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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.

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.

BK polyomavirus (BKV) persists lifelong in the urinary tract with asymptomatic urinary shedding in healthy individuals. In immunocompromised persons after transplantation of hematopoietic stem cells (HSCT) the BKV high-rate replication is associated with haemorrhagic cystitis (HC) with a reported incidence of 17 %. Numerous studies of reconstitution of the immune system after HSCT have established the principal role of T cell effectors in the control of viral replication and reactivation. The value of pretransplant BKV-specific antibodies in transplanted patients for the protection from viral disease was long considered insignificant. We hypothesized that the status of BKV immunity prior to HSCT could provide evidence for the BKV tendency to reactivate and that examining the level of subtype-specific antibodies and T-cell response in individual patients could help to predict the risk of BKV reactivation and HC. Evaluation of the risk of HC in relation to pretransplant anti-BKV1,2,4 IgG levels together with clinical factors known before transplantation revealed that patients with „medium” anti-BKV IgG and significant clinical risk (SR) have a very significantly increased HC risk in comparison with the reference group of “low” anti-BKV IgG level and low clinical risk (LR) (P=0.0009). Predictive value of pretransplant BKV specific IgG was confirmed on the level of virus genotypes. Analysis of pretransplant T cell immunity to BKV antigens VP1 and LTag has shown that magnitude of IFN-gamma T cell response inversely corelated with posttransplant DNAuria. We hypothesize that the control of BKV latency by BKV specific T cells before HSCT would be one of the factors that influence BKV reactivation after HSCT. Our study has shown that prediction using a combination of clinical and immunological pretransplant risk factors can help early identification of patients who are at risk of developing BKV disease after HSCT.

Human polyomaviruses HPyV6, HPyV7, TSPyV, HPyV9, MWPyV, and KIPyV have been discovered between 2007 and 2012. TSPyV causes a rare skin disease trichodysplasia spinulosa in immunocompromised patients, the role of remaining polyomaviruses in human pathology is not clear. In this study, we assessed the occurrence of serum antibodies against above polyomaviruses in healthy blood donors. Serum samples were examined by enzyme-linked immunoassay (ELISA), using virus-like particles (VLPs) based on the major VP1 capsid proteins of these viruses. Overall, serum antibodies against HPyV6, HPyV7, TSPyV, HPyV9, MWPyV, and KIPyV were found in 88.2%, 65.7%, 63.2%, 31.6%, 84.4%, and 58%, respectively, of this population. The seroprevalence generally increased with age, the highest rise we observed for HPyV9 and KIPyV specific antibodies. The levels of anti-HPyV antibodies remained stable across the age-groups, except for TSPyV and HPyV9, where we saw change with age. ELISAs based on VLP and GST-VP1 gave comparable seroprevalence for HPyV6 antibodies (88.2% vs.85.3%) but not for HPyV7 antibodies (65.7% vs. 77.2%), suggesting some degree of crossreactivity between HPyV6 and HPyV7 VP1 proteins. In conclusion, these results provide evidence that human polyomaviruses HPyV6, HPyV7, TSPyV, HPyV9, MwPyV, and KIPyV circulate widely in the Czech population and their seroprevalence is comparable to other countries.

JC and BK polyomaviruses (JCV and BKV) infect humans and can cause severe illnesses in immunocompromised patients. Merkel cell polyomavirus (MCPyV) can be found in skin carcinomas. In this study, we assessed the occurrence of serum antibodies against MCPyV, BKV, and JCV polyomaviruses in a healthy population of the Czech Republic. Serum samples from 991 healthy individuals (age range: 6-64 years) were examined by enzyme-linked immunoassay (ELISA) using virus-like particles (VLPs) based on the major VP1 capsid proteins of these viruses. Overall, serum antibodies against MCPyV, JCV, and BKV were found in 63%, 57%, and 69%, respectively, of this population. For all three viruses, these rates were associated with age; the occurrence of antibodies against MCPyV and JCV was highest for those older than 59 years, while the occurrence of antibodies against BKV was highest in those aged 10-19 years and 20-29 years. This is the first large study to determine the seroprevalence rates for BKV, JCV, and MCPyV polyomaviruses in the general Czech Republic population.

