Tina Schmidt

Evolutionarily conserved herpesviral protein interaction networks.

Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV) and Kaposis sarcoma-associated herpesvirus (KSHV). In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1), murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H), and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species.

Altered Phenotype and Functionality of Varicella Zoster Virus–Specific Cellular Immunity in Individuals With Active Infection

BACKGROUND Varicella zoster virus (VZV) establishes lifelong persistence and may reactivate in individuals with impaired immune function. To investigate immunologic correlates of protection and VZV reactivation, we characterized specific immunity in 207 nonsymptomatic immunocompetent and 132 immunocompromised individuals in comparison with patients with acute herpes zoster. METHODS VZV-specific CD4 T cells were quantified flow cytometrically after stimulation and characterized for expression of interferon-γ, interleukin 2, and tumor necrosis factor α and surface markers for differentiation (CD127) and anergy (cytotoxic T lymphocyte antigen 4 [CTLA-4] and programmed death [PD]-1). Immunoglobulin G and A levels were quantified using an enzyme-linked immunosorbent assay. RESULTS In healthy individuals, VZV-specific antibody and T-cell levels were age dependent, with the highest median VZV-specific CD4 T-cell frequencies of 0.108% (interquartile range, 0.121%) during adolescence. VZV-specific T-cell profiles were multifunctional with predominant expression of all 3 cytokines, CD127 positivity, and low expression of CTLA-4 and PD-1. Nonsymptomatic immunocompromised patients had similar VZV-specific immunologic properties except for lower T-cell frequencies (P<.001) and restricted cytokine expression. In contrast, significantly elevated antibody- and VZV-specific CD4 T-cell levels were found in patients with zoster. Their specific T cells showed a shift in cytokine expression toward interferon γ single positivity, an increase in CTLA-4 and PD-1, and a decrease in CD127 expression (all P<.001). This phenotype normalized after resolution of symptoms. CONCLUSIONS VZV-specific CD4-T cells in patients with zoster bear typical features of anergy. This phenotype is reversible and may serve as adjunct tool for monitoring VZV reactivations in high-risk patients.

CD4+T‐cell immunity after pandemic influenza vaccination cross‐reacts with seasonal antigens and functionally differs from active influenza infection

Antigen‐specific antibodies are well characterized after vaccination with pandemic H1N1 or seasonal influenza vaccines. However, knowledge on cellular immunity toward pandemic H1N1 after vaccination and infection and cross‐reactivities toward seasonal antigens is limited. Nineteen individuals were vaccinated with the pandemic H1N1 vaccine. Among those, ten had been prevaccinated against seasonal influenza. CD4+ T cells specific for pandemic H1N1 and for seasonal vaccine, and antibodies were monitored using flow cytometry and ELISA/neutralization assays, respectively. In addition, seven patients with acute pandemic influenza infection were analyzed. Pandemic H1N1 vaccination induced a strong 4.63‐fold (IQR 4.16) increase in antigen‐specific CD4+ T cells that was more pronounced in individuals not prevaccinated with seasonal influenza (p = 0.01). T‐cell levels toward seasonal vaccine concomitantly rose by 2.71‐fold (IQR 2.26). Likewise, prevaccination with seasonal influenza induced a less pronounced increase in specific antibodies. Influenza‐specific T cells in vaccinees had a Th1 phenotype mainly coexpressing IFN‐γ and IL‐2, whereas patients with active pandemic influenza showed a shift toward cells predominantly expressing IFN‐γ. In conclusion, T cells toward seasonal influenza antigens cross‐react with pandemic H1N1 antigens and affect induction of specific T cells after pandemic influenza vaccination. In addition, the cytokine patterns of specific T cells during acute H1N1 infection and after vaccination differ, and the predominantly dual‐positive cytokine profile of vaccine‐induced T cells suggests sufficient functionality to confer successful virus control.

The heterogeneous nuclear ribonucleoprotein K is important for Herpes simplex virus‐1 propagation

The heterogeneous nuclear ribonucleoprotein (hnRNP) K is an evolutionarily conserved protein with roles in signal transduction and gene expression. An impact of hnRNP K on the life cycle of a broad range of viral pathogens was reported while functional data for herpesviruses were lacking. In this study we show that hnRNP K is important for Herpes simplex virus 1 egress. In absence of hnRNP K, viral entry, gene expression, viral DNA replication, and maturation of nuclear particles appear normal whereas release of infectious virions to the extracellular space was significantly affected. Our results indicate that hnRNP K has an impact on a late step of herpesviral propagation making it a potential antiviral target.

