Cíl: Zaměřili jsme se na stanovení prevalence infekce SARS-CoV-2 se symptomatickým nebo asymptomatickým průběhem a na identifikaci prediktorů symptomatické nebo asymptomatické infekce SARS-CoV-2 u pacientů během sedmi měsíců následujících po transplantaci alogenních hematopoetických kmenových buněk (alo-HSCT) v období cirkulace varianty omikron. Metody: Prevalence proběhlé infekce SARS-CoV-2 byla detekována u pacientů během sedmi měsíců po allo-HSCT v omikronovém období pomocí buněčné a humorální imunitní odpovědi proti nukleoproteinu SARS-CoV-2 (NCP). Výsledky: Pozitivní markery prodělané infekce byly identifikovány u 45,2 % pacientů (n = 42). Infekce byla asymptomatická u 68,4 % pacientů s anti-NCP pozitivitou. Hledání rizikových faktorů pro symptomatickou infekci SARS-CoV-2 u příjemců alo-HSCT odhalilo, že nízká úroveň rekonstituce B buněk byla jediným signifikantně souvisejícím rizikovým faktorem. Závěr: Vysoký podíl příjemců alo-HSCT, kteří byli asymptomaticky infikováni do sedmi měsíců po transplantaci v letech 2022–2023, přestože byli imunokompromitovaní a neočkovaní, ukazuje na oslabení cirkulujícího viru a může signalizovat pro pacienty po transplantaci menší riziko onemocnění SARS-CoV-2 v omikronovém období. Ukázalo se, že očkování těchto pacientů proti SARS- -CoV-2 je spojeno s nízkým, ale významným rizikem exacerbace vyléčené chronické reakce štěpu proti hostiteli (GVHD – Graft Versus Host Disease) a s rizikem de novo GVHD. Nízká úroveň rekonstituce B-buněk byla jediným významným rizikovým faktorem pro symptomatickou infekci SARS-CoV-2 u příjemců alo-HSCT.

Aim: We aimed to determine the prevalence of SARS-CoV-2 infection, including both symptomatic and asymptomatic courses, and to identify predictors of asymptomatic or symptomatic SARS-CoV-2 infection in patients within seven months after allo-HSCT (allogenic hematopoietic stem cell transplantation) in the Omicron period. Methods: Prevalence of the past SARS-CoV-2 infection was determined in patients within seven months after allo-HSCT in the Omicron period using the cellular and humoral immune response against the SARS-CoV-2 nucleoprotein (NCP). Results: Positive markers of past infection were identified in 45.2% of patients (n = 42). The infection was asymptomatic in 68.4% of anti-NCP positive patients. The search for risk factors for symptomatic SARS-CoV-2 infection in allo-HSCT recipients revealed that a low level of B cell reconstitution was the only significantly associated risk factor. Conclusion: A high proportion of allo-HSCT recipients who were asymptomatically infected within up to seven months after transplantation from 2022 to 2023 despite being immunosuppressed and unvaccinated indicates an attenuation of the circulating virus and may signal less risk for transplanted patients from SARS-CoV-2 infection in the Omicron period. Vaccination of these patients against SARS-CoV-2 was shown to be associated with a low but significant risk of exacerbation of cured chronic GVHD (graft versus host disease) and the risk of de novo GVHD. The low level of B-cell reconstitution was the only significant risk factor for symptomatic SARS-CoV-2 infection in HSCT recipients.

• Klíčová slova
• Omikron,
• MeSH
• asymptomatické infekce * epidemiologie   MeSH
• B-lymfocyty imunologie   mikrobiologie   transplantace   MeSH
• COVID-19 * komplikace   mikrobiologie   MeSH
• homologní transplantace MeSH
• kohortové studie MeSH
• lidé MeSH
• nemoc štěpu proti hostiteli etiologie   imunologie   MeSH
• rizikové faktory MeSH
• SARS-CoV-2 patogenita   MeSH
• transplantace hematopoetických kmenových buněk * MeSH
• vakcíny proti COVID-19 škodlivé účinky   MeSH
• Check Tag
• lidé MeSH