Background aims Clinical-grade chimeric antigenic receptor (CAR)19 T cells are routinely manufactured by lentiviral/retroviral (LV/RV) transduction of an anti-CD3/CD28 activated T cells, which are then propagated in a culture medium supplemented with interleukin (IL)-2. The use of LV/RVs for T-cell modification represents a manufacturing challenge due to the complexity of the transduction approach and the necessity of thorough quality control. Methods We present here a significantly improved protocol for CAR19 T-cell manufacture that is based on the electroporation of peripheral blood mononuclear cells with plasmid DNA encoding the piggyBac transposon/transposase vectors and their cultivation in the presence of cytokines IL-4, IL-7 and IL-21. Results We found that activation of the CAR receptor by either its cognate ligand (i.e., CD19 expressed on the surface of B cells) or anti-CAR antibody, followed by cultivation in the presence of cytokines IL-4 and IL-7, enables strong and highly selective expansion of functional CAR19 T cells, resulting in >90% CAR+ T cells. Addition of cytokine IL-21 to the mixture of IL-4 and IL-7 supported development of immature CAR19 T cells with central memory and stem cell memory phenotypes and expressing very low amounts of inhibitory receptors PD-1, LAG-3 and TIM-3. Conclusions Our protocol provides a simple and cost-effective method for engineering high-quality T cells for adoptive therapies.

Human polyomaviruses HPyV6, HPyV7, TSPyV, HPyV9, MWPyV, and KIPyV have been discovered between 2007 and 2012. TSPyV causes a rare skin disease trichodysplasia spinulosa in immunocompromised patients, the role of remaining polyomaviruses in human pathology is not clear. In this study, we assessed the occurrence of serum antibodies against above polyomaviruses in healthy blood donors. Serum samples were examined by enzyme-linked immunoassay (ELISA), using virus-like particles (VLPs) based on the major VP1 capsid proteins of these viruses. Overall, serum antibodies against HPyV6, HPyV7, TSPyV, HPyV9, MWPyV, and KIPyV were found in 88.2%, 65.7%, 63.2%, 31.6%, 84.4%, and 58%, respectively, of this population. The seroprevalence generally increased with age, the highest rise we observed for HPyV9 and KIPyV specific antibodies. The levels of anti-HPyV antibodies remained stable across the age-groups, except for TSPyV and HPyV9, where we saw change with age. ELISAs based on VLP and GST-VP1 gave comparable seroprevalence for HPyV6 antibodies (88.2% vs.85.3%) but not for HPyV7 antibodies (65.7% vs. 77.2%), suggesting some degree of crossreactivity between HPyV6 and HPyV7 VP1 proteins. In conclusion, these results provide evidence that human polyomaviruses HPyV6, HPyV7, TSPyV, HPyV9, MwPyV, and KIPyV circulate widely in the Czech population and their seroprevalence is comparable to other countries.

Immunogenicity of Varicella-zoster virus glycoproteins gE, gB, gH, and gL expressed by recombinant vaccinia viruses (VV) separately or simultaneously was determined in mice and guinea pigs by ELISA, Western blotting, radioimmunoprecipitation, plaque reduction assay, and skin test. Single VV-gE and VV-gB recombinants and double VV-gH/gL recombinant elicited specific antibodies with VZV neutralizing activity in mice. Co-expression of gE and gB by one recombinant VV resulted in an increased antibody response in comparison with immunization with single recombinants or their mixtures. Unlike anti-gB and anti-gH/gL antibodies, the gE-specific antibodies had no virus neutralizing activity in absence of complement, and when used alone, they even caused considerable increase of VZV infectious units. Moreover, immune sera containing anti-gE antibodies antagonized complement independent virus-neutralizing activity of anti-gB- and anti-gH/gL-positive sera. The ability to induce delayed hypersensitivity reaction to VZV antigens was observed after immunization of guinea pigs with gE- and/or gB-expressing VVs.