PD-1 Analysis on CD28−CD27− CD4 T Cells Allows Stimulation-Independent Assessment of CMV Viremic Episodes in Transplant Recipients

Expression of the inhibitory receptor programmed death 1 (PD‐1) on cytomegalovirus (CMV)‐specific CD4 T cells defines a phenotype associated with CMV viremia in transplant recipients. Moreover, CD28−CD27− double negativity is known as a typical phenotype of CMV‐specific CD4 T cells. Therefore, the co‐expression of inhibitory receptors on CD28−CD27− CD4 T cells was assessed as a rapid, stimulation‐independent parameter for monitoring CMV complications after transplantation. Ninety‐three controls, 67 hemodialysis patients and 81 renal transplant recipients were recruited in a cross‐sectional and longitudinal manner. CMV‐specific CD4 T cell levels quantified after stimulation were compared to levels of CD28−CD27− CD4 T cells. PD‐1 and cytotoxic T lymphocyte–associated antigen 4 (CTLA‐4) expression on CD28−CD27− CD4 T cells were related to viremia. A percentage of ≥0.44% CD28−CD27− CD4 T cells defined CMV seropositivity (93.3% sensitivity, 97.1% specificity), and their frequencies correlated strongly with CMV‐specific CD4 T cell levels after stimulation (r = 0.73, p < 0.0001). Highest PD‐1 expression levels on CD28−CD27− CD4 T cells were observed in patients with primary CMV viremia and reactivation (p < 0.0001), whereas CTLA‐4 expression was only elevated during primary CMV viremia (p < 0.05). Longitudinal analysis showed a significant increase in PD‐1 expression in relation to viremia (p < 0.001), whereas changes in nonviremic patients were nonsignificant. In conclusion, increased PD‐1 expression on CD28−CD27− CD4 T cells correlates with CMV viremia in transplant recipients and may serve as a specific, stimulation‐independent parameter to guide duration of antiviral therapy.

Cytomegalovirus-specific T cells are detectable in early childhood and allow assignment of the infection status in children with passive maternal antibodies.

Serological identification of the cytomegalovirus (CMV) status in children less than 18 months of age is complicated by the variable persistence of maternal antibodies. As T cells are not passively transferred, we attempted to assess whether CMV‐specific cellular immunity may be superior to determine the actual CMV status; we also performed a functional characterization of T‐cell immunity in childhood. Antibodies from 59 mothers and 168 children were determined, and specific CD4+ T cells were identified by induction of IFN‐γ, IL‐2, TNF‐α, IL‐4, and IL‐17 after CMV‐specific and polyclonal stimulation. Agreement between both tests was perfect for mothers and children more than 18 months. Among infants less than 18 months, 17/30 were concordantly negative. Interestingly, 8/13 seropositive children had detectable CMV‐specific T cells, whereas only 5/13 were T‐cell negative, indicating passive immunity. CMV‐specific T cells from young infants differed in cytokine profiles from that of older age groups, and polyclonal effector T‐cell frequencies were higher in young infants with detectable CMV‐specific T cells compared with those without. In conclusion, the majority of young infants with CMV‐specific antibodies show evidence of true infection, which indicates that passive immunity is overestimated. Our data may have important implications for improved risk stratification and CMV management in infants in the setting of transplantation.

The "ABC" of Virus-Specific T Cell Immunity in Solid Organ Transplantation.

Transplant patients are at increased risk of viral complications due to impaired control of viral replication, resulting from HLA mismatching between graft and host and the immunosuppression needed to avert alloimmune reactions. In the past decade, quantitative viral load measurements have become widely available to identify patients at risk and to inform treatment decisions with respect to immunosuppressive drugs and antiviral therapies. Because viral loads are viewed as the result of viral replication and virus‐specific immune control, virus‐specific T cell monitoring has been explored to optimize management of adenovirus, BK polyomavirus and cytomegalovirus (“ABC”) in transplant patients. Although most studies are descriptive using different technologies, the overall results show that the quantity and quality of virus‐specific T cells inversely correlate with viral replication, whereby strong cellular immune responses are associated with containment of viral replication. The key obstacles to the introduction of assays for virus‐specific T cells into clinical practice is the definition of reliable cutoffs for clinical decision making, the poor negative predictive value of some assays, and the absence of interventional trials justifying changes of antiviral treatment or immunosuppression. More clinical research is needed using optimized assays and targets before standardization and commutability can be envisaged as achieved for viral load testing.