BACKGROUND: Allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients are at high risk of complications associated with COVID-19 infection due to dysfunction of their immune system. Vaccination can protect from the adverse consequences of COVID-19. However, studies on the efficacy of COVID-19 vaccines in HSCT recipients with insufficient post-HSCT immune reconstitution are still scarce. In our study, we determined how immunosuppressive medication and the reconstitution of the cellular immune system influenced T cell responses specific for the surface glycoprotein of SARS-CoV-2 virus (S antigen) after two doses of mRNA vaccine against COVID-19 in patients with myeloid malignancies treated with HSCT. METHODS: Vaccination outcomes were followed in 18 (allo-HSCT) recipients and 8 healthy volunteers. The IgG antibodies against SARS-CoV-2 spike (S) and nucleocapsid (NCP) protein were determined in ELISA and S-specific T cells were detected using a sensitive ELISPOT-IFNγ based on in vitro expansion and restimulation of T cells in pre- and post-vaccination blood samples. Multiparametric flow cytometry analysis of peripheral blood leukocyte differentiation markers was employed for determination of reconstitution of the main subpopulations of T cells and NK cells at month 6 after HSCT. RESULTS: S- specific IgG antibody response detected in 72% of the patients was lower than in healthy vaccinees (100%). Vaccine-induced T-cell responses to S1 or S2 antigen were significantly reduced in HSCT recipients, which were treated with corticosteroids in dose 5 mg of prednisone- equivalents or higher during the vaccination period or in preceeding 100 days in comparison with recipients un-affected with corticosteroids. A significant positive correlation was found between the level of anti-SARS-Cov-2 spike protein IgG antibodies and the number of functional S antigen-specific T cells. Further analysis also showed that the specific response to vaccination was significantly influenced by the interval between administration of vaccine and transplantation. Vaccination outcomes were not related to age, sex, type of mRNA vaccine used, basic diagnosis, HLA match between HSC donor and recipient, and blood counts of lymphocytes, neutrophils, and monocytes at the time of vaccination. Multiparametric flow cytometry analysis of peripheral blood leukocyte differentiation markers showed that good humoral and cellular S-specific immune responses induced by vaccination were associated with well-reconstituted CD4+ T cells, mainly CD4+ effector memory subpopulation at six months after HSCT. CONCLUSIONS: The results showed that both humoral and cellular adaptive immune responses of HSCT recipients to the SARS-CoV-2 vaccine were significantly suppressed by corticosteroid therapy. Specific response to the vaccine was significantly affected by the length of the interval between HSCT and vaccination. Vaccination as early as 5 months after HSCT can lead to a good response. Immune response to the vaccine is not related to age, gender, HLA match between HSC donor and recipient, or type of myeloid malignancy. Vaccine efficacy was dependent on well-reconstituted CD4+ T cells, at six months after HSCT.

• MeSH
• COVID-19 * prevence a kontrola   MeSH
• imunita MeSH
• imunoglobulin G MeSH
• imunosupresivní léčba MeSH
• lidé MeSH
• mRNA vakcíny MeSH
• nádory * MeSH
• SARS-CoV-2 MeSH
• transplantace hematopoetických kmenových buněk * škodlivé účinky   MeSH
• vakcíny proti COVID-19 MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The aim of the proposed project is to analyse risk factors contributing to recently recorded elevation in number of cases of haemorrhagic cystitis (HC) in oncohematological patients after allogeneic hematopoietic stem cell transplantation (HSCT) what could help to find subjects at high risk of this type of morbidity. The main analysed factor will include the type of used posttransplant immunosuppressive prevention of GvHD, connection with haploidentical HSCT and association with BKV and AdV infection and the state of antiviral cellular immune response. Analysis of anti-viral effector T cells in HC patients with BK or AdV infection will result in determination of protective levels of anti-viral effector T cells and in characterisation of phenotype, functional activity and antigenic specificity of T cells responsible for virological and clinical response. The results of the study are essential for adoption and optimization of protocols for expansion of T cells for immunotherapy of opportunistic viral infections that endanger adult and children HSCT patients.

Cílem je analyzovat rizikové faktory zodpovědné za růst případů hemoragické cystitidy (HC) u onkohematologických pacientů po alogenní transplantaci hematopoetických kmenových buněk (HSCT), což by mělo pomoci při hledání osob s vysokým rizikem HC. Hlavními analyzovanými faktory budou typ imunosupresivní terapie pro prevenci reakce štěpu proti hostiteli (GvHD) aplikované po transplantaci, spojitost s haploidentickou HSCT, přítomnost infekce viry BKV a AdV a stav protivirové buněčné imunity. Analýza anti-virové specifické T buněčné odpovědi u pacientů s HC s infekcí BKV a AdV umožní určit protektivní hladinu anti-virových efektorových T buněk a charakterizovat fenotyp, funkční aktivitu a antigenní specifitu T buněk odpovědných za virologickou a klinickou odpověď. Výsledky studie jsou nepostradatelné pro zavedení a optimalizaci protokolů pro expanzi antivirových T buněk využitelných pro adoptivní imunoterapii oportunních virových infekcí ohrožujících dospělé i dětské pacienty po transplantaci HSCT.

• Klíčová slova
• haploidentická transplantace kmenových buněk, hemoragická cystitida, BKV, adenovirus, protektivní buněčná imunita, T buňka, haploidentical stem cell transplantation, haemorrhagic cystitis, BKV, adenovirus, protective cellular response, effector T cell,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Nucleophosmin (NPM1, B23) is a multifunctional phosphoprotein expressed in all tissues. The protein is mainly localized in nucleoli. In hematological malignancies, NPM1 belongs to commonly altered genes. Its mutation, always heterozygous, leads to the re-localization of the NPM1 protein from the nucleolus to the cytoplasm (NPM1c+). NPM1c+ is found in 30% of acute myeloid leukemia (AML). Our study showed that an AML patient, whose leukemia cells carried the NPM1c+ mutation and who was the recipient of allogeneic HSCT from a haploidentical donor, raised a robust allorestricted CD8+ T cell response directed against the NPM1wt protein. Favourably, the response against NPM1wt was not accompanied by side effects such as GvHD. Moreover, the induction of a high NPM1wt-specific response coincided with the decrease in NPM1c+ transcripts detected, implying a beneficial graft versus leukemia effect. On the basis of these results, we suppose that TCRs from allorestricted NPM1wt-specific T cells are worth studying in other recipients of grafts from haploidentical donors as a possible tool for TCR gene therapy.

