Specific immune response to mRNA vaccines against COVID-19 in patients receiving allogeneic stem cell transplantation for myeloid malignancy was altered by immunosuppressive therapy.

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.

Non-Mutated Nucleophosmin 1 Is Recognized by the CD8+ T Lymphocytes of an AML Patient after the Transplantation of Hematopoietic Stem Cells from an HLA-Haploidentical Donor.

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.

Antigenic competition in the generation of multi-virus-specific cell lines for immunotherapy of human cytomegalovirus, polyomavirus BK, Epstein-Barr virus and adenovirus infection in haematopoietic stem cell transplant recipients.

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.

Frequent Recurrences of Genital Herpes Are Associated with Enhanced Systemic HSV-Specific T Cell Response.

OBJECTIVES: Genital herpes simplex virus (HSV) infection is controlled by HSV-specific T cells in the genital tract, and the role of systemic T cell responses is not fully understood. Thus, we analysed T cell responses in patients with recurrent genital herpes (GH). METHODS: T cell responses to HSV-1 and HSV-2 native antigens and the expression of HLA-DR and CD38 molecules on circulating CD8+ T cells were analysed in adults with high frequency of GH recurrences (19 patients) and low frequency of GH recurrences (7 patients) and 12 HSV-2 seronegative healthy controls. The study utilized the interferon-γ Elispot assay for measurement of spot-forming cells (SFC) after ex vivo stimulation with HSV antigens and flow cytometry for analysis of the expression of activation markers in unstimulated T cells. RESULTS: The patients with high frequency of GH recurrences (mean number of recurrences of 13.3 per year) had significantly enhanced HSV-specific T cell responses than the HSV-2 seronegative healthy controls. Moreover, a trend of higher numbers of SFC was observed in these patients when compared with those with low frequency of GH recurrences (mean number of recurrences of 3.3 per year). Additionally, no differences in CD38 and HLA-DR expression on circulating CD8+ T cells were found among the study groups. CONCLUSIONS: Frequency of GH recurrences positively correlates with high numbers of systemic HSV-specific T cells.

Prevalence of antibodies against BKPyV subtype I and IV in kidney transplant recipients and in the general Czech population.

Active infection with BK polyomavirus (BKPyV) may cause serious complications in transplantation settings. Recently, the level of BKPyV IgG seroreactivity in graft donors has been shown to predict viremia and BKPyV-associated nephropathy in kidney transplant (KTx) recipients. Pretransplantation testing of the donor and recipient BKPyV serostatus could, therefore, identify patients at high risk. For the development of serological immunoassays, antibody response to the predominant BKPyV subtypes (BKPyV-I and BKPyV-IV) was studied using virus-like particle (VLP)-based enzyme-linked immunosorbent assay (ELISA). VLPs made from the capsid protein, VP1, derived from BKPyV-I and BKPyV-IV subtypes were produced using a baculovirus expression system and used as antigens. The tests were used for IgG antibody determination in 50 KTx recipients and 111 healthy blood donors. While 87% of samples reacted with mixed BKPyV-I and BKPyV-IV antigens, only 49% of samples were reactive in both ELISA tests when using BKPyV-I or BKPyV-IV antigens separately. Twenty-seven percent of healthy blood donors and 26% of KTx recipients were reactive only with BKPyV-I, while 9% and 20% were reactive only with BKPyV-IV, respectively. To determine the specificities of the antigens, selected seropositive samples were retested after preadsorption with soluble BKPyV-I, BKPyV-IV, or JC polyomavirus antigens. The experiments confirmed that recombinant VP1 VLP-based ELISAs predominantly detected BKPyV type-specific antibodies. The results imply that anti-BKPyV antibody ELISA tests should contain a mixture of subtype-specific VLP-based antigens instead of antigen derived from the most prevalent BKPyV-I subtype. The tests can be used for serological surveys of BKPyV infection and improved KTx patient management.

Presence of herpesvirus DNA in cerebrospinal fluid of patients with tick-borne encephalitis and enteroviral meningoencephalitis.