Comparative analysis of assays for detection of cell-mediated immunity toward cytomegalovirus and M. tuberculosis in samples from deceased organ donors.

Cell‐mediated immunity assays could be valuable for risk assessment of organ donors, but no data exist on their feasibility in deceased donors. In this study, 105 deceased donors (52.3 ± 16.9 years) were screened at the time of organ procurement. Pathogen‐specific stimulation was performed using a cytomegalovirus (CMV) lysate, tuberculin (purified protein derivative [PPD]) and soluble Mycobacterium tuberculosis‐specific ESAT‐6/CFP‐10 proteins in combination with an in‐house fluorescence‐activated cell sorting (FACS) assay or commercial assay formats (QuantiFERON‐CMV/TB for ELISA, T‐SPOT.TB for ELISPOT). CMV‐IgG antibody titers were determined as gold standard for CMV infection; 51.4% of samples were CMV seropositive. Indeterminate results were observed in 47.6% of ELISA, 12.5% of FACS and 0% of ELISPOT assays. Agreement with serology was highest for FACS (95.6%, κ = 0.91), followed by ELISPOT (84.0%, κ = 0.68) and ELISA (80.0%, κ = 0.60). Agreement between ELISA and serology increased if the CMV lysate was used as stimulus (96.7%, κ = 0.92). Among the T cell assays, agreement between ELISPOT and FACS was highest (κ = 0.70). PPD‐positive results among valid samples differed between assays (26.5% for ELISA, 23.1% for FACS and 50.5% for ELISPOT); 2.0% were QuantiFERON‐TB positive, 3.3% were ESAT‐6/CFP‐10‐positive in FACS and 13.4% were positive in the T‐SPOT.TB assay. In conclusion, cellular immunity may be analyzed from samples of deceased donors, although the assays differ in the rate of positivity and indeterminate results.

Cytomegalovirus-specific T-cell immunity to assign the infection status in individuals with passive immunity: A proof of principle

BACKGROUND Serological analysis of the infection status with the human cytomegalovirus (CMV) may be inaccurate after transfusion of blood products due to the variable content of CMV-specific antibodies. OBJECTIVES In this situation, analysis of cellular immunity may represent a more accurate parameter to assign the individual CMV-infection status. This hypothesis was assessed in a sequence of clinically defined events where a CMV-seronegative patient received human immunoglobulins before AB0 incompatible transplantation of a graft from his CMV-seropositive mother and developed CMV-primary infection thereafter. STUDY DESIGN Humoral immunity was analyzed using ELISA, and CMV-specific CD4 T-cells were flow-cytometrically quantified using intracellular cytokine staining after a 6 h-stimulation with a CMV-antigen lysate. RESULTS Prior to transplantation, both CMV-specific antibody-titers and T-cell frequencies were below detection limit. After plasma infusion, the patient was temporarily seropositive but remained T-cell negative indicating passive immunity. CMV-specific T-cells became stably detectable after graft-related primary infection, thereby confirming a truly positive infection status. CONCLUSION This case provides an instructive proof of principle to show that CMV-specific CD4 T-cells may serve as an accurate marker to define the true CMV-infection status in situations where serological testing is limited by the presence of passively administered antibodies.

Detection of Antigen-Specific T Cells Based on Intracellular Cytokine Staining Using Flow-Cytometry

CMV-specific T cells may be detected and quantified after antigen-specific stimulation based on the induction of cytokines as a readout system. Secreted cytokines may be detected from the supernatant of stimulated cells using ELISA. Alternatively, antigen-specific cytokine-secreting cells may be enumerated using an ELISPOT assay. These assays generally rely on the detection of IFNγ and do not allow for a simultaneous assessment of several cytokines on a single cell basis. Here we describe a flow-cytometry based method to analyze CMV-specific CD4 T cells after specific stimulation with a whole antigen lysate. In this assay, cytokine secretion from stimulated cells is blocked by the addition of brefeldin A. Using a panel of fluorescently labeled antibodies, not only intracellularly accumulated cytokines but also phenotypical characteristics of specifically activated T cells may be quantified in a multiparameter staining approach.