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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
2. Gandhi MK, Khanna R. Human cytomegalovirus: clinical aspects, immune regulation, and emerging treatments. Lancet Infect Dis 2004; 3. Humar A, Gillingham K, Payne WD, Sutherland DE, Matas AJ.
Increased incidence of cardiac complications in kidney transplant recipients with cytomegalovirus disease. Transplantation 2000; 70: 4. Boeckh M, Nichols WG, Papanicolaou G, Rubin R,Wingard JR, Zaia J.
Cytomegalovirus in hematopoietic stem cell transplant recipients: current status, known challenges, and future strategies. Biol BloodMarrow Transplant 2003; 9: 543^558.
5. Boeckh M, Nichols WG. Immunosuppressive effects of beta- herpesviruses. Herpes 2003; 10: 12^16.
6. Elkington R,Walker S, Crough T, et al. Ex vivo pro¢ling of CD8 1 T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers. J Virol 2003; 77: 7. Riddell SR, Greenberg PD. Tcell therapy of human CMV and EBV infection in immunocompromised hosts. Rev Med Virol 1997; 8. Sylwester AW, Mitchell BL, Edgar JB, et al. Broadly targeted human cytomegalovirus-specific CD4 1 and CD8 1 T cells dominate memory compartment of exposed subjects. J Exp Med 2005; 202: 673^ 9. Ghanekar SA, Nomura LE, Suni MA, Picker LJ, Maecker HT, Maino VC. Gamma interferon expression in CD8(1) T cells is a marker forcirculating cytotoxic T lymphocytes that recognize an HLA A2- restricted epitope of human cytomegalovirus phosphoprotein pp65.
Clin Diagn Lab Immunol 2001; 8: 628^631.
10. Akiyama Y, Maruyama K, Mochizuki T, Sasaki K,Takaue Y, Yamaguchi K. Identi¢cation of HLA-A24 -restricted CTL epitope Fig. 2. (A and B) Comparison of QuantiFERONs-CMV (A) and ELI- encoded by the matrix protein pp65 of human cytomegalovirus.
SPOT (B) assays in solid organ transplant patients. Data from 4 di¡erent solid organ transplant patients (T1, T2, T3 and T4) are presented in this 11. Kern F, Faulhaber N, Frommel C, et al. Analysis of CD8 T cell ¢gure. The QuantiFERONs-CMV assay was carried out as described in reactivity to cytomegalovirus using protein-spanning pools of the ‘‘Material and methods’ section. The results for ELISPOT assay are overlapping pentadecapeptides. Eur J Immunol 2000; 30: 1676^1682.
expressed as mean Æ SEM of spot-forming cells (SFC) per 106 peripheral 12. Wills MR, Carmichael AJ, Mynard K, et al. The human cytotoxic blood mononuclear cells (PBMC). Sample collection time points for each of T-lymphocyte (CTL) response to cytomegalovirus is dominated by the transplant patients are shown on x-axis.
structural protein pp65: frequency, speci¢city, and T-cell receptorusage of pp65 -specific CTL. J Virol 1996; 70: 7569^7579.
13. Masuoka M,Yoshimuta T, Hamada M, et al. Identi¢cation of the HLA- opes can be detected well before the anti-viral IgM response A24 peptide epitope within cytomegalovirus protein pp65 recognized can be observed using standard serological assays (unpub- by CMV-specific cytotoxic T lymphocytes.Viral Immunol 2001; 14: lished data).We propose that the QuantiFERONs-CMV as- say in combination with viral DNA load should be 14. Gratama JW, van Esser JW, Lamers CH, et al. Tetramer-based quanti¢cation of cytomegalovirus (CMV)-specific CD8 1 T considered for the routine monitoring of HCMV-associated lymphocytes in T-cell-depleted stem cell grafts and after complications in various clinical settings. It will be import- transplantation may identify patients at risk for progressive CMV ant to conduct formal clinical studies to assess whether the infection. Blood 2001; 98: 1358^1364.
QuantiFERONs-CMVassay can be used to identify or pre- 15. Kern F, Faulhaber N, Khatamzas E, et al. Measurement of anti-human dict individuals who are likely to develop HCMV-associated cytomegalovirus T cell reactivity in transplant recipients and itspotential clinical use: a mini-review. Intervirology 1999; 42: 16. Harrop R, Ryan MG, Golding H, Redchenko I, Carroll MW. Monitoring of human immunological responses to vaccinia virus. Methods MolBiol 2004; 269: 243^266.
17. Lacey SF, Diamond DJ, Zaia JA. Assessment of cellular immunity to human cytomegalovirus in recipients of allogeneic stem celltransplants. Biol Blood Marrow Transplant 2004; 10: 433^447.
1. Alford CA, Stagno S, Pass RF, Britt WJ. Congenital and perinatal 18. Waldrop SL, Davis KA, Maino VC, Picker LJ. Normal human CD4 1 cytomegalovirus infections. Rev Infect Dis 1990; 12 (Suppl 7 ): memory T cells display broad heterogeneity in their activation threshold for cytokine synthesis. J Immunol 1998; 161: 5284^5295.
Walker et al: Immune monitoring for HCMV infection 19. Grint PC, Ronalds CJ, Kangro HO, et al. Screening tests for antibodies repertoire in CMV-exposed individuals. J Infect Dis 2002; 185: to cytomegalovirus: an evaluation of ¢ve commercial products. J Clin 22. Gratama JW, Cornelissen JJ. Diagnostic potential of tetramer-based 20. Weekes MP, Wills MR, Mynard K, Carmichael AJ, Sissons JG.
monitoring of cytomegalovirus-specific CD8 1 T lymphocytes in The memory cytotoxic T-lymphocyte (CTL) response to human allogeneic stem cell transplantation. Clin Immunol 2003; 106: cytomegalovirus infection contains individual peptide-specific CTL clones that have undergone extensive expansion in vivo. J Virol 1999; 23. Kern F, LiPira G, Gratama JW, Manca F, Roederer M. Measuring Ag- specific immune responses: understanding immunopathogenesis 21. Kern F, Bunde T, Faulhaber N, et al. Cytomegalovirus (CMV) and improving diagnostics in infectious disease, autoimmunity and phosphoprotein 65 makes a large contribution to shaping the T cell cancer. Trends Immunol 2005; 26: 477^484.

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