Reactivation of HHVs in the CNS due to inflammation has not been well described yet. The primary aim of this study was to investigate the frequency of HHV DNA detection in the cerebrospinal fluid (CSF) of immunocompetent patients with meningoencephalitis of other than HHV origin. The secondary aim of this study was to evaluate the impact of herpesvirus co-infection on the clinical course and patient outcome. Ninety-six patients with clinically and laboratory proven tick-borne encephalitis (TBE) and 77 patients with a confirmed diagnosis of enteroviral meningitis (EVM), along with a control group of 107 patients without evidence of inflammation in the CSF were retrospectively tested by nested PCR for the presence of DNA of the neurotropic herpesviruses HSV1, HSV2, VZV, and HHV6 in the CSF. The clinical course, laboratory tests, antiviral treatment, and neurological complications in a 6-month follow-up were compared between the groups positive or negative for HHV DNA in the CSF. HHV DNA was found in the CSF of 12 (6.9%) patients (6.3% and 7.8% in the TBE and EVM groups, respectively) and in 1 (0.9%) control patient. None of the patients had recent blisters or rash. The clinical course was comparably mild in all patients. No permanent neurological sequelae were observed. Only the CSF total protein level was significantly higher in HHV DNA-positive than in HHV-negative patients.

[Recurrent genital herpes]. / Recidivující genitální herpes.

In recent years, the rate of genital infections caused by Herpes simplex virus types 1 and 2 (HSV-1, HSV-2) has increased. Following primary infection with HSV-2, recurrent genital herpes (GH) often develops or asymptomatic virus shedding occurs. HSV-1-induced GH recurrencies are significantly less frequent. The options for diagnosing HSV infections have improved markedly: they include determination of type-specific antibodies in the blood (anti-HSV-1 and anti-HSV-2), culture of HSV from lesions or detection of viral antigens by immunohistochemistry methods; it is also possible to detect viral DNA by polymerase chain reaction. At present, recurrent genital herpes is treated by the so-called episodic therapy or suppressor antiviral therapy, in the Czech Republic based on acyclovir or valacyclovir. Preventive measures are possible only to a limited extent, particular attention is paid to pregnant women and the risk of disseminated herpes infection in newborns. New preventive and therapeutic options continue to be developed: a preventive vaccine against HSV-2 has been successfully tested, but this is effective only in HSV-1 negative women; experimental therapeutic vaccination stimulating specific immunity has not yet been successful.

[Detection of active varicella-zoster virus (VZV) infection in patients with neurological complications]. / Prukaz aktivní infekce virem varicella zoster (VZV) u pacientu s neurologickými komplikacemi.

OBJECTIVES: When introduced into routine virological diagnosis of nervous system infections, PCR detection of viral DNA revealed the varicella-zoster virus (VZV) in cerebrospinal fluid (CSF) at much higher rates than expected. The aim of the study was to evaluate the frequency of VZV DNA detection in CSF of patients with neurological symptoms in correlation with their VZV-specific serological findings and clinical symptoms. MATERIAL AND METHODS: A total of 438 patients followed up in the neurology departments of the Motol and Královské Vinohrady University Hospitals and the Department of Infectious Diseases of the Bulovka University Hospital were screened for the presence of VZV-specific antibodies in serum and intrathecal antibodies in CSF. A home-brew nested PCR assay was used for detection of VZV DNA in CSF. Positive results were correlated with clinical findings. RESULTS: Intrathecal antibodies against VZV were detected in 19.6 % of the studied patients, VZV-specific IgM antibodies were present in serum of 17.3 % of the patients and VZV DNA was recorded in CSF of 9.4 % of the patients. The clinical diagnosis was confirmed in 16 patients positive for VZV DNA in CSF: encephalitis as a complication of neonatal varicella in a 2-week child; encephalitis or meningoencephalitis in 5 adult patients of whom three had a history of herpes zoster, one suffered from severe haemorrhagic focal encephalitis with fatal complications and one had encephalitis and myelitis; neuropathies in 4 patients, two with inflammatory polyneuropathy of unknown origin and two with brachial plexopathy, in one case preceded by herpes zoster; epileptic symptoms in 2 patients; multiple sclerosis in 3 patients and nonspecific symptoms of chronic fatigue in one patient. CONCLUSIONS: 1) PCR proved to be a suitable method for diagnosing VZV-mediated nervous system infections. 2) VZV DNA can be present in CSF of patients with a wide range of neurological symptoms, even with no history of either herpes zoster or varicella. 3) VZV DNA detection in CSF needs to be interpreted with caution and in correlation with case histories, clinical findings and electrophysiological and imaging data, especially in patients with chronic inflammatory disease receiving immunosuppressive therapy.