• MeSH
• akutní myeloidní leukemie * genetika   metabolismus   terapie   MeSH
• CD8-pozitivní T-lymfocyty metabolismus   patologie   MeSH
• hematopoetické kmenové buňky metabolismus   patologie   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• nukleofosmin * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BK polyomavirus (BKPyV) persists lifelong in renal and urothelial cells with asymptomatic urinary shedding in healthy individuals. In some immunocompromised persons after transplantation of hematopoietic stem cells (HSCT), the BKPyV high-rate replication is associated with haemorrhagic cystitis (HC). We tested whether the status of BKPyV immunity prior to HSCT could provide evidence for the BKPyV tendency to reactivate. We have shown that measurement of pretransplant anti-BKPyV 1 and 4 IgG levels can be used to evaluate the HC risk. Patients with anti-BKPyV IgG in the range of the 1st-2nd quartile of positive values and with positive clinical risk markers have a significantly increased HC risk, in comparison to the reference group of patients with "non-reactive" anti-BKPyV IgG levels and with low clinical risk (LCR) (p = 0.0009). The predictive value of pretransplant BKPyV-specific IgG was confirmed by determination of genotypes of the shed virus. A positive predictive value was also found for pretransplant T-cell immunity to the BKPyV antigen VP1 because the magnitude of IFN-γ T-cell response inversely correlated with posttransplant DNAuria and with HC. Our novel data suggest that specific T-cells control BKPyV latency before HSCT, and in this way may influence BKPyV reactivation after HSCT. Our study has shown that prediction using a combination of clinical and immunological pretransplant risk factors can help early identification of HSCT recipients at high risk of BKPyV disease.

• Publikační typ
• časopisecké články MeSH

• MeSH
• virologie * MeSH
• Publikační typ
• biografie MeSH

• O autorovi
• Vonka, Vladimír, 1930- Autorita

Significant progress in the field of tumor immunotherapy has been recently shown to complement available treatment modalities of hematological malignancies. This novel treatment method is based on the use adoptively transferred T lymphocytes which were modified in vitro prior to transfer to express artificial signaling molecule designated Chimeric Antigen Receptor (CAR) which redirects the specificity of modified lymphocytes to surface antigens expressed by malignant cells. In this project we propose to develop methods for CAR-based therapy of lymphomas and leukemia. Next, we propose to develop methods for selective expansion of T cells specific for EBV, HCMV or adenovirus from donor lymphocytes for the use in patients who received allogeneic stem cell transplantation and as a result of immunosupresion developed acute viral infection. The goal of the project is the manufacture of GMP-grade cells and their pre-clinical testing. Proposed project meets criteria of national programme for the support of applied medical research in Aims 1.3.1., 1.6.1., 2.5.1. a 2.5.2.

Významné úspěchy v oblasti nádorové imunoterapie, kterých bylo dosaženo v nedávné době úspěšně doplňují zavedené léčebné metody hematologických malignit. Tyto nové metody jsou založené na použití modifikovaných T lymfocytů, které exprimují arteficielní signalizační molekulu označovanou jako Chimerický antigenní receptor (CAR), která přesměruje specifitu T lymfocytů na povrchové antigeny přítomné na nádorových buňkách. V tomto projektu navrhujeme vývoj metod léčby leukemií a lymfomů pomocí CAR-lymfocytů. Dále navrhujeme vývoj metody selektivní expanze T lymfocytů specifických na viry EBV, HCMV a adenoviry z dárcovských lymfocytů, které by bylo možné použít pro léčbu akutní infekce u pacientů po transplantaci kostní dřeně. Cílem projektu je příprava buněk v GMP-kvalitě a preklinická fáze jejich testování. Předkládaný projekt je plně v souladu s prioritami a cíli “Programu na podporu zdravotnického aplikovaného výzkumu a vývoje” v oblasti 1. Vznik a rozvoj chorob a oblasti 2. Nové diagnostické a terapeutické metody, kde plní dílčí cíle 1.3.1., 1.6.1., 2.5.1. a 2.5.2.

• MeSH
• buněčná a tkáňová terapie metody   MeSH
• chimerické antigenní receptory terapeutické užití   MeSH
• hematologické nádory genetika   terapie   MeSH
• imunoterapie metody   MeSH
• lidé MeSH
• T-lymfocyty MeSH
• vyvíjení léků metody   MeSH
• Check Tag
• lidé MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• hematologie a transfuzní lékařství
• onkologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

• MeSH
• virologie * MeSH
• Publikační typ
• biografie MeSH

• O autorovi
• Vonka, Vladimír, 1930- Autorita

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
• virologie * MeSH
• Publikační typ
• biografie MeSH

• O autorovi
• Vonka, Vladimír, 1930- Autorita