Adoptive transfer of multivirus-specific T cell lines (MVST) is an advanced tool for immunotherapy of virus infections after hematopoietic stem cell transplantation (HSCT). Their preparation includes activation of donor virus-specific T cells by the mixture of oligopeptides derived from immunodominant antigens of several most harmful viruses, i.e. human cytomegalovirus (HCMV), polyomavirus BK (BKV), Epstein-Barr virus (EBV) and adenovirus (ADV). The aim of our study was to find out whether antigenic competition may have an impact on the expansion of virus-specific T cells. MVST from several heathy blood donors were generated using a pulse of overlapping oligopeptides (PepMixes™, derived from the IE1 and pp65 CMV antigens, VP1 and LTAG BKV antigens, BZLF1 and EBNA1 proteins of EBV and hexon protein from ADV) and short time culture in the presence of IL-7 and IL-4. The amount of virus-specific T cells in MVST was measured by ELISPOT and flow cytometry after re-stimulation with individual antigens. To evaluate antigenic competition, MVST were expanded either with a complete set of antigens or with the mixture lacking some of them. MVST expanded with the antigen mixture including CMV antigens contained a lower proportion of the T cells of other antigen specificities. A similar inhibitory effect was not apparent for EBV-derived peptides. The competitive effect of CMV antigens was most pronounced in MVST from CMV-seropositive donors and was mediated by both IE1 and pp65-derived peptides. Antigenic competition did not influence the phenotype of either CMV- or BKV-specific T cells. Both T cell populations had an effector memory phenotype (CD45RO+, CD27-, CCR7-). However, CMV-specific T cells preferentially consist of CD8+ while in BKV-specific T cells, the CD4+ phenotype predominated. Modification of the MVST manufacture protocol to prevent antigenic competition may increase the efficacy of MVST in therapy of BKV infections in HSCT recipients.
- MeSH
- Adenoviridae imunologie patogenita MeSH
- adenovirové infekce lidí imunologie terapie virologie MeSH
- aktivace lymfocytů MeSH
- antigeny virové imunologie MeSH
- cytomegalovirové infekce imunologie terapie virologie MeSH
- Cytomegalovirus imunologie patogenita MeSH
- fenotyp MeSH
- imunodominantní epitopy MeSH
- imunoterapie adoptivní * MeSH
- infekce onkogenními viry imunologie terapie virologie MeSH
- infekce virem Epsteina-Barrové imunologie terapie virologie MeSH
- interakce hostitele a patogenu MeSH
- kultivované buňky MeSH
- lidé MeSH
- polyomavirové infekce imunologie terapie virologie MeSH
- T-lymfocyty imunologie transplantace virologie MeSH
- transplantace hematopoetických kmenových buněk škodlivé účinky MeSH
- virové nemoci imunologie terapie virologie MeSH
- virus BK imunologie patogenita MeSH
- virus Epsteinův-Barrové imunologie patogenita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- biologické markery analýza krev MeSH
- imunoblotting statistika a číselné údaje MeSH
- lidé MeSH
- senzitivita a specificita MeSH
- sérologické testy metody statistika a číselné údaje MeSH
- testování odbornosti laboratoří * MeSH
- virus Epsteinův-Barrové * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- tabulky MeSH
- MeSH
- lidský herpesvirus 1 izolace a purifikace MeSH
- lidský herpesvirus 2 izolace a purifikace MeSH
- mikrobiologické techniky MeSH
- prediktivní hodnota testů MeSH
- senzitivita a specificita MeSH
- testování odbornosti laboratoří * metody statistika a číselné údaje MeSH
- virus varicella zoster izolace a purifikace MeSH
- Publikační typ
- tabulky MeSH
Development of highly sensitive quantitative assays for cytomegalovirus (CMV) DNA detection is crucial for identification of immunodeficient patients at high risk of CMV disease. We designed 2 internally controlled competitive quantitative assays, enzyme-linked immunosorbent assay (ELISA)-based and real-time polymerase chain reaction (PCR) tests, using amplification of the same segment of the CMV genome. The aim of this study was to compare sensitivity, specificity, and laboratory performance characteristics of these assays. In both assays, a 159-bp segment of UL83 gene was amplified. External and internal controls were constructed by cloning the amplification product and heterogenous DNA segment flanked by target sequences for CMV-derived primers into bacterial plasmids, respectively. Real-time PCR was performed on LightCycler (Roche Diagnostics, Mannheim, Germany), and amplicons were detected using fluorescence resonance energy transfer probes. Alternatively, PCR products were labeled by digoxigenin, hybridized to immobilized probes, and detected by ELISA. The assays were tested on genomic DNA isolated from laboratory strains of CMV, QCMD control panel, and CMV DNA-positive peripheral blood DNA samples from hematopoietic stem cell transplant recipients, previously characterized by pp65 antigenemia and qualitative nested PCR. Real-time and ELISA-based PCR assays showed a linear course of 1-10(8) and 10-10(5) copies of CMV DNA per reaction, respectively. When compared with ELISA-based PCR, real-time PCR showed superiority in inter- and intra-assay reproducibility. Both assays were highly specific in detecting CMV DNA. No difference in amplification efficiency of internal or external standards and wild-type CMV DNA was found. The assays exhibited 83% concordance in CMV DNA detection from clinical samples, all discrepant samples having low CMV DNA copy numbers. There was a good correlation between viral DNA loads measured by the 2 assays. Statistically significant correlation was observed between the numbers of CMV DNA copies and pp65-positive leukocytes in the samples tested. Both variants of competitive PCR are adequately sensitive to be used for CMV DNA quantitation in clinical samples. LightCycler PCR, having superior performance characteristics and being less time-consuming, seems to be more suitable for routine diagnosis.