Comparison of quantitative competitive polymerase chain reaction-enzyme-linked immunosorbent assay with LightCycler-based polymerase chain reaction for measuring cytomegalovirus DNA in patients after hematopoietic stem cell transplantation.

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.

Probable neuroimmunological link between Toxoplasma and cytomegalovirus infections and personality changes in the human host.

BACKGROUND: Recently, a negative association between Toxoplasma-infection and novelty seeking was reported. The authors suggested that changes of personality trait were caused by manipulation activity of the parasite, aimed at increasing the probability of transmission of the parasite from an intermediate to a definitive host. They also suggested that low novelty seeking indicated an increased level of the neurotransmitter dopamine in the brain of infected subjects, a phenomenon already observed in experimentally infected rodents. However, the changes in personality can also be just a byproduct of any neurotropic infection. Moreover, the association between a personality trait and the toxoplasmosis can even be caused by an independent correlation of both the probability of Toxoplasma-infection and the personality trait with the third factor, namely with the size of living place of a subject. To test these two alternative hypotheses, we studied the influence of another neurotropic pathogen, the cytomegalovirus, on the personality of infected subjects, and reanalyzed the original data after the effect of the potential confounder, the size of living place, was controlled. METHODS: In the case-control study, 533 conscripts were tested for toxoplasmosis and presence of anti-cytomegalovirus antibodies and their novelty seeking was examined with Cloninger's TCI questionnaire. Possible association between the two infections and TCI dimensions was analyzed. RESULTS: The decrease of novelty seeking is associated also with cytomegalovirus infection. After the size of living place was controlled, the effect of toxoplasmosis on novelty seeking increased. Significant difference in novelty seeking was observed only in the largest city, Prague. CONCLUSION: Toxoplasma and cytomegalovirus probably induce a decrease of novelty seeking. As the cytomegalovirus spreads in population by direct contact (not by predation as with Toxoplasma), the observed changes are the byproduct of brain infections rather than the result of manipulation activity of a parasite. Four independent lines of indirect evidence, namely direct measurement of neurotransmitter concentration in mice, the nature of behavioral changes in rodents, the nature of personality changes in humans, and the observed association between schizophrenia and toxoplasmosis, suggest that the changes of dopamine concentration in brain could play a role in behavioral changes of infected hosts.

[A comparison of the detection of IgM antibodies against the viral capsid antigen (VCA) of EBV (Epstein-Barr virus) in various groups of patients, using indirect immunofluorescence, indirect ELISA and reverse ELISA. Study of the diagnostic efficacy of the anti-VCA EBV IgM ELISA-VIDITEST kit]. / Porovnání záchytu IgM protilátek proti virovému kapsidovému antigenu (VCA) viru Epsteina a Barrové (EBV) u ruzných skupin pacientu pomocí neprímé imunofluorescence, neprime ELISA a reversní ELISA. Studie diagnostické úcinnosti soupravy ELISA-VIDITEST anti-VCA EBV IgM.

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 reliable 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.

[Incidence of human herpes virus 8 (HHV 8) infections in risk groups of the population].

OBJECTIVE: To identify and to examine the population groups at highest risk of acquiring HHV8 infection. MATERIAL AND METHODS: Indirect immunofluorescence test (IIF) for detection of IgG antibodies to the lytic antigen of HHV 8. RESULTS: In total 101 STD patients of a dermatovenerological clinic, 129 HIV - negative homosexual men - who have asked for voluntary HIV testing and 37 patients with repeated blood transfusions were tested. The highest number of patients - 13 (10 %) with IgG antibodies to the lytic antigen of HHV 8 was found in the group of HIV - negative homosexual men. CONCLUSIONS: In the Czech Republic are at highest risk of HHV 8 infection (except HIV positive persons), HIV negative men.