adoptive transfer sponses to the HCMV phosphoprotein pp65 (10^13). In this or reconstitution of CD8 1 HCMV-specific cytotoxicT lym- study, we measure the IFN-g responses to a range of T-cell Walker et al: Immune monitoring for HCMV infection epitopes of HCMV viral proteins including pp65 and pp50, line (PBS) for use in ELISPOT or QuantiFERON-CMV the glycoprotein gB, and the immediate early IE-1 antigen that are specific for a wide range of human leukocyte anti-gen (HLA) class I speci¢cities (6). The development of a testthat uses a number of different HCMV antigen T-cell epi- topes and HLA class I alleles, such as QuantiFERONs (Cel- The assay was conducted in 2 parts, an overnight culture of lestis Ltd., Melbourne, Australia)-CMV will potentially blood with HCMV CD8 1 T-cell synthetic peptide epitopes have wide clinical diagnostic applications including moni- and the subsequent quanti¢cation of IFN-g production by toring HCMV infection and HCMV CD8 1 T-cell responses an enzyme-linked immunosorbent assay (ELISA). Initially, in immunosuppressed transplant patients during therapy.
1 mL aliquots of heparinized whole blood were incubated in In this study, we compare ELISPOT, which is routinely Linbro 24 -well plates with either HCMV peptide epitopes used as a laboratory research-based test, with the Quant- (2 mg/mL of each of 21 peptides in Table 1), sterilem PBS iFERONs CMV whole blood test to measure the IFN-g re- (no-antigen control), and phytohemagglutinin (PHA, posi- sponses using previously de¢ned CD8 1 CTL peptide tive mitogen control). Following an overnight incubation at epitopes for HCMV in healthy individuals and transplant 371C in a humidi¢ed atmosphere, the supernatant plasmas were harvested and analyzed for IFN-g by standard ELISA.
Results were calculated using Analysis Software v1.51(Cellestis Ltd.). Absorbances for the standards and test samples are entered into the computer and, based on thequality control acceptance criteria software, indicate pass or fail of the assay. Results were considered positive whenthe peptide response was greater than 0.2 IU/mL of IFN-g.
Peripheral blood samples from 37 healthy volunteer donors(age ranging from 29 to 51, median 40 years) were collected into heparinized collection tubes. These blood sampleswere used for HLA class I typing, HCMV serology, and The ELISPOT assay was used to detect IFN-g expression T-cell assays. HLA typing for healthy volunteers was per- by virus-specific Tcells following stimulation with HCMV formed by the V|ctorian Immunogenetics Service (Austral- epitope-specific CD8 1 T-cell epitopes (6). All the peptide ian Red Cross Blood Transfusion Service, V|ctoria, epitopes used in the ELISPOT assays are shown in Table 1.
Australia). In addition, a total of 25 solid organ transplant(heart and/or lung) (SOT) patients (age ranging from 15 to74, median 50 years) from the Prince Charles Hospital, Bris- bane, Australia, were recruited in this study. All sampleswere collected following informed consent, and the study Linear regression analysis (GraphPad Prism v4.00) was was approved by the human ethics committees of The used to compare the magnitude of IFN-g responses to mito- Queensland Institute of Medical Research and The Prince gen and HCMV peptide-stimulated samples in SOT pa- Charles Hospital. Anti-HCMV serological testing was per- tients. In addition, the non-parametric Mann^Whitney formed using the Abbott CMV total AB EIA (Abbott Labo- ratories, Abbott Park, Illinois, USA) by the V|rus SerologyDepartment, Australian Red Cross Blood Transfusion Ser-vice,V|ctoria, Australia.
Measurement of cell-mediated immune (CMI) responses is becoming increasingly important for many aspects of med-ical diagnosis, medical monitoring, vaccine development, HCMV epitopes restricted through various HLA class I al- and research in general (14, 15). Whereas historically, leles (HLA-A1, HLA-A2, HLA-A23, HLA-A24, HLA-B8, immune responses to stimuli such as infection and HLA-B35, HLA-B41, and HLA-B57 ) were used in this study vaccination have been generally detected by measuring a (see Table 1) (6). These peptides were synthesized using the specific antibody response, it has become apparent that Merri¢eld solid phase method and purchased from Chiron for many situations the measurement of CMI response is Mimotopes (Melbourne, Australia). All peptides were dis- solved in 10% dimethyl sulfoxide (DMSO) and diluted in In the ¢rst set of experiments we assessed HCMV- serum-free RPMI-1640 medium or phosphate-bu¡ered sa- specific T-cell responses in 10 healthy virus carriers using Walker et al: Immune monitoring for HCMV infection List of HCMV T-cell epitopes used in this study peptide epitopes resulted in high levels of IFN-g produc-tion which were readily detected by the QuantiFERONs assay. The sensitivity of the QuantiFERONs assay forHCMV epitopes was at least equivalent and in some cases more sensitive than the ELISPOT (Table 2). Most notably the QuantiFERONs assay does not require separation of peripheral blood lymphocytes (PBLs) and the assay can be carried out on whole blood. Thus, it is possible that the presence of additional ‘professional’ antigen presenting cells in the whole blood may improve the activation thresh-old for cytokine synthesis by virus-specificTcells resulting Based on the results obtained from the preliminary anal- ysis, we designed a QuantiFERONs-CMVassay kit that in- cluded a pool of HCMV peptide epitopes restricted through a range of HLA class I alleles (Table 1). These peptide epitopes were derived from multiple HCMV antigens. Theef¢cacy of this kit was assessed in a panel of 37 healthy volunteers at 2 different centers. A comprehensive sum- mary of data from these individuals is presented in F|g.