It has been shown recently that the residual virulence of vaccinia virus (VV) is an important factor that influences the outcome of immunization with VV recombinants. This study focused on the correlation of the residual virulence of several VV recombinants with antibody responses against the strongly immunogenic extrinsic glycoprotein E of varicella-zoster virus and the weakly immunogenic extrinsic protein preS2–S of hepatitis B virus and against VV proteins, with mice used as a model organism. Furthermore, the effects of mixing different recombinants on the antibody response were studied. The results obtained indicated that: (i) the antibody response depended on the residual virulence of the recombinants, more so in the case of the weakly immunogenic protein; (ii) the residual virulence, the growth rate of the VV recombinants in extraneural tissues and the immunogenicity were associated features; (iii) immunization with mixtures of two differently virulent recombinants or with unequal amounts of two similarly virulent recombinants sometimes led to the suppression of antibody response. The appearance of this suppression was dependent on three factors: the residual virulence of the recombinants, the immunogenicity of the extrinsic proteins and the ratio of the recombinants in the mixtures. Thus, the data obtained demonstrate that there are various limitations to the use of replicating VV recombinants for immunization purposes.

ICR mice were immunized intraperitoneally with two doses (10 6 PFU per dose) of vaccinia virus (VV) recombinants of variable virulence expressing either the strongly immunogenic glycoprotein E (gE) of varicella zoster virus (VZV) or weakly immunogenic hepatitis B virus (HBV) preS2-S (S) antigen. Recombinants expressing gE were able to elicit primary and secondary anti-gE antibody irrespective of their residual virulence; after the second dose they did so even in the presence of VV antibody resulting from primary vaccination dose or under other conditions limiting VV replication. As for the S-recombinants, pronounced anti-S antibody development was only observed in mice which had received the more virulent recombinant virus as the first dose. A repeated dose of S-recombinants was unable to elicit a secondary anti-S antibody response. The present findings do not support the assumption that the poor immunogenicity of some extrinsic antigens could be overcome by administering repeated doses of the particular VV recombinant.

Varicella-zoster virus (VZV) glycoproteins gH and gL were examined in a recombinant vaccinia virus system. Single expression of glycoprotein gL produced two molecular forms: an 18 kDa form and a 19 kDa form differing in size by one endoglycosidase H-sensitive N-linked oligosaccharide. Coexpression of gL and gH resulted in binding of the 18 kDa gL form with the mature form of gH, while the 19 kDa gL form remained uncomplexed. The glycosylation processing of gL was not dependent on gH; however, gL was required for the conversion of precursor gH (97 kDa) to mature gH (118 kDa). Subsequent analyses indicated that gL (18 kDa) was a more completely processed gL (19 kDa). Screening of the culture media revealed that gH and gL were secreted, but only if coexpressed and complexed together. The secreted form of gL was 18 kDa while that of gH was 114 kDa. The fact that secreted gH was smaller than intracytoplasmic gH suggested a proteolytic processing event prior to secretion. The 19 kDa form of gL was never secreted. These findings support a VZV gL recycling pathway between the endoplasmic reticulum and the cis-Golgi apparatus.

Using the Praha strain of vaccinia virus (VV) two double recombinant VVs expressing the surface and capsid HBV proteins (HBsAg and HBcAg) under the control of the P 7.5 promoter were constructed. In the first construct the gene coding for HBsAg was inserted into the Hin dIll J fragment (TK gene) and the gene coding for HBcAg was inserted into the Hin dIll M fragment (host range, K1L gene) of the VV genome. To test whether the expression of the foreign genes was influenced by the insertion site, in the second construct their locations were inversely changed. When compared with single VV-HBV recombinants expressing either HBsAg or HBcAg, the double recombinants expressed in vitro approximately the same amounts of the respective antigens. The particles formed by either HBsAg or HBcAg expressed by recombinant viruses, were isolated and examined by electron microscopy. Particles composed of both HBsAg and HBcAg were not detected in cultures infected with one of the double recombinants. The residual virulence in 3-week-old mice of the single recombinants was not markedly altered by the insertion of the second gene. The immunogenicity in mice of both the single and double recombinants was comparable and was not influenced by the location of the HBV genes in VV genome, as revealed by antibodies developed against the respective antigens.

The safety and efficacy of a subunit herpes simplex virus (HSV) type 1 vaccine were tested in a small-scale double-blind trial carried out in a group of 42 volunteers suffering from frequent recurrences of herpetic lesions. The patients were paired according to sex, age, type of virus isolated, previous history of the disease and some non-specific immunological markers. One member of each pair received repeated doses of HSV vaccine, the other a placebo. Clinical reactions were mild. Antibody responses following the vaccination were generally low and were almost entirely limited to subjects suffering from HSV-2 lesions. A majority of the patients exhibited improvement of their condition during the postvaccination period. These improvements were, however, nearly equally distributed between the vaccine and placebo groups.