- MeSH
- cytomegalovirové infekce diagnóza MeSH
- Cytomegalovirus genetika izolace a purifikace MeSH
- DNA virů krev MeSH
- ELISA metody MeSH
- fosfoproteiny imunologie krev MeSH
- lidé MeSH
- polymerázová řetězová reakce metody přístrojové vybavení MeSH
- proteiny virové matrix imunologie krev MeSH
- senzitivita a specificita MeSH
- transplantace hematopoetických kmenových buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- validační studie MeSH
Cíl práce: Ověření diagnostické účinnosti nové metody na stanovení IgM protilátek proti VCA EBV. Porovnání záchytu těchto protilátek ve vzorcích sér od pacientů v různých stadiích infekce EBV pomocí různých sérologických metod. Materiál a metodika: Ve čtyřech různých diagnostických laboratořích byly pomocí nového testu (Viditest anti-VCA EBV IgM, Vidia, ČR) vyšetřeny soubory sér: 1) od pacientů s infekční mononukleózou, 2) od pacientů se sérologickými znaky reaktivace infekce EBV, 3) od séropozitivních jedinců bez známek aktivní infekce, 4) od séronegativních jedinců, 5) vzorky pozitivní na IgM protilátky proti jiným herpetickým virům, 6) vzorky pozitivní na revmatoidní faktor. Jako srovnávací metody byly použity testy nepřímé imunofluorescence, nepřímé ELISA (Human, SRN) a reversní ELISA (DiaSorin, Itálie). Vzorky s rozdílnými výsledky ve Viditestu a srovnávacím testu byly přetestovány v referenční laboratoři metodou nepřímé imunofluorescence a reversní ELISA. Celkem bylo hodnoceno 854 IgM-pozitivních, či IgM-negativních vzorků. Výsledky: Citlivost testu Viditest anti-VCA EBV IgM byla 94,7 % a specifita 96,1 %. Srovnávací studie stanoveni IgM protilátek proti EBV různými sérologickými metodami ukázala, že všechny tři posuzované testy jsou srovnatelně spolehlivé při vyšetření vzorků z infekčních mononukleóz. U vzorků pacientů se vzorcem protilátek charakteristickým pro reaktivace však byl však záchyt IgM protilátek proti EBV metodou nepřímé imunofluorescence výrazně vyšší, než záchyt metodami ELISA. Zjištěné titry IgM protilátek proti EBV byly v této skupině vzorků podstatně nižší, než u vzorků z primoinfekcí. Závěr. Multilaboratorni studie účinnosti soupravy ELISA-Viditest anti-VCA EBV IgM (Vidia, s. r. o.), určené ke stanovení IgM protilátek proti VCA EBV ukázala, že účinnost této soupravy je srovnatelná s účinností jiných metod, které jsou pro tyto účely u nás běžně používány, a je proto vhodná pro sérologickou diagnostiku EBV.
Objectives: To test diagnostic efficiency of a novel method for detection of IgM antibodies to VCA EBV. To compare sensitivity and specificity of detection of IgM antibodies to VCA EBV from patients at various stages of EBV infection by various serological methods. Material and Methods: IgM antibodies to VCA EBV were detected using IgM ELISA Viditest anti-VCA EBV IgM assay (Vidia, Ltd., Czech Republic) and comparative serological methods (indirect immunofluorescence assay, indirect ELISA (Human, SRN) and reverse ELISA (DiaSorin, Italy) in four independent diagnostic laboratories on panels of sera from 1) infectious mononucleosis patients, 2) patients with serological markers of EBV reactivation, 3) seropositive individuals lacking serological markers of active EBV infection, 4) seronegative individuals, 5) patients with IgM antibodies against another herpesvirus and 6) patients positive for rheumatoid factor. The sera yielding discrepant results were retested in the reference laboratory using the reference methods (indirect immunofluorescence and reverse ELISA). Overall, 854 IgM anti-VCA-positive or -negative sera were evaluated. Results: The sensitivity and specificity of the IgM Viditest anti-VCA EBV assay were 94.7 and 96.1 %, respectively. All of the three tests compared were similarly reUable in detecting IgM anti -VCA EBV antibodies in the samples from infectious mononucleosis patients. On the other hand, indirect fluorescence assay proved clearly superior to ELISAs for detection of IgM anti-VCA antibodies in the samples with serological pattern of EBV reactivation, typically showing significantly lower IgM anti-VCA titers compared with those from primary infection. Conclusions: The multilaboratory comparative study proved that the novel IgM ELISA-Viditest anti-VCA EBV assay (Vidia, Ltd., Czech Republic) which shows comparable diagnostic efficiency for detection of IgM EBV antibodies to viral capsid antigen (VCA) as compared to the other assays tested, i.e. ELISA (Human, Germany) and reverse ELISA (DiaSorin, Italy), is suitable for use in serological diagnosis of EBV.