1A. QuantiFERONs-CMV assay showed 97% (36/37 indi- viduals; k 5 0.95) agreement with the anti-HCMV serology test of healthy individuals, and none of the seronegative do- nors showed reactivity using this assay. One seropositiveindividual did not show any reactivity with the QuantiFE- RONs-CMV assay. This may have been a false anti-HCMV serological test (19) or may be the result of undetectable levels of HCMV-specific T-cell precursors (20, 21).
In the next set of experiments we assessed the ef¢cacy of QuantiFERONs-CMVassay in a cohort of 25 SOT patients.
Peripheral blood samples from these patients were collect- ed at different time points posttransplant (range days 3^900). All 8 seronegative transplant patients showed IFN-g response below 0.1 IU/mL (mean Æ SD 5 0.0 Æ 0.1 IU/mL).
Of the 17 HCMV seropositive transplant recipients tested, all had a positive IFN-g response (19.0 Æ 22.5 IU/mL) (F|g.
1B).The magnitude of IFN-grk gamma responses to HCMV peptides positively correlated (r 5 0.70, P 5 0.0004) with that of the mitogen response. Importantly the reactivity to-wards the HCMV peptide epitopes was not affected ad- versely by antiviral or immunosuppressive therapies (cyclosporine A, mycopheloate mofetil, prednisolone, and tacrolimus [FK506]). It was observed that the levels of the *These peptides were not included in the QuantiFERONs-CMV kit.
IFN-g secretion following stimulation with HCMV peptide HCMV, human cytomegalovirus; HLA, human leukocyte antigen.
epitopes in the QuantiFERONs-CMVassay correlated withthe ELISPOT responses (F|g. 2).
Taken together the data demonstrate that QuantiFE- RONs-CMV assay is a sensitive and specific test for the de-tection of virus-specific T-cell responses in both healthy QuantiFERONs assay and compared the ef¢cacy of this individuals and SOT patients previously exposed to infec- assay with ELISPOT technology. Data from these donors tion. Over the last few years a number of technologies have are presented in Table 2. This analysis consistently showed been explored to monitor immune responses in various dis- that stimulation of peripheral blood with HCMV T-cell eases such as cancer, HIV infection, and transplantation Walker et al: Immune monitoring for HCMV infection Ex vivo analysis of virus-specificT-cell responses to HCMV T-cell epitopes Fig. 1. (A and B) Assessment of QuantiFERONs-CMV assay in healthy virus carriers (A) and solid organ transplant patients (B). It is important to note that multiple blood samples from di¡erent time intervals were ana- Data are presented as Mean Æ SD for both ELISPOT and QuantiFERON lyzed for some transplant patients. QuantiFERONs-CMV assay results assays.
HCMV, human cytomegalovirus; HLA, human leukocyte antigen; NT, not are expressed as interferon-gamma (IFN-g) (IU/mL) compared with the anti-human cytomegalovirus (HCMV) result from serological testing.
Data bars represent median Æ interquartile range of IFN-g production following stimulation with HCMV peptide epitopes or mitogen (PHA).
Number of patients tested for each of these groups is shown on x 5 axis.
(22, 23). The application of these techniques in the hospital No statistically signi¢cant di¡erence was observed between any groups.
setting is often constrained by the requirement of special-ized equipment or trained personnel.The QuantiFERONs- fection. Indeed, studies carried out in our laboratory have CMV assay overcomes many of these limitations, by using shown that many transplant recipients who acquire prima- whole blood and required minimal laboratory processing of ry HCMV infection post-transplantation can be identi¢ed clinical samples. Another application of this assay is its for active HCMV infection using QuantiFERONs-CMVas- ability to identify patients undergoing primary HCMV in- say. We have found that IFN-g response against HCMVepit- Walker et al: Immune monitoring for HCMV infection QuantiFERON® -CMV